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.     

Sensory physiology and behavior of blue crab (Callinectes sapidus) postlarvae during horizontaltransport

Adult blue crabs (Callinectes sapidus) live in estuaries and release larvae near the entrances to estuaries. Larvae are then transported offshore to continental shelf areas where they undergo development. Postlarvae, or megalopae, remain near the surface and undergo reverse diel vertical migration. The behaviors underlying this migration pattern are responses to light and a solar day rhythm in activity, in which megalopae are active during the day and inactive at night. Onshore transport probably occurs by wind‐generated surface currents. Once in the vicinity of an estuary, megalopae move up the estuary by selective tidal stream transport, in which they swim in the water column on rising tides at night and are on or near the bottom at all other times. Light inhibits swimming during the day. The ascent into the water column on nocturnal rising tides does not result from a biological rhythm in activity, but rather is cued by the rate of increase in salinity during rising tides. Megalopae have separatebehavioural responses in coastal/shelf areas and in estuaries, which are induced by chemical cues in offshore and estuarine waters.     

Purification and characterization of an iron-binding protein from the blue crab (Callinectes sapidus)

The presence of an iron-binding protein in the hemolymph of the blue crab (Callinectes sapidus) was detected by gel filtration of 59Fe-labeled hemolymph. The iron-binding protein was purified to homogeneity by ion exchange chromatography. 2. This protein has a mol. wt of 155,000 and consists of a single polypeptide chain with an isoelectric point of 5.0. 3. Analysis of the iron-loaded protein indicates that it has a high affinity for iron and the capacity to bind approximately 10 atoms iron/molecule protein. 4. The isolation of a specific iron-binding protein from the blue crab (Callinectes sapidus) provides additional support for the proposal that such proteins are an ancient evolutionary development not necessarily linked to the appearance of iron proteins (hemoglobin and hemerythrin) as a means for oxygen transport.     

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.     

Hemolymph ecdysteroids during the last three molt cycles of the blue crab,Callinectes sapidus: quantitative and qualitative analyses and regulation

The profiles of circulating ecdysteroids during the three molt cycles prior to adulthood were monitored from the juvenile blue crab, Callinectes sapidus. Ecdysteroid patterns are remarkably similar in terms of peak concentrations ranging between 210–330 ng/ml hemolymph. Analysis of hemolymph at late premolt stage revealed six different types of ecdysteroids with ponasterone A (PoA) and 20‐OH ecdysone (20‐OH E) as the major forms. This ecdysteroid profile was consistent in all three molt cycles. Bilateral eyestalk ablation (EA) is a procedure that removes inhibitory neurohormones including crustacean hyperglycemic hormone (CHH) and molt‐inhibiting hormone (MIH) and often results in precocious molting in crustaceans. However, the inhibitory roles of these neuropeptides in vivo have not yet been tested in C. sapidus. We determined the regulatory roles of CHH and MIH in the circulating ecdysteroid from ablated animals through daily injection. A daily administration of purified native CHH and MIH at physiological concentration maintained intermolt levels of ecdysteroids in the EA animals. This suggests that Y organs (YO) require a brief exposure to CHH and MIH in order to maintain the low level of ecdysteroids. Compared to intact animals, the EA crabs did not exhibit the level of peak ecdysteroids, and the major ecdysteroid turned out to be 20‐OH E, not PoA. These results further underscore the important actions of MIH and CHH in ecdysteroidogenesis, as they not only inhibit, but also control the composition of output of the YO activity. © 2009 Wiley Periodicals, Inc.     

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.     

Synthesis of a high-density lipoprotein in the developing blue crab (Callinectes sapidus)

An important lipoprotein in the hemolymph of crustaceans is LpI. It transports lipid to peripheral tissues and also has a role in crustacean immune recognition. We employed a monoclonal antibody specific for the LpI peptide to demonstrate by ELISA, western blot and immunohistochemistry the appearance of LpI during development of Callinectes sapidus, the blue crab. LpI was first found in stage 5 embryos and appeared to be synthesized by lateral basophilic cuboidal cells that demonstrated cytoplasmic immunoreactivity for LpI at their interface with the yolk mass. The embryonic cuboidal cells bore a strong cytologic resemblance to the hepatopancreas cells of later stages (zoea, megalopae, adults), which were also immunoreactive for LpI.     

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.     

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.     

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.     

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.     

Comparative properties of channel catfish (Ictalurus punctatus) and blue crab (Callinectes sapidus) acetylcholinesterases

1. Acetylcholinesterases (AChEs) from channel catfish and blue crabs were examined for substrate preference, KmS, effects of inhibitors, and pH and osmotic activity profiles. 2. Similarities were noted for substrate preference along with pH and osmotic optima. 3. Crab AChE had a lower Km (9 x 10(-5) vs 2 x 10(-4) M) and was more sensitive in terms of KI50S than fish AChE to eserine (2.6 x 10(-7) vs 3 x 10(-7) M), malathion (4.5 x 10(-5) vs 1.6 x 10(-4) M) and parathion (6.9 x 10(-5) vs 7 x 10(-4) M). 4. Fish AChE appeared easier to solubilize using Triton X-100.     

Growth of blue crab, Callinectes sapidus, in the Yumurtalik Cove, Turkey: a molt process approach

The blue crab (Callinectes sapidus) is native to the western Atlantic, but is an invasive species in the Mediterranean. This study examined the dynamics of growth in an invasive population of blue crab in the Yumurtal?k Cove, Turkey (North Eastern Mediterranean). Growth was quantified using a discontinuous growth model, a molt process model. Crab growth histories were observed for individual crabs held in field enclosures in summer 2010 and 2011. Carapace widths ranged from 14.13 to 80.07 mm. A mean growth per molt of 120.6% increase in carapace width was observed. Chronological inter-molt periods ranging between 3 days and 67 days were observed. The average IMP was 16 days in Yumurtal?k Cove. The mean physiological IMP was 270±163 degree-days, ranging from 72–781 degree-days.     

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.     

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.     

Concentration of rotavirus and enteroviruses from blue crabs (Callinectes sapidus)

A simple method for concentration and detection of rotavirus and enteroviruses in the blue crab is described. Virus was separated from tissue homogenates at pH 9.5, concentrated by adsorption to protein precipitates at pH 3.5, and recovered by elution of precipitates at pH 9.2. Test samples of 12 to 15 ml were produced from an initial 100 g of crab tissues. Cat-floc precipitation was used to remove sample toxicity for cell cultures. Recovery effectiveness averaged 52% with poliovirus 1, echovirus 7, and coxsackievirus B5 and 23% with rotavirus SA11.