Circadian rhythms of crawling and swimming in the nudibranch mollusc Melibe leonina

Abstract

Daily rhythms of activity driven by circadian clocks are expressed by many organisms, including molluscs. We initiated this study, with the nudibranch Melibe leonina, with four goals in mind: (1) determine which behaviors are expressed with a daily rhythm; (2) investigate which of these rhythmic behaviors are controlled by a circadian clock; (3) determine if a circadian clock is associated with the eyes or optic ganglia of Melibe, as it is in several other gastropods; and (4) test the hypothesis that Melibe can use extraocular photoreceptors to synchronize its daily rhythms to natural light-dark cycles. To address these goals, we analyzed the behavior of 55 animals exposed to either artificial or natural light-dark cycles, followed by constant darkness. We also repeated this experiment using 10 animals that had their eyes removed. Individuals did not express daily rhythms of feeding, but they swam and crawled more at night. This pattern of locomotion persisted in constant darkness, indicating the presence of a circadian clock. Eyeless animals also expressed a daily rhythm of locomotion, with more locomotion at night. The fact that eyeless animals synchronized their locomotion to the light-dark cycle suggests that they can detect light using extraocular photoreceptors. However, in constant darkness, these rhythms deteriorated, suggesting that the clock neurons that influence locomotion may be located in, or near, the eyes. Thus, locomotion in Melibe appears to be influenced by both ocular and extraocular photoreceptors, although the former appear to have a greater influence on the expression of circadian rhythms.

Figure 1

Locomotion, but not feeding, was expressed rhythmically in LD. (A) A comparison of the percentage of each hour Melibe (n = 3) spent engaged in two different behaviors, feeding alone vs. simultaneously crawling and feeding. Values represent the average for five consecutive days. Error bars indicate standard error of the mean (SEM). Note that the rate of feeding was fairly consistent throughout the day and night, while simultaneous crawling and feeding occurred more often at night. Black bars at top indicate periods of darkness. (B) A comparison of the mean percentage of time (± SEM) spent on three activities between day and night demonstrates significantly more (*) crawling and crawling while feeding during the night than the day, while there was no significant difference in the amount of feeding (when not exhibited with any locomotion) between day and night.

Figure 2

Double-plotted actogram showing the pattern of crawling expressed by one Melibe specimen in artificial LD for 4 days, followed by 6 days in DD. One line on the actogram represents 2 days. Black bars at top indicate periods of darkness in LD. While exhibiting some activity during the day, this animal was much more active during the early portion of the evening. In DD, this individual expressed a tau value of 21.3 h. With a tau value less than 24 h, the activity clearly shifts to the left on successive days.

Figure 3

Actogram showing the daily rhythm of swimming expressed by a Melibe specimen in artificial LD for 3 days, followed by DD for 4 days. Black bars at top of figure indicate periods of darkness in LD. In LD, this animal consistently swam around sunset and sunrise. In DD, a circadian rhythm of swimming around subjective sunset persisted, with a tau value of 21.7 h.

Figure 4

Actogram showing a clear nocturnal pattern of crawling by a Melibe specimen exposed to a natural LD cycle, followed by DD (tau = 22.9 h). Black bars indicate periods of darkness in LD. Note that there was a period of inactivity around the transition from LD to DD. However, this inactivity began before the shift to DD, and other animals did not exhibit such periods of inactivity around this transition, suggesting that this period of quiescence was not related to the transition to DD.

Figure 5

The relationship between Melibe locomotion and ambient light levels. Data from 5 consecutive days are averaged and plotted (± SEM) for one sham-operated individual. Activity is calculated in terms of the percentage of each 10-min time interval that the animal was active. Note how strongly this animal’s behavior was influenced by the change in light intensity at sunset and sunrise.

Figure 6

Representative actogram from a Melibe specimen that had its eyes removed, showing activity both in LD and DD. Black bars indicate periods of darkness in LD. This eyeless animal’s activity in LD was entrained to sunset, indicating an ability to still detect light. The pattern of activity became more arrhythmic in DD.

Figure 7

The relationship between changes in ambient light levels and the activity of an eyeless Melibe specimen. The percentage of each 10-min time interval that a single animal was active, from 5 consecutive days, was averaged along with light data, for the same time intervals. Error bars represent SEM. Data were obtained in February 2013, so the days were relatively short. Note how nocturnal activity does not increase until after sunset is complete, and how sedentary this animal became after sunrise, indicating that the animal still coordinated its locomotor activity to light levels.