Morphology of the gastric mill teeth in dotillid crabs (Crustacea: Brachyura: Dotillidae) from Indonesia.

The gastric mill is a prominent structure in the digestive system of brachyuran crabs, consisting of a median tooth plate and a pair of lateral tooth plates. Among crab species that are deposit feeders, the morphology and size of the gastric mill teeth are correlated with the preferred substrate types and food spectrum. In this study, we provide a detailed description of the morphology of the median and lateral teeth of the gastric mills in eight species of dotillid crabs from Indonesia, and compare them in relation to habitat preferences and molecular phylogeny. Ilyoplax delsmani, Ilyoplax orientalis, and Ilyoplax strigicarpus have comparatively simple shapes of their median and lateral teeth, with fewer teeth on each lateral tooth plate compared to Dotilla myctiroides, Dotilla wichmanni, Scopimera gordonae, Scopimera intermedia, and Tmethypocoelis aff. ceratophora, which have more complexly shaped median and lateral teeth, with a greater number of teeth on each lateral tooth plate. The number of teeth on lateral tooth correlates with habitat preference, that is, dotillid crabs inhabiting muddy substrata have fewer teeth on the lateral tooth plate, and those inhabiting sandy substrata have a more teeth. Phylogenetic analysis using partial COI and 16S rRNA genes supports that teeth morphology is similar among closely related species. Therefore, the description of median and lateral teeth of the gastric mill is expected to contribute to the systematic study of dotillid crabs.

Mechanical properties, degree of sclerotisation and elemental composition of the gastric mill in the red swamp crayfish Procambarus clarkii (Decapoda, Crustacea).

The gastric mill of Decapoda is a unique feature, which comprises teeth, stabilizing ossicles, and particle sorting setae. Involved in the fragmentation and sorting of the food, this structure serves as interface between the organism and its environment. As material properties complement morphology and hold information about function and trophic preferences, we here provide a basis for more comparative research on gastric mills. For gastric mill components of the adult red swamp crayfish Procambarus clarkii, we studied (a) the micro-structure via scanning electron microscopy, (b) the elemental composition by energy-dispersive X-ray spectroscopy, (c) the heterogeneities in material properties and degree of tanning (autofluorescence) by confocal laser scanning microscopy, and (d) the mechanical properties hardness and elasticity by nanoindentation technique. The morphology and micro-structure were previously described for this species, but the mechanical properties and the autofluorescence were not studied before. As epicuticle and exocuticle could be analyzed individually, material property gradients, with values decreasing from the interacting surface towards interior, could be determined. Finally, we were able to relate the mechanical property data with the elemental composition and the degree of tanning. We found that the epicuticle of the teeth is among the hardest and stiffest biological materials in invertebrates due to the incorporations of high proportions of silicon.

Unusual bromine enrichment in the gastric mill and setae of the hadal amphipod Hirondellea gigas.

The hadal amphipod Hirondellea gigas is an emblematic animal of the Pacific trenches, and has a number of special adaptations to thrive in this 'extreme' environment, which includes the deepest part of the Earth's ocean. One such adaptation that has been suggested is the presence of an 'aluminum gel shield' on the surface of its body in order to prevent the dissolution of calcitic exoskeleton below the carbonate compensation depth. However, this has not been investigated under experimental conditions that sufficiently prevent aluminum artefacts, and the possibility of other elements with similar characteristic X-ray energy as aluminum (such as bromine) has not been considered. Here, we show with new electron microscopy data gathered under optimized conditions to minimize aluminum artefacts that H. gigas actually does not have an aluminum shield-instead many parts of its body are enriched in bromine, particularly gastric ossicles and setae. Results from elemental analyses pointed to the use of calcite partially substituted with magnesium by H. gigas in its exoskeleton, in order to suppress dissolution. Our results exemplify the necessity of careful sample preparation and analysis of the signals in energy-dispersive X-ray spectroscopic analysis, and the importance of analyses at different electron energies.

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 to 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 h, or 2 h after 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 that the fed hemolymph also enhanced the influence of a projection neuron that 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.NEW & NOTEWORTHY Little is known about behavior-linked modulation of microcircuit activity. We show that the VCN-triggered gastric mill (chewing) and pyloric (food filtering) rhythms in the isolated crab Cancer borealis stomatogastric nervous system were changed by applying hemolymph from recently fed but not unfed crabs. This included some distinct parameter changes during each examined post-fed hemolymph time-point. These results suggest the presence of feeding-related changes in circulating hormones that regulate consummatory microcircuit activity.

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.

Growling from the gut: co-option of the gastric mill for acoustic communication in ghost crabs.

Animal acoustic communication systems can be built upon co-opted structures that become specialized for sound production or morphological novelties. The ghost crab, Ocypode quadrata, evolved a novel stridulation apparatus on the claws that is used during agonistic interactions, but they also produce a rasping sound without their claw apparatus. We investigated the nature of these sounds and show that O. quadrata adopted a unique and redundant mode of sound production by co-opting the gastric mill (grinding teeth of the foregut). Acoustic characteristics of the sound are consistent with stridulation and are produced by both male and female crabs during aggressive interactions. Laser Doppler vibrometry localized the source of maximum vibration to the gastric region and fluoroscopy showed movement of the gastric mill that coincided with stridulation. The lateral teeth of the gastric mill possess a series of comb-like structures that rub against the median tooth to produce stridulation with dominant frequencies below 2 kHz. This previously undescribed gastric stridulation can be modulated and provide a means of assessment during aggressive interactions, similar to the use of the claw stridulation apparatus. This functional redundancy of stridulation in crabs offers unique insights into the mechanisms of evolution of acoustic communication systems.

Neuronal morphologies built for reliable physiology in a rhythmic motor circuit.

It is often assumed that highly-branched neuronal structures perform compartmentalized computations. However, previously we showed that the Gastric Mill (GM) neuron in the crustacean stomatogastric ganglion (STG) operates like a single electrotonic compartment, despite having thousands of branch points and total cable length >10 mm (Otopalik et al., 2017a; 2017b). Here we show that compact electrotonic architecture is generalizable to other STG neuron types, and that these neurons present direction-insensitive, linear voltage integration, suggesting they pool synaptic inputs across their neuronal structures. We also show, using simulations of 720 cable models spanning a broad range of geometries and passive properties, that compact electrotonus, linear integration, and directional insensitivity in STG neurons arise from their neurite geometries (diameters tapering from 10-20 µm to < 2 µm at their terminal tips). A broad parameter search reveals multiple morphological and biophysical solutions for achieving different degrees of passive electrotonic decrement and computational strategies in the absence of active properties.

Flapper Valve and Hayfork: Functional anatomy and taxonomic potential of the Gastric Mill of Bairdioidea (Ostracoda, Podocopida).

The chewing apparatus of the Bairdioidea has been described just once and is rarely illustrated, but it might have more taxonomic significance than commonly supposed. It is constructed as a flapper valve (hinged check valve), which is unique among Ostracoda and unusual among animals. It projects into the midgut and is substantially enveloped by it. It serves three functions: to move bites of food into the stomach, to close the esophagus against back-flow, and to pack strands of food and mucus onto the rotating food ball. It is probably less effective for macerating the food to reduce particle size. Two braces anchor this structure to the lateral wall of the forehead. It is lined by cuticle that is shed at each molt, and the formation of food balls is interrupted during molting. In its construction and action, this apparatus is quite unlike the gastric mill of decapod crustaceans, and it shows only distant homology to the dorsal Wulst of Cypridoidea. Some architectural details differ among families and genera. The well-sclerotized plate has some potential for fossil preservation in exceptional circumstances. A revised anatomical analysis is presented, together with an annotated glossary of terms.

The transient potassium outward current has different roles in modulating the pyloric and gastric mill rhythms in the stomatogastric ganglion.

The crustacean stomatogastric nervous system is a classic model for understanding the effects of modulating ionic currents and synapses at both the cell and network levels. The stomatogastric ganglion in this system contains two distinct central pattern generators: a slow gastric mill network that generates flexible rhythmic outputs (8-20 s) and is often silent, and a fast pyloric network that generates more consistent rhythmic outputs (0.5-2 s) and is always active in vitro. Different ionic conductances contribute to the properties of individual neurons and therefore to the overall dynamics of the pyloric and gastric mill networks. However, the contributions of ionic currents to different dynamics between the pyloric and gastric mill networks are not well understood. The goal of this study is to evaluate how changes in outward potassium current (I A) in the stomatogastric ganglion affect the dynamics of the pyloric and gastric mill rhythms by interfering with normal I A activity. We bath-applied the specific I A blocker 4-aminopyridine to reduce I A's effect in the stomatogastric ganglion in vitro and evaluated quantitatively the changes in both rhythms. We found that blocking I A in the stomatogastric ganglion alters the synchronization between pyloric neurons, and consistently activates the gastric mill rhythm in quiescent preparations.

When complex neuronal structures may not matter.

Much work has explored animal-to-animal variability and compensation in ion channel expression. Yet, little is known regarding the physiological consequences of morphological variability. We quantify animal-to-animal variability in cable lengths (CV = 0.4) and branching patterns in the Gastric Mill (GM) neuron, an identified neuron type with highly-conserved physiological properties in the crustacean stomatogastric ganglion (STG) of Cancer borealis. We examined passive GM electrotonic structure by measuring the amplitudes and apparent reversal potentials (Erevs) of inhibitory responses evoked with focal glutamate photo-uncaging in the presence of TTX. Apparent Erevs were relatively invariant across sites (mean CV ± SD = 0.04 ± 0.01; 7-20 sites in each of 10 neurons), which ranged between 100-800 µm from the somatic recording site. Thus, GM neurons are remarkably electrotonically compact (estimated λ > 1.5 mm). Electrotonically compact structures, in consort with graded transmission, provide an elegant solution to observed morphological variability in the STG.

Network feedback regulates motor output across a range of modulatory neuron activity.

Modulatory projection neurons alter network neuron synaptic and intrinsic properties to elicit multiple different outputs. Sensory and other inputs elicit a range of modulatory neuron activity that is further shaped by network feedback, yet little is known regarding how the impact of network feedback on modulatory neurons regulates network output across a physiological range of modulatory neuron activity. Identified network neurons, a fully described connectome, and a well-characterized, identified modulatory projection neuron enabled us to address this issue in the crab (Cancer borealis) stomatogastric nervous system. The modulatory neuron modulatory commissural neuron 1 (MCN1) activates and modulates two networks that generate rhythms via different cellular mechanisms and at distinct frequencies. MCN1 is activated at rates of 5-35 Hz in vivo and in vitro. Additionally, network feedback elicits MCN1 activity time-locked to motor activity. We asked how network activation, rhythm speed, and neuron activity levels are regulated by the presence or absence of network feedback across a physiological range of MCN1 activity rates. There were both similarities and differences in responses of the two networks to MCN1 activity. Many parameters in both networks were sensitive to network feedback effects on MCN1 activity. However, for most parameters, MCN1 activity rate did not determine the extent to which network output was altered by the addition of network feedback. These data demonstrate that the influence of network feedback on modulatory neuron activity is an important determinant of network output and feedback can be effective in shaping network output regardless of the extent of network modulation.

Convergent neuromodulation onto a network neuron can have divergent effects at the network level.

Different neuromodulators often target the same ion channel. When such modulators act on different neuron types, this convergent action can enable a rhythmic network to produce distinct outputs. Less clear are the functional consequences when two neuromodulators influence the same ion channel in the same neuron. We examine the consequences of this seeming redundancy using a mathematical model of the crab gastric mill (chewing) network. This network is activated in vitro by the projection neuron MCN1, which elicits a half-center bursting oscillation between the reciprocally-inhibitory neurons LG and Int1. We focus on two neuropeptides which modulate this network, including a MCN1 neurotransmitter and the hormone crustacean cardioactive peptide (CCAP). Both activate the same voltage-gated current (I MI ) in the LG neuron. However, I MI-MCN1 , resulting from MCN1 released neuropeptide, has phasic dynamics in its maximal conductance due to LG presynaptic inhibition of MCN1, while I MI-CCAP retains the same maximal conductance in both phases of the gastric mill rhythm. Separation of time scales allows us to produce a 2D model from which phase plane analysis shows that, as in the biological system, I MI-MCN1 and I MI-CCAP primarily influence the durations of opposing phases of this rhythm. Furthermore, I MI-MCN1 influences the rhythmic output in a manner similar to the Int1-to-LG synapse, whereas I MI-CCAP has an influence similar to the LG-to-Int1 synapse. These results show that distinct neuromodulators which target the same voltage-gated ion channel in the same network neuron can nevertheless produce distinct effects at the network level, providing divergent neuromodulator actions on network activity.

Mucous contribution to gut nutrient content in American gizzard shad Dorosoma cepedianum.

This study developed and applied an approach to calculate the proportion of fish gut content composed of mucus secreted by the oropharyngeal cavity and gut. The amount of nitrogen in the contents of the foregut (oesophagus and gizzard) and the epibranchial organs of suspension-feeding American gizzard shad Dorosoma cepedianum was significantly higher than the nitrogen in the homogeneous food source. Using data collected from suspension-feeding experiments and the nitrogen content of D. cepedianum mucus, a series of equations illustrated that mucus constituted c. 10% of D. cepedianum foregut content and 12% of epibranchial organ content by dry mass. Future quantification of fish feeding selectivity and absorption efficiency can use this approach to take into account the contribution of fish mucus to the nutrients in the gut contents. This study supports the conclusion that suspension-feeding D. cepedianum in a heterogeneous environment selectively ingest nutrient-rich particles, even when gut nutrient content is adjusted to take into account the contribution of mucus.

Phylogeographical pattern of Mazocraeoides gonialosae (Monogenea, Mazocraeidae) on the dotted gizzard shad, Konosirus punctatus, along the coast of China.

In the present study, we examined the phylogeographical pattern of the monogenean, Mazocraeoides gonialosae, which parasitises the dotted gizzard shad (Konosirus punctatus) along the coast of China. Fragments of 756 bp of the mitochondrial cytochrome c oxidase subunit I gene were sequenced for 147 individuals from seven localities along the coast of China. Phylogenetic analysis revealed no significant genealogical clades of samples corresponding to sampling localities. Analyses of molecular variance and pairwise F(ST) suggested a high rate of gene flow and the lack of a predictable genetic structure between different populations of this parasite. Both neutrality tests and mismatch distribution analyses indicated a recent population expansion in M. gonialosae after the last glacial maximum. Gradually decreasing genetic diversity in more northerly populations implied a historical south-to-north expansion of this parasite. Dispersal of eggs and larvae with ocean currents was considered to be associated with the genetic homogeneity of this species. The limited time to accumulate genetic variation after the last glacial maximum may also account in part for the lack of phylogeographical structure in the studied region.

Activities and functions of peripheral neurons in the enteric nervous system of Aplysia and Lymnaea.

In order to explore the functions of the peripheral neurons in the enteric nervous system (ENS) of the gastropods, Aplysia and Lymnaea, we investigated the correlation between peripheral neuronal activities and movements of the digestive tract. In Aplysia, movements of the gizzard were distinguished into two types of contraction: a large constriction of the whole gizzard following bursting activities of the neurons on the gizzard and EJP-like potentials in the musculature; and a small contraction of a restricted part of the gizzard following a slow muscle potential. When TTX was applied to isolated gizzard preparation, the bursting activities were blocked and the EJP-like potentials and the subsequent constriction disappeared, whereas the slow potentials in the musculature and partial contractions appeared to be unaffected. Therefore, it was suggested that the peripheral neurons on the gizzard were motor neurons for constriction, while the partial contraction was thought to be myogenic. In Lymnaea, we recorded periodic bursting activities in the enteric nervous system that were followed by EJP-like potentials and gastrointestinal movements. The results show that, in both species, there may exist motor neurons in the ENS that are responsible for neurogenic movements of the digestive tract.

Correlative morphometric and electrochemical measurements of serotonin content in earthworm muscles.

Distribution of serotonin (5-HT) content of nervous fibers in both the somatic and the visceral muscle of Eisenia fetida have been investigated using immunocytochemical staining and voltammetric measurements. The somatic muscles in the body wall are richer innervated with serotoninergic fibers than the visceral ones in the pharynx and gizzard. The relative density of immunopositive fibers in the circular muscle layer of the body wall was found to be 2.73% while in the prostomium it was 1.02%. In the case of the muscle in pharynx 1.12% and in gizzard 1.28% density values were found. Differential Pulse Voltammetric (DPV) measurements with carbon fiber electrodes in the above mentioned muscle layers gave 272.5 nA, 135.0 nA, 122.5 nA, 137.5 nA peak heights, respectively. In the statistical analysis T-test was used at a confidence level of 95% (p<0.05). DPV current peak (i(p)) values reflect clearly the 5-HT concentration differences. Significant correlation was found between the innervation density and the i(p) values recorded in different areas. The i(p) values recorded at different times in different locations are determined by instantaneous serotonin concentration of the living tissue. As far as we know this is the first report using in vivo voltammetry investigating serotonin content in earthworm, E. fetida.

Analytical methods used in the United Kingdom Wildlife Incident Investigation Scheme for the detection of animal poisoning by pesticides.

The United Kingdom Wildlife Incident Investigation Scheme (WIIS) investigates cases of suspected poisoning of wildlife, honey bees, and companion animals by pesticides. Together with field inquiries and veterinary post-mortem examinations, the analytical procedures presented here provide a comprehensive approach to the investigation of these cases. The paper covers selection of animal tissues for analysis and methods suitable for the analysis of honey bees and for various types of bait. Seven multiresidue methods cover around 130 pesticides, and methods are also described for a further 8 compounds. These methods are currently used on samples submitted to the Scheme in England and Wales.

Non-muscle myosin II and myosin light chain kinase are downstream targets for vasopressin signaling in the renal collecting duct.

We have previously demonstrated that vasopressin increases the water permeability of the inner medullary collecting duct (IMCD) by inducing trafficking of aquaporin-2 to the apical plasma membrane and that this response is dependent on intracellular calcium mobilization and calmodulin activation. Here, we address the hypothesis that this water permeability response is mediated in part through activation of the calcium/calmodulin-dependent myosin light chain kinase (MLCK) and regulation of non-muscle myosin II. Immunoblotting and immunocytochemistry demonstrated the presence of MLCK, the myosin regulatory light chain (MLC), and the IIA and IIB isoforms of the non-muscle myosin heavy chain in rat IMCD cells. Two-dimensional electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry identified two isoforms of MLC, both of which also exist in phosphorylated and non-phosphorylated forms. 32P incubation of the inner medulla followed by autoradiography of two-dimensional gels demonstrated increased 32P labeling of both isoforms in response to the V2 receptor agonist [deamino-Cys1,D-Arg8]vasopressin (DDAVP). Time course studies of MLC phosphorylation in IMCD suspensions (using immunoblotting with anti-phospho-MLC antibodies) showed that the increase in phosphorylation could be detected as early as 30 s after exposure to vasopressin. The MLCK inhibitor ML-7 blocked the DDAVP-induced MLC phosphorylation and substantially reduced [Arg8]vasopressin (AVP)-stimulated water permeability. AVP-induced MLC phosphorylation was associated with a rearrangement of actin filaments (Alexa Fluor 568-phalloidin) in primary cultures of IMCD cells. These results demonstrate that MLC phosphorylation by MLCK represents a downstream effect of AVP-activated calcium/calmodulin signaling in IMCD cells and point to a role for non-muscle myosin II in regulation of water permeability by vasopressin.

Trifluoperazine inhibits the MgATPase activity and in vitro motility of conventional and unconventional myosins.

Trifluoperazine, a calmodulin antagonist, has recently been shown to inhibit the MgATPase activity of scallop myosin in the absence of light chain dissociation (Patel et al. (2000) J Biol Chem 275: 4880-4888). To investigate the generality of this observation and the mechanism by which it occurs, we have examined the ability of trifluoperazine to inhibit the enzymatic properties of other conventional and unconventional myosins. We show that trifluoperazine can inhibit the actin-activated MgATPase activity of rabbit skeletal muscle myosin II heavy meromyosin (HMM), phosphorylated turkey gizzard smooth muscle myosin II HMM, phosphorylated human nonmuscle myosin IIA HMM and myosin V subfragment-1 (S1). In all cases half maximal inhibition occurred at 50-75 microM trifluoperazine while light chains (myosin II) or calmodulin (myosin V) remained associated with the heavy chains. In vitro motility of all myosins tested was completely inhibited by trifluoperazine. Chymotryptic digestion of baculovirus-expressed myosin V HMM possessing only two calmodulin binding sites yielded a minimal motor fragment with no bound calmodulin. The MgATPase of this fragment was inhibited by trifluoperazine over the same range of concentrations as the S1 fragment of myosin.