Older but not wider. Impacts of molt frequency variation on age and growth dynamics of Newfoundland & Labrador snow crab (Chionoecetes opilio) toward corroborating gastric mill 2 age estimations

Aging specimen and survey data from a heterogeneous stock range are combined to investigate effects of molt frequency variation on age and growth dynamics and efficacy of gastric mill age band estimations in snow crab. A central assumption that bands form independent of molting is examined through analyses of molt frequency variation with an overall result of support for the hypothesis that gastric mill bands form independent of molting. This is based on an applied review approach, demonstrating blind age estimation results within and across population units featuring different rates of molt frequency interruptions in context of literature-based expected outcomes of age and growth dynamics. The comprehensive analyses overall supports the ability of gastric mill bands to track chronological ages, but non-fully explained outcomes of growth delays into sexual maturity stages in males and potential estimation error in 1-3 year old crab preclude a conclusion that absolute ages are consistently tracked throughout ontogeny. The results suggest gastric mill bands continue to form during both skip-molts and after terminal molt. Despite uncertainties, the research highlights that males originating from populations where skip-molting is prominent are older reaching fishery size than previously thought (9-10 years), with tentative average estimates of 10-12 years. The study presents novel observations of size-at-age and age-at-maturity in Newfoundland & Labrador (NL) snow crab and broadens life history knowledge for the species. Along with corroborating gastric mill age band estimations, the study reveals future research streams to further help advance the discipline of aging crustaceans [247].

Neuromodulation enables temperature robustness and coupling between fast and slow oscillator circuits in Cancer borealis

Acute temperature changes can disrupt neuronal activity and coordination with severe consequences for animal behavior and survival. Nonetheless, two rhythmic neuronal circuits in the crustacean stomatogastric ganglion (STG) and their coordination are maintained across a broad temperature range. However, it remains unclear how this temperature robustness is achieved. Here, we dissociate temperature effects on the rhythm generating circuits from those of upstream ganglia. We demonstrate that heat-activated factors extrinsic to the rhythm generators are essential to the slow gastric mill rhythm's temperature robustness and contribute to the temperature response of the fast pyloric rhythm. The gastric mill rhythm crashed when only the STG circuits were heated. It could be restored when upstream ganglia were heated in addition, and the activity of the peptidergic modulatory projection neuron (MCN1) increased. Correspondingly, MCN1's neuropeptide transmitter stabilized the rhythm and maintained it over a broad temperature range. Extrinsic neuromodulation is thus essential for the oscillatory circuits in the STG and enables neural circuits to maintain function in temperature-compromised conditions. In contrast, integer coupling between pyloric and gastric mill rhythms was independent of whether extrinsic inputs and STG pattern generators were temperature-matched or not, demonstrating that the temperature robustness of the coupling is enabled by properties intrinsic to the rhythm generators. However, at near-crash temperature, integer coupling was maintained only in some animals but was absent in others. This was true despite regular rhythmic activity in all animals, supporting that degenerate circuit properties result in idiosyncratic responses to environ-mental challenges.

Feeding State-Dependent Modulation of Feeding-Related Motor Patterns

Studies elucidating modulation of microcircuit activity in isolated nervous systems have revealed numerous insights regarding neural circuit flexibility, but this approach limits the link between experimental results and behavioral context. To bridge this gap, we studied feeding behavior-linked modulation of microcircuit activity in the isolated stomatogastric nervous system (STNS) of male Cancer borealis crabs. Specifically, we removed hemolymph from a crab that was unfed for ≥24 h ('unfed' hemolymph) or fed 15 min - 2 h before hemolymph removal ('fed' hemolymph). After feeding, the first significant foregut emptying occurred >1 h later and complete emptying required ≥6 h. We applied the unfed or fed hemolymph to the stomatogastric ganglion (STG) in an isolated STNS preparation from a separate, unfed crab to determine its influence on the VCN (ventral cardiac neuron)-triggered gastric mill (chewing)- and pyloric (filtering of chewed food) rhythms. Unfed hemolymph had little influence on these rhythms, but fed hemolymph from each examined time-point (15 min, 1- or 2 h post-feeding) slowed one or both rhythms without weakening circuit neuron activity. There were also distinct parameter changes associated with each time-point. One change unique to the 1 h time-point (i.e. reduced activity of one circuit neuron during the transition from the gastric mill retraction to protraction phase) suggested the fed hemolymph also enhanced the influence of a projection neuron which innervates the STG from a ganglion isolated from the applied hemolymph. Hemolymph thus provides a feeding state-dependent modulation of the two feeding-related motor patterns in the C. borealis STG.

Dual modulatory effects on feedback from a proprioceptor in the crustacean stomatogastric nervous system

Neuromodulatory actions that change the properties of proprioceptors or the muscle movements to which they respond necessarily affect the feedback provided to the central network. Here we further characterize the responses of the gastropyloric receptor 1 (GPR1) and gastropyloric receptor 2 (GPR2) neurons in the stomatogastric nervous system of the crab Cancer borealis to movements and contractions of muscles, and we report how neuromodulation modifies those responses. We observed that the GPR1 response to contractions of the gastric mill 4 (gm4) muscle was absent, or nearly so, when the neuron was quiescent but robust when it was spontaneously active. We also found that the effects of four neuromodulatory substances (GABA, serotonin, proctolin and TNRNFLRFamide) on the GPR1 response to muscle stretch were similar to those previously reported for GPR2. Finally, we showed that an excitatory action on gm4 due to proctolin combined with an inhibitory action on GPR2 due to GABA can allow for larger muscle contractions without increased proprioceptive feedback.

Investigating Possible Gastroliths in a Referred Specimen of Bohaiornis guoi (Aves: Enantiornithes)

Gastroliths, where preserved, can provide indirect evidence regarding diet in extinct avian and non-avian dinosaurs. Masses of gastroliths consistent with the presence of a gastric mill are preserved in many Early Cretaceous Jehol birds mostly belonging to the Ornithuromorpha. Gastroliths are also present in basal birds Sapeornis and Jeholornis in which herbivory is supported by direct evidence these taxa consumed seeds in the form of crop or stomach contents. Although gastroliths have been correlated with herbivory in non-avian dinosaurs, the presence of gastroliths and bone together in Ambopteryx calls this association in to question. Despite being known from greater numbers of specimens than other avian lineages, no unequivocal direct or indirect evidence of diet has been recovered from Jehol deposits for the Enantiornithes. A referred specimen of Bohaiornis guoi IVPP V17963 was described as preserving a small number of gastroliths interpreted as rangle, gastroliths whose function is cleaning the stomach in extant raptorial birds. However, based on comparison with gastroliths in other Jehol birds, it has alternatively been suggested that the identified structures are not ingested stones at all but some unusual mineral precipitate. Considering the limited evidence regarding diet in Enantiornithes and the importance of accurately identifying the traces in Bohaiornis in order to understand the enantiornithine digestive system, we extracted two samples of these purported gastroliths and explored these traces using computerized laminography scanning, scanning electron microscopy, energy dispersive x-ray spectroscopy, ground sections, and body size to gastral mass regressions. Similar analyses were conducted on gastroliths extracted from undisputed gastral masses of two Jehol ornithuromorphs and the non-avian pennaraptoran Caudipteryx. The combined results contradict the hypothesis that these traces are gastroliths and supports the interpretation they are mineral precipitate, most likely authigenic quartz (chalcedony). Although authigenesis is commonly responsible for the preservation of soft tissues, it is unclear if these traces record part of the tissues of this Bohaiornis. This study highlights the importance of a multidisciplinary approach in understanding unusual traces in the fossil record and reveal a previously unidentified taphonomic phenomenon in fossils from Jehol deposits.

Neuronal Switching Between Single- and Dual-Network Activity via Modulation of Intrinsic Membrane Properties

Oscillatory networks underlie rhythmic behaviors (e.g. walking, chewing), and complex behaviors (e.g. memory formation, decision making). Flexibility of oscillatory networks includes neurons switching between single- and dual-network participation, even generating oscillations at two distinct frequencies. Modulation of synaptic strength can underlie this neuronal switching. Here we ask whether switching into dual-frequency oscillations can also result from modulation of intrinsic neuronal properties. The isolated stomatogastric nervous system of male Cancer borealis crabs contains two well-characterized rhythmic feeding-related networks (pyloric, ∼1 Hz; gastric mill, ∼0.1 Hz). The identified modulatory projection neuron MCN5 causes the pyloric-only LPG neuron to switch to dual pyloric/gastric mill bursting. Bath applying the MCN5 neuropeptide transmitter Gly1-SIFamide only partly mimics the LPG switch to dual activity, due to continued LP neuron inhibition of LPG. Here, we find that MCN5 uses a co-transmitter, glutamate, to inhibit LP, unlike Gly1-SIFamide excitation of LP. Thus, we modeled the MCN5-elicited LPG switching with Gly1-SIFamide application and LP photoinactivation. Using hyperpolarization of pyloric pacemaker neurons and gastric mill network neurons, we found that LPG pyloric-timed oscillations require rhythmic electrical synaptic input. However, LPG gastric mill-timed oscillations do not require any pyloric/gastric mill synaptic input and are voltage dependent. Thus, we identify modulation of intrinsic properties as an additional mechanism for switching a neuron into dual-frequency activity. Instead of synaptic modulation switching a neuron into a second network as a passive follower, modulation of intrinsic properties could enable a switching neuron to become an active contributor to rhythm generation in the second network.Significance StatementNeuromodulation of oscillatory networks can enable network neurons to switch from sing<bacle- to dual-network participation, even when two networks oscillate at distinct frequencies. We used small, well-characterized networks to determine whether modulation of synaptic strength, an identified mechanism for switching, is necessary for dual-network recruitment. We demonstrate that rhythmic electrical synaptic input is required for continued linkage with a “home” network, but that modulation of intrinsic properties is sufficient to switch a neuron into dual-frequency oscillations, linking it to a second network. Neuromodulator-induced switches in neuronal participation between networks occurs in motor, cognitive, and sensory networks. Our study highlights the importance of considering intrinsic properties as a pivotal target for enabling parallel participation of a neuron in two oscillatory networks.

Coupling between fast and slow oscillator circuits in Cancer borealis is temperature-compensated

Coupled oscillatory circuits are ubiquitous in nervous systems. Given that most biological processes are temperature-sensitive, it is remarkable that the neuronal circuits of poikilothermic animals can maintain coupling across a wide range of temperatures. Within the stomatogastric ganglion (STG) of the crab, Cancer borealis, the fast pyloric rhythm (~1 Hz) and the slow gastric mill rhythm (~0.1 Hz) are precisely coordinated at ~11°C such that there is an integer number of pyloric cycles per gastric mill cycle (integer coupling). Upon increasing temperature from 7°C to 23°C, both oscillators showed similar temperature-dependent increases in cycle frequency, and integer coupling between the circuits was conserved. Thus, although both rhythms show temperature-dependent changes in rhythm frequency, the processes that couple these circuits maintain their coordination over a wide range of temperatures. Such robustness to temperature changes could be part of a toolbox of processes that enables neural circuits to maintain function despite global perturbations.

Age-at-size relationships of the American lobster (Homarus americanus) from three contrasting thermal regimes using gastric mill band counts as a direct aging technique

Direct age determination of crustaceans has remained a long-standing challenge because all calcified structures are shed with each molt. Cuticle bands in the ossicles of the gastric mill have shown promise as age indicators. We validated the one-to-one relationship between known age and number of cuticle bands for 15 hatchery-raised juvenile American lobsters (Homarus americanus). Additionally, we applied this method to 308 lobsters from three contrasting thermal regimes in New England, USA. Band counts matched our expectations of differences in age-at-size across this thermal gradient; lobsters at harvestable size in southern New England were estimated to be 5.5 (±1.5) years old compared with 7.5 (±1.6) years in the Gulf of Maine. We found 81% of our band count estimates of age fell within 2 years of independent, regionally specified growth model estimates of age-at-size for lobster. Notwithstanding remaining uncertainties regarding the mechanism of band formation, our findings indicate the method may provide an independent and direct means to determine the age of individual American lobsters, which will improve estimates of essential life history parameters.

Different microcircuit responses to comparable input from one versus both copies of an identified projection neuron

ABSTRACTNeuronal inputs to microcircuits are often present as multiple copies of apparently equivalent neurons. Thus far, however, little is known regarding the relative influence on microcircuit output of activating all or only some copies of such an input. We examine this issue in the crab (Cancer borealis) stomatogastric ganglion, where the gastric mill (chewing) microcircuit is activated by modulatory commissural neuron 1 (MCN1), a bilaterally paired modulatory projection neuron. Both MCN1s contain the same co-transmitters, influence the same gastric mill microcircuit neurons, can drive the biphasic gastric mill rhythm, and are co-activated by all identified MCN1-activating pathways. Here, we determine whether the gastric mill microcircuit response is equivalent when stimulating one or both MCN1s under conditions where the pair are matched to collectively fire at the same overall rate and pattern as single MCN1 stimulation. The dual MCN1 stimulations elicited more consistently coordinated rhythms, and these rhythms exhibited longer phases and cycle periods. These different outcomes from single and dual MCN1 stimulation may have resulted from the relatively modest, and equivalent, firing rate of the gastric mill neuron LG (lateral gastric) during each matched set of stimulations. The LG neuron-mediated, ionotropic inhibition of the MCN1 axon terminals is the trigger for the transition from the retraction to protraction phase. This LG neuron influence on MCN1 was more effective during the dual stimulations, where each MCN1 firing rate was half that occurring during the matched single stimulations. Thus, equivalent individual- and co-activation of a class of modulatory projection neurons does not necessarily drive equivalent microcircuit output.

Degenerate circuits use distinct mechanisms to respond similarly to the same perturbation

There is considerable flexibility embedded within neural circuits. For example, separate modulatory inputs can differently configure the same underlying circuit but these different configurations generate comparable, or degenerate, activity patterns. However, little is known about whether these mechanistically different circuits in turn exhibit degenerate responses to the same inputs. We examined this issue using the crab (Cancer borealis) stomatogastric nervous system, in which stimulating the modulatory projection neuron MCN1 and bath applying the neuropeptide CabPK II elicit similar gastric mill (chewing) rhythms in the stomatogastric ganglion, despite differentially configuring the same neural circuit. We showed previously that bath applying the peptide hormone CCAP or stimulating the muscle stretch-sensitive sensory neuron GPR during the MCN1-elicited gastric mill rhythm selectively prolongs the protraction or retraction phase, respectively. Here, we found that these two influences on the CabPK-rhythm elicited some unique and unexpected consequences compared to their actions on the MCN1-rhythm. For example, in contrast to its effect on the MCN1-rhythm, CCAP selectively decreased the CabPK-rhythm retraction phase and thus increased the rhythm speed, whereas the CabPK-rhythm response to stimulating GPR during the retraction phase was similar its effect on the MCN1-rhythm (i.e. prolonging retraction). Interestingly, despite the comparable GPR actions on these degenerate rhythms, the underlying synaptic mechanism was distinct. Thus, degenerate circuits do not necessarily exhibit degenerate responses to the same influence, but when they do, it can occur via different underlying mechanisms.Significance StatementCircuits generating seemingly identical behaviors are often thought to arise from identical circuit states, as that is the most parsimonious explanation. Here we take advantage of an alternate scenario wherein a well-defined circuit with known connectivity generates similar activity patterns using distinct circuit states, via known mechanisms. The same peptide hormone modulation of these distinct circuit states produced divergent activity patterns, whereas the same sensory feedback altered these circuit outputs similarly but via different synaptic pathways. The latter observation limits the insights available from comparable studies in systems lacking detailed access to the underlying circuit.