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Modulation of ecdysis in the moth Manduca sexta : the roles of the suboesophageal and thoracic ganglia

Megumi Fuse^* and James W. Truman

Department of Zoology, University of Washington, Seattle, WA 98195-38100, USA
^* Present address: Department of Biology, San Francisco State University, San Francisco, CA 94132, USA

View larger version (43K): [in a new window]   Fig. 1. Time course of cyclic GMP immunoreactivity (cGMP-IR) in abdominal neurons during larval ecdysis after injection of ecdysis-triggering hormone (ETH). Animals were injected with 200 pmol of ETH. (A) The intensity of cGMP-IR was quantified, as described in Materials and methods, for 5-15 samples per time point. Standard error bars were generally small, but have been omitted for clarity. Blue lines represent immunoreactivity in cells 27 and 704. The orange line represents immunoreactivity in paired cells L2,5. The green column represents the time of eclosion hormone (EH) release, based on Ewer and Truman (1997). The black arrow represents the mean time of the start of ecdysis (E). (B) Video micrographs show the cGMP response in neurons. Each photograph is a video montage of 2-3 optical sections to show neuron morphology optimally. Cells 27 and 704 are marked with a blue arrow, and the L2,5 cells are marked with an orange arrow. Times of dissection (in minutes) are noted in the lower left-hand corners. Scale bar, 150 µm.

View larger version (130K): [in a new window]   Fig. 2. Photographs of single-labeled (A) and double-labeled (B-D) preparations of ganglion A3 from ecdysis-triggering hormone (ETH)-injected larvae dissected at ecdysis. Arrows in all panels point to the L2,5 cells. (A) Video montage of 2-3 optical sections of a cGMP-stained preparation, as visualized with a color reaction. Axons of the L2,5 cells can be seen exiting the ventral nerve (black arrowhead). (B-D) Collapsed Z-series confocal images of double-labeled preparations. Red is FMRFamide (FMRFa), leucokinin (LK) or Manduca sexta diuretic hormone (DH) staining in panels B-D, respectively. cGMP is indicated by green (B-D) and is marked by white arrows. Co-localization was not noted in cells L2,5 in any panel. Scale bars, 100 µm.

View larger version (17K): [in a new window]   Fig. 3. Timing of the commencement of ecdysis in larvae and pupae decapitated at various times after injection of 200 pmol of ecdysis-triggering hormone (ETH). Animals were injected with ETH (at 0 min) and decapitated at the times listed on the y-axis (indicated by white arrowheads). Control larvae (C) were not decapitated. Animals decapitated 10 min after ETH injection did not ecdyse (`No ecdysis'), which is shown by a broken bar. Bars are mean times + S.E.M. Sample sizes are indicated in parentheses. Letters represent significant differences compared with controls within groups (P<0.001), as determined by analysis of variance using the Tukey test for differences (=0.05).

View larger version (18K): [in a new window]   Fig. 4. The effects of surgical manipulations on the initiation of ecdysis in larvae and pupae. Animals were injected with 200 pmol of ecdysis-triggering hormone (ETH) (at 0 min), and surgery was performed 20 min later (black arrow). Controls were not treated surgically. ShamBr had their head capsule punctured and forceps inserted into the head region. ShamLig had a ligature placed and tightened between the suboesophageal ganglion (SOG) and the thoracic ganglia (TG); the ligature was then removed. Major ganglia were eliminated as described in the Materials and methods. Decerebration, —Brain; removal of the brain and SOG, —Br/SOG; removal of the brain, SOG and thoracic ganglia, —Br-TG; removal of the terminal abdominal ganglion, —Terminal. Bars are mean times + S.E.M. Sample sizes are indicated in parentheses. Letters represent significant differences from controls within groups (P<0.001), as determined by analysis of variance using the Tukey test for differences (=0.05). Different letters denote significantly different values within a group (P<0.001).

View larger version (8K): [in a new window]   Fig. 5. Commencement and duration of the ecdysis motor program in larvae. Animals were injected with ecdysis-triggering hormone (ETH) (at 0 min) and ligated 20 min later (white arrowheads). Ligatures removed the brain and suboesophageal ganglion (SOG) (—Br/SOG) or the brain, SOG and thoracic ganglia (—Br-TG), as described in Materials and methods. The solid bars represent the mean time of onset of ecdysis, while the open bars reflect the mean duration of the motor program. Values are means + S.E.M. A letter represents a significant difference between the two treatments, as assessed with the unpaired t-test (P<0.005).

View larger version (43K): [in a new window]   Fig. 6. The timing of the onset of ecdysis-triggering hormone (ETH)-induced ecdysis in larvae injected with the cGMP-specific phosphodiesterase inhibitor Zaprinast. (A) Animals were injected with Zaprinast 10 min prior to injection with 200 pmol of ETH (at 0 min), at the amounts listed on the y-axis. Controls (C) were injected with the Zaprinast solubilizing agent, 100% dimethylsulfoxide (DMSO), before injection of ETH. The timing of ecdysis was not significantly different in these controls from that in untreated controls (data not shown). The bars represent mean times + S.E.M. Letters represent significant differences from the control and from each other, as determined by analysis of variance (P<0.001) using the Tukey test for differences (=0.05). (B) Representative micrographs of cGMP immunoreactivity in abdominal ganglion 3 in DMSO- (C) and Zaprinast-treated (Z) animals 60 min after injection with ETH. Scale bar, 100 µm.

View larger version (12K): [in a new window]   Fig. 7. The commencement of ecdysis-triggering hormone (ETH)-induced ecdysis in larvae after treatment with CO2 or N2. (A) Larvae were injected with 200 pmol of ETH (at 0 min) and 20 min later were treated with CO2 or N2 for 1 min (white arrowheads). (B) Larvae were treated with CO2 for 1 min, at various times after ETH injection, as noted on the y-axis and by arrowheads. Controls (C) were handled at 20 min after ETH injection. The bars represent mean times + S.E.M. Letters represent significant differences from controls and from each other within groups (P<0.001), as determined by analysis of variance using the Tukey test for differences (=0.05).

View larger version (18K): [in a new window]   Fig. 9. Distribution of entrained adult eclosion (A) and responses to CO2 during adult eclosion (B—E). (A) The entrainment period consisted of 2 weeks in a 17 h:7 h light:dark cycle coupled to a thermoperiod of 28 °C:25 °C. The start of the scotophase is depicted by the shaded column at 15:00 h Pacific Standard Time (PST). The frequency of eclosion is depicted for 189 animals. (B—E) The ability of CO2 to induce premature eclosion in entrained pharate adults. Animals were treated with CO2 for 1 min at various times (pink arrows) before lights out. Each pair of points (open and filled circle) and a cross represent eclosion behaviours from one animal (sample). Since the onset of the abdominal and thoracic components of ecdysis (pink open and filled circles, respectively) often occurred separately in CO2-treated animals, they are separated temporally on the graph. When behaviours occurred in immediate succession, the dots were overlaid as a circle with a dotted center. Controls, in which abdominal and thoracic behaviours always occurred in immediate succession (thus open circles containing black dots), are shown in black. The time when the wings are fully expanded and move to the `moth' position is depicted by a cross for each animal.

View larger version (114K): [in a new window]   Fig. 8. Photographic sequence of adult abdominal peristalsis during normal eclosion (A—D) and CO2-induced premature eclosion (E—H). A lateral view of the abdomen showing the retraction (B; upward-pointing blue arrow) and extension (C; downward-pointing blue arrow) of normal eclosion. These movements are lacking in CO2-treated animals (E—H). Peristaltic contractions of segments are identified by green circles and black arrows.

View larger version (17K): [in a new window]   Fig. 10. Schematic diagram illustrating the interactions between inhibitors and activators of ecdysis behaviours and the role of cGMP levels in neurons regulating these behaviours. (A) Diagram of eclosion hormone (EH) neurons activating (+) both descending inhibitors (DI) from the suboesophageal ganglion or thoracic ganglia and crustacean cardioactive peptide (CCAP)-containing abdominal neurons (Ab). (B) Representational plots of cell excitability of the DI (Bi) and abdominal 27/704 neurons (Ab27/704) (Bii). The excitability of DI neurons declines below threshold (black dashed line) sooner than that of the abdominal 27/704 neurons. Thus, inhibition is lifted and CCAP is released from the abdominal 27/704 neurons. If a phosphodiesterase inhibitor (ZAP) is present (gray curve), cGMP levels are elevated and cell excitability stays high for longer (gray dashed line). Release of CCAP, and subsequently the onset of the ecdysis motor pattern, is therefore delayed.