(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, muscle; Ov, ovary; Tg, thoracic ganglia. (B) Expression levels of the ACP gene in the brain during the time course of sustained flighting of adult locusts. The mRNA level of ACP significantly increased after 1 h of sustained flighting. Different letters indicate that there is a significant difference between the two groups (one-way ANOVA, F = 20.69, df = 3, p<0.0001, n = 4 replicates). (C) Gene structure and designed sgRNA-targeted site in exon 2 of the locust ACP gene. Three exons are represented in different colors. The sgRNA targeted site in exon two is highlighted in blue. The pink region in exon two indicates the mature ACP peptide-encoding region. (D) Genome PCR product sequencing of the wild-type (WT) and 13 bp-deletion ACP mutants (ACP13/13, referring to ACP-/- in the following section), which could not give rise to mature ACP peptide. (E) Detection of ACP peptide in WT and ACP-/- locusts by immunohistochemistry assay. The bar indicates 100 μm. (F) Morphology of females and males of WT and ACP-/- locusts. Both females and males of ACP mutant adults show increased body size compared to that of WT controls. Bars represent 1 cm. Measurement of (G) total flight time, (H) total flight distance, and (I) average flight velocity in females and males of WT and ACP-/- locusts. Each dot represents a single individual (Student’s t-test for G, p=0.8499, t = 0.1904, df = 44 for 15 min♀, p=0.0183, t = 2.460, df = 40 for 60 min♀; p=0.9504, t = 0.0626, df = 46 for 15 min♂, p=0.0088, t = 2.755, df = 40 for 60 min♂; For H, p=0.6416, t = 0.4687, df = 44 for 15 min♀, p=0.0097, t = 2.718, df = 40 or 60 min♀; p=0.7489, t = 0.332, df = 46 for 15 min♂, p=0.01, t = 2.704, df = 40 for 60 min♂; For I, p=0.6067, t = 0.5186, df = 44 for 15 min♀, p=0.654, t = 0.4516, df = 40 for 60 min♀; p=0.7644, t = 0.3015, df = 46 for 15 min♂, p=0.9122, t = 0.1109, df = 40 for 60 min♂; n = 23 (WT♀ 15 min), 23 (ACP-/-♀ 15 min), 22 (WT♀ 60 min), 20 (ACP-/-♀ 60 min), 24 (WT♂ 15 min), 24 (ACP-/-♂ 15 min), 21 (WT♂ 60 min), 21 (ACP-/-♂ 60 min), *p<0.05, **p<0.01, n.s. indicates not significant). See Figure 2—source data 1 for details.