Modulation of behavior by biogenic amines and peptides in the blue crab,Callinectes sapidus

Using the blue crab Callinectes sapidus as a model system, we have investigated the effects of potential neuromodulators on freely behaving animals. Of interest is the modulatory effect of a number of drugs on three rhythmic behaviors of the blue crab: courtship display (CD) of the male crab, sideways swimming and backward swimming. The drugs tested were proctolin, dopamine, octopamine, serotonin, and norepinephrine. Injection of each drug elicited a unique posture or combination of limb movements. These experiments showed two results pertinent to CD behavior: A posture identical to the CD posture was displayed after dopamine injection; and rhythmic leg waving similar to CD was evoked by proctolin. An unusual combination of flexion and extension of all limbs and movements of some limbs occurred after serotonin injection. Injection of octopamine led to a posture antagonistic to CD posture. The effects of these drugs were concentration- and time-dependent. Injection of dopamine, octopamine, or serotonin produced effects that were seasonally-dependent, and the influence of proctolin proved to be dependent on developmental stage. Quantitative analysis of leg waving movements after proctolin injection allowed for comparison of these movements to naturally-occurring behavior.Abbreviations CD courtship display - DA dopamine - OA octopamine - 5-HT serotonin - NE norepinephrine - PROC proctolin     

Coordination and neuromuscular control of rhythmic behaviors in the blue crab,Callinectes sapidus

The stereotypical courtship display (CD) behavior of the male blue crab, Callinectes sapidus, includes an unusual component: the rhythmic waving of the swimming appendages above the carapace. This behavior occurs in a unique context but it resembles two other rhythmic behaviors performed using the swimming legs: sideways swimming and backward swimming. As a first step to understanding the mechanisms that allow the expression of apparently different rhythmic motor patterns, we have examined these behaviors using slow motion video analysis and electromyography of the basal muscles of the swimming legs in freely behaving crabs. The results show that these behaviors are distinguished by four parameters: the frequency of leg waving, the phase relationship between the legs, the presence of a stationary pause in basal muscle activity combined with rotation of the distal leg during CD, and an extended range of motion of these legs during CD and backward swimming, relative to sideways swimming. EMG analysis revealed that during sideways swimming, the sequence of muscular activity between the two legs was different. In contrast, during CD and backward swimming the sequence of activity for these legs is identical.Abbreviations CD courtship display - EMGs electromyograms - CD AMP courtship display in crabs with amputated fifth legs - CD1 crabs that voluntarily used one leg to perform courtship display waving - CD 1–3 courtship waving in cycles 1–3 - CD MID courtship waving after cycles 1–3 - M-C meral-carpal joint     

Physiological aspects of molting in the blue crab Callinectes sapidus

From a respiratory and metabolic standpoint, a blue crab isin an extremely precarious condition during a molt. A molt isphysiologically possible for two major reasons. First, a premoltalkalosis anticipates the acidosis that arises during actualexuviation when the gas exchanger and ventilatory appendageare impaired and thus anaerobic metabolism must be activated.Second, the crab is able to revert to more primitive forms ofskeletal support and, immediately after exuviation, gas exchange.These mechanisms are very fragile, however, as are the cardiovascularmechanisms that provide the force for exuviation; any one mayfail. Changes in various enzyme activities and in free aminoacid content of the tissues, which are usually associated withosmotic challenges, are associated specifically with a moltas well. I suggest that they are related to isosmotic wateruptake and cell volume regulation.     

Gas-bubble disease in the blue crab, Callinectes sapidus.

Exposure of crabs to water supersaturated with air resulted in formation of gas emboli in the hemal system, which in turn caused localized ischemia. More than one-third of the exposed crabs died during the 2 days following the episode. In surviving crabs, the most severely affected organs and tissues were the gills, heart, and antennal gland. Especially in gills, emboli were still present in apparently healthy crabs 35 days following exposure to water supersaturated with air. Evidence of ischemic injury was focal in character except in the antennal gland, where the epithelium of the labyrinth was sometimes extensively degenerate. Repair processes apparently did not involve hemocytes except for occasional fibroblastic infiltration in damaged gill lamellae.     

Silicification of the medial tooth in the blue crab Callinectes sapidus

Observations of cuticular structures mineralized with silica within the Crustacea have been limited to the opal teeth of copepods, mandibles of amphipods, and recently the teeth of the gastric mill in the blue crab Callinectes sapidus. Copepod teeth are deposited during premolt, with sequential elaboration of organic materials followed by secretion of silica into the tooth mold. The timing of mineralization is in stark contrast to that of the general integument of crustaceans in which calcification is completely restricted to the postmolt period. To determine the timing of molt‐related deposition and silicification of the teeth of the gastric mill, the medial tooth of the blue crab C. sapidus was examined histologically and ultrastructurally across the molt cycle. Histological data revealed deposition of the organic matrix of the epicuticle and exocuticle during premolt. No evidence of postmolt changes in the thickness of the epicuticle and exocuticle, or any deposition of endocuticle, was observed. Scanning electron microscopy revealed degradation of the outer surface of the old tooth during premolt. During premolt, epithelial structures resembling papilla appeared to secrete a fibrous web that coalesces to become the matrix of the new tooth. Semi‐quantitative elemental analyses indicated simultaneous deposition of silica and organic matrix, and demonstrated a homogeneous distribution of silicon throughout the epicuticle of the tooth at all stages. However, there is evidence of deposition (presumably silicification) during postmolt as spaces between the papillae become filled in. Thus, the pattern and timing of deposition and silicification of the tooth are different from both teeth of copepods and the general exoskeleton of decapods, and may facilitate rapid resumption of feeding and consumption of the exuvia in early postmolt. J. Morphol. 277:1648–1660, 2016. © 2016 Wiley Periodicals, Inc.     

Formation of the arthrodial membrane in the blue crab,Callinectes sapidus

In this study the pattern of arthrodial membrane deposition in Callinectes sapidus was determined by histological and ultrastructural examination of tissues from the carpus joint of the cheliped collected during premolt, ecdysis, postmolt, and intermolt. Apolysis in the arthrodial membrane occurs at stage D(0) and is synchronous with apolysis of the calcified cuticle. Epicuticle formation begins at early stage D(1) and is completed in late stage D(1). Procuticle deposition starts at D(2) and continues until ecdysis. Numerous cytoplasmic extensions occur throughout the lamellae. Component fibers of the arthrodial membrane are intimately associated with dense plaques on the apical membrane of the underlying hypodermal cells, suggesting a site for fiber polymerization. Deposition of the arthrodial membrane continues after ecdysis, with most of the cuticle thickening occurring during stage C. When stained with PAS and counterstained with hematoxylin, a difference can be discerned between preecdysial and postecdysial procuticle of the arthrodial membrane, a distinction not made in previous studies. The boundary between the arthrodial membrane and calcified cuticle is thicker than either of the two layers and the layers overlap rather than butting up against one another. This pattern suggests that underlying hypodermal cells have to produce multiple types of cuticle over the molt cycle. A summary of the various molting patterns in C. sapidus suggests that the control of these diverse events may prove to be complex.     

The identification and structure-activity relations of a cardioactive FMRFamide-related peptide from the blue crab Callinectes sapidus.

The pericardial organs and thoracic ganglia of the blue crab Callinectes sapidus were resected and extracted. The extracts were fractionated by HPLC and the fractions analyzed by a radioimmunoassay (RIA) to FMRFamide. Multiple peaks of immunoreactivity were present and one of these, upon fast atom bombardment mass spectrometry (FAB-ms) and microsequencing, yielded the sequence GYNRSFLRFamide. The amount of this peptide in each crab is between 7 and 13 pmol. Several incomplete sequences were also characterized, suggesting a precursor with multiple copies of peptides related to GYNRSFLRFamide might occur. The peptide caused a dose-dependent increase in heart rate; threshold was 10 to 30 nM, and the EC₅₀ was 323±62 nM. A structure-activity study of GYNRSFLRFamide on the crab heart suggests that, for full potency, a peptide should be at least a heptapeptide with the sequence XXZFLRFamide, where X is any amino acid and Z is either asparagine or serine.     

Neuromodulation of flight initiation by octopamine in the cockroach Periplaneta americana

We have tested the effect of a known insect neuromodulator, octopamine, on flight initiation in the cockroach. Using minimally dissected animals, we found that octopamine lowered the threshold for windevoked initiation of flight when applied to either of two major synaptic sites in the flight circuitry: 1) the last abdominal ganglion, where wind-sensitive neurons from the cerci excite dorsal giant interneurons, or 2) the metathoracic ganglion, where the dorsal giant interneurons activate interneurons and motoneurons which are involved in producing the rhythmic flight motor pattern in the flight muscles (Fig. 2).Correlated with this change in flight initiation threshold, we found that octopamine applied to the last abdominal ganglion increased the number of action potentials produced by individual dorsal giant interneurons when recruiting the cereal wind-sensitive neurons with wind puffs (Figs. 3, 4, 5) or with extracellular stimulation of their axons (Fig. 6). Octopamine increases the excitability of the giant interneurons (Figs. 7, 8). Also, when we stimulated individual dorsal giant interneurons intracellularly, the number of action potentials needed to initiate flight was reduced when octopamine was applied to the metathoracic ganglion (Fig. 9).Abbreviations EMG electromyogram - dGIs dorsal giant interneurons - GI giant interneuron - A6 sixth abdominal ganglion - T3 third thoracic ganglion - EPSP excitatory postsynaptic potential     

Hepatopancreas alteration of the blue crab Callinectes sapidus by the rhizocephalan barnacle Loxothylacus texanus

A histological study of the hepatopancreas of the blue crab Callinectes sapidus parasitized by the rhizocephalan barnacle Loxothylacus texanus was conducted to explore if the degree of development of the parasite’s rootlet system was correlated to its maturation process as seen by external characters of its reproductive body or externa. Four types of crabs were examined: control, with virgin and mature externa, and scarred. A clear progression with an increase in number and size of the parasite’s rootlets in the hosts’ hepatopancreas can be seen. Although the hepatopancreatic tubules remain functional, the hepatopancreas appears as a loose structure, completely infiltrated with L. texanus rootlets, in advanced stages of the parasitism.     

Glycosidase activity in the post-ecdysial cuticle of the blue crab, Callinectes sapidus

We have previously demonstrated a marked change in sugar moieties of glycoproteins of the cuticle of the blue crab, Callinectes sapidus, between 0.5 and 3 h post-ecdysis. The present study has identified a glycosidase that appears in the cuticle during the early post-ecdysial hours. The enzyme has affinities for p-nitrophenyl derivatives of both N-acetylglucosamine and N-acetylgalactosamine. Both activities are competitively inhibited by chitobiose, suggesting that the enzyme could be a N-acetylhexosaminidase (EC 3.2.1.52). Atypical of N-acetylhexosaminidases described to date, this enzyme has a pH optimum of 7.0. The enzyme activity is high during the post-ecdysial period coincident with the changes in glycoprotein profiles observed in vivo. Partial purification of the enzyme has been accomplished by Sephacryl size-exclusion chromatography followed by concanavalin A affinity chromatography.     

A new enveloped helical virus from the blue crab,Callinectes sapidus

Quite different ultrastructural changes were observed in the columnar cell and the goblet cell of the silkworm midgut after administration of the crystalline toxin of Bacillus thuringiensis. Shortly after the ingestion of the toxin, the deep infoldings of the basal cell membrane of some columnar cells became very irregular in shape and the mitochondria near the basal region were transformed into a condensed form. A few goblet cells showed relatively high electron density in the cytoplasm. The earliest pathological changes were slight and located in a region lying between the first and second thirds of the midgut. With the passage of time, they spread anteriorly and posteriorly to include the entire anterior two thirds of the midgut and became more profound. The cytoplasm of columnar cells became very electron transparent. Most mitochondria were transformed into a condensed form and the endoplasmic reticulum assumed a vacuole-like configuration. The basal infoldings of the cell membrane almost disappeared. On the other hand, the cytoplasm of the goblet cells became very electron dense and granular. The clear basal infoldings of the cell membrane were enlarged making a striking contrast with the dense cytoplasm. However, the mitochondria and the endoplasmic reticulum did not show any pathological deformation.     

The function of hemocyanin in respiration of the blue crab Callinectes sapidus.

Blood PO2 in the blue crab Callinectes sapidus, a very active species of tropical origin, is lower at 22 degrees C than that of larger crabs in colder waters. These low oxygen levels permit its hemocyanin to be highly oxygenated at the gill, and to deliver almost half of its oxygen to the tissues in resting animals. Sustained muscular activity results in conspicuous decreases in blood PO2, pH and hemocyanin oxygenation. Although the venous reserve is fully utilized, hemocyanin oxygenation at the gill decreases so much that there is no change in its total quantitative function. The large Bohr shift becomes functional during activity, but its quantitative importance is not clear.     

Uptake and survival of enteric viruses in the blue crab, Callinectes sapidus.

Uptake of poliovirus 1 by the blue crab, Callinectes sapidus, was measured to assess the likelihood of contamination by human enteric viruses. Virus was found in all parts of the crab within 2 h after the crab was placed in contaminated artificial seawater. The highest concentrations of virus were found in the hemolymph and digestive tract, but the meat also contained virus. The concentration of virus in the crabs was generally less than in the surrounding water. Changes in salinity did not substantially affect the rate of accumulation. An increase in temperature from 15 to 25 degrees C increased the rates of both uptake and removal. Poliovirus survived up to 6 days in crabs at a temperature of 15 degrees C and a salinity of 10 g/kg. When contaminated crabs were boiled, 99.9% of poliovirus 1, simian rotavirus SA11, and a natural isolate of echovirus 1 were inactivated within 8 min. These data demonstrate that viruses in crabs should not pose a serious health hazard if recommended cooking procedures are used.     

Colonization of the gut of the blue crab (Callinectes sapidus) by Vibrio cholerae

Attachment of Vibrio cholerae to the mucosal surface of the intestine is considered to be an important virulence characteristic. Vibrio cholerae, an autochthonous member of brackish water and estuarine bacterial communities, also attaches to crustacea, a significant factor in multiplication and survival of V. cholerae in nature. The ability of V. cholerae to attach to the gut wall of the blue crab (Callinectes sapidus) was examined, and attachment was observed only in the hindgut and not the midgut of crabs, confirming a requirement for chitin in the attachment of V. cholerae to invertebrate and zooplankton surfaces. The new finding of attachment of V. cholerae to the hindgut of crabs may be correlated with the epidemiology and transmission of cholera in the aquatic environment. The crab model may also prove useful in elucidating the mechanism(s) of ion transport in crustacea.     

Respiration and phosphorylation by mitochondria from the hepatopancreas of the blue crab (Callinectes sapidus)

Mitochondria isolated from the hepatopancreas of the blue crab Callinectes sapidus show high rates of oxidation of pyruvate + proline and of various intermediates of the tricarboxylic acid cycle in a 280- to 380-mOsm sucrose-mannitol medium supplemented with bovine serum albumin. The respiratory control ratio ranged from 6 to 10. Respiration was accompanied by phosphorylation of ADP, with the expected ADP:O ratio for all substrates tested except α-ketoglutarate, indicating that all three energy-conserving sites were functional. Fatty acids were also oxidized, but no oxidation of β-hydroxybutyrate, glycerol 3-phosphate, or NADH was observed. The crab mitochondria showed a relatively low affinity for phosphate, but a normal affinity for ADP. Respiration and phosphorylation gave the normal responses to respiratory chain inhibitors, uncoupling agents, oligomycin, and ionophores. Crab mitochondria have an exceptionally high content of phosphate, exceeding 1000 nmoles per mg protein, but a normal energy charge of the adenylic system. An unusual feature is the presence of considerable arginine kinase activity, which is usually thought to be restricted to muscle and nerve tissue in anthropods. This enzyme allows arginine to act as secondary phosphate acceptor. The arginine kinase is located on the cytosol side of the atractyloside-sensitive barrier and is thus unable to transfer the terminal phosphate group of matrix ATP directly to arginine.     

Immune defense reduces respiratory fitness in Callinectes sapidus, the Atlantic blue crab

Crustacean gills function in gas exchange, ion transport, and immune defense against microbial pathogens. Hemocyte aggregates that form in response to microbial pathogens become trapped in the fine vasculature of the gill, leading to the suggestion by others that respiration and ion regulation might by impaired during the course of an immune response. In the present study, injection of the pathogenic bacterium Vibrio campbellii into Callinectes sapidus, the Atlantic blue crab, caused a dramatic decline in oxygen uptake from 4.53 to 2.56 micromol g-1 h-1. This decline in oxygen uptake is associated with a large decrease in postbranchial PO2, from 16.2 (+/-0.46 SEM, n=7) to 13.1 kPa (+/-0.77 SEM, n=9), while prebranchial PO2 remains unchanged. In addition, injection of Vibrio results in the disappearance of a pH change across the gills, an indication of reduced CO2 excretion. The hemolymph hydrostatic pressure change across the gill circulation increases nearly 2-fold in Vibrio-injected crabs compared with a negligible change in pressure across the gill circulation in saline-injected, control crabs. This change, in combination with stability of heart rate and branchial chamber pressure, is indicative of a significant increase in vascular resistance across the gills that is induced by hemocyte nodule formation. A healthy, active blue crab can eliminate most invading bacteria, but the respiratory function of the gills is impaired. Thus, when blue crabs are engaged in the immune response, they are less equipped to engage in oxygen-fueled activities such as predator avoidance, prey capture, and migration. Furthermore, crabs are less fit to invade environments that are hypoxic.