Neuropeptide ACP facilitates lipid oxidation and utilization during long-term flight in locusts
- State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, China
- CAS Center for Excellence in Biotic Interactions, University of Chinese Academy of Sciences, China
- Beijing Institutes of Life Science, Chinese Academy of Sciences, China
Figures
Figure 1 with 7 supplements
Identification of AKH/Corazonin-related neuropeptide (ACP) as a potential neuroendocrine modulator for flight activity in the locust.
Volcano plot of neuropeptidomes from (A) brains (Br) and (B) the retrocerebral complex (RC) of the final instar nymphs (5th) and mature adult locusts. Each circle represents a neuropeptide. …
-
Figure 1—source data 1
Raw data for comparisons of peptide contents, gene expression, and flight activity.
- https://cdn.elifesciences.org/articles/65279/elife-65279-fig1-data1-v3.xlsx
Figure 1—figure supplement 1
Tissue-specific abundance of neuropeptides identified in the neuropeptidome.
The abundant level of neuropeptides generated from 37 precursors were log transformed for heat map drawing. Unsupervised hierarchical clustering was performed using Clustal 3.0 under centered …
Figure 1—figure supplement 2
Comparison of neuropeptide abundance in Brain and retrocerebral complex between 5th instar nymphs and adult locusts.
Log2 (FC adult/5th) of neuropeptide abundances are shown in the figure. Each line represents the content of neuropeptides produced from a single precursor. n.s. indicates that the difference is not …
Figure 1—figure supplement 3
Expression levels of 10 neuropeptide precursor genes in Brain and retrocerebral complex of adult locusts.
The data are presented as the mean ± SEM (Student’s t-test, p<0.0001, t = 11.65, df = 6 for ACP, p<0.0001, t = 13.59, df = 6 for DH46; p<0.0001, t = 13.23, df = 6 for GPB5; p=0.013, t = 3.477, df = 6…
Figure 1—figure supplement 4
Expression levels of six neuropeptide precursor genes in Brain or retrocerebral complex upon 1 h-sustained flighting.
The data are presented as the mean ± SEM (Student’s t-test, p=0.242, t = 1.298, df = 6 for AKH3; p=0.606, t = 0.5436, df = 6 for AST-A; p=0.4925, t = 0.7307, df = 6 for CAPA; p=0.949, t = 0.06548, …
Figure 1—figure supplement 5
RNAi efficiency of four flight-related neuropeptide precursor genes examined by qPCR.
The data are presented as the mean ± SEM (Student’s t-test, p<0.0001, t = 15.17, df = 6 for ACP; p=0.0019, t = 5.274, df = 6 for AKH2; p<0.0001, t = 9.053, df = 6 for NPF1; p=0.0002, t = 8.403, df = …
Figure 1—figure supplement 6
Measurement of (A) average flight velocity and (B) maximum flight velocity after the knockdown of four candidate neuropeptide precursor genes, ACP, AKH2, NPF1, and GPB, in adult locusts.
Significant differences between different groups are denoted by letters (one-way ANOVA, F = 2.365, df = 4, p=0.0566, for A; F = 2.423, df = 4, p=0.0522 for B). Each blot represents a single …
Figure 1—figure supplement 7
Measurement of (A) total flight time, (B) total flight distance, (C) average flight velocity, and (D) maximum flight velocity after the injection of synthetic ACP peptide in wide type locusts.
Each blot represents a single individual. The mean ± SEM are shown (Student’s t-test, p=0.0267, t = 0.8592, df = 44 for A; p=0.0186, t = 2.444, df = 44 for B; p=0.3949, t = 2.294, df = 44 for C; …
Figure 2 with 4 supplements
Functional role of ACP in long-term flight validated by CRISPR/Cas9 system-mediated gene knockout.
(A) Tissue-specific expression pattern of the ACP precursor gene in adult locusts analyzed by qPCR (n = 4 replicates, 6–8 locusts/replicate). Br, brain; RC, retrocerebral complex; FB, fat body; MS, …
-
Figure 2—source data 1
Raw data for gene expression and flight activity.
- https://cdn.elifesciences.org/articles/65279/elife-65279-fig2-data1-v3.xlsx
Figure 2—figure supplement 1
Construction of the ACP loss-of-function mutant line of the migratory locust.
(A) Determination of the gene type of the ACP mutation in the G0 generation by Sanger sequencing. (B) Schematic of screening strategies to obtain 13 bp-deleted homozygous ACP mutants.
Figure 2—figure supplement 2
Localization of ACP peptide in the locust brain detected by IHC.
ACP peptide is abundantly detected in neurons in the pars intercerebralis (PI) and bilateral forebrain of the locust brain. Scale bars represent 100 μm.
Figure 2—figure supplement 3
Survival rate of females and males of WT and ACP-/- locusts.
ACP mutants show a normal survival rate similar to that of WT locusts. Gehan-Breslow-Wilcoxon test, p=0.154 and 0.172 for females and males, respectively (n > 50).
Figure 2—figure supplement 4
Measurement of maximum flight velocity in (A) females and (B) males of WT and ACP-/- locusts.
Each dot represents a single individual (Student’s t-test for A, p=0.4996, t = 0.6808, df = 44 for 15 min ♀, p=0.5075, t = 0.6688, df = 40 for 60 min♀; p=0.9957, t = 0.005381, df = 46 for 15 min ♂, …
Figure 3 with 2 supplements
CRISPR/Cas9-mediated knockout of ACPR impairs long-term flight in the locust.
(A) Phylogenic relationship of AKHR, ACPR, CRZR from the migratory locust and their homologs from other representative insect species. The phylogenetic tree is constructed using the neighbor-joining …
-
Figure 3—source data 1
Raw data for gene expression and flight activity.
- https://cdn.elifesciences.org/articles/65279/elife-65279-fig3-data1-v3.xlsx
Figure 3—figure supplement 1
Immunostaining of ACP peptide in the brain of ACPR13/13 locusts.
Intense signal of ACP peptide is detected in the brain of ACPR mutants. Scale bars represent 100 μm.
Figure 3—figure supplement 2
Measurement of (A) average flight velocity and (B) maximum flight velocity in females and males of WT and ACPR13/13 locusts.
Each dot represents a single individual (Student’s t-test for A, p=0.2153, t = 1.252, df = 60 for 15 min ♀, p=0.1522, t = 1.45, df = 60 for 60 min♀; p=0.1556, t = 1.437, df = 63 for 15 min ♂, …
Figure 4 with 4 supplements
Transcriptome analysis reveals significant downregulation of genes and pathways associated with lipid transport and oxidation in the flight muscle of ACP-/- locusts.
(A) Ven diagram of differentially expressed genes (DEGs) in flight muscle and fat body between WT and ACP-/- locusts. There were 212 upregulated and 349 downregulated genes in the flight muscle and …
-
Figure 4—source data 1
Raw data for RNA-seq and gene expression.
- https://cdn.elifesciences.org/articles/65279/elife-65279-fig4-data1-v3.xlsx
Figure 4—figure supplement 1
Numbers of differentially expressed genes in the flight muscle and fat body between WT control and ACP-/- locusts.
Red indicates upregulated, blue indicates downregulated.
Figure 4—figure supplement 2
Expression levels of genes related to fatty acid transport and beta-oxidation are downregulated in the flight muscle of ACP-/- locusts.
(A) mRNA levels of eight genes related to fatty acid transport and metabolism in the flight muscle of WT and ACP-/- locusts examined by qPCR. The data are presented as the mean ± SEM (Student’s …
Figure 4—figure supplement 3
Expression levels of genes related to fatty acid transport and oxidation after ACP peptide injection and 1 h-sustained flight in WT locusts.
mRNA levels of eight genes related to fatty acid transport and oxidation in the flight muscle (A) after injection of ACP peptide and (B) 1 h-sustained flight. The data are presented as the mean ± SEM…
Figure 4—figure supplement 4
RNAi efficiency of FABP in the muscle examined by qPCR.
(Student’s t-test, p=0.0012, t = 4.038, df = 14, **p<0.01, n = 8 replicates, 3–4 locusts/replicate).
Figure 5 with 4 supplements
Metabolomic analysis reveals impaired lipid metabolism in the flight muscle of ACP mutants.
(A) Metabolite distribution in the flight muscle of WT and ACP-/- locusts, as determined by orthogonal partial least squares discriminant analysis (OPLS-DA). Red blocks indicate WT samples, blue …
-
Figure 5—source data 1
Raw data for metabolic and metabolite contents.
- https://cdn.elifesciences.org/articles/65279/elife-65279-fig5-data1-v3.xlsx
Figure 5—figure supplement 1
Pie chart of metabolite chemical classes structurally annotated in this experiment.
In total, 881 metabolites were characterized in 12 samples (n = 6 for both WT and ACP-/- samples).
Figure 5—figure supplement 2
Unsupervised PCA score plots of metabolic phenotypes between the ACP-/- and WT groups.
Metabolomics data were log transformed and scaled to unit variance for modeling. Model parameter: R2X = 0.55 (cumulative variance proportion of three principal components).
Figure 5—figure supplement 3
Heat map of TG, PC, and PE in the flight muscle of WT and ACP-/- locusts.
Content of metabolites are highlighted with red (upregulated) and blue (downregulated), respectively.
Figure 5—figure supplement 4
Measurements of acetyl CoA and NADH levels in the flight muscle after ACP peptide injection and 1 h-sustained flight in WT locusts.
(A) Abundance detection of acetyl CoA and NADH in the flight muscle after injection of ACP peptide in WT locusts. (B) Abundance detection of acetyl CoA and NADH in the flight muscle upon 1 …
Figure 6 with 1 supplement
FABP mediates the regulatory effects of ACP on lipid metabolism and flight activity.
(A) Total flight time and (B) total flight distance after knockdown of FABP in adult locusts (Student’s t-test for A, p=0.451, t = 0.7609, df = 44, n = 24 (dsGFP) and 22 (dsFABP) for 15 min, …
-
Figure 6—source data 1
Raw data for flight activity, metabolite contents, and gene expression.
- https://cdn.elifesciences.org/articles/65279/elife-65279-fig6-data1-v3.xlsx
Figure 6—figure supplement 1
Flight speed measurement after knockdown of FABP in the locust.
Measurements of (A) average flight velocity and (B) maximum flight velocity after knockdown of FABP in the locust. Each dot represents a single individual (Student’s t-test, p=0.2705, t = 1.116, df =…
Figure 7
Schematic model showing that the neuropeptide.
ACP regulates long-term flight by affecting FABP-mediated fatty acid transport and subsequent β-oxidation in the flight muscle. Neuropeptide ACP is produced from the brain and could be secreted into …
Author response image 1
Author response image 2
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Locust migratoria) | ACP | Yang et al., 2019 | http://www.locustmine.org:8080/locustmine (LOCMI05723) | |
| Gene (Locust migratoria) | ACPR | Yang et al., 2019 | http://www.locustmine.org:8080/locustmine (comp340809_c0_seq1) | |
| Genetic reagent (Locust migratoria) | GeneArtPrecision gRNA Synthesis Kit | ThermoFisher | A29377 | |
| Genetic reagent (Locust migratoria) | Cas9 protein | Invitrogen | A36496 | |
| Antibody | (anti-ACP rabbit polyclonal) | This paper | Produced by ABclone, China | (1:200) |
| Antibody | Alexa Fluor-488 goat anti-rabbit IgG | Life Technologies | Cat. A-11008 | (1:500) |
| Antibody | anti-FABP | This paper | Developed by ABclone, China | (1:5000) |
| Antibody | anti-CPT2 | Abcam | ab153869 | (1:1000) |
| Antibody | anti-ACDM | Abcam | ab92461 | (1:1000) |
| Antibody | Goat anti-rabbit IgG secondary antibody | EASYBIO | BE0101-100 | (1:5000) |
| Antibody | Polyclonal antibody against tubulin | This paper | Produced by ABclone, China | (1:5000) |
| Peptide, recombinant protein | ACP peptide | ABclone | pQVTFSRDWSPGamide | |
| Commercial assay or kit | Acetyl-CoA assay kit | Sigma-Aldrich | MAK039 | |
| Commercial assay or kit | NAD/NADH Assay Kit | Abcam | ab65348 | |
| Commercial assay or kit | Pyruvate Colorimetric/Fluorometric Assay Kit | BioVison | K609 | |
| Commercial assay or kit | Citric acid content detection kit | Solarbio | BC2150 | |
| Chemical compound, drug | LightCycler 480 SYBR Green I Master | Roche | 04887352001 | |
| Software, algorithm | GraphPad Prism 5 | GraphPad Software | RRID:SCR_002798 |
Additional files
-
Supplementary file 1
Neuropeptides identified in the brain and retrocerebral complex by peptidome.
- https://cdn.elifesciences.org/articles/65279/elife-65279-supp1-v3.xlsx
-
Supplementary file 2
Mutation efficiency of the ACP gene in G0 and G1 generation locusts.
- https://cdn.elifesciences.org/articles/65279/elife-65279-supp2-v3.docx
-
Supplementary file 3
Mutation efficiency of the ACPR gene in G0 and G1 generation locusts.
- https://cdn.elifesciences.org/articles/65279/elife-65279-supp3-v3.docx
-
Supplementary file 4
Primers for qPCR, gRNA synthesis, and RNAi experiment.
- https://cdn.elifesciences.org/articles/65279/elife-65279-supp4-v3.docx
-
Transparent reporting form
- https://cdn.elifesciences.org/articles/65279/elife-65279-transrepform-v3.pdf
