Molecular and phylogenetic analyses reveal mammalian-like clockwork in the honey bee (Apis mellifera) and shed new light on the molecular evolution of the circadian clock

Abstract

The circadian clock of the honey bee is implicated in ecologically relevant complex behaviors. These include time sensing, time-compensated sun-compass navigation, and social behaviors such as coordination of activity, dance language communication, and division of labor. The molecular underpinnings of the bee circadian clock are largely unknown. We show that clock gene structure and expression pattern in the honey bee are more similar to the mouse than to Drosophila. The honey bee genome does not encode an ortholog of Drosophila Timeless (Tim1), has only the mammalian type Cryptochrome (Cry-m), and has a single ortholog for each of the other canonical "clock genes." In foragers that typically have strong circadian rhythms, brain mRNA levels of amCry, but not amTim as in Drosophila, consistently oscillate with strong amplitude and a phase similar to amPeriod (amPer) under both light-dark and constant darkness illumination regimes. In contrast to Drosophila, the honey bee amCYC protein contains a transactivation domain and its brain transcript levels oscillate at virtually an anti-phase to amPer, as it does in the mouse. Phylogenetic analyses indicate that the basal insect lineage had both the mammalian and Drosophila types of Cry and Tim. Our results suggest that during evolution, Drosophila diverged from the ancestral insect clock and specialized in using a set of clock gene orthologs that was lost by both mammals and bees, which in turn converged and specialized in the other set. These findings illustrate a previously unappreciated diversity of insect clockwork and raise critical questions concerning the evolution and functional significance of species-specific variation in molecular clockwork.

Molecular Dynamics and Social Regulation of Context-Dependent Plasticity in the Circadian Clockwork of the Honey Bee

“…1 B, C). We further analyzed brain transcript levels for Cry and Per, which are the clock genes that show the most robust oscillations in honey bees (Rubin et al, 2006;Shemesh et al, 2007). We found that both Cry and Per transcript levels oscillated with higher levels during the night in foragers, but not in nurses, which is consistent with previous analyses of honey bees (Toma et al, 2000;Bloch et al, , 2004Shemesh et al, 2007) (Fig.…”

“…6 A). Cosine models typically account for much of the variation in transcript levels during the day for genes that are regulated by the circadian clock (Rubin et al, 2006;Krupp et al, 2008). We normalized the obtained amplitude and R 2 for each group relative to those in nurse bees from the same colony and experiment (e.g., the normalized value is 1 if the amplitude for the nurse age bees is similar to that of nurse bees from the same colony).…”

“…3B). Given that Per and Cry are canonical clock genes in the honey bee and show robust cycling in the brain of foragers and other bees with strong circadian rhythms (Toma et al, 2000;Bloch et al, , 2004Rubin et al, 2006;Shemesh et al, 2007), we assume that the whole brain expression of these genes stems largely from clock cells that are part of the circadian network. It is noteworthy that the appearance of circadian rhythms in locomotor activity was faster than the molecular oscillations.…”

“…We assessed whether brain clock gene mRNA levels differed between bees collected at different time points with a one-way ANOVA (SPSS software) with time as a factor, and a Fisher LSD post hoc test. For more details see Rubin et al (2006).…”

“…To minimize RNA degradation, we collected the bees directly into liquid nitrogen, and stored the frozen samples at Ϫ80°C until brain dissection. We measured mRNA levels with real-time reverse transcription (RT)-PCR as described previously by Rubin et al (2006). Briefly, we removed and freeze-dried the bee head, and dissected out the brain on a frozen dissecting dish that was immersed in dry ice.…”