Figures and data
Determination of the toxicity of emamectin benzoate on BPH in systemic and topical application bioassays.
Fecundity of BPH following exposure to sub-lethal (LC15) and median lethal (LC50) concentrations of emamectin benzoate following system application bioassays (A: ♀t ×♂t; B: ♀t ×♂ck; C: ♀ck ×♂t) and topical application bioassays (D: ♀t ×♂t; E: ♀t ×♂ck; F: ♀ck ×♂t), respectively. The letter “t” represents treatment with insecticide, while “ck” indicates controls that was not treated with insecticide. These were exposed as 4th instar nymphs. All data are presented as the mean ± s.e.m. Different lower-case letters above the bars indicate significant differences (One-way ANOVA with Tukey’s Multiple Range Test, p < 0.05).
(A) Fecundity of BPH when newly emerged adults were treated with sub-lethal (LD15) and median lethal (LD50) concentrations of emamectin benzoate via topical application. (B) Fecundity of BPH when 4th instar nymphs were treated with sub-lethal (LC15) and median lethal (LC50) concentrations of abamectin via systemic exposure. All data are presented as the mean ± s.e.m. Different lower-case letters above the bars indicate significant differences (One-way ANOVA with Tukey’s Multiple Range Test, p < 0.05).
Fecundity of small brown planthopper, Laodelphax striatellus, (A-C) white backed planthopper, Sogatella furcifera (D-F) and fruit fly, Drosophila melanogaster (G and H) when larvae and newly emerged adults were treated with sub-lethal concentrations of emamectin benzoate. All data are presented as the mean ± s.e.m. Different lower-case letters above the bars indicate significant differences (One-way ANOVA with Tukey’s Multiple Range Test, p < 0.05).
The impact of emamectin benzoate on the reproductive fitness of BPH. Fourth instar nymphs were treated with the LC50 concentration of emamectin benzoate in systemic bioassays. (A) Preoviposition period: Preoviposition refers to the period in an insect’s life cycle between the time it becomes an adult and the time it starts laying eggs. (B) Emergence rate: the rate of emergence of adults; (C) Female ratio: the ratio of female (to male) insects in the total of emerged adults; (D) Female adult longevity. (E) Brachypterism female ratio: the ratio of short-winged to long-winged adults; (F) The weight per female. All data are presented as the mean ± s.e.m. Different lower-case letters above the bars indicate significant differences (Student’s t test, p < 0.05).
The impact of emamectin benzoate on ovarian maturation in BPH. Fourth instar nymphs were treated with the LC50 concentration of emamectin benzoate in systemic bioassays. (A) Ovarian development in EB treated BPH at 1, 3, 5 and 7 days after eclosion (DAE) compared to untreated controls. Scale bar = 1,000 μm. (B) Number of mature eggs in the ovaries of EB treated 4th instar BPH nymphs measured at 1, 3, 5 and 7 DAE compared to controls. All data are presented as the mean ± s.e.m. Asterisks indicate values significantly different from the control using student t test (ns, no significant; *p < 0.05 and **p < 0.01). (C) Different developmental stages of BPH eggs. (D) No impairment of emamectin benzoate on oogenesis of BPH. Scale bar = 100 μm.
Number of mature eggs in the ovaries of EB treated BPH female adults compared to controls. The data are presented as the mean ± s.e.m. Asterisks indicate values significantly different from the control using student t test (*p < 0.05).
The oogenesis of BPHs. See the main text for detailed information.
Amounts of Glycogen (A), TAG (B), total protein content (C), cholesterol (D) and four circulating sugars including sucrose, glucose, fructose and trehalose (E-H) after BPH exposure to EB. All data are presented as the mean ± s.e.m. The differences between the EB-treated and solvent-treated BPH were analyzed using unpaired student t-test (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).
EB induced reproduction in BPHs is mediated by components of the JH signaling pathway.
(A) The titer of JH III (as measured by ELISA assay) at different developmental stages of BPH when 4th instar nymphs were treated with the median lethal concentrations of EB. (B and C) The titer of JH III (as measured by HPLC-MS/MS) in BPH females at 4L and 3 DAE when treated with median lethal concentrations of EB. (D) Oviposition rate of BPH when 4th instar nymphs were treated with 4 ppm methoprene or 10 ppm pyriproxyfen. (E-J) Expression of selected JH-related genes (FAMeT, JHAMT, Met, Kr-h1, Vg, and JHE) in EB-treated BPH. (K) Egg production following silencing of JHAMT with or without EB application. (L) Egg production following silencing of met with or without EB application. (M) Egg production after silencing Kr-h1 with or without EB application. (N) Schematic illustrating the proposed impact of EB on the JH signaling pathway leading to enhanced reproduction. The question mark indicates one or more potential additional signals. All data are presented as means ± s.e.m. Student’s t test was used to compare the two samples. One-way ANOVA with Tukey’s multiple comparisons test was used to compare more than two samples. ns, no significant difference; Asterisks indicate values significantly different from the control (ns, no significant; *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001). Different lower-case letters above the bars indicate significant differences (p < 0.05).
Ecdysone titer at different developmental stages of BPH when 4th instar nymphs were treated with median lethal concentrations of EB.
(A) Expression of Kr-h1 following RNAi knockdown. (B) Expression of Vg when Kr-h1 was silenced in BPH. All data are presented as means ± s.e.m. *p < 0.05; Mann–Whitney test.
(A) The titer of JH III (as measured by HPLC-MS/MS) in BPH females at adult stages when treated with median lethal concentrations of EB. (B) The titer of ecdysone titer of BPH when female adults were treated with median lethal concentrations of EB. (C and D) Expression of JHAMT and Kr-h1 in EB-treated BPH female adults. All data are presented as means ± s.e.m. Student’s t test was used to compare the two samples. Asterisks indicate values significantly different from the control (ns, no significant; *p < 0.05).
EB induced reproduction in BPHs is mediated by the AstA/AstAR and JH signaling pathway.
(A and B) Expression of AstA and AstAR in different stages of BPH following EB treatment. (C) Downregulation of AstAR using RNAi leads to a reduction in mRNA expression level. (D) Egg production in female BPH following silencing of AstAR gene. (E-I) Expression of selected JH signaling pathway related genes (JHAMT, Met, Kr-h1 and JHE) in AstAR silenced BPH. (J) JHIII titer of BPH females after AstAR gene silencing determined by HPLC-MS/MS. (K) Number of eggs laid per female in 48h following injection of the six mature AstA1-AstA6 peptides and one mature AT peptide. Fifty nanoliter of PBS (as control) and seven different peptides (20 pmol/insect) were injected into female BPHs three days after eclosion. (L and M) The JH III titer of BPH females at different time points following AstA or AT injection. (N) Schematic of the proposed role of AstA/AstAR in the regulation of JH following EB exposure. The question mark indicates one or more possible additional signals. All data are presented as means ± s.e.m. Student’s t test was used to compare the two samples. One-way ANOVA with Tukey’s multiple comparisons test was used to compare more than two samples. ns, no significant difference; Asterisks indicate values significantly different from the control (ns, no significant; *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001). Different lower-case letters above the bars indicate significant differences (p < 0.05).
Alignments of the amino acid sequences of: (A) AT, (B) AstA, (C) AstB/MIP, (D) AstCC and (E) AstCCC peptides from select species. AT, AstA, AstB/MIP and AstCCC are predicted to have a C-terminal amide. The mature peptides belonging to the same species have been highlighted with the same color. Species names are as follows: Nillu (Nilaparvata lugens), Locmi (Locusta migratoria), Scham (Schistocerca americana), Homvi (Homalodisca vitripennis), Manse (Manduca sexta), Spofr (Spodoptera frugiperda), Drome (Drosophila melanogaster), Spoex (Spodoptera exigua), Nasvi (Nasonia vitripennis), Grybi (Gryllus bimaculatus), Bommi (Bombyx mori); Mesma (Mesobuthus martensii), Stear (Stegodyphus araneomorph), Limpo (Limulus polyphemus), Carma (Carcinus maenas), Strma (Strigamia maritima), Athro (Athalia rosae), Apime (Apis mellifera), Dapma (Daphnia magna). Black lines under the sequences indicate the locations of the disulfide bridges in the mature peptides. The accession numbers of the sequences are listed in Figure 4-figure supplement 1 source data.
Phylogenetic tree of the predicted BPH (*) allatotropin receptor (A16, ATR), allatostatins A receptor (A2, AstAR), AstB (MIP) receptor (A10, AstBR or MIPR) and allatostatins C receptor (A1, AstCR) with other insect species. The tree was generated using the maximum likelihood method. Drosophila melanogaster metabotropic glutamate receptor was included as an outgroup. The accession numbers of the sequences used for this phylogenetic tree are listed in Figure 4-figure supplement 2 source data.
EB induced changes in the expression of AT, AstB, AstCC, AstCCC, ATR, AstBR and AstCR in BPH.
All data are presented as means ± s.e.m. Student’s t test was used to compare the two samples. ns, no significant difference; Asterisks indicate values significantly different from the control (*p < 0.05, **p < 0.01, and ***p < 0.001).
EB induced reproduction in brown planthoppers through its molecular target protein GluCl.
(A) Expression of GluCl in EB-treated and untreated BPH. (B) Expression of GluCl following injection of dsGluCl in BPH. (C) Egg production after GluCl gene knockdown in EB-treated and untreated BPH. (D) The JH III titer of BPH females after GluCl gene silencing as quantified using the ELISA method. (E-I) Expression patterns of selected JH-related genes (JHAMT, Met, Kr-h1 and JHE) in GluCl silenced BPH. (J) Schematic of the proposed role of GluCl as a molecular target of EB and EB-enhanced reproduction in BPH. The question mark indicates one or more possible additional signals. All data are presented as means ± s.e.m. Student’s t test was used to compare the two samples. One-way ANOVA with Tukey’s multiple comparisons test was used to compare more than two samples. ns, no significant difference; Asterisks indicate values significantly different from the control (ns, no significant; *p < 0.05 and **p < 0.01). Different lower-case letters above the bars indicate significant differences (p < 0.05).
Phylogenetic analysis of glutamate-gated chloride channels in different species. The numbers at the nodes of the branches represent the percentage bootstrap support (1000 replications) for each branch. The Sogatella furcifera GABA-gated chloride channel and Nilaparvata lugens nAchR were used as outgroup. Alignment was performed with amino acid sequences from TM1-7. The receptor names are listed in the tree. The accession numbers of the sequences used for this phylogenetic tree are listed in Figure 5-figure supplement 1 source data.
The expression of AT, AstA, AstB, AstCC, AstCCC, AstAR, AstBR and AstCR in BPH injected with dsGluCl or dsgfp. All data are presented as means ± s.e.m. Student’s t test was used to compare the two samples. ns, no significant. Different lower-case letters above the bars indicate significant differences (p < 0.05).
The expression of GluCl after BPH female adult treated with EB. All data are presented as means ± s.e.m. Student’s t test was used to compare the two samples, *, p < 0.05.
Schematic of the proposed regulatory pathway of EB-enhanced fecundity in BPH.
Emamectin benzoate (EB) exposure results in the upregulation of genes that promote JH signaling pathway (JHAMT and Kr-h1) and the downregulation of genes that inhibit it (allatostatin, AstA and allatostatin A receptor, AstAR). This transcriptome reprograming is dependent on the action of EB on its molecular target the glutamate-gated chloride channel (GluCl) receptor. The resulting increased JH titer promotes vg synthesis and increased fecundity in EB exposed insects. We observe significant cross-talk in the expression of genes that inhibit JH production and those that promote it, with AstAR inhibiting the expression of JHAMT, Met and Kr-h1 and GluCl activating the expression of JHAMT which is responsible for JH synthesis, and the JH signalling downstream genes Met and Kr-h1.
