Circadian rhythms of five wild caught species of drosophila

Abstract

Living organisms have evolved circadian clocks to time their physiological and behavioural activities to stay in tuned with the geophysical world. Signatures of such timing systems can be observed at various levels of complexity and organization. The fruit fly Drosophila melanogaster has been extensively used for understanding the structure and function of circadian clocks. Some of the best-studied circadian rhythms in Drosophila include those in adult emergence, activity/rest, mating, and egg laying behaviours. Many researchers have used different species of Drosophila to study variability in circadian rhythms in order to understand how circadian clocks have evolved. For my thesis, I have studied circadian rhythms in five wild caught species of Drosophila to investigate if closely related species have similar circadian rhythms. The flies belong to three separate genuses. D. melanogaster and D. ananassae belong to the subgenus Sophophora, while D. malerkatliana belongs to a complex subgroup within D. ananassae. D. nasuta belongs to the subgenus Drosophila, and species group lmmigrans and Zaprionus indianus belongs to the genus Zaprionus and sub-genus Zaprionus and species group Armatus. These populations were maintained on cornmeal medium under constant light and at 25 "C temperature and •'90% relative humidity. I began by studying the activity/rest rhythm of five wild caught species of Drosophila. The main objective of my study was to ass<lY the activity/rest behaviour of five different species of Drosophila, and the distribution of activity/rest patterns in males and females under 12:12 hr light/dark (LD) cycles and in constant darkness (DD).

Drosophila Activity Monitors, for the first 8 days under LD cycle and for subsequent 8 days under DD. The results of the experiment revealed species- and sex- specific differences. Males of all five species were found to show bimodal activity patterns, while the activity of the females is evenly distributed throughout day. A closer look of the activity profiles suggests sex-specific differences in the overall activity patterns with females showing higher activity than males. Furthermore, D. ananassae (both males and females} flies were found to be more active during morning hours than evening hours compared with other related species of Drosophila. The anticipation index (AI} for the "morning" and "evening" activity peaks in D. me/anogaster, D. annanasse, D. nasuta was significantly different from those in Zaprionus indianus, while the AI in D. melanogaster, D. onanassae, D. nasuta and D. malerkotliana did not differ statistically. The morning peak in D. ananassae is higher than the evening peak, while in D. melanogaster and D. malerkotliana the evening peaks were higher than the morning peaks. The morning and evening peaks in Zaprionus indianus occurred on an average 2 hr after "dawn" and "dusk", respectively. The activity in males and females was higher during the light phase than dark phase, and females exhibited higher overall daytime activity than males. Analyses of the sleep data revealed that duration of sleep is shorter in females than in males Further analysis revealed that D. nasuta females show higher sleep duration compared to other related species. Night time sleep in D. ananassae females was significantly greater than D. melanogaster and D. malerkotliana but lesser than D. nasuta. The circadian period could only be calculated for three of the five wild caught

species and sex specific; with D. onanassae having significantly shorter periodicity than other two species of the same genus (D. melanogaster and D. malerkotliana). The circadian periodicity of the males is significantly shorter periodicity than females. The results from the activity/rest behaviour experiments suggest that male-female dimorphism in circadian phenotypes is consistent across a wide range of Drosophila species. In my second experiment, I studied the mating rhythm in five wild caught species

of Drosophila. Male-female pairs from each species were introduced in glass vials loaded with corn meal, and mating was assayed continuously for 20 minute, every 3 hours. The results of the assay indicate that the time course and waveform of mean mating frequency in different species, under LD cycles was significantly different; D. ananassae has its mating peak (with -40% mating) during "dawn" (ZTOO), D. nasuta (-70%) four hours later (ZT04), whereas D. malerkotliana, D. melanogaster and Z. indianus (-30 to 40%) during mid-to-late afternoon hours with peaks of mating between ZTOO and ZT09. While most Drosophila species mate only during day, D. malerkotliana was found to mate even in the night. The results suggest that D. ananassae and D. malerkotliana which belong to the same sub-genus and species group mate at different timEs of the day • D. ananassae prefers morning hours, while D. malerkotliana evening hours, leading to temporal isolation. Finally I studied the egg laying behaviour in only four of the five wild caught

three days and subsequently transferred to DD for the egg-laying assay. To estimate the time course and waveform of egg-laying behaviour, the number of eggs laid over a period of two hours was counted for a minimum of seven days. The results suggest that D. melanogast-er lays maximum number of eggs soon after "dusk" ( zn2), D. ananassae and D. nasuta prior to "dusk" ( zno), whereas no such preference for egg-laying was observed in Zaprionus indianus. These results suggest that different species of Drosophila have different preference for time of egg-laying under 12:12 hr light/dark cycle. The percentage of flies in which egg-laying rhythm entrained to 12:12 hr LD cycles varied from one species to another- egg-laying rhythm in ss.O% of D. ananassae flies, 18.2% of Zaprionus indianus flies entrained to LD cycles, whereas the percentage was

61.53% and 40% in D. nasuta and D. melanogaster, respectively. These results indicate that entrainment of egg-laying rhythm to 12:12 hr LD cycles is highest in D. ananassae and lowest in Zaprionus indianus, suggesting that egg-laying rhythm of different species of Drosophila respond differently to LD cycles. Based on the results of our studies we conclude that five wild caught species of Drosophila show species-specific differences in their circadian rhythms in activity/rest, mating and egg-laying behaviours. The results are in conformity with the findings from previous studies done in other species of Drosophila, and suggest that during the course of evolution different species of Drosophila evolve different clock mechanisms to regulate activity/rest, mating and egg-laying behaviours. While five wild caught species

mating and egg-laying rhythms, the differences do not correlate to relatedness between species. In other words, in Drosophila even phylogenetically related species show significant differences in circadian phenotype. My study suggests that wild caught species of Drosophila living in sympatry may have evolved different circadian clock mechanisms for activity, mating and egg-laying rhythms over the course of time for better adaptability and survivorship.