Karen G. Burnett

Effects of Hypercapnic Hypoxia on the Clearance of Vibrio campbellii in the Atlantic Blue Crab, Callinectes sapidus Rathbun

Callinectes sapidus, the Atlantic blue crab, encounters hypoxia, hypercapnia (elevated CO2), 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 Po2 = 4 kPa; Pco2 = 1.8 kPa; and pH = 6.7–7.1) and were injected with 2.5 × 104 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.

Effects of hypoxia and hypercapnic hypoxia on the localization and the elimination of Vibrio campbellii in Litopenaeus vannamei, the Pacific white shrimp.

Low oxygen (hypoxia) and elevated CO2 (hypercapnia, are characteristic of estuarine environments. Although hypoxia and hypercapnic hypoxia decrease the resistance of shrimp to bacterial pathogens, their direct effects on the immune system are unknown. Here we present evidence 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 elimination 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 reaction was used to determine the number of intact V. campbellii in hemolymph, gills, hepatopancreas, heart, and lymphoid organ. Selective plating was used to quantify the injected bacteria that remained culturable. We found that both hypercapnic hypoxia and hypoxia increased the percentage of culturable bacteria recovered from the hemolymph and tissues, suggesting an overall decrease in bacteriostatic 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.

Immune Defense Reduces Respiratory Fitness in Callinectes sapidus, the Atlantic Blue Crab

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 immune response. In the present study, injection of the pathogenic bacterium Vibrio campbellii into Callinectes sapidus, the Atlantic blue crab, caused a dramatic decline in oxygen uptake from 4.53 to 2.56 μmol g−1 h−1. This decline in oxygen uptake is associated with a large decrease in postbranchial Po2, from 16.2 (±0.46 SEM, n=7) to 13.1 kPa (±0.77 SEM, n=9), while prebranchial Po2 remains unchanged. In addition, injection of Vibrio results in the disappearance of a pH change across the gills, an indication of reduced CO2 excretion. The hemolymph hydrostatic pressure 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 formation. A healthy, active blue crab can eliminate most invading 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 environments that are hypoxic.

Energy metabolism and metabolic depression during exercise in Callinectes sapidus, the Atlantic blue crab: effects of the bacterial pathogen Vibrio campbellii.

SUMMARY Callinectes 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.

Effects of hypercapnic hypoxia on inactivation and elimination of Vibrio campbellii in the Eastern oyster, Crassostrea virginica.

ABSTRACT The Eastern oyster, Crassostrea virginica, inhabits shallow coastal waters that frequently experience periods of low dissolved oxygen (hypoxia) and elevated CO2 (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 O2 pressure [PO2] = 20.7 kPa, CO2 < 0.06 kPa, pH 7.8 to 8.0) or HH (PO2 = 4 kPa, CO2 = 1.8 kPa, pH 6.5 to 6.8) seawater at 25°C for 4 h before being injected in the adductor muscle with 105 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.

Impact of Exposure to Bacteria on Metabolism in the Penaeid Shrimp Litopenaeus vannamei

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 μmol 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 μmol 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 μmol 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.

Clearance of Vibrio campbellii injected into the hemolymph of Callinectes sapidus, the Atlantic blue crab: the effects of prior exposure to bacteria and environmental hypoxia.

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)<0.06 kPa; pH 7.8-8.0) or hypercapnic hypoxic (HH; P O2=4 kPa; CO(2)=1.8 kPa; pH 6.9-7.2) seawater received an injection (pre-challenge dose) of 1 x 10(5)Vibrio campbellii g(-1) crab. Control animals were injected with an equivalent dose of HEPES-buffered saline (1 microl g(-1) crab). At 2h or 24h after the pre-challenge injection, both Vibrio and saline-pre-challenged animals were injected with a dose of live V. campbellii (1 x 10(5)g(-1) crab). This second injection will be referred to as a second injection or challenge injection. Degradation in or physical removal of intact bacteria from hemolymph was quantified using real-time PCR; bacteriostasis was quantified as the percentage of intact bacteria that could not be recovered by selective plating. We demonstrated that bacteriostasis occurs in the hemolymph of blue crabs. Furthermore, blue crabs that received a challenge injection 2h after a pre-challenge dose of V. campbellii cleared culturable bacteria from their hemolymph more rapidly when compared to animals that received a pre-challenge dose of saline. This enhanced clearance of culturable bacteria was associated with an increase in antibacterial activity in the cell-free hemolymph. However, the enhanced clearance of culturable bacteria disappeared when the time interval between the pre-challenge and challenge dose was extended to 24h and when crabs were held in HH seawater throughout the experiment. Neither the time interval between the pre-challenge and the challenge dose nor exposure to HH altered the pattern of intact bacterial clearance in blue crabs. These results demonstrate that prior exposure to bacteria does not increase the susceptibility of C. sapidus to a second, sublethal dose of V. campbellii. In fact, a recent exposure to V. campbellii enhances the ability of blue crabs to render bacteria non-culturable and the immune mechanisms/effectors responsible for this are short lived and appear to be sensitive to low dissolved oxygen and high carbon dioxide concentrations in the environment.

Differential localization and bacteriostasis of Vibrio campbellii among tissues of the Eastern oyster, Crassostrea virginica.

In bivalve mollusks the roles of individual tissues in antimicrobial defense remain unclear. In this study, Crassostrea virginica were injected in the adductor muscle with 10(5) live Vibrio campbellii. Major tissues were dissected at 10, 30, 60 or 120 min postinjection (PI); in each tissue undegraded (intact) bacteria were quantified by real-time PCR and culturable bacteria were enumerated by selective plating. At 10 min PI, accumulation of bacteria varied among tissues from approximately 2.4 x 10(3) (labial palps, digestive gland) to 24.2 x 10(3) (gonads) intact Vibrio g(-1). Neither distribution nor accumulation of intact bacteria changed with time except in the hemolymph. In most tissues, more than 80% of intact bacteria were culturable at 10 min PI and culturability decreased with time. In contrast, only 19% of intact bacteria in gonadal tissue could be cultured at 10 min PI, pointing to a major role for the gonadal tissues in antibacterial defense of molluscs.

Time-course analysis of peroxinectin mRNA in the shrimp Litopenaeus vannamei after challenge with Vibrio campbellii.

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.

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.