Figures and data
CRZ signaling affects the post-mating response (PMR) and oviposition in female N. lugens.
Wild-type males were used in all behavioral assays. (A) Experimental design for panels B-C. The white/black segments on the experimental time lines denote light/dark periods, respectively. (B) Receptivity of virgin females 6 h post-injection, using different doses of CRZ (sCRZ as control), each virgin female BPH was injected with a calibrated glass capillary needle directly into the abdomen with 100 ng/10 ng/1 ng/0.1 ng of peptide dissolved in 50 nL of 1 × PBS balanced salt solution. The graph shows percentage females copulating within 30 min. **P < 0.01 vs. control; ns (non-significant): P > 0.05 (Mann-Whitney test). The numbers above the curves denote total number of animals. (C) Eggs laid per mated female 12 h post-copulation. We used the highest CRZ dose from panel B (sCRZ, control). Each mated female BPH was injected with a calibrated glass capillary needle directly into the abdomen with 100 ng of peptide dissolved in 50 nL of 1 × PBS balanced salt solution. *P < 0.05 (Student’s t-test). Data: mean ± s.e.m (≥3 biological replicates, ≥8 insects/replicate). The numbers below the bars denote total number of animals. (D) Experimental design for panels E-F. (E) Receptivity of virgin (1st mating) and mated females after Crz-RNAi (dsgfp as control). Graph displays the percentage of females copulating within 30 min. The small circles denote the number of replicates; the numbers below the bars denote total number of animals. Data are shown as mean ± s.e.m. *P < 0.05, and ns (non-significant), P > 0.05, two-way ANOVA followed by Holm-Šídák’s multiple comparisons test. (F) Total numbers of eggs laid per female over 72 h after Crz-RNAi (dsgfp as control). ****P < 0.0001 (Student’s t-test). The small circles and the numbers below the bars denote total number of animals. Data are shown as mean ± s.e.m. (≥3 biological replicates, ≥8 insects/replicate). (G) Schematic diagram of the Crz gene and sgRNA (single-guide RNA) design for Crz mutant production. The Crz gene consists of 2 exons, and ATG and TAG are located on Exon 1. The target site of the 20 bp sgRNA on Exon 1 is highlighted in pink. The PAM sites are indicated in yellow. The two knockout strains ΔCrz1and ΔCrz2 were unable to produce mature CRZ peptides. (G’) The amino acid sequences obtained by translating the mutated base sequences. (H) Absence of CRZ immunoreactivity in homozygous ΔCrz1 and ΔCrz2 mutants compared to wild type (WT) expression. Scale bars: 50 μm. (I) Experimental design for panels J-K. (J) Receptivity of virgin/mated females across genotypes. No effect was seen on virgin receptivity, only the re-mating was affected. The small circles denote the number of replicates; the numbers below the bars denote total number of animals. Data are shown as mean ± s.e.m. **P < 0.01; ***P < 0.001, and ns (non-significant), P > 0.05, two-way ANOVA followed by Holm-Šídák’s multiple comparisons test. (K) Eggs laid 72 h post-mating. The numbers below the bars denote total number of animals. Data are shown as mean ± s.e.m. ****P < 0.0001; Student’s t test.
Evolutionary conservation of CRZ peptide sequences in Nilaparvata lugens and other arthropods.
(A) Alignment of mature CRZ peptide sequences across species. Abbreviations of species names are as follows: Bmori (Bombyx mori), Gmole (Grapholita molesta), Cpomo (Cydia pomonella), Sexig (Spodoptera exigua), Harmi (Helicoverpa armigera), Dplex (Danaus plexippus), Dmela (Drosophila melanogaster), Csupp (Chilo suppressalis), Dmagn (Daphnia magan), Dviri (Drosophila virilis), Cnodu (Catagly nodus), Carcu (Callinectes arcuatus), Nnorv (Nephrops norvegicus), Cmaen (Carcinus maenas), Mrose (Macrobrachium rosenbergii), Mscal (Megaselia scalaris), Cglom (Cotesia glomerata), Rmicr (Rhipicephalus microplus), Derec (Drosophila erecta), Agamb (Anopheles gambiae), Pstal (Plautia stali), Nluge (Nilaparvata lugens), Cdraw (Caerostris darvini), Cextr (Caerostris extrusa), Dpule (Daphnia pulex), Hitam (Heterotrigona itama), Lmigr (Locusta migratoria), Cmoro (Carausius morosus), Sgreg (Schistocerca gregaria), Lsalm (Lepeophtheirus salmonis), Alabo (Apis laboriosa). Species within the blue area possess highly conserved mature peptide sequences, whereas those in the magenta zone exhibit relatively less conservation in their mature peptides. (B) Schematic organization of CRZ precursors in representative species. Signal peptides (blue) and mature CRZ peptides (red) are indicated.
The Crz gene-silencing efficacy in female insects following dsRNA injection assayed by qPCR.
The small circles denote the number of replicates. Data are shown as mean ± s.e.m. Mann–Whitney test. ***P<0.001.
Phenotypic effects of CRZ peptide injection and Crz gene knockout.
(A) Receptivity of virgin females at indicated time points after CRZ injection (sCRZ as control). Each virgin female BPH was injected with a calibrated glass capillary needle directly into the abdomen with 100 ng of peptide dissolved in 50 nL of 1 × PBS balanced salt solution. Graph shows percentage females copulating within 30 min. ****P < 0.0001; ns: not significant (Chi-square test). The numbers associated with the curves denote total number of animals. (B) Eggs laid per virgin female after neuropeptide injection (sCRZ, control). Females were mated with wild-type males for 12 h prior to assessment. Each virgin female BPH was injected with a calibrated glass capillary needle directly into the abdomen with 100 ng of peptide dissolved in 50 nL of 1 × PBS balanced salt solution. Data are shown as mean ± s.e.m. ns: P > 0.05 (Student’s t-test). The numbers below the bars denote total number of animals. We used at least four biological replicates with at least ten insects per replicate for each experiment.
Characterization of Crz mutants in N. lugens.
(A) Sequencing of Crz mutant (ΔCrz1 and ΔCrz2) and wild type (WT) PCR products from BPHs. The exact indel types of the G0 mutant individuals were confirmed by cloning and sequencing. The wild-type sequences are shown at the top with the target site marked by in pink shading and PAM in yellow shading. The change in the length of the mutant sequence is shown on the right side of the sequence (+, insertion; -, deletion). (B) The Sanger sequence chromatograms flanking the sgRNA target site for wild type (WT). (C and D) The Sanger sequence chromatograms flanking the sgRNA target site for wild type (WT), heterozygous mutant (ΔCrz1/+and ΔCrz2/+). (C’ and D’) The Sanger sequence chromatograms flanking the sgRNA target site for wild type (WT), homozygous mutant (ΔCrz1 and ΔCrz2). (E) Developmental duration (egg to adult) across genotypes. ****P < 0.0001 (Student’s t-test). The numbers below the bars denote total number of animals. (F) Survival curves of adult Crz mutants vs. WT. *P < 0.05 (Log-rank Mantel-Cox test; df = 1; n ≥ 50/group).
Expression of Crz and CRZ in the nervous system of the brown planthopper (BPH).
(A) Schematic of primer design for Crz detection (primer sequences provided in Table S1). (B) RT-PCR analysis of Crz mRNA levels in various BPH tissues. Actin served as the loading control. Tissues: Nervous system (NS), fat body (FB), Epidermis (EP), digestive system (DS), female reproductive organs (FRO), male reproductive organs (MRO). Note: Tissues except reproductive organs were pooled from both sexes. (C) qPCR analysis of Crz mRNA levels in BPH tissues (abbreviations as in B). (D and E) Immunolocalization of CRZ peptide in the nervous system of male (D) and female (E) BPHs. Four pairs of CRZ neurons in the brain and one pair in the subesophageal ganglion are shown. Note that some of the CRZ neurons are likely to be neurosecretory cells with terminations in the corpora cardiaca, others may be interneurons. The extensive branches within the brain suggest CRZ signaling in brain circuits. VNC, ventral nerve cord. Scale bars: 50 μm. (E) CRZ immunolabeling in the nervous system of female BPHs. Scale bar: 50 μm.
CRZ immunolabeling in abdominal ganglion and reproductive organs of male and female BPHs.
(A) Anti-CRZ immunolabeling in the abdominal ganglia of the ventral nerve cord (VNC) in N. lugens and Drosophila melanogaster (D. Melan.). Scale bar: 50 μm. Note: Male-specific CRZ-positive neurons are present in D. melanogaster, as previously reported. (B) Lack of CRZ immunoreactivity in BPH reproductive organs. (B1-B3) Male reproductive organs (MRO): testes (test). (B4-B6) Female reproductive organs (FRO): lateral oviduct (Lat. ov), common oviduct (Com. ov). Scale bars: 100 μm.
The CRZ receptor (CrzR) is essential for the female PMR and is required for action of MAG-derived factors.
(A) Experimental design for B and C. (B) Receptivity after CrzR-RNAi (injection of dsCrzR; dsGFP as control). Percentage females copulating within 30 min. The small circles denote the number of replicates; the numbers below the bars denote total number of animals. Data are shown as mean ± s.e.m. *P < 0.05, and ns (non-significant), P > 0.05, two-way ANOVA followed by Holm-Šídák’s multiple comparisons test. (C) Numbers of eggs laid within 72 h post-mating (dsCrzR females × WT males). Data are shown as mean ± s.e.m. ****P < 0.0001 (Student’s t-test). The numbers below the bars denote total number of animals. (D) Experimental design for E-F. (E) Receptivity after CRZ injection in CrzR-knockdown virgins (dsGFP + CRZ control). Graph shows percentage females copulating within 30 min. Each virgin female BPH was injected with a calibrated glass capillary needle directly into the abdomen with 10 ng of peptide dissolved in 50 nL of 1 × PBS balanced salt solution. The small circles denote the number of replicates; the numbers below the bars denote total number of animals. Data are shown as mean ± s.e.m. **P < 0.01 (Mann-Whitney test). (F) Receptivity after MAG extract (with seminal fluid proteins) injection in CrzR-knockdown virgins (dsGFP + SFP control). Percentage copulating within 30 min. The small circles denote the number of replicates; the numbers below the bars denote total number of animals. Data are shown as mean ± s.e.m. **P < 0.01 (Mann-Whitney test). (G) CrzR targeting strategy for generation of mutants. Top: Genomic structure (ATG/TAG in Exons 2/7). sgRNA target (pink), PAM (yellow). Bottom: CRISPR alleles. (G’) Translated receptor mutant sequences. (H) Experimental design for I-J. (I) Receptivity of CrzR mutants compared to wild type animals (WT). The small circles denote the number of replicates; the numbers below the bars denote total number of animals. Data are shown as mean ± s.e.m. *P < 0.05, ***P < 0.001, two-way ANOVA followed by Holm-Šídák’s multiple comparisons test. (J) Number of eggs laid by mutants mated with WT males within 72 h. The numbers below the bars denote total number of animals. Data are shown as mean ± s.e.m. ****P < 0.0001; Student’s t test. (K) Experimental design for L-M. (L) Receptivity after CRZ injection in CrzR mutants compared to WT. Each virgin female BPH was injected with a calibrated glass capillary needle directly into the abdomen with 10 ng of peptide dissolved in 50 nL of 1 × PBS balanced salt solution. The small circles denote the number of replicates; the numbers below the bars denote total number of animals. Data are shown as mean ± s.e.m. **P < 0.01, Mann–Whitney test. (M) Receptivity after SFP injection in CrzR mutants compared to WT. The small circles denote the number of replicates; the numbers below the bars denote total number of animals. Data are shown as mean ± s.e.m. **P < 0.01, Mann–Whitney test. (N) Receptivity after macc injection in CrzR mutants compared to WT. The small circles denote the number of replicates; the numbers below the bars denote total number of animals. Data are shown as mean ± s.e.m. **P < 0.01, Mann–Whitney test.
Structural conservation of the CrzR across insect species.
(A) Multiple sequence alignment highlighting conserved transmembrane domains (TM1-TM7, shaded) in CRZ receptor orthologs. The species shown are: N. lugens: Nilaparvata lugens; M. sexta: Manduca sexta; B. mori: Bombyx mori; A. gamb: Anopheles gambiae; D. mel: Drosophila melanogaster. (B) Phylogenetic analysis of CrzR and AkhR based on the alignment of amino acid sequences of insect species. Phylogenetic tree was constructed using MEGA 12 software with the Maximum Likelihood Method and bootstrapped with 10000 replications. The numbers at the nodes of the branches represent the numbers of bootstrap replications supporting that branch.
Characterization of CrzR mutants in N. lugens.
(A) Sanger sequencing of CrzR mutants. WT sequence with target (pink), PAM (yellow). Indels: + (insertion), – (deletion). (B) Sanger sequencing chromatograms of CrzR alleles: WT (wild-type). (C) Sanger sequencing chromatograms of CrzR alleles: heterozygous (CrzRM/+). (C’) Sanger sequencing chromatograms of CrzR alleles: homozygous (CrzRM) mutants. (D) Developmental duration (egg to adult eclosion) in CrzR mutants compared to WT. Data: mean ± SD. ****P < 0.0001 (Student’s t-test; n ≥ 40/group). (E) Survival curves of adult CrzR mutants compared to WT. ***P < 0.001 (Log-rank Mantel-Cox test; df = 1; n ≥ 50/group).
Spatial expression profiling of CrzR in the female N. lugens reproductive tract.
(A) CrzR genomic structure with primer design for experiments in panels B-C. (B) RT-PCR analysis of CrzR tissue distribution. Note weaker expression in central nervous system and fat body. Actin loading control is shown in top row. Tissue abbreviations: NS: central nervous system; FB: fat body; EP: Epidermis; DS: digestive system, FRO: female reproductive organs, MRO: male reproductive organs. Except for the reproductive system, the rest of the tissues come from a mixture of female and male. (C) qPCR quantification of CrzR transcript levels (abbreviations as in B). Data are shown as mean ± s.e.m. Groups that share at least one letter are statistically indistinguishable. One-way ANOVA followed by Tukey’s multiple comparisons test. (D1 - D2’) CrzR mRNA localization via fluorescent in situ hybridization (antisense probe): D1: There is no signal in lateral/common oviducts; D2/D2’: Specific signal is detected in spermatheca (SP) and pouched gland (PG). (D3 - D4’) Negative controls (sense probe) showing background-level signal. Scale bars: 100 μm.
Developmental and tissue-specific expression profiling of CrzR in N. lugens determined by qPCR.
(A) CrzR transcript levels across developmental stages. One-way ANOVA followed by Tukey’s multiple comparisons test. (B) CrzR expression in distinct body regions. Data are shown as mean ± SEM shown. One-way ANOVA followed by Tukey’s multiple comparisons test.
CRZ signaling in male brown planthoppers affects the induction of PMR in females.
(A, D, G, J) Experimental protocols for the behavioral assays presented in panels B/C, E/F, H/I, and K/L, respectively. (B) Receptivity of virgin and mated wild-type females, scored as the percentage copulating within 30 min when paired with males injected with CRZ (sCRZ as control). Only the re-mating is affected. Small circles indicate the number of replicates; numbers below bars denote total animals. Data are mean ± s.e.m. *P < 0.05, **P < 0.01 (two-way ANOVA followed by Holm-Šídák’s multiple comparisons test). (C) Number of eggs laid per wild-type female within 72 h after mating with males injected with CRZ (sCRZ as control). Numbers below bars denote total animals. Data are mean ± s.e.m. ***P < 0.001 (Student’s t-test). Experiments involved ≥4 biological replicates with ≥ 8 insects per replicate. (E) Receptivity of virgin and mated wild-type females, scored as the percentage copulating within 30 min when paired with Crz-RNAi-treated males (dsGFP as control). Again, only re-mating is affected. Small circles indicate the number of replicates; numbers below bars denote total animals. Data are mean ± s.e.m. **P < 0.01, ns (P > 0.05) (two-way ANOVA followed by Holm-Šídák’s multiple comparisons test). (F) Number of eggs laid per wild-type female within 72 h after mating with Crz-RNAi-treated males (dsGFP as control). Data are mean ± s.e.m. ****P < 0.0001 (Student’s t-test). Experiments involved ≥4 biological replicates with ≥ 8 insects per replicate. (H) Receptivity of virgin and mated wild-type females, scored as the percentage copulating within 30 min when paired with males of wild type (WT) and CRZ mutants (ΔCrz1, ΔCrz2 and ΔCrz1/2). Small circles indicate the number of replicates; numbers below bars denote total animals. Data are mean ± SEM. **P < 0.01, ***P < 0.001, ns (P > 0.05) (two-way ANOVA followed by Holm-Šídák’s multiple comparisons test). (I) Number of eggs laid per wild-type female within 72 h after mating with males of different genotypes (as in H). Numbers below bars denote total animals. Data are mean ± SEM. ****P < 0.0001 (Student’s t-test). Experiments involved ≥ 4 biological replicates with ≥ 8 insects per replicate. (K) Crossing male and female Crz mutant insects yield a stronger phenotype than when only males are mutants. Receptivity of virgin and mated females when using Crz mutants of both males and females, with wild-type as controls, scored as the percentage copulating within 30 min when paired with males of different genotypes. Compared to Fig. 2H and 2I, double Crz knockouts exhibit a stronger effect on receptivity. Small circles indicate the number of replicates; numbers below bars denote total animals. Data are mean ± SEM. ***P < 0.001, ****P < 0.0001, ns (P > 0.05) (two-way ANOVA followed by Holm-Šídák’s multiple comparisons test). (L) Number of eggs laid per female in different genotypic mating combinations. Again phenotypes are stronger compared to those observed with only male mutants (Fig. 2I). Numbers below bars denote total animals. Data are mean ± SEM. ****P < 0.0001 (Student’s t-test). Experiments involved ≥4 biological replicates with ≥ 8 insects per replicate.
The effects of CRZ neuropeptide injection on the behavior and physiology of male BPHs.
(A) CRZ peptide injection into the terminal abdomen of male BPHs does not affect ejaculation. The table shows the ejaculation frequency within 30 minutes post-injection. (B) Morphology of male BPH accessory glands following Crz gene knockout. Representative images show accessory glands from Crz mutants and wild-type (WT) controls. (C) Accessory gland protein content (determined by BCA method assay) after CRZ neuropeptide injection (sCRZ control). Data show mean ± SEM; ns (P > 0.05, Mann–Whitney U test).
Manipulation of Crz in male BPHs does not affect male courtship behavior.
(A) Courtship rate (number of males that exhibit courtship behavior) in males after CRZ peptide injection (sCRZ control) and mating with wild type (WT) females. Numbers below bars denote total animals. Data represent mean; ns (P > 0.05, χ² test). (B) Mating duration of CRZ-injected males paired with WT females (sCRZ control). Data show mean ± SEM; ns (P > 0.05, Student’s t-test). (C) Courtship rate after Crz knockdown via RNAi in males (dsGFP control). Data represent mean; ns (P > 0.05, χ² test). (D) Mating duration of Crz-knockdown males paired with wild-type females (dsGFP control). Data show mean ± SEM; ns (P > 0.05, Student’s t-test). (E) Courtship rate of Crz mutant males (WT controls). Data represent mean; ns (P > 0.05, χ² test). (F) Mating duration of males of different Crz mutants paired with WT females. Data show mean ± SEM; ns (P > 0.05, Student’s t-test).
CRZ signaling affects seminal fluid protein composition in male BPHs
(A) Spermathecae of WT females mated to WT or ΔCrz1males. Scale bar: 1 mm. No morphological differences were observed (n = 15 pairs (B) DAPI-stained testes (males) and spermathecae (females mated within 4 h) of WT or ΔCrz1BPHs. Scale bar: 100 μm. Sperm counts showed no genotype-dependent differences (testes: P = 0.32; spermathecae: P = 0.41; n = 12; Mann–Whitney U test). (C) Volcano plot of differentially expressed genes in male accessory glands (MAGs) from ΔCrz1 mutants versus wild-type (WT) males. Seminal protein genes with significant differential expression (|log2FC| > 1, P < 0.05) are highlighted (black line). Gene IDs for seminal proteins are annotated. Upregulated genes are shown in red and downregulated in black. (D) Top 20 enriched Gene Ontology (GO) terms among downregulated genes. Rich factor indicates enrichment strength; input number denotes enriched genes per term. (E) MAG protein content in WT and ΔCrz1 males measured by BCA method assay. Data: mean ± SEM; **P < 0.01 (Mann–Whitney U test; n= 8 MAG pairs). (F-I) qPCR validation of seminal fluid genes 43558, 53903, macc and ITP-L in MAGs. Data: mean ± SEM (n = 4 biological replicates). ns: not significant (P > 0.05); *P < 0.05 (Mann– Whitney U test).
CRZ regulates part of the PMR in Drosophila melanogaster.
(A) Schematic of experimental design. White segments on the time axis indicate light periods; black segments indicate dark periods. (B) Receptivity of virgin and mated Crz mutant (Crzattp) females, scored as the percentage copulating within 30 min. ***P < 0.001 (Chi-square test). (C) Mated Crz mutants (Crzattp) lay more eggs. Graph shows eggs laid per female after mating. ****P < 0.0001 (Student’s t-test). (D) Number of eggs laid per virgin female Crz mutants , n.s.: P > 0.05 (Student’s t-test). (E) Anti-CRZ immunostaining in brains of Crz mutant (Crzattp), control (Cs) and after Crz -RNAi). Note CRZ-expressing neurons at arrows in control. Scale bar: 100 μm. (F) Receptivity of virgin and mated females after Crz-RNAi. ****P < 0.0001; Chi quare test. (G) Crz-RNAi reduces the number of eggs laid by mated females. ****P < 0.0001 (Student’s t-test). (H) Receptivity of virgin females following CRZ peptide injection (monitored 6 h after injection). *P < 0.05 (Mann-Whitney test).
