Pesticide-induced resurgence in brown planthoppers is mediated by action on a suite of genes that promote juvenile hormone biosynthesis and female fecundity
- State Key Laboratory of Agricultural and Forestry Biosecurity, College of Plant Protection, Nanjing Agricultural University, China
- Department of Zoology, Stockholm University, Sweden
- College of Life and Environmental Sciences, Biosciences, University of Exeter, United Kingdom
- College of Sciences, Nanjing Agricultural University, China
Figures
Figure 1 with 3 supplements
Fecundity of brown planthopper (BPH) following exposure to sublethal (LC15) and median lethal (LC50) concentrations of emamectin benzoate following systemic 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 were not treated with insecticide. Insects were exposed as fourth-instar nymphs. (A) Left to right, n = 16, 16, and 16; (B) Left to right, n = 30, 17, and 31; (C) Left to right, n = 16, 16, and 16; (D) Left to right, n = 16, 16, and 16; (E) Left to right, n = 16, 15, and 16; (F) Left to right, n = 16, 16, and 16. All data are presented as the mean ± s.e.m. Different lowercase letters above the bars indicate significant differences (one-way ANOVA with Tukey’s multiple range test, p<0.05).
-
Figure 1—source data 1
Numerical source data for Figure 1.
- https://cdn.elifesciences.org/articles/91774/elife-91774-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
Effects of emamectin benzoate and abamectin on fecundity and residue dynamics in brown planthoppers.
(A) Fecundity of brown planthopper (BPH) when newly emerged adults were treated with sublethal (LD15) and median lethal (LD50) concentrations of emamectin benzoate (EB) via topical application. Left to right, n = 13, 13, and 13. (B) Fecundity of BPH when fourth-instar nymphs were treated with sublethal (LC15) and median lethal (LC50) concentrations of abamectin via systemic exposure. Left to right, n = 14, 14, and 13. (C) Residual levels of EB in BPH following treatment. Nymphs (fourth instar) were exposed to EB, and residues were quantified by HPLC-MS/MS method in nymphs (immediately post-treatment) and adults (after eclosion). No detectable EB residues were observed in adults (p = 0.0037, two-tailed t-test), indicating complete metabolic clearance or degradation during development. Left to right, n = 4 and 4. All data are presented as the mean ± s.e.m. Different lowercase letters above the bars indicate significant differences (one-way ANOVA with Tukey’s multiple range test, p<0.05).
-
Figure 1—figure supplement 1—source data 1
Numerical source data for Figure 1—figure supplements 1–3.
- https://cdn.elifesciences.org/articles/91774/elife-91774-fig1-figsupp1-data1-v1.xlsx
Figure 1—figure supplement 2
Fecundity of small brown planthopper, Laodelphax striatellus (A–C) white-backed planthopper, Sogatella furcifera (D–F) and vinegar fly, Drosophila melanogaster (G, H) when larvae and newly emerged adults were treated with sublethal concentrations of emamectin benzoate.
(A) Left to right, n = 18, 16, and 12; (B) Left to right, n = 15, 14, and 15; (C) Left to right, n = 15, 15, and 15; (D) Left to right, n = 23, 23, and 21; (E) Left to right, n = 23, 11, and 11; (F) Left to right, n = 23, 13, and 11; (G) Left to right, n = 9, 6, 6, and 9; (H) Left to right, n = 50, 56, and 47. All data are presented as the mean ± s.e.m. Different lowercase letters above the bars indicate significant differences (one-way ANOVA with Tukey’s multiple range test, p<0.05).
Figure 1—figure supplement 3
The impact of emamectin benzoate on the reproductive fitness of brown planthopper (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. Left to right, n = 10 and 10. (B) Emergence rate: the rate of emergence of adults. Left to right, n = 6 and 6. (C) Female ratio: the ratio of female (to male) insects in the total of emerged adults. Left to right, n = 6 and 6. (D) Female adult longevity. Control: n = 100 and emamectin benzoate: n = 100. (E) Brachypterism female ratio: the ratio of short-winged to long-winged adults. Left to right, n = 8 and 6. (F) The weight per female.Left to right, n = 51 and 41. All data are presented as the mean ± s.e.m. Different lowercase letters above the bars indicate significant differences (Student’s t-test, p<0.05).
Figure 2 with 3 supplements
The impact of emamectin benzoate (EB) on ovarian maturation in brown planthopper (BPH).
Fourth-instar nymphs were treated with the LC50 concentration of EB 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 = 1000 μm. (B) Number of mature eggs in the ovaries of EB-treated fourth-instar BPH nymphs measured at 1, 3, 5, and 7 DAE compared to controls. Left to right, n = 20, 19, 21, 21, 20, 20, 21, and 21. All data are presented as the mean ± s.e.m. Asterisks indicate values significantly different from the control using Student’s t-test (ns, no significant; *p<0.05 and **p<0.01). (C) Different developmental stages of BPH eggs. (D) No impairment of EB on oogenesis of BPH. Scale bar = 100 μm.
-
Figure 2—source data 1
Numerical source data for Figure 2.
- https://cdn.elifesciences.org/articles/91774/elife-91774-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Number of mature eggs in the ovaries of emamectin benzoate (EB)-treated brown planthopper (BPH) female adults compared to controls.
Left to right, n = 14, and 15.The data are presented as the mean ± s.e.m. Asterisks indicate values significantly different from the control using Student’s t-test (*p<0.05).
-
Figure 2—figure supplement 1—source data 1
Numerical source data for Figure 2—figure supplements 1 and 3.
- https://cdn.elifesciences.org/articles/91774/elife-91774-fig2-figsupp1-data1-v1.xlsx
Figure 2—figure supplement 2
The oogenesis of brown planthopper (BPH).
See the main text for detailed information. Stage 1: connected to the germarium, the egg is trapezoidal. Stage 2: the egg is larger than in stage 1 and is square to rectangular. Stage 3: the egg begins to elongate toward both poles, taking on an oval shape. Stage 4: the egg continues to enlarge and elongate, becoming spindle-shaped. Due to rapid growth, follicle cells on the egg surface start dividing, indicating nuclear division. Stage 5: the egg further enlarges, and most follicle cells display binucleation, the most notable feature of this stage. Small amounts of lipids start entering the egg. Stage 5 is the final stage before maturation, after which the egg enters the pedicel, a structure primarily formed by muscle fibers. The expanded mature egg stretches the muscle fibers in the pedicel, increasing the spacing between them. Stage 6: this is the mature egg stage, which occurs after the egg passes through the pedicel. During this stage, the egg undergoes rapid growth, continuously absorbing vitellogenin and lipids (Nile red staining confirmed that Stage 6 has the highest lipid content). This absorption causes the egg’s surface color to transition from the transparency seen in Stage 5 to mature eggs' dark, opaque appearance. Once the egg enters the pedicel and becomes completely opaque, it reaches full maturity. The mature egg then moves into the lateral oviduct for oviposition, completing the egg-laying process.
Figure 2—figure supplement 3
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 brown planthopper (BPH) exposure to emamectin benzoate (EB).
N = 3 for each point. 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’s t-test (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).
Figure 3 with 4 supplements
Emamectin benzoate (EB)-induced reproduction in brown planthopper (BPH) is mediated by components of the juvenile hormone (JH) signaling pathway.
(A) The titer of JH III (as measured by ELISA) at different developmental stages of whole-body BPH when fourth-instar nymphs were treated with the median lethal concentrations of EB. N = 3 for each point. (B, C) The titer of JH III (as measured by HPLC-MS/MS) in whole-body BPH females at 4L and 3 days after eclosion (DAE) when treated with median lethal concentrations of EB. N = 5 for panel B and n = 17 for panel C. (D) Oviposition rate of BPH when fourth-instar nymphs were treated with 4 ppm methoprene or 10 ppm pyriproxyfen. Left to right, n = 22, 14 and 21. (E–J) Expression of selected JH-related genes (FAMeT, JHAMT, Met, Kr-h1, Vg, and JHE) in EB-treated BPH. N = 8 for panel E-I and n = 10 for panel J. (K) Egg production following silencing of JHAMT with or without EB application. Left to right, n = 7, 7, 7 and 7. (L) Egg production following silencing of met with or without EB application. Left to right, n = 7, 8, 7 and 7. (M) Egg production after silencing Kr-h1 with or without EB application. Left to right, n = 7, 7, 7 and 7.All data are presented as means ± s.e.m. Student’s t-test was used to compare controls and treatments. 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 lowercase letters above the bars indicate significant differences (p<0.05).
-
Figure 3—source data 1
Numerical source data for Figure 3.
- https://cdn.elifesciences.org/articles/91774/elife-91774-fig3-data1-v1.xlsx
Figure 3—figure supplement 1
20-Hydroxyecdysone titer at different developmental stages of whole-body brown planthopper (BPH) when fourth-instar nymphs were treated with median lethal concentrations of emamectin benzoate (EB).
N = 3 for each point.
-
Figure 3—figure supplement 1—source data 1
Numerical source data for Figure 3—figure supplements 1–4.
- https://cdn.elifesciences.org/articles/91774/elife-91774-fig3-figsupp1-data1-v1.xlsx
Figure 3—figure supplement 2
Effects of Kr-h1 RNAi knockdown on Kr-h1 and Vg expression in brown Planthopper (BPH).
(A) Expression of Kr-h1 following RNAi knockdown. Left to right, n = 8 and 8. (B) Expression of Vg when Kr-h1 was silenced in brown planthopper (BPH). Left to right, n = 4 and 4. All data are presented as means ± s.e.m. *p<0.05; Mann–Whitney test.
Figure 3—figure supplement 3
Effects of emamectin benzoate on hormone titers and gene expression in brown planthopper female adults.
(A) The titer of juvenile hormone (JH) III (as measured by HPLC-MS/MS) in brown planthopper (BPH) females at adult stages after treatment with median lethal concentrations of emamectin benzoate (EB). Left to right, n = 4 and 4.(B) The titer of 20-hydroxyecdysone in BPH when female adults were treated with median lethal concentrations of EB. Left to right, n = 4 and 6. (C, D) Expression of JHAMT and Kr-h1 in EB-treated BPH female adults. Left to right, n = 6 and 5 for panel C. Left to right, n = 6 and 6 for panel D. All data are presented as means ± s.e.m. Student’s t-test was used to compare controls and treatments. Asterisks indicate values significantly different from the control (ns, no significant; *p<0.05).
Figure 3—figure supplement 4
Evaluation of the potential of emamectin benzoate (EB) to reverse dsRNA-mediated silencing by quantifying Kr-h1 gene expression.
Left to right, n = 4, 4, 4, and 4. Different lowercase letters above the bars indicate significant differences (one-way ANOVA with Tukey’s multiple comparisons test, p<0.05).
Figure 4 with 3 supplements
Emamectin benzoate (EB)-induced reproduction in brown planthopper (BPH) is mediated by the AstA/AstAR and juvenile hormone (JH) signaling pathway.
(A, B) Expression of AstA and AstAR in different stages of BPH following EB treatment. N = 8 for panel A and n = 12 for panel B. (C) Downregulation of AstAR using RNAi leads to a reduction in mRNA expression level. Left to right, n = 8 and 8. (D) Egg production in female BPH following silencing of AstAR gene. Left to right, n = 47 and 49. (E–I) Expression of selected JH signaling pathway related genes (JHAMT, Met, Kr-h1, and JHE) in AstAR silenced BPH. N = 4 for panel E-H. Left to right, n = 11 and 12 for panel I. (J) JH III titer of BPH females after AstAR gene silencing determined by HPLC-MS/MS. Left to right, n = 18 and 18. (K) Number of eggs laid per female in 48 h following injection of the six mature AstA1-AstA6 peptides and one mature AT peptide. Fifty nanoliters of PBS (as control) and seven different peptides (20 pmol/insect) were injected into female BPH 3 days after eclosion. Left to right, n = 16, 20, 22, 17, 18, 13, and 14. (L, M) The JH III titer of whole-body BPH females at different time points following AstA or AT injection. N = 15 for panel L and n = 16 for panel M. All data are presented as means ± s.e.m. Student’s t-test was used to compare controls and treatments. 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 lowercase letters above the bars indicate significant differences (p<0.05).
-
Figure 4—source data 1
Numerical source data for Figure 4.
- https://cdn.elifesciences.org/articles/91774/elife-91774-fig4-data1-v1.xlsx
Figure 4—figure supplement 1
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), and 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 Supplementary file 3.
Figure 4—figure supplement 2
Phylogenetic tree of the predicted brown planthopper (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 Supplementary file 3.
Figure 4—figure supplement 3
Emamectin benzoate (EB)-induced changes in the expression of AT (A, n = 8), AstB (B, n = 4), AstCC (C, n = 4), AstCCC (D, n = 4), ATR (E, n = 4), AstBR (F, n = 4), and AstCR (G and H, n = 4) in brown planthopper (BPH).
All data are presented as means ± s.e.m. Student’s t-test was used to compare controls and treatments. ns, no significant difference; asterisks indicate values significantly different from the control (*p<0.05, **p<0.01, and ***p<0.001).
-
Figure 4—figure supplement 3—source data 1
Numerical source data for Figure 4—figure supplement 3.
- https://cdn.elifesciences.org/articles/91774/elife-91774-fig4-figsupp3-data1-v1.xlsx
Figure 5 with 3 supplements
Role of emamectin benzoate (EB) and the GluCl channel in fecundity and juvenile hormone signaling in brown planthopper (BPH).
(A) Expression of GluCl in EB-treated and untreated BPH. N = 8 for each point. (B) Expression of GluCl following injection of dsGluCl in BPH. Left to right, n = 6 and 8. (C) Egg production after GluCl gene knockdown in EB-treated and untreated BPH. Left to right, n = 28, 28, 27, and 29. (D) The juvenile hormone (JH) III titer of whole-body BPH females after GluCl gene silencing as quantified using the ELISA method. Left to right, n = 15 and 15. (E–I) Expression patterns of selected JH-related genes (JHAMT, Met, Kr-h1, FAMeT and JHE) in GluCl silenced BPH. N = 8 for panel E–G and n = 4 for panel H and I. 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 lowercase letters above the bars indicate significant differences (p<0.05).
-
Figure 5—source data 1
Numerical source data for Figure 5.
- https://cdn.elifesciences.org/articles/91774/elife-91774-fig5-data1-v1.xlsx
Figure 5—figure supplement 1
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 nAChRα6 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 Supplementary file 4.
Figure 5—figure supplement 2
The expression of AT (A, n = 8), AstA (B, n = 8), AstB (C, n = 4), AstCC (D, n = 4), AstCCC (E, n = 4), AstAR (F, n = 8), AstBR (G, n = 4), and AstCR (H, n = 4) 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 lowercase letters above the bars indicate significant differences (p<0.05).
-
Figure 5—figure supplement 2—source data 1
Numerical source data for Figure 5—figure supplements 2 and 3.
- https://cdn.elifesciences.org/articles/91774/elife-91774-fig5-figsupp2-data1-v1.xlsx
Figure 5—figure supplement 3
The expression of GluCl after female adult brown planthopper (BPH) was treated with emamectin benzoate (EB).
N = 6 for each point. All data are presented as means ± s.e.m. Student’s t-test was used to compare the two samples, *p<0.05.
Figure 6
Schematic of the proposed regulatory pathway of emamectin benzoate (EB)-enhanced fecundity in brown planthopper (BPH).
EB exposure results in the upregulation of genes that promote juvenile hormone (JH) signaling pathway (JHAMT and Kr-h1) and the downregulation of genes that inhibit it (allatostatin, AstA and allatostatin A receptor, AstAR). This transcriptome reprogramming is dependent on the allosteric action of EB on its molecular target the glutamate-gated chloride channel (GluCl) receptor. Note that the mechanism of action on the GluCl is unknown in BPH, but likely the channel conformation changes and renders the receptor dysfunctional. Importantly, we do not suggest that the GluCl upregulation is due to direct action of EB on the channel. The resulting increased JH titer promotes vitellogenin (vg) biosynthesis 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 signaling downstream genes Met and Kr-h1.
Author response image 1
This illustration provides a visual representation of the brown planthopper (BPH), a major rice pest.
Additional files
-
Supplementary file 1
Determination of the toxicity of emamectin benzoate on BPH in systemic and topical application bioassays.
- https://cdn.elifesciences.org/articles/91774/elife-91774-supp1-v1.docx
-
Supplementary file 2
Sequences of oligonucleotide primers used in this study.
- https://cdn.elifesciences.org/articles/91774/elife-91774-supp2-v1.docx
-
Supplementary file 3
Amino acid sequences of neuropeptide G Protein-Coupled Receptors (GPCRs) and related receptors from multiple arthropod species.
- https://cdn.elifesciences.org/articles/91774/elife-91774-supp3-v1.docx
-
Supplementary file 4
Amino acid sequences of Glutamate-gated Chloride channels (GluCls) and acetylcholine receptors from diverse invertebrate species.
- https://cdn.elifesciences.org/articles/91774/elife-91774-supp4-v1.docx
-
MDAR checklist
- https://cdn.elifesciences.org/articles/91774/elife-91774-mdarchecklist1-v1.docx
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Pesticide-induced resurgence in brown planthoppers is mediated by action on a suite of genes that promote juvenile hormone biosynthesis and female fecundity
eLife 12:RP91774.
https://doi.org/10.7554/eLife.91774.4
