Characterization of the <i>mod</i> mutant.

<p>(A) Precocious metamorphosis observed in <i>mod</i> larvae. (left panel) Lateral and dorsal views and (middle panel) a magnified view of a larval-pupal intermediate. In intermediate animals, the new head capsule of the next instar (fifth) is formed (arrowhead). Beneath the old cuticles (asterisk), a new exoskeleton with larval eye spot markings (arrows) and brown-colored pupal cuticles are formed. (Right panel) Late-maturing trimolters form small cocoons and are able to develop into small but normal adults with normal fertility. (B) The developmental profiles of two batches of <i>mod</i> larvae (t011 strain). All of the larvae underwent precocious metamorphosis in the fourth instar, and no dimolters or tetramolters were observed. Larvae could be classified into two groups (early- and late-maturing trimolters) on the basis of the timing of onset of spinning. The numbers in parentheses indicate the sex of the moths (male/female). (C) Timing of the onset of spinning in <i>mod</i> (red, n = 178) and p50T (black, n = 28) strains after final larval molting. As highlighted by the grey ellipses, spinning was induced at two distinct timings in the <i>mod</i> strain, unlike the p50T strain. (D) Comparison of timings of the onset of spinning among early- and late-maturing trimolters of the <i>mod</i> strain and normal strain larvae that had been allatectomized (CAX) at the beginning of the fourth instar. Data on CAX larvae are from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002486#pgen.1002486-Fukuda1" target="_blank">[17]</a>; these larvae were reared at relatively low temperatures (23.0–25.5°C), which delays the timing of the onset of spinning to some extent. (E) Methoprene treatment of <i>mod</i> larvae. Selected doses of methoprene (0.01–10 µg/larva) were topically applied to newly molted third and fourth instar larvae (8–12 h after molting). As highlighted in blue, precocious pupation could be blocked by methoprene treatment. (F) Measurement of the JH titer in the hemolymph of third instar larvae of p50T and <i>mod</i> strains at 24 h after molting. Hemolymph was collected from ∼400 larvae using a microsyringe and the pooled sample was analyzed. JH in the hemolymph was converted to its corresponding methoxyhydrin derivatives and analyzed by GC-MS. JHs were not detected (ND) in the hemolymph of <i>mod</i> larvae.</p

Transgenic rescue of <i>mod</i>.

<p>(A) Visualization of <i>GAL4</i> expression in CA of the enhancer trap line <i>ET14</i> carrying the <i>UAS-GFP</i> construct. GFP expression (green) is limited to CA (arrowhead). Red fluorescence in the optic nerve is due to DsRed2 expression driven by the 3xP3 promoter <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002486#pgen.1002486-Horn1" target="_blank">[26]</a>. Br, brain; SOG, suboesophageal ganglion; and CA, corpus allatum. (B) Developmental profiles of binary GAL4/UAS transgenic lines. Male moths with a <i>w-1</i>; <i>mod</i> background and carrying <i>UAS-CYP15C1</i> were crossed with <i>w-1; mod</i> female moths carrying <i>ET14</i>, and their progenies were analyzed. Tetramolters appeared in GAL4/UAS transgenic lines, but not in nonbinary lines. (C) Images of pupae and moths of GAL4/UAS transgenic lines. Larvae carrying both <i>ET14</i> and <i>UAS-CYP151</i> constructs entered the fifth larval instar and eventually formed larger adults. Control animals did not carry transgenic vectors. (D) Measurement of the JH titer in the hemolymph of GAL4/UAS transgenic lines on the <i>w-1</i>; <i>mod</i> background. Hemolymph was collected from fourth instar larvae at 24 h after molting and analyzed. JH was detected only in GAL4/UAS lines, but not in nonbinary lines. ND, not detected.</p

Temporal and spatial expression of <i>CYP15C1</i>.

<p>(A) qRT-PCR analysis of the spatial expression of <i>CYP15C1</i> in the silkworm strain Kinshu×Showa. “<i>CYP15C1</i>/<i>rp49</i>” on the vertical axis indicates the level of <i>CYP15C1</i> mRNA normalized to that of internal <i>rp49</i> mRNA. RNAs were collected from larvae on day 1 of the fourth instar (4th D1), fourth instar larvae showing head capsule slippage (4th HCS), larvae on day 2 of the fifth instar (5th D2), and larvae on day 1 after the onset of spinning (Spin+1). CC-CA, corpus cardiacum-corpus allatum complex; PG, prothoracic gland; Br, brain; FB, fat body; MG, midgut; Ep, epidermis; Ms, muscle; Mp, Malpighian tubule; SiG, silk gland; SaG, salivary gland; Ts, testis; and Ov, ovary. (B) <i>In situ</i> mRNA hybridization of <i>CYP15C1</i> and <i>JHAMT</i>. Br-CC-CA complexes on day 2 of the fourth instar and day 4 of the fifth instar were used for analysis. Signals of both genes were limited to CA as indicated by arrows, but <i>JHAMT</i> was not detected on day 4 of the fifth instar. The purple coloration in the brain is primarily due to ommochrome pigments and does not reflect gene expression. The result of control experiments using sense probes are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002486#pgen.1002486.s002" target="_blank">Figure S2</a>. (C) Developmental changes in the rate of JH biosynthesis by <i>B. mori</i> CA <i>in vitro</i>. The data are based on Kinjoh et al. (2007). Black, red, and blue lines indicate CA from unsexed larvae, female and male animals, respectively. The activity in CA on day 1 of the fourth instar was set as 100. (D) Temporal expression patterns of <i>JHAMT</i> (upper) and <i>CYP15C1</i> (lower) in the Br-CC-CA (first and second instar larvae) or CC-CA (third to fifth instar larvae, pupae, and adults) complex. Developmental stages are defined as h/days after certain developmental events [i.e., molting, head capsule slippage (HCS), spinning, or emergence] or by a spiracle index (si) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002486#pgen.1002486-Kiguchi1" target="_blank">[56]</a>. Animals were unsexed during larval stages, while sexed during pupal and adult stages (female in red and male in blue). The expression profile of <i>JHAMT</i> after the second larval instar is based on published data (20). Expression levels measured on day 2 of the 4th larval instar are arbitrarily set at 100 (for actual transcript numbers per <i>rp49</i>) and are shown in a log scale. Asterisks indicate that data were not available.</p

Enzymatic properties of <i>B. mori</i> CYP15C1.

<p>(A) Enzymatic activity against FA. Medium containing FA was incubated with <i>Drosophila</i> S2 cells transiently expressing CYP15C1 (middle) or GFP (bottom), and analyzed by HPLC. Standard JHA III (top). Arrows indicate peaks of JHA III. (B) Stereospecificity. JHA III generated from FA by Sf9 cells stably expressing CYP15C1 (Sf9/CYP15C1) was chemically methylated and analyzed by a Chiral-HPLC. R and S indicate peaks of (<i>R</i>)- and (<i>S</i>)-JH III enantiomers, respectively. The <i>R</i>∶<i>S</i> ratio of standard racemic JH III (top) was 50∶50, while that of CYP15C1-produced JH III (bottom) was 97∶3. (C) The late JH biosynthetic step in <i>B. mori</i>, in which major JHs in the hemolymph are JH I and II <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002486#pgen.1002486-Kimura1" target="_blank">[28]</a>. Ethyl-branched farnesyl diphosphates (homo-FPPs) are converted to homo-FAs, epoxidized to JHAs by the cytochrome P450 epoxidase CYP15C1 (this study), and then methylated by the JHA methyltransferase (JHAMT) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002486#pgen.1002486-Shinoda1" target="_blank">[21]</a>. JH I: R1 = R2 = C₂H₅, JH II: R1 = C₂H₅, R2 = CH₃.</p

A model for JH biosynthetic pathway in the CA of wt and <i>mod</i> silkworms.

<p>(A) In the <i>B. mori</i> CA, constitutive CYP15C1 expression allows the consistent conversion of homo-FAs to JHAs (predominantly JHA I and II in Lepidoptera). When JHAMT is expressed in CA, JHAs are further converted to JHs, and released from CA, thereby preventing precocious metamorphosis. When JHAMT expression is shut off (e.g., in the prepupal stage), JHAs are likely to be released from CA. (B) In CA of the <i>mod</i> strain, homo-FAs are not converted to JHAs because of the loss of CYP15C1, but instead, homo-FAs are converted to ethyl-branched homologs of MF (homo-MFs, i.e., unepoxidized JH I and II) by JHAMT. The loss of CYP15C1 does not allow the conversion of homo-MFs to the authentic JHs. Therefore, neither JHs is synthesized in nor released from CA of the <i>mod</i> strain, thereby causing precocious metamorphosis. The synthesized homo-MFs might be released from CA of the <i>mod</i> strain, similar to that of higher dipteran insects <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002486#pgen.1002486-Jones1" target="_blank">[57]</a>. JH I: R1 = R2 = C₂H₅, JH II: R1 = C₂H₅, R2 = CH₃.</p