Effect of salinity on osmoregulatory patch epithelia in gills of the blue crab Callinectes sapidus

In euryhaline crabs, ion-transporting cells are clustered into osmoregulatory patches on the lamellae of the posterior gills. To examine changes in the branchial osmoregulatory patch in the blue crab Callinectes sapidus in response to change in salinity and to correlate these changes with other osmoregulatory responses, crabs were acclimated to a range of salinities between 10 and 35 ppt. When crabs that had been acclimated to 35 ppt were subsequently transferred to 10 ppt, both the size of the osmoregulatory patch on individual gill lamellae and the specific activity of Na+, K+-ATPase in whole-gill homogenates increased only after the first 24 h of exposure to dilute seawater. Enzyme activity and size of patch area increased gradually and reached their maxima (increasing by 200% and 60%, respectively) 6 days following transfer to 10 ppt seawater and then remained at these levels. Patch size at acclimation varied inversely with the salinity for seawater dilutions below 26 ppt (the isosmotic point of the crab), although it did not vary in salinities at or above 26 ppt. Thus, the size of the patch clearly is modulated with acclimation salinity, but it increases only in those salinities in which the crab hyperosmoregulates. An increase in the total RNA/DNA ratio in gill homogenates, the lack of mitotic figures in the lamellae, and the lack of incorporation of bromodeoxyuridine into nuclei of lamellar epithelial cells during acclimation to dilute seawater were interpreted as evidence that no cell proliferation had occurred and that increases in the size of the osmoregulatory patch occurred through differentiation of existing gas exchange cells or of undifferentiated epithelial cells into ion-transporting cells.     

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.     

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.     

The influence of environmental salinity on hemocyanin function in the blue crab, Callinectes sapidus.

The effects of inorganic ions and of the hydrogen ion on oxygen-binding properties of most respiratory pigments are opposite. The addition of salt to the medium increases oxygen affinities, and the addition of H+ decreases oxygen affinities of crustacean hemocyanins. These oxygenation properties, as observed in vitro, suggest that the oxygen-transport system must adapt to ionic changes in the blood. In fact, decreases in the salt concentration of the blood of estaurine blue crabs are accompanied by increases in pH, probably resulting from the input of ammonia produced in deamination of the intracellular pool of free amino acids as the cells conform to osmotic changes in body fluids. The result is a stability of hemocyanin function until the blood becomes very dilute. As the acclimation salinity is reduced from 35 to 15 o/ooo, the ionic effects on respiratory transport are balanced and there is no change in total oxygen uptake. At 5 o/ooo salinity, however, the higher blood pH is manifested in an elevation of the total oxygen concentration of prebranchial blood, probably because the Bohr shift is no longer opposed by a critical level of salt in the blood. Under these conditions the role of hemocyanin in aerobic respiration is reduced at high environmental oxygen levels, but it may be enhanced in hypoxic uaters.     

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.     

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.     

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.     

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.     

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.     

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.     

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     

A viral disease of the blue crab,Callinectes sapidus: Histopathology and differential diagnosis

A disease of the blue crab caused by a cytoplasmic virus morphologically resembling reovirus is described. Crabs infected with the virus were collected both from a low-salinity tributary of Chesapeake Bay, Maryland, and from Chicoteague Bay, Virginia, a high-salinity area. Virus occurs with greatest frequency in cells of the hemopoietic tissue and in hyaline hemocytes. It is also found in epidermis and gill epithelia and within the neurilemma where it occurs in granulocytes and other cells of uncertain identity. The following diseases are considered in the differential diagnosis of the viral infection: paramoebiasis, bacterial infection, microsporidan infection, Hematodinium infection, and disease caused by a herpes-like virus.     

Formation of the inner branchiostegal cuticle of the blue crab,Callinectes sapidus

The noncalcified inner branchiostegal cuticle, which lines the branchial chamber, was examined histologically and ultrastructurally over the molt cycle in the blue crab, Callinectes sapidus. In intermolt crabs (stage C₄) the epithelium underlying the inner cuticle is cuboidal and has abundant intercellular spaces and a prominent basement membrane. Apolysis occurs at stage D₀ and dissolution of the cuticle is accompanied by the formation of numerous lysosomes in the epithelium. During stage D₁, cells increase in height, apical mitochondria become more abundant, and the cuticle continues to be resorbed. An epicuticle is formed in early D₂, arising from a fusion of small subunits apparently attached to short apical microvilli. Cuticle deposition continues through D₂ and is complete by stage D₃. By the time cuticle deposition is complete, the epithelium has become extremely columnar and cells are filled with bundles of microtubules. In stage D₄, an amorphous electron‐dense core appears in the microtubule‐filled cells, which are attached to the cuticle at their apical end and anchored to their basement membrane at the basal surface. These microtubule‐filled cells persist through ecdysis, stage E, but during stage A₁ the cores disappear and some organelles begin to reappear in the cytoplasm. By stage A₂, the cells return to the cuboidal morphology seen in intermolt and remain so throughout the remainder of the molt cycle. This new pattern of cuticle deposition resembles that observed in the gills of crustaceans in that the cuticle is uncalcified and there is no postecdysial cuticle formation. However, instead of apolysis being delayed until just before ecdysis, the inner cuticle is formed during the first half of premolt, allowing the epithelial cells time to differentiate into a morphology that provides tensile strength for the stress of ecdysis. These new observations demonstrate that cuticle formation can follow very diverse structural and temporal patterns. In order to integrate and coordinate these diverse patterns, it is suggested that a suite of feedback mechanisms must be present. J. Morphol. 240:267–281, 1999. © 1999 Wiley‐Liss, Inc.