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.     

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.     

The subunit characterization of Callinectes sapidus hemocyanin.

Hemocyanin from the blue crab, Callinectes sapidus, sediments at 25.7 S and has a native molecular weight of 940 000 +/- 20 000. Under solution conditions of increased pH (approximately 10) or ionic strength, the native molecule dissociates to a 17 S species. Reversal of this dissociation was unsuccessful. At pH 10 and with the removal of Mg2+, the 17 S species reversibly dissociates to form a subunit species which sediments at 6 S. A comparison of the circular dichroic spectra of the 25.7 S and 6 S hemocyanins suggests that little happens to the structural integrity of the polypeptide backbone upon the two dissociations. Molecular weight estimations under reducing and denaturing conditions indicate that the 6 S hemocyanin species represents the constituent polypeptide chain of the protein molecule. Chemical analysis suggests the presence of a small amount, less than 3%, of carbohydrate bound to the polypeptide chain. Electrophoresis of the hemocyanin in the presence of sodium dodecyl sulfate or urea reveals two major electrophoretic species of either slightly different chemical composition or slightly different polypeptide chain length.     

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.     

Changes in ecdysteroids during embryogenesis of the blue crab, Callinectes sapidus rathbun.

Total ecdysteroid titers [estimated by radioimmunoassay (RIA)] in embryos of the blue crab increased from ～6 ng 20-hydroxyecdysone equivalents/g in the immature embryo to a maximum of ～500 ng 20-hydroxyecdysone equivalents/g in maturing embryos; titers dropped to ～300 ng 20-hydroxyecdysone equivalents/g in prehatch embryos. High-pressure reverse-phase chromatography of the embryo extracts resolved five RIA-active components. α-Ecdysone and the polar conjugate of 20-hydroxyecdysone were present in low quantities. The concentration of 20-hydroxyecdysone increased during embryogenesis to a maximum of ～160 ng/g in maturing embryos and decreased only slightly in the prehatch embryos. Two unidentified components were also detected and the changes in their concentrations were estimated. One, an apolar component (peak III), accounted for as much as 63% of the total RIA activity as the embryos matured. The estimated concentration of this component increased from 85 ng/g in early embryos to 475 ng/g in maturing embryos, then decreased by 50% in the prehatch embryos. The level of the other, more polar component (peak II) increased from 7.5 to 75 ng/g as the embryos developed. The increase in the concentration of ecdysteroids during embryogenesis indicates that crab embryos have the capacity to synthesize ecdysteroids and suggests that these hormones may have a physiological role in the embryonic development of crustaceans.     

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.     

The utilization of lipovitellin during blue crab (Callinectes sapidus) embryogenesis

Embryos of the blue crab Callinectes sapidus develop in egg sacs carried on the abdomen of the female. They develop over a period of 10-13 days at 28 degrees C and are nutritionally dependent on yolk until they emerge from the egg sacs as free-swimming zoeae. The principal component of blue crab yolk is lipovitellin (LpII), a water-soluble lipoprotein composed of approximately equal amounts of lipid and protein. We followed changes in the concentration of apoproteins of LpII during embryogenesis by ELISA and Western blots, using monoclonal antibodies against two LpII apoprotein associated peptides identified as Protein A (107 kDa) and Protein B (75 kDa). During embryogenesis there was a decrease in Protein B but an increase in two smaller peptides (52 and 35 kDa) that reacted with the Protein B antibody. Utilization of LpII during embryogenesis was also followed morphologically by immunohistochemistry. Utilization of LpII was slow in early embryonic stages, followed by rapid utilization in late embryonic stages, such that only traces of LpII were present at the end of embryogenesis. The cells of the developing hepatopancreas appear to play an important role in the utilization of LpII.     

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.     

Epizootiology of the parasitic dinoflagellate Hematodinium sp. in the American blue crab Callinectes sapidus

Hematodinium sp. is a parasitic dinoflagellate that infects and kills blue crabs Callinectes sapidus. Periodic outbreaks of dinoflagellate infections with subsequent high host mortalities prompted a study of the epizootiology and distribution of the crab pathogen. Hemolymph samples from over 13000 crabs were assessed for infections over 8 yr. Moderate to high prevalences were found at several locations along the Atlantic and Gulf coasts of the United States. In the coastal bays of Maryland and Virginia, prevalence followed a seasonal pattern, with a sharp peak in late autumn. Infections were significantly more prevalent in crabs measuring less than 30 mm carapace width; host sex did not influence prevalence. Prevalences were highest in crabs collected from salinities of 26 to 30%o; no infected crabs were found in salinities below 11%o. Intensity of infection did not vary among crab sizes, molt stages, or sexes. Naturally and experimentally infected crabs died over 35 and 55 d in captivity, with a mean time to death of approximately 13 and 42 d, respectively. Several other crustaceans, including gammaridean amphipods, xanthid (mud) crabs, and the green crab Carcinus maenus, were found with Hematodinium-like infections. Considering its widespread distribution and high pathogenicity, we suggest that Hematodinium sp. represents a significant threat to blue crab populations in high salinity estuaries along the Atlantic and Gulf coasts of the USA.     

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 physiological basis for pitch-induced antennule movements in the blue crab,Callinectes sapidus

• 1.1. Measurements of the compensatory response of antennular basal segments to pitch were made in the crab Callinectes sapidus.
• 2.2. Electrical recordings were obtained from efferent fibers in promotor and remoter bundles of the antennular basal segments.
• 3.3. Most of the phasic response to pitch seen in these fibers is the result of input from thread hairs in the ipsilateral statocyst.
• 4.4. Most of the tonic activity occurring during static placement of crabs in positions about the pitch axis is the result of hook hair input from the ipsilateral statocyst.

     

Lack of transmission of Hematodinium sp. in the blue crab Callinectes sapidus through cannibalism

Hematodinium spp. are parasitic dinoflagellates of marine crustaceans. Outbreaks of Hematodinium sp. have impacted commercial landings of the blue crab Callinectes sapidus in the coastal bays of Virginia and Maryland (USA), with seasonal peaks in prevalence reaching 85%. The life cycle and transmission routes of the parasite in blue crabs are poorly understood. Cannibalism and waterborne transmission may be routes of transmission, although little conclusive evidence has been reported for these modes. We examined cannibalism as a route by a series of experiments wherein we repeatedly fed adult and juvenile crabs the tissues of crabs infected with Hematodinium. In each experiment, feeding was done 3 times over the course of 1 wk. Only 2 of 120 crabs were infected within 7 to 9 d after feeding, and these 2 were likely infected prior to the experimental exposures. Crabs inoculated with hemolymph from infected donors served as positive controls. They developed infections over 11 to 21 d, indicating that the Hematodinium sp. used in the cannibalism trials was infectious at the time of inoculation. Because amphipods also harbor Hematodinium-like infections, we fed tissues of infected crabs to the estuarine amphipod Leptocheirus plumulosus. Hematodinium DNA was detected in amphipods shortly after feeding, but not in animals held for longer periods, nor was it observed in histological preparations. Amphipods did not obtain infections by scavenging infected crab tissues. Our results show that Hematodinium sp. is not effectively transmitted through ingestion of diseased tissues, indicating that cannibalism may not be a major route of transmission for Hematodinium sp. in blue crabs.