A Lunar Clock Changes Shielding Pigment Transparency in Larval Ocelli ofClunio marinus

Abstract

Living in the tidal zones of the sea requires synchronization with the dominant environmental influences of tidal, solar, and lunar periodicity. Endogenous clocks anticipate those geoclimatic changes and control the respective rhythms of vital functions. But the underlying mechanisms are only partly understood. While the circadian clocks in animals are investigated employing neurobiological, molecular, and genetic approaches, clocks with a lunar periodicity have been studied with reference to development and behavior only. Sites of their pacemakers, zeitgeber receptors, and coupled endocrine components are unknown. Here, a lunar-rhythmic change of shielding pigment transparency in the larval ocelli of the intertidal midge Clunio marinus is demonstrated for the first time as a possible access to the neurobiology of lunar timing mechanisms. We studied third instar larvae (Vigo strain) throughout the lunar cycle by light- and electron-microscopy as well as by x-ray fluorescence analysis for the identification of the pigment. Moonlight detection is a prerequisite for photic synchronization of the lunar clock. The larval ocelli of Clunio putatively may function as moonlight receptors and are also controlled by the circalunar clock itself, hence being primary candidates for tracing input and output pathways of the lunar pacemaker. Additionally, the demonstration of a reversible optical change of shielding pigment transparency in Clunio is a novel finding, not reported so far in any other animal species, and reveals a mechanism to enhance photosensitivity under the condition of very dim light. It represents a remarkable change of a sense organ from an imaging device to a radiometer. Its restriction to the developmental stage susceptible to lunar timing elucidates a unique sensory strategy evolved at the level of sensory input. It also raises basic questions about the biochemistry of optically active pigments, like melanin, and their intracellular control.

Correlative Imaging of Melanosoms with Ptychography, X-ray Fluorescence and Light Microscopy

“…The interplay of the image contrasts optical density, trace element concentration and electron density on the basis of individual melanosomes within the cell and tissue environment of a Clunio larva ocellus reflect the complexity of the melanization process. The ptychography measurements support the model of loading and unloading of previously empty vesicles for melanogenesis [1,6].…”

“…Clunio is known as a unique model system to decipher mechanisms underlying the complex moon-related rhythms. Shielding pigment granules within the photoreceptor cells of their primitive eyes (ocelli) have been shown to change appearance in the same rhythm [1]. The larvae are equipped with simple pigment-cup eyes (ocelli) in which the dense shielding pigment permits light access to the photosensitive rhabdomers only through a narrow pinhole resulting in a diffuse image of the environment.…”

Common features in diverse insect clocks

“…Fleissner et al [ 125 ] suggested that the larval ocelli of C. marinus function as moonlight receptors for entrainment of the circalunar clock, and are also controlled by the circalunar clock itself, based on the finding that the shielding pigment granules in larval ocelli changed reversibly between black-brown and transparent during lunar phases. Therefore, the larval ocelli are primary candidates for use in the identification of the circalunar clock by tracing input and output pathways of the circalunar clock.…”

“…Therefore, the larval ocelli are primary candidates for use in the identification of the circalunar clock by tracing input and output pathways of the circalunar clock. Although Fleissner et al [ 125 ] considered that a lunar-rhythmic change in the larval ocelli provided a possible handle for approaching the neuronal basis of the circalunar (or circasemilunar) clock, further reports on the location of the clock in C. marinus have not appeared.…”

Chronobiology by moonlight

“…The function of tim3 is still unknown, but it has sequence similarities to tim2 and is also present in mosquitoes as Aedes aegypti and Culex quinquefasciatus [93]. The photoreceptor responsible for moonlight detection is most probably located in the larval ocelli [95]. The latter show a lunar-rhythmic rspb.royalsocietypublishing.org Proc R Soc B 280: 20123088 change of shielding pigment transparency indicating that they do not only function as moonlight receptors, but that they are also controlled by the circalunar clock itself, hence being primary candidates for tracing input and output pathways of the lunar pacemaker.…”