—Reporter gene expression mediated by dAkh promoter. (A) Schematics of dAkh gene structure and dAkh-gal4 construct. (B–I) X-gal histochemistry or gfp expression of the offspring of [dAkh-gal4 × UAS-lacZ or UAS-mCD8-gfp] cross. (B) Third instar larval central nervous system. LacZ expression is restricted to the CC. Processes innervating the prothoracic gland (PG) are indicated by arrows. (C–E) LacZ expression in adults. (C) Somata of adult AKHergic neurons are present exclusively in the CC. Projections to the brain are denoted by white arrows and ones to the crop by black arrows. (D) Projections arising from the posterior side of the CC (arrows). A white arrowhead designates a projection leading to the crop. (E) Nerve terminals at the crop duct indicated by an arrowhead. (F) Expression of gfp in a “live” third instar larval progeny from a [dAkh-gal4 × UAS-mCD8-gfp] cross. Specific GFP-mediated fluorescence signals are clearly visible in a paired structure (arrowhead) at a position where the CC locate. (G and H) LacZ expression (arrowheads) during embryonic development. The expression (arrowhead) was first seen in stage-14 embryos (G) and became stronger in older embryos (stage 15; H). (I) CC-specific lacZ expression (arrowhead) in first instar larva. Bars, 100 μm.

—Cellular characteristics of AKHergic neurons. (A and B) Dissected ring glands from the progeny of a [dAkh-gal4 × UAS-NZ] cross were processed for X-gal histochemistry. Each nuclear lacZ expression (arrowheads) represents individual neurons in third instar larval (A) and adult CC (B). Dotted lines in A contour the brain. Bars, 100 μm. (C and D) The CC of larval offspring from a [dAkh-gal4 × UAS-mCD8-gfp] cross were stained by DAPI, and the fluorescence signals were captured for either gfp (C) or DAPI (D). Note that most of the CC cells are AKHergic neurons. (E) A bright-field image of the same specimen shown in C and D. Bar, 25 μm. (F and G) AKH-immunoreactive projections in larval CC. Images were captured from the same specimen at different focal plans to show projections (arrows) innervating the prothoracic gland (PG) and the aorta (a). Bar, 50 μm.

—Transgenic ablation of AKHergic neurons. The AKHergic neurons (arrowheads) were visualized by X-gal histochemistry and anti-AKH immunohistochemistry in the CC of third instar larvae (L3) or adults. (A) Control (dAkh-gal4 × UAS-lacZ). (B) Reaper (rpr)-induced apoptosis of AKHergic neurons was confirmed by the lack of lacZ expression and AKH immunoreactivity (arrowheads). (C) Incomplete rescue of AKHergic neurons by coexpression of p35. Bar, 100 μm.

—Effect of altered dAkh expression on larval hemolymph sugar levels. Numbers in parentheses indicate the number of samples tested for each genotype. (A) Ablation of AKHergic neurons (dAkh-gal4/UAS-rpr) caused significant reduction in trehalose levels. (B) Partial rescue of rpr-mediated ablation by p35 (dAkh-gal4, UAS-p35/UAS-rpr) incompletely restored trehalose titers. (C) Hemolymph trehalose concentrations were unchanged by overexpression of dAkh in AKHergic neurons (UAS-dAkh/+; dAkh-gal4/+), whereas (D) ∼34% increase was triggered by ectopic dAkh expression in the fat body (UAS-dAkh/+; r⁴-gal4/+). (E) Fat body-specific lacZ expression driven by r⁴-gal4 in larvae. (F and G) AKH immunohistochemistry of the fat body. (F) Control, nonspecific autofluorescence in AKH-CD fat body. (G) AKH immunofluorescence in AKH-EE fat body. Note that the signals shown here are much stronger than those in AKH-CD. (H) A summary for the trehalose phenotypes affected by various transgenic manipulations of the dAkh gene.

—Effect of AKH-EE on lipid contents within larval fat cells. Lipid droplets were stained by Sudan Black. The sizes and amounts of the droplets (arrowheads) are significantly reduced in AKH-EE (D) compared to those of genetic controls (A–C). Bar, 50 μm. (E and F) Quantitative measurements of triglyceride (TG) content in the fat body (E) or glycerol in the hemolymph (F). Numbers in parentheses designate the numbers of samples tested. (E) Significant reduction of TG or (F) elevation of glycerol is observed in AKH-EE (UAS-dAkh/+; r⁴-gal4/+). Asterisks indicate statistical significance (P < 0.001).

—Survival curves in response to starvation. (A) At 48 hr after starvation initiation, ∼50% of control flies died, whereas most of the AKH-CD flies (dAkh-gal4/UAS-rpr) were still alive. (B) Both male (m) and female (f) AKH-CD flies display similar levels of tolerance to starvation. (C) Rescue of AKH-CD by coexpression of p35 (dAkh-gal4, UAS-p35/UAS-rpr) shows intermediate levels of resistance to starvation. (D) Degrees of resistance to starvation-induced death are indistinguishable between young (<30 hr after eclosion) and old AKH-CD flies. At least 40 flies were tested for each genotype or sex.

—Circadian locomotor activity rhythms of fed flies. Flies were entrained to four cycles of 12 hr:12 hr light:dark (LD) and then proceeded to 7 days of constant darkness (DD). Bars represent average numbers of activity events per half-hour bin for the number of flies tested. Horizontal open and solid boxes indicate the light-on and light-off phases, respectively, and the shaded box denotes subjective day under DD condition. Numbers on the x-axis are Zeitgeber (ZT) times (light is on at ZT-0 and off at ZT-12). Chi-square periodogram analysis revealed free-running rhythms with the following periodicities: 24.0 ± 0.5 (mean ± SEM) for wild-type and 24.2 ± 0.5 for AKH-CD flies. Most of flies tested in each group were rhythmic.