Fig 1.
Microplate simulation of juvenile hormone III (JH III)- or juvenile hormone analog (JHA)-dependent Met-Taiman or GCE-Taiman binding.
Yeast Y197 cells were transformed using yeast two-hybrid (Y2H) plasmids that contained Met-Taiman or GCE-Taiman and were incubated with different concentrations of JH III or JHAs. The JH III/JHA-mediated binding of Met-Taiman and GCE-Taiman was measured by absorbance values at 420 nm (OD420) after assaying β-galactosidase activity. Values and error bars indicate means ± SD (n = 3).
Fig 2.
Juvenile hormone disruptor (JHD) activity of 53 plant species against Met-Taiman and GCE-Taiman binding.
The JHD activities of 53 high-JHD plant species against Met-Taiman and GCE-Taiman binding were calculated as the average JHD activity of four independent experiments. Values in the rectangular box indicate the coefficients of determination (R2) for JHD activity of Met-Taiman and GCE -Taiman binding. 1, Lindera erythrocarpa; 2, Pinus densiflora; 3, Solidago serotina.
Fig 3.
Effect of Lindera erythrocarpa, Solidago serotina, and Pinus densiflora extracts and three juvenile hormone disruptor (JHD) diterpenes from L. erythrocarpa on the larval development of Drosophila melanogaster.
(A) Effect of L. erythrocarpa on the development of D. melanogaster larvae at a concentration-dependent manner. (B) Effect of 2% (w/v) S. serotina and P. densiflora extracts on the development of D. melanogaster larvae. (C) Effect of 2% (w/v) methyl lucidone, a JHD diterpene from L. erythrocarpa, on the development of D. melanogaster larvae.
Fig 4.
Juvenile hormone disruptor (JHD) activities of three Lindera erythrocarpa diterpenes against Met-Taiman and GCE-Taiman binding.
Dose-dependent inhibition of pyriproxyfen-mediated Met-Taiman binding or constitutive GCE-Taiman binding was observed for two L. erythrocarpa JHD diterpenes, i.e., methyl lucidone and methyl linderone. Kanakugiol did not significantly disrupt either Met-Taiman or GCE-Taiman binding. Values and error bars indicate means ± SD (n = 4).
Fig 5.
Larval and pupal development of Drosophila melanogaster fed sublethal doses of juvenile hormone analog (JHA) or disruptor (JHD).
Values and error bars indicate means ± SD (n = 3). *, p < 0.05 (t-test).
Fig 6.
Illumina RNA-seq transcriptome analysis of juvenile hormone analog (JHA, methoprene)- and disruptor (JHD, methyl lucidone)-treated Drosophila melanogaster larvae.
JHA, genes significantly affected by JHA; JHD, genes significantly affected by JHD; JHA/JHD, genes significantly affected by both JHA and JHD; JHA↑, genes significantly activated by JHA; JHD↓, genes significantly repressed by JHD; JHA↑JHD↓, genes significantly activated by JHA and significantly repressed by JHD.
Fig 7.
Transcriptome analysis of genes that were differentially expressed in juvenile hormone analog (JHA, methoprene)- and disruptor (JHD, methyl lucidone)-treated Drosophila melanogaster larvae.
JHA↑, genes significantly activated by JHA treatment; JHA↓, genes significantly repressed by JHA treatment; JHD↑, genes significantly activated by JHD treatment; JHD↓, genes significantly repressed by JHD treatment. (A) JHA↑JHD↓, genes significantly activated by JHA and significantly repressed by JHD; (B) JHA↓JHD↑, genes significantly repressed by JHA and significantly activated by JHD; (C) JHA↑JHD↑, genes significantly activated by both JHA and JHD; and (D) JHA↓JHD↓, genes significantly repressed by both JHA and JHD.
Table 1.
Overrepresented gene ontology groups among juvenile hormone (JH)-regulated genes.
Fig 8.
Gene ontology analysis of gene cohorts that were differentially expressed in juvenile hormone analog (JHA)- and disruptor (JHD)-fed D. melanogaster larvae.
Red indicates ontology groups with significant overrepresentation (P<0.01 in a hypergeometric distribution). The functional groups with corresponding abbreviations and colors are indicated. (A) JHA↑JHD↓, genes significantly activated by JHA and significantly repressed by JHD; (B) JHA↓JHD↑, genes significantly repressed by JHA and significantly activated by JHD; (C) JHA↑JHD↑, genes significantly activated by both JHA and JHD; and (D) JHA↓JHD↓, genes significantly repressed by both JHA and JHD.
Fig 9.
Tissue-specificity of genes that were differentially expressed in juvenile hormone analog (JHA)- and disruptor (JHD)-fed D. melanogaster larvae.
The genes significantly activated by JHA and significantly repressed by JHD (S2 Table) were applied to gene expression database constructed using Affymetrix microarray results to characterize tissue-specific expression. (A) Tissue-specificity of JHA↑JHD↓ genes which were significantly activated by JHA and significantly repressed by JHD; (B) Tissue-specificity of JHA↓JHD↑ genes which were significantly repressed by JHA and significantly activated by JHD; (C) Tissue-specificity of JHA↑JHD↑ genes which were significantly activated by both JHA and JHD; and (D) Tissue-specificity of JHA↓JHD↓ genes which were significantly repressed by both JHA and JHD.
Fig 10.
Validation of genes significantly activated by juvenile hormone analog (JHA) and significantly repressed by juvenile hormone disruptor (JHD).
(A) JHD- or JHA-dependent regulation of five randomly selected JHA↑JHD↓ genes: ocnus (ocn), janus B (janB), Glycine N-methyltransferase (Gnmt), Odorant-binding protein 99b (Obp99b), and Sperm-Leucylaminopeptidase 1 (S-Lap1). (B) JHD- or JHA-dependent regulation of three tTAF-dependent JHA↑JHD↓ genes: Male-specific RNA 87F (Mst87F), don juan (dj), and don juan-like (djl). Total RNA was extracted from the wandering third-instar larvae that were fed ethanol (control)-, methoprene (JHA)-, or methyl lucidone (JHD)-supplemented diet and analyzed using qPCR. Values and error bars indicate means ± SD (n = 3). *, p < 0.01 and **, p < 0.05 (t-test).
Fig 11.
Stage-specific regulation of Krüppel homolog 1 (Kr-h1) by juvenile hormone analog (JHA) and disruptor (JHD).
Total RNA samples were prepared from second-, early third-, and wandering third-instar larvae fed ethanol (control)-, methoprene (JHA)-, or methyl lucidone (JHD)-supplemented diets and analyzed using qPCR. Values and error bars indicate means ± SD (n = 3). *, p < 0.01 and **, p < 0.05 (t-test).