Myzobdella platensis (Hirundinida: Piscicolidae) is true parasite of blue crabs (Crustacea: Portunidae)

Leeches exhibit a marked scope of diversity, including different kinds of symbiosis. The aim of the present study was to demonstrate through biochemical and histological analysis that a species of piscicolid leech, Myzobdella platensis, is a true parasite of blue crabs, feeding on their hemolymph and using them as a site for cocoon deposition. In a total of 48 blue crabs collected on October 2007 at 3 sites of the S?o Vicente Estuary, 12 specimens were infested with leeches. Callinectes bocourti (n = 7) was the most infested species with leeches and cocoons; it was chosen for biochemical and histological assays. The immunoblotting assays showed a positive reaction of the proteins in the intestinal samples of leeches collected from crabs using antihemocyanin polyclonal antibody of Ampullaria canaliculata. In addition, leech intestinal samples were recognized by antihemolymph polyclonal antibody of nonparasitized blue crabs. Histological sections of leech gut showed hemocytes and a granular matrix similar to those found in crab blood vessels. Collectively, this evidence strongly suggests a parasitic interaction between the leech M. platensis and the blue crab C. bocourti, in which the former utilizes the latter as a site for cocoon deposition and possibly for dispersal similar to that proposed for Myzobdella lugubris in Callinectes sapidus in North America.     

Molt-inhibiting hormone immunoreactive neurons in the eyestalk neuroendocrine system of the blue crab, Callinectes sapidus

The production of ecdysteroid molting hormones by crustacean Y-organs is negatively regulated by a neuropeptide, molt-inhibiting hormone. It is generally agreed that molt-inhibiting hormone is produced and released by the eyestalk neuroendocrine system. In the present study, immunocytochemical methods were used to detect molt-inhibiting hormone immunoreactive neurons in eyestalk ganglia of the blue crab, Callinectes sapidus. The primary antiserum used was generated against molt-inhibiting hormone of the green shore crab, Carcinus maenas. A preliminary Western blot analysis indicated the antiserum binds molt-inhibiting hormone of Callinectes sapidus. Using confocal and conventional immunofluorescence microscopy, molt-inhibiting hormone immunoreactivity was visualized in whole mounts and thin sections of Callinectes sapidus eyestalk ganglia. Immunoreactivity was detected in 15-25 neurosecretory cell bodies in the medulla terminalis X-organ, their associated axons and collateral branches, and their axon terminals in the neurohemal sinus gland. The cellular organization of molt-inhibiting hormone immunoreactive neurons in blue crabs is generally similar to that reported for other crab species. The combined results suggest the cellular structure of the molt-inhibiting hormone neuroendocrine system is highly conserved among brachyurans.     

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.     

Clearance of Vibrio campbellii injected into the hemolymph of Callinectes sapidus, the Atlantic blue crab: the effects of prior exposure to bacteria and environmental hypoxia

The Atlantic blue crab, Callinectes sapidus (Rathbun), lives in a bacteria-rich environment that experiences daily fluctuations in water quality. In the present study, we tested the hypothesis that crustaceans with prior or ongoing exposure to bacteria in their hemolymph have an increased susceptibility to subsequent infections, and that acute exposure to low dissolved oxygen (hypoxia) and elevated carbon dioxide levels (hypercapnia) may further confound the ability of blue crabs to counter a subsequent infection. Adult male blue crabs held in well-aerated (normoxic; P O2=20.7 kPA; CO(2)<0.06 kPa; pH 7.8-8.0) or hypercapnic hypoxic (HH; P O2=4 kPa; CO(2)=1.8 kPa; pH 6.9-7.2) seawater received an injection (pre-challenge dose) of 1 x 10(5)Vibrio campbellii g(-1) crab. Control animals were injected with an equivalent dose of HEPES-buffered saline (1 microl g(-1) crab). At 2h or 24h after the pre-challenge injection, both Vibrio and saline-pre-challenged animals were injected with a dose of live V. campbellii (1 x 10(5)g(-1) crab). This second injection will be referred to as a second injection or challenge injection. Degradation in or physical removal of intact bacteria from hemolymph was quantified using real-time PCR; bacteriostasis was quantified as the percentage of intact bacteria that could not be recovered by selective plating. We demonstrated that bacteriostasis occurs in the hemolymph of blue crabs. Furthermore, blue crabs that received a challenge injection 2h after a pre-challenge dose of V. campbellii cleared culturable bacteria from their hemolymph more rapidly when compared to animals that received a pre-challenge dose of saline. This enhanced clearance of culturable bacteria was associated with an increase in antibacterial activity in the cell-free hemolymph. However, the enhanced clearance of culturable bacteria disappeared when the time interval between the pre-challenge and challenge dose was extended to 24h and when crabs were held in HH seawater throughout the experiment. Neither the time interval between the pre-challenge and the challenge dose nor exposure to HH altered the pattern of intact bacterial clearance in blue crabs. These results demonstrate that prior exposure to bacteria does not increase the susceptibility of C. sapidus to a second, sublethal dose of V. campbellii. In fact, a recent exposure to V. campbellii enhances the ability of blue crabs to render bacteria non-culturable and the immune mechanisms/effectors responsible for this are short lived and appear to be sensitive to low dissolved oxygen and high carbon dioxide concentrations in the environment.     

Novel protein inhibits in vitro precipitation of calcium carbonate.

Organic molecules both coexist and interact with inorganic crystal lattices in biomineralizing tissues. Mineral precipitation and crystal morphology are tightly regulated by the actions of these molecules. Polyacrylamide gel electrophoresis studies on water soluble extracts from the cuticle of Callinectes sapidus (Atlantic blue crab) reveal the presence, in unmineralized nascent premolt cuticle, of proteins which are absent in the mineralized postmolt cuticle. In the present studies, homogenates from both premolt and postmolt C. sapidus cuticles have been tested for their effect on the in vitro precipitation of calcium carbonate. The role of protein in this process was determined by heat pretreatment and trypsin pretreatment of the cuticle homogenates prior to the precipitation assay. The results from these experiments indicate that proteins, with molecular weights of approximately 75,000 and between 10,000 and 20,000, concentrated in the C. sapidus premolt cuticle, inhibit calcium carbonate precipitation in vitro. The inhibitory activity of these proteins appears to be a result of specific interactions since trypsin, myoglobin, and ovalbumin are not inhibitory. The presence of lower amounts of these inhibitory proteins in C. sapidus postmolt cuticle may be responsible for the subsequent mineralization of this tissue.     

Expression of crustacean (Callinectes sapidus) molt-inhibiting hormone in insect cells using recombinant baculovirus.

Molt-inhibiting hormone (MIH) negatively regulates the synthesis of ecdysteroid molting hormones by crustacean Y-organs. We report here the expression of blue crab (Callinectes sapidus) MIH in insect cells using recombinant baculovirus. Insect Sf9 cells were transfected with recombinant baculovirus containing a DNA insert encoding the C. sapidus MIH prohormone (signal sequence plus mature hormone). The construct was designed to yield a mature, fully processed recombinant MIH (recMIH). Several baculovirus recombinants showing no contamination with wild-type viral DNA were subsequently analyzed for their ability to direct expression of recMIH. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins from infected cells revealed time-dependent expression of two proteins of approximately the predicted size for the C. sapidus MIH prohormone and mature hormone. Western blot results (using antiserum against MIH of Carcinus maenas) indicated that the proteins were MIH-immunoreactive. N-Terminal amino acid sequence data and mass spectral analysis indicated the expressed proteins were of the correct sequence and molecular mass. Cell lysates containing the recombinant protein dose-dependently suppressed the synthesis of ecdysteroids by Y-organs in vitro. We anticipate the recombinant peptide will prove useful for studies of the structure and function of MIH.     

Incidence of bacteremia in stressed and unstressed populations of the blue crab, Callinectes sapidus

The incidence of bacteremia in the blue crab, Callinectes sapidus, is reported to be in excess of 80%. Because these results have been controversial, a field study was initiated to determine the effect of commercial capture and handling stresses on the incidence and levels of infection in blue crabs. The majority (75%) of "unstressed" crabs which were captured individually and bled immediately upon removal from the water were bacteremic, with a geometric mean level of infection of 14 CFU/ml of hemolymph. Crabs collected by crab pot, confined within these pots for as long as 24 h, and sampled immediately after removal from the water had a similar mean level of infection. Crabs subjected to the stresses of commercial capture, handling, and transport showed a higher incidence of infection (91%) and a mean infection level of 46 CFU/ml. Injuries sustained by crabs during commercial handling are thought to be associated with the higher incidence of infection. Vibrio spp. were primarily responsible for progressive infections in commercially stressed crabs and were the predominant bacterial type in heavily infected crabs. Our results indicated that uninjured healthy crabs do not have sterile hemolymph but instead harbor low-level bacterial infections.     

Incidence of bacteremia in stressed and unstressed populations of the blue crab, Callinectes sapidus.

The incidence of bacteremia in the blue crab, Callinectes sapidus, is reported to be in excess of 80%. Because these results have been controversial, a field study was initiated to determine the effect of commercial capture and handling stresses on the incidence and levels of infection in blue crabs. The majority (75%) of "unstressed" crabs which were captured individually and bled immediately upon removal from the water were bacteremic, with a geometric mean level of infection of 14 CFU/ml of hemolymph. Crabs collected by crab pot, confined within these pots for as long as 24 h, and sampled immediately after removal from the water had a similar mean level of infection. Crabs subjected to the stresses of commercial capture, handling, and transport showed a higher incidence of infection (91%) and a mean infection level of 46 CFU/ml. Injuries sustained by crabs during commercial handling are thought to be associated with the higher incidence of infection. Vibrio spp. were primarily responsible for progressive infections in commercially stressed crabs and were the predominant bacterial type in heavily infected crabs. Our results indicated that uninjured healthy crabs do not have sterile hemolymph but instead harbor low-level bacterial infections.     

Detection of protein variation in human serum by externally released precipitins from the blue crab, Callinectes sapidus

1. A number of invertebrate species releases precipitins into the environment. This process is apparently part of an externally directed immunologic system. 2. A saline solution of precipitins released from the blue crab, Callinectes sapidus, provided a reagent that produced two different precipitation reactions with proteins from the sera of human individuals. 3. One of the reaction variants was also detected with mammalian antiserum, the other was not. The former variant was associated with a medical condition: hypergammaglobulinemia. 4. Saline reagents from different invertebrate species may prove useful in clinical diagnosis and for revealing new human protein polymorphisms.     

Antipeptide antibodies for detecting crab (Callinectes sapidus) molt-inhibiting hormone

In crustaceans, the synthesis of ecdysteroid molting hormones is regulated by molt-inhibiting hormone (MIH), a neuropeptide produced by an eyestalk neuroendocrine system, the X-organ/sinus gland complex. Using sequence analysis software, two regions of the blue crab (Callinectes sapidus) MIH peptide were selected for antibody production. Two 14-mer peptides were commercially synthesized and used to generate polyclonal antisera. Western blot analysis revealed that each antiserum bound to proteins of the predicted size in extracts of C. sapidus sinus glands, and lysates of insect cells containing recombinant MIH. Thin section immunocytochemistry using either antiserum showed specific immunoreactivity in X-organ neurosecretory cell bodies, their associated axons and collaterals, and their axon terminals in the sinus gland.     

Taste the Traditions: Crabs, Crab Cakes, and the Chesapeake Bay Blue Crab Fishery

For centuries, people in the Chesapeake Bay watershed have harvested and consumed blue crabs (Callinectes sapidus). Historically, the production of the crabs was intimately connected to the work and knowledge of commercial watermen. In recent years, declining crab populations have resulted in an increased local use of pasteurized crab meat imported from Asia and South America. Also emerging is an ecological discourse that emphasizes pollution reduction to save crabs to eat. In this article, I analyze these production and consumption changes for Chesapeake Bay blue crabs within a broad-ranging framework of cultural models and environmental anthropology. Explicit textual information increasingly suggests that the cultural model of Chesapeake blue crabs as food is one of crab cakes made (with imported crab meat) in the "local tradition" and, to a lesser degree so far, is an emerging discourse presenting blue crabs as a culinary poster child for antipollution campaigns.     

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 effects of hypoxia and pH on phenoloxidase activity in the Atlantic blue crab, Callinectes sapidus

In its natural coastal and estuarine environments, the blue crab, Callinectes sapidus, often encounters hypoxia, accompanied by hypercapnia (increased CO2) and an associated decrease in water pH. Previous studies have shown that exposure to hypercapnic hypoxia (HH) impairs the crab's ability to remove culturable bacteria from its hemolymph. In the present study we demonstrate that the activity of phenoloxidase (PO), an enzyme critical to antibacterial immune defense in crustaceans, is decreased at the low levels of hemolymph O2 and pH that occur in the tissues of blue crabs exposed to HH. Hemocyte PO activity was measured at tissue O2 levels that occur in normoxic (5% and 15% O2, approximate venous and arterial hemolymph, respectively) and hypoxic (1% O2) crabs and compared to PO activity in air-saturated conditions (21% O2). PO activity decreased by 33%, 49% and 70% of activity in air at 15%, 5% and 1% O2, respectively. When O2 was held at 21% and pH lowered within physiological limits, PO activity decreased with pH, showing a 16% reduction at pH 7.0 as compared with a normoxic pH of 7.8. These results suggest that decreased PO activity at low tissue O2 and pH compromises the ability of crustaceans in HH to defend themselves against microbial pathogens.     

Response of isolated axonal preparations to hyperosmoticity: evidence for a cellular volume regulation.

When submitted to a hyperosmotic stress, isolated axons of Callinectes sapidus shrink. Volume regulation is observed if the experiment is carried out by using serum from a crab adapted to sea water. 3' : 5'-AMP and dibutyryl AMP are also effective. A compound isolated from the hemolymph and having a molecular weight close to 10 000 mimics the effects of the serum and induces a partial regulation of the axonal volume.     

Trimeric G proteins in crustacean (Callinectes sapidus) Y-organs: occurrence and functional link to protein synthesis

Crustacean Y-organs produce ecdysteroid molting hormones. Regulation of ecdysteroidogenesis appears to be complex, involving regulatory ligands (including but not limited to molt-inhibiting hormone, an eyestalk neurohormone) and the capacity of the Y-organs to respond to those ligands. Available data indicate cell signaling pathways involving cAMP, cGMP, or both may be involved in regulation of Y-organ function. Trimeric G proteins link receptor occupancy to regulation of intracellular cAMP levels. In studies reported here, we have assessed the occurrence of G proteins in blue crab (Callinectes sapidus) Y-organs, and the link of G proteins to Y-organ function. Bacterial toxin-catalyzed ADP-ribosylation revealed a PTX-sensitive (alpha i-like) protein in Y-organ membranes, but failed to reveal a CTX-sensitive (alpha s-like) protein in Y-organ membranes. Western blotting with primary antibodies raised against conserved regions of mammalian G proteins detected an alpha i-immunoreactive protein (approximately 40 kDa) and two alpha s-immunoreactive proteins (approximately 50 and approximately 57 kDa) in Y-organ membrane preparations. Incubation of Y-organ membrane fractions with cholera toxin significantly suppressed incorporation of [35S]-methionine into TCA-precipitable Y-organ proteins, but had no detectable effect on ecdysteroidogenesis in short-term (6 h) incubations. The combined results indicate that C. sapidus Y-organs possess both Gi and Gs proteins, and that alpha s is functionally linked to regulation of glandular protein synthesis.     

The genomic organization of the open reading frame of the red pigment concentrating hormone gene in the blue crab Callinectes sapidus

The open reading frame (ORF) of the gene for the precursor of the octapeptide Red Pigment Concentrating Hormone (RPCH) from the blue crab Callinectes sapidus was cloned by PCR with oligonucleotides targeted to the initiation and the end of the translation coding sequences. A 272 bp intron was characterized between nucleotides 343 and 344 of the reported cDNA, present in the region coding for the last amino acids of the precursor related peptide of RPCH. The intron genomic structure here described is similar to that reported for the gene coding for the Adipokinetic Hormone (AKH) of the grasshopper Schistocerca nitans.     

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.     

The role of alternate hosts in the ecology and life history of Hematodinium sp., a parasitic dinoflagellate of the blue crab (Callinectes sapidus)

Hematodinium sp. infections are relatively common in some American blue crab (Callinectes sapidus) populations in estuaries of the western Atlantic Ocean. Outbreaks of disease caused by Hematodinium sp. can be extensive and can cause substantial mortalities in blue crab populations in high salinities. We examined several species of crustaceans to determine if the same species of Hematodinium that infects C. sapidus is found in other crustaceans from the same localities. Over a 2-yr period, 1,829 crustaceans were collected from the Delmarva Peninsula, Virginia, examined for the presence of infections. A portion of the first internal transcribed spacer (ITS1) region of the ribosomal RNA (rRNA) gene complex from Hematodinium sp. was amplified and sequences were compared among 35 individual crustaceans putatively infected with the parasite, as determined by microscopic examination, and 4 crustaceans putatively infected based only on PCR analysis. Of the 18 crustacean species examined, 5 were infected with Hematodinium sp. after microscopic examination and PCR analysis, including 3 new host records, and an additional species was positive only via PCR analysis. The ITS1 rRNA sequences of Hematodinium sp. from the infected crustaceans were highly similar to each other and to that reported from C. sapidus (>98%). The similarity among these ITS1 sequences and similarities in the histopathology of infected hosts is evidence that the same species of Hematodinium found in C. sapidus infects a broad range of crustaceans along the Delmarva Peninsula. Our data indicate that the species of Hematodinium found in blue crabs from estuaries along the east coast of North America is a host generalist, capable of infecting hosts in different families within the Order Decapoda. Additionally, evidence indicates that it may be capable of infecting crustaceans within the Order Amphipoda.