Figure 3
Phenotypic analyses of ΔDHP and ΔDHR mutants.
(A) Representative images of eggs in wt (25DD), ΔDHP33 (25DD) and ΔDHP96 (25DD), 2 d or 10 d after oviposition. (B) Pigmented non-diapause eggs were observed in mutants. At 2 d after oviposition, pigmented eggs were observed (black asterisk); these eggs had delayed development (white asterisk). (C) Percentages of diapause eggs are shown for wt and four mutants incubated under various environmental conditions. The percentages of diapause eggs were estimated by counting the numbers of eggs in diapause and those not in diapause after non-diapause eggs hatched in 50 egg batches. (D) Morphological observation using thionin staining of egg. Embryos were collected 10 d after oviposition and thionin staining was performed. We observed arrested development at diapause stage in early embryonic development in wt and in ΔDHP33 (25DD) and ΔDHR96 (25DD). (E) Rescue experiments by injection of synthetic FXPRLa peptides (DH, α-, β- and γ-SGNPs and PBAN) into female pupa of mutants. The peanut oil (PO) or each peptide (10, 100 and 1000 pmol) were injected into wt (15DD), ΔDHP33 (25DD), ΔDHP531 (25DD), ΔDHR96 (25DD) and ΔDHR111 (25DD) and the diapause eggs inducing activity was measured. Each bar represents the mean ± SD of 6–10 animals. *P < 0.05; **P < 0.01. Asterisks indicate significant differences vs. PO injection of each strain. (F) Developmental timing of 100 larvae was observed in the wt and four mutants. The developmental period was considered the period when all larvae molted to the next instar (instars I–IV) and when all larvae initiated spinning (instar V). The number of parenthesis shows the number of larva died at this stage. (G–H) Eggs were incubated at 25 °C (25DD) or 15 °C (15DD) in the dark. Pupae (G) and cocoon-shell (H) weights after 4 d of pupation are shown in female. Each bar represents the mean ± SD of 35 animals. n.s., non-significant; *P < 0.05; ***P < 0.001. Orange asterisks indicate significant differences vs. wt (25DD); blue asterisks indicate significant differences vs. each 25DD strain. (Scale bar, 2 mm).
