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 micromol 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.     

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.     

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 CO(2)), 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 PO(2) = 4 kPa; PCO(2) = 1.8 kPa; and pH = 6.7-7.1) and were injected with 2.5 x 10(4) 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.     

Heart rate changes caused by varying the oxygen supply to isolated hind legs of rats

In a rat with an isolated hind leg circulation perfused with varying tyrode solutions, heart rate (HR) changes were studied in dependence of VO2 in the isolated hind leg and of PCO2, [K+], pH and lactic acid concentration ([Lac]) measured in the venous outflow of the isolated hind leg. In experimental series I the inflow PO2 (PiO2) was kept constantly high (either about 65 or 72 kPa). The perfusion pressure alternated between 16 and 24 kPa leading to flow rates in isolated hind legs (Qa) from 30 to 50 ml . 100 g-1 . min-1. The VO2 depended on the momentary Qa (flow-limited oxygen uptake). The [K+] and [Lac], the pH and the AVDO2 remained nearly constant while the PCO2 was lower at small flow rates. The HR decreases some 4 min after initial enhancement of Qa and VO2. Series II comprised experiments with low flow rates and a medium oxygen supply (Qa = 2.5-17.4 ml . 100 g-1 . min-1), PiO2 = 17.5-62.7 kPa). The VO2 ranged between 0.02 and 0.2 ml . 100 g-1 . min-1. The [K+] and [Lac], the PCO2 and the HR increased while the pH decreased. The [Lac] in the outflow showed a strong dependence on oxygen uptake and--at a weak oxygen supply--on the time. Cross-correlation analyses between the parameters confirmed that the HR was best temporally correlated to the [Lac] in the outflow.(ABSTRACT TRUNCATED AT 250 WORDS)     

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

The Eastern oyster, Crassostrea virginica, inhabits shallow coastal waters that frequently experience periods of low dissolved oxygen (hypoxia) and elevated CO(2) (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 O(2) pressure [P(O2)] = 20.7 kPa, CO(2) < 0.06 kPa, pH 7.8 to 8.0) or HH (P(O2) = 4 kPa, CO(2) = 1.8 kPa, pH 6.5 to 6.8) seawater at 25 degrees C for 4 h before being injected in the adductor muscle with 10(5) 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 ( approximately 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.     

Nonnutritive flow impairs uptake of fatty acid by white muscles of the perfused rat hindlimb

Triglyceride hydrolysis by the perfused rat hindlimb is enhanced with serotonin-induced nonnutritive flow (NNF) and may be due to the presence of nonnutritive route-associated connective tissue fat cells. Here, we assess whether NNF influences muscle uptake of 0.55 mM palmitate in the perfused hindlimb. Comparisons were made with insulin-mediated glucose uptake. NNF induced during 60 nM insulin infusion inhibited hindlimb oxygen uptake from 22.0 +/- 0.5 to 9.7 +/- 0.8 micromol x g(-1) x h(-1) (P < 0.001), 1-methylxanthine metabolism (indicator of nutritive flow) from 5.8 +/- 0.4 to 3.8 +/- 0.4 nmol x min(-1) x g(-1) (P = 0.004), glucose uptake from 29.2 +/- 1.7 to 23.1 +/- 1.8 micromol x g(-1) x h(-1) (P = 0.005) and muscle 2-deoxyglucose uptake from 82.1 +/- 4.6 to 41.6 +/- 6.7 micromol x g(-1) x h(-1) (P < 0.001). Palmitate uptake, unaffected by insulin alone, was inhibited by NNF in extensor digitorum longus, white gastrocnemius, and tibialis anterior muscles; average inhibition was from 13.9 +/- 1.2 to 6.9 +/- 1.4 micromol x g(-1) x h(-1) (P = 0.02). Thus NNF impairs both fatty acid and glucose uptake by muscle by restricting flow to myocytes but, as shown previously, favors triglyceride hydrolysis and uptake into nearby connective tissue fat cells. The findings have implications for lipid partitioning in limb muscles between myocytes and attendant adipocytes.     

Gluconeogenesis predominates in periportal regions of the liver lobule

Rates of gluconeogenesis from lactate were calculated in periportal and pericentral regions of the liver lobule in perfused rat livers from increases in O2 uptake due to lactate. When lactate (0.1-2.0 mM) was infused into livers from fasted rats perfused in either anterograde or the retrograde direction, a good correlation (r = 0.97) between rates of glucose production and extra O2 uptake by the liver was observed as expected. Rates of oxygen uptake were determined subsequently in periportal and pericentral regions of the liver lobule by placing miniature oxygen electrodes on the liver surface and measuring the local change in oxygen concentration when the flow was stopped. Basal rates of oxygen uptake of 142 +/- 11 and 60 +/- 4 mumol X g-1 X h-1 were calculated for periportal and pericentral regions, respectively. Infusion of 2 mM lactate increased oxygen uptake by 71 mumol X g-1 X h-1 in periportal regions and by 29 mumol X g-1 X h-1 in pericentral areas of the liver lobule. Since the stoichiometry between glucose production and extra oxygen uptake is well-established, rates of glucose production in periportal and pericentral regions of the liver lobule were calculated from local changes in rates of oxygen uptake for the first time. Maximal rates of glucose production from lactate (2 mM) were 60 +/- 7 and 25 +/- 4 mumol X g-1 X h-1 in periportal and pericentral zones of the liver lobule, respectively. The lactate concentrations required for half-maximal glucose synthesis were similar (0.4-0.5 mM) in both regions of the liver lobule in the presence or absence of epinephrine (0.1 microM). In the presence of epinephrine, maximal rates of glucose production from lactate were 79 +/- 5 and 59 +/- 3 mumol X g-1 X h-1 in periportal and pericentral regions, respectively. Thus, gluconeogenesis from lactate predominates in periportal areas of the liver lobule during perfusion in the anterograde direction; however, the stimulation by added epinephrine was greatest in pericentral areas. Differences in local rates of glucose synthesis may be due to ATP availability, as a good correlation between basal rates of O2 uptake and rates of gluconeogenesis were observed in both regions of the liver lobule in the presence and absence of epinephrine. In marked contrast, when livers were perfused in the retrograde direction, glucose production was 28 +/- 5 mumol X g-1 X h-1 in periportal areas and 74 +/- 6 mumol X g-1 X h-1 in pericentral regions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Expression and applications of recombinant crustacean hyperglycemic hormone from eyestalks of white shrimp (Litopenaeus vannamei) against bacterial infection

Crustacean hyperglycemic hormone (CHH) has many functions to regulate carbohydrate metabolism, ecdysis and reproduction including ion transport in crustaceans. The cDNA encoding CHH peptides containing 369 bp open reading frame encoding 122 amino acids was cloned from eyestalk of white shrimp (Litopenaeus vannamei) and was produced by a bacterial expression system. The biological activity of recombinant L. vannamei crustacean hyperglycemic hormone (rLV-CHH) was tested. The hemolymph glucose level of shrimp increased two-fold at 1h after the rLV-CHH injection and then returned to normal after 3h. In addition to the effect of rLV-CHH administration (25 μg/shrimp) on immunological responses of white shrimp against pathogenic bacteria, Vibrio harveyi was studied. Results showed that the blood parameters of shrimp injected with rLV-CHH; the THC, PO activity, serum protein level and clearance ability to V. harveyi, were also higher than those of Neg-protein and PBS-injected shrimp. The survival of shrimp injected with rLV-CHH was significantly higher (66.0%) than shrimp that injected with Neg-protein (33.3%) and PBS (28.9%) after 14 days. It is possible that the administration of rLV-CHH in L. vannamei exhibited a higher immune response related to resistance against V. harveyi infection.     

Lactic Acid efflux as a mechanism of hypoxic acclimation of maize root tips to anoxia

Hypoxic pretreatment (3 kPa oxygen) of maize (Zea mays L.) root tips improved their survival time in a subsequent anoxic incubation from 10 h to more than 3 d, provided that glucose was added to the medium to sustain metabolism. The glycolytic flux (lactate + ethanol) was the same in both pretreated and untreated root tips during the 1st h after transfer to anoxia. It was only after 2 h that it declined sharply in untreated tips, but was sustained in pretreated ones. Right after the transition from normoxia to anoxia of untreated root tips, the only fermentative product detected was lactic acid, which accumulated in a 7:1 proportion after 30 min in tissue and medium, respectively. It took 10 min before ethanol could be detected and 20 min for it to be produced at its maximum rate at the expense of lactate production, which slowed down. In contrast, in hypoxically pretreated root tips, ethanol was produced at a maximum rate right after the transfer to anoxia. Concurrently, low amounts of lactic acid were produced that accumulated in a 1:1 proportion after 30 min in tissue and medium, respectively. This large efflux of lactic acid could account for the higher cytoplasmic pH values always found in pretreated tissues. The presence of cycloheximide during pretreatment abolished this difference, suggesting that the greater efficiency of lactate efflux was linked to protein synthesis. The role of lactate in cytosolic pH regulation and in sensitivity to anoxia is discussed.     

The immune response of white shrimp Litopenaeus vannamei following Vibrio alginolyticus injection

White shrimp Litopenaeus vannamei injected with saline, and injected with tryptic soy broth (TSB)-grown Vibrio alginolyticus at 1.0 x 10(5) and 1.8 x 10(5) colony-forming units (cfu) shrimp(-1) were examined for hyaline cell (HC) counts, granular cell (GC) counts, total haemocyte counts (THCs), phenoloxidase (PO) activity, respiratory burst (RB) and superoxide dismutase (SOD) activity after 1-168 h. Shrimp that received no injection served as the control. The shrimps which received V. alginolyticus at both doses showed significant decreases in these parameters after 6-96 h. The values for HC and SOD activity decreased earlier and then RB. The time to cause maximum depletion of haemocytes (haemocytopenia), PO activity, RB, and SOD activity were 12, 72, 48, and 24 h post-injection, respectively. The HC, GC, and RB returned to the original values earlier at 72 h, followed by SOD activity at 96 h, and then PO activity at 168 h post-infection. It was concluded that an injection of V. alginolyticus rapidly reduced the shrimp's immunity by decreasing HC, GC, SOD activity, RB, and PO activity within 3-24 h, followed by a slow recovery during 72-168 h post-injection. Furthermore, white shrimp L. vannamei which received V. alginolyticus showed a 6-9 h later response in PO activity, and a 72-96 h later recovery of PO activity, compared to the responses in RB and SOD activity indicating their roles in shrimp defence and immunity.     

Brain oxygen utilization is unchanged by hypoglycemia in normal humans: lactate, alanine, and leucine uptake are not sufficient to offset energy deficit

During hypoglycemia, substrates other than glucose have been suggested to serve as alternate neural fuels. We evaluated brain uptake of endogenously produced lactate, alanine, and leucine at euglycemia and during insulin-induced hypoglycemia in 17 normal subjects. Cross-brain arteriovenous differences for plasma glucose, lactate, alanine, leucine, and oxygen content were quantitated. Cerebral blood flow (CBF) was measured by Fick methodology using N(2)O as the dilution indicator gas. Substrate uptake was measured as the product of CBF and the arteriovenous concentration difference. As arterial glucose concentration fell, cerebral oxygen utilization and CBF remained unchanged. Brain glucose uptake (BGU) decreased from 36.3+/-2.6 to 26.6+/-2.1 micromol.100 g of brain(-1).min(-1) (P<0.001), equivalent to a drop in ATP of 291 micromol.100 g(-1).min(-1). Arterial lactate rose (P<0.001), whereas arterial alanine and leucine fell (P<0.009 and P<0.001, respectively). Brain lactate uptake (BLU) increased from a net release of -1.8+/- 0.6 to a net uptake of 2.5+/-1.2 micromol.100 g(-1).min(-1) (P<0.001), equivalent to an increase in ATP of 74 micromol.100 g(-1).min(-1). Brain leucine uptake decreased from 7.1+/-1.2 to 2.5 +/- 0.5 micromol.100 g(-1).min(-1) (P<0.001), and brain alanine uptake trended downward (P<0.08). We conclude that the ATP generated from the physiological increase in BLU during hypoglycemia accounts for no more than 25% of the brain glucose energy deficit.     

V(O2) max is unaffected by altering the temporal pattern of stimulation frequency in rat hindlimb in situ.

It might be anticipated that fatiguing contractions would impair the aerobic metabolic response in skeletal muscle if significant fatigue developed before full activation of aerobic metabolism. On the basis of this premise, we examined two groups of rats to test the hypothesis that a gradual increase in stimulation frequency would yield a higher maximal O2 uptake (Vo2 max) than beginning immediately with an intense stimulation frequency because of a slower progression of fatigue under the former conditions. In one group of animals, the distal hindlimb muscles were electrically stimulated at a frequency of 60 tetani/min for 4 min (F60; n = 6 rats); in the other group, the muscles were incrementally stimulated for 1 min at each of 7.5, 15, 30, and 60 tetani/min and for 2 min at 90 tetani/min (FInc; n = 5 rats). Despite large differences in rate of fatigue [time to 60% of initial force was 47 +/- 3 (SE) vs. 188 +/- 1 s in F60 and FInc, respectively] and the time at which Vo2 max occurred (120 +/- 15 vs. 264 +/- 6 s), Vo2 max was not different (419 +/- 24 vs. 381 +/- 44 micromol x min-1. 100 g-1). Furthermore, time x tension integral at Vo2 max (3.82 +/- 0.41 vs. 4.07 +/- 0.31 N. s) and peak lactate efflux (910 +/- 45 vs. 800 +/- 98 micromol x min-1. 100 g-1) were not different between groups. Thus our results show that the more rapid progression of fatigue in F60 did not compromise the aerobic metabolic response in electrically stimulated rat hindlimb muscles. However, in both groups, O2 uptake and lactate efflux declined after Vo2 max was attained in similar proportion to a further fall in force, suggesting that ongoing fatigue with intense contractions reduced ATP demand below that requiring maximal aerobic and glycolytic metabolic responses once Vo2 max was reached.     

Increased susceptibility of white spot syndrome virus-infected Litopenaeus vannamei to Vibrio campbellii

The concept of polymicrobial disease is well accepted in human and veterinary medicine but has received very little attention in the field of aquaculture. This study was conducted to investigate the synergistic effect of white spot syndrome virus (WSSV) and Vibrio campbellii on development of disease in specific pathogen-free (SPF) shrimp Litopenaeus vannamei. The juvenile shrimp were first injected with WSSV at a dose of 30 SID(50) shrimp(-1) (SID(50) = shrimp infectious dose with 50% endpoint) and 24 h later with 10(6) colony-forming units (cfu) of V. campbellii shrimp(-1). Controls receiving just one of the pathogens or negative inocula were included. In the treatment with WSSV only, shrimp started to die at 48-108 h post injection (hpi) and cumulative mortality reached 100% at 268-336 hpi. In the treatment with only V. campbellii injection (10(6) cfu shrimp(-1)), cumulative mortality reached 16.7%. Shrimp in the dual treatment died very quickly after V. campbellii injection and 100% cumulative mortality was obtained at 72-96 hpi. When WSSV-injected shrimp were given sonicated V. campbellii instead of live V. campbellii, no synergistic effect was observed. Density of V. campbellii in the haemolymph of co-infected moribund shrimp collected 10 h after V. campbellii injection was significantly higher than in shrimp injected with V. campbellii only (P < 0.01). However, there was no difference in WSSV replication between shrimp inoculated with WSSV only compared with dually inoculated ones. This study revealed that prior infection with WSSV enhances the multiplication and disease inducing capacity of V. campbellii in shrimp.     

Virulence of Vibrio harveyi responsible for the "Bright-red" Syndrome in the Pacific white shrimp Litopenaeus vannamei

Vibrio harveyi (Vh) CAIM 1792 strain was isolated from Litopenaeus vannamei affected with "Bright-red" Syndrome (BRS). The strain grew in 1-10% NaCl, at 15-35°C and was resistant to ampicillin (10 μg), carbenicillin (100 μg) and oxytetracycline (30 μg). The lowest MIC was for enrofloxacine (0.5 μgml(-1)). The in vivo and in vitro toxicity of bacterial cells and the extracellular products (ECPs) of Vh CAIM 1792 grown at 1.0%, 2.0% and 4.0% NaCl were evaluated. Adherence ability, enzymatic activities and siderophore production of bacterial cell was tested. The ECPs exhibited several enzymatic activities, such as gelatinase, amylase, lipase, phospholipase and caseinase. These ECPs displayed a strong cytotoxic effect on HELA cell line at 6 and 24 h. Challenges using 10(3) CFU g(-1) caused opacity at the site of injection and over 80% shrimp mortality before 24 h p.i. (post-injection). Mortality caused by the ECPs was higher than mortalities with bacteria, especially in the first hours p.i. Bacteria were re-isolated from hemolymph samples of moribund shrimp and identified as Vh CAIM 1792 by rep-PCR. Histological analysis of shrimp L. vannamei injected with Vh CAIM 1792 revealed generalized necrosis involving skeletal muscle (MU) at the injection site, the lymphoid organ (LO), heart and connective tissues. Melanization within the MU at the site of injection was also observed as well as hemocytic nodules within the hearth and MU at 168 h p.i. LO was the target organ of BRS. Necrosis of the MU at the injection site was the main difference in comparison to other shrimp vibriosis.     

The immunostimulatory effect of hot-water extract of Gracilaria tenuistipitata on the white shrimp Litopenaeus vannamei and its resistance against Vibrio alginolyticus

The total haemocyte count (THC), phenoloxidase activity, respiratory burst, superoxide dismutase (SOD) activity, phagocytic activity and clearance efficiency to the pathogen Vibrio alginolyticus were examined in the white shrimp Litopenaeus vannamei (10.3+/-1.5 g) injected individually with hot-water extract of Gracilaria tenuistipitata at 4 or 6 microg g-1. L. vannamei receiving hot-water extract of G. tenuistipitata at either dose increased significantly its THC, phenoloxidase activity, and respiratory burst after 2 days. L. vannamei received hot-water extract of G. tenuistipitata at 6 microg g-1 increased its phagocytic activity and clearance efficiency to V. alginolyticus after 1 day. In another experiment, L. vannamei which had been injected with hot-water extract of G. tenuistipitata were challenged with V. alginolyticus at 2x10(6) colony-forming units (cfu) shrimp-1 and then placed in seawater of 34 per thousand. The survival of shrimp that received hot-water extract of G. tenuistipitata at 6 microg g-1 was significantly higher than that of shrimp that received saline and the control shrimp after 3 days, and at the termination of the experiment (6 days after the challenge). It is therefore concluded that L. vannamei receiving the hot-water extract of G. tenuistipitata at 6 microg g-1 or less increased its immune ability and resistance to V. alginolyticus infection.     

Effects of rutin from Toona sinensis on the immune and physiological responses of white shrimp (Litopenaeus vannamei) under Vibrio alginolyticus challenge

Rutin is a bioflavonoid with strong antioxidant activity. To investigate the regulatory roles of rutin in various functions in crustaceans, we examined physiological (haemolymph glucose, lactate, and lipid) and innate non-specific immune responses (total haemocyte count (THC), phenoloxidase activity (PO), respiratory bursts (release of superoxide anion, O(2)(-)) and superoxide dismutase (SOD) activity) to the pathogen Vibrio alginolyticus in white shrimp (Litopenaeus vannamei) that were individually injected with rutin extracted from Toona sinensis at 10, 20, or 50 microg g(-1). Results showed that PO activity and respiratory burst of L. vannamei increased obviously (P<0.05) when injected with rutin at a dose of 20 and 50 microg g(-1) after 12 and 24 h, respectively. Both the THC and SOD activities of experimental and control groups revealed no significant difference at all doses. L. vannamei injected with rutin at either dose maintained lower glucose, lactate, and lipid levels in response to V. alginolyticus challenge after 12-36, 24-48, and 24-60 h, respectively. The survival rate of L. vannamei that received rutin at either dose was significantly higher than that received saline after 48-72 h. It was, therefore, concluded that the immune ability and resistance against V. alginolyticus infection of L. vannamei receiving rutin at > or = 10 microg g(-1) increased.     

Innate immunity of the white shrimp Litopenaeus vannamei weakened by the combination of a Vibrio alginolyticus injection and low-salinity stress

White shrimp Litopenaeus vannamei were reared at a salinity of 35‰ without a Vibrio alginolyticus injection (unchallenged group), and other shrimp were reared at 35‰, injected with tryptic-soy broth (TSB)-grown V. alginolyticus at 1.8 × 10⁵ colony-forming units (cfu) shrimp^?1 (challenged group), and then examined for the hyaline cell (HC) count, granular cell (GC, including semi-granular cell) count, total haemocyte count (THC), phenoloxidase (PO) activity, respiratory burst (RB) and superoxide dismutase (SOD) activity after transfer to 35‰ (control), 25‰, 20‰, and 15‰ for 1, 6, 12, 24, 72, and 120 h. Results indicated that the haemocyte count, PO activity, RB, and SOD activity of unchallenged shrimp and challenged shrimp that were transferred to low-salinity levels all began to significantly decrease at 6, 6, 6, and 1 h, respectively, and reached the lowest levels at 12 h. HC, GC, the THC, PO activity, RB, and SOD activity of unchallenged shrimp that were transferred to 15‰ decreased by 53%, 41%, 49%, 68%, 39%, and 62%, whereas those parameters of challenged shrimp that were transferred to 15‰ decreased by 79%, 78%, 79%, 82%, 54%, and 72%, respectively after 12 h compared to control shrimp. These immune parameters began to recover after 24–72 h for both unchallenged shrimp and challenged shrimp. We concluded that the innate immunity was weakened in white shrimp L. vannamei that received combined stresses of a V. alginolyticus injection, and low-salinity transfer. It was also concluded that shrimp with respectively 21%, 18%, 46%, and 28% lower THC, PO activity, RB, and SOD activity of the original values would be killed due to decreases in their immunity, and resistance to V. alginolyticus infection. Shrimp farming should be maintained at a constant high salinity level to prevent exacerbated decreases in innate immune parameters of shrimp when infected by a pathogen coupled with low-salinity stress leading to mortality.     

Vibrio parahaemolyticus and V. harveyi cause detachment of the epithelium from the midgut trunk of the penaeid shrimp Sicyonia ingentis

Shrimp Sicyonia ingentis were either injected with Vibrio parahaemolyticus (10(4) CFU) or V. harveyi (10(6) CFU) or immersed in ASW containing either species at 10(5) CFU ml(-1). These densities were shown in preliminary experiments to kill approximately half the population by 7 d. On Day 7, surviving shrimp were classified as either diseased or apparently healthy, and their midgut trunks (MGT) were examined by light and electron microscopy. All shrimp immersed in ASW containing either species of Vibrio showed detachment of the epithelium in the MGT. In shrimp injected with either species of Vibrio, epithelial detachment was common in diseased shrimp but not in apparently healthy animals. Experiments with live shrimp were supported by in vitro experiments where MGTs were removed, tied off at both ends, and injected with either pathogenic bacteria (V. parahaemolyticus or V. harveyi), non-pathogenic bacteria (Bacillus subtilis or Escherichia coli), or ASW. After 2 h incubations in ASW at 15 degrees C, the MGTs were processed and examined. The epithelium consistently detached from isolated MGTs injected with either species of Vibrio, but not from MGTs injected with non-pathogenic bacteria or ASW. Because the MGT epithelium secretes the peritrophic membrane, loss of the epithelium eliminates 2 layers that may restrict penetration of ingested pathogens into the shrimp body and may disrupt the osmoregulatory function of the MGT. A second finding was that fixed, large-granule hemocytes associated with the basal lamina degranulated in the presence of the 2 species of Vibrio, but not with the non-pathogenic bacteria or ASW. These blood cells may help fight specific bacteria penetrating the MGT.     

Examination of Lactobacillus plantarum lactate metabolism side effects in relation to the modulation of aeration parameters

AIMS: The characterization of global aerobic metabolism of Lactobacillus plantarum LP652 under different aeration levels, in order to optimize acetate production kinetics and to suppress H2O2 toxicity. METHODS AND RESULTS: Cultures of L. plantarum were grown on different aeration conditions. After sugar exhaustion and in the presence of oxygen, lactate was converted to acetate, H2O2 and carbon dioxide with concomitant ATP production. Physiological assays were performed at selected intervals in order to assess enzyme activity and vitality of the strain during lactic acid conversion. The maximal aerated condition led to fast lactate-to-acetate conversion kinetics between 8 and 12 h, but H2O2 immediately accumulated, thus affecting cell metabolism. Pyruvate oxidase activity was highly enhanced by oxygen tension and was responsible for H2O2 production after 12 h of culture, whereas lactate oxidase and NADH-dependent lactate dehydrogenase activities were not correlated to metabolite production. Limited NADH oxidase (NOX) and NADH peroxidase (NPR) activities were probably responsible for toxic H2O2 levels in over-aerated cultures. CONCLUSION: Modulating initial airflow led to the maximal specific activity of NOX and NPR observed after 24 h of culture, thus promoting H2O2 destruction and strain vitality at the end of the process. SIGNIFICANCE AND IMPACT OF THE STUDY: Optimal aeration conditions were determined to minimize H2O2 concentration level during growth on lactate.