A Characterization of Quiescent Behavior and Profiling of the Brain Transcriptome During Larval Development of the Holometabolous Insect, Manduca sexta

Author(s): MacWilliam, Dyan | Advisor(s): Adams, Michael E | Abstract: The holometabolous insect Manduca sexta has served as one of the primary model systems for the study of the neuroendocrine regulation of the insect molt. It has long been observed that during the molting period prior to cuticle shedding during its larval-larval stage transitions, M. sexta displays an extended period of quiescent behavior. This behavior has been called the "molt-sleep", and has been described anecdotally as a non-moving, non-feeding state; but it has never been characterized. We observed that molting larvae respond to gentle handling and noxious stimuli with behavior resembling that observed for inter-molt larvae, and hypothesized that the quiescent behavior of larvae is similar to the sleep-like behavior observed for many adult insects, and for molting C. elegans. We used time-lapse digital recordings to profile the rest-activity patterns of M. sexta across several larval stages, and found that larval quiescent behavior shows developmental rhythms that are reproducible both across individuals and across larval stages. Quiescent behavior is reversible both during the inter-molt and molting periods. Once aroused, molting larvae can locomote at rates comparable to inter-molt larvae. Larvae that are quiescent also have increased response latencies following exposure to a noxious stimulus, resembling (in both stages) the sleep-like states of other invertebrates. However, there are differences between inter-molt and molting quiescence, in that both feeding and quiescence seem to be homeostatically regulated during the inter-molt, but not the molting period. We propose that molting larvae engage in an adaptive quiescent state that promotes energy conservation while retaining the ability to respond to a changing environment. The correlation between the timing of molt-sleep onset with the estimated peak of the steroid molting hormones suggests that molting hormones might act directly or indirectly on the nervous system to bring about changes in gene expression that alter general arousal state. To investigate possible gene products that might be involved in the induction and maintenance of the molt-sleep, we used Illumina RNA-seq to profile the brain transcriptome at several developmental time points both during and surrounding the onset of the molt-sleep. This study is the first to profile the transcriptome of any M. sexta nervous tissue. We developed a novel method for identifying transcripts, which entailed the mapping of translated reads onto the proteome of Bombyx mori, the most closely related species for which a genome is known. 10,664 unique proteins were mapped by Illumina reads during at least one stage of development. Between 2.8%-7.8% of all genes were differentially-expressed in the brain at a given stage when compared to mid-inter-molt reference levels. Cluster and enrichment analysis reveals a dominance of processes related to trachea cuticle synthesis and degradation. In addition, many of the differentially-expressed transcripts encode neuropeptides, enzymes involved in the synthesis of small neurotransmitters, ligand-gated ion channels, G-protein-coupled receptors, ion channels and other products associated with neural activity. Possible roles for these gene products in the modulation of larval arousal-state are described.