Energy metabolism and metabolic depression during exercise

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Crab metabolism exercise and immunity 3429, McMahon 1981 Gaessar and Brooks 1984 Gleeson and Hancock Bacterial preparation. 2001 Hancock and Gleeson 2002 Highly aerobic crustaceans The bacterial pathogen used was V campbellii 90 69B3 stably. such as the blue crab typically have a small oxygen deficit recovery transfected with the Vibrio derived plasmid EVS146 that expresses. e g Booth et al 1982 and oxygen uptake often returns to pre green fluorescent protein and resistance to antibiotics kanamycin. activity levels within 20 45 min Herreid et al 1979 Booth et al and chloramphenicol Dr E Stabb University of Georgia The. 1982 Full and Herreid 1983 Houlihan and Innes 1984 Hamilton parental strain of V campbellii 90 69B3 was isolated from diseased. and Houlihan 1992 However in organisms more reliant on shrimp Litopenaeus vannamei by D Lightner and L Mahone. anaerobic pathways to fuel energy demands during activity this University of Arizona V campbellii is an opportunistic pathogen. recovery may take as long as 5 24 h McDonald et al 1979 and its pathogenicity in L vannamei is comparable to that of other. McMahon et al 1979 Wood and Randall 1981 Of course this marine Vibrio spp that are commonly isolated from marine waters. also partially depends on the duration and intensity of the activity Mikulski et al 2000 V campbellii was selected for the present. performed study because its distribution inactivation and elimination from. It is perhaps surprising that aerobic metabolism in shrimp tissues of shrimp and crabs have been extensively documented. Scholnick et al 2006 and in blue crabs Burnett et al 2006 Holman et al 2004 Burgents et al 2005b Stability of the plasmid. decreases profoundly following injection of a sublethal dose of and growth characteristics of the transfected strain have been. bacteria This metabolic depression is especially interesting since described previously Burgents et al 2005b. the gills are implicated in the immune response Smith and Ratcliffe One day prior to injection V campbellii was streaked from a. 1980a White et al 1985 Martin et al 2000 during which time frozen glycerol working stock onto a TSA plate containing 2 5. their respiratory function declines Burnett et al 2006 The decline NaCl 100 g ml 1 kanamycin A Sigma Aldrich St Louis MO. is rapid and in the case of the shrimp it persists for 24 h We were USA and 5 g ml 1 chloramphenicol Sigma Aldrich and incubated. interested in investigating this phenomenon further in the blue crab at 25 C for 24 h Bacteria from this plate were then transferred to. and determining whether bacterial injection impaired the response a tube containing sterile 10 mmol l 1 saline using a sterile inoculating. of the blue crab to exercise loop and the bacterial concentration was adjusted to an optical. density of 0 1 0 005 at 540 nm which equals a bacterial. MATERIALS AND METHODS concentration of 108 CFU ml 1 Mikulski et al 2000 Burgents et. Animal collection and maintenance al 2005b The prepared bacterial suspension was adjusted with. Adult male blue crabs Callinectes sapidus 60 140 g were sterile saline to obtain the desired concentration for injection. collected locally in Charleston SC USA and held at the Hollings Injection volumes of saline or saline containing Vibrio never. Marine Laboratory in well aerated recirculating seawater 30 exceeded 300 l. 20 22 C pH 7 5 8 5 on a 12 h 12 h dark light cycle for a. minimum of 3 days but no longer than 2 weeks before use in Determination of bacterial LD50. experiments Crabs were fed daily with squid that were previously Lethality of V campbellii 90 69B3 in the blue crab was assessed. frozen but food was withheld at least 1 day prior to and during by performing three 48 h LD50 bacterial challenges In each replicate. each experiment All experiments were performed in filtered experiment crabs 60 125 g N 7 for each dose were injected with. seawater 30 salinity saline control or one of three doses 2 5 104 2 5 105 or. 2 5 106 CFU g 1 crab of V campbellii The inoculum of bacteria. Animal preparation and assessment for each injection was prepared as described above to obtain a. Crabs were prepared for injection as described by Holman et al concentration of 108 CFU ml 1 saline To prepare the lowest dose. Holman et al 2004 At least 24 h before injecting bacteria this bacterial suspension was diluted 1 3 with sterile saline to obtain. referred to as a bacterial challenge a 1 mm hole was drilled through a concentration of 2 5 107 CFU ml 1 This concentration when. the carapace directly over the heart as a port for injection of bacteria injected as 1 l g 1 crab delivered a dose of 2 5 104 Vibrio g 1 crab. or saline into the ventricle Injection of bacteria directly into the Animals receiving the mid range dosage were injected with. ventricle ensured rapid and even distribution of the injected bacteria 2 5 l g 1 crab of the original bacterial suspension 108 CFU ml 1. by the circulatory system as described by Macey et al Macey et saline which delivered the desired dose of 2 5 105 CFU g 1 crab. al 2008 Two similar holes were drilled over the pericardial sinus To prepare the highest bacterial challenge dose 10 ml of the. adjacent to the heart for sampling hemolymph from the pericardial original bacterial suspension of 108 CFU ml 1 saline was pelleted by. space A thin layer of latex rubber was secured over the drilled holes centrifugation 2000 g for 5 min the supernatant discarded and the. with cyanoacrylate glue and acted as a diaphragm to allow for pellet resuspended in 1 ml of saline to obtain a concentration of. injection or sampling of hemolymph with a needle Each crab was 109 CFU ml 1 Each animal was injected with 2 5 l g 1 crab of the. placed individually in a small container with 1 5 l well aerated 30 concentrated bacteria to deliver a final dose of 2 5 106 CFU g 1 crab. seawater and a thin layer of gravel Control animals received an injection of 1 l g 1 crab of sterile saline. To assess crabs for a pre existing systemic infection with In each replicate trial crabs were injected directly into the. culturable bacteria 150 l of hemolymph was sampled from the ventricle with the appropriate dose of saline containing Vibrio or. pericardial sinus diluted 1 10 in sterile 10 mmol l 1 Hepes buffered saline control within their individual containers ensuring that the. 2 5 NaCl hereafter referred to as saline suspended in marine gill chambers stayed fully submerged while doing so The water. agar and overlaid onto a sterile tryptic soy agar TSA Difco Becton within each container was changed every 24 h during the experiment. Dickenson and Co Sparks MD USA microbial culture plate Mortality was recorded at 0 2 4 8 12 24 and 48 h following. supplemented with 2 NaCl These plates were incubated for 24 h bacterial challenge. at 25 C and then examined for colony forming units CFU of. bacteria Only those crabs with no culturable bacteria present in Bacterial doses for experiments. the hemolymph as detected by this assay were used for Based on our determination of an LD50 dose of V campbellii. experimentation methods described above and reported in Results we used a. THE JOURNAL OF EXPERIMENTAL BIOLOGY,3430 L K Thibodeaux K G Burnett and L E Burnett. sublethal dose of 2 5 104 CFU g 1 crab for subsequent experiments Oxygen uptake and hemolymph lactate. This is equivalent to a circulating dose of 1 105 CFU ml 1 during and after exercise. hemolymph assuming a hemolymph volume of 25 ml O2 uptake was measured at 25 C in the treadmill respirometer as. 100 g 1 body mass Gleeson and Zubkoff 1977 described above Flow rates through the chamber ranged between. 80 and 200 ml min 1 Instantaneous measurements of O2 uptake. Oxygen uptake and hemolymph lactate in resting crabs can be obtained by the difference in O2 pressures in the water. Oxygen uptake following the injection of a sub lethal dose of V flowing into and out of the chamber at a known water flow rate. campbellii was measured at 25 C using a flow through respirometry when the rate of O2 uptake is constant as observed under resting. system The PO2 of seawater entering and leaving a sealed 1 8 l conditions However when the oxygen uptake rate of the animal. circular plexiglass respirometry chamber 17 cm wide 8 cm high changes to a new steady state level there is a delay in the. lined with sterile gravel was measured every 10 s using an O2 establishment of a new steady state PO2 of the water exiting the. electrode Yellow Springs Instruments Yellow Springs OH USA respirometer The approach to a new steady state level is an. and a Sable System Las Vegas NV USA data acquisition system exponential function of time This delay or washout time has. Seawater entering the chamber was gassed with 30 O2 31 kPa been described by Frappell et al Frappell et al 1989 and was. to ensure that the partial pressure within the chamber did not drop accounted for in the present study by applying the Z transformation. below 80 air saturation 16 5 kPa O2 when a crab was present in Bartholomew et al 1981 In order to minimize any washout. the chamber Flow rates through the chamber usually ranged effects associated with the transition from resting O2 uptake to. between 80 and 150 ml min 1 that which occurs during walking and the early stages of recovery. Typically a crab was placed in the respiratory chamber then the water flow through the respirometer was increased during activity. chamber was sealed covered and the animal left undisturbed for Increasing the flow rate during the activity period decreases the. 2 h After this time the chamber was opened and the crab was washout time and insures that the O2 pressure within the chamber. injected with saline control or saline containing V campbellii stays reasonably constant and does not drop below 80 air. 2 5 104 CFU g 1 crab The chamber was then resealed and O2 saturation In fact PO2 of the water exiting the treadmill chamber. uptake was measured continuously for 4 h Afterwards the crab was during walking changed very little from resting values as a result. removed from the respiratory chamber and held overnight in an of increasing the water flow rate. individual container of well aerated 30 seawater at 25 C Each crab was placed in the treadmill chamber and left. Approximately 22 h after injection the crab was returned to the undisturbed for 2 h during which time well oxygenated seawater. passed through the chamber The lid of the chamber was then. sealed respiratory chamber and left undisturbed for 2 h A final. removed and without removing the crab from the water saline. measurement of O2 uptake over 30 min was recorded, control or saline containing V campbellii was injected directly. In a separate group of crabs changes in anaerobic metabolism. into the ventricle though the injection port The chamber was. were assessed by measuring hemolymph lactate levels over time. resealed and the animal was allowed to rest in the treadmill chamber. after injection of saline or V campbellii Hemolymph 30 l was. for 1 h Each crab then walked on the treadmill for 30 min as. sampled from the pericardial space of each crab at 30 min 2 h 4 h. described above Subsequently the treadmill was stopped and O2. and 24 h following injection and immediately deproteinized in ice. uptake for each animal was recorded during a 3 h recovery period. cold 12 perchloric acid 1 10 dilution The samples were then Excess post exercise oxygen consumption EPOC was calculated. centrifuged and lactate concentrations measured enzymatically by integrating the area described by the elevation of O2 uptake above. using a NADH linked colorimetric assay Sigma Technical Bulletin the base line value from 0 to 80 min after exercise by which time. No 862 UV lactate dehydrogenase Sigma L2500 and modified O2 uptake had returned to base line levels. according to Graham et al Graham et al 1983 Hemolymph lactate concentration was measured on a separate. group of crabs injected with saline control or saline containing V. Exercise protocol campbellii and exercised according to the same regimen Each crab. Walking was initiated and sustained on a variable speed was injected with saline control or saline containing V campbellii. treadmill equipped with a flow through respirometry system into the ventricle while remaining submerged in its individual. The treadmill is enclosed in a 4 3 l clear watertight plexiglass container Hemolymph 30 l was sampled from the pericardial. chamber and powered with an external DC motor The crabs sinus after 1 h at rest in the treadmill chamber immediately after. were able to move freely about in the 18 cm 17 cm 7 cm high 30 min of walking and at 1 2 and 3 h during the recovery period. compartment above the tread In each experiment crabs were Hemolymph samples were immediate deproteinized and lactate was. injected with saline control or saline containing V campbellii measured as described above. placed within the treadmill chamber and allowed to rest for 1 h. Animals then walked on the treadmill at a speed of 8 m min 1 Whole body and muscle metabolites during and after exercise. for 30 min After this activity was completed each crab was An additional 84 crabs were injected exercised and rested following. allowed to recover for 3 h the same regimen described above To measure metabolites in the. Three different sets of experiments employed this apparatus whole crab and separately in muscle tissues at different times before. and activity regime In the first set of experiments O2 uptake was during and after the exercise period crabs were removed rapidly. measured in 14 crabs N 7 each for saline injected and Vibrio from the apparatus at the appropriate time and plunged into liquid. The Atlantic blue crab Callinectes sapidus Rathbun an important species for commercial and recreational fishing Whitaker et al 1998 lives in estuarine and other coastal environments along the Atlantic and Gulf coasts of North America In these habitats seasonal and diurnal fluctuations of salinity temperature oxygen and pH deFur et al 1990 Mikulski et al 2000 can profoundly

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