Effects of Feeding Horseshoe Crabs () on Their Recovery after Being Bled.

AbstractBleeding of horseshoe crabs () for the biomedical industry can have both sublethal and lethal impacts. Bleeding induces a significant drop in the concentration of hemolymph hemocyanin, as well as decreased levels of activity. Furthermore, horseshoe crabs with low hemocyanin prior to being bled have been found to be more likely to die after the procedure.

Long-term circatidal rhythms of heart rate, gill ventilation, and locomotion in the American horseshoe crab, (Arthropoda: Chelicerata: Merostomata: Limulidae).

While several marine species exhibit biological rhythms of heart rate, gill ventilation, or locomotion, the relationship between these three measures in any species remains unexplored. The American horseshoe crab, , Linnaeus, 1758, expresses circalunidian locomotor rhythms and circadian eye sensitivity rhythms but it is not clear if either heart and ventilation rates are controlled on a circadian or circatidal basis or the nature of the relationship between these three measures. The goal of this study was to determine the extent to which the heart and ventilation rates of are coordinated with its endogenous rhythms of locomotion.

A Circadian Rhythm of Visual Sensitivity in the American Lobster, .

AbstractTo determine whether eyes of American lobsters () are more sensitive to light at night than during the day, electroretinograms were continuously recorded from 23 adult lobsters for at least 3 days (range: 3 to 9 days) in constant darkness. A green light-emitting diode, mounted 10 cm away from the eyes, was briefly flashed every 2 minutes to evoke the electroretinogram. The average increase in the response to a light flash, between the minimum during the subjective day and the maximum during the subjective night, was 105.

In the sea slug the posterior nerves communicate stomach distention to inhibit feeding and modify oral hood movements.

The sea slug is an excellent model system for the study of the neural basis of satiation, and previous studies have demonstrated that stomach distention attenuates feeding. Here we expanded on this work by examining the pathway communicating stomach distention to the central nervous system and the effects of distention on motor output. We found that the posterior nerves (PN), which extend posteriorly from the buccal ganglia and innervate the stomach, communicate stomach distention in Melibe.

Effects of the Biomedical Bleeding Process on the Behavior of the American Horseshoe Crab, , in Its Natural Habitat.

Horseshoe crabs are harvested by the biomedical industry in order to create amebocyte lysate to test medical devices and pharmaceutical drugs for endotoxins. Most previous studies on the impacts of the biomedical bleeding process on horseshoe crabs have focused on mortality rates and sublethal impacts in the laboratory. In this study, we investigated the effects of the bleeding process on the behavior of horseshoe crabs after they had been released back into their natural environment.

Computational biomechanical analyses demonstrate similar shell-crushing abilities in modern and ancient arthropods.

The biology of the American horseshoe crab, , is well documented-including its dietary habits, particularly the ability to crush shell with gnathobasic walking appendages-but virtually nothing is known about the feeding biomechanics of this iconic arthropod. is also considered the archetypal functional analogue of various extinct groups with serial gnathobasic appendages, including eurypterids, trilobites and other early arthropods, especially from the mid-Cambrian (508 Myr) Burgess Shale of Canada. Exceptionally preserved specimens of show evidence suggestive of durophagous (shell-crushing) tendencies-including thick gnathobasic spine cuticle and shelly gut contents-but the masticatory capabilities of this fossil species have yet to be compared with modern durophagous arthropods.

Sequences of Circadian Clock Proteins in the Nudibranch Molluscs Hermissenda crassicornis, Melibe leonina, and Tritonia diomedea.

While much is known about the genes and proteins that make up the circadian clocks in vertebrates and several arthropod species, much less is known about the clock genes in many other invertebrates, including nudibranchs. The goal of this project was to identify the RNA and protein products of putative clock genes in the central nervous system of three nudibranchs, Hermissenda crassicornis, Melibe leonina, and Tritonia diomedea. Using previously published transcriptomes (Hermissenda and Tritonia) and a new transcriptome (Melibe), we identified nudibranch orthologs for the products of five canonical clock genes: brain and muscle aryl hydrocarbon receptor nuclear translocator like protein 1, circadian locomotor output cycles kaput, non-photoreceptive cryptochrome, period, and timeless.

Localization and expression of putative circadian clock transcripts in the brain of the nudibranch Melibe leonina.

The nudibranch, Melibe leonina, expresses a circadian rhythm of locomotion, and we recently determined the sequences of multiple circadian clock transcripts that may play a role in controlling these daily patterns of behavior. In this study, we used these genomic data to help us: 1) identify putative clock neurons using fluorescent in situ hybridization (FISH); and 2) determine if there is a daily rhythm of expression of clock transcripts in the M. leonina brain, using quantitative PCR.

Local tidal regime dictates plasticity of expression of locomotor activity rhythms of American horseshoe crabs, .

While horseshoe crabs from regions with two daily tides express endogenous circatidal (~ 12.4 h) activity rhythms, much less is known about locomotor rhythm expression in horseshoe crabs from other tidal regimes. This study investigated whether horseshoe crabs (1) always express activity rhythms consistent with their natural tides, and (2) can alter activity rhythm expression in response to novel tide cycles.

Circalunidian clocks control tidal rhythms of locomotion in the American horseshoe crab, .

While many intertidal animals exhibit circatidal rhythms, the nature of the underlying endogenous clocks that control these rhythms has been controversial. In this study American horseshoe crabs, , were used to test the circalunidian hypothesis by exposing them to four different tidal regimes. Overall, the results obtained support the circalunidian hypothesis: each of the twice-daily rhythms of activity appears to be controlled by a separate clock, each with an endogenous period of approximately 24.

Identification of putative circadian clock genes in the American horseshoe crab, Limulus polyphemus.

While the American horseshoe crab, Limulus polyphemus, has robust circadian and circatidal rhythms, virtually nothing is known about the molecular basis of these rhythms in this species or any other chelicerate. In this study, next generation sequencing was used to assemble transcriptomic reads and then putative homologs of known core and accessory circadian genes were identified in these databases. Homologous transcripts were discovered for one circadian clock input gene, five core genes, 22 accessory genes, and two possible output pathways.

Influence of natural inshore and offshore thermal regimes on egg development and time of hatch in American lobsters, Homarus americanus.

Some egg-bearing (ovigerous) American lobsters (Homarus americanus) make seasonal inshore-to-offshore movements, subjecting their eggs to different thermal regimes than those of eggs carried by lobsters that do not make these movements. Our goal was to determine if differences in thermal regimes influence the rate of egg development and the subsequent time of hatch. We subjected ovigerous lobsters to typical inshore or offshore water temperatures from September to August in the laboratory (n=8 inshore and 8 offshore, each year) and in the field (n=8 each, inshore and offshore), over 2 successive years.

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

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.

A noninvasive method for in situ determination of mating success in female American lobsters (Homarus americanus).

Despite being one of the most productive fisheries in the Northwest Atlantic, much remains unknown about the natural reproductive dynamics of American lobsters. Recent work in exploited crustacean populations (crabs and lobsters) suggests that there are circumstances where mature females are unable to achieve their full reproductive potential due to sperm limitation. To examine this possibility in different regions of the American lobster fishery, a reliable and noninvasive method was developed for sampling large numbers of female lobsters at sea.

Sublethal behavioral and physiological effects of the biomedical bleeding process on the American horseshoe crab, Limulus polyphemus.

The hemolymph of the American horseshoe crab, Limulus polyphemus, is harvested from over 500,000 animals annually to produce Limulus amebocyte lysate (LAL), 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%, sublethal 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.

Patterns of activity expressed by juvenile horseshoe crabs.

Adult American horseshoe crabs, Limulus polyphemus, possess endogenous circadian and circatidal clocks controlling visual sensitivity and locomotion, respectively. The goal of this study was to determine the types of activity rhythms expressed by juvenile horseshoe crabs (n = 24) when exposed to a 14:10 light/dark cycle (LD) for 10 days, followed by 10 days of constant darkness (DD). Horseshoe crab activity was recorded with a digital time-lapse video system that used an infrared-sensitive camera so animals could be monitored at night.

Evidence that potential fish predators elicit the production of carapace vibrations by the American lobster.

American lobsters (Homarus americanus) will on rare occasions produce sounds by vibrating their dorsal carapace. Although this behavior can be elicited in the laboratory by handling lobsters, the stimulus that triggers the production of sounds in the lobster's natural habitat is not known. We investigated the influence of two fish that are known to prey on lobsters, cod (Gadus morhua) and striped bass (Morone saxatilis), on the production of sounds by American lobsters.

Rhythms of locomotion expressed by Limulus polyphemus, the American horseshoe crab: II. Relationship to circadian rhythms of visual sensitivity.

In the laboratory, horseshoe crabs express a circadian rhythm of visual sensitivity as well as daily and circatidal rhythms of locomotion. The major goal of this investigation was to determine whether the circadian clock underlying changes in visual sensitivity also modulates locomotion. To address this question, we developed a method for simultaneously recording changes in visual sensitivity and locomotion.

Rhythms of locomotion expressed by Limulus polyphemus, the American horseshoe crab: I. Synchronization by artificial tides.

Limulus polyphemus, the American horseshoe crab, has an endogenous clock that drives circatidal rhythms of locomotor activity. In this study, we examined the ability of artificial tides to entrain the locomotor rhythms of Limulus in the laboratory. In experiments one and two, the activity of 16 individuals of L.

Mechanisms underlying the production of carapace vibrations and associated waterborne sounds in the American lobster, Homarus americanus.

American lobsters produce carapace vibrations, which also lead to waterborne acoustic signals, by simultaneously contracting the antagonistic remotor and promotor muscles located at the base of the second antenna. These vibrations have a mean frequency of 183.1 Hz (range 87-261 Hz), range in duration from 68 to 1720 ms (mean 277.

Central pattern generator for swimming in Melibe.

The nudibranch mollusc Melibe leonina swims by bending from side to side. We have identified a network of neurons that appears to constitute the central pattern generator (CPG) for this locomotor behavior, one of only a few such networks to be described in cellular detail. The network consists of two pairs of interneurons, termed 'swim interneuron 1' (sint1) and 'swim interneuron 2' (sint2), arranged around a plane of bilateral symmetry.

Neural correlates of swimming behavior in Melibe leonina.

The nudibranch Melibe leonina swims by rhythmically bending from side to side at a frequency of 1 cycle every 2-4 s. The objective of this study was to locate putative swim motoneurons (pSMNs) that drive these lateral flexions and determine if swimming in this species is produced by a swim central pattern generator (sCPG). In the first set of experiments, intracellular recordings were obtained from pSMNs in semi-intact, swimming animals.

Modulation of swimming in the gastropod Melibe leonina by nitric oxide.

Nitric oxide (NO) is a gaseous intercellular messenger produced by the enzyme nitric oxide synthase. It has been implicated as a neuromodulator in several groups of animals, including gastropods, crustaceans and mammals. In this study, we investigated the effects of NO on the swim motor program produced by isolated brains and by semi-intact preparations of the nudibranch Melibe leonina.