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Oxygen bioavailability is declining in aquatic systems worldwide as a result of climate change and other anthropogenic stressors. For aquatic organisms, the consequences are poorly known but are likely to reflect both direct effects of declining oxygen bioavailability and interactions between oxygen and other stressors, including two—warming and acidification— that have received substantial attention in recent decades and that typically accompany oxygen changes. Drawing on the collected papers in this symposium volume (“An Oxygen Perspective on Climate Change”), we outline the causes and consequences of declining oxygen bioavailability. First, we discuss the scope of natural and predicted anthropogenic changes in aquatic oxygen levels. Although modern organisms are the result of long evolutionary histories during which they were exposed to natural oxygen regimes, anthropogenic change is now exposing them to more extreme conditions and novel combinations of low oxygen with other stressors. Second, we identify behavioral and physiological mechanisms that underlie the interactive effects of oxygen with other stressors, and we assess the range of potential organismal responses to oxygen limitation that occur across levels of biological organization and over multiple timescales. We argue that metabolism and energetics provide a powerful and unifying framework for understanding organism-oxygen interactions. Third, we conclude by out-lining a set of approaches for maximizing the effectiveness of future work, including focusing on long-term experiments using biologically realistic variation in experimental factors and taking truly cross-disciplinary and integrative approaches to understanding and predicting future effects.

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

Coastal acidification in southeastern U.S. estuaries and coastal waters is influenced by biological activity, run-off from the land, and increasing carbon dioxide in the atmosphere. Acidification can negatively impact coastal resources such as shellfish, finfish, and coral reefs, and the communities that rely on them. Organismal responses for species located in the U.S. Southeast document large negative impacts of acidification, especially in larval stages. For example, the toxicity of pesticides increases under acidified conditions and the combination of acidification and low oxygen has profoundly negative influences on genes regulating oxygen consumption. In corals, the rate of calcification decreases with acidification and processes such as wound recovery, reproduction, and recruitment are negatively impacted. Minimizing the changes in global ocean chemistry will ultimately depend on the reduction of carbon dioxide emissions, but adaptation to these changes and mitigation of the local stressors that exacerbate global acidification can be addressed locally. The evolution of our knowledge of acidification, from basic understanding of the problem to the emergence of applied research and monitoring, has been facilitated by the development of regional Coastal Acidification Networks (CANs) across the United States. This synthesis is a product of the Southeast Coastal and Ocean Acidification Network (SOCAN). SOCAN was established to better understand acidification in the coastal waters of the U.S. Southeast and to foster communication among scientists, resource managers, businesses, and governments in the region. Here we review acidification issues in the U.S. Southeast, including the regional mechanisms of acidification and their potential impacts on biological resources and coastal communities. We recommend research and monitoring priorities and discuss the role SOCAN has in advancing acidification research and mitigation of and adaptation to these changes.

Atlantic horseshoe crabs, Limulus polyphemus (HSC), are commercially harvested along the eastern U.S. coast and bled for hemolymph used in pharmaceutical safety testing. In South Carolina, some HSCs are held in outdoor ponds before transport to facilities where they are bled and then released to the wild. This study examined whether the time HSCs are held before bleeding, bleeding itself, or the duration of the recovery after bleeding affects HSC mortality and physiological condition. Female HSCs were collected from Coffin Point Beach, South Carolina (April 22–24, 2016), held in ponds for 2, 4, 6, or 8 weeks, then bled or held as controls. Body weights, hemocyanin concentrations, and hemocyte densities were measured prior to treatment (bled/control) and at 2, 6, and 12 days of recovery. Hemocyanin concentrations declined significantly in HSCs held in ponds for 8 weeks prior to bleeding and were excluded from further analyses. Compared to some studies, HSC mortalities were low (11%). Impacts of time in holding ponds, bleeding, and recovery from bleeding on physiological measures were assessed using 3-way fixed-effects ANOVA. While duration of recovery had main effects on physiological measures, significant interactions were also present. There was an interaction of treatment and recovery duration, with control crabs having higher hemocyte densities than bled animals at days 2 and 6 of recovery. There were two significant two-way interactions influencing hemocyanin concentration: pond time and recovery, and treatment and recovery. Our study suggests both main and synergistic effects are important when assessing the physiology and mortality of HSCs harvested for biomedical purposes.

The responses of estuarine invertebrates to hypoxic conditions are well established. However, many studies have investigated hypoxia as an isolated condition despite its frequent co-occurrence with hypercapnia (elevated CO2 ). Although many studies suggest deleterious effects, hypercapnia has been observed to improve blue crab walking performance in hypoxia. To investigate the physiological effects of combined hypercapnic hypoxia, we measured Po2 , pH, [l-lactate], Pco2 , and total O2 in pre- and postbranchial hemolymph sampled from blue crabs during walking exercise. Crabs walked at 8 m min-1 on an aquatic treadmill in normoxic (100% air saturation), moderately hypoxic (50%), and severely hypoxic (20%) seawater with and without the addition of hypercapnia (about 2% CO2 ). Respiration was almost completely aerobic in normoxic conditions, with little buildup of lactate. During exercise under severe hypoxia, lactate increased from 1.4 to 11.0 mM, indicating a heavy reliance on anaerobic respiration. The O2 saturation of arterial hemocyanin was 47% in severe hypoxia after 120 min, significantly lower than in normoxia (80%). However, the addition of hypercapnia significantly increased the percentage saturation of arterial hemocyanin in severe hypoxia to 92% after 120 min of exercise, equivalent to normoxic levels. Hypercapnia in severe hypoxia also caused a marked increase in hemolymph Pco2 (around 1.1 kPa), but caused only a minor decrease in pH of 0.1 units. We suggest that the improved O2 saturation at the gills results from a specific effect of molecular CO2 on hemocyanin oxygen binding affinity, which works independently of and counter to the effects of decreased pH.

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 (Rathbun), is a highly mobile crustacean that must locomote to find food, evade predators, find mates, and avoid adverse conditions such as hypoxia. In this study we tested the effects of two levels of hypoxia (10.4 kPa, 50% air saturation = moderate hypoxia; 4 kPa, 20% air saturation = severe hypoxia) and hypercapnic hypoxia (50% air saturation O(2) with Pco(2) = 2 kPa) on fatigue during sustained continuous exercise. Fatigue was induced by an exercise trial that entailed continuous sideways hexapedal walking on an underwater treadmill. Fatigue was quantified using two methods: (1) a pull force test that measures the holding strength of the legs, and (2) the number of fatigue-resisting behaviors (180° turns and stopping). Fatigue was defined as a pull force of 67% or less of the initial pre-exercise pull force and was reached after 6.12 h of walking for crabs in well-aerated normoxic seawater, 4 h in 50% air saturation, 2.07 h in 20% air saturation, and 4.58 h in 50% air saturation and hypercapnia. The number of fatigue-resisting behaviors increased with walking time in all treatments. Performance decreased in hypoxia, with fatigue being reached more quickly as the level of hypoxia intensified. Hypercapnia in moderate hypoxia did not have a deleterious influence on behavior and lengthened slightly the time it took crabs to fatigue. In addition, severe hypoxia exacerbated changes in gait kinematics as crabs became fatigued, by significantly increasing stride length and decreasing stride frequency.

Size is an important factor for the Atlantic blue crab, Callinectes sapidus (Rathbun), with large male crabs often having an advantage over small crabs in mating, molting, and locomoting. The ability of larger crabs to resist fatigue may be another benefit of being big. We describe a novel pull force test that measures the force crabs use to hold onto a mesh grid, mimicking the way a male holds a female during mate guarding. In this study, we used this pull force test to determine whether larger male crabs have an advantage over smaller crabs by being able to sustain a greater pull force even after fatigue from sustained walking. Fatigue was induced by a 6-h exercise trial that entailed continuous sideways hexapedal walking on an underwater treadmill. Larger crabs maintained an advantage over smaller crabs even after fatigue. In crabs, including the Atlantic blue crab, increased size seems to have advantages. These include reproduction

Aquatic crustaceans can experience low levels of O2 alone but more often in combination with high levels of CO2 both in natural estuaries and in aquaculture ponds. Hemocyanin, the respiratory pigment in many crustacean species, facilitates O2 transport and is documented to change in abundance, structure, and function in response to low O2 The impacts of high CO2 on the respiratory pigment are less clear. In this synthesis we bring together data from recently published and new RNA-Seq studies toward the aims of defining the full repertoire of hemocyanin subunits, as well as their differential expression and regulation in the Penaeoidea family in response to low O2 with or without high CO2 RNA-Seq data were collected from the hepatopancreas tissues of aquacultured Pacific whiteleg shrimp Litopenaeus vannamei, wild-caught L. vannamei, and wild-caught Atlantic brown shrimp Farfantepenaeus aztecus. De novo assembly yielded high-quality stranded transcriptomes. Manual curation of the hemocyanin subunits from all three groups of penaeid shrimp confirmed the existence of a small γ-type hemocyanin subunit (HcS), greater sequence diversity in the large γ-type hemocyanin subunit than previously identified (HcL1-3 isoforms) and expression of a β-type hemocyanin subunit (HcB) previously unidentified in Penaeid shrimp. Relative abundance of transcripts encoding these hemocyanin isoforms differed within and among the three species/strains. Exposure to low O2 induced expression of all of the subunits in aquacultured L. vannamei With concurrent exposure to high CO2, the number of γ-type hemocyanin transcripts decreased while the expression of β-type transcripts remained unchanged. Together with functional data for hemocyanins in the same shrimp species/strains, the RNA-Seq approach shows great promise to provide new insights into the connection between sequence, protein structure, and physiological function of respiratory pigments in this decapod crustacean family.

Hypoxia inducible factor (HIF) is a transcription factor that under low environmental oxygen regulates the expression of suites of genes involved in metabolism, angiogenesis, erythropoiesis, immune function, and growth. Here, we isolated and sequenced partial cDNAs encoding hif-α and arnt/hif-β from the Atlantic blue crab, Callinectes sapidus, an estuarine species that frequently encounters concurrent hypoxia (low O(2)) and hypercapnia (elevated CO(2)). We then examined the effects of acute exposure (1h) to hypoxia (H) and hypercapnic hypoxia (HH) on relative transcript abundance for hif-α and arnt/hif-β in different tissues (glycolytic muscle, oxidative muscle, hepatopancreas, gill, and gonads) using quantitative real-time RT-PCR. Our results indicate that hif-α and arnt/hif-β mRNAs were constitutively present under well-aerated normoxia (N) conditions in all tissues examined. Further, H and HH exposure resulted in both tissue-specific and muscle fiber type-specific effects on relative hif-α transcript abundance. In the gill and glycolytic muscle, relative hif-α mRNA levels were significantly lower under H and HH, compared to N, while no change (or a slight increase) was detected in oxidative muscle, hepatopancreas and gonadal tissues. H and HH did not affect relative transcript abundance for arnt/hif-β in any tissue or muscle fiber type. Thus, in crustaceans the HIF response to H and HH appears to involve changes in hif transcript abundance, with variation in hif-α and arnt/hif-β transcriptional dynamics occurring in both a tissue- and muscle fiber type-dependent manner.

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.

Peroxinectin (Pox), which promotes cell adhesion and encapsulation of bacteria in crustaceans, is synthesized in granular and semigranular hemocytes. In this study, real-time PCR was used to quantify Pox transcripts in individual tissues of the Pacific white shrimp, Litopenaeus vannamei, over 48 h following injection of a sublethal dose of the shrimp pathogen Vibrio campbellii. The resulting data were used to infer the movements of hemocytes among the tissues in response to bacterial challenge. Over all times and treatments, Pox transcripts (ng total RNA)(-1) varied by orders of magnitude among individual tissues, such that circulating hemocytes >> gills >> heart lymphoid organ hepatopancreas approximately muscle. Relatively low constitutive expression of Pox in the lymphoid organ compared to circulating hemocytes, gills, and heart supports a primary role for this organ in bacteriostasis and degradation, rather than encapsulation of invasive bacteria. Numbers of Pox transcripts increased significantly at the injection site within 4 h and remained significantly elevated for 48 h, consistent with a rapid and sustained recruitment of hemocytes to the site of injection. Transcripts increased significantly in the gill but not in other tissues over the time-course of this experiment. These expression data reinforce the role of the gill in trapping and encapsulating invasive bacteria as a primary strategic focus during the early phase of the crustacean immune response and, by comparison with earlier studies of lysozyme expression in the same tissues, suggest differential roles for various tissues in a successful immune response.

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 .

Extensive similarities in the molecular architecture of the crustacean immune system to that of insects give credence to the current view that the Hexapoda, including Insecta, arose within the clade Pancrustacea. The crustacean immune system is mediated largely by hemocytes, relying on suites of pattern recognition receptors, effector functions, and signaling pathways that parallel those of insects. In crustaceans, as in insects, the cardiovascular system facilitates movement of hemocytes and delivery of soluble immune factors, thereby supporting immune surveillance and defense along with other physiological functions such as transport of nutrients, wastes, and hormones. Crustaceans also rely heavily on their cardiovascular systems to mediate gas exchange; insects are less reliant on internal circulation for this function. Among the largest crustaceans, the decapods have developed a condensed heart and a highly arteriolized cardiovascular system that supports the metabolic demands of their often large body size. However, recent studies indicate that mounting an immune response can impair gas exchange and metabolism in their highly developed vascular system. When circulating hemocytes detect the presence of potential pathogens, they aggregate rapidly with each other and with the pathogen. These growing aggregates can become trapped in the microvasculature of the gill where they are melanized and may be eliminated at the next molt. Prior to molting, trapped aggregates of hemocytes also can impair hemolymph flow and oxygenation at the gill. Small shifts to anaerobic metabolism only partially compensate for this decrease in oxygen uptake. The resulting metabolic depression is likely to impact other energy-expensive cellular processes and whole-animal performance. For crustaceans that often live in microbially-rich, but oxygen-poor aquatic environments, there appear to be distinct tradeoffs, based on the gill's multiple roles in respiration and immunity. Insects have developed a separate tracheal system for the delivery of oxygen to tissues, so this particular tradeoff between oxygen transport and immune function is avoided. Few studies in crustaceans or insects have tested whether mounting an immune response might impact other functions of the cardiovascular system or alter integrity of the gut, respiratory, and reproductive epithelia where processes of the attack on pathogens, defense by the host, and physiological functions play out. Such tradeoffs might be fruitfully addressed by capitalizing on the ease of molecular and genetic manipulation in insects. Given the extensive similarities between the insect and the crustacean immune systems, such models of epithelial infection could benefit our understanding of the physiological consequences of immune defense in all of the Pancrustacea.

Acclimation to low O2 in many organisms involves changes at the level of the transcriptome. Here we used high-throughput RNA sequencing (RNA-Seq) to explore the global transcriptomic response and specific involvement of a suite of hemocyanin (Hc) subunits to low O2 alone and in combination with high CO2, which naturally co-occurs with low O2. Hepatopancreas mRNA of juvenile L. vannamei exposed to air-saturated water, low O2, or low O2/high CO2 for 4 or 24 h was pooled, sequenced (HiSeq 2500) and assembled (Trinity: 52,190 contigs) to create a deep strand-specific reference transcriptome. Annotation of the assembly revealed sequences encoding the previously described small Hc subunit (HcS), and three full-length isoforms of the large subunit (HcL1-3). In addition to this, a previously unidentified full-length Hc subunit was discovered. Phylogenetic analysis demonstrated the subunit to be a β-type Hc subunit (denoted HcB), making this the first report of a β-type hemocyanin subunit in the Penaeoidea. RNAs of individual shrimp were sequenced; regulated genes identified from pairwise comparisons demonstrated a distinct pattern of regulation between prolonged low O2 and low O2/high CO2 treatments by GO term enrichment analysis (Roff-Bentzen, P < 0.0001), showcasing the stabilization of energetically costly translational machinery, mobilization of energy stores, and downregulation of the ubiquitin/proteasomal degradation machinery. Exposure to hypoxia for 24 h resulted in an increase in all of the full-length hemocyanin subunits (HcS, HcL1, HcL2, HcL3, and HcB). The addition of CO2 to hypoxia muted the transcriptomic response of all the Hc subunits to low O2, except for the β-type subunit.

Low oxygen (hypoxia) and elevated CO2 (hy- percapnia, are characteristic of estuarine environments. Al- though hypoxia and hypercapnic hypoxia decrease the re- sistance of shrimp to bacterial pathogens, their direct effects on the immune system are unknown. Here we present evi- dence demonstrating in the penaeid shrimp Litopenaeus vannamei that both hypoxia and hypercapnic hypoxia affect the localization of bacteria, their conversion from culturable to non-culturable status (bacteriostasis), and their elimina- tion from hemolymph and selected tissues. Shrimp were injected with a sublethal dose of a pathogenic strain of Vibrio campbellii expressing green fluorescent protein and resistance to kanamycin. Real-time polymerase chain reac- tion was used to determine the number of intact V. camp- bellii in hemolymph, gills, hepatopancreas, heart, and lym- phoid organ. Selective plating was used to quantify the injected bacteria that remained culturable. We found that both hypercapnic hypoxia and hypoxia increased the per- centage of culturable bacteria recovered from the hemo- lymph and tissues, suggesting an overall decrease in bacte- riostatic activity. Hypoxia and hypercapnic hypoxia generally increased the distribution of intact V. campbellii to the hepatopancreas and the gills, which are major targets for the pathogenic effects of Vibrio spp., without affecting the number of intact bacteria in the lymphoid organ, a main site of bacterial accumulation and bacteriostatic activity.

Although numerous mechanisms of immune defense have been described in crustaceans, the tissue distribution and fate of live bacteria introduced into the host remain unclear. In the present study, Litopenaeus vannamei were injected with a sub-lethal dose of kanamycin-resistant Vibrio campbellii expressing green fluorescent protein. Accumulation of intact bacteria was quantified by real-time PCR, while bacteriostasis was quantified as the percentage of intact bacteria that could not be recovered by selective plating. Over the 240 min examined, the lymphoid organ contained the greatest number of intact V. campbellii per gram tissue as well as the lowest percentage of culturable V. campbellii compared to other tissues, including the hemolymph. In contrast, the gills and hepatopancreas accumulated intact bacteria, but contained a significantly greater percentage of culturable bacteria than the hemolymph after 240 min. These data suggest that the lymphoid organ plays a major role in bacterial uptake and bacteriostasis in penaeid shrimp.

Callinectes sapidus, the Atlantic blue crab, encounters hypoxia, hypercapnia (elevated CO(2)), and bacterial pathogens in its natural environment. We tested the hypothesis that acute exposure to hypercapnic hypoxia (HH) alters the crab's ability to clear a pathogenic bacterium, Vibrio campbellii 90-69B3, from the hemolymph. Adult male crabs were held in normoxia (well-aerated seawater) or HH (seawater with PO(2) = 4 kPa; PCO(2) = 1.8 kPa; and pH = 6.7-7.1) and were injected with 2.5 x 10(4) Vibrio g(-1) body weight. The animals were held in normoxia or in HH for 45, 75, or 210-240 min before being injected with Vibrio, and were maintained in their respective treatment conditions for the 120-min duration of the experiment. Vibrio colony-forming units (CFU) ml(-1) hemolymph were quantified before injection, and at 10, 20, and 40 min afterward. Total hemocytes (THC) ml(-1) of hemolymph were counted 24 h before (-24 h), and at 10 and 120 min after injection. Sham injections of saline produced no change in the bacterial or hemocyte counts in any treatment group. Among the groups that received bacterial injections, Vibrio was almost completely cleared within 1 h, but at 10-min postinjection, Vibrio CFU ml(-1) hemolymph was significantly higher in animals held in HH for 75 and 210-240 min than in those held in normoxia. Within 10 min after crabs were injected with bacteria, THC ml(-1) significantly decreased in control and HH45 treatments, but not in the HH75 and HH210-240 treatments. By 120 min after injection of bacteria, hemocyte counts decreased in all but the HH45 group. These data demonstrate that HH significantly impairs the ability of blue crabs to clear Vibrio from the hemolymph. These results also suggest that HH alters the normal role of circulating hemocytes in the removal of an invading pathogen.

A yeast culture feed supplement (Diamond V XP Yeast Culture®, Diamond V Mills, Cedar Rapids, Iowa [IA]) was assessed for its impact on disease resistance in the Pacific white shrimp, Litopenaeus vannamei. Animals were fed a standard shrimp pellet diet supplemented with 0% (control with 1% grain carrier), 0.5% (with 0.5% carrier), or 1.0% XP daily for 4 weeks. To assess resistance to bacterial disease, at 1-week intervals 21 shrimp (0.5–2.5 g) from each test diet were injected intramuscularly with an LD50 dose (2.0×105/g body weight) of a gram-negative shrimp pathogen, Vibrio sp. 90-69B3. Survival was monitored every 4 h for 48-h post injection. Each week, three independent bacterial challenges were performed for each diet and the results expressed as the mean percent survival±standard error (S.E.). A two-way analysis of variance (ANOVA) showed a significant effect of diet (p=0.003, df=31), but not duration of feeding, on survival. A one-way ANOVA showed no differences among the treatment groups after 1 or 2 weeks. After 3 weeks, the mean survival of 1% XP-fed shrimp (74.2±1.4%) was significantly higher than that of controls (42.9±5.5%), while mean survival of shrimp fed 0.5% XP (54.8±11.9%) was not significantly different from controls. After 4 weeks, mean survival of 1.0% XP-fed shrimp (63.4±8.8%) remained higher than that of controls but the difference was not significant (p=0.07). An insufficient number of animals were available from the 0.5% XP-fed group to perform bacterial challenges at this timepoint. Mean survival of control shrimp declined significantly over the 4 weeks of study (slope of linear regression ≠0, p=0.005, df=11), but no decline was observed in animals fed the 0.5% or 1.0% XP diets. After 4 weeks L. vannamei fed standard shrimp pellets, 0% XP control, or 1.0% XP diets showed no significant differences in weight, suggesting that the changes in disease resistance did not correlate with changes in growth rate among the treatment groups. These results indicate that dietary administration of Diamond V XP Yeast Culture® can protect shrimp against a decline in resistance to bacterial disease.

Acute hypoxia can cause massive fish and shellfish mortality. Less clear is the role that chronic sublethal hypoxia might play in aquatic animal health. This study tested whether production of reactive oxygen species (ROS) and bactericidal activity of fish phagocytic cells are suppressed under the conditions of decreased oxygen and pH and increased carbon dioxide which occur in the blood and tissue of animals exposed to sublethal hypoxia. Anterior head kidney (AHK) cells of the mummichog, Fundulus heteroclitus , were exposed in parallel to normoxic (pO 2 =45 torr, pCO 2 =3·8 torr, pH=7·6) or hypoxic (pO 2 =15 torr, pCO 2 =8·0 torr, pH=7·0) conditions and stimulated with a yeast cell wall extract, zymosan, or live Vibrio parahaemolyticus . Hypercapnic hypoxia suppressed zymosan-stimulated ROS production by 76·0% as measured in the chemiluminescence assay and by 58·5% in the nitroblue tetrazolium (NBT) assay. The low O 2 , high CO 2 and low pH conditions also suppressed superoxide production by 75·0 and 47·3% as measured by the NBT assay at two different challenge ratios of cells:bacteria (1:1 and 1:10, respectively). In addition to its effects on ROS production, hypercapnic hypoxia also reduced bactericidal activity by 23·6 and 72·5% at the 1:1 and 1:10 challenge ratios, respectively. Low oxygen levels alone (pO 2 =15 torr, pCO 2 =0·76 torr, pH=7·6) did not significantly compromise the killing activity of cells challenged with equal numbers of V. parahaemolyticus . At the higher 1:10 AHK:bacteria challenge ratio, low oxygen caused a small (26·3%) but significant suppression of bactericidal activity as compared to aerial conditions (pO 2 =155 torr, pCO 2 =0·76 torr, pH=7·6). This study demonstrates that while hypoxia alone has detrimental effects on immune function, suppression of phagocytic cell activity is compounded by naturally occurring conditions of hypercapnia and low pH, creating conditions that might be exploited by opportunistic pathogens. These results indicate that the adverse health effects of chronic hypercapnic hypoxia might greatly exceed the effects of low oxygen alone.

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 species frequently encounter areas of simultaneously low dissolved O2(hypoxia) and high CO2(hypercapnia). Organisms exposed to hypoxia experience a metabolic depression that serves to decrease ATP utilization and O2demand during stress. This downregulation is typically facilitated by a reduction in protein synthesis, a process that can be responsible for up to 60% of basal metabolism. The added effects of hypercapnia, however, are unclear. Certain decapods also exhibit a metabolic depression in response to bacterial challenges, leading us to hypothesize that protein synthesis may also be reduced during infection. In the present study, we examined the effects of hypoxia (H), hypercapnic hypoxia (HH), and bacterial infection ( Vibrio campbellii) on tissue-specific (muscle and hepatopancreas) fractional protein synthesis rates ( ks) in Litopenaeus vannamei. We observed a significant decrease in ksin muscle after 24 h exposure to both H and HH, and in hepatopancreas after 24 h exposure to HH. Thus ksis responsive to changes in O2, and the combined effect of hypercapnic hypoxia on ksis more severe than hypoxia alone. These reductions in ksappear to be driven by changes in RNA translational efficiency ( kRNA), and not RNA capacity ( Cs). Bacterial infection, however, had no significant effect on ksin either tissue. These results suggest that crustaceans reduce metabolic demand during environmental hypoxia by reducing global protein synthesis, and that this effect is magnified when hypercapnia is concomitantly present. Conversely, an immune-mediated metabolic depression is not associated with a decrease in overall protein production.

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.

In the blue crab Callinectes sapidus, injection with the bacterial pathogen Vibrio campbellii causes a de- crease in oxygen consumption. Histological and physiolog- ical evidence suggests that the physical obstruction of he- molymph flow through the gill vasculature, caused by aggregations of bacteria and hemocytes, underlies the de- crease in aerobic function associated with bacterial infec- tion. We sought to elucidate the bacterial properties suffi- cient to induce a decrease in circulating hemocytes (hemocytopenia) as an indicator for the initiation of hemo- cyte aggregation and subsequent impairment of respiration. Lipopolysaccharide (LPS), the primary component of the gram-negative bacterial cell wall, is known to interact with crustacean hemocytes. Purified LPS was covalently bound to the surfaces of polystyrene beads resembling bacteria in size. Injection of these "LPS beads" caused a decrease in circulating hemocytes comparable to that seen with V. campbellii injection, while beads alone failed to do so. These data suggest that in general, gram-negative bacteria could stimulate hemocytopenia. To test this hypothesis, crabs were injected with different bacteria—seven gram- negative and one gram-positive species—and their effects on circulating hemocytes were assessed. With one excep- tion, all gram-negative strains caused decreases in circulat- ing hemocytes, suggesting an important role for LPS in the induction of this response. However, LPS is not necessary to provoke the immune response given that Bacillus coral, a gram-positive species that lacks LPS, caused a decrease in circulating hemocytes. These results suggest that a wide range of bacteria could impair metabolism in C. sapidus.

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.

Heavy metals, such as copper, zinc and cadmium, represent some of the most common and serious pollutants in coastal estuaries. In the present study, we used a combination of linear and artificial neural network (ANN) modelling to detect and explore interactions among low-dose mixtures of these heavy metals and their impacts on fundamental physiological processes in tissues of the Eastern oyster, Crassostrea virginica. Animals were exposed to Cd (0.001-0.400 microM), Zn (0.001-3.059 microM) or Cu (0.002-0.787 microM), either alone or in combination for 1 to 27 days. We measured indicators of acid-base balance (hemolymph pH and total CO(2)), gas exchange (Po(2)), immunocompetence (total hemocyte counts, numbers of invasive bacteria), antioxidant status (glutathione, GSH), oxidative damage (lipid peroxidation; LPx), and metal accumulation in the gill and the hepatopancreas. Linear analysis showed that oxidative membrane damage from tissue accumulation of environmental metals was correlated with impaired acid-base balance in oysters. ANN analysis revealed interactions of metals with hemolymph acid-base chemistry in predicting oxidative damage that were not evident from linear analyses. These results highlight the usefulness of machine learning approaches, such as ANNs, for improving our ability to recognize and understand the effects of sub-acute exposure to contaminant mixtures.

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.

Changes in vascular pressures, blood respiratory properties and blood gas transport induced by swimming were investigated in the leopard shark Triakis semifasciata (Girard). In resting sharks, the mean ventral and dorsal aortic pressures (systolic/diastolic) were 6.8/5.6 kPa and 4.5/3.9 kPa, respectively, and only the former were increased significantly during swimming. Swimming also caused a significant decline in venous , (1.6 to 0.9kPa), O2 content (0.9 to 0.4 mmol l−1) and percentage O2 saturation (, 39 to 18%) but the arterial variables were not affected. A significant decline in venous pH and an increase in venous also occurred during swimming but lactate concentration did not increase during or after swimming. An in vivo dissociation curve compiled from blood and data for sharks in the resting, swimming and post-swimming recovery phases shows a mean P50 of 2.04 kPa, as determined by Hill transformation. The pH-bicarbonate plot for this fish shows a weak blood buffer capacity of 9.3 mmol I−1 pH unit−1 and during swimming the average blood pH and bicarbonate concentration follow the buffer line which was not compensated in recovery. Neither oxygenated nor deoxygenated blood pH values were affected by CO2 equilibration, suggesting the absence of a Haldane effect. Thus, at the expense of respiratory acidosis, Triakis can aerobically sustain long (up to 60 min) and moderately intense (0.45 Ls−1, where L is body length) periods of swimming by increasing cardiac output and tapping its venous reserve.

Sodium ion uptake in the isolated perfused posterior gill of the blue crab, Callinectes sapidus Rathbun, acclimated to 5‰ salinity and 20°C was measured using 22Na as a tracer. Unidirectional influxes across individual gills occurred against large sodium concentration differences (internal perfusate=273 mmol l−1; external perfusate=l–80 mmol l−1) and depended strongly on the concentration of sodium ions present in the external perfusate. The maximum rate of influx was 3.24 μmol g−1 min−1 and the Km for this process was 23.0 mmol l−1. Ouabain added to the internal perfusate reduced the influx to an average of 51.3 % of the control values and had its maximal effect at 1 mmol l−1. Ouabain (1 mmol l−1) added to the external perfusate had no significant effect on sodium influx. Amiloride (0.1 mmol l−1) added to the external perfusate reduced the influx to an average of 38.9 % of the control values. During all treatments, perfusion pressures showed no treatment-dependent effects, indicating that the observed changes in fluxes were due to specific effects of the treatment on the epithelium and not due to some indirect effect on perfusion pattern within the gill. Treatment of the gill with the metabolic poisons cyanide and iodoacetate resulted in a rapid decline of sodium influx to 20–30% of the control values. This was correlated with a large reduction in gill ATP content to 13% of the control. The overall results of this study are consistent with the general picture of epithelial sodium uptake across gills, supporting the existence of a ouabain-sensitive Na+ pump on the basolateral membrane and an amiloride-sensitive Na+/H+ exchanger on the apical membrane.

Open circulatory systems have been portrayed aspoorly designed systems with poor performance characteristics, lacking in adequate tissue perfusion or fine control mechanisms. Recent studies cast doubt on these assumptions. Open circulatory systems of at least the higher Malacostraca have elaborate capillary beds in many tissues. Cardiac outputs are generally higher than those of the closed systems of poikilothermic vertebrates of equivalent size and activity potential. Although arterial pressures and flows are often lower than those characteristic of poikilothermic vertebrates, the crustacean arterial system is adapted to deliver equivalent flows under these conditions. The control systems of the crustacean neurogenic heart appear capable offine graded control over cardiac output. In addition, although crustaceans lack the arteriolar smooth muscle upon which much of theperipheral circulatory control of vertebrate closed systems depends, cardioarterial valvular mechanisms under neural and neurohormonal con...

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.

Although numerous mechanisms of immune defense have been described in crustaceans, the tissue distribution and fate of live bacteria introduced into the host remain unclear. In the present study, Litopenaeus vannamei were injected with a sub-lethal dose of kanamycin-resistant Vibrio campbellii expressing green fluorescent protein. Accumulation of intact bacteria was quantified by real-time PCR, while bacteriostasis was quantified as the percentage of intact bacteria that could not be recovered by selective plating. Over the 240 min examined, the lymphoid organ contained the greatest number of intact V. campbellii per gram tissue as well as the lowest percentage of culturable V. campbellii compared to other tissues, including the hemolymph. In contrast, the gills and hepatopancreas accumulated intact bacteria, but contained a significantly greater percentage of culturable bacteria than the hemolymph after 240 min. These data suggest that the lymphoid organ plays a major role in bacterial uptake and bacteriostasis in penaeid shrimp.

Upon exposure to air (emersion), the purple sea urchin Strongylocentrotus purpuratus releases an "emersion fluid" from its esophagus. Release of this fluid causes air to appear within the test (or calcareous theca), most likely inside the intestine. The air space is large, occupying 33.5% of the volume of the intrathecal space. The intestine containing air forms a facultative lung and contributes to the oxygenation of the perivisceral coelomic fluid (PCF) during emersion. During emersion, the mean partial pressure of oxygen (PO(2)) of the PCF declined from 56 to 24 torr (1 torr = 0.1333 kPa) after 2 h, remained relatively unchanged after 4 h, and rose to 39 torr after 8 h. The partial pressure of carbon dioxide (PCO(2)) rose from 2.6 to 3.8 torr after 2 h, remained unchanged after 4 h, and declined to 2.7 after 8 h. Due to the elevation of PCO(2) PCF pH declined from 7.41 to 7.17. PCF osmotic concentration, calcium ion concentration, chloride ion concentration, ammonium ion concentration, and protein concentration were unchanged by air exposure. Lactate levels in the PCF were undetectable. S. purpuratus was an osmoconformer and a chloride ion conformer at salinities down to 20.9 ppt. Below this salinity, the sea urchins died. The respiratory acidosis resulting from air exposure was uncompensated, supporting the hypothesis that compensation for a respiratory acidosis induced by air exposure does not occur in organisms that are unable to regulate ions in a dilute environment. We suggest that the facultative lung ensures a minimal PO(2) in the PCF, which may be especially important when the intrathecal space is full of ripe gonads, allowing the gonads to be more reliant on aerobic metabolism.