Ecdysis behavior allows insects to shed their old exoskeleton at the end of every molt. It is controlled by a suite of interacting hormones and neuropeptides, and has served as a useful behavior for understanding how bioactive peptides regulate CNS function. Previous findings suggest that crustacean cardioactive peptide (CCAP) activates the ecdysis motor program; the hormone bursicon is believed to then act downstream of CCAP to inflate, pigment, and harden the exoskeleton of the next stage. However, the exact roles of these signaling molecules in regulating ecdysis remain unclear. Here we use a genetic approach to investigate the functions of CCAP and bursicon in Drosophila ecdysis. We show that null mutants in CCAP express no apparent defects in ecdysis and postecdysis, producing normal adults. By contrast, a substantial fraction of flies genetically null for one of the two subunits of bursicon [encoded by the partner of bursicon gene (pburs)] show severe defects in ecdysis, with escaper adults exhibiting the expected failures in wing expansion and exoskeleton pigmentation and hardening. Furthermore, flies lacking both CCAP and bursicon show much more severe defects at ecdysis than do animals null for either neuropeptide alone. Our results show that the functions thought to be subserved by CCAP are partially effected by bursicon, and that bursicon plays an important and heretofore undescribed role in ecdysis behavior itself. These findings have important implications for understanding the regulation of this vital insect behavior and the mechanisms by which hormones and neuropeptides control the physiology and behavior of animals.

Insect body color is an easily assessed and visually engaging trait that is informative on a broad range of topics including speciation, biomaterial science, and ecdysis. Mutants of the fruit fly Drosophila melanogaster have been an integral part of body color research for more than a century. As a result of this long tenure, backlogs of body color mutations have remained unmapped to their genes, all while their strains have been dutifully maintained, used for recombination mapping, and part of genetics education. Stemming from a lesson plan in our undergraduate genetics class, we have mapped sable1, a dark body mutation originally described by Morgan and Bridges, to Yippee, a gene encoding a predicted member of the E3 ubiquitin ligase complex. Deficiency/duplication mapping, genetic rescue, DNA and cDNA sequencing, RT-qPCR, and two new CRISPR alleles indicated that sable1 is a hypomorphic Yippee mutation due to a mdg4 element insertion in the Yippee 5’-UTR. Further analysis revealed additional Yippee mutant phenotypes including curved wings, ectopic/missing bristles, delayed development, and failed adult emergence. RNAi of Yippee in the ectoderm phenocopied sable body color and most other Yippee phenotypes. Although Yippee remains functionally uncharacterized, the results presented here suggest possible connections between melanin biosynthesis, copper homeostasis, and Notch/Delta signaling; in addition, they provide insight into past studies of sable cell nonautonomy and of the genetic modifier suppressor of sable.

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We used P-element transposase-mediated ''male recombination'' between two P elements in trans to create genetic deletions that removed a number of loci, including the gene encoding the neuropeptide crustacean cardioactive peptide (CCAP). Two classes of recombinant chromosomes were produced. Approximately one-quarter were viable when homozygous or hemizygous, whereas the remaining lines caused homozygous and hemizygous lethality. Preliminary analyses using PCR and CCAP immunohistochemistry suggested that, whereas the DNA of the viable lines was largely intact, most lethal lines contained chromosomal deletions that were roughly bounded by the insertion sites of the two P elements used. Southern blot analyses of select lethal lines showed that the DNA flanking the deletion was indeed grossly intact whereas the intervening DNA could not be detected. Sequencing across the deletion in three of these lethal lines identified a single line bearing intact genomic DNA on either side of the deletion separated by 30 bp of P-element DNA. The method described here suggests a simple procedure for creating deletions with defined end points. Importantly, it can use preexisting P-element insertion strains and does not rely on the use of transposable elements that are engineered to cause specific DNA rearrangements.

The bang-sensitive (BS) mutants of Drosophila are an important model for studying epilepsy. We recently identified a novel BS locus, julius seizure (jus), encoding a protein containing two transmembrane domains and an extracellular cysteine-rich loop. We also determined that jus, a previously identified BS mutation, is an allele of jus by recombination, deficiency mapping, complementation testing, and genetic rescue. RNAi knockdown revealed that jus expression is important in cholinergic neurons and that the critical stage of jus expression is the mid-pupa. Finally, we found that a functional, GFP-tagged genomic construct of jus is expressed mostly in axons of the neck connectives and of the thoracic abdominal ganglia. In this Extra View article, we show that a MiMiC GFP-tagged Jus is localized to the same nervous system regions as the GFP-tagged genomic construct, but its expression is mostly confined to cell bodies and it causes bang-sensitivity. The MiMiC GFP-tag lies in the extracellular loop while the genomic construct is tagged at the C-terminus. This suggests that the alternate position of the GFP tag may disrupt Jus protein function by altering its subcellular localization and/or stability. We also show that a small subset of jus-expressing neurons are responsible for the BS phenotype. Finally, extending the utility of the BS seizure model, we show that jus mutants exhibit cold-sensitive paralysis and are partially sensitive to strobe-induced seizures.