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

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

Visually impaired flies do not avoid the caged pantropical jumping spider.

<p>The mean % time spent in the middle zone adjacent to the central cage is shown for wild type and the indicated sensory mutants. Each genotype was examined with and without the presence of a pantropical jumping spider within the central cage. Significance differences are indicated by letters above each experimental groups. Groups sharing a letter are not different according to Dunn’s test (α = 0.0018). The middle line of each box plot represents the median, while upper and lower box display the 3^rd and 2^nd quartile respectively. The whiskers show the 90% confidence intervals.</p

<i>Drosophila</i> increase activity in the presence of pantropical jumping spiders and moving mock spiders.

<p>The changes in activity of the fly (cm/sec) and the probability for continued forward motion (P++) as a function of the minimum number of visits to each discrete section of the arena boundary (coverage) is shown. Both activity and P++ decrease as the fly habituates to the novel arena boundary [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180749#pone.0180749.ref022" target="_blank">22</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180749#pone.0180749.ref023" target="_blank">23</a>]. (a) In the presence of the pantropical jumping spider, Canton-S males significantly increase their activity during the early exploration of the arena, coverage 1–2. (b) Canton-S flies do not alter their P++ in the presence of the pantropical jumping spider. (c) The activity or (d) the P++ of Canton-S flies in the presences or absence of a mock spider were not significantly different. (e) Canton-S males display significantly greater activity in the presence of a moving mock spider as compare to an immobile mock spider. (f) The moving mock spider does not significantly change the P++ of Canton-S flies compared to the still mock spider. Shown are the mean +/- standard error of the mean for each data point. The lines show the regressions fit to <i>y</i> = <i>a</i> * (1 + <i>x</i>/<i>b</i>)^<i>c</i> for each experimental group.</p

<i>Drosophila</i> predator avoidance in a circular arena with an alcove.

<p>A diagram of the 8.2 cm diameter arena with a central cage and an alcove is shown in (a). To measure avoidance in this arena, the % time spent in the cove zone during the 10 min assay is shown. Canton-S males spend significantly more time in the cove zone when (b) a pantropical jumping spider (<i>Plexippus paykulli</i>) is present. In this arena, Canton-S flies did not significantly avoid (c) the twin-flagged jumping spider (<i>Anasaitis canosa</i>), (d) the Texas unicorn mantis (<i>Phyllovates chlorophaea</i>) nymph, nor (e) the non-predator milkweed bug (<i>Oncopeltus fasciatus</i>). ** p < 0.01. The middle line of each box plot represents the median, while upper and lower box display the 3^rd and 2^nd quartile respectively. The whiskers show the 90% confidence intervals.</p

Vision is needed to avoid a moving mock-spider.

<p>The avoidance of the artificial predator is measured as the mean % time spent in the middle zone adjacent to the central cage. (a) The presence of an immobile mock-spider in the central cage significantly reduced the % time in the middle zone. (b) Canton-S flies are placed in the arena with either a centrally located mock-spider or a blackened stir bar. The avoidance of the middle zone by Canton-S is then measured with these objects immobile (still) or rotating (mobile). The movement of the mock spider and the stir bar generate a significantly greater avoidance of the middle zone than the still objects. The avoidance of the moving mock spider is even greater than the moving stir bar. (c) The blind <i>norpA</i>^<i>7</i> mutants do not avoid the moving mock spider, suggesting a visual detection of the moving mock-spider is required for this avoidance response. The middle point of each box plot represents the median, while upper and lower box display the 3^rd and 2^nd quartile respectively. The whiskers show the 90% confidence intervals. Groups with different letters situated above the column are significantly different from each other.</p

Visually impaired flies do not avoid the caged pantropical jumping spider.

<p>The mean % time spent in the middle zone adjacent to the central cage is shown for wild type and the indicated sensory mutants. Each genotype was examined with and without the presence of a pantropical jumping spider within the central cage. Significance differences are indicated by letters above each experimental groups. Groups sharing a letter are not different according to Dunn’s test (α = 0.0018). The middle line of each box plot represents the median, while upper and lower box display the 3^rd and 2^nd quartile respectively. The whiskers show the 90% confidence intervals.</p

<i>Drosophila</i> predator avoidance in a circular arena.

<p>(a) A diagram of the 8.2 cm diameter arena with a central cage of 4.5 cm diameter to hold the predators is shown. To measure avoidance, the time spent in the middle zone adjacent to the central cage was measured. Wild type Canton-S flies spend significantly less time in the middle zone when (b) a pantropical jumping spider (<i>Plexippus paykulli</i>) is present. Canton-S did not significantly avoid (c) the twin-flagged jumping spider (<i>Anasaitis canosa</i>). (d) Wild type Canton-S males did avoid a Texas unicorn mantis (<i>Phyllovates chlorophaea</i>) nymph, (e) but not a non-predator milkweed bug (<i>Oncopeltus fasciatus</i>). ** p < 0.01, *p <0.05. The middle line of each box plot represents the median, while upper and lower box display the 3^rd and 2^nd quartile respectively. The whiskers show the 90% confidence intervals.</p