A comparison of the predatory impacts of an invasive and native crab species using a functional response approach

The European green crab (Carcinus maenas) is invasive on the West coast of North America, but the ecological consequences of this invasion remain poorly understood. Comparative functional response analysis has arisen as a method of elucidating ecological consequences of invasive species by comparing the impact of these species to native analogues. Through comparative functional response experiments of green crabs and native red rock crabs (Cancer productus) we found that green crab predation increased asymptotically (Type II functional response) when fed increasing densities of Pacific oysters (Magallana gigas), while red rock crab predation displayed a sigmoidal (Type III) response. At high oyster densities red rock crabs consume more Pacific oysters than green crabs do, due to their reduced handling time, though green crabs consume more Pacific oysters relative to their size than red rock crabs. However, compared to red rock crabs, green crabs consume more oysters at low prey densities, which implies that they have a larger, potentially destabilizing impact on low densities of Pacific oysters. As green crabs continue to spread across the West coast of North America, Pacific oysters will face increased predation pressure. Our results show the advantage of using functional response analysis to compare density dependent predation between an invasive species and a native species to predict the ecological consequences of invasions.

     

Adjuvant Action of Capsular Polysaccharide of Klebsiella pneumoniae on Antibody Response

The sequence of histological changes in the regional lymph node and other lymphoid organs of mice injected with the capsular polysaccharide of Klebsiella pneumoniae (CPS-K) or bacterial lipopolysaccharide (LPS) was followed. Injection of CPS-K, but not LPS, induced the following characteristic histological changes in the regional lymph node. In the early stage there was a marked decrease in the number of small lymphocytes, accompanied by the appearance of scattered fragmented nuclei and infiltration of polymorphonuclear neutrophilic leukocytes, and in the late stage there was marked proliferation of macrophage-like cells and pyroninophilic cells. Histological changes in the thymus and spleen and changes in cell populations in the bone marrow and peripheral blood after CPS-K injection were essentially the same as after LPS injection. Since CPS-K has a much stronger adjuvant action on antibody response than does LPS, it is suggested that the characteristic histological changes in the regional lymph node after injection of CPS-K are closely related to its extraordinarily strong adjuvant action.     

In Vitro Propagation of the Protozoan Perkinsus Marinus, A Pathogen of the Eastern Oyster, Crassostrea Virginica

ABSTRACT. Perkinsus marinus , a pathogen of eastern oysters ( Crassostrea virginica ), has been successfully propagated in vitro. Cultures of the parasite were initiated from heart fragments of an infected oyster. the cultured protozoan (designated Parkinsus -1) was similar in morphology at both the light and transmission electron microscopy levels to histozoic stages of P. marinus in naturally infected oysters. In addition, cultured cells incubated in fluid thioglycollate medium produced enlarged cells (prezoosporangia) that stained blue-black in Lugol's solution, a response characteristic to Perkinsus spp. and used in routine diagnosis. Polyclonal antibodies raised against P. marinus prezoosporangia reacted positively to Perkinsus -1. Finally, the cultured cells infected susceptible oysters and reisolation of Perkinsus -1 cells was possible from the hearts of experimentally infected oysters. the culture medium contained most of the known constituents of cell-free hemolymph of oysters. the success achieved in culturing P. marinus will allow further investigations aimed at reducing mortalities caused by this important oyster pathogen and at addressing many unanswered questions about its biology and pathobiology.     

Ontogeny and water temperature influences the antiviral response of the Pacific oyster,Crassostrea gigas

Disease is caused by a complex interaction between the pathogen, environment, and the physiological status of the host. Determining how host ontogeny interacts with water temperature to influence the antiviral response of the Pacific oysters, Crassostrea gigas, is a major goal in understanding why juvenile Pacific oysters are dying during summer as a result of the global emergence of a new genotype of the Ostreid herpesvirus, termed OsHV-1 μvar. We measured the effect of temperature (12 vs 22 °C) on the antiviral response of adult and juvenile C. gigas injected with poly I:C. Poly I:C up-regulated the expression of numerous immune genes, including TLR, MyD88, IκB-1, Rel, IRF, MDA5, STING, SOC, PKR, Viperin and Mpeg1. At 22 °C, these immune genes showed significant up-regulation in juvenile and adult oysters, but the majority of these genes were up-regulated 12 h post-injection for juveniles compared to 26 h for adults. At 12 °C, the response of these genes was completely inhibited in juveniles and delayed in adults. Temperature and age had no effect on hemolymph antiviral activity against herpes simplex virus (HSV-1). These results suggest that oysters rely on a cellular response to minimise viral replication, involving recognition of virus-associated molecular patterns to induce host cells into an antiviral state, as opposed to producing broad-spectrum antiviral compounds. This cellular response, measured by antiviral gene expression of circulating hemocytes, was influenced by temperature and oyster age. We speculate whether the vigorous antiviral response of juveniles at 22 °C results in an immune-mediated disorder causing mortality.     

Fate of Bacillus sphaericus 2362 spores following ingestion by nontarget invertebrates.

Elimination of Bacillus sphaericus spores ingested by midge larvae, snails, and oysters was most rapid among midge larvae. Spores remained in oysters up to 21 days and in snails up to 49 days. Viable spores were recovered in snail and oyster feces for these same periods. There was no indication of actively growing B. sphaericus in the animals. Passage through oyster gut detoxified the B. sphaericus mosquito larval toxin, but there was a 33% retention of toxicity following snail gut passage. Midge larvae reared to adults in spore-containing water carried spores in/on the adult body. This suggests that these animals could carry the bacteria to sites beyond the application area.     

Integration of Vibrio vulnificus into Marine Aggregates and Its Subsequent Uptake by Crassostrea virginica Oysters

Marine aggregates are naturally forming conglomerations of larvacean houses, phytoplankton, microbes, and inorganics adhered together by exocellular polymers. In this study, we show in vitro that the bacterial pathogen Vibrio vulnificus can be concentrated into laboratory-generated aggregates from surrounding water. We further show that environmental (E-genotype) strains exhibit significantly more integration into these aggregates than clinical (C-genotype) strains. Experiments where marine aggregates with attached V. vulnificus cells were fed to oysters (Crassostrea virginica) resulted in greater uptake of both C and E types than nonaggregated controls. When C- and E-genotype strains were cocultured in competitive experiments, the aggregated E-genotype strains exhibited significantly greater uptake by oyster than the C-genotype strains.     

Predator-prey dynamics between recently established stone crabs (Menippe spp.) and oyster prey (Crassostrea virginica)

Range expansion and population establishment of individual species can have significant impacts on previously established food webs and predator-prey dynamics. The stone crab (Menippe spp.) is found throughout southwestern North Atlantic waters, from North Carolina through the Gulf of Mexico and the Central American Caribbean, including the Greater Antilles. Recent observations suggest that stone crabs have become better established on certain oyster reefs in North Carolina than in the early 1900s when they we first observed in NC. To assess the predatory impact of stone crabs on oysters, we (1) quantified stone crab densities on subtidal oyster reefs in Pamlico Sound, NC using scuba surveys, and (2) conducted laboratory predation experiments to assess the functional response of stone crabs to varying densities of oysters. We then (3) analyzed previously unpublished functional response data on another important oyster predator, the mud crab Panopeus herbstii. Finally, we (4) compared and contrasted potential predatory impacts of stone, mud and blue crabs (Callinectes sapidus). The functional response data and analyses for both stone crabs and mud crabs were consistent with a type II functional response. Mud crabs, on a m² basis, inflicted the highest proportional mortality on oysters over a 24 hour period, followed by stone and then blue crabs. Proportional mortality did not vary significantly with oyster size; however, relatively small and large oysters were consumed disproportionately less than medium-sized oysters, likely due to the mechanical inability of stone crabs to handle small oysters, and the inability to crush large oysters. Although stone crabs appear to be established in Pamlico Sound at densities equivalent to densities in other systems such as the U.S. Florida Panhandle, their predatory activities on oysters are not expected to have as significant a negative impact on oyster populations compared to other resident predators such as mud crabs.     

Effect of repeated sampling on haemolymph pH,PO2 and haemocyte activity in the Pacific oyster,Crassostrea gigas (Thunberg)

The effects of repeat bleeding via a novel cannulation technique, on the physiology of the Pacific oyster, Crassostrea gigas (Thunberg), were compared to control animals from which haemolymph was sampled by syringe and needle. The effects of surgery and cannulation were characterized by an initial alkalosis (up to 8 h) in the haemolymph of cannulated animals followed by an acidosis of up to 12 h; which occurred in both cannulated and control oysters. A decline in partial pressure of oxygen of oyster haemolymph, observed in both cannulated and control oysters, was directly related to shell closure following handling. Furthermore, a haemocyte concentration observed at 4 h followed by haemocyte dilution at 12 h post-surgery, suggested a handling-related stress response in both cannulated and control oysters. A heightened phagocytic activity of haemocytes at 4 h also supported the occurrence of handling-related stress response in all animals. The cannulation technique described here provides the investigator with a convenient tool for chronic, and repeated sampling of haemolymph with a transient disturbance to the animal.     

Modulation of the human in vitro antibody response by human leukocyte interferon preparations

The ability of human leukocyte Interferon to modulate the plaque-forming-cell response of human peripheral blood leukocytes to horse red blood cells was examined. Human peripheral blood mononuclear cells were cultured in vitro with the addition of varying doses of human leukocyte interferon 24 hr prior to, simultaneously with, and 24 hr after sensitization of the cultures with horse red blood cells. Plaque-forming-cell responses were measured 5 days after sensitization with antigen using poly-L-lysine-coupled horse red blood cell monolayers. When human leukocyte interferon preparations were added 24 hr prior to sensitization with antigen, a significant enhancement of the plaque-forming-cell response was observed. When the interferon was added simultaneously with antigen, the plaque-forming-cell response was significantly suppressed. Therefore, human leukocyte interferon appears to have a time-dependent immunomodulatory activity. The kinetics of immunomodulation appear to be different from those of previously described mouse models.     

Preparation of 111in leukocytes after hemolytic removal of erythrocytes

An optimized procedure is described for isolation and highefficiency radiolabeling of leukocytes using ¹¹¹In-oxine. The chief advantages over conventional methods include virtually no loss of leukocytes during washing and separation steps; a significant reduction in the time required to prepare leukocytes for radiolabeling compared to non-hemolytic preparations; a 28% increase in the average labeling efficiency obtained using ¹¹¹In-oxine; >95% cell viability as measured by the trypan blue exclusion test; elimination of contaminating red blood cells from the leukocyte pellet prior to labeling; and 80% survivability at 15 min post injection (measured as per cent of blood activity on leukocyte fraction).     

Role of inducible nitric oxide synthase in leukocyte extravasation in vivo

Several recent studies have suggested that nitric oxide (NO) derived from the inducible isoform of NO synthase (NOS) may act as an endogenous modulator of the inflammatory response by inhibiting adhesion of leukocytes to endothelial cells in vitro. Few studies have addressed specifically the role of iNOS in regulating leukocyte recruitment in vivo in a model of acute inflammation. Thus, the objective of this study was to assess the role of iNOS in modulating neutrophil (PMN) extravasation in an oyster glycogen-induced model of acute peritonitis in rats. Data obtained in the present study demonstrates that injection (IP) of oyster glycogen induces massive and selective PMN recruitment into the peritoneal cavity of rats at 6 hrs following OG administration. These extravasated cells were found to contain significant amounts of iNOS protein as assessed by Western blot analysis. Treatment of rats with the selective iNOS inhibitor L-iminoethyl-lysine (L-NIL) dramatically reduced NO levels in lavage fluid as measured by decreases in nitrate and nitrite concentrations without significantly affecting iNOS protein levels. Although L-NIL inhibited NO production by >70%, it did not alter oyster glycogen-induced PMN recruitment when compared to vehicle-treated rats. We conclude that PMN-associated, iNOS-derived NO does not play an important role in modulating extravasation of these leukocytes in this model of acute inflammation.     

Biolocalization and cell labeling properties of neutral-lipophilic 99mTc-amine-phenol chelates

^99mTc-diamine-diphenol chelates are neutral lipophilic chelates exhibiting good stability in aqueous solutions. The cell labeling and biolocalization properties of four different ^99mTc-amine-phenol complexes were determined. All four chelates readily labeled leukocytes and RCBs in high yields. Even though ^99mTc was retained by the cells, the elution rate of ^99mTc from the labeled cells in plasma at 37 °C was unacceptably high for potential utility in scintigraphic imaging. The uptake of ^99mTc in brain or heart following i.v. injection of the chelates in rats was low and clearance of activity from the blood was slow.     

Antimicrobial Histones and DNA Traps in Invertebrate Immunity: EVIDENCES IN CRASSOSTREA GIGAS*

Although antimicrobial histones have been isolated from multiple metazoan species, their role in host defense has long remained unanswered. We found here that the hemocytes of the oyster Crassostrea gigas release antimicrobial H1-like and H5-like histones in response to tissue damage and infection. These antimicrobial histones were shown to be associated with extracellular DNA networks released by hemocytes, the circulating immune cells of invertebrates, in response to immune challenge. The hemocyte-released DNA was found to surround and entangle vibrios. This defense mechanism is reminiscent of the neutrophil extracellular traps (ETs) recently described in vertebrates. Importantly, oyster ETs were evidenced in vivo in hemocyte-infiltrated interstitial tissues surrounding wounds, whereas they were absent from tissues of unchallenged oysters. Consistently, antimicrobial histones were found to accumulate in oyster tissues following injury or infection with vibrios. Finally, oyster ET formation was highly dependent on the production of reactive oxygen species by hemocytes. This shows that ET formation relies on common cellular and molecular mechanisms from vertebrates to invertebrates. Altogether, our data reveal that ET formation is a defense mechanism triggered by infection and tissue damage, which is shared by relatively distant species suggesting either evolutionary conservation or convergent evolution within Bilateria.     

Enzyme-linked immunosorbent assay (ELISA) used in quantification of reproductive output in the pacific oyster,Crassostrea gigas,in Korea

Polyclonal antibody developed from egg proteins of the Pacific oysters, Crassostrea gigas, was used in quantification of the eggs. Quantity of the egg protein was measured using ELISA and a weight-normalized gonadosomatic index (GSI) was then established. Histology indicated that the oysters collected from Goseong Bay, Korea, in 2000 initiated gametogenesis as early as late February and spawned mostly in late June to early July when water temperature reached 23–25 °C. A second spawning peak was also observed in late August and the spawning activity continued to late October. Oyster egg proteins could be detected by ELISA in all months except March and November; a few oysters collected during December and January contained a measurable amount of eggs. The highest monthly mean GSI, 0.423, was observed in mid-June when the oysters were ready to spawn. The maximum GSI recorded in this study was 0.667. A positive correlation was found between oyster size and fecundity; the number of eggs increased as oyster biomass increased (r²=0.7497). Our data suggest that oysters in Goseong Bay produce 3–196 million eggs during the spawning season. The immunological method developed in this study was sensitive enough to measure a small quantity of eggs and considered to be method of choice for studying bivalve reproduction.     

Assessment of Human Health Risks of Consumption of Cadmium Contaminated Cultured Oysters

With farmed British Columbia (BC) oysters containing higher cadmium concentrations than wild oysters, long-term exposure to cadmium through consumption of oysters has the potential to cause health risks. This study reports on a risk assessment for cadmium intake resulting from the consumption of BC-cultured oyster. The study concludes that Health Canada's current recommended BC-cultured oyster consumption rate for Canadians of 12 oysters per month exceeds the Agency for Toxic Substances and Disease Registry chronic oral minimal risk levels (MRL) of 0.2 μ g·kg^{? 1}·day^{? 1} by approximately 4-to 5-fold and reaches the Food and Agriculture Organization/World Health Organization (FAP/WHO) reference dose of 1 μg·kg^?1·day^?1 for cadmium consumption for Canadians. This suggests that although the current recommended maximum oyster consumption rates is consistent with the FAO/WHO and U.S. Environmental Protection Agency limits for acceptable risk, it leaves little or no room for error or uncertainty. This is noteworthy as recent studies demonstrate toxicological effects at cadmium intakes of 0.43 to 0.71 μ gCd·kg^?1·day^?1. This study indicates that a lower maximum BC-cultured oyster rate should be considered, particularly for high risk groups, including women with low iron stores, people with renal impairment, smokers, children, and indigenous people who consume organ meats of games and wildlife other than shellfish.     

Preparation and characterization of an 125I-labeled Sendai virus ligand

In an attempt to prepare a radioviral ligand that was effective both as an antigen in binding to antibody and as a ligand that effectively binds to receptor-bearing cells, inactivated Sendai virus was adsorbed to immobilized fetuin at 4°C and recovered by temperature elevation at 37°C. The eluted virus was iodinated using the chloramine-T procedure and free iodine and labeled virus were separated on a Sepharose 4B column. Radiolabeted virus contained 1000 to 5000 cpm per hemagglutination unit and over 90% of the counts were sedimented following high-speed centrifugation. Radioviral ligand was 80% reactive with immobilized Sendai virus antibody, 70% reactive with sheep red blood cells,and 1% reactive with receptor-negative horse red blood cells. Pretreatment of the cells with neuraminidase completely inhibited the virus binding reaction. The reaction with receptor-bearing cells was competitively inhibited by unlabeled Sendai virus, but not by murine leukemia virus or T-2 coliphage. Radioviral ligand binding to human lymphoblastic cell line CCL-119 was a saturable reaction, a result that demonstrates the absence of virus-virus aggregates. The preparation of an effective cell-reactive radioviral ligand was dependent on the initial purification from immobilized receptor-containing proteins.