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

The sea slug Melibe leonina 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. PN lesions led to increased feeding duration and food consumption, and PN activity increased in response to stomach distention. Additionally, the percentage of incomplete feeding movements increased with satiation, and PN stimulation had a similar impact in the nerves that innervate the oral hood. These incomplete movements may be functionally similar to the egestive, food rejecting motions seen in other gastropods and enable Melibe to remain responsive to food, yet adjust their behavior as they become satiated. Such flexibility would not be possible if the entire feeding network were inhibited.

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

AbstractTo determine whether eyes of American lobsters (Homarus americanus) 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 105.6% ± 38.8%; and there was a statistically significant difference between day and night responses. This change in visual sensitivity took place while lobsters were held in constant darkness, suggesting that it was due to the influence of a circadian clock. The average period (tau) for the 10 animals that expressed significant circadian rhythms was 23.4 ± 0.8 hours. Previous studies have demonstrated that lobsters have circadian clocks that influence their locomotor activity; and the present data suggest that this is also true for their eyes, leading to an increase in their visual sensitivity at night, when they are typically most active.

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

Horseshoe crabs are harvested by the biomedical industry in order to create Limulus 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. A total of 28 horseshoe crabs (14 control and 14 bled) were fitted with acoustic transmitters and released into the Great Bay Estuary, New Hampshire, during the spring of 2016. The acoustic tags transmitted information about the activity and depth of each animal, and these data were logged by an array of passive acoustic receivers. These data were collected from May to December 2016 and from March to October 2017. Bled animals approached mating beaches less than control animals during the first week after release, with the greatest differences between bled and control females. Bled animals also remained significantly deeper during the spawning season than control animals. However, overall, bled and control animals expressed similar biological rhythms and seasonal migrations. Thus, it appears as if the most obvious impacts of the bleeding process take place during the first one to two weeks after crabs are bled.

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

The biology of the American horseshoe crab, Limulus polyphemus, 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. Limulus polyphemus is also considered the archetypal functional analogue of various extinct groups with serial gnathobasic appendages, including eurypterids, trilobites and other early arthropods, especially Sidneyia inexpectans from the mid-Cambrian (508 Myr) Burgess Shale of Canada. Exceptionally preserved specimens of S. inexpectans 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. Here, we use advanced computational techniques, specifically a unique application of 3D finite-element analysis (FEA), to model the feeding mechanics of L. polyphemus and S. inexpectans: the first such analyses of a modern horseshoe crab and a fossil arthropod. Results show that mechanical performance of the feeding appendages in both arthropods is remarkably similar, suggesting that S. inexpectans had similar shell-crushing capabilities to L. polyphemus This biomechanical solution to processing shelly food therefore has a history extending over 500 Myr, arising soon after the first shell-bearing animals. Arrival of durophagous predators during the early phase of animal evolution undoubtedly fuelled the Cambrian 'arms race' that involved a rapid increase in diversity, disparity and abundance of biomineralized prey species.

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. Additionally, orthologous sequences for the products of five related genes-aryl hydrocarbon receptor nuclear translocator like, photoreceptive cryptochrome, cryptochrome DASH, 6-4 photolyase, and timeout-were determined. Phylogenetic analyses confirmed that the nudibranch proteins were most closely related to known orthologs in related invertebrates, such as oysters and annelids. In general, the nudibranch clock proteins shared greater sequence similarity with Mus musculus orthologs than Drosophila melanogaster orthologs, which is consistent with the closer phylogenetic relationships recovered between lophotrochozoan and vertebrate orthologs. The suite of clock-related genes in nudibranchs includes both photoreceptive and non-photoreceptive cryptochromes, as well as timeout and possibly timeless. Therefore, the nudibranch clock may resemble the one exhibited in mammals, or possibly even in non-drosopholid insects and oysters. The latter would be evidence supporting this as the ancestral clock for bilaterians.

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. FISH indicated the presence of the clock-related transcripts clock, period, and photoreceptive and non-photoreceptive cryptochrome (pcry and npcry, respectively) in two bilateral neurons in each cerebropleural ganglion and a group of <10 neurons in the anterolateral region of each pedal ganglion. Double-label experiments confirmed colocalization of all four clock transcripts with each other. Quantitative PCR demonstrated that the genes clock, period, pcry and npcry exhibited significant differences in expression levels over 24 h. These data suggest that the putative circadian clock network in M. leonina consists of a small number of identifiable neurons that express circadian genes with a daily rhythm.

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

While horseshoe crabs Limulus polyphemus 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. Activity rhythms of animals from environments with two daily tides (Gulf of Maine, 43°6' N/70°52' W, and Massachusetts, 41°32' N/70°40'W), one dominant daily tide (Apalachee Bay, Florida, 29°58' N/84°20' W), and microtides (Indian River Lagoon, Florida, 28°5' N/80°35' W) were recorded in 2011-2013 during three artificial tide conditions: no tides, a 12.4 h tidal cycle, and a 24.8 h tidal cycle. Interestingly, L. polyphemus from the microtidal site (n = 7) appeared "plastic" in their responses; they were able to express both bimodal and unimodal rhythms in response to different tide cycles. In contrast, the other two populations exhibited more fixed responses: regardless of the tides they were exposed to, animals from areas with one dominant daily tide (n = 18) consistently expressed unimodal rhythms, while those from areas with two daily tides (n = 28) generally expressed bimodal rhythms. Rhythms expressed by L. polyphemus thus appear to be a function of endogenous clocks, the tidal cues to which animals are exposed, and tidal cues that animals experience throughout ontogeny.

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

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, Limulus polyphemus, 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 24.8h. First, spontaneous "skipping" of one of the daily bouts was observed under several different conditions. Second, the presence of two bouts of activity/day, with different periods, was observed. Lastly, we were able to separately synchronize bouts of activity to two artificial tidal regimes with different periods. These results, taken together, argue in favor of two separate circalunidian clocks in Limulus, each of which controls one of the two bouts of their daily tidal activity rhythms.

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. Alignments and functional domain analyses showed generally high conservation between the putative L. polyphemus clock genes and homologs from Drosophila melanogaster and Daphnia pulex. The presence of both cry1 and cry2 in the L. polyphemus transcriptome would classify its system as an "ancestral", type 2 clock system. In addition, a novel duplication of CYCLE, and a novel triplication of PERIOD were found. Investigations are currently underway to determine if any of these "circadian" genes also participate in the molecular processes that drive the Limulus circatidal clock.

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. Although the rate of egg development did not differ significantly between treatments in the fall (P∼0.570), eggs exposed to inshore thermal regimes developed faster in the spring (P<0.001). "Inshore" eggs hatched about 30 days earlier (mean=26 June) than "offshore" eggs (mean=27 July), and their time of development from the onset of eyespot to hatch was significantly shorter (inshore=287±11 days vs. offshore: 311.5±7.5 days, P=0.034). Associated growing degree-days (GDD) did not differ significantly between inshore and offshore thermal treatments (P=0.061). However, eggs retained by lobsters exposed to offshore thermal regimes accumulated more GDD in the winter than did eggs carried by inshore lobsters, while eggs exposed to inshore temperatures acquired them more rapidly in the spring. Results suggest that seasonal movements of ovigerous lobsters influence the time and location of hatching, and thus the transport and recruitment of larvae to coastal and offshore locations.

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. This method involves inserting a blunt-tipped needle into the female's seminal receptacle to determine the presence or absence of a sperm plug and to withdraw a sample that can be examined for the presence of sperm. A series of control studies were conducted at the dock and in the laboratory to test the reliability of this technique. These efforts entailed sampling 294 female lobsters to confirm that the presence of a sperm plug was a reliable indicator of sperm within the receptacle and thus, mating. This paper details the methodology and the results obtained from a subset of the total females sampled. Of the 230 female lobsters sampled from George's Bank and Cape Ann, MA (size range = 71-145 mm in carapace length), 90.3% were positive for sperm. Potential explanations for the absence of sperm in some females include: immaturity (lack of physiological maturity), breakdown of the sperm plug after being used to fertilize a clutch of eggs, and lack of mating activity. The surveys indicate that this technique for examining the mating success of female lobsters is a reliable proxy that can be used in the field to document reproductive activity in natural populations.

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. 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.

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. In LD, 15 animals expressed daily patterns of activity, 6 displayed a circatidal pattern, and the remaining 3 were arrhythmic. Of the 15 animals with daily patterns of locomotion, 7 had a significant preference (P < 0.05) for diurnal activity and 3 for nocturnal activity; the remainder did not express a significant preference for day or night activity. In DD, 13 horseshoe crabs expressed circatidal rhythms and 8 maintained a pattern of about 24 h. Although these results suggest the presence of a circadian clock influencing circatidal patterns of locomotion, these apparent circadian rhythms may actually represent the expression of just one of the two bouts of activity driven by the putative circalunidian clocks that control their tidal rhythms. Overall, these results indicate that, like adults, juvenile horseshoe crabs express both daily and tidal patterns of activity and that at least one, and maybe both, of these patterns is driven by endogenous clocks.

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. 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 LAL may decrease female fitness, and thus may contribute to the current population decline.

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. In addition, we examined the response of the same fish to the sounds the lobsters produced. Although solitary lobsters spontaneously produced sounds at a low rate of 1.2 ± 0.23 sound events per 30 min, the presence of a single cod or striped bass led to an increase in the rate of sound production (cod: 51.1 ± 13.1 events per 30 min; striped bass: 17.0 ± 7.0 events per 30 min). Most (74.6 ± 6.6%) of the 292 sound events recorded occurred when a fish came within 0.5 m of a lobster, but a fish did not have to come into contact with a lobster to elicit sounds. Immediately following the production of a sound by a lobster, fish turned and swam away significantly faster than when they encountered a lobster that did not make a sound. Moreover, after striped bass (but not cod) experienced a number of these sound events, they subsequently tended to avoid swimming close to the lobsters. These data, taken together, suggest that sound production by American lobsters may serve to deter potential fish predators.

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. polyphemus was monitored with activity boxes and "running wheels." When the crabs were exposed to artificial tides created by changes in water depth, circatidal rhythms were observed in animals exposed to 12.4-h "tidal" cycles of either water depth changes (8 of 8 animals) or inundation (7 of 8 animals). In experiment three, an additional 8 animals were exposed to water depth changes under cyclic conditions of light and dark and then monitored for 10 days with no imposed artificial tides. Most animals (5) clearly synchronized their activity to the imposed artificial tidal cycles, and 3 of these animals showed clear evidence of entrainment after the artificial tides were terminated. Overall, these results demonstrate that the endogenous tidal clock that influences locomotion in Limulus can be entrained by imposed artificial tides. In the laboratory, these tidal cues override the influence of light/dark cycles. In their natural habitat, where both tidal and photoperiod inputs are typically always present, their activity rhythms are likely to be much more complex.

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. Although every animal (24) expressed consistent circadian rhythms of visual sensitivity, rhythms of locomotion were more variable: 44% expressed a tidal rhythm, 28% were most active at night, and the rest lacked statistically significant rhythms. When exposed to artificial tides, 8 of 16 animals expressed circatidal rhythms of locomotion that continued after tidal cycles were stopped. However, rhythms of visual sensitivity remained stable and showed no tendency to be influenced by the imposed tides or locomotor activity. These results indicate that horseshoe crabs possess at least two biological clocks: one circadian clock primarily used for modulating visual sensitivity, and one or more clocks that control patterns of locomotion. This arrangement allows horseshoe crabs to see quite well while mating during both daytime and nighttime high tides.

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 277.1 ms) and lead to waterborne sounds of similar frequencies. Lobsters most often produce these signals using only one pair of muscles at a time and alternate between the muscles of the left and right antennae when making a series of vibrations. Occasionally, they vibrate their carapace by simultaneously contracting both sets of muscles. While the remotor muscle is required for producing carapace vibrations, the promotor appears to play a secondary role. Electrical stimulation of the remotor, but not the promotor, results in the production of vibrations, while lesions of the remotor, but not promotor, eliminate the ability of lobsters to vibrate their carapace. Lobsters of all sizes and both sexes produce these signals when startled, grasped or threatened. However, at this time, the behavioral significance of vibration and/or sound production by American lobsters is not known.

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. Interneurons on one side of the brain, which includes the paired cerebral, pleural and pedal ganglia, coordinate bending movements toward the same side and communicate via non-rectifying electrical synapses. Interneurons on opposite sides of the brain coordinate antagonistic movements and communicate over mutually inhibitory synaptic pathways. Several criteria were used to identify members of the swim CPG, the most important being the ability to shift the phase of swimming behavior in a quantitative fashion by briefly altering the firing pattern of an individual neuron. Strong depolarization of any of the interneurons produces an ipsilateral swimming movement during which the several components of the motor act occur in sequence. Strong hyperpolarization causes swimming to stop and leaves the animal contracted to the opposite side for the duration of the hyperpolarization. The four swim interneurons make appropriate synaptic connections with motoneurons, exciting synergists and inhibiting antagonists. Finally, these are the only neurons that were found to have this set of properties in spite of concerted efforts to sample widely in the Melibe CNS. This led us to conclude that these four cells constitute the CPG for swimming. While sint1 and sint2 work together during swimming, they play different roles in the generation of other behaviors. Sint1 is normally silent when the animal is crawling on a surface but it depolarizes and begins to fire in strong bursts once the foot is dislodged and the animal begins to swim. Sint2 also fires in bursts during swimming, but it is not silent in non-swimming animals. Instead activity in sint2 is correlated with turning movements as the animal crawls on a surface. This suggests that the Melibe motor system is organized in a hierarchy and that the alternating movements characteristic of swimming emerge when activity in sint1 and sint2 is bound together.