The hemolymph of the American horseshoe crab, Limulus polyphemus, is harvested from over 500,000 animals annually to produce Limulus Amebocyte Lysate, a medically important product used to detect pathogenic bacteria. Declining abundance of spawning Limulus females in heavily harvested regions suggests deleterious effects of this activity and, while mortality rates of the harvest process are known to be 10–30%, sub-lethal behavioral and physiological effects are not known. In this study, we determined the impact of the harvest process on locomotion and hemocyanin levels of 28 female horseshoe crabs. While mortality rates after bleeding (18%) were similar to previous studies, we found significant decreases in the linear and angular velocity of freely moving animals, as well as changes in their activity levels and expression of circatidal behavioral rhythms. Further, we found reductions in hemocyanin levels, which may alter immune function and cuticle integrity. These previously unrecognized behavioral and physiological deficits suggest that the harvest of Limulus Amebocyte Lysate may decrease female fitness, and thus may contribute to the current population decline.
The role of the hormone melatonin in the circadian system of pigeons (Columba livia) was investigated. Using an automatic infusion system, melatonin at physiological levels was delivered for 10 h each day to cannulated, pinealectomized (P-X) pigeons in constant darkness. These cyclic infusions of melatonin entrained feeding rhythms in P-X pigeons while vehicle infusions were ineffective entraining agents. When the retinae of P-X pigeons were removed (E-X), feeding rhythms were abolished in constant darkness. When cyclic melatonin infusions were delivered to these birds (E-X and P-X), feeding rhythmicity was restored whereas vehicle infusions alone did not restore rhythmicity. When melatonin infusions were terminated in E-X/P-X pigeons, feeding rhythms persisted for several days but eventually decayed. Blood melatonin levels were measured in both P-X and E-X/P-X birds infused cyclically with exogenous melatonin and were found to be within the physiological range both in level and pattern. These results strongly suggest that endogenous melatonin, released by the pineal gland and the retinae, regulates the timing of feeding rhythms by entraining other oscillators in the circadian system of the pigeon.
While eye sensitivity in the American horseshoe crab Limulus polyphemus has long been known to be under the control of an endogenous circadian clock, only recently has horseshoe crab locomotion been shown to be controlled by a separate clock system. In the laboratory, this system drives clear activity rhythms throughout much of the year, not just during the mating season when horseshoe crabs express clear tidal rhythms in the field. Water temperature is a key factor influencing the expression of these rhythms: at 17̊C tidal rhythms are expressed by most animals, while at 11̊C expression of circatidal rhythms is rarely seen, and at 4̊C rhythms are suppressed. Neither long (16:8 Light:Dark) nor short (8:16) photoperiods modify this behavior at any of these temperatures. Synchronization of these circatidal rhythms can be most readily effected by water pressure cycles both in situ and in the lab, while temperature and current cycles play lesser, but possibly contributory, roles. Interestingly, Light:Dark cycles appear to have synchronizing as well as “masking” effects in some individuals. Evidence that each of two daily bouts of activity are independent suggests that the Limulus circatidal rhythm of locomotion is driven by two (circalunidian) clocks, each with a period of 24.8h. While the anatomical locations of either the circadian clock, that drives fluctuations in visual sensitivity, or the circatidal clock, that controls tidal rhythms of locomotion, are currently unknown, preliminary molecular analyses have shown that a 71 kD protein that reacts with antibodies directed against the Drosophila PERIOD (PER) protein is found in both the protocerebrum and the subesophageal ganglion.
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