Louis E. Burnett

Respiratory responses of the salt marsh animals, Fundulus heteroclitus, Leiostomus xanthurus, and Palaemonetes pugio to environmental hypoxia and hypercapnia and to the organophosphate pesticide, azinphosmethyl☆

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; Dunns 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.

The Crustacean Open Circulatory System: A Reexamination

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 control appear to be capable ofselective distribution of this output between the several separate arterial systems. Thus, although open circulatory systems differ greatly in anatomyfrom closed blood systems, they are, nonetheless, functionally equivalent.

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.

Gas Exchange, Hemolymph Acid-Base Status, and the Role of Branchial Water Stores during Air Exposure in Three Littoral Crab Species

O₂ uptake and hemolymph acid-base status, together with branchial water volume, CO₂ content, and titratable alkalinity, were measured in three species of intertidal crabs. In Pachygrapsus crassipes, a grapsid crab that actively moves between air and water, O₂ uptake increased on emersion. In Eurytium albidigitum, a mud-burrowing xanthid crab that is air exposed by tidal action, O₂ uptake declines dramatically on emersion. There is no significant lactate production by either crab following emersion. An emersion-induced respiratory acidosis was fully compensated in P. crassipes and another grapsid, Hemigrapsus nudus, but uncompensated in E. albidigitum. Branchial water volume 10 min after emersion was 0.013 ml/g crab weight in P. crassipes and 0.072 ml/g in E. albidigitum. The CO₂ content of branchial water in P. crassipes increased rapidly during air exposure and was accompanied by an increase in titratable alkalinity (TA). The CO₂ content of branchial water in E. albidigitum remained constant for at least 4 h and increased slightly after 8 h. TA remained unchanged for up to 8 h. We suggest that the ability of the crabs to compensate for the respiratory acidosis and to increase branchial water TA is correlated with osmoregulation in P. crassipes and H. nudus. On the other hand, E. albidigitum is an osmoconformer and neither compensates for the respiratory acidosis nor changes its TA during air exposure. Possible adaptive advantages of the two different strategies may be related to the relatively short duration of emersion and active habits of P. crassipes and the longer periods of air exposure and inactivity of E. albidigitum.

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.

Facilitation of CO2 excretion by carbonic anhydrase located on the surface of the basal membrane of crab gill epithelium

An isolated perfused crab gill preparation was used to test the hypotheses that crab gill carbonic anhydrase (CA) catalyzes the efflux of CO2 from the hemolymph, which lacks the enzyme, to the ambient medium and that the CA is localized on the luminal surface of the basal membrane. It was found that the efflux of CO2 from the internal perfusate was sensitive to the flow rate of the internal perfusate through the gill (and thus the residence time within the gill). The sensitivity of the CO2 efflux to residence time was nearly abolished upon treatment of the gill with an impermeable dextran-bound CA inhibitor. It is concluded that CA present on the luminal surface of the gill epithelium facilitates CO2 excretion by catalyzing the dehydration of the large hemolymph bicarbonate pool to the more diffusible molecular CO. The action of the enzyme is important in maintaining a CO2 gradient between hemolymph and water in a situation where hemolymph PCO2 is normally low, water PCO2 is variable, and the gills themselves are a source of metabolic CO2.

Respiratory and Acid-Base Physiology of the Purple Sea Urchin, Strongylocentrotus purpuratus, During Air Exposure: Presence and Function of a Facultative Lung

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 (Po2) 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 (Pco2) 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 Pco2 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 Po2 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.

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.