Immune Defense in Hypoxic Waters: Impacts of CO2 Acidification.

AbstractPeriodic episodes of low oxygen (hypoxia) and elevated CO2 (hypercapnia) accompanied by low pH occur naturally in estuarine environments. Under the influence of climate change, the geographic range and intensity of hypoxia and hypercapnic hypoxia are predicted to increase, potentially jeopardizing the survival of economically and ecologically important organisms that use estuaries as habitat and nursery grounds. In this review we synthesize data from published studies that evaluate the impact of hypoxia and hypercapnic hypoxia on the ability of crustaceans and bivalve molluscs to defend themselves against potential microbial pathogens. Available data indicate that hypoxia generally has suppressive effects on host immunity against bacterial pathogens as measured by in vitro and in vivo assays. Few studies have documented the effects of hypercapnic hypoxia on crustaceans or bivalve immune defense, with a range of outcomes suggesting that added CO2 might have additive, negative, or no interactions with the effects of hypoxia alone. This synthesis points to the need for more partial pressure of O2 × low pH factorial design experiments and recommends the development of new host∶pathogen challenge models incorporating natural transmission of a wide range of viruses, bacteria, and parasites, along with novel in vivo tracking systems that better quantify how pathogens interact with their hosts in real time under laboratory and field conditions.

Integrative Approaches to Understanding Organismal Responses to Aquatic Deoxygenation.

AbstractOxygen 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 outlining 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.

Acidification in the U.S. Southeast: Causes, Potential Consequences and the Role of the Southeast Ocean and Coastal Acidification Network.

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.

Physiological impacts of time in holding ponds, biomedical bleeding, and recovery on the Atlantic horseshoe crab, Limulus polyphemus.

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.

Effects of Hypoxia and Hypercapnic Hypoxia on Oxygen Transport and Acid-Base Status in the Atlantic Blue Crab, Callinectes sapidus, During Exercise.

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.

Uncovering Hemocyanin Subunit Heterogeneity in Penaeid Shrimp using RNA-Seq.

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 ß-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 ß-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.

High CO2 alters the hypoxia response of the Pacific whiteleg shrimp (Litopenaeus vannamei) transcriptome including known and novel hemocyanin isoforms.

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 ß-type Hc subunit (denoted HcB), making this the first report of a ß-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 ß-type subunit.

Respiratory Properties of Hemocyanin From Wild and Aquacultured Penaeid Shrimp and the Effects of Chronic Exposure to Hypoxia.

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.

Respiratory and Metabolic Impacts of Crustacean Immunity: Are there Implications for the Insects?

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.

Effects of salinity on the accumulation of hemocyte aggregates and bacteria in the gills of Callinectes sapidus, the Atlantic blue crab, injected with Vibrio campbellii.

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.

Recovery from hypoxia and hypercapnic hypoxia: impacts on the transcription of key antioxidants in the shrimp Litopenaeus vannamei.

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 4h or 24h followed by recovery in air-saturated water (normoxia) for 0, 1, 6 or 24h, 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 4h 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 (24h) 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.

Effect of hypercapnic hypoxia and bacterial infection (Vibrio campbellii) on protein synthesis rates in the Pacific whiteleg shrimp, Litopenaeus vannamei.

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 O2 demand 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 ks in muscle after 24 h exposure to both H and HH, and in hepatopancreas after 24 h exposure to HH. Thus ks is responsive to changes in O2, and the combined effect of hypercapnic hypoxia on ks is more severe than hypoxia alone. These reductions in ks appear to be driven by changes in RNA translational efficiency (kRNA), and not RNA capacity (Cs). Bacterial infection, however, had no significant effect on ks in 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.

Transcriptomic responses of juvenile Pacific whiteleg shrimp, Litopenaeus vannamei, to hypoxia and hypercapnic hypoxia.

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 CO2 may be important to understanding the survival of this and other estuarine species.

Locomotory fatigue during moderate and severe hypoxia and hypercapnia in the Atlantic blue crab, Callinectes sapidus.

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.

Gene transcripts encoding hypoxia-inducible factor (HIF) exhibit tissue- and muscle fiber type-dependent responses to hypoxia and hypercapnic hypoxia in the Atlantic blue crab, Callinectes sapidus.

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-ß 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-ß 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-ß 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-ß 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-ß transcriptional dynamics occurring in both a tissue- and muscle fiber type-dependent manner.

Properties of bacteria that trigger hemocytopenia in the Atlantic blue crab, Callinectes sapidus.

In the blue crab Callinectes sapidus, injection with the bacterial pathogen Vibrio campbellii causes a decrease in oxygen consumption. Histological and physiological evidence suggests that the physical obstruction of hemolymph flow through the gill vasculature, caused by aggregations of bacteria and hemocytes, underlies the decrease in aerobic function associated with bacterial infection. We sought to elucidate the bacterial properties sufficient to induce a decrease in circulating hemocytes (hemocytopenia) as an indicator for the initiation of hemocyte 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 exception, all gram-negative strains caused decreases in circulating 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.

Modelling interactions of acid-base balance and respiratory status in the toxicity of metal mixtures in the American oyster Crassostrea virginica.

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.