Artemia has been proposed as a model organism to study the biology of infections, host-microbes interaction, the regulation of gene expression during embryonic development or the effect of chemotherapy agents against diseases on penaeid shrimp, lobsters and other crustaceans (Marques, 2005; Marques et al., 2006; Nunes et al., 2006; Soltanian, 2007).
According to Soltanian (2007), Artemia possesses several unique characteristics, enabling their advantages in using as model system for basic research in animal biology, such as: (i) ability to be cultured under different axenic and gnotobiotic conditions with a simple experiment apparatus; (ii) the possibility to be cultured with a wide range of feed sources due to the non-selective continuous filter feeding; (iii) short generation time (2-3 weeks) although they can live for several months, and under favorable conditions they can grow from nauplius to adult in only 8 days; (iv) high fecundity that a female can reproduce 300 nauplii or cysts every 4 days (FAO, 2013; Van Stappen, 1996); (v) availability of large quantities of cysts, different species and strains from all continents (and hence different genetic background (Bossier et al., 2004)); (vi) small body size that can be cultured easily at high density and/or on a small scale with very simple systems; (vii) high adaptability to a variety of testing conditions; (viii) bisexual versus parthenogenetic reproduction strategies, high hatchability, simple availability, and low costs of the tests (Dvorak et al., 2012).
23 3. MATERIALS AND METHODS
3.1. Materials
3.1.1. Bacteria and growth conditions
V. harveyi BB120 was used in all experiments. The strain was grown at 28⁰C in marine broth (Difco, Detroit, MI, USA) under constant agitation 100min-1. Density was measured spectrophotometrically at 600 nm.
3.1.2. Hormones
Norepinephrine (Sigma-Aldrich) and dopamine (Sigma-Aldrich) were added to the media at a concentration of 50 µM and 100 µM. The 10 mM stock solution of norepinephrine and dopamine were made by dissolving in hydrocholoride acid (HCl 0.1N) and distilled water, respectively. The stock solution was stored at -20⁰C.
3.1.3. Hormone antagonists
The catecholamine antagonists used in this study are listed in Table 5.
Table 5. Catecholamine antagonists used in this study
Antagonist Specificity Solvent Stock
concentration
Labetalol hydrochloride α and β-adrenergic (α1, β) Ethanol 10mM
Chlorpromazine hydrochloride Dopaminergic Water 10mM
LED209
24 3.2. In vitro experiments
3.2.1. Swimming motility assays
Swimming motility assays were performed on soft agar (Marine broth containing 0.3%
agar) as described previously (Rui et al., 2008). V. harveyi BB120 was grown overnight in marine broth, and 5 µl of the culture (diluted to OD600 = 0.5) was spotted in the center of the soft agar plates. Plates were incubated for 24 hours, after which the diameters of the motility halos were measured. All assays were done with fresh media in at least 5 replicates.
3.2.2. Effect of hormones on other virulence factors
In order to observe the effect of hormones on other virulence factors, some enzymatic activities of V. harveyi BB120 including lipase, phosphilipase, gelatinase, hemolysin and casinase were carried out according to Natrah et al. (2011).
3.2.2.1. Lipase
Lipase activity was tested by adding 1% Tween 80 (Sigma – Aldrich) into marine agar.
Two concentrations of hormones at 50µM and 100µM were examined. The development of opalescent zones around the colonies was observed and the diameter of the zones was measured after 3 days of incubation at 28⁰C.
3.2.2.2. Phospholipase
Phospholipase activity was assessed according to Natrah et al. (2011). Similarly, marine agar was supplemented with 1% egg yolk emulsion (Sigma – Aldrich). Two concentrations of 50µM and 100µM for both norepinephrine and dopamine were used. The development of opalescent zones around the colonies was observed and the diameters of the zones were measured after 3days of incubation at 28⁰C.
3.2.2.3. Gelatinase
Gelatinase assay plates were done by mixing 0.5% gelatin (Sigma – Aldrich) with marine agar. Two different concentrations of norepinephrine and dopamine were added into gelatinase plates. After 7 days of incubation at 28⁰C, saturated ammonium sulfate (80%) in distilled water was poured over the plates and after 2 min, the diameters of the clearing zones around the colonies were measured.
25 3.2.2.4. Hemolysin
Hemolytic activity was determined by supplementing marine agar with 5% defibrinated sheep blood (Oxoid, Basingstoke, Hampshire, UK). In this assay, two concentrations of hormones were used to evaluate if there are some effects of hormones on hemolytic activity of V. harveyi BB120. The clearing zones were measured after 2 – 3 days of incubation at 28⁰C.
3.2.2.5. Caseinase
In this assay, two concentrations of norepinephrine and dopamine were examined whether if they impacts caseinase activity of V. harveyi or not. The caseinase assay plates were prepared by mixing double strength marine agar with 4% skim milk powder (Oxoid, Basingstoke, Hampshire, UK), autoclaved separately at 121⁰C for 5minutes to prevent denaturation of protein (milk). Clearing zones surrounding the bacterial colonies were measured after 2-3 days of incubation.
3.3. In vivo experiments - Effect of hormones and their antagonists on virulence of V.
harveyi towards brine shrimp (A. franciscana) larvae
3.3.1. Axenic hatching of brine shrimp larvae
Two hundred milligrams of high-quality hatching cysts of A. franciscana (EG® Type;
INVE Aquaculture, Belgium) were hydrated in 18mL of tap water for 1hour with aeration.
Sterile cysts were obtained by decapsulation based on the method described by Marques et al. (2004). Briefly 660µL of NaOH (32%) and 10mL of NaOCl (50%) were added into the hydrated cyst suspension to facilitate decapsulation. Then, 14mL of Na2S2O3 (10gL-1) was added to stop the decapsulating process after maximum 2 minutes. The decapsulated cysts were collected by washing over 100µm sieve with autoclaved (moist heat at 121°C for 15 minutes) artificial seawater (35gL-1 of Instant Ocean synthetic sea salt (Aquarium Systems Inc., Sarrebourg, France). The cysts were resuspended again in a 50mL falcon tube containing 30mL of autoclaved artificial seawater and incubated for 24-28 hours on a rotor (4 min-1) at 28⁰C with constant illumination (approximately 2000lux). The axenity of cysts was verified by inoculating 1mL of culture water into 9mL of marine broth and incubating at 28°C for 24 hours. After 28 hours of hatching, batches of 30 nauplii were counted and transferred to fresh, sterile 50-mL tubes containing 30mL of filtered and autoclaved seawater. Finally, the tubes were returned to the rotor and kept at 28°C. All manipulations were performed in a laminar flow to maintain sterility of the cysts and nauplii.
26 3.3.2. Brine shrimp challenge tests
Challenge tests were performed as described by Defoirdt et al. (2005) with some modifications (Defoirdt et al., 2005). The animals were challenged with 107CFUmL-1 of the vibrios per mL Artemia culture water. The hormones and hormone antagonists were added directly to the culture water at different concentrations. A suspension of autoclaved LVS3 bacteria (Verschuere et al., 1999) in filtered and autoclaved seawater was added as feed at the start of the challenge test equivalent to 107 cells mL-1 culture water. After that these tubes were put back on the rotor and kept at the same conditions as the hatching’s.
The survival rate of the brine shrimp was counted after 2 days. Each treatment was carried out in quadruplicate and each experiment was repeated twice to verify the reproducibility.
In each test, the sterility of the control treatments were checked at the end of the challenge by inoculating 1mL of Artemia culture water to 9mL of marine broth and incubating the mixture for 2 days at 28°C. Detail treatments are listed in the tables below:
Table 6. Experimental set-up for the in vivo experiment with norepinephrine and its antagonists
Treatment of norepinephrine and the antagonists V. harveyi BB120
V. harveyi BB120 + Norepinephrine
V. harveyi BB120 + Norepinephrine + Phentolamine V. harveyi BB120 + Norepinephrine + Phenoxybenzamine V. harveyi BB120 + Norepinephrine + Labetalol
V. harveyi BB120 + Norepinephrine + LED209 Norepinephrine
Phentolamine Phenoxybenzamine Labetalol
LED209 Control
27
(The concentration of norepeniphrine and its antagonists would be indicated in the experiment of effect of hormones and their antagonists on swimming motility as described in 3.2.2.)
Table 7. Experimental set-up for the in vivo experiment with dopamine and its antagonists
Treatment of dopamine and the antagonists
V. harveyi BB120
V. harveyi BB120 + Dopamine
V. harveyi BB120 + Dopamine + LED209
V. harveyi BB120 + Dopamine + Chlorpromazine
Dopamine LED209
Chlorpromazine Control
(The concentration of dopamine and its antagonists would be indicated in the experiment of effect of hormones and their antagonists on swimming motility as described in 3.2.2.)
3.4. Statistical analysis
Data analysis was carried out using the Statistical Package for the Social Sciences SPSS statistical software (version 16.0). All data were compared with one-way ANOVA, followed by a Ducan’s post hoc test. Two-way ANOVA was applied to evaluate the effect of V. harveyi, the hormones or an interaction between hormone and pathogen on survival of the brine shrimp. A 1% significance level was used to analyse statically for all virulence factors of V. harveyi and survivals of Artemia nauplii in challenge tests.
28 4. RESULTS
4.1. Effects of stress hormones on virulence factors of V.harveyi BB120 4.1.1. Swimming motility
Swimming motility of V. harveyi was observed using soft marine agar by supplementing 0.3% agar powder into marine broth. The results of the swimming motility assays showed that both norepinephrine (NE) and dopamine (Dop) could increase the swimming motility of V. harveyi compared to the controls (Table 8). Norepinephrine seemed to show a bit stronger effect than dopamine (P<0.05), however it did not show a significant difference at the significance level 1% (P>0.01).
Table 8. Effects of the catecholamine stress hormones dopamine and norepinephrine on the swimming motility of V. harveyi on soft agar
Treatments Motility halo diameter (mm)
Norepinephrine 50µM 30.8 ± 1.6d
Norepinephrine 100µM 24.0 ± 1.6bc
Dopamine 50µM 27.6 ± 2.5cd
Dopamine 100µM 22.6 ± 3.3b
Control (soft agar) 16.8 ± 0.8a
Results are expressed as average ± standard deviation of five replicates
Values with a different superscript letter are significantly different from each other (P< 0.01).
We hypothesized that the higher concentration of stress hormone would result in stronger swimming activity, but actually the results were not shown like the hypothesis. The lower motility in the presence of 100 µM norepinephrine might partly be due to the solvent (HCl) as the solvent control also showed lower motility (see Table A1 in appendix). Based on these results, 50µM was selected for the later experiments with stress hormones and antagonists.
4.1.2. Lipase and phospholipase activity
Lipase and phospholipase activities were examined by mixing 1% of Tween 80 and 1%
egg yolk emulsion into autoclaved marine agar, respectively. Lipase or phospholipase
29
activity resulted in the formation of an opalescent zone surrounding colonies on the respective agars. The ratio between opalescent zone and colony diameter was recorded after 3 days incubation at 28⁰C. As can be seen in Table 9 and Table 10, there were no significant differences between treatments.
Table 9. Lipase activity of V. harveyi BB120 towards Tween 80 after 3 days of incubation, with and without catecholamines. CD: Colony Diameter, OZ: Opalescent
Zone
Treatments CD (mm) OZ (mm) Ratio OZ/CD
Norepinephrine 50µM 9.8 ± 0.4 20.4 ± 1.0 2.09 ± 0.10a
Norepinephrine 100µM 10.0 ± 0.5 20.0 ± 0.7 2.00 ± 0.07a
Dopamine 50µM 10.6 ± 0.7 22.0 ± 1.0 2.09 ± 0.13a
Dopamine 100µM 10.3 ± 1.4 20.9 ± 2.0 2.04 ± 0.14a
Control 9.7 ± 0.5 19.7 ± 1.1 2.04 ± 0.11a
Results are expressed as average ± standard deviation of nine replicates
Values with a different superscript letter are significantly different from each other (P< 0.01).
Table 10. Phospholipase activity of V. harveyi BB120 towards egg yolk after 3 days of incubation, with and without catecholamines. CD: Colony Diameter, OZ: Opalescent
Zone
Treatments CD (mm) OZ (mm) Ratio OZ/CD
Norepinephrine 50µM 7.7 ± 0.7 13.2 ± 0.8 1.73 ± 0.10a
Norepinephrine 100µM 7.9 ± 0.3 13.2 ± 0.4 1.68 ± 0.09a
Dopamine 50µM 7.8 ± 0.7 13.7 ± 0.9 1.76 ± 0.14a
Dopamine 100µM 7.9 ± 0.3 13.0 ± 0.3 1.65 ± 0.1a
Control 7.6 ± 0.5 13.1 ± 0.6 1.74 ± 0.16a
Results are expressed as average ± standard deviation of nine replicates
Values with a different superscript letter are significantly different from each other (P< 0.01).
30 4.1.3. Gelatinase activity
Gelatinase assay plates were obtained by autoclaving marine agar supplemented with 0.5%
gelatin. After incubation at 28⁰C for 7 days, clearing zone and colony diameter were measured after washing with saturated ammonium sulfate 80%. Gelatinase activity results in the formation of a clearing zone surrounding colonies on this medium. Interestingly, the presence of both stress hormones resulted in a significant reduction of gelatinase activity (Table 11). At the same concentration, it can be found that the data did not reveal any significant differences between norepinephrine and dopamine (P>0.01). Table 11 also shows that the effect of the catecholamines is concentration-dependent.
Table 11. Gelatinase activity of V. harveyi after 7 days of incubation, with and without catecholamines. CD: Colony Diameter, CZ: Clearing Zone
Treatments CD (mm) CZ (mm) Ratio CZ/CD
Norepinephrine 50µM 9.9 ± 0.3 52.4 ± 1.9 5.31 ± 0.20ab
Norepinephrine 100µM 10.8 ± 1.0 52.7 ± 1.4 4.91 ± 0.32a
Dopamine 50µM 8.7 ± 0.7 47.9 ± 1.2 5.57 ± 0.59b
Dopamine 100µM 10.2 ± 1.1 49.1 ± 1.1 4.83 ± 0.33a
Control 7.8 ± 0.4 48.9 ± 1.1 6.30 ± 0.34c
Results are expressed as average ± standard deviation of nine replicates
Values with a different superscript letter are significantly different from each other (P< 0.01).
4.1.4. Hemolytic activity
In order to evaluate the effects of stress hormones on hemolytic activity of V. harveyi, hemolytic assay plates were prepared by mixing 5% sheep blood with autoclaved marine agar. Hemolytic activity is manifested by degradation of the blood cells. As depicted in Figure 3, V. harveyi exhibited β-hemolytic activity because the red blood cells surrounding the colony were completely lysed, resulting in a clearing zone. The ratio between clearing zone and colony diameter was determined after 72 hours of incubation.
31
Figure 3. Hemolytic activity of V. harveyi against sheep blood
According to table 12, it can be concluded that the stress hormones could increase hemolytic activity of V. harveyi. Increasing the concentration of both norepinephrine and dopamine to 100µM resulted in an increased activity. Further, the data did not show any differences between the two stress hormones (P > 0.01).
Table 12. Hemolytic activity of V. harveyi after 72 hours incubation, with and without catecholamines. CD: Colony Diameter, CZ: Clearing Zone
Treatments CD (mm) CZ (mm) Ratio CZ/CD
Norepinephrine 50µM 11.0 ± 0.0 23.3 ± 0.8 2.12 ± 0.07a
Norepinephrine 100µM 11.0 ± 0.0 25.0 ± 0.9 2.27 ± 0.08b
Dopamine 50µM 11.0 ± 0.0 23.5 ± 0.6 2.14 ± 0.05a
Dopamine 100µM 11.0 ± 0.0 25.0 ± 0.6 2.27 ± 0.06b
Control 12.0 ± 0.0 24.3 ± 1.0 2.03 ± 0.09a
Results are expressed as average ± standard deviation of six replicates
Values with a different superscript letter are significantly different from each other (P< 0.01).
32 4.1.5. Caseinase activity
Caseinase assays were done by autoclaving double strange of marine agar and mixing it afterwards with 4% skim milk powder heated at 121⁰ for 5 minutes. The data expressed in Table 13 indicated that although the presence of stress hormones seemed to increase caseinase activity of V. harveyi, the data did not show significant differences between treatments and the control except for norepinephrine 100µM.
Table 13. Caseinase activity of V. harveyi after 72 hours incubation, with and without catecholamines. CD: Colony Diameter, CZ: Clearing Zone
Treatments CD (mm) CZ (mm) Ratio CZ/CD
Norepinephrine 50µM 10.7 ± 0.8 22.3 ± 1.0 2.11 ± 0.21ab
Norepinephrine 100µM 10.5 ± 0.6 22.7 ± 1.2 2.16 ± 0.13b
Dopamine 50µM 12.0 ± 0.0 24.8 ± 1.0 2.07 ± 0.08ab
Dopamine 100µM 11.8 ± 0.8 25.0 ± 0.6 2.12 ± 0.17ab
Control 11.5 ± 0.6 21.5 ± 1.1 1.87 ± 0.09a
Results are expressed as average ± standard deviation of six replicates
Values with a different superscript letter are significantly different from each other (P< 0.01).
4.2. Effects of hormone antagonists on swimming motility of V. harveyi
As swimming motility is the phenotype that is most strongly affected by the catecholamines in V. harveyi BB120, in further experiments, we investigated whether catecholamine antagonists could neutralise the effects of the catecholamines (added at 50µM) on swimming motility.
4.2.1. Phentolamine
Phentolamine is a reversible α-adrenergic antagonist which competes with eukaryotic α- adrenergic receptors. In order to evaluate the impact of phentolamine, different concentrations were added into soft agar containing 50µM norepinephrine. Table 14 indicates that phentolamine could inhibit swimming motility of V. harveyi induced by norepinephrine at 100µM or more. At the lower concentration (50µM), it did not exhibit any antagonistic effects on swimming motility. In order to determine whether these
33
inhibitions come from the effects of antagonist or the solvent (methanol), we also checked the swimming motility of bacteria under the presence of different volumes of methanol used in dissolving phentolamine. Our data showed that there were not any effects on swimming motility of using methanol as a phentolamine solvent, except for the highest concentration (see Table A2 in appendix).
Table 14. Swimming motility of V. harveyi in the presence of norepinephrine and the α- adrenergic antagonist phentolamine
Treatments Motility halo diameter (mm)
Norepinephrine 50µM 33.6 ± 1.1c
Norepinephrine 50µM + Phentolamine 50µM 35.8 ± 1.8c
Norepinephrine 50µM + Phentolamine 100µM 26.2 ± 2.2ab
Norepinephrine 50µM + Phentolamine 200µM 28.8 ± 4.2b
Control 24.4 ± 0.6a
Results are expressed as average ± standard deviation of five replicates
Values with a different superscript letter are significantly different from each other (P< 0.01).
4.2.2. Phenoxybenzamine
Similar to phentolamine, phenoxybenzamine is also able to inhibit swimming motility of bacteria in the presence of norepinephrine (Table 15). As can be seen in Table 15, phenoxybenzamine could inhibit bacterial swimming motility induced by norepinephrine already at 10µM (P<0.01). These inhibitory effects of phenoxybenzamine were confirmed by excluding the possibility of the toxicity of the solvent DMSO (see Table A3 in the appendix). Interestingly, there were no significantly different effects of the three concentrations of phenoxybenzamine (10, 20 nor 50µM) (P>0.01).
34
Table 15. Swimming motility of V. harveyi in the presence of norepinephrine and the α- adrenergic antagonist phenoxybenzamine
Treatments Motility halo diameter (mm)
Norepinephrine 50µM 39.6 ± 1.5c
Norepinephrine 50µM + Phenoxybenzamine 10µM 31.0 ± 1.0ab Norepinephrine 50µM + Phenoxybenzamine 25µM 31.0 ± 1.0ab Norepinephrine 50µM + Phenoxybenzamine 50µM 32.2 ± 3.0b
Control 28.0 ± 1.2a
Results are expressed as mean ± standard deviation of five replicates
Values with a different superscript letter are significantly different from each other (P< 0.01).
4.2.3. S- propanolol
In a further experiment the effect of the nonselective β - adrenergic antagonist S-propanolol on norepinephrine-induced swimming motility was determined.
Table 16. Swimming motility of V. harveyi in the presence of norepinephrine and the β- adrenergic antagonist S-propanolol
Treatments Motility halo diameter (mm)
Norepinephrine 50µM 30.6 ± 1.95d
Norepinephrine 50µM + S-propanolol 100µM 30.8 ± 3.35d Norepinephrine 50µM + S-propanolol 200µM 27.4 ± 3.85cd Norepinephrine 50µM + S-propanolol 300µM 17.2 ± 1.10b
Norepinephrine 50µM + S-propanolol 500µM 0a
Control 25.6 ± 1.52c
Results are expressed as average ± standard deviation of five replicates
Values with a different superscript letter are significantly different from each other (P< 0.01).
35
At 200µM, S-propanolol did not show any effect on norepinephrine-induced motility (Table 16). Although S-propanolol exhibited the inhibitory effects at higher concentrations (300 and 500µM), our data showed that these effects were not due to S-propanolol but the toxicity of ethanol by adding very large volumes (see Table A4 in the appendix).
Therefore, it might be conclude that β-antagonist S-propanolol has no effect in V. harveyi.
4.2.4. Labetalol
Labetalol competes with norepinephrine through both of α- and β-adrenergic receptors.
According to table 17, labetalol was able to neutralise the effect of norepinephrine from 25µM (P<0.01).
Table 17. Swimming motility of V. harveyi in the presence of norepinephrine and the α- and β- adrenergic antagonist labetalol
Treatments Motility halo diameter (mm)
Norepinephrine 50µM 39.8 ± 0.5d
Norepinephrine 50µM + Labetalol 10µM 37.8 ± 1.6cd
Norepinephrine 50µM + Labetalol 25µM 36.6 ± 1.1c
Norepinephrine 50µM + Labetalol 50µM 33.0 ± 1.4b
Norepinephrine 50µM + Labetalol 100µM 29.2 ± 2.2a
Control 30.4 ± 2.1a
Results are expressed as average ± standard deviation of five replicates
Values with a different superscript letter are significantly different from each other (P< 0.01).
4.2.5. Chlorpromazine
Chlorpromazine is a dopaminergic antagonist competing for the eukaryotic dopamine receptor. Thus, chlorpromazine was added together with dopamine into soft agar to assess its effect. As can be seen in Table 18, chlorpromazine exhibited inhibitory effect on swimming motility of V. harveyi. Swimming activity was recorded to reduce sharply when the concentration of chlorpromazine increased from 10µM to higher. Importantly, this was probably due to toxicity as chlorpromazine at 25 µM and 50 µM completely inhibited the growth of V. harveyi.
36
Table 18. Swimming motility of V. harveyi in the presence of dopamine and the dopaminergic antagonist chlorpromazine
Treatments Motility halo diameter (mm)
Dopamine 50µM 40.6 ± 0.6d
Dopamine 50µM + chlorpromazine 10µM 34.2 ± 2.4c
Dopamine 50µM + chlorpromazine 25µM 0.00 ± 0.0a
Dopamine 50µM + chlorpromazine 50µM 0.00 ± 0.0a
Control 30.4 ± 2.1b
Results are expressed as average ± standard deviation of five replicates
Values with a different superscript letter are significantly different from each other (P< 0.01).
4.2.6. LED 209
LED 209 has been reported to block the bacterial adrenergic receptor QseC, which mediates the activation of virulence gene expression, thus in a further experiment we tested whether it can inhibit swimming motility of V. harveyi in the presence of norepinephrine and dopamine. In this experiment, LED209 was tested at three concentrations (0.05, 0.1 and 0.5µM).
Table 19. Swimming motility of V. harveyi in the presence of norepinephrine and the bacterial adrenergic antagonist LED209
Treatments Motility halo diameter (mm)
Norepinephrine 50µM 30.8 ± 1.9d
Norepinephrine 50µM + LED209 0.05µM 26.2 ± 1.5c
Norepinephrine 50µM + LED209 0.1µM 19.8 ± 1.3ab
Norepinephrine 50µM + LED209 0.5µM 18.6 ± 0.9a
Control 21.8 ± 2.4b
Results are expressed as average ± standard deviation of five replicates
Values with a different superscript letter are significantly different from each other (P< 0.01).
37
Table 19 shows that LED209 was able to inhibit norepinephrine-induced swimming motility of V. harveyi already at a very low concentration of 0.05µM. At higher concentration, the inhibitory effect was stronger, however, there was no significant difference between these two concentrations (P>0.01).
Similarly, LED209 was also able to inhibit dopamine-induced motility at 0.05µM and higher concentrations, but the strongest antagonistic effect was recorded at 0.5µM.
Table 20. Swimming motility of V. harveyi in the presence of dopamine and the bacterial adrenergic antagonist LED209
Treatments Motility halo diameter (mm)
Dopamine 50µM 30.6 ± 1.5c
Dopamine 50µM + LED209 0.05µM 24.6 ± 4.3b
Dopamine 50µM + LED209 0.1µM 21.4 ± 1.5b
Dopamine 50µM + LED209 0.5µM 16.8 ± 1.6a
Control 21.8 ± 2.4b
Results are expressed as average ± standard deviation of five replicates
Values with a different superscript letter are significantly different from each other (P< 0.01).
4.3. Brine shrimp challenge tests
Challenge experiments were performed to investigate the effects of norepinephrine, dopamine and their antagonists on survival of brine shrimp larvae in the presence of V.
harveyi BB120. The survival rates were counted after 48hours. The treatments of norepinephrine or dopamine together with V. harveyi BB120 resulted in the lowest survival (Table 21). On the contrary, in the presence of only norepeniphrine or dopamine (without pathogen), the larval survival rates were as high as in the control treatment, indicating that norepinephrine and dopamine are not toxic for Artemia. Consequently, it can be concluded
harveyi BB120. The survival rates were counted after 48hours. The treatments of norepinephrine or dopamine together with V. harveyi BB120 resulted in the lowest survival (Table 21). On the contrary, in the presence of only norepeniphrine or dopamine (without pathogen), the larval survival rates were as high as in the control treatment, indicating that norepinephrine and dopamine are not toxic for Artemia. Consequently, it can be concluded