Risk of Handling as a Route of Exposure to Infectious Waterborne Cryptosporidium parvum Oocysts via Atlantic Blue Crabs (Callinectes sapidus)

Commercial Atlantic blue crabs (Callinectes sapidus) were exposed to 2.0 × 10⁴ infectious waterborne oocysts of Cryptosporidium parvum. The study demonstrated that blue crabs can transfer C. parvum oocysts to persons involved in handling or preparing crabs and that they may contaminate other surfaces or products during storage.     

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.     

Trophic relationships of juvenile blue crabs (Callinectes sapidus) in estuarine habitats

Salt marshes and shallow-water macroalgal beds are known to provide nursery habitat for many species of fish and invertebrates. The role of these habitats as refuge from predation is well established, but the degree to which indigenous primary production within the nursery provides food for growth and development of estuarine species remains unresolved. In this study, we tested the hypothesis that juvenile blue crabs depend on indigenous primary production, directly or indirectly, during their entire stay within the nursery. To test this hypothesis, we conducted isotopic studies and stomach content analyses of juveniles from habitats near the mouth of Delaware Bay and from an adjacent lagoonal estuary (ca. 39.5° N, 75.1° W). Primary producers, marsh detritus, various life-history stages of blue crabs and potential prey species were sampled in the main estuary and in an adjacent marsh during the summer and early fall of two consecutive years. Newly settled juveniles (<15 mm carapace width) from the marsh were about 1.8‰ lighter in carbon (−17.2‰) relative to larger juveniles from the marsh (15–30 mm carapace width) and appeared to have retained a carbon isotopic signature indicative of the phytoplankton-based food web associated with larval stages. However, the signature of juveniles changed as a function of size. Large juveniles and crabs >60 mm were enriched in δ¹³C (−14.7 ± 0.1‰) compared to small crabs, suggesting a gradual shift in diet from a planktonic to a detritus-based food web with increasing size. As with crabs from Delaware Bay, the δ¹³C signature of juvenile crabs sampled from macroalgal beds in the lagoonal estuary (Rehoboth Bay) changed as a function of size. Also, δ¹³C ratios of crabs varied among the various species of macroalgae. The δ¹⁵N composition of primary producers in the marsh and main estuary also was reflected in the δ¹⁵N values of crabs and other benthic consumers in the respective habitats. Results of stomach-content analysis in this study were consistent with isotope data. Observed changes in prey preferences were related to changes in size of juvenile crabs and also differed among habitats. Gut content analyses of the three size classes of juveniles in macroalgal beds from Rehoboth Bay indicated that the crabs depend heavily on various amphipod species that occur on the seaweeds. These amphipods graze directly on the macroalgae and are among the most abundant invertebrates in the macroalgal beds. This implies a direct trophic relationship between the juvenile crabs and the macroalgae. In summary, our study provides strong evidence that the value of nursery areas such as salt marshes and macroalgal beds goes beyond that of providing refuge from predation, and that species using these nurseries (e.g. juvenile blue crabs) are ultimately dependent on primary production originating in benthic plants indigenous to the nursery.     

Diet selectivity of juvenile blue crabs (Callinectes sapidus) in Chesapeake Bay

Shallow coves in Chesapeake Bay have abundant food and serve as nursery grounds for juvenile blue crabs. In this study, we examined the relationships between the diet of very small (4-40?mm CW) juvenile blue crabs and the benthic infauna in shallow, unvegetated nursery coves. We compared infauna in benthic samples with gut contents of juvenile blue crabs from six shallow coves in each of two sub-estuaries (Rappahannock and York Rivers) in Chesapeake Bay, Virginia, USA. Benthic communities differed depending on river and location, with abundant clams in upriver regions and abundant polychaetes in downriver regions. Juvenile crabs, like adults, appeared to be opportunistic feeders, with gut contents including clams, amphipods, polychaetes, small crustaceans, plant matter, and detritus. There was a positive relationship between polychaetes in the benthic samples and in crab guts, suggesting that juvenile crabs are opportunistic feeders on polychaetes in the benthos. Moreover, Ivlev's electivity index and foraging ratio showed that clams and polychaetes were selectively eaten at all locations. Alternatively, crabs selectively rejected amphipods. Crab densities corresponded positively with polychaete densities, which suggests that there may be bottom-up control of crab distributions and that food resources are important in nursery habitats.     

Water fluxes and urine production in blue crabs (Callinectes sapidus) as a function of environmental salinity

• 1.1. Water efflux and urine production rates were measured in blue crabs acclimated to several salinities.
• 2.2. In 100% seawater the mean rate of water efflux (31.3 ml/100g hr⁻¹) was significantly greater than that in 50% seawater (18.9 ml/100 g hr⁻¹.
• 3.3. Water efflux was directly related to body weight.
• 4.4. The mean urine production rate was significantly greater in crabs acclimated to 50% and 30% seawater (0.17 and 0.18 ml/100g hr⁻¹) than in animals conditioned to 100% seawater (0.09 ml/100 g hr⁻¹).
• 5.5. The difference between theoretical net water fluxes for crabs exposed to 100% seawater and 50% seawater was similar to the difference in urine output in the same salinities, demonstrating the importance of the antennal gland in volume regulation.

     

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.     

Factors influencing planktonic, post-settlement dispersal of early juvenile blue crabs (Callinectes sapidus Rathbun)

An emerging body of literature points to post-settlement, planktonic dispersal as a key determinant of distribution and abundance patterns of aquatic organisms, yet little is known about mechanisms inducing such dispersal. Recent evidence suggests that early juvenile blue crabs (Callinectes sapidus Rathbun) may use planktonic emigration as a means of post-settlement dispersal. The goal of this study was to identify mechanisms inducing post-settlement, planktonic dispersal of early juvenile blue crabs. A combination of field mark-recapture experiments in large seagrass beds within a 2x3 km region near Oregon Inlet, North Carolina, USA, and a series of laboratory flume experiments examined the effects of day vs. night, crab size (first-second juvenile benthic instars: J1-J2 vs. third-fifth juvenile benthic instars: J3-J5), crab density and current speed on planktonic dispersal of early juvenile blue crabs. Transport of dead crabs in the flume experiment identified that planktonic dispersal was an active behavioral response rather than a passive response to increasing current speed. The experimental results demonstrated that planktonic dispersal can range from 4 to 18% under medium to high flow conditions. Planktonic dispersal of juvenile crabs is (1) an active behavioral response, (2) increased significantly with current speeds above 20 cms(-1), and (3) was higher for relatively large (J3-J5) than small (J1-J2) instars. There was a non-significant trend towards greater dispersal at night than during the day in the field experiment, and no effect of crab density on dispersal in the flume experiment. The results from this study highlight the need to consider mechanisms inducing post-settlement, planktonic dispersal when attempting to understand and predict recruitment and population dynamics of aquatic organisms, as well as when linking hydrodynamics, animal behavior and planktonic dispersal.     

Cardiac frequency compensation responses of adult blue crabs (Callinectes sapidus Rathbun) exposed to moderate temperature increases

• 1.1. Cardiac frequency patterns of Callincctes sapidus Rathbun were used to evaluate potential thermal stress after exposure to 5°C increases over a range of acclimation temperatures from 5° to 30°C.
• 2.2. An acclimated rate-temperature curve (R-T curve), acute R-T curves of the stabilized rates at the increased temperatures and Q₁₀ temperature coefficients were used to assess the significance of the changes in rate frequency.
• 3.3. The acclimated R-T curve showed that blue crabs go through a series of seasonal adaptation types characterized by a plateau of perfect adaptation for both cold and warm adapted organisms. Paradoxical adaptation occurred between the transition from cold to warm acclimation temperatures.
• 4.4. The acute R-T curves showed that cardiac frequency was highly responsive to a 5°C increase when the organisms were acclimated to low temperatures.
• 5.5. The Q₁₀'s of the acute R-T curves at the warm acclimation temperatures approximated those values derived for the acclimated R-T curve.
• 6.6. This suggests that the temperature increase had a negligible effect on the warm adapted crabs, that is, little or no thermal stress occurred.

     

Respiratory and digestive responses of postprandial Dungeness crabs, Cancer magister, and blue crabs, Callinectes sapidus, during hyposaline exposure

Respiratory responses and gastric processing were examined during hyposaline exposure in two crab species of differing osmoregulatory ability. The efficient osmoregulator, Callinectes sapidus, displayed an immediate increase in oxygen uptake when exposed to low salinity in isolation. In contrast, the weak osmoregulator, Cancer magister, showed no change in oxygen uptake upon acute exposure (<6 h), but slight increases in oxygen uptake tended to occur over longer time scales (12–24 h). These changes were likely attributable to an increase in avoidance activity after 6 h hyposaline exposure. Following feeding in 100% SW, oxygen uptake doubled for both species and remained elevated for 15 h. When postprandial crabs were exposed to low salinities, C. sapidus were able to sum the demands of osmoregulation and digestion. Thus, gastric processes continued unabated in low salinity. Conversely, postprandial C. magister prioritized responses to low salinity over those of digestion, resulting in a decrease in oxygen uptake when exposed to low salinity. This decrease in oxygen uptake corresponded to a reduction in the rate of contraction of the pyloric stomach and a subsequent doubling of gastric evacuation time. The current study is one of the few to illustrate how summation or prioritization of competing physiological systems is manifested in digestive processes.     

Incidence of Vibrio species associated with blue crabs (Callinectes sapidus) collected from Galveston Bay, Texas.

Bacteria were readily isolated from the hemolymph of a majority (88%) of the blue crabs collected from Galveston Bay, Texas. The hemolymph of most crabs contained moderate (greater than 10(3) bacteria/ml) to heavy (greater than (10(5) bacteria/ml) infections. Large variances were observed in the bacterial number associated with individual crabs, but no significant difference was observed between the mean bacterial levels in the hemolymph of crabs collected during different seasons of the sampling year. Vibrio spp. were the predominant bacterial types in the hemolymph of infected crabs and increased in number significantly during the summer season. Warmer water temperatures were thought to be responsible for this increase. Bacterial numbers and the percentage of Vibrio spp. were highest in the interior of the crab bodies, especially in the digestive tract. The exterior of the crabs did not appear to be the source of the hemolymph's bacterial flora. Bacteria taxonomically identical to Vibrio cholerae. V. vulnificus, and V. parahaemolyticus were routinely isolated from the crab hemolymph and external carapace. V. parahaemolyticus was the most prevalent of the pathogenic Vibrio spp. and was isolated from 23% of the hemolymph samples. V. vulnificus (7%) and V. cholerae (2%) occurred less commonly in the hemolymph. The incidences of V. parachaemolyticus and V. vulnificus were related and increased in the summer months. Both organisms were frequently isolated from the same crab.     

Detection of infectious Cryptosporidium parvum oocysts in mussels (Mytilus galloprovincialis) and cockles (Cerastoderma edule)

Infective Cryptosporidium parvum oocysts were detected in mussels (Mytilus galloprovincialis) and cockles (Cerastoderma edule) from a shellfish-producing region (Gallaecia, northwest Spain, bounded by the Atlantic Ocean) that accounts for the majority of European shellfish production. Shellfish were collected from bay sites with different degrees of organic pollution. Shellfish harboring C. parvum oocysts were recovered only from areas located near the mouths of rivers with a high density of grazing ruminants on their banks. An approximation of the parasite load of shellfish collected in positive sites indicated that each shellfish transported more than 10(3) oocysts. Recovered oocysts were infectious for neonatal mice, and PCR-restriction fragment length polymorphism analysis demonstrated a profile similar to that described for genotype C or 2 of the parasite. These results demonstrate that mussels and cockles could act as a reservoir of C. parvum infection for humans. Moreover, estuarine shellfish could be used as an indicator of river water contamination.     

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.     

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 effect of salinity on experimental infections of a Hematodinium sp. in blue crabs, Callinectes sapidus

The parasitic dinoflagellate Hematodinium sp. parasitizes blue crabs along the Atlantic seaboard of the United States. Infections in blue crabs have only been reported from waters where salinity is >11 practical salinity units (psu). Blue crabs maintain a hyperosmotic internal concentration at low salinities (0-5 psu), roughly comparable to 24 psu, and should be capable of maintaining an infection in low-salinity waters even if Hematodinium spp. cells are intolerant of low salinities. We tested this notion by observing the effect of low salinity on the progression of disease in crabs experimentally infected with the parasite. Blue crabs were acclimated to 5 psu or 30 psu salinity treatments. They were inoculated with Hematodinium sp. and necropsied 3, 7, 10, and 15 days post-inoculation. The low-salinity treatment did not have an effect on the proliferation of Hematodinium sp. infections in blue crabs; moreover, a greater proportion of infections in crabs in the low-salinity treatment developed dinospore stages than did those in the high-salinity treatment, indicating that salinity may affect the development of the parasite. However, dinospores from in vitro cultures rapidly became inactive when held in salinities <15 psu. Our experiments indicate that Hematodinium spp. can develop in blue crabs at low salinities, but that the parasite is incapable of transmission in this environment, which explains the lack of natural infections in crabs at low salinities.     

Trophic ecology of the Atlantic blue crab Callinectes sapidus as an invasive non-native species in the Aegean Sea

Invasive non-native crustaceans are a biodiversity and management concern in the Mediterranean Sea. The Atlantic blue crab (Callinectes sapidus) was first recorded in the Mediterranean Sea in 1949, but may have arrived as early as in the 1930’s. Blue crabs in the Mediterranean Sea are of concern due to their presumed potential for negative consumptive and competitive interactions with native fauna. The aim of this study was to provide a first assessment of the trophic ecology of non-native blue crab in the Northern Aegean Sea using stable carbon (δ¹³C) and nitrogen isotope (δ¹⁵N) analysis. We found limited isotopic niche overlap between blue crabs and seven native species examined at Gökçeada Island in April, June, and August of 2017. In addition, the range of calculated trophic positions of blue crabs at Gökçeada Island (2.0 to 4.4), while broad, is in general agreement with prior studies in both native and non-native ranges. We also observe that trophic position declined and the relative importance of pelagic carbon sources to blue crabs increased from April to August. However, we also found that differing assumptions as to the number and type of food web baselines and trophic discrimination factors led to differing estimates of trophic position in blue crabs at Gökçeada Island by as much as one to two trophic levels. These methodical differences make it challenging to directly compare results within and between studies, and thus limit our ability to assess negative consumptive and competitive interactions of invasive blue crab with native coastal species in the Mediterranean Sea.

     

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.     

Characterization and potential use of a Cryptosporidium parvum virus (CPV) antigen for detecting C. parvum oocysts

The purpose of this study was to characterize the viral symbiont (CPV) of Cryptosporidium parvum sporozoites and evaluate the CPV capsid protein (CPV40) as a target for sensitive detection of the parasite. Recombinant CPV40 was produced in Escherichia coli, purified by affinity chromatography, and used to prepare polyclonal rabbit sera specific for the viral capsid protein. Anti-rCPV40 recognized a 40 kDa and a 30 kDa protein in C. parvum oocysts and appeared to localize to the apical end of the parasite. Anti-rCPV40 serum was capable of detecting as few as 1 C. parvum oocyst in a dot blot assay, the sensitivity being at least 1000-fold greater than sera reactive with total native C. parvum oocyst protein or specific for the 41 kDa oocyst surface antigen. Water samples were seeded with C. parvum oocysts and incubated at 4, 20, or 25 degrees C for greater than 3 months to determine if CPV levels were correlated with oocyst infectivity. Samples were removed monthly and subjected to mouse and cell culture infectivity, as well as PCR analysis for infectivity and viral particle presence. While sporozoite infectivity declined by more than 75% after 1 month at 25 degrees C, the CPV signal was similar to that of control samples at 4 degrees C. By 3 months at 20 degrees C, the C. parvum oocysts were found to be non-infectious, but retained a high CPV signal. This study indicates that CPV is an excellent target for sensitive detection of C. parvum oocysts in water, but may persist for an indefinite time after oocysts become non-infectious.