Ecdysis behaviors and circadian rhythm of ecdysis in the stick insect, Carausius morosus

Abstract

Successful ecdysis in insects depends on proper timing and sequential activation of an elaborate series of motor programs driven by a relatively conserved network of neuropeptides. The behaviors must be activated at the appropriate times to ensure successful loosening and shedding of the old cuticle, and can be influenced by environmental cues in the form of immediate sensory feedback and by circadian rhythms. We assessed the behaviors, components of the neural network and the circadian basis of ecdysis in the stick insect, Carausius morosus. C. morosus showed many of the characteristic pre-ecdysis and ecdysis behaviors previously described in crickets and locusts. Ecdysis was described in three phases, namely the (i) preparatory or pre-ecdysis phase, (ii) the ecdysial phase, and (iii) the post-ecdysis or exuvial phase. The frequencies of push-ups and sways during the preparatory phase were quantified as well as durations of all the phases. The regulation of ecdysis appeared to act via elevation of cGMP, as described in many other insects, although eclosion hormone-like immunoreactivity was not noted using a lepidopteran antiserum. Finally, C. morosus showed a circadian rhythm to the onset of ecdysis, with ecdysis occurring just prior to or at lights on. Ecdysis could be induced precociously with mechanical stimulation.

Keywords: Circadian rhythm; Ecdysis; Eclosion hormone; Mechanical stimulation; cGMP.

Figure 1

The average body length of C. morosus at each nymphal stage, immediately after ecdysis. Lengths of stick insects were measured after each ecdysis event since hatching. An exponential increase was noted with a linear regression of 0.98. The majority of insects experienced 6 ecdysis events before reaching reproductive maturity, with only four insects reaching a 7^th stage (data omited).

Figure 2

The average duration between molts (days) ± sem for each given sample size noted on the respective histogram bar. Timing started at E3 (molting from L2 to L3), and continued until E6. On occasion nymphs molted a 7^th time, but this was rare and the sample size was very low.

Figure 3

Two behaviors from the stick insect “Preparatory (pre-ecdysis) phase”, which included “pushups” (A) and “sways” (B). (A) Up and down motions, noted by arrows, are due to bilaterally symmetrical leg extensions (up) and flexions (down) of the coxae-trochanter (ct), the trochanter-femur (tf) and the femur-tibia joints (ft). Extensions and flexions resulted in up and down bouncing movements of the body. Dashed lines represent the original position of the leg. (B) The left diagram shows the stationary median position when the insect stayed attached to its perch in between sways. The right diagram denotes the swaying mechanism for a left sway, denoted by the arrow. The swaying motion was due to fore, abdominal, and hind leg flexions and/or extensions. The dashed lines denote original positions of the legs in the perch position (see left diagram) prior to a left sway: generally, the left legs flexed and the right legs extended. Swaying to the right was a mirror image of the left sway (not shown).

Figure 4

Stick insect ecdysis. (A) During ecdysis, C. morosus began a series of stereotyped swaying motions while its head remained tucked forward with front legs caught in the old cuticle. (B) Immediately after the front legs and antennae were extricated from the old cuticle, the thorax remained bent. The middle and hind legs were not yet extracted from the old cuticle. (C) The remaining legs and posterior end were extricated from the old cuticle via peristaltic abdominal waves.

Figure 5

Cyclic GMP immunoreactivity in the ventral nerve cord of C. morosus. (A) Metathoracic ganglion during the intermolt period. Dashed lines denote areas of expected immunoreactive cells. (B) Cyclic GMP immunoreactivity in lateral neurosecretory cells (thick arrows), interneurons (thin arrows) and neurites (right arrowhead) and axon (left arrowhead) of the metathoracic ganglion during ecdysis. (C) Abdominal ganglion 3 during the intermolt period. Dashed lines denote areas of expected immunoreactive cells. (D) Cyclic GMP immunoreactivity in lateral neurosecretory cells and interneurons (asterisks) and axons (arrowheads) of abdominal ganglion 3 during ecdysis. Scale bar = 100 μm for all panels.

Figure 6

Ecdysis profiles of C. morosus in short and long day photoperiods. (A) The first 2-3 molts from 32 nymphs were monitored in a “long day” (17L:7D) light:dark photoperiod. (B) These insects were then transitioned to a “short day” (7L:17D) light:dark photoperiod and timing of the last two nymphal ecdyses were noted.

Figure 7

Time of ecdysis, in real time, in an all-dark photoperiod. Hatchlings were placed in an all-dark environment (0L:24D) with temperature and humidity maintained as described in the methods. Three ecdysis events were recorded for 15 stick insects during this photoperiod. The 1^st (E1), 2^nd (E2) and 3^rd (E3) ecdysis events are plotted for each insect at the time of occurance, with symbols noted in the legend.

Figure 8

Ecdysis profiles of C. morosus in an all dark (0L:24D) photoperiod. Data are plotted as the difference in time from a 24 hour period from the previous molt. Time to ecdyse to (A) the second stage nymph (E2) relative to the first, and (B) the third stage nymph (E3) relative to E2.

Figure 9

Effects of mechanical stimulation on ecdysis behaviors. Stick insects were reared in a 12L:12D photoperiod (light and dark period indicated by black bars on the x-axis). Animals were handled every hour, at times between the arrows. (A) Animals were checked during the lights on period. (B) Animals were checked during the lights off period.