Melibe Leonina Research Articles

The distribution and evolutionary dynamics of dopaminergic neurons in molluscs.

Dopamine is one of the most versatile neurotransmitters in invertebrates. It's distribution and plethora of functions is likely coupled to feeding ecology, especially in Euthyneura (the largest clade of molluscs), which presents the broadest spectrum of environmental adaptations. Still, the analyses of dopamine-mediated signaling were dominated by studies of grazers. Here, we characterize the distribution of dopaminergic neurons in representatives of two distinct ecological groups: the sea angel - obligate predatory pelagic mollusc Clione limacina (Pteropoda, Gymnosomata) and its prey - the sea devil Limacina helicina (Pteropoda, Thecosomata) as well as the plankton eater Melibe leonina (Nudipleura, Nudibranchia). By using tyrosine hydroxylase-immunoreactivity (TH-ir) as a reporter, we showed that the dopaminergic system is moderately conservative among euthyneurans. Across all studied species, small numbers of dopaminergic neurons in the central ganglia contrast to significant diversification of TH-ir neurons in the peripheral nervous system, primarily representing sensory-like cells, which predominantly concentrated in the chemotactic areas and projecting afferent axons to the central nervous system. Combined with α-tubulin immunoreactivity, this study illuminates the unprecedented complexity of peripheral neural systems in gastropod molluscs, with lineage-specific diversification of sensory and modulatory functions.

Pairing cellular and synaptic dynamics into building blocks of rhythmic neural circuits. A tutorial.

In this study we focus on two subnetworks common in the circuitry of swim central pattern generators (CPGs) in the sea slugs, Melibe leonina and Dendronotus iris and show that they are independently capable of stably producing emergent network bursting. This observation raises the question of whether the coordination of redundant bursting mechanisms plays a role in the generation of rhythm and its regulation in the given swim CPGs. To address this question, we investigate two pairwise rhythm-generating networks and examine the properties of their fundamental components: cellular and synaptic, which are crucial for proper network assembly and its stable function. We perform a slow-fast decomposition analysis of cellular dynamics and highlight its significant bifurcations occurring in isolated and coupled neurons. A novel model for slow synapses with high filtering efficiency and temporal delay is also introduced and examined. Our findings demonstrate the existence of two modes of oscillation in bicellular rhythm-generating networks with network hysteresis: i) a half-center oscillator and ii) an excitatory-inhibitory pair. These 2-cell networks offer potential as common building blocks combined in modular organization of larger neural circuits preserving robust network hysteresis.

Error Function Optimization to Compare Neural Activity and Train Blended Rhythmic Networks.

We present a novel set of quantitative measures for "likeness" (error function) designed to alleviate the time-consuming and subjective nature of manually comparing biological recordings from electrophysiological experiments with the outcomes of their mathematical models. Our innovative "blended" system approach offers an objective, high-throughput, and computationally efficient method for comparing biological and mathematical models. This approach involves using voltage recordings of biological neurons to drive and train mathematical models, facilitating the derivation of the error function for further parameter optimization. Our calibration process incorporates measurements such as action potential (AP) frequency, voltage moving average, voltage envelopes, and the probability of post-synaptic channels. To assess the effectiveness of our method, we utilized the sea slug Melibe leonina swim central pattern generator (CPG) as our model circuit and conducted electrophysiological experiments with TTX to isolate CPG interneurons. During the comparison of biological recordings and mathematically simulated neurons, we performed a grid search of inhibitory and excitatory synapse conductance. Our findings indicate that a weighted sum of simple functions is essential for comprehensively capturing a neuron's rhythmic activity. Overall, our study suggests that our blended system approach holds promise for enabling objective and high-throughput comparisons between biological and mathematical models, offering significant potential for advancing research in neural circuitry and related fields.

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.

Comparative Analysis of Neuropeptides in Homologous Interneurons and Prohormone Annotation in Nudipleuran Sea Slugs.

Despite substantial research on neuronal circuits in nudipleuran gastropods, few peptides have been implicated in nudipleuran behavior. In this study, we expanded the understanding of peptides in this clade, using three species with well-studied nervous systems, Hermissenda crassicornis, Melibe leonina, and Pleurobranchaea californica. For each species, we performed sequence homology analysis of de novo transcriptome predictions to identify homologs to 34 of 36 prohormones previously characterized in the gastropods Aplysia californica and Lymnaea stagnalis. We then used single-cell mass spectrometry to characterize peptide profiles in homologous feeding interneurons: the multifunctional ventral white cell (VWC) in P. californica and the small cardioactive peptide B large buccal (SLB) cells in H. crassicornis and M. leonina. The neurons produced overlapping, but not identical, peptide profiles. The H. crassicornis SLB cells expressed peptides from homologs to the FMRFamide (FMRFa), small cardioactive peptide (SCP), LFRFamide (LFRFa), and feeding circuit activating peptides prohormones. The M. leonina SLB cells expressed peptides from homologs to the FMRFa, SCP, LFRFa, and MIP-related peptides prohormones. The VWC, previously shown to express peptides from the FMRFa and QNFLa (a homolog of A. californica pedal peptide 4) prohormones, was shown to also contain SCP peptides. Thus, each neuron expressed peptides from the FMRFa and SCP families, the H. crassicornis and M. leonina SLB cells expressed peptides from the LFRFa family, and each neuron contained peptides from a prohormone not found in the others. These data suggest each neuron performs complex co-transmission, which potentially facilitates a multifunctional role in feeding. Additionally, the unique feeding characteristics of each species may relate, in part, to differences in the peptide profiles of these neurons. These data add chemical insight to enhance our understanding of the neuronal basis of behavior in nudipleurans and other gastropods.

The Digestive Diverticula in the Carnivorous Nudibranch, Melibe leonina, Do Not Contain Photosynthetic Symbionts.

SynopsisA number of nudibranchs, including Melibe engeli and Melibe pilosa, harbor symbiotic photosynthetic zooxanthellae. Melibe leonina spends most of its adult life on seagrass or kelp, capturing planktonic organisms in the water column with a large, tentacle-lined oral hood that brings food to its mouth. M. leonina also has an extensive network of digestive diverticula, located just beneath its translucent integument, that are typically filled with pigmented material likely derived from ingested food. Therefore, the focus of this project was to test the hypothesis that M. leonina accumulates symbiotic photosynthetic dinoflagellates in these diverticula. First, we conducted experiments to determine if M. leonina exhibits a preference for light, which would allow chloroplasts that it might be harboring to carry out photosynthesis. We found that most M. leonina preferred shaded areas and spent less time in direct sunlight. Second, we examined the small green circular structures in cells lining the digestive diverticula. Like chlorophyll, they exhibited autofluorescence when illuminated at 480 nm, and they were also about the same size as chloroplasts and symbiotic zooxanthellae. However, subsequent electron microscopy found no evidence of chloroplasts in the digestive diverticula of M. leonina; the structures exhibiting autofluorescence at 480 nm were most likely heterolysosomes, consistent with normal molluscan digestion. Third, we did not find evidence of altered oxygen consumption or production in M. leonina housed in different light conditions, suggesting the lack of any significant photosynthetic activity in sunlight. Fourth, we examined the contents of the diverticula, using HPLC, thin layer chromatography, and spectroscopy. The results of these studies indicate that the diverticula did not contain any chlorophyll, but rather harbored other pigments, such as astaxanthin, which likely came from crustaceans in their diet. Together, all of these data suggest that M. leonina does sequester pigments from its diet, but not for the purpose of symbiosis with photosynthetic zooxanthellae. Considering the translucent skin of M. leonina, the pigmented diverticula may instead provide camouflage.

The Distribution and Possible Roles of Small Cardioactive Peptide in the Nudibranch Melibe leonina.

SynopsisThe neuropeptide small cardioactive peptide (SCP) plays an integrative role in exciting various motor programs involved in feeding and locomotion in a number of gastropod species. In this study, immunohistochemistry, using monoclonal antibodies against SCPB, was used to localize SCPB-like-immunoreactive neurons in the central nervous system, and map their connections to various tissues, in the nudibranch, Melibe leonina. Approximately 28–36 SCPB-like-immunoreactive neurons were identified in the M. leonina brain, as well as one large neuron in each of the buccal ganglia. The neuropil of the pedal ganglia contained the most SCPB-like-immunoreactive varicosities, although only a small portion of these were due to SCPB-like-immunoreactive neurons in the same ganglion. This suggests that much of the SCPB-like immunoreactivity in the neuropil of the pedal ganglia was from neurons in other ganglia that projected through the pedal–pedal connectives or the connectives from the cerebral and pleural ganglia. We also observed extensive SCPB innervation along the length of the esophagus. Therefore, we investigated the impact of SCPB on locomotion in intact animals, as well as peristaltic contractions of the isolated esophagus. Injection of intact animals with SCPB at night led to a significant increase in crawling and swimming, compared to control animals injected with saline. Furthermore, perfusion of isolated brains with SCPB initiated expression of the swim motor program. Application of SCPB to the isolated quiescent esophagus initiated rhythmic peristaltic contractions, and this occurred in preparations both with and without the buccal ganglia being attached. All these data, taken together, suggest that SCPB could be released at night to arouse animals and enhance the expression of both feeding and swimming motor programs in M. leonina.

Command or Obey? Homologous Neurons Differ in Hierarchical Position for the Generation of Homologous Behaviors.

In motor systems, higher-order neurons provide commands to lower-level central pattern generators (CPGs) that autonomously produce rhythmic motor patterns. Such hierarchical organization is often thought to be inherent in the anatomical position of the neurons. Here, however, we report that a neuron that is member of a CPG in one species acts as a higher-order neuron in another species. In the nudibranch mollusc, Melibe leonina, swim interneuron 1 (Si1) is in the CPG underlying swimming, firing rhythmic bursts of action potentials as part of the swim motor pattern. We found that its homolog in another nudibranch, Dendronotus iris, serves as a neuromodulatory command neuron for the CPG of a homologous swimming behavior. In Dendronotus, Si1 fired irregularly throughout the swim motor pattern. The burst and spike frequencies of Dendronotus swim CPG neurons correlated with Si1 firing frequency. Si1 activity was both necessary and sufficient for the initiation and maintenance of the swim motor pattern. Each Si1 was electrically coupled to all of the CPG neurons and made monosynaptic excitatory synapses with both Si3s. Si1 also bilaterally potentiated the excitatory synapse from Si3 to Si2. "Virtual neuromodulation" of both Si3-to-Si2 synapses using dynamic clamp combined with depolarization of both Si3s mimicked the effects of Si1 stimulation on the swim motor pattern. Thus, in Dendronotus, Si1 is a command neuron that turns on, maintains, and accelerates the motor pattern through synaptic and neuromodulatory actions, thereby differing from its homolog in Melibe in its functional position in the motor hierarchy.SIGNIFICANCE STATEMENT Cross-species comparisons of motor system organization can provide fundamental insights into their function and origin. Central pattern generators (CPGs) are lower in the functional hierarchy than the neurons that initiate and modulate their activity. This functional hierarchy is often reflected in neuroanatomical organization. This paper definitively shows that an identified cerebral ganglion neuron that is a member of a CPG underlying swimming in one nudibranch species serves as a command neuron for the same behavior in another species. We describe and test the synaptic and neuromodulatory mechanisms by which the command neuron initiates and accelerates rhythmic motor patterns. Thus, the functional position of neurons in a motor hierarchy can shift from one level to another over evolutionary time.

Actor or Director? Homologous Neurons Differ in Hierarchical Position for the Generation of Homologous Behaviors

Nervous systems are hierarchically organized; higher order neurons provide commands to central pattern generators (CPGs), which autonomously produce rhythmic motor patterns. Such hierarchies are generally thought to be inherent in the structure of nervous systems. Here, however, we report that a neuron that is a CPG member in one species acts as a higher order neuron in another species in the production of a homologous motor behavior. In the nudibranch mollusc, Melibe leonina, swim interneuron 1 (Si1) is in the CPG underlying swimming, firing bursts in phase with the motor pattern. In contrast, its homolog in another nudibranch, Dendronotus iris, serves as a neuromodulatory command neuron for the CPG of a homologous swimming behavior. In Dendronotus, Si1 fired irregularly throughout the swim motor pattern. The burst and firing frequencies of Dendronotus swim CPG neurons correlated with the firing frequency of Si1. Si1 activity was both necessary and sufficient for the initiation and maintenance of the swim motor pattern. Each Si1 was electrically coupled to all of the CPG neurons and made monosynaptic excitatory synapses with both Si3s. Si1 also potentiated the excitatory synapses from Si3 to Si2. Artificial synaptic enhancement of the Si3‐to‐Si2 synapses using dynamic clamp combined with depolarization of Si3s mimicked the effects of Si1 stimulation on the swim motor pattern. Thus, in Dendronotus, Si1 is a command neuron that turns on, maintains, and accelerates the motor pattern through synaptic and neuromodulatory actions, thereby differing from its homolog in Melibe in its hierarchical position in the motor system.

Characterization of the complete mitochondrial genome of Melibe japonica (Eliot, 1913) collected from Korean water

Melibe japonica is the rarely identified nudibranch species in Korean and Japanese waters. Total mitochondrial genome of M. japonica collected from the coastal water of Busan, Korea, was determined by the bioinformatic assembly of the contigs generated by Illumina Miseq platform. The circular complete mitochondrial genome of M. japonica was 13, 216 bp in length, which contains 13 proteins, 2 ribosomal RNAs, and 22 tRNAs. Mitochondrial gene order of M. japonica is considerably different from the mitochondrial genome of Melibe leonina in which 6 genes (ND1, COX2, CYTB, ND4L, ND5, and ND6) were differently located. Phylogenetic analysis with COI regions showed that M. japonica is most closely related to Melibe viridis. However, not all Melibe species formed a single clade based on the phylogenetic analysis by COI region and more mitochondrial genomes in this genus should be further studied to have a better knowledge of the evolutional relationship of the nudibranchs.

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.

The influence of stomach distention on feeding in the nudibranch mollusk Melibe leonina

Although research on satiation has revealed much about the effect of sensory inputs on motivational state, we have yet to fully understand exactly how satiating signals influence the neural circuits underlying specific behaviors. One organism that is well suited for addressing this question is the nudibranch Melibe leonina, because its feeding activity is easily quantified, it has translucent skin that makes the stomach easy to observe, and it has large, identifiable neurons that are very suitable for subsequent analysis of the neural correlates of satiation. In this study our goal was to document the time course of satiation in Melibe, and determine if stomach distention contributes to satiation. When exposed to brine shrimp (Artemia), Melibe immediately commenced stereotypic oral hood movements to capture prey, and continued to do so for approximately five hours. Individuals eventually stopped, despite the continued presence of food, and the slowing and eventual termination of oral hood closures was correlated with distension of the stomach caused by the ingested Artemia. We obtained further evidence that stomach distension is one of the underlying causes of satiation by injecting artificial non-nutritive food into the stomach, and by cutting open part of the stomach wall to prevent it from filling and distending. The first treatment caused satiation to occur more rapidly, while the second treatment delayed satiation. Both results demonstrate that in Melibe stomach distention has a major impact on the motivation to feed. These findings provide the framework for subsequent studies designed to determine precisely how stomach distention influences feeding circuits.

Making a Swim Central Pattern Generator Out of Latent Parabolic Bursters

We study the rhythmogenesis of oscillatory patterns emerging in network motifs composed of inhibitory coupled tonic spiking neurons represented by the Plant model of R15 nerve cells. Such motifs are argued to be used as building blocks for a larger central pattern generator network controlling swim locomotion of sea slug Melibe leonina.

The Mitochondrial Genomes of the Nudibranch Mollusks, Melibe leonina and Tritonia diomedea, and Their Impact on Gastropod Phylogeny.

The phylogenetic relationships among certain groups of gastropods have remained unresolved in recent studies, especially in the diverse subclass Opisthobranchia, where nudibranchs have been poorly represented. Here we present the complete mitochondrial genomes of Melibe leonina and Tritonia diomedea (more recently named T. tetraquetra), two nudibranchs from the unrepresented Cladobranchia group, and report on the resulting phylogenetic analyses. Both genomes coded for the typical thirteen protein-coding genes, twenty-two transfer RNAs, and two ribosomal RNAs seen in other species. The twelve-nucleotide deletion previously reported for the cytochrome oxidase 1 gene in several other Melibe species was further clarified as three separate deletion events. These deletions were not present in any opisthobranchs examined in our study, including the newly sequenced M. leonina or T. diomedea, suggesting that these previously reported deletions may represent more recently divergent taxa. Analysis of the secondary structures for all twenty-two tRNAs of both M. leonina and T. diomedea indicated truncated d arms for the two serine tRNAs, as seen in some other heterobranchs. In addition, the serine 1 tRNA in T. diomedea contained an anticodon not yet reported in any other gastropod. For phylogenetic analysis, we used the thirteen protein-coding genes from the mitochondrial genomes of M. leonina, T. diomedea, and seventy-one other gastropods. Phylogenetic analyses were performed for both the class Gastropoda and the subclass Opisthobranchia. Both Bayesian and maximum likelihood analyses resulted in similar tree topologies. In the Opisthobranchia, the five orders represented in our study were monophyletic (Anaspidea, Cephalaspidea, Notaspidea, Nudibranchia, Sacoglossa). In Gastropoda, two of the three traditional subclasses, Opisthobranchia and Pulmonata, were not monophyletic. In contrast, four of the more recently named gastropod clades (Vetigastropoda, Neritimorpha, Caenogastropoda, and Heterobranchia) were all monophyletic, and thus appear to be better classifications for this diverse group.

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.

Two interconnected kernels of reciprocally inhibitory interneurons underlie alternating left-right swim motor pattern generation in the mollusk Melibe leonina

The central pattern generator (CPG) underlying the rhythmic swimming behavior of the nudibranch Melibe leonina (Mollusca, Gastropoda, Heterobranchia) has been described as a simple half-center oscillator consisting of two reciprocally inhibitory pairs of interneurons called swim interneuron 1 (Si1) and swim interneuron 2 (Si2). In this study, we identified two additional pairs of interneurons that are part of the swim CPG: swim interneuron 3 (Si3) and swim interneuron 4 (Si4). The somata of Si3 and Si4 were both located in the pedal ganglion, near that of Si2, and both had axons that projected through the pedal commissure to the contralateral pedal ganglion. These neurons fulfilled the criteria for inclusion as members of the swim CPG: 1) they fired at a fixed phase in relation to Si1 and Si2, 2) brief changes in their activity reset the motor pattern, 3) prolonged changes in their activity altered the periodicity of the motor pattern, 4) they had monosynaptic connections with each other and with Si1 and Si2, and 5) their synaptic actions helped explain the phasing of the motor pattern. The results of this study show that the motor pattern has more complex internal dynamics than a simple left/right alternation of firing; the CPG circuit appears to be composed of two kernels of reciprocally inhibitory neurons, one consisting of Si1, Si2, and the contralateral Si4 and the other consisting of Si3. These two kernels interact with each other to produce a stable rhythmic motor pattern.

Comparative mapping of GABA‐immunoreactive neurons in the central nervous systems of nudibranch molluscs

The relative simplicity of certain invertebrate nervous systems, such as those of gastropod molluscs, allows behaviors to be dissected at the level of small neural circuits composed of individually identifiable neurons. Elucidating the neurotransmitter phenotype of neurons in neural circuits is important for understanding how those neural circuits function. In this study, we examined the distribution of γ-aminobutyric-acid;-immunoreactive (GABA-ir) neurons in four species of sea slugs (Mollusca, Gastropoda, Opisthobranchia, Nudibranchia): Tritonia diomedea, Melibe leonina, Dendronotus iris, and Hermissenda crassicornis. We found consistent patterns of GABA immunoreactivity in the pedal and cerebral-pleural ganglia across species. In particular, there were bilateral clusters in the lateral and medial regions of the dorsal surface of the cerebral ganglia as well as a cluster on the ventral surface of the pedal ganglia. There were also individual GABA-ir neurons that were recognizable across species. The invariant presence of these individual neurons and clusters suggests that they are homologous, although there were interspecies differences in the numbers of neurons in the clusters. The GABAergic system was largely restricted to the central nervous system, with the majority of axons confined to ganglionic connectives and commissures, suggesting a central, integrative role for GABA. GABA was a candidate inhibitory neurotransmitter for neurons in central pattern generator (CPG) circuits underlying swimming behaviors in these species, however none of the known swim CPG neurons were GABA-ir. Although the functions of these GABA-ir neurons are not known, it is clear that their presence has been strongly conserved across nudibranchs.

Toward robust phase-locking in Melibe swim central pattern generator models

Small groups of interneurons, abbreviated by CPG for central pattern generators, are arranged into neural networks to generate a variety of core bursting rhythms with specific phase-locked states, on distinct time scales, which govern vital motor behaviors in invertebrates such as chewing and swimming. These movements in lower level animals mimic motions of organs in higher animals due to evolutionarily conserved mechanisms. Hence, various neurological diseases can be linked to abnormal movement of body parts that are regulated by a malfunctioning CPG. In this paper, we, being inspired by recent experimental studies of neuronal activity patterns recorded from a swimming motion CPG of the sea slug Melibe leonina, examine a mathematical model of a 4-cell network that can plausibly and stably underlie the observed bursting rhythm. We develop a dynamical systems framework for explaining the existence and robustness of phase-locked states in activity patterns produced by the modeled CPGs. The proposed tools can be used for identifying core components for other CPG networks with reliable bursting outcomes and specific phase relationships between the interneurons. Our findings can be employed for identifying or implementing the conditions for normal and pathological functioning of basic CPGs of animals and artificially intelligent prosthetics that can regulate various movements.

Developmental Mode in Opisthobranch Molluscs from the Northeast Pacific Ocean: Additional Species from Southern California and Supplemental Data

Abstract We document development type for 33 species of benthic opisthobranch gastropods – 15 for the first time – collected mainly from the Southern California Bight. Fourteen of the newly examined species had planktotrophic development, while the dorid nudibranch Atagema alba had capsular metamorphic development, the first example of direct development in a non-dendrodoridid nudibranch known from the northeast Pacific Ocean. For the remaining 18 species our new data are either consistent with earlier determinations of development type, or confirm previous inferences. The new data also broaden geographic coverage for some species, and for the sacoglossan Stiliger fuscovittatus and the nudibranch Melibe leonina, suggest that egg size is inversely related to temperature. We correct the previous erroneous identification of nephrocysts as eyespots in the hatching planktotrophic larvae of the nudibranchs Tritonia festiva and Janolus fuscus. These results further highlight the predominance of planktotrophic de...