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A suite of blood chemistry parameters (including acid–base indicators and plasma electrolytes) was serially measured in blacktip sharks (Carcharhinus limbatus), captured via rod-and-reel, to gain a more thorough understanding of the physiological stress response to recreational capture. Sharks were caught both from the shore and from fishing vessels and experienced varying degrees of air exposure during handling. While all captured sharks exhibited a metabolic acidosis during the fight on the line (increasing lactate and decreasing pH and bicarbonate), the observed acidosis was compounded by a respiratory component (increasing pCO2) in sharks removed from the water during handling. Vessel-caught sharks handled in the water exhibited significantly greater increases in lactate and glucose (0.73 ± 0.21 mmoll−1 min−1 and 0.81 ± 1.07 mg dl−1 min−1, respectively) than sharks handled out of water (0.21 ± 0.17 mmoll−1 min−1 and −0.32 ± 1.05 mg dl−1 min−1; p

Properties of hemocyanins vary greatly among crustaceans due to environmental conditions, lifestyle, and genetic variation. These properties can also be modified to maintain aerobic respiration in response to ambient hypoxia, as experienced by both aquacultured and wild populations of penaeid shrimp. Under normoxic conditions, hemocyanin concentrations were significantly higher (P < 0.001) in aquacultured Pacific whiteleg shrimp, Litopenaeus vannamei (10.3 g/100 ml ± 0.23 SEM, n = 49), compared to those in individuals of wild-caught L. vannamei (7.0 g/100 ml ± 0.52 SEM, n = 10), wild Farfantepenaeus aztecus (7.10 g/100 ml ± 0.48 SEM, n = 28), and wild Litopenaeus setiferus (8.0 g/100 ml ± 0.22 SEM, n = 37). Oxygen affinity of hemocyanin at 25 °C in both populations of L. vannamei was higher (Kruskal-Wallis ANOVA on ranks, P < 0.001) (aquacultured P50 = 1.47 kPa ± 0.03 SEM; wild P50 = 1.72 kPa ± 0.01 SEM at pH 7.4) than that of both Atlantic species (F. aztecus P50 = 3.94 kPa ± 0.06 SEM, L. setiferus P50 = 3.98 kPa ± 0.04 SEM at pH 7.4). The effect of l-lactate on oxygen affinity was similar among all wild groups, but significantly smaller in the aquacultured L. vannamei. Total hemocyanin concentration and oxygen binding properties were measured after exposure to 12 days and 25-31 days of hypoxia (30% air saturation). Aquacultured L. vannamei showed no change in hemocyanin concentration for up to 31 days, but both wild F. aztecus and wild L. setiferus displayed a significant increase over the same time period. No discernible change in oxygen affinity of hemocyanin was detected in any of the three species. Hypoxia tolerance appears to differ among these species of penaeid shrimp, due to either an inherent difference among the species, domestication by aquaculture, or a combination of both.

In addition to respiration and ion regulation, crustacean gills accumulate and eliminate injected particles, along with hemocyte aggregates that form in response to those particles. Here we report that the dose of Vibrio campbellii previously shown to induce a decrease in respiration and hemolymph flow across the gill in the Atlantic blue crab, Callinectes sapidus, also triggered the formation of aggregates containing four or more hemocytes in the gills, compared with saline-injected controls. More bacteria were trapped and rendered non-culturable per unit weight by anterior respiratory gills than posterior gills specialized for ion regulation. Further, more bacteria accumulated in the anterior gills of animals held at 30 ppt than those at 10 ppt. Thus, the role of the gills in immune defense comes at an energetic cost to this and likely to other crustaceans; this cost is influenced by acclimation salinity and the position and specialized function of individual gills.

The Atlantic blue crab Callinectes sapidus is an important fisheries resource that is subject to mortality and morbidity from hemolymph infections. We used culture-independent methods based on the analysis of 16S rRNA genes to characterize and quantify the microflora from the carapace, gut, and hemolymph of C. sapidus with the goals of (1) characterizing the C. sapidus microbial assemblage and (2) identifying the reservoirs of potential pathogens associated with the crab. We found that the carapace, gut and hemolymph microflora have a core Proteobacteria community with contributions from other phyla including Bacteroidetes, Firmicutes, Spirochetes, and Tenericutes. Within this Proteobacteria core, γ-Proteobacteria, including the members of the Vibrionaceae that are closely related to potential pathogens, dominate. Bacteria closely related to hemolymph pathogens were found on the carapace, supporting the hypothesis that punctures, molting damage, or broken dactyls may be routes for hemolymph infections. These results provide some of the first data on the blue crab microbial assemblage obtained with culture-independent techniques and offer insights into the routes of infection and potential bacterial pathogens associated with blue crabs.

The Atlantic blue crab, Callinectes sapidus, is an economically, ecologically, and recreationally valuable decapod crustacean that inhabits estuaries along the Atlantic and Gulf coasts of the United States. In their natural environment, blue crabs are exposed to many stressors including anthropogenic contaminants, viruses and bacteria. Bacterial infection results in the depression of oxygen uptake, and impairs normal metabolic function in a manner that has not yet been fully elucidated. Our laboratory is developing NMR-based metabolomic tools for environmental research to discover metabolomic biomarkers of stress in marine organisms. We have used NMR spectroscopy to compare the response of the crab metabolome to depression of aerobic metabolism by injection of the bacterium Vibrio campbellii, versus elevation of aerobic metabolism by treatment with 2,4-dinitrophenol (DNP), a known uncoupler of oxidative phosphorylation. The corresponding NMR spectral variations between treatments were evaluated using chemometric tools for pattern recognition and biomarker identification, including principal components analysis and partial least-squares analysis. Metabolic changes were identified in crab hemolymph 30 min after injection with V. campbellii and DNP. Glucose, considered a reliable indicator for biological stress in crustaceans, and lactate, a metabolite indicating anaerobic respiration, provided the largest variations in the metabolomes, respectively. While biological variability and/or tight regulation of the hemolymph masked subtle metabolic changes at individual time-points, metabolic trajectory analysis revealed clear differences between the two modes of oxidative stress, providing insight into the biochemical pathways involved.

SUMMARYCallinectes sapidus (Rathbun), the Atlantic blue crab, commonly harbors low to moderate amounts of bacteria in hemolymph and other tissues. These bacteria are typically dominated by Vibrio spp., which are known to cause mortality in the blue crab. The dose-dependent lethality of an isolate of Vibrio campbellii was determined in crabs; the mean 48 h LD50 (half-maximal lethal dose) was 6.2×105 colony forming units g–1 crab. Injection of a sublethal dose of V. campbellii into the hemolymph of the crab resulted in a rapid and large depression (30–42%) of metabolic rate, which persisted for 24 h. Because gills are an organ of immune function as well as respiration, we were interested in how bacteria injected into the crab would affect the energetic costs associated with walking. Overall metabolism (aerobic and anaerobic) more than doubled in crabs walking for 30 min at 8 m min–1. The metabolic depression resulting from bacterial injection persisted throughout the exercise period and patterns of phosphagen and adenylate consumption within walking leg muscle were not affected by treatment. The ability of crabs to supply required energy for walking is largely unaffected by exposure to Vibrio; however, Vibrio-injected crabs are less aerobic while doing so. This depressed metabolic condition in response to bacteria,present during moderate activity, could be a passive result of mounting an immune response or may indicate an actively regulated metabolic depression. A compromised metabolism can affect the performance of daily activities, such as feeding and predator avoidance or affect the ability to cope with environmental stressors, such as hypoxia.

The occurrence of summer mortalities of the commercially important Pacific oyster, Crassostrea gigas , has increased in recent years. These mortality events occur during the late summer when water temperatures are at their highest. Many theories have been proposed concerning the causes including reproductive stress, environmental stress, disease, or synergistic interactions of these factors. C. gigas are grown intertidally and are exposed to the air (emersed) for hours at a time. These organisms can experience extreme changes in temperature during the course of a day. An oyster closed during emersion depletes the oxygen stores to near zero within the shell and builds up CO 2 causing a decrease in tissue pH. The focus of this study is to determine the respiratory (pH, Po 2 , Pco 2 and total CO 2 ) and immune responses of oysters exposed to air at normal seasonal temperatures, and to determine whether these stresses associated with emersion inhibit the immune system of the oyster and contribute to the summer mortalities. The respiratory variables of the hemolymph of oysters submerged at 18 °C (pH = 7.52 ± 0.04 S.E.M., Po 2 = 7.09 ± 0.53 S.E.M. kPa and Pco 2 = 0.20 ± 0.03 S.E.M. kPa) varied significantly from oysters emersed for four hours at 22°C (pH = 7.11 ± 0.03 S.E.M., Po 2 = 3.83 ± 0.15 S.E.M. kPa, Pco 2 = 0.36 ± 0.03 S.E.M. kPa) and those emersed for four hours at 30 °C (pH = 6.84 ± 0.02 S.E.M., Po 2 = 3.10 ± 0.12 S.E.M. kPa, Pco 2 = 1.31 ± 0.06 S.E.M. kPa). The ability of hemocytes to kill the bacterium Vibrio campbellii was assessed using an in vitro assay to generate a killing index. There was no significant difference in the killing index between pH treatment groups ( p = 0.856): at pH 7.6 killing index = 50.2% ± 2.33 S.E.M., at pH 6.6 killing index = 52.3% ± 3.67 S.E.M.. Temperature was the only factor to significantly affect the killing indices among temperature and oxygen treatment groups. The killing index was lowest (29.3% ± 3.25 S.E.M.) at 30 °C and 7% oxygen, simulating in vivo oxygen pressure in well-aerated conditions and 30 °C and 3% oxygen, simulating in vivo oxygen pressure in hypoxia (30.5% ± 3.25 S.E.M.), compared with the index in 7% oxygen at low temperature (18 °C) (44.4% ± 4.50 S.E.M.) or compared with low oxygen (3%) at low temperature (18 °C) (39.7% ± 2.51 S.E.M.). The seasonal and diurnal rise in temperature may, therefore, be an important factor contributing to summer mortalities of C. gigas .

The Eastern oyster, Crassostrea virginica , lives in shallow coastal waters and experiences many different environmental extremes including hypoxia, hypercapnia and air exposure and many oysters are infected with the protozoan parasite Perkinsus marinus . The effects of these conditions on oyster metabolism, as measured by oxygen uptake, were investigated. Mild hypercapnia had no effect on the ability of oysters to regulate oxygen uptake in hypoxic water, as measured by the B2 coefficient of oxygen regulation. The average B2 was −0.060×10 −3 (±0.01×10 −3 S.E.M.; n =20; low and high CO 2 treatments combined) in oysters uninfected with P. marinus and −0.056×10 −3 (±0.01×10 −3 S.E.M.; n =16; low and high CO 2 treatments combined) in infected oysters. There was no significant effect of light to moderate infections of P. marinus on oxygen regulation. Nor did the presence of P. marinus have an effect on the rate of oxygen uptake of whole animals in well-aerated water. In well-aerated conditions, oxygen uptake was significantly reduced by moderate hypercapnia in oysters when data from uninfected and infected oysters were combined. Mean oxygen uptake of infected oysters under hypercapnia ( p CO 2 =6–8 Torr; pH 7) was 9.10 μmol O 2 g ww −1 h −1 ±0.62 S.E.M. ( n =9), significantly different from oxygen uptake under normocapnia ( p CO 2 ≤1 Torr; pH 8.2) (10.71 μmol O 2 g ww −1 h −1 ±0.62 S.E.M.; n =9). Similar to what occurred in infected whole animals, mean oxygen uptake of uninfected gill tissues under high CO 2 , low pH conditions was 9.44 μmol O 2 g ww −1 h −1 ±0.95 S.E.M. ( n =10), significantly different from oxygen uptake under low CO 2 , high pH conditions (12.30 μmol O 2 g ww −1 h −1 ±0.95 S.E.M.; n =10). This result is due primarily to the low pH induced by hypercapnia rather than a CO 2 -specific effect. The presence of P. marinus had no effect on oxygen uptake in gill tissues. Intertidal oysters from South Carolina take up very little oxygen from the air when they are air exposed. Mean oxygen uptake in air at 25°C (5.66×10 −4 μmol O 2 g ww −1 h −1 ±2.65×10 −4 S.E.M.; n =11) is less than 0.1% of oxygen uptake in seawater, suggesting that upon air exposure, oysters close their valves and isolate themselves from air. Oxygen uptake in air is slightly elevated at 35°C (9.28×10 −4 μmol O 2 g ww −1 h −1 ±5.57×10 −4 S.E.M.; n =11). There was not a strong correlation between oxygen uptake and P. marinus infection intensity at either 25 or 35°C.

Organisms living in coastal waters, and especially estuaries, have long been known to have behavioral or physiological mechanisms that enable them toexist in water containing low amounts of oxygen. However, the respiratory consumption of oxygen that generates hypoxia is also responsible for producing significant amounts of carbon dioxide. An elevation of carbon dioxide pressure in water will cause a significant acidosis in most aquatic organisms. Thus, the combination of low oxygen and elevated carbon dioxide that occurs in estuaries represents a significant environmental challenge to organisms living in this habitat. Organisms may maintain oxygen uptake in declining oxygen conditions by using a respiratory pigment and/or by making adjustments in the convective flow of water and blood past respiratory surfaces ( i.e. , increase cardiac output and ventilation rate). Severe hypoxia may result in an organism switching partially or completely to anaerobic biochemical pathways to sustain metabolic rate. There is also evidence to suggest that organisms lower their metabolism during hypoxic stress. Elevated water CO2 (hypercapnia) produces an acidosis in the tissues of organisms that breathe it. This acidosis may be wholly or partially compensated ( i.e. , mechanisms return pH to pre-exposure levels), or may be uncompensated. Some studies have examined the effects on organisms of exposure simultaneously to hypoxia and hypercapnia. This article reviews some of the specific adaptations and responses of organisms to low oxygen, to high carbon dioxide, and to the cooccurrence of low oxygen and high carbon dioxide

Estuarine waters are prone to regular bouts of low oxygen (hypoxia) and high carbon dioxide (hypercapnia). In vertebrates, tissue hypoxia followed by reoxygenation can generate high levels of reactive oxygen species (ROS) that exceed cellular antioxidant capacity, leading to tissue damage. Here we quantified the expression of several antioxidant genes in the hepatopancreas of Pacific whiteleg shrimp, Litopenaeus vannamei , after exposure to hypoxia or hypercapnic hypoxia for 4 h or 24 h followed by recovery in air-saturated water (normoxia) for 0, 1, 6 or 24 h, as compared to time-matched controls maintained only in normoxia. Transcripts of cytoplasmic Mn-superoxide dismutase (cMnSOD), glutathione peroxidase (GPX) and peptide-methionine (R)-S-oxide reductase (MsrB) increased after 4 h exposure to either hypoxia or hypercapnic hypoxia; these elevated transcript levels persisted longer in animals recovering from hypercapnic hypoxia than hypoxia alone. cMnSOD transcripts generally increased, but GPX, MsrB, glutathione-S-transferase (GST), and thioredoxin 1 (TRX-1) decreased or did not change in most long-term (24 h) treatment-recovery groups. Thus, the transcriptional responses of several antioxidant genes during recovery from tidally-driven hypoxia and hypercapnic hypoxia decrease or are muted by more persistent exposure to these conditions, leaving L. vannamei potentially vulnerable to ROS damage during recovery.

Estuarine crustaceans are often exposed to low dissolved O2(hypoxia) accompanied by elevated CO2(hypercapnia), which lowers water pH. Acclimatory responses to hypoxia have been widely characterized; responses to hypercapnia in combination with hypoxia (hypercapnic hypoxia) are less well known. Here we used oligonucleotide microarrays to characterize changes in global gene expression in the hepatopancreas of Pacific whiteleg shrimp, Litopenaeus vannamei, exposed to hypoxia or hypercapnic hypoxia for 4 or 24 h, compared with time-matched animals held in air-saturated water (normoxia). Unigenes whose expressions were significantly impacted by treatment and/or time were used to build artificial neural networks (ANNs) to identify genes with the greatest sensitivity in pairwise discriminations between treatments at each time point and between times for each treatment. ANN gene sets that discriminated hypoxia or hypercapnic hypoxia from normoxia shared functions of translation, mitochondrial energetics, and cellular defense. GO terms protein modification/phosphorylation/cellular protein metabolism and RNA processing/apoptosis/cell cycling occurred at highest frequency in discriminating hypercapnic hypoxia from hypoxia at 4 and 24 h, respectively. For 75.4% of the annotated ANN genes, exposure to hypercapnic hypoxia for 24 h reduced or reversed the transcriptional response to hypoxia alone. These results suggest that high CO2/low pH may interfere with transcriptionally based acclimation to hypoxia or elicit physiological or biochemical responses that relieve internal hypoxia. Whether these data reflect resilience or sensitivity of L. vannamei in the face of expanding hypoxic zones and rising levels of atmospheric CO2may be important to understanding the survival of this and other estuarine species.

Hemolymph acid-base variables were investigated in the Eastern oyster, Crassostrea virginica, to determine its responses to air exposure and to infections by the parasite Perkinsus marinus. Infected and uninfected oysters were subjected to two treatments of temperature (21° and 30°C) and air exposure (5 and 24 h). Upon exposure to air, oysters underwent a respiratory acidosis that remained uncompensated in uninfected oysters but was partially compensated in highly infected oysters at both 21° and 30°C. The acidosis was significantly greater in oysters with high infections. Hemolymph in uninfected oysters had a greater buffering capacity (-6.80 +/- 0.76 SEM slykes) than hemolymph in highly infected oysters (-3.30 +/- 0.50 SEM slykes). Calcium ion concentrations in hemolymph increase when the hemolymph becomes acidic, suggesting that shell decalcification plays a role in buffering the acid. During air exposure, although oysters do not visibly gape, they access air and are apparently not completely anaerobic.

In tidal saltmarshes in South Carolina hypoxic (low O2) and hypercapnic (high CO2) conditions occur frequently. In the summer, water Po2 was measured in the upper marshes and over a 24-h period ranged from 9 to 170 torr and Pco2 ranged from 0.3 to 12 torr. These conditions depend on the stage of the tide and the time of day. The respiratory responses to different levels of Po2 and Pco2 of the grass shrimp, Palaemonetes pugio, the spot fish, Leiostomus xanthurus, and the killifish or mummichog, Fundulus heteroclitus living and feeding in the saltmarsh were investigated. Mean oxygen uptake in P. pugio, L. xanthurus, and F. heteroclitus at normoxic Po2 (130–150 torr) and low Pco2 (< 0.6 torr) was 17.5, 17.1, and 9.5 μmol · g−1 · h−1 and 16.3, 24.5, and 10.5 μmol · g−1 · h−1 at high Pco2 (= 7 torr), respectively. The critical Po2 for all species was between 30 and 35 torr. Mean whole body lactate concentrations in P. pugio, L. xanthurus, and F. heteroclitus at Pco2 < 0.6 torr are 3.5, 2.4, and 2.3 μmol · g−1, respectively, in normoxia and 12.3, 4.5, and 11.0 μmol · g−1 (p < 0.05; Dunn's pairwise test) in hypoxia and Pco2 < 0.6 torr. In these saltmarsh animals there appears in general to be no specific effects on oxygen uptake of environmental fluctuations in CO2 over a wide range of Po2. The organophosphate pesticide, azinphosmethyl, appears to have no effect on the oxygen uptake of these three species at concentrations of 10 μg · l−1 for fish and 2 μg · l−1 for shrimp.

A large number of factors are now known to influence the oxygen affinity of hemocyanins in crabs. In this article I review several case studies in which a number of different variables, including pH, CO2, calcium ions, lactate, urate, dopamine, and temperature, act together on hemocyanin oxygen affinity to bring about adaptive changes in oxygen transport. These changes act in tandem with ventilatory and cardiovascular adjustments to stabilize oxygen uptake during hypoxia or to increase oxygen uptake during exercise in a swimming crab. It is important, however, that the effects of the different modulators be examined at very low concentrations, where their effects are large, to assess their importance in resting crabs where their concentrations may be low. Temperature effects among hemocyanins in crabs are variable and the magnitude may be inversely related to the magnitude of the Bohr effect. Several examples are presented of acclimation effects where oxygen affinity increases significantly at cold temperatures. The acclimation response may be important in partially regulating the metabolic responses of crabs to seasonal changes in temperature.

The green or shore crab Carcinus maenas (Linnaeus) is believed to have emigrated by ship from its native Europe to the Atlantic coast of North America. The chronological sequence of new records suggests the arrival in New York of pioneer individuals in the early 19th century, followed by gradual northward extension of the species range (Almaca, 1963; J. T. Carlton, personal communication). The number of immigrations and the present genetic relationships of various populations are unknown.

The Eastern oyster, Crassostrea virginica , inhabits shallow coastal waters that frequently experience periods of low dissolved oxygen (hypoxia) and elevated CO 2 (hypercapnia) levels. Bacteria are extremely abundant in these environments and accumulate in large numbers in filter-feeding oysters, which can act as passive carriers of human pathogens. Although hypercapnic hypoxia (HH) can affect certain specific immune mechanisms, its direct effect on the inactivation, degradation and elimination of bacteria in oysters is unknown. This research was conducted to determine whether exposure to HH reduces the ability of C. virginica to inactivate and eliminate Vibrio campbellii following its injection into the adductor muscle. Oysters were held in fully air-saturated (normoxic; partial O 2 pressure [P O2 ] = 20.7 kPa, CO 2 < 0.06 kPa, pH 7.8 to 8.0) or HH (P O2 = 4 kPa, CO 2 = 1.8 kPa, pH 6.5 to 6.8) seawater at 25°C for 4 h before being injected in the adductor muscle with 10 5 live Vibrio campbellii bacteria and remained under these conditions for the remainder of the experiment (up to 24 h postinjection). Real-time PCR was used to quantify the number of intact V. campbellii bacteria, while selective plating was used to quantify the number of injected bacteria remaining culturable in whole-oyster tissues, seawater, and feces/pseudofeces at 0, 1, 4, and 24 h postinjection. We found that oysters maintained under normoxic conditions were very efficient at inactivating and degrading large numbers of injected bacteria within their tissues. Moreover, a small percentage (∼10%) of injected bacteria were passed into the surrounding seawater, while less than 1% were recovered in the feces/pseudofeces. In contrast, HH increased the percentage of culturable bacteria recovered from the tissues of oysters, suggesting an overall decrease in bacteriostasis. We suggest that poor water quality may increase the risk that oysters will harbor and transmit bacterial pathogens hazardous to human and ecosystem health.

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)

Crustacean gills function in gas exchange, ion transport, and immune defense against microbial pathogens. Hemocyte aggregates that form in response to microbial pathogens become trapped in the fine vasculature of the gill, leading to the suggestion by others that respiration and ion regulation might by impaired during the course of an im- mune response. In the present study, injection of the patho- genic bacterium Vibrio campbellii into Callinectes sapidus, the Atlantic blue crab, caused a dramatic decline in oxygen uptake from 4.53 to 2.56 mol g 1 h 1 . This decline in oxygen uptake is associated with a large decrease in post- branchial Po2, from 16.2 (0.46 SEM, n7) to 13.1 kPa (0.77 SEM, n9), while prebranchial Po2 remains un- changed. In addition, injection of Vibrio results in the dis- appearance of a pH change across the gills, an indication of reduced CO2 excretion. The hemolymph hydrostatic pres- sure change across the gill circulation increases nearly 2-fold in Vibrio-injected crabs compared with a negligible change in pressure across the gill circulation in saline- injected, control crabs. This change, in combination with stability of heart rate and branchial chamber pressure, is indicative of a significant increase in vascular resistance across the gills that is induced by hemocyte nodule forma- tion. A healthy, active blue crab can eliminate most invad- ing bacteria, but the respiratory function of the gills is impaired. Thus, when blue crabs are engaged in the immune response, they are less equipped to engage in oxygen-fueled activities such as predator avoidance, prey capture, and migration. Furthermore, crabs are less fit to invade environ- ments that are hypoxic.

We hypothesized that aggregation of bacteria and hemocytes at the gill, which occurs as part of the shrimp's antibacterial immune defenses, would impair normal respiratory function and thereby disrupt aerobic metabolism. Changes in oxygen uptake and lactate accumulation were determined in Litopenaeus vannamei, the Pacific white shrimp, following injection with either saline (control) or a strain of the gram-negative bacterium Vibrio campbellii that is pathogenic in crustaceans. The rate of oxygen uptake was determined during the first 4 h after injection and after 24 h. Injection of bacteria decreased oxygen uptake by 27% (from 11.0 to 8.2 micromol g-1 h-1) after 4 h, while saline-injected shrimp showed no change. Decreased oxygen uptake persisted 24 h after Vibrio injection. In well-aerated water, resting whole-animal lactic acid levels increased in shrimp injected with bacteria (mean=2.59 micromol lactate g-1+/-0.39 SEM, n=8) compared to saline-injected control shrimp, but this difference did not persist at 24 h. Exposure to hypercapnic hypoxia (PCO2=1.8 kPa, PO2=6.7 kPa) also resulted in significant whole-body lactic acid differences (mean=3.99 and 1.8 micromol g-1 tissue in Vibrio and saline-injected shrimp, respectively). Our results support the hypothesis that the crustacean immune response against invading bacteria impairs normal metabolic function, resulting in depression of oxygen uptake and slightly increased anaerobic metabolism.

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.

Oysters are frequently exposed to severely hypoxic conditions, especially during summer months. During the summer, there are also large numbers of disease-related oyster mortalities. This research was conducted to determine whether exposure to environmental hypoxia reduces the ability of oyster hemocytes to produce reactive oxygen intermediates (ROIs), an important part of their defense system. Oysters of the species Crassostrea virginica were held in normoxic (P(O)(2)=20.0-20.7 kPa, pH 7.8-8.0) and hypoxic conditions (P(O)(2)=4.0-6.7 kPa, pH 7.1-7.4). In vivo hemolymph variables (P(O)(2), P(CO)(2) and pH) were measured after both 1 hour and 2 days in each treatment to determine the appropriate environment for subsequent hemocyte experiments. Production of reactive oxygen intermediates by hemocytes was measured using luminol-enhanced chemiluminescence (CL). During CL tests, hemocytes were held under the following conditions: air (P(O)(2)=20.7, P(CO)(2)

The effect of CO2 on hemocyanin-oxygen binding is not generally related to the effect of Cl-. Some hemocyanins respond to both and some to either one alone. The direction of the responses of O2 affinity of the various hemocyanins to CO2 is poorly correlated with the direction of responses to other effectors. The influence of CO2 on Busycon and Limulus hemocyanins reaches its maximum at high pH. Since the effect can be abolished by restoring divalent cation activities to the control levels prior to the addition of CO2, we suggest that the effect is not specific but rather indirect, by the pairing of the allosteric effectors Ca+2 and Mg+2 with the CO2 anions. In contrast the effect of CO2 on crustacean hemocyanins is greaterat low pH and it can be enhanced by maintaining HCO3- levels within narrow limits and permitting PCO2 to vary by a large factor. This finding suggests that the effective species is molecular CO2. While a influences only oxygen affinity, CO2 may influence cooperativity as well. Indifferen...

The respiratory function of the hemocyanin (Hcy)-containing blood in the aquatic spider crab, Libinia emarginata, and the terrestrial ghost crab, Ocypode guadrata, was studied in animals exposed to normoxic and hypoxic conditions a t 25°C. In L. emarginata low concentrations of Hcy, although 90% O2, saturated at the gill, necessitate a high blood and water convection strategy. O. quadrata has blood with a higher concentration of hemocyanin which is 96% O2 saturated at the gill, obviating a very high rate of blood circulation. During progressive hypoxia convection initially increases on both sides of the gill in L. emarginata while in O. quadrata cardiac output decreases. Blood pH increases with decreasing ambient P, below 60 torr in L. emarginata, inducing a greater hemocyanin O2 affinity.

The temperature sensitivity of hemocyaninoxygen affinity and cooperativity was measured at 5, 15, 25, and 35°C in a variety of manine molluscs and arthropods from different thermal environments. These environments included a subtidal habitat in which the temperature is generally less than 15°C and the diurnal temperature variation is small, and an intertidal habitat in which the temperature varies more than 30°C. The temperature sensitivity of P50 showed considerable variation (▵H = 0 to ▵H = -67 kJ/mol) depending on species and experimental temperatures. Sensitivity generally decreased as temperature increased. In several species temperature sensitivity was either absent or greatly reduced above 15°C. The horseshoe crab Limulus polyphemus showed a minimum temperature sensitivity between 15 and 25°C but higher sensitivity above and below this range. The hypothesis that a greater interaction between hemocyanin molecules and calcium ions at high temperatures offsets the temperature effect, resulting in a pi...

The CO2-specific sensitivity of hemocyanin oxygen affinity and oxygen binding site cooperativity were investigated in five species of crabs. The hypothesis that CO2-specific effects on hemocyanin are related to the molecular CO2 concentrations that occur naturally in crab hemolymph was tested in crabs that experience a variety of hemolymph CO2 pressures. Crab species were chosen from subtidal, intertidal, and semiterrestrial habitats. Hemolymph samples from each species were added to physiological salines buffered with 50 mM HEPES. O2 equilibrium curves were determined spectrophotometrically by gassing the hemolymph-saline mixtures with different P at constant P and temperature. We found no CO2-specific effects on oxygen equilibrium properties of hemocyanin in any species under the conditions investigated. It is concluded that molecular CO2 and bicarbonate ions are not important in directly modulating the oxygen equilibrium properties of crustacean hemocyanins.

O₂ uptake and hemolymph acid-base status, together with branchial water volume, CO₂ content, and titratable alkalinity, were measured in three species of intertidal crabs. In Pachygrapsus crassipes, a grapsid crab that actively moves between air and water, O₂ uptake increased on emersion. In Eurytium albidigitum, a mud-burrowing xanthid crab that is air exposed by tidal action, O₂ uptake declines dramatically on emersion. There is no significant lactate production by either crab following emersion. An emersion-induced respiratory acidosis was fully compensated in P. crassipes and another grapsid, Hemigrapsus nudus, but uncompensated in E. albidigitum. Branchial water volume 10 min after emersion was 0.013 ml/g crab weight in P. crassipes and 0.072 ml/g in E. albidigitum. The CO₂ content of branchial water in P. crassipes increased rapidly during air exposure and was accompanied by an increase in titratable alkalinity (TA). The CO₂ content of branchial water in E. albidigitum remained constant for at least...

SYNOPSIS. Intertidal organisms usually exhibit one of two behaviors when they are air exposed. They either isolate themselves from the aerial environment or they interact with the aerial environment. Among the animals using the first behavior, body fluid acid-base balance is partially maintained by dissolution of the calcium carbonate shell in the bivalve molluscs to buffer the metabolic acids produced anaerobically. Calcium ions compensate for the acidosis by increasing the strong ion difference. The release of carbonate from the shell causes an increase in molecular CO2 which offsets the compensation somewhat, but this effect is minimized by distributing the CO2 among the other fluids within the shell and/or by venting the shell to the air. In animals which have a fairly high concentration of a respiratory pigment, such as a lugworm, an anaerobically induced acidosis is minimized by a large Haldane effect. Among the animals which interact with the aerial environment, the decapod crustaceans by and large maintain their metabolism aerobically, although it may be greatly reduced. A respiratory acidosis due to elevated hemolymph Pco2 may be either fully compensated or not at all. Compensation involves an increase in hemolymph calcium, probably from the calcium carbonate exoskeleton. Compensatory mechanisms may also include branchial water stores which accumulate a titratable base. It is suggested that the alkalinization of the branchial water maintains a steeper Pco2 gradient across the gill and reduces the magnitude of the acidosis for a short period of time. The ability to use branchial water stores in this way may be tied to the ability of the animal to osmoregulate.

The sublethal and lethal effects of chlorine produced oxidants (CPO) on juveniles of the estuarine teleost,Leiostomus xanthurus, were investigated in flowing water tests conducted at 30 ±1°C and 26 to 31‰ salinity. Short-term LT50 tests were conducted at two nominal concentrations of NaOCl, 1.0 and 1.4 mg/l (respective measured CPO concentrations 0.09 and 0.12 mg/l) which were sublethal in 2,880 minute exposures; and three nominal concentrations, 1.6, 1.8 and 3.2 mg/l NaOCl (respective measured CPO concentrations 0.13, 0.20 and 0.37 mg/l) which were acutely toxic. Opercular ventilation rates in exposed spot were much higher than in control fish, but returned to rates only slightly above those of controls during the latter portion of the 2,880 minute exposure to the two sublethal CPO concentrations. Opercular rates at the three acutely toxic CPO concentrations remained much higher than control rates until the exposed fish died. Blood pH after 2,880 minutes of exposure to the sublethal concentrations of CPO; or at the respective estimated LT50 for lethal concentrations, showed significant decreases ( $$\bar X$$ as low as 6.84) compared to controls $$(\bar X 7.35)$$ . No significant changes in the % methemoglobin were observed. Oxygen uptake by spot was depressed at all of the measured concentrations of CPO tested. Histopathological examination showed that gill respiratory epithelial tissues sloughed away from the underlying pillar cells. Complete denudation of circulatory tissues and hemangiectic secondary lamellae were observed in gill tissues from fish exposed to the highest CPO concentration of 0.37 mg/l.