Most organisms rely on a molecular circadian clock to orchestrate a wide range of physiological processes to match the 24-hour day. These molecular clocks are typically based on a negative feedback loop among a small set of proteins that govern the circadian output. Light or other environmental conditions can reset the circadian clock, but true circadian behaviors continue to cycle even in constant darkness, with an intrinsic period called the free-running period (FRP). Spiders have unusual FRPs, with some species having extremely short FRP (e.g. 18 hours for trashline orb weaver), and many having highly variable FRPs (intraspecific variation of up to 10 hours). In the absence of any genetic model of circadian rhythms in spiders, we developed a mathematical model to optimize experimental conditions for identifying circadian genes that also respond to light cues.
Our mathematical model involved a single gene that encodes a protein that inhibits its own transcription. In our model, light degrades the circadian transcript, which allows a broad range of FRPs to be entrained to a 24-hour day. Our model predicted that exposing spiders to a pulse of light in the middle of the night would cause a pattern of expression between two later time points that was opposite the pattern exhibited by spiders who did not receive a pulse of light. RNA-sequencing of four groups of adult female orb weaving spiders, Metazygia wittfeldae, under these experimental conditions resulted in 528 significantly differentially expressed (DE) transcripts between the two collection times or between the light pulse and no light pulse. Consistent with our model, we found a cluster of transcripts with the flipped pattern of expression between the two collection times, dependent on the application of light.
Our DE transcripts represent the first genetic evidence for circadian output in spiders. Furthermore, those transcripts with a flipped pattern of expression represent prime candidates for light-sensitive circadian genes, which may be involved in entraining the circadian clock to light. Functions of these genes varied from growth and development to reproduction to gene regulation, consistent with other circadian systems.