What Is Ecdysis? And Why Study It?
Our behavioral studies focus on the motor sequences used by flies to molt. Molting in flies, as in all insects, is performed several times during development to permit continued growth, and the stereotyped motor programs used to remove the old exoskeleton are called “ecdysis sequences.”
Ecdysis sequences are designed first to loosen and then remove the old exoskeleton in addition to expanding the new one. The motor programs that comprise ecdysis sequences vary dramatically across insect species, but they are governed by a conserved set of hormones, and their study permits one to address several fundamental questions in behavioral neurobiology:
- How do hormones and neuromodulators induce transitions in brain states that alter an animal’s behavior?
- How are brain networks organized to convert hormonal inputs into complex motor sequences?
- How are such sequences assembled and coordinated with the physiological changes needed to support behavioral execution?
- How are brain circuits rendered sensitive to environmental input so that the behavioral sequences they produce are adaptive?
- How can neural circuits be reconfigured to allow them to generate widely different behaviors at different developmental stages?
The pupal molt in Drosophila is unusual in that it involves casting off only the portions of the larval exoskeleton that line the gut and trachea. Instead of being shed, most of the exoskeleton is instead hardened in place to form the pupal case (or puparium), which protects the animal during metamorphosis. The pupal ecdysis sequence is likewise recruited to serve the purposes of metamorphosis, with its component motor programs initiating critical changes in body morphology, such as the extension of the appendages and the ejection of the adult head capsule from the larval body.
To elucidate the neural circuitry underlying the pupal ecdysis sequence, we have been mapping the sites of action of the key ecdysis hormones, including Ecdysis Triggering Hormone (ETH), Eclosion Hormone (EH), Crustacean Cardioactive Peptide (CCAP), and Bursicon. We have used our “Trojan exon” technique (Diao et al., 2015 ) to map neurons that express the hormone receptors and we find that subsets of such neurons define essential layers of a hierarchically organized circuit. ETH targets an “input layer” that includes neurons that co-express CCAP and Bursicon (Diao et al., 2016 ), while the latter hormones target distinct downstream layers (Diao et al., 2017 ), including a multifunctional central pattern generator and an output layer consisting of the CPGs target motor neurons. Remarkably, the pattern of neuromodulatory connectivity in the ecdysis circuit thus reveals its neuroarchitecture. We are currently capitalizing on this insight to understand how information is transformed as it flows through the different hierarchical layers of the pupal ecdysis circuit. We do so by using light-sheet microscopy and GCaMP-mediated Ca++ imaging to comprehensively monitor neural activity within different circuit layers during the generation of an ecdysis sequence.
This video shows the muscular activity underlying all three phase of the pupal ecdysis sequence. Note the alternating left-right contractions during Phase II, which generate the back-and-forth swinging of the abdomen.
Neurons in the abdominal ganglia that express the bursicon receptor (RK) are involved in central pattern generation. This video shows the Ca++ activity of these neurons during Phase II of pupal ecdysis. The alternating left-right activity is responsible for abdominal swinging.
While pupal ecdysis is executed within the puparium, the adult ecdysis sequence mediates the animal’s escape from this protective casing and brings it into contact with the outside world. It is no surprise then that components of the adult ecdysis sequence are sensitive to environmental input. Light levels influence initiation of the motor programs used to break out of the puparium, and environmental conditions encountered by the fly after emergence influence its execution of motor programs used to expand the adult exoskeleton. Expansion and hardening of the exoskeleton, which completes the adult ecdysis sequence, requires the hormone Bursicon and includes expansion and hardening of the newly formed wings. Failure to expand the wings spells early death for a fly, and an animal emerging into a confined environment from which it cannot readily escape will delay expansion and instead search for more favorable conditions. The mechanisms by which environmental and hormonal influences are weighed to generate a behavioral decision are thus naturally open to investigation in this system and appear to converge on a pair of Bursicon-expressing neurons called the “BSEG” (Luan et al., 2012 ). These neurons are targets of ETH, which is required for emergence from the puparium, and their activation before emergence is clearly suppressed (Peabody & White, 2013 ) However, after emergence, activation of the BSEG induces wing expansion even under adverse environmental circumstances.
Current work is aimed at identifying neurons upstream of the BSEG that participate in the wing expansion decision as well as neurons downstream that dictate the motor output. More generally, we are interested in the circuitry that organizes the execution of all phases of the adult ecdysis sequence and in understanding how this circuitry differs from that governing pupal ecdysis.
The adult ecdysis sequence is characterized by behaviors that first extricate the animal from the pupal case and then allow it to expand and harden the wings, as shown in this video.