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The virulence of Enterococcus to freshwater prawn Macrobrachium rosenbergii and its immune resistance under ammonia stress
Fish & Shellfish Immunology (2002) 12, 97–109 doi:10.1006/fsim.2001.0363 Available online at http://www.idealibrary.com on
The virulence of Enterococcus to freshwater prawn Macrobrachium rosenbergii and its immune resistance under ammonia stress WINTON CHENG1 AND JIANN-CHU CHEN2* 1
Department of Aquaculture, National Pingtung University of Science and Technology, Pingtung, Taiwan, 912, Republic of China, and 2Department of Aquaculture, National Taiwan Ocean University, Keelung, Taiwan, 202, Republic of China (Received 19 October 2000, accepted after revision 14 May 2001) Growth of pathogen bacterium, Enterococcus was not a#ected in tryptic soy broth (TSB) medium containing ammonia-N concentration in the range of 0–5·14 mg l 1. Giant freshwater prawn Macrobrachium rosenbergii (8–12 g) were challenged with Enterococcus which had been incubated for 24 h in TSB medium containing di#erent concentrations of ammonia-N at 0–5·14 mg l 1. Cumulative mortality of M. rosenbergii was higher for the bacteria incubated in TSB medium having ammonia-N at 0 and 0·26 mg l 1, than those incubated in TSB medium having 1·28, 2·57 and 5·14 mg l 1 ammonia-N after 24 h of challenge. However, cumulative mortality of prawn was significantly higher for the bacteria incubated in TSB medium with no ammonia added after 120 h of challenge. The prawns (8–12 g) were challenged with Enterococcus previously incubated in TSB medium for 24 h, then placed in water having concentrations of ammonia-N at control (0·06 mg l 1), 0·55, 1·01, 1·68 and 3·18 mg l 1. Mortality of prawns increased directly with ammonia-N concentrations after 72 h challenge. The pranws (20–30 g) which had been exposed to control, 0·55, 1·68 and 3·18 mg l 1 ammonia-N for 7 days were examined for the total haemocyte count (THC), di#erential haemocyte count (DHC), phenoloxidase activity and respiratory burst of haemocytes. Phenoloxidase activity decreased when the prawns were exposed to ammonia-N greater than 0·55 mg l 1. The respiratory burst increased significantly at 0·55 mg l 1, but decreased significantly at 1·68 and 3·18 mg l 1 ammonia-N. No significant di#erence in haemocyte count was observed among the prawns at di#erent ammonia-N concentrations. It is suggested that ammonia in water decreases the virulence of Enterococcus, and reduces the immune resistance of M. rosenbergii. 2002 Academic Press Key words: Macrobrachium rosenbergii, Enterococcus, ammonia, challenge, virulence, haemocyte count, phenoloxidase activity, superoxide anions.
I. Introduction The giant freshwater prawn Macrobrachium rosenbergii is commercially important in the world as a primary inland cultured species [1]. Disease *Corresponding author. E-mail: [email protected] 97 1050–4648/02/020097+12 $35.00/0
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outbreaks caused by yeast in the cool season and bacteria in the hot season results in declined production of farmed prawns in Taiwan [2]. The bacterium Enterococcus isolated from diseased prawns has been documented as a cause of muscular necrosis and mass mortality of prawns [2]. In decapoda crustaceans, there are in general three types of circulating haemocytes, hyaline cells, semi-granular cells and large granular cells [3]. They are associated with cellular defence [4, 5]. Haemocytes are also associated with proteins like prophenoloxidase (proPO) which are involved in encapsulation, melanisation and functions as a non-self recognition system [6]. Phenoloxidase is the terminal enzyme in the proPO activation system and is activated by several microbial polysaccharides, including -1,3-glucan from fungal cell walls [7]. Phagocytosis is generally recognised as a central and important way to eliminate micro-organisms or foreign particles [8]. Reactive oxygen species are produced during phagocytosis. This phenomenon, known as respiratory burst, plays an important role in microbicidal activity [9]. The generation of O2 (superoxide anions) has been reported in the haemocyte of shore crab Carcinus maenas [10], tiger shrimp Penaeus monodon [9] and blue shrimp Penaeus stylirostris [11]. It is known that temperature changes, poor water quality and handling a#ect fish health by suppressing the immune system and increasing vulnerability to invading pathogens [12, 13]. Physiological stress has been reported to decrease the defence mechanisms of molluscs [14]. Enterococcus infection in M. rosenbergii is exacerbated by high pH (8·8–9·5) and temperature (33–34 C), but reduced by low salinity (5–10 ppt) [15]. Exposing M. rosenbergii to water at di#erent pH, salinity and temperature levels, Cheng & Chen [16] reported that the decreases in both THC (total haemocyte count) and phenoloxidase activity at pH 9·0–9·5, 33–34 C and salinity at freshwater indicating susceptibility of M. rosenbergii infected with Enterococcus. The conditions in brain heart infusion broth (BHIB) medium are near optimal conditions for the growth of Enterococcus (pH 7–8, 27–30 C and 0·5–1·0% NaCl) and have been reported to increase virulence to M. rosenbergii [17]. Therefore, environmental parameters are considered to trigger disease outbreaks not only by a#ecting the health and defence mechanisms of the host, but also the virulence and density of bacterial pathogens. Ammonia, the end product of protein catabolism, accounts for more than half the nitrogenous waste released by decapod crustaceans [18]. Elevated concentrations of environmental ammonia have been reported to a#ect growth and molting [19], oxygen consumption and ammonia excretion [20], haemocyanin and protein levels in haemolymph [21], and osmotic regulation, ion concentration and Na + -, K + -ATPase activity of penaeid shrimps and American lobster Homarus americanus [22–24]. The 24 and 144 h LC50 of ammonia on M. rosenbergii larvae was 115 and 40 mg l 1, respectively at pH 7·6 [25]. However, no data is available on the immune response and resistance to pathogen in prawns under ammonia stress. The purpose of the present study was to determine the e#ects of di#erent ammonia-N concentrations on the immune response of M. rosenbergii and its resistance to Enterococcus. The haemocyte counts (total and di#erential),
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phenoloxidase activity and respiratory burst were used as indicators of health status under the stress of ammonia. In addition, growth of Enterococcus and its virulence to M. rosenbergii were studied when exposed to di#erent concentrations of ammonia. II. Materials and Methods EFFECT OF AMMONIA ON THE GROWTH OF ENTEROCOCCUS
The bacterium Enterococcus which had been isolated from the diseased M. rosenbergii, and demonstrated to cause opaque and whitish musculature in experimental infections, was used in the study [2]. Stocks were cultured on tryptic soy agar (TSA, Difco, Sparks, MD, U.S.A.) for 24 h at 28 C before being transferred to 10 ml tryptic soy broth (TSB, Difco) for 24 h at 30 C, as a stock bacterial broth for growth tests. Inoculum for the growth test consisted of 0·5 ml of this stock broth culture. The bacteria were then incubated in the 50 ml TSB medium having di#erent concentrations of ammonia-N (control, 0·26, 1·28, 2·57 and 5·14 mg l 1) in 250 ml flasks at 30 C. Ammonia-N concentration was prepared by dissolving 38·2 g of NH4Cl (Merck reagent grade) in 1 l distilled water to make 10 000 mg l 1 as stock solution. Each test was conducted in triplicate and bacterial growth was monitored at 12, 24, 48 and 120 h incubation by measuring absorbency at 601 nm using a Model U-2000 spectrophotometer (Hitachi, Tokyo, Japan). EFFECT OF AMMONIA ON VIRULENCE OF ENTEROCOCCUS
After 24 h cultivation, Enterococcus in test media was harvested by centrifugation at 7155g for 15 min at 4 C. The pellet was resuspended in saline solution (0·85% NaCl) at 1107 cfu ml 1, as the stock bacterial suspension for challenges. Bacteria from the TSB medium, containing ammonia-N concentrations of control, 0·26, 1·28, 2·57 and 5·14 mg l 1, were served as test media and were tested for virulence of Enterococcus. M. rosenbergii (8–12 g in the intermolt stage) were obtained from a commercial farm in Pingtung, Taiwan, and acclimated in the laboratory for 2 weeks prior to experimentation. Bacterial suspensions (20 l) were injected into the ventral cephalothoracic sinus of each prawn. The trial was conducted in triplicate with a dose of 2105 cfu prawn 1. The test and control groups comprised of 10 prawns each (180 tests in all). Challenge tests were conducted following the method of Cheng & Chen [17]. Prawns were kept in water at 281 C at pH 7·3–7·8 with aeration. Control prawns were injected with an equal volume of sterile saline solution. EFFECT OF AMMONIA ON THE RESISTANCE OF M. ROSENBERGII TO ENTEROCOCCUS
Stocks were cultured on TSB medium for 24 h at 30 C, and then centrifuged at 7155g for 15 min at 4 C. The supernatant was removed and the bacterial pellet was resuspended in saline solution (0·85% NaCl) at 1107 cfu ml 1 as the stock bacterial suspension for injection challenges.
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M. rosenbergii (8–12 g in the intermolt stage) used in this study were obtained from a commercial farm at Pingtung, and acclimated in the laboratory for 2 weeks prior to experimentation, as described above. Challenge tests at 2105 cfu prawn 1 were conducted with test and control groups comprising 10 prawns for each replicate. Bacterial suspensions (20 l) were injected into the ventral cephalothoracic sinus of each prawn. After injection, prawns were kept in 60 l glass aquaria (10 prawns each) containing 40 l water with di#erent concentrations of ammonia-N at control, 0·5, 1·0, 1·5 and 3·0 mg l 1, as test solutions, and exposed for 7 days. The measured concentration of ammonia-N at every test solution was renewed every 24 h and was 0·06, 0·55, 1·01, 1·68 and 3·18 mg l 1 ammonia-N for the treatment in control, 0·5, 1·0, 1·5 and 3·0 mg l 1, respectively [26]. The prawns were kept in water at 281 C with aeration. Prawns injected with equal volumes of sterile saline solution and exposed to 3·18 mg l 1 ammonia-N served as unchallenged control (Table 1). EFFECT OF AMMONIA ON IMMUNE PARAMETERS OF M. ROSENBERGII
M. rosenbergii (20–30 g in the intermolt stage) used in this study were harvested and acclimated, as described above. Two prawns were kept in 60 l glass aquaria containing 40 l of test solution with di#erent ammonia-N concentrations. The prawns were exposed to each test solution for 7 days with daily water replacement. Each test solution was conducted in triplicate. The mean measured concentration of ammonia-N in each test solution was the same as that above (Table 1). Haemolymph was sampled individually at the beginning and after 7 days exposure to di#erent test solutions. Haemolymph (100 l) was withdrawn from the ventral sinus of each prawn into a 1 ml sterile syringe (25 G) containing 0·9 ml anti-coagulant solution (trisodium citrate 0·114 M, sodium chloride 0·1 M, pH 7·45; osmolarity 490 mOsm kg 1). A drop of haemolymph suspension was placed on a haemocytometer and the THC and di#erential haemocyte count (DHC) measured using an inverted phase contrast microscope (Leica DMIL, Leica Microsystems Wetzlar GmbH, Germany). Phenoloxidase activity was measured spectrophotometrically by recording the formation of dopachrome from L-dihydroxyphenylalanine (L-DOPA) [27]. The diluted haemolymph was centrifuged at 300g at 4 C for 10 min, the supernatant discarded and the pellet rinsed, resuspended gently in cacodylatecitrate bu#er (sodium cacodylate 0·01 M, sodium chloride 0·45 M, trisodium citrate 0·10 M, pH 7·0), and then centrifuged again. The pellet was then resuspended with 200 l cacodylate bu#er (sodium cacodylate 0·01 M, sodium chloride 0·45 M, calcium chloride 0·01 M, magnesium chloride 0·26 M, pH 7·0). The cell suspension (100 l) was incubated with 50 l of trypsin (1 mg ml 1), which served as an elicitor, for 10 min at 25–26 C. L-DOPA (50 l) was added and then 800 l of cacodylate bu#er was added 5 min later. The optical density at 490 nm was measured using a Hitachi U-2000 spectrophotometer (Tokyo, Japan). The control solution, which consisted of 100 l of cell suspension, 50 l cacodylate bu#er (to replace the trypsin) and 50 l of L-DOPA, was used for the background phenoloxidase activity in all test conditions. The background
3·18 Control (0·06) 0·55 1·01 1·68 3·18
Ambient ammonia-N (mg l 1) 30 30 30 30 30 30
Number of prawns 0 13·30·3a 20·05·8a 26·70·9a 26·70·3a 26·70·7a
24 h 0 23·30·3b 40·05·8ab 40·00·6ab 53·30·7a 50·01·0a
48 h 0 23·30·3c 43·36·7bc 50·01·0ab 70·00·6a 70·00·6a
72 h 0 23·30·3c 46·70·3b 56·70·3b 80·00·6a 83·30·3a
96 h
0 23·30·3c 46·70·3b 56·70·3b 80·00·6a 86·70·3a
120 h
Cumulative mortality (%)
At 281 C and pH 7·3–7·8. Data in the same column with di#erent letters are significantly di#erent (P<0·05) among treatments. Values are mean S.E.
Control 2105 2105 2105 2105 2105
Bacterial dose (cfu prawn 1)
0 26·70·3c 53·30·3d 66·70·3c 83·30·3b 96·70·3a
144 h
Table 1. Susceptibility of Macrobrachium rosenbergii to Enterococcus in di#erent concentrations of ammonia-N
0 26·70·3e 53·30·3d 66·70·3c 83·30·3b 96·70·3a
168 h
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phenoloxidase activity was subtracted from the phenoloxidase activity of prawns for all test conditions and ranged from 0·02 to 0·08. Respiratory burst activity of haemocytes was quantified using the reduction of nitroblue tetrazolium (NBT) to formazan as a measure of superoxide anion (O2 ) production, as described by several scientists [9–11]. Haemolymph (100 l) in anti-coagulant solution were deposited in l plate wells previously coated with 100 l poly-L-lysine solution (0·2%) to improve cell adhesion. Microplates were centrifuged at 300g for 15 min. Plasma was removed and then 100 l zymosan (0·1% in Hank’s solution minus phenol red) were added and allowed to react for 30 min at room temperature. Zymosan was discarded and the haemocytes were washed three times with 100 l Hank’s solution then stained with 100 l NBT solution (0·3%) for 30 min at room temperature. NBT solution was removed and the haemocytes were fixed and washed three times with 70% methanol (100 l) and air-dried. The formazan was dissolved by addition of 120 l KOH and 140 l dimethyl sulfoxide (DMSO). The optical density at 630 nm was measured in triplicate in a micrplate reader (Dynex Mrx II, U.S.A.). STATISTICAL ANALYSIS
All data were subjected to one-way ANOVA [28]. If significant di#erences were indicated at the 0·05 level, then the Duncan Multiple Range test was used to identify significant di#erences among the treatments [29]. III. Results EFFECT OF AMMONIA ON THE GROWTH OF ENTEROCOCCUS
The bacterium Enterococcus grew well in the TSB medium containing ammonia-N concentration in the range of 0 to 5·14 mg l 1. The log phase occurred at 12 and 24 h, and the bacterial density was highest after 24 h of incubation in all test media. EFFECT OF AMMONIA ON VIRULENCE OF ENTEROCOCCUS
All the unchallenged control prawns survived. In contrast, the onset of mortality occurred at 16–24 h in the challenged prawns. Cumulative mortality after 24 h was significantly higher for the bacteria incubated in control medium and the medium with 0·26 mg l 1 ammonia-N, than those incubated in other medium with 1·28, 2·57 and 5·14 mg l 1 ammonia-N. After 120 h of challenge until the end of the experiment at 168 h, the cumulative mortality of the prawns was significantly higher (P<0·05) for the bacteria incubated in control medium than those incubated in other media with ammonia-N. Over 168 h, the cumulative mortality of the prawns was 53·3, 36·7, 40·0, 36·7 and 33·3% for the bacteria in control solution, or medium with 0·26, 1·28, 2·57 and 5·14 mg l 1 ammonia-N, respectively. EFFECT OF AMMONIA ON THE RESISTANCE OF M. ROSENBERGII TO ENTEROCOCCUS
Results from Enterococcus challenge tests in test water with di#erent ammonia-N concentrations are shown in Table 1. The unchallenged control
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Phenoloxidase activity
0.6
0.4
*
*
*
0.55
1.68
3.18
0.2
0.0
Control
–1
Ammonia-N concentration (mg l )
Fig. 1. Mean ( S.E.) phenoloxidase activity in the haemocytes of M. rosenbergii at the beginning ( ), and after 7 days exposure () to di#erent concentrations of ammonia-N. Each bar represents mean value from six determinations with standard error. Asterisk indicates a significant di#erence from the initial sampling.
prawns survived. In contrast to saline-injected controls, all challenged prawns ceased feeding after injection. The onset of mortality occurred between 10 and 16 h in all challenge prawns. Cumulative mortality of prawns in the control solution after 48 h was significantly lower than the test water with ammonia-N at 1·68 and 3·18 mg l 1. The cumulative mortality increased directly with ammonia-N concentration and exposure time after 96 h, with a significant dose e#ect between all ammonia-N concentrations by the end of the experiment (Table 1). EFFECT OF AMMONIA ON THE IMMUNE PARAMETERS OF M. ROSENBERGII
No significant di#erence in THC and DHC was observed among the prawns at the beginning, and following 7 days exposure, to ammonia-N concentration in the range of 0·07–3·18 mg l 1. The mean S.E. THC varied from 59·112·2105 to 92·718·2105 cell ml 1. The mean S.E. DHC varied from 52·311·9105 to 84·217·4105 cell ml 1 for hyaline cells, 2·80·6105 to 4·61·4105 cell ml 1 for semi-granular cells, and 3·80·7105 to 4·961·4105 cell ml 1 for large granular cells. No significant di#erence in phenoloxidase activity was observed between the prawns in the control solution at the beginning and 7 days later. Phenoloxidase activity decreased significantly (P<0·05) when the prawns were exposed to ammonia-N at 0·55, 1·68 and 3·18 mg l 1 (Fig. 1). The relative phenoloxidase activity of prawns at the beginning and after 7 days exposure to 0·55, 1·68 and 3·18 mg l 1 ammonia-N decreased by 36%, 37% and 47% compared to that of prawns in the control solution (Fig. 1). No significant di#erence in the respiratory burst, which was expressed as NBT-reduction per 100 l haemolymph, was observed for the prawns in the control solution between the beginning and 7 days. A significant increase (P<0·05) of NBT-reduction (about 30%) occurred for the prawns following
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0.10 *
Respiratory burst
0.08
* *
0.06
0.04
0.02
0.00
Control
0.55
1.68
3.18 –1
Ammonia-N concentration (mg l )
Fig. 2. Mean respiratory burst in the haemocytes of M. rosenbergii at the beginning ( ), and after 7 days exposure () to di#erent concentrations of ammonia-N. Each bar represents mean value from six determinations. See Fig. 1 for statistical information.
7 days exposure to 0·55 mg l 1 ammonia-N. However, the NBT-reduction decreased significantly, by 26 and 23%, for the prawns following 7 days exposure to 1·68 and 3·18 mg l 1 ammonia-N, respectively (Fig. 2). IV. Discussion It is well known that environmental parameters in media a#ect the growth of pathogens and their production of toxins [30, 31]. Ramesh et al. [32] also suggested that environmental parameters influence the growth of luminous bacteria. Smith [33] indicated that fast-growing pathogens could overwhelm the initial, non-specific defense and cause disease before more powerful immune defences can operate fully. Slow-growing pathogens are more prone to both types of defence. Previous research indicated that Enterococcus incubated in BHIB medium grew from pH 3 to 10 with optimum growth at pH 7–8, and indicated that the Enterococcus grew from 5 to 45 C with optimum growth at 25–30 C in BHIB medium [17]. The present study indicated that Enterococcus grew well in TSB medium with ammonia-N at a concentration of 5·14 mg l 1. Riquelme et al. [34] reported that water temperature, and other parameters, a#ected the pathogenicity of Vibrio strains in Chilean scallop Argopecten purpuratus larvae. Prayitno & Latchford [35] demonstrated that exposure of luminous bacteria Vibrio harveyi to low salinity levels (10 and 15 ppt) for 12 h, before use in immersion challenge tests with tiger shrimp Penaeus monodon larvae, resulted in higher mortality. They also reported that exposure of V. harveyi to pH 5·5 reduced its pathogenicity. Previous research indicated that the optimal level for the growth of Enterococcus incubated at its optimal growth levels (pH 7–8, 25–30 C and 0·5–1·0% NaCl) enhanced the virulence to M. rosenbergii [17]. The fact that M. rosenbergii challenged with Enterococcus
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incubated in TSB medium with ammonia-N had lower mortality indicated ammonia decreases the virulence of Enterococcus. Several scientists have investigated the e#ects of environmental parameters on crustacean defence mechanisms. An increased prevalence in the shell disease of marine decapod crustaceans has been reported to result from polluted environments, also suggesting a decrease in immunocompetence [36, 37]. In decapod crustaceans, circulating haemocytes are associated with cellular defence [4, 5]. It is well known that life cycle, food intake, disease outbreak, pollutants and environmental stress a#ect the circulating haemocyte count of crustaceans, both in quantity and quality [38–41]. Circulating haemocytes of M. rosenbergii at di#erent size, season and molting cycle have been reported by Cheng & Chen [42]. Le Moullac et al. [5] reported that the THC was significantly higher during the premolt stage than during the intermolt stage, whereas phenoloxidase activity was significantly higher during the intermolt stage than during the premolt stage for the blue shrimp Penaeus stylirostris. Cheng & Chen [42] indicated that the molting cycle is one of the main intrinsic factors a#ecting THC, and reported that the THC was significantly higher at stage C and lower at stage D3, with no significant di#erence between male and female [43]. In the present study the M. rosenbergii used was stage C, therefore they are considered to have the same defence condition. Circulating haemocytes are also a#ected by extrinsic factors like temperature, pH, salinity and dissolved oxygen. An increase in water temperature has been reported to increase THC in several crustaceans [44]. M. rosenbergii reared at temperatures of 27–28 C and 30–31 C had significantly higher THC than those reared at 20–21 C and 33–34 C, and the prawns reared at pH 7·5– 7·7 have significantly higher THC than those reared at pH 4·6–5·0 and 9·0–9·5 [16]. Blue shrimp Penaeus stylirostris, following 24 h exposure to dissolved oxygen as low as 1 mg O2 l 1, decreased its THC [11]. The decrease of THC was considered to be mainly due to the decrease of hyaline cells. P. stylirostris, following exposure to ammonia at 3·0 mg l 1, decreased its THC by 30% [41]. Unfortunately, they did not report the exposure time. No changes in THC were observed in shore crab Carcinus maenas following 30 days exposure to 50 g l 1 mercury [44]. The present study indicated that no significant di#erence of THC and DHC was observed between the prawns in the control solution and following 7 days exposure to ammonia-N as high as 3·18 mg l 1. Le Moullac et al. [11] reported that P. stylirostris following hypoxia exposure increased its phenoloxidase activity, but decreased its THC. Negative correlation between phenoloxidase activity and THC was also observed in P. stylirostris when ambient temperature dropped from 27 C to 18 C [41]. Smith & Johnston [40] reported that common shrimp Crangon crangon, following exposure to PCB 15 (polychlorinated biphenyl 15), produced significantly decreased THC and phenoloxidase activity. Cheng & Chen [16] reported that both phenoloxidase activity and THC of M. rosenbergii were significantly higher at pH 7·5–7·7 and 30–31 C, and were significantly lower at pH 4·6–5·0, 9·0–9·5 and 33–34 C, and at freshwater. The present study indicated that the phenoloxidase activity of M. rosenbergii was significantly lower for the prawns exposed to ammonia-N at 0·55, 1·68 and 3·18 mg l 1 after 7 days.
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This fact indicated that the decrease of phenoloxidase activity under ammonia stress was not a consequence of a reduced THC or altered DHC. NBT staining has been used for both qualitative and quantitative analyses of O2 (superoxide anions) generated by haemocytes, which are the first product of the respiratory burst. Song & Hsieh [9] reported that -glucan had the strongest stimulative e#ect on haemocytes in P. monodon in terms of generating O2 and H2O2 (hydrogen peroxide), which were considered to play an important role in shrimp microbicidal activity when compared to ClO (hypochlorites) and MPO (myeloperoxidase). Le Moullac & Ha#ner [41] reported that white shrimp Penaeus vannamei, following injection of propiconazole (a fungicide) induced an increase of respiratory burst at day 6, but caused dose-dependent decrease of the respiratory burst at day 13. Le Mollac et al. [11] indicated that the production of superoxide radicals, decreased in hypoxic P. stylirostris, was due to the decrease of THC, suggesting that NADPH oxidase responsible for production of superoxide was not a#ected under hypoxia. The present study indicated that M. rosenbergii following 7 days exposure to 0·55 mg l 1 ammonia-N stimulated the total production of superoxide radicals, whereas the prawns following 7 days exposure to ammonia-N greater than 1·68 mg l 1 decreased the total production of superoxide radicals. This fact indicated that the NADPH oxidase is a#ected under the stress of ammonia. Further research is needed to examine the activities of superoxide dismutase, catalase and peroxidase to understand the production of superoxide radicals under the stress of ambient ammonia. A relationship between salinity and infectious hypodermal and haematopoietic necrosis (IHHN) has been observed in white shrimp Penaeus vannamei [45]. Le Moullac et al. [11] reported that hypoxia reduced the resistance of shrimp to experimental infection with Vibrio alginolyticus. Previous research has indicated that Enterococcus infected M. rosenbergii is exacerbated by high pH (8·8–9·5) and high temperature (33–34 C), but reduced by low salinity (5–10 ppt) [15], indicating the susceptibility of crustacean to pathogen. The present study indicated that the susceptibility of M. rosenbergii to Enterococcus increased directly with ammonia-N concentration, when the prawns were exposed to ammonia. LeMoullac & Ha#ner [41] indicated that the defence functions belonging to the phenoloxidase activity and the peroxinectin are reduced at the level of gene expression in P. stylirostris when exposed to ammonia. In conclusion, the present study reports that ambient ammonia a#ects immune competence and the degree of protection to Enterococcus infection in M. rosenbergii. This increased susceptibility is considered to be related to the decrease in phenoloxidase activity, which is more relevant than haemocyte count and the respiratory burst products in the resistance of prawns infected by Enterococcus. Ammonia decreases the virulence of Enterococcus pathogen, and increases the susceptibility to Enterococcus by reducing the immune ability of M. rosenbergii. Further research is needed to determine other immune competence activities like phagocytosis, lectins and agglutinin and the product of immune inhibitors like 2-macroglobulin in the haemolymph of M. rosenbergii under ammonia stress.
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The paper was supported by the National Science Council (NSC 89-2313-B-020-10), Republic of China. We appreciate Mr P. C. Leu and Miss C. S. Wang for their assistance in the experiment.
References 1 New, M. B. (1995). Status of freshwater prawn farming: a review. Aquaculture Research 26, 1–54. 2 Cheng, W. & Chen, J. C. (1998). Isolation and characterization of Enterococcus-like bacterium causing muscle necrosis and mortality with Macrobrachium rosenbergii in Taiwan. Diseases of Aquatic Organisms 34, 93–101. 3 Sung, H. H., Yang, Y. L. & Song, Y. L. (1996). Enhancement of microbicidal activity in the tiger shrimp Penaeus monodon vis immunostimulation. Journal of Crustacean Biology 16, 278–284. 4 Hose, J. E., Martin, G. G. & Gerard, A. S. (1990). A decapod hemocyte classification scheme integrating morphology, cytochemistry, and function. Biological Bulletin 178, 33–45. 5 Le Moullac, G., Le Groumellec, M., Ansquer, D., Froissard, S., Levy, P. & Aquacop (1997). Haematological and phenoloxidase activity changes in the shrimp Penaeus stylirostris in relation with the moult cycle: protection against vibrosis. Fish & Shellfish Immunology 7, 227–234. 6 Johansson, M. W. & Söderhäll, K. (1989). Cellular immunity in crustaceans and the proPO system. Parasitology Today 5, 171–176. 7 Smith, V. J., Söderhäll, K. & Hamilton, M. (1984). -1,3-glucan induced cellular defence reactions in the shore crab, Carcinus maenas. Comparative Biochemistry and Physiology 77, 635–639. 8 Bachère, E., Miahle, E. & Rodriguez, J. (1995). Identification of defence e#ectors in the haemolymph of crustaceans with particular reference to the shrimp Penaeus japonicus (Bate): prospects and application. Fish & Shellfish Immunology 5, 597–612. 9 Song, Y. L. & Hsieh, Y. T. (1994). Immunostimulation of tiger shrimp (Penaeus monodon) hemocytes for generation of microbicidal substances: analysis of reactive oxygen species. Developmental and Comparative Immunology 18, 201–209. 10 Bell, K. L. & Smith, V. J. (1993). In vitro superoxide production by hyaline cells of the shore crab Carcinus maenas (L.). Developmental and Comparative Immunology 17, 211–219. 11 Le Moullac, G., Soyez, C., Saulnier, D., Ansquer, D., Avarre, J. C. & Levy, P. (1998). E#ect of hypoxia stress on the immune response and the resistance to vibriosis of the shrimp Penaeus stylirostris. Fish & Shellfish Immunology 8, 621–629. 12 Snieszko, S. F. (1974). The e#ects of environmental stress on outbreaks of infectious diseases of fishes. Journal of Fish Biology 6, 197–208. 13 Zeeman, M. (1986). Modulation of the immune response in fish. Veterinary Immunology Immunopathology 12, 235–241. 14 Cheng, T. C. & Combes, C. (1990). Influence of environmental factors on the invasion of molluscs by parasites: with special reference to Europe. In Biological Invasions in Europe and the Mediterranean Basin (F. Castri, A. J. Hansen & M. Debussche, eds) pp. 307–332. Dordrecht: Kluwer Academic Publishers. 15 Cheng, W. & Chen, J. C. (1998). Enterococcus-like infections Macrobrachium rosenbergii are exacerbated by high pH and temperature but reduced by low salinity. Diseases of Aquatic Organisms 34, 103–108. 16 Cheng, W. & Chen, J. C. (2000). E#ects of pH, temperature and salinity on immune parameters of the freshwater prawn Macrobrachium rosenbergii. Fish & Shellfish Immunology 10, 387–391. 17 Cheng, W. & Chen, J. C. (1999). E#ect of cultivation broth pH, temperature and NaCl concentration on virulence of an Enterococcus-like bacterium to the giant freshwater prawn Macrobrachium rosenbergii. Diseases of Aquatic Organisms 36, 233–237.
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18 Regnault, M. (1987). Nitrogen excretion in marine and freshwater crustacea. Biology Review 62, 1–24. 19 Chen, J. C. & Kou, Y. Z. (1992). E#ects of ammonia on growth and molting of Penaeus japonicus juveniles. Aquaculture 104, 249–260. 20 Chen, J. C. & Lin, C. Y. (1992). Oxygen consumption and ammonia-N excretion of Penaeus chinensis juveniles exposed to ambient ammonia at di#erent salinity levels. Comparative Biochemistry and Physiology 102 C, 287–291. 21 Chen, J. C. & Cheng, S. Y. (1993). Hemolymph PCO2, hemocyanin, protein levels and urea excretions of Penaeus monodon exposed to ambient ammonia. Aquatic Toxicology 27, 281–292. 22 Young-Lai, W. W., Charmantier-Daures, M. & Charmantier, G. (1991). E#ect of ammonia on survival and osmoregulation in di#erent life stages of the lobster Hormarus americanus. Marine Biology 110, 293–300. 23 Chen, J. C. & Nan, F. H. (1992). E#ect of ambient ammonia on ammonia-N excretion and ATPase activity of Penaeus chinensis. Aquatic Toxicology 23, 1–10. 24 Chen, J. C. & Chen, C. T. (1996). Changes of osmotic and electrolyte concentrations in the hemolymph of Penaeus japonicus exposed to ambient ammonia. Comparative Biochemistry and Physiology 114C, 35–38. 25 Armstrong, D. A., Chippendale, D., Knight, A. W. & Colt, J. E. (1978). Interaction of ionized and un-ionized ammonia on short-term survival and growth of prawn larvae Macrobrachium rosenbergii. Biology Bulletin 154, 15–31. 26 Solorzano, L. (1969). Determination of ammonia in natural waters by the phenolhypochlorite method. Limnology and Oceanography 14, 799–801. 27 Herández-López, J., Gollas-Galván, T. S. & Vargas-Albores, F. (1996). Activation of the prophenoloxidase system of the brown shrimp (Penaeus californiensis Holmes). Comparative Biochemistry and Physiology 113, 61–66. 28 Steel, R. G. D. & Torrie, J. H. (1980). Principles and Procedures of Statistics. New York, NY: McGraw-Hill. 633 pp. 29 Duncan, D. B. (1955). Multiple-range and multiple F test. Biometrics 11, 1–42. 30 Weinberg, E. D. (1985). Enzymes, nutrition, and virulence. In Bacterial Enzymes and Virulence (I. A. Holder, ed.) pp. 1–16. Boca Raton, FL: CRC Press. 31 Arp, L. H. (1988). Bacterial infection of mucosal surfaces: an overview of cellular and molecular mechanisms. In Virulence Mechanisms of Bacterial Pathogens (J. A. Roth, ed.) pp. 3–27. Washington, DC: American Society for Microbiology. 32 Ramesh, A., Loganthan, B. G. & Venugopalan, V. K. (1989). Seasonal distribution of luminous bacteria in the sediments of tropical estuary. Journal of General and Applied Microbiology 35, 265–268. 33 Smith, H. (1990). Pathogenicity and the microbe in vivo. The Journal of General Microbiology 136, 377–383. 34 Riquelme, C., Hayashida, G., Toranzo, A. E., Vilches, J. & Chavez, P. (1995). Pathogenicity studies on a Vibrio anguillarum-related (VAR) strain causing an epizootic in Argopecten purpuratus larvae cultured in Chile. Diseases of Aquatic Organisms 22, 135–141. 35 Prayitno, S. B. & Latchford, J. W. (1995). Experimental infections of crustaceans with luminous bacteria related to Photobacterium and Vibrio. E#ect of salinity and pH on infectiosity. Aquaculture 132, 105–112. 36 Gopalan, U. K. & Young, J. S. (1975). Incidence of shell disease in shrimp in the New York Bight. Marine Pollution Bulletin 6, 149–153. 37 Young, J. S. & Pearce, J. B. (1975). Shell disease in crabs and lobsters from New York Bight. Marine Pollution Bulletin 6, 101–105. 38 Persson, M., Cerenius, L., Söderhäll, K. (1987). The influence of haemocytes number on the resistance of the freshwater crayfish Pacifastacus leniusculus Dana, to the parasitic fungus, Aphanomyces astaci. Journal of Fish Diseases 10, 471–477. 39 Söderhäll, K., Johansson, M. W. & Smith, V. J. (1988). Internal defence mechanisms. In Freshwater Crayfish: Biology, Management and Exploitation (D. M. Holdich & R. S. Lowery, eds) pp. 213–235. London: Croom Helm.
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40 Smith, V. J. & Johnston, P. A. (1992). Di#erential haemotoxic e#ect of PCB congeners in the common shrimp Crangon crangon. Comparative Biochemistry Physiology 101, 641–649. 41 LeMoullac, G. & Ha#ner, P. (2000). Environmental factors a#ecting immune response in Crustacea. Aquaculture 191, 121–131. 42 Cheng, W. & Chen, J. C. (2001). E#ects of intrinsic and extrinsic factors on the haemocyte profile of the prawn, Macrobrachium rosenbergii. Fish & Shellfish Immunology 11, 53–63. 43 Peebles, J. B. (1977). A rapid technique for molt staging in live Macrobrachium rosenbergii. Aquaculture 12, 173–180. 44 Truscott, R. & White, K. N. (1990). The influence of metal and temperature stress on the immune system of crabs. Functional Ecology 4, 455–461. 45 Bray, W. A., Lawrence, A. L. & Leung-Trujillo, J. R. (1994). The e#ect of salinity on growth and survival of Penaeus vannamei, with observations on the interaction of IHHN virus and salinity. Aquaculture 122, 133–146.
The virulence of Enterococcus to freshwater prawn Macrobrachium rosenbergii and its immune resistance under ammonia stress WINTON CHENG1 AND JIANN-CHU CHEN2* 1
Department of Aquaculture, National Pingtung University of Science and Technology, Pingtung, Taiwan, 912, Republic of China, and 2Department of Aquaculture, National Taiwan Ocean University, Keelung, Taiwan, 202, Republic of China (Received 19 October 2000, accepted after revision 14 May 2001) Growth of pathogen bacterium, Enterococcus was not a#ected in tryptic soy broth (TSB) medium containing ammonia-N concentration in the range of 0–5·14 mg l 1. Giant freshwater prawn Macrobrachium rosenbergii (8–12 g) were challenged with Enterococcus which had been incubated for 24 h in TSB medium containing di#erent concentrations of ammonia-N at 0–5·14 mg l 1. Cumulative mortality of M. rosenbergii was higher for the bacteria incubated in TSB medium having ammonia-N at 0 and 0·26 mg l 1, than those incubated in TSB medium having 1·28, 2·57 and 5·14 mg l 1 ammonia-N after 24 h of challenge. However, cumulative mortality of prawn was significantly higher for the bacteria incubated in TSB medium with no ammonia added after 120 h of challenge. The prawns (8–12 g) were challenged with Enterococcus previously incubated in TSB medium for 24 h, then placed in water having concentrations of ammonia-N at control (0·06 mg l 1), 0·55, 1·01, 1·68 and 3·18 mg l 1. Mortality of prawns increased directly with ammonia-N concentrations after 72 h challenge. The pranws (20–30 g) which had been exposed to control, 0·55, 1·68 and 3·18 mg l 1 ammonia-N for 7 days were examined for the total haemocyte count (THC), di#erential haemocyte count (DHC), phenoloxidase activity and respiratory burst of haemocytes. Phenoloxidase activity decreased when the prawns were exposed to ammonia-N greater than 0·55 mg l 1. The respiratory burst increased significantly at 0·55 mg l 1, but decreased significantly at 1·68 and 3·18 mg l 1 ammonia-N. No significant di#erence in haemocyte count was observed among the prawns at di#erent ammonia-N concentrations. It is suggested that ammonia in water decreases the virulence of Enterococcus, and reduces the immune resistance of M. rosenbergii. 2002 Academic Press Key words: Macrobrachium rosenbergii, Enterococcus, ammonia, challenge, virulence, haemocyte count, phenoloxidase activity, superoxide anions.
I. Introduction The giant freshwater prawn Macrobrachium rosenbergii is commercially important in the world as a primary inland cultured species [1]. Disease *Corresponding author. E-mail: [email protected] 97 1050–4648/02/020097+12 $35.00/0
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outbreaks caused by yeast in the cool season and bacteria in the hot season results in declined production of farmed prawns in Taiwan [2]. The bacterium Enterococcus isolated from diseased prawns has been documented as a cause of muscular necrosis and mass mortality of prawns [2]. In decapoda crustaceans, there are in general three types of circulating haemocytes, hyaline cells, semi-granular cells and large granular cells [3]. They are associated with cellular defence [4, 5]. Haemocytes are also associated with proteins like prophenoloxidase (proPO) which are involved in encapsulation, melanisation and functions as a non-self recognition system [6]. Phenoloxidase is the terminal enzyme in the proPO activation system and is activated by several microbial polysaccharides, including -1,3-glucan from fungal cell walls [7]. Phagocytosis is generally recognised as a central and important way to eliminate micro-organisms or foreign particles [8]. Reactive oxygen species are produced during phagocytosis. This phenomenon, known as respiratory burst, plays an important role in microbicidal activity [9]. The generation of O2 (superoxide anions) has been reported in the haemocyte of shore crab Carcinus maenas [10], tiger shrimp Penaeus monodon [9] and blue shrimp Penaeus stylirostris [11]. It is known that temperature changes, poor water quality and handling a#ect fish health by suppressing the immune system and increasing vulnerability to invading pathogens [12, 13]. Physiological stress has been reported to decrease the defence mechanisms of molluscs [14]. Enterococcus infection in M. rosenbergii is exacerbated by high pH (8·8–9·5) and temperature (33–34 C), but reduced by low salinity (5–10 ppt) [15]. Exposing M. rosenbergii to water at di#erent pH, salinity and temperature levels, Cheng & Chen [16] reported that the decreases in both THC (total haemocyte count) and phenoloxidase activity at pH 9·0–9·5, 33–34 C and salinity at freshwater indicating susceptibility of M. rosenbergii infected with Enterococcus. The conditions in brain heart infusion broth (BHIB) medium are near optimal conditions for the growth of Enterococcus (pH 7–8, 27–30 C and 0·5–1·0% NaCl) and have been reported to increase virulence to M. rosenbergii [17]. Therefore, environmental parameters are considered to trigger disease outbreaks not only by a#ecting the health and defence mechanisms of the host, but also the virulence and density of bacterial pathogens. Ammonia, the end product of protein catabolism, accounts for more than half the nitrogenous waste released by decapod crustaceans [18]. Elevated concentrations of environmental ammonia have been reported to a#ect growth and molting [19], oxygen consumption and ammonia excretion [20], haemocyanin and protein levels in haemolymph [21], and osmotic regulation, ion concentration and Na + -, K + -ATPase activity of penaeid shrimps and American lobster Homarus americanus [22–24]. The 24 and 144 h LC50 of ammonia on M. rosenbergii larvae was 115 and 40 mg l 1, respectively at pH 7·6 [25]. However, no data is available on the immune response and resistance to pathogen in prawns under ammonia stress. The purpose of the present study was to determine the e#ects of di#erent ammonia-N concentrations on the immune response of M. rosenbergii and its resistance to Enterococcus. The haemocyte counts (total and di#erential),
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phenoloxidase activity and respiratory burst were used as indicators of health status under the stress of ammonia. In addition, growth of Enterococcus and its virulence to M. rosenbergii were studied when exposed to di#erent concentrations of ammonia. II. Materials and Methods EFFECT OF AMMONIA ON THE GROWTH OF ENTEROCOCCUS
The bacterium Enterococcus which had been isolated from the diseased M. rosenbergii, and demonstrated to cause opaque and whitish musculature in experimental infections, was used in the study [2]. Stocks were cultured on tryptic soy agar (TSA, Difco, Sparks, MD, U.S.A.) for 24 h at 28 C before being transferred to 10 ml tryptic soy broth (TSB, Difco) for 24 h at 30 C, as a stock bacterial broth for growth tests. Inoculum for the growth test consisted of 0·5 ml of this stock broth culture. The bacteria were then incubated in the 50 ml TSB medium having di#erent concentrations of ammonia-N (control, 0·26, 1·28, 2·57 and 5·14 mg l 1) in 250 ml flasks at 30 C. Ammonia-N concentration was prepared by dissolving 38·2 g of NH4Cl (Merck reagent grade) in 1 l distilled water to make 10 000 mg l 1 as stock solution. Each test was conducted in triplicate and bacterial growth was monitored at 12, 24, 48 and 120 h incubation by measuring absorbency at 601 nm using a Model U-2000 spectrophotometer (Hitachi, Tokyo, Japan). EFFECT OF AMMONIA ON VIRULENCE OF ENTEROCOCCUS
After 24 h cultivation, Enterococcus in test media was harvested by centrifugation at 7155g for 15 min at 4 C. The pellet was resuspended in saline solution (0·85% NaCl) at 1107 cfu ml 1, as the stock bacterial suspension for challenges. Bacteria from the TSB medium, containing ammonia-N concentrations of control, 0·26, 1·28, 2·57 and 5·14 mg l 1, were served as test media and were tested for virulence of Enterococcus. M. rosenbergii (8–12 g in the intermolt stage) were obtained from a commercial farm in Pingtung, Taiwan, and acclimated in the laboratory for 2 weeks prior to experimentation. Bacterial suspensions (20 l) were injected into the ventral cephalothoracic sinus of each prawn. The trial was conducted in triplicate with a dose of 2105 cfu prawn 1. The test and control groups comprised of 10 prawns each (180 tests in all). Challenge tests were conducted following the method of Cheng & Chen [17]. Prawns were kept in water at 281 C at pH 7·3–7·8 with aeration. Control prawns were injected with an equal volume of sterile saline solution. EFFECT OF AMMONIA ON THE RESISTANCE OF M. ROSENBERGII TO ENTEROCOCCUS
Stocks were cultured on TSB medium for 24 h at 30 C, and then centrifuged at 7155g for 15 min at 4 C. The supernatant was removed and the bacterial pellet was resuspended in saline solution (0·85% NaCl) at 1107 cfu ml 1 as the stock bacterial suspension for injection challenges.
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M. rosenbergii (8–12 g in the intermolt stage) used in this study were obtained from a commercial farm at Pingtung, and acclimated in the laboratory for 2 weeks prior to experimentation, as described above. Challenge tests at 2105 cfu prawn 1 were conducted with test and control groups comprising 10 prawns for each replicate. Bacterial suspensions (20 l) were injected into the ventral cephalothoracic sinus of each prawn. After injection, prawns were kept in 60 l glass aquaria (10 prawns each) containing 40 l water with di#erent concentrations of ammonia-N at control, 0·5, 1·0, 1·5 and 3·0 mg l 1, as test solutions, and exposed for 7 days. The measured concentration of ammonia-N at every test solution was renewed every 24 h and was 0·06, 0·55, 1·01, 1·68 and 3·18 mg l 1 ammonia-N for the treatment in control, 0·5, 1·0, 1·5 and 3·0 mg l 1, respectively [26]. The prawns were kept in water at 281 C with aeration. Prawns injected with equal volumes of sterile saline solution and exposed to 3·18 mg l 1 ammonia-N served as unchallenged control (Table 1). EFFECT OF AMMONIA ON IMMUNE PARAMETERS OF M. ROSENBERGII
M. rosenbergii (20–30 g in the intermolt stage) used in this study were harvested and acclimated, as described above. Two prawns were kept in 60 l glass aquaria containing 40 l of test solution with di#erent ammonia-N concentrations. The prawns were exposed to each test solution for 7 days with daily water replacement. Each test solution was conducted in triplicate. The mean measured concentration of ammonia-N in each test solution was the same as that above (Table 1). Haemolymph was sampled individually at the beginning and after 7 days exposure to di#erent test solutions. Haemolymph (100 l) was withdrawn from the ventral sinus of each prawn into a 1 ml sterile syringe (25 G) containing 0·9 ml anti-coagulant solution (trisodium citrate 0·114 M, sodium chloride 0·1 M, pH 7·45; osmolarity 490 mOsm kg 1). A drop of haemolymph suspension was placed on a haemocytometer and the THC and di#erential haemocyte count (DHC) measured using an inverted phase contrast microscope (Leica DMIL, Leica Microsystems Wetzlar GmbH, Germany). Phenoloxidase activity was measured spectrophotometrically by recording the formation of dopachrome from L-dihydroxyphenylalanine (L-DOPA) [27]. The diluted haemolymph was centrifuged at 300g at 4 C for 10 min, the supernatant discarded and the pellet rinsed, resuspended gently in cacodylatecitrate bu#er (sodium cacodylate 0·01 M, sodium chloride 0·45 M, trisodium citrate 0·10 M, pH 7·0), and then centrifuged again. The pellet was then resuspended with 200 l cacodylate bu#er (sodium cacodylate 0·01 M, sodium chloride 0·45 M, calcium chloride 0·01 M, magnesium chloride 0·26 M, pH 7·0). The cell suspension (100 l) was incubated with 50 l of trypsin (1 mg ml 1), which served as an elicitor, for 10 min at 25–26 C. L-DOPA (50 l) was added and then 800 l of cacodylate bu#er was added 5 min later. The optical density at 490 nm was measured using a Hitachi U-2000 spectrophotometer (Tokyo, Japan). The control solution, which consisted of 100 l of cell suspension, 50 l cacodylate bu#er (to replace the trypsin) and 50 l of L-DOPA, was used for the background phenoloxidase activity in all test conditions. The background
3·18 Control (0·06) 0·55 1·01 1·68 3·18
Ambient ammonia-N (mg l 1) 30 30 30 30 30 30
Number of prawns 0 13·30·3a 20·05·8a 26·70·9a 26·70·3a 26·70·7a
24 h 0 23·30·3b 40·05·8ab 40·00·6ab 53·30·7a 50·01·0a
48 h 0 23·30·3c 43·36·7bc 50·01·0ab 70·00·6a 70·00·6a
72 h 0 23·30·3c 46·70·3b 56·70·3b 80·00·6a 83·30·3a
96 h
0 23·30·3c 46·70·3b 56·70·3b 80·00·6a 86·70·3a
120 h
Cumulative mortality (%)
At 281 C and pH 7·3–7·8. Data in the same column with di#erent letters are significantly di#erent (P<0·05) among treatments. Values are mean S.E.
Control 2105 2105 2105 2105 2105
Bacterial dose (cfu prawn 1)
0 26·70·3c 53·30·3d 66·70·3c 83·30·3b 96·70·3a
144 h
Table 1. Susceptibility of Macrobrachium rosenbergii to Enterococcus in di#erent concentrations of ammonia-N
0 26·70·3e 53·30·3d 66·70·3c 83·30·3b 96·70·3a
168 h
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phenoloxidase activity was subtracted from the phenoloxidase activity of prawns for all test conditions and ranged from 0·02 to 0·08. Respiratory burst activity of haemocytes was quantified using the reduction of nitroblue tetrazolium (NBT) to formazan as a measure of superoxide anion (O2 ) production, as described by several scientists [9–11]. Haemolymph (100 l) in anti-coagulant solution were deposited in l plate wells previously coated with 100 l poly-L-lysine solution (0·2%) to improve cell adhesion. Microplates were centrifuged at 300g for 15 min. Plasma was removed and then 100 l zymosan (0·1% in Hank’s solution minus phenol red) were added and allowed to react for 30 min at room temperature. Zymosan was discarded and the haemocytes were washed three times with 100 l Hank’s solution then stained with 100 l NBT solution (0·3%) for 30 min at room temperature. NBT solution was removed and the haemocytes were fixed and washed three times with 70% methanol (100 l) and air-dried. The formazan was dissolved by addition of 120 l KOH and 140 l dimethyl sulfoxide (DMSO). The optical density at 630 nm was measured in triplicate in a micrplate reader (Dynex Mrx II, U.S.A.). STATISTICAL ANALYSIS
All data were subjected to one-way ANOVA [28]. If significant di#erences were indicated at the 0·05 level, then the Duncan Multiple Range test was used to identify significant di#erences among the treatments [29]. III. Results EFFECT OF AMMONIA ON THE GROWTH OF ENTEROCOCCUS
The bacterium Enterococcus grew well in the TSB medium containing ammonia-N concentration in the range of 0 to 5·14 mg l 1. The log phase occurred at 12 and 24 h, and the bacterial density was highest after 24 h of incubation in all test media. EFFECT OF AMMONIA ON VIRULENCE OF ENTEROCOCCUS
All the unchallenged control prawns survived. In contrast, the onset of mortality occurred at 16–24 h in the challenged prawns. Cumulative mortality after 24 h was significantly higher for the bacteria incubated in control medium and the medium with 0·26 mg l 1 ammonia-N, than those incubated in other medium with 1·28, 2·57 and 5·14 mg l 1 ammonia-N. After 120 h of challenge until the end of the experiment at 168 h, the cumulative mortality of the prawns was significantly higher (P<0·05) for the bacteria incubated in control medium than those incubated in other media with ammonia-N. Over 168 h, the cumulative mortality of the prawns was 53·3, 36·7, 40·0, 36·7 and 33·3% for the bacteria in control solution, or medium with 0·26, 1·28, 2·57 and 5·14 mg l 1 ammonia-N, respectively. EFFECT OF AMMONIA ON THE RESISTANCE OF M. ROSENBERGII TO ENTEROCOCCUS
Results from Enterococcus challenge tests in test water with di#erent ammonia-N concentrations are shown in Table 1. The unchallenged control
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Phenoloxidase activity
0.6
0.4
*
*
*
0.55
1.68
3.18
0.2
0.0
Control
–1
Ammonia-N concentration (mg l )
Fig. 1. Mean ( S.E.) phenoloxidase activity in the haemocytes of M. rosenbergii at the beginning ( ), and after 7 days exposure () to di#erent concentrations of ammonia-N. Each bar represents mean value from six determinations with standard error. Asterisk indicates a significant di#erence from the initial sampling.
prawns survived. In contrast to saline-injected controls, all challenged prawns ceased feeding after injection. The onset of mortality occurred between 10 and 16 h in all challenge prawns. Cumulative mortality of prawns in the control solution after 48 h was significantly lower than the test water with ammonia-N at 1·68 and 3·18 mg l 1. The cumulative mortality increased directly with ammonia-N concentration and exposure time after 96 h, with a significant dose e#ect between all ammonia-N concentrations by the end of the experiment (Table 1). EFFECT OF AMMONIA ON THE IMMUNE PARAMETERS OF M. ROSENBERGII
No significant di#erence in THC and DHC was observed among the prawns at the beginning, and following 7 days exposure, to ammonia-N concentration in the range of 0·07–3·18 mg l 1. The mean S.E. THC varied from 59·112·2105 to 92·718·2105 cell ml 1. The mean S.E. DHC varied from 52·311·9105 to 84·217·4105 cell ml 1 for hyaline cells, 2·80·6105 to 4·61·4105 cell ml 1 for semi-granular cells, and 3·80·7105 to 4·961·4105 cell ml 1 for large granular cells. No significant di#erence in phenoloxidase activity was observed between the prawns in the control solution at the beginning and 7 days later. Phenoloxidase activity decreased significantly (P<0·05) when the prawns were exposed to ammonia-N at 0·55, 1·68 and 3·18 mg l 1 (Fig. 1). The relative phenoloxidase activity of prawns at the beginning and after 7 days exposure to 0·55, 1·68 and 3·18 mg l 1 ammonia-N decreased by 36%, 37% and 47% compared to that of prawns in the control solution (Fig. 1). No significant di#erence in the respiratory burst, which was expressed as NBT-reduction per 100 l haemolymph, was observed for the prawns in the control solution between the beginning and 7 days. A significant increase (P<0·05) of NBT-reduction (about 30%) occurred for the prawns following
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0.10 *
Respiratory burst
0.08
* *
0.06
0.04
0.02
0.00
Control
0.55
1.68
3.18 –1
Ammonia-N concentration (mg l )
Fig. 2. Mean respiratory burst in the haemocytes of M. rosenbergii at the beginning ( ), and after 7 days exposure () to di#erent concentrations of ammonia-N. Each bar represents mean value from six determinations. See Fig. 1 for statistical information.
7 days exposure to 0·55 mg l 1 ammonia-N. However, the NBT-reduction decreased significantly, by 26 and 23%, for the prawns following 7 days exposure to 1·68 and 3·18 mg l 1 ammonia-N, respectively (Fig. 2). IV. Discussion It is well known that environmental parameters in media a#ect the growth of pathogens and their production of toxins [30, 31]. Ramesh et al. [32] also suggested that environmental parameters influence the growth of luminous bacteria. Smith [33] indicated that fast-growing pathogens could overwhelm the initial, non-specific defense and cause disease before more powerful immune defences can operate fully. Slow-growing pathogens are more prone to both types of defence. Previous research indicated that Enterococcus incubated in BHIB medium grew from pH 3 to 10 with optimum growth at pH 7–8, and indicated that the Enterococcus grew from 5 to 45 C with optimum growth at 25–30 C in BHIB medium [17]. The present study indicated that Enterococcus grew well in TSB medium with ammonia-N at a concentration of 5·14 mg l 1. Riquelme et al. [34] reported that water temperature, and other parameters, a#ected the pathogenicity of Vibrio strains in Chilean scallop Argopecten purpuratus larvae. Prayitno & Latchford [35] demonstrated that exposure of luminous bacteria Vibrio harveyi to low salinity levels (10 and 15 ppt) for 12 h, before use in immersion challenge tests with tiger shrimp Penaeus monodon larvae, resulted in higher mortality. They also reported that exposure of V. harveyi to pH 5·5 reduced its pathogenicity. Previous research indicated that the optimal level for the growth of Enterococcus incubated at its optimal growth levels (pH 7–8, 25–30 C and 0·5–1·0% NaCl) enhanced the virulence to M. rosenbergii [17]. The fact that M. rosenbergii challenged with Enterococcus
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incubated in TSB medium with ammonia-N had lower mortality indicated ammonia decreases the virulence of Enterococcus. Several scientists have investigated the e#ects of environmental parameters on crustacean defence mechanisms. An increased prevalence in the shell disease of marine decapod crustaceans has been reported to result from polluted environments, also suggesting a decrease in immunocompetence [36, 37]. In decapod crustaceans, circulating haemocytes are associated with cellular defence [4, 5]. It is well known that life cycle, food intake, disease outbreak, pollutants and environmental stress a#ect the circulating haemocyte count of crustaceans, both in quantity and quality [38–41]. Circulating haemocytes of M. rosenbergii at di#erent size, season and molting cycle have been reported by Cheng & Chen [42]. Le Moullac et al. [5] reported that the THC was significantly higher during the premolt stage than during the intermolt stage, whereas phenoloxidase activity was significantly higher during the intermolt stage than during the premolt stage for the blue shrimp Penaeus stylirostris. Cheng & Chen [42] indicated that the molting cycle is one of the main intrinsic factors a#ecting THC, and reported that the THC was significantly higher at stage C and lower at stage D3, with no significant di#erence between male and female [43]. In the present study the M. rosenbergii used was stage C, therefore they are considered to have the same defence condition. Circulating haemocytes are also a#ected by extrinsic factors like temperature, pH, salinity and dissolved oxygen. An increase in water temperature has been reported to increase THC in several crustaceans [44]. M. rosenbergii reared at temperatures of 27–28 C and 30–31 C had significantly higher THC than those reared at 20–21 C and 33–34 C, and the prawns reared at pH 7·5– 7·7 have significantly higher THC than those reared at pH 4·6–5·0 and 9·0–9·5 [16]. Blue shrimp Penaeus stylirostris, following 24 h exposure to dissolved oxygen as low as 1 mg O2 l 1, decreased its THC [11]. The decrease of THC was considered to be mainly due to the decrease of hyaline cells. P. stylirostris, following exposure to ammonia at 3·0 mg l 1, decreased its THC by 30% [41]. Unfortunately, they did not report the exposure time. No changes in THC were observed in shore crab Carcinus maenas following 30 days exposure to 50 g l 1 mercury [44]. The present study indicated that no significant di#erence of THC and DHC was observed between the prawns in the control solution and following 7 days exposure to ammonia-N as high as 3·18 mg l 1. Le Moullac et al. [11] reported that P. stylirostris following hypoxia exposure increased its phenoloxidase activity, but decreased its THC. Negative correlation between phenoloxidase activity and THC was also observed in P. stylirostris when ambient temperature dropped from 27 C to 18 C [41]. Smith & Johnston [40] reported that common shrimp Crangon crangon, following exposure to PCB 15 (polychlorinated biphenyl 15), produced significantly decreased THC and phenoloxidase activity. Cheng & Chen [16] reported that both phenoloxidase activity and THC of M. rosenbergii were significantly higher at pH 7·5–7·7 and 30–31 C, and were significantly lower at pH 4·6–5·0, 9·0–9·5 and 33–34 C, and at freshwater. The present study indicated that the phenoloxidase activity of M. rosenbergii was significantly lower for the prawns exposed to ammonia-N at 0·55, 1·68 and 3·18 mg l 1 after 7 days.
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This fact indicated that the decrease of phenoloxidase activity under ammonia stress was not a consequence of a reduced THC or altered DHC. NBT staining has been used for both qualitative and quantitative analyses of O2 (superoxide anions) generated by haemocytes, which are the first product of the respiratory burst. Song & Hsieh [9] reported that -glucan had the strongest stimulative e#ect on haemocytes in P. monodon in terms of generating O2 and H2O2 (hydrogen peroxide), which were considered to play an important role in shrimp microbicidal activity when compared to ClO (hypochlorites) and MPO (myeloperoxidase). Le Moullac & Ha#ner [41] reported that white shrimp Penaeus vannamei, following injection of propiconazole (a fungicide) induced an increase of respiratory burst at day 6, but caused dose-dependent decrease of the respiratory burst at day 13. Le Mollac et al. [11] indicated that the production of superoxide radicals, decreased in hypoxic P. stylirostris, was due to the decrease of THC, suggesting that NADPH oxidase responsible for production of superoxide was not a#ected under hypoxia. The present study indicated that M. rosenbergii following 7 days exposure to 0·55 mg l 1 ammonia-N stimulated the total production of superoxide radicals, whereas the prawns following 7 days exposure to ammonia-N greater than 1·68 mg l 1 decreased the total production of superoxide radicals. This fact indicated that the NADPH oxidase is a#ected under the stress of ammonia. Further research is needed to examine the activities of superoxide dismutase, catalase and peroxidase to understand the production of superoxide radicals under the stress of ambient ammonia. A relationship between salinity and infectious hypodermal and haematopoietic necrosis (IHHN) has been observed in white shrimp Penaeus vannamei [45]. Le Moullac et al. [11] reported that hypoxia reduced the resistance of shrimp to experimental infection with Vibrio alginolyticus. Previous research has indicated that Enterococcus infected M. rosenbergii is exacerbated by high pH (8·8–9·5) and high temperature (33–34 C), but reduced by low salinity (5–10 ppt) [15], indicating the susceptibility of crustacean to pathogen. The present study indicated that the susceptibility of M. rosenbergii to Enterococcus increased directly with ammonia-N concentration, when the prawns were exposed to ammonia. LeMoullac & Ha#ner [41] indicated that the defence functions belonging to the phenoloxidase activity and the peroxinectin are reduced at the level of gene expression in P. stylirostris when exposed to ammonia. In conclusion, the present study reports that ambient ammonia a#ects immune competence and the degree of protection to Enterococcus infection in M. rosenbergii. This increased susceptibility is considered to be related to the decrease in phenoloxidase activity, which is more relevant than haemocyte count and the respiratory burst products in the resistance of prawns infected by Enterococcus. Ammonia decreases the virulence of Enterococcus pathogen, and increases the susceptibility to Enterococcus by reducing the immune ability of M. rosenbergii. Further research is needed to determine other immune competence activities like phagocytosis, lectins and agglutinin and the product of immune inhibitors like 2-macroglobulin in the haemolymph of M. rosenbergii under ammonia stress.
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The paper was supported by the National Science Council (NSC 89-2313-B-020-10), Republic of China. We appreciate Mr P. C. Leu and Miss C. S. Wang for their assistance in the experiment.
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