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Influence of hypoxia on bacteremia in the dungeness crab, Cancer magister.

Introduction

Marine crustaceans encounter diurnal, tidal, and seasonal fluctuations in dissolved oxygen in coastal areas. The Dungeness crab. Cancer magister, experiences low oxygen levels in estuaries and muddy bays, and encounters episodic hypoxia offshore due to upwelling events along the Pacific west coast (Grantham et at, 2004; Chan et al., 2008; Turner et al., 2008). In addition, low levels of bacteremia are common in marine decapod crustaceans, with more than 70% of populations of "unstressed" animals infected with bacteria (Tublah et al., 1975; Faghri et al., 1984; Welsh and Sizemore. 1985). While many studies have investigated the physiological effects of hypoxia on marine crustaceans, few have considered the effects of persistent bacteria in the hemolymph and how these bacteria may be affected by hypoxic conditions.

In well-aerated water, crustaceans are able to rapidly remove bacteria from the hemolymph. After bacterial injection, the number of circulating hemocytes declines within minutes and aggregates of bacteria and hemocytes accumulate at the gill (Martin et al., 2000; Burnett et al., 2006). In the Atlantic blue crab, Callinectes sapidus, the majority of the culturahle bacterium Vibrio campbelli is removed from the hemolymph within 10 min of injection, and the bacterium becomes undetectable after 40 min in well-aerated water (Holman et al, 2004).

Low oxygen reduces immune response in marine crustaceans (Mikulski et al., 2000; Burgents et al., 2005; Paschke et al., 2010), thereby limiting the rate at which bacteria are eliminated from the hemolymph (Holman et al., 2004; Burgents et al., 2005). The mechanism of hypoxic immune suppression varies among marine invertebrates: reduced phagocytic activity of hemocytes (Direkbusarakom and Danayadol, 1998), decreased total circulating hemocytes (Le Moullac and Haffner, 2000; Cheng et al., 2002), limited production of reactive oxygen species and reduced phenoloxidase activity (Tanner et al., 2006), and impaired respiratory burst activity (Boyd and Burnett, 1999). Ultimately, low environmental oxygen can increase mortality, as reported in the shrimp Litopenaeus stylirostris exposed to Vibrio alginolyticus (Le Moullac et al., 1998).

Limited environmental oxygen may be particularly important when bacteria are present in the hemolymph, given the critical role the crustacean gill plays in immune defense (Smith and Ratcliffe, 1981; White et al., 1985; Martin et al., 2000). The gill accumulates aggregates of bacteria and hemocytes, and recent reports suggest that the localization of pathogens at the gill limits respiratory function (Burnett et al., 2006). Bacterial injection is accompanied by a large decrease in oxygen uptake, a reduction in post-branchial partial pressures of oxygen, and an increase in the change in hydrostatic pressure across the gills in C, sapidus (Burnett et al., 2006). Bacterial injection has also been shown to reduce aerobic metabolism and elevate anaerobic metabolism for up to 24 h in the penaeid shrimp Litopenaeus vannamei (Scholnick et al., 2006). Thus, when crustaceans experience environmental hypoxia while bacteria are present in the hemolymph, there could be the potential for an associated increase in susceptibility to bacteremia and a reduction in respiratory function.

In the current investigation, we examined the influence of decreased levels of environmental oxygen on indigenous bacteria in the hemolymph of Cancer magister. In view of the impact of low oxygen on the immune response, we hypothesized that environmental hypoxia will impair the ability of crabs to suppress persistent bacteria, thereby elevating bacteremia and reducing respiratory ability. Considering the increasing occurrence of low oxygen conditions in coastal waters (Diaz and Rosenberg, 1995), the current study may better elucidate the possible link between low environmental oxygen and increased susceptibility of marine organisms to infectious disease.

Materials and Methods

Animal collection and care

Male Dungeness crabs, Cancer magister (mean mass = 287.5 g [+ or -] 18.3 SEM), were collected off of the central Oregon coast using 81-cm-diameter crab rings. The crabs were transported to Pacific University where they were held in UV-sterilized recirculating seawater at 32 ppt and 9 [+ or -] 1 [degrees]C. Crabs were fed frozen shrimp every 3 days, and food was withheld for at least 24 h before all experiments.

Hemolymph sampling

At least one day prior to experimentation, a sterile dremel bit was used to drill two 1-mm holes into the dorsal carapace. One hole was drilled directly over the ventricle for injection, and the other was drilled over the pericardium adjacent to the heart for hemolymph sampling and oxygen measurements. A latex patch (0.76-mm thick) pre-soaked in topical antiseptic (10% povidone iodine) was sealed over the holes with cyanoacrylate glue. The procedure usually lasted less than 2 min and there was no hemolymph loss, indicating that the epidermis was not disrupted. The patch served as a diaphragm through which saline or bacteria could be injected or hemolymph sampled without bacterial contamination, hemolymph loss, or the large changes in hemocyte number that can result from repeated sampling through the uncalcified membrane of a leg joint (D. Schol-nick, pers. obs.). By lowering water levels to just below the latex patch, we were able to inject and sample without removing the crabs from the individual flow-through containers that were used for hypoxia experiments. Thus, the latex patch eliminated handling, limited disturbance, and prevented air exposure during hemolymph sampling.

Influence of hypoxia on innate bacteria

To determine the effect of hypoxia on bacteremia, we placed animals into individual flow-through containers (11.5 1 at 450 ml [min.sup.-1]) and exposed them to either air-saturation (normoxia > 20 kPa, n = 8) or hypoxia (50% saturation = 10 kPa, n = 8) for 72 h by bubbling a reservoir with air or a mix of air and nitrogen. Effluent water was UV-sterilized and returned to the reservoir before recirculation.

Levels of bacteria in the hemolymph were measured as the total numbers of colony-forming units (CFU) present per milliliter of hemolymph. About 100 [micro]l of hemolymph was drawn into syringes that were partially filled with sterile buffered saline (2.5% NaCl with 10 mmol [l.sup.-1] HEPES) to minimize clotting of the hemolymph. Three aliquots of this solution were suspended in melted marine agar and then poured over plates of 2.5% NaCl trypticase soy agar (TSA). Plates were incubated at 25 [degrees]C for 72 h, and the number of bacterial colonies was counted under a dissecting microscope to determine CFU [ml.sup.-1].

To assess changes in the number of circulating hemocytes, we drew a separate hemolymph sample (< 100 [micro]l) into a syringe containing cold 10% neutral buffered formalin. Cells from three separate aliquots were counted in a hemocytometer (two slides per aliquot) and averaged.

Bacterial challenge

One bacterial colony type was identified by morphological characteristics as the most common and persistent hemolymph bacterium (> 95% of CFU from experiments described above) both from crabs exposed to normoxia and those exposed to hypoxia. More than 20 different colonies were picked and cultured at a variety of temperatures (10 to 25 [degrees]C) from crabs exposed to both normoxic and hypoxic conditions. The Clinical Microbiology Laboratory at the University of Washington Medical Center identified all colonies examined as Psychrobacter cibarius, a gram-negative, non-motile coccobacilli, by using the first 500 base pairs on the 16S ribosomal gene and universal primers (5'-AGAGTTTGATCCTGGCTCAG and 5'-TTACCGCG-GCTGCTGGCA). Isolated P. cibarius was used in all subsequent challenge experiments.

Crabs in individual flow-through containers were exposed to normoxia or hypoxia at 10 [degrees]C for 3 h prior to bacterial challenge and for 80 min post-injection. Crabs were injected with either live P. cibarius mixed in buffered saline or an equal volume of bacteria-free buffered saline (sterile 2.5% NaCl with 10 mmol [1.sup.-1] HEPES adjusted to pH 7.6). The concentration of P. cibarius was determined spectrophotometrically at 540 nm and diluted with buffered saline to obtain an injection dose of 2.5 X [10.sup.-4] CFU [g.sup.-1] crab (adapted from Mikulski et al., 2000). The circulating dose for normoxic and hypoxic crabs approximated 4000 CFU [ml.sip.-1] 10 min post-injection.

Hemolymph P[o.aub.2]

Hemolymph oxygen levels were measured from animals in a small oxygen-controlled recirculating seawater system at 10 [degrees]C. Crabs were placed into a plastic chamber that contained 660 ml of well-aerated (20.9 kPa Po ) sterile seawater. Seawater was pumped at 8.8 1 [min.sup.-1] from a reservoir in which oxygen was controlled by means of bubbling seawater with a mixture of air and nitrogen. Seawater oxygen levels were continuously monitored using a beveled Orion Start dissolved oxygen electrode (Thermo Electron Corp., Beverly, MA). Partial pressures of oxygen in hemolymph were measured continuously using an Ocean Optics 0.5-mm phase fluorescence optic sensor (Dunedin, FL). The probe was inserted through the sterilized latex patch covering a pre-drilled hole above the pericardium, and partial pressures of hemolymph oxygen were recorded every 5 s for at least 30 min once readings were stable.

Statistical analysis

The effect of hypoxia on the number of culturable bacteria, number of hemocytes, or hemolymph partial pressures of oxygen over time was analyzed by two-way repeated-measures analysis of variance (RM ANOVA). If statistical differences between treatments were found, the Holm-Sidak (HS) method was used for post hoc comparisons between hypoxia and anoxia at individual time points. To determine whether hypoxia influenced the amount of bacteria in the hemolymph after injection, a two-way RM ANOVA was performed on CFU values over lime for normoxic and hypoxic exposure. If statistical differences between treatments were found, post hoc comparisons between oxygen treatments at individual time points were performed using the HS method. Separate RM ANOVA analyses were used to compare saline and bacterial injections over time at a given oxygen level. All data are reported as mean [+ or -] standard error unless noted otherwise. Statistical analyses were done using SigmaStat 3.5 (Systat Software Inc., San Jose,

Results

Hypoxic bacteremia

Exposing Cancer magister to 50% air-saturation (10 kPa) significantly increased the levels of pre-existing culturable bacteria in the hemolymph when compared to normoxic crabs (Fig. 1A; RM ANOVA, P < 0.01, n = 8 for each treatment). Bacterial levels were elevated after 24 h and remained above initial levels for 72 h of hypoxia. CFU values increased from less than 30 to over 4000 [ml.sup.-1] hemolymph during 72 h of hypoxia. Hypoxia increased the number of culturable bacteria in hemolymph after 24 h? and it remained significantly elevated for 72 h when compared to that of crabs exposed to air-saturation (HS; P < 0.001). Crabs exposed to air-saturation had low and persistent levels of cultivable pre-existing hemolymph bacteria over the course of the experiment (n = 6),

The total number of circulating hemocytes was unaltered after 48 h of hypoxia and significantly decreased from initial levels only after 72 h (from about 15 to 8 million cells ml l; RM ANOVA P = 0.014; Fig. 1B). Hemocyte number remained constant in crabs exposed to air-saturated water for 72 h and sampled every 24 h.

Bacterial challenge

The ability of Cancer magister to remove injected Psychrobacter from the hemolymph was measured after 3 h of exposure to hypoxia or air-saturation. The number of culturable bacteria in hemolymph was significantly larger in crabs exposed to hypoxia (n = 8) than in those with normoxic exposure (n = 1) after 80 min (HS; P < 0.05; Fig. 2). Elevated CFU values persisted for 80 min after injection of Psychrobacter in both hypoxic and air-saturated crabs compared to saline-injected animals (RM ANOVA, P < 0.01). As many as 2500 CFU [ml.sup.-1] remained in the hemolymph of hypoxic crabs compared to about 1000 for normoxic animals 80-min post-injection. Saline injection had no influence on the number of culturable bacteria in hemolymph for animals exposed to hypoxia (n = 6) or air-saturation (n = 6).

Hemolymph oxygen

We were able to measure real-time changes in hemolymph oxygen tensions after injections with saline or bacteria during normoxia and hypoxia (Fig. 3)

Bacteria-injected crabs had lower oxygen pressures than saline-injected animals in air-saturated and hypoxic water (Fig. 3, P < 0.01 for both treatments). A significant difference was found between controls and bacteria-injected animals after 72 h for crabs exposed to air-saturated water and after 48 h for crabs exposed to hypoxia (Fig. 4). Oxygen pressures decreased more than 30% after 72 h for normoxia and hypoxia. We were able to observe periodic and rapid depressions in oxygen tensions after injection with saline or saline containing Psychrobacter; such depressions are indicative of ventilator pauses (Fig. 3).

Discussion

Sustained reduction of ambient oxygen increased bacteremia in Cancer magister. Culturable hemolymph bacteria increased from about 30 to over 4000 CFU [ml.sup.-1] after 72 h of relatively mild hypoxia (Fig. 1A). Hypoxia-induced bacteremia appears to result from elevations in innate bacteria. Previous studies have reported that lobsters and shrimp contain chronic low levels of hemolymph bacteria (Stewart et al., 1966; Lightner, 1977), whereas the blue crab Calanus sapidus maintains low levels of predominately Vibrio species (12% of the crabs are bacteria-free) that are influenced by temperature and physiologic stress (Davis and Sizemore, 1982; Welsh and Sizemore, 1985).

A gram-negative, psychrotolerant coccobacillus is present in hemolymph of Cancer magister. Psychrobacter cibarius was previously isolated and described from Korean fermented seafood (Jung et al., 2005). Two strains of Psychrobacter, KC 40 and KC 65, designated Psychrobacter fulvigenes sp. nov., have been previously reported from the marine crab Paralithodes camtschatica collected from the Sea of Japan (Romanenko et ml, 2009), and Psychrobacter proteolyticus was isolated from the Antarctic krill Euphausia (Denner et al., 2001). The pathophysiology of Psychrobacter in crustaceans remains unstudied, although the genus has been isolated from a wide range of marine animals, marine waters, and sediments (Maruyama et al., 2000; Romanenko et al., 2002; Yoon et at., 2005a, b), suggesting a ubiquitous presence in marine systems and a potentially important source for what appears to be a persistent bacterium in C magister. It should be noted that the present study was not designed to identify or quantify all bacteria present in the hemolymph. Thus, the increase in culturable bacteria due to hypoxia reported in the current study may in part be explained by the presence of P. cibarius, but other bacteria may also be present in the hemolymph and influenced by hypoxia.

The results presented in the current study support the hypothesis that reduced environmental oxygen tensions can limit the role of hemocytes in preventing elevated bacteremia. Hypoxia can restrict a number of immune responses in crustaceans, including respiratory burst activity (Le Moullac et al., 1998). rate of hemocyte phagocytosis, and prophenoloxidase activity (Cheng and Chen, 2000). In the current study, bacterial levels increased 24 h after initiation of hypoxia, but there was no significant change in total number of circulating hemocytes until 72 h (Fig. 1B). The limited change in hemocyte number suggests hypoxic suppression of hemocyte bactericidal and phagocytic activity. The number of circulating hemocytes in crustaceans has previously been reported to decline rapidly after injection of bacteria or foreign substances in well-aerated water (Martin et al., 1993), whereas exposure to hypercapnic hypoxia (80% reduction in oxygen) reduced clearance of injected bacteria and constrained the immune response (Holman et al., 2004). In the present study, the ambient P[o.sub.2] was reduced by only 50%, yet we found large increases in bacteremia and limited changes in hemocyte number, suggesting that immune function may be particularly oxygen-sensitive in C. megister.

Eighty minutes after Psychrobacter injection, large numbers of culturable bacteria remain in the hemolymph of C. magister (Fig. 2). Marine invertebrates are capable of rapid immune response; oysters eliminated Escherichia coli within 40 min (Alvarez et al., 1995), and decapod crustaceans are capable of removing circulating foreign particles from the hemolymph within 15 min (Martin et al, 2000). In the blue crab, hemocyte aggregates begin to appear in the gills within 30 min after bacterial injection (Burnett et al., 2006). and injected Vibrio, a known crustacean pathogen, is cleared within 10 min at 25 [degrees]C (Holman et al., 2004). Cancer magister individuals injected with Psychrobacter at 10 [degrees]C were not able to completely remove eulturable bacteria in normoxic or hypoxic conditions; 80 min after injection, the hemolymph of hypoxic crabs had a CFU value of more than 2500.

In hypoxia- and normoxia-exposed crabs, we measured large declines in postbranchial hemolymph P[o.sub.2] 48 and 72 h, respectively, after bacteria injection (Fig. 4). Bacteremia has previously been reported to accompany reduced respiratory function in the blue crab C sapidus (Burnett et al., 2006), and metabolic depression occurs in the penaeid shrimp Litopenaeus vannamei (Scholnick et al., 2006). Reduced oxygen tensions may be a consequence of localization of bacteria and hemocytes at the gill into nodules, which may ultimately limit hemolymph flow and oxygen uptake. Martin et al, (2000) reported that gill nodules resulting from particle injection in various crustaceans begin to fuse and increase in size several days after injection. In the present study, Po2 declined in normoxia-exposed crabs 72 h after they were injected with bacteria, suggesting that it may take several days for gill nodules to become large enough to occlude blood flow and disrupt respiratory function under normoxic conditions.

We observed periodic rapid depressions, downward spikes, in Po2 that appeared to be sensitive to bacterial infection (Fig. 3) and may be linked to episodic reversal or cessation of scaphognathite (gill-pump) beating common in marine crustaceans (Taylor, 1982). Crustaceans exhibit a variety of ventilatory responses to environmental oxygen, yet few studies have examined the effect of hemolymph bacteria on those responses. Burnett et at. (2006) determined scaphognathite activity from pressure recordings and found no difference in rate due to Vibrio injection in C sapidus. Given the apparent sensitivity of scaphognathite activity to hypoxia (Fig. 3) and the different P[o.sub.2] tensions associated with bacteremia determined in the present study, further examination of the effects of infection on cardiorespiratory function appear warranted.

In the present study, prostbranchial oxygen tensions for saline-injected C. magister (17.8 kPa, Fig. 4) were much higher than those reported in an earlier C. magister study (10 kPa, McMahon et al., 1979) and slightly higher than in more recent studies using Carcinus maenas (14.3 kPa, Lallier and Truchot, 1989) and blue crab (16.2 kPa, Burnett et al., 2006). We noted that although crabs were not handled during insertion of the oxygen probe, oxygen tensions initially declined rapidly, 5 to 10 kPa for the first several minutes of measurement, before increasing and stabilizing. We recorded oxygen tensions only after P[o.sub.2] had increased and stabilized, which may explain in part the higher values found in this study.

Overall, our results suggest a potential link between low environmental oxygen in coastal waters and increased susceptibility of marine crustaceans to infectious disease. We report that Psychrobacter cibarius is present in Dungeness crabs and that sustained mild hypoxia elevates CFU values in crab hemolymph. Injections of P. cibarius diminish C. magister hemolymph oxygen tensions after 48 h in hypoxic water and after 72 h in air-saturated water.

Acknowledgments

The work was supported in part by Pacific Research Institute for Science and Math to support undergraduate research. The authors thank Lou and Karen Burnett for their technical advice and support. Gyorgyi Nyerges and Kelsey Schweitzer assisted with animal collections and bacteria isolation.

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DAVID A. SCHOLNICK * AND VENA N. HAYNES

Pacific University, Department of Biology, 2043 College Way, Forest Grove, Oregon 97116

Received 20 September 2011; accepted 6 February 2012.

* To whom correspondence should be addressed. E-mail: david.scholnick@pacificu.edu
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Author:Scholnick, David A.; Haynes, Vena N.
Publication:The Biological Bulletin
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Geographic Code:1CANA
Date:Feb 1, 2012
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