Expression of JHAMT-RNAi lowers the courtship index and can be rescued by application of the JH analog Methoprene.

<p>Graphs show the courtship index CI (fraction of time males spend courting during the observation period) ± SEM of the indicated genotypes (A, C), or the performance of males in a control activity assay (# of line crossings ± SEM) (B). Data were analyzed by ANOVA followed by Bonferroni multiple comparisons. A) Expression of <i>UAS-JHAMT RNAi</i> using our CA-specific <i>JHAMT-Gal4</i> driver significantly reduces male courtship in comparison to the control males. N = 20. (p < 0.001). Genotypes used were <i>UAS-dicer/Y; UAS-JHAMT-RNAi</i>/+; <i>JHAMT-GAL4/+;</i> and control genotypes <i>UAS-dicer/Y; UAS-JHAMT-RNAi/+; +/+</i> and <i>X/Y; +/+; JHAMT-GAL4/+</i>. B) Activity levels in the mutants and in control flies (genotypes as in A). Mutants are not different from the normally courting X/Y; +/+; <i>JHAMT-Gal4</i> control. (N = 10). C) Mature males of the genotypes described in (A) were treated with Methoprene in acetone or acetone alone, and tested four hours later. The experimental genotype shows complete rescue of the courtship index following Methoprene treatment compared to acetone treatment alone. (N = 20).</p

Conditional adult ablation of the corpora allata (CA) causes courtship defects that can be rescued by application of the JH analog Methoprene.

<p>The conditional Gal80^ts system was used to express UAS-DTI in adult males. The flies were created and maintained at 18°C. For induction, mature males were placed at 30°C for 2 days and then kept at room temperature for one day. The induced and un-induced experimental and control genotypes were subjected to courtship assay. Graphs show the courtship index CI (fraction of time males spend courting during the observation period) ± SEM of the indicated genotypes (A, C), or the performance of males in a control activity assay (# of line crossings ± SEM (B). Data were analyzed by ANOVA followed by Bonferroni multiple comparisons. (A) Courtship assay of induced and un-induced experimental <i>X/Y; Gal80</i>^<i>ts</i><i>/Gal80</i>^<i>ts</i><i>; JHAMT-GAL4/ UAS-DTI</i> and control genotypes <i>X/Y; Gal80</i>^<i>ts</i><i>/+; UAS-DTI/ +;</i> and <i>X/Y; Gal80</i>^<i>ts</i><i>/+; JHAMT-GAL4/+</i>. (N = 15). Experimental flies had significantly lower courtship than the controls (p<0.01). (B) Activity assay of the flies described in (A) (N = 10). (C) Methoprene in acetone or acetone alone was applied to induced males four hours prior to testing for courtship. Methoprene application completely rescued the courtship defect of experimental flies (P<0.001). (N = 20). (Un-induced (-), Induced (+).</p

Conditional reduction of JHAMT in mature males causes courtship defects that can be rescued by application of the JH analog Methoprene.

<p>Graphs show the courtship index CI (fraction of time males spend courting during the observation period) ± SEM of the indicated genotypes (A, C), or the performance of males in a control activity assay (# of line crossings ± SEM (B). Data were analyzed by ANOVA followed by Bonferroni multiple comparisons. (A) Courtship index of induced and un-induced experimental (<i>X/Y; UAS-JHAMT-RNAi/+; hsp70-GAL4/+</i>) and control (<i>X/Y; +/+; hsp70-GAL4/+;</i> and <i>X/Y; UAS-JHAMT-RNAi/+;+/+</i>) genotypes. The experimental genotype shows a significant reduction in courtship index (p = 0.0007). (N = 20). Induced flies were heat-shocked at 37°C for 1 hour and let recover for four hours. (B) Activity assay of the induced and un-induced genotypes; genotypes as in (A) (n = 10). (C) Genotypes as in (A); one hour after induction, flies were treated with Methoprene in acetone, or with acetone alone, and courtship was examined 4 hours later. The experimental genotype shows complete rescue (p< 0.003) (N = 20). (Un-induced (-), Induced (+).</p

JHAMT-Gal4 driver directs expression in the corpora allata (CA).

<p>Frozen sections of adult <i>JHAMT-Gal4/UAS-lacZ</i> males were incubated with an anti-ßGal antibody (red) and with an anti-JHAMT antibody (green). Overlapping expression in the CA (marked by arrow) was observed. H:Head; T:Thorax</p

Feminization of the Adult, but Not the Larval, Fat Body Affects Male Courtship

<p>(A and B) CIs of various males toward <i>Canton-S</i> virgin females. Males carrying the <i>UAS-traF</i> (black bar) or the <i>Lsp2-Gal4</i> (white bars) transgenes individually are compared to <i>0.68Lsp2-Gal4/UAS-traF</i> males (A, gray bars) and <i>3.1Lsp2-Gal4/UAStraF</i> males (B, red bars). Courtship was reduced only in <i>3.1Lsp2-Gal4/UAS-traF</i> males. The results from two different transgenic <i>Lsp2-Gal4</i> lines (designated a and b) are shown for each transgene (<i>n</i> = 10 for each group; **indicates indices that were significantly different from those of parental strains, <i>p</i> < 0.001). Performance in a short-term activity assay (number of line crossings) is shown for <i>3.1Lsp2-Gal4/UAS-traF</i> males and the corresponding parental lines (C) (<i>n</i> = 10 for each group). No differences between genotypes were observed.</p

Stage-Specific Fat Body Drivers

<p>Fat body (fb)-specific Gal4 activity driven by the 3.1-kb <i>Lsp2</i> promoter fragment (A–D) or the 0.68-kb <i>Lsp2</i> promoter fragment (E–H) was detected in larvae and in sections from adult heads and bodies using a <i>UAS-dsRed</i> (A and E) or a <i>UAS-lacZ</i> reporter gene (B–D and F–H). Staining in larvae and flies younger than 2 d [representing larval fat body staining (A, B, E, and F)] and in 1-wk-old flies [adult fat body staining (C, D, G, and H)] is shown. For reference, the positions of the central brain (cb), optical layers of the brain (ol), mouthparts (mp), gut (g), salivary glands (sg), and fat body (fb) are indicated.</p

Masculinization of the Fat Body Enhances the Courtship of <i>Fru^M</i> Females

<p>CIs of various females toward <i>Canton-S</i> virgin females are shown. All animals were <i>Fru^M</i>, carried the <i>Lsp3.1-Gal4</i> (Lsp) fat body driver on a <i>fru⁴</i> chromosome, and were female (except for column 5). The presence of two copies of <i>tra2-IR</i> significantly enhanced courtship, which was further increased when the females were heterozygous for <i>tra-2^B/+</i> at the same time. Column 5: Control male siblings of the females in column 4. Animals were kept at 29 °C following eclosion until testing which was done at room temperature (<i>n</i> = 10 for each group; **indicates indices that were significantly different from those of <i>Fru^M</i> females [column 1], <i>p</i> < 0.05). The CI of <i>tra2-IR/tra2-IR; tra-2^B/+; Lsp, fru^4/Fru^M</i> females is statistically not different from that of the control males.</p

Takeout Is Present in the Hemolymph and Is Functional when Expressed from Oenocytes

<div><p>(A) Takeout is present in the hemolymph. Western blot of isolated hemolymph from wild-type (<i>Canton-S [CS]</i>) males and females and of <i>to¹</i> mutant males probed with anti-Takeout antiserum as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0030016#pgen-0030016-g004" target="_blank">Figure 4</a>A. Hemolymph from an equal number of animals was loaded in each lane. An approximately 27-kDa protein was recognized in hemolymph from male flies, but not from females and <i>to¹</i> mutant males. A background band (*) is shown to visualize comparable protein amounts in all lanes.</p><p>(B) Takeout expression in oenocytes rescues the <i>takeout</i> courtship defect. CIs of various males towards <i>Canton-S</i> virgin females are shown. Males carrying the <i>UAS-takeout (UAS-to)</i> or the <i>Oenocyte-Gal4 (Oen-Gal4)</i> transgenes individually in a <i>fru⁴, to¹/+, to¹</i> background (columns 1 and 2) are compared to <i>Oen-Gal4/UAS-to</i> males in the same genetic background (columns 4 and 5). Results for two different <i>Oen-Gal4</i> drivers are shown (red bars, columns 4 and 5) (<i>n</i> = 10 for each group; ** indicates indices that were significantly different from those of mutant <i>fru⁴, to¹/+, to¹</i> males [column 3], <i>p</i> < 0.01). Column 6: Control males. All males had wild-type eye color. The better performance of males with the <i>Oen-Gal4</i> driver only (compared to just the mutant alone) is likely due to background effects.</p></div

Reduction of <i>CG4395</i> RNA in <i>GH146</i> neurons reduces courtship.

<p>Graphs show the courtship index CI (fraction of time males spend courting during the observation period) ± SEM of the indicated genotypes, N = 10. Expression of <i>4395-RNAi</i> in <i>GH146</i> projection neurons reduces male courtship in both white and red light (A, B). (C) Optical sections showing co-expression of <i>UAS-mcD8::GFP/4395-Gal4</i> (magenta) and <i>UASQ-tomato::GH146-Q</i> (green) in antennal glomeruli. (D) <i>CG4395^f06077</i> mutant males missing the third antennal segment court less than control males.</p

Analysis of courtship elements in the mutants.

<p>Individual courtship steps in a standard courtship assay were analyzed for males of the indicated genotypes paired with a wild-type virgin female. Values are mean ± SEM (N = 10). Latency: The time to first orientation toward the female is indicated. For wing extension, the relative time engaged in this behavior relative to the total time spent courting was calculated. For attempted copulation, the total number of events is given. Copulation was not scored since the females were only a few hours old and resisted copulation.</p><p>*Values that were significantly different from those of the control flies.</p