The immune response of tiger shrimp Penaeus monodon and its susceptibility to Photobacterium damselae subsp. damselae under temperature stress

Aquaculture 258 (2006) 34 – 41 www.elsevier.com/locate/aqua-online

The immune response of tiger shrimp Penaeus monodon and its susceptibility to Photobacterium damselae subsp. damselae under temperature stress Feng-I Wang, Jiann-Chu Chen ⁎ Department of Aquaculture, College of Life Sciences, National Taiwan Ocean University, Keelung, Taiwan, 202, ROC Received 19 December 2005; received in revised form 12 March 2006; accepted 19 March 2006

Abstract Tiger shrimp Penaeus monodon held in 25‰ seawater and 26 °C seawater were injected with Photobacterium damselae subsp. damselae grown in TSB at a dose of 8.48 × 104 colony-forming units (cfu) shrimp− 1, and then reared onward at water temperatures of 22, 26 (control), 30 and 34 °C. Over 24–96 h, the cumulative mortalities of P. damselae subsp. damselae-injected shrimp held in 22 and 34 °C were significantly higher than those for injected-shrimp held in 26 and 30 °C. In a separate experiment, shrimp held in 25‰ seawater and 26 °C, and then cultured onward at 22, 26 (control), 30 and 34 °C were examined for immune parameters, phagocytic activity and clearance efficiency of P. damselae subsp. damselae at 12–96 h. THC (total hemocyte count), DHC (differential hemocyte count), phenoloxidase (PO) activity, respiratory burst, superoxide dismutase (SOD) activity, phagocytic activity and clearance efficiency in shrimp decreased significantly after 24–96 h transfer to 22 and 34 °C. It was concluded that transfer of tiger shrimp P. monodon from 26 °C to 22 and 34 °C reduced their resistance against P. damselae subsp. damselae infection. © 2006 Elsevier B.V. All rights reserved. Keywords: Penaeus monodon; Photobacterium damselae subsp. damselae; Temperature; Susceptibility; Immune parameters; Phagocytic activity; Clearance efficiency

1. Introduction Tiger shrimp Penaeus monodon, which is naturally distributed from the east coast of Africa, Red Sea to Pakistan, Malay Archipelago, Philippines and Australia, is commercially important in several Pacific rim countries. This species is euryhaline, and has a tolerance for salinity

⁎ Corresponding author. Tel.: +886 2 2462 0295; fax: +886 2 2462 0295. E-mail address: [email protected] (J.-C. Chen). 0044-8486/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2006.03.043

ranging from 3‰ to 45‰ with an iso-osmotic point of 750 mOsm kg− 1 which is equivalent to 25‰ (Cheng and Liao, 1986). Salinities in the range of 10–35‰ and temperatures in the range of 25–35 °C were suitable levels for growth of P. monodon (Liao, 1986; Chen, 1990); seasonal ranges of water temperature at shrimp farms may vary from 15 to 32 °C in Taiwan (Chen, 1990). Commercial farming of tiger shrimp P. monodon has been badly hit by an epidemic of viruses, monodon baculoviros virus (MBV), white spot syndrome virus (WSSV), yellow head virus (YHV) and infectious hypodermal and hematopoietic necrosis virus (IHHNV) (Lo et al., 2003).

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The bacteria, Vibrio alginolyticus, Vibrio harveyi and Photobacterium damselae subsp. damselae (also known as Vibrio damsela) are considered to be secondary and opportunistic pathogens, and have been demonstrated to cause disease outbreaks of vibriosis associated with poor environmental conditions (Lee and Chen, 1994; Lee et al., 1996; Liu et al., 1996). These diseases have also been reported to be associated with increases of Vibrio population in culture pond waters (Sung et al., 2001). Decapod crustaceans have three types of circulating hemocytes: hyaline cell (HC), semi-granular cell (SGC) and granular cell (GC) (Hose et al., 1990). They are involved not only in phagocytosis, an important process in eliminating microorganisms or foreign particles (Bayne, 1990), but also in the production of melanin via the prophenoloxidase (proPO) system which is an important component of the cellular defense reaction (Söderhäll and Cerenius, 1998). Phenoloxidase is the terminal enzyme in the proPO system and is activated by cell polysaccharides like β-1,3-glucan, lipopolysaccharide or peptidoglycan from microorganisms through pattern recognition proteins (Smith et al., 1984). Once bacteria or foreign particles are engulfed by hemocytes, the host's NADPH-oxidase is activated, which in turn increases oxygen consumption and subsequently produces several reactive oxygen species (ROS) such as superoxide anion (O2−), hydrogen peroxide (H2O2), hydroxyl radical (OH), and singlet oxygen (1O2) (Roch, 1999). These oxidants including superoxide anion can cause cytotoxic problems (Warner, 1994). Superoxide dismutase (SOD) catalyses the rapid two-step dismutations of the toxic superoxide anion, and scavenges it to molecular oxygen and hydrogen peroxide through the alternate reduction and oxidation of the active-site metal ion (Zelick et al., 2005). It is known that temperature affects the development of WSSV in kuruma shrimp Marsupenaeus japonicus (Guan et al., 2003), in white shrimp Litopenaeus vannamei (Vidal et al., 2002) and in freshwater crayfish Pacifastacus leniusculus (Jiravanichpaisal et al., 2004). It is also known that changes in temperature affect the phagocytic activity of freshwater prawn Macrobrachium rosenbergii against Lactococcus garvieae (Cheng et al., 2003), and white shrimp L. vannamei against V. alginolyticus (Cheng et al., 2005). It is assumed that changes in temperature may weaken the immune system of P. monodon, and lead to its susceptibility to vibriosis. Accordingly, this study examined 1) changes in temperature on the susceptibility of P. monodon to P. damselae subsp. damselae, and 2) changes in temperature on the immune responses of P. monodon.

35

2. Materials and methods 2.1. P. monodon P. monodon were obtained from a commercial farm in Iilan, Taiwan, and acclimated in the laboratory for two weeks; only shrimp in the intermolt stage were used for the study. The molt stage was determined by the examination of uropoda in which partial retraction of the epidermis could be distinguished (Robertson et al., 1987). In all tests, the shrimp were fed twice daily with a formulated shrimp diet (Tairoun Feed Company, Taipei, Taiwan). The shrimp ranged from 15.7 to 23.2 g, averaging 18.75 ± 3.60 g (mean ± SD) with no significant size difference among the treatments. During experiments, salinity was maintained at 25‰, pH 7.9 to 8.2 while temperature was maintained at 26 ± 1 °C. 2.2. P. damselae subsp. damselae P. damselae subsp. damselae obtained from Bioresources Collection and Research Center, Food Industry and Development Institute (Hsinchu, Taiwan) was used for the study. This species was demonstrated to cause high pathogenicity in P. monodon (Lee and Chen, 1994). Stocks were on tryptic soy agar (TSA supplemented with 2.5% NaCl, Difco) for 24 h at 26 °C, and transferred to 10 ml tryptic soy broth (TSB supplemented with 2.5% NaCl, Difco) for 24 h at 26 °C for use as a stock bacterial broth. Stock cultures were centrifuged at 7155 ×g for 20 min at 4 °C. The supernatant fluid was removed and the bacterial pellet was re-suspended in saline solution (0.85% NaCl) at 4.24 × 106 cfu ml− 1 for the susceptibility test, and 4.70 × 106 cfu ml− 1 for phagocytic activity and clearance efficiency tests. 2.3. Effect of temperature change on the susceptibility of P. monodon to P. damselae subsp. damselae Challenge tests were conducted in triplicate with ten shrimp per replicate following the methods described before (Liu and Chen, 2004). Into the ventral sinus of the cephalothorax of each shrimp, 20 μl of bacterial suspension (4.24 × 106 cfu ml − 1 ) was injected resulting in 8.48 × 10 4 cfu shrimp− 1 . After injection, shrimp were kept in a separate 60 l glass aquarium (10 shrimp each) containing 40 l of aerated water (25‰) at 22, 26 (control), 30 and 34 °C. The experiment lasted 96 h. Shrimp injected with an equal volume of sterile saline solution and kept in 22, 26, 30 and 34 °C served as the unchallenged controls (Table 1).

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Table 1 Susceptibility of Penaeus monodon to Photobacterium damselae subsp. damselae for the shrimp transferred from 26 °C to different temperatures (22, 26, 30 and 34 °C) Bacterial dose (cfu shrimp− 1)

Temperature (°C)

No. shrimp

Cumulative mortality (%), and number of shrimp killed (in each group), time after challenge (h) 6

12

24

48

72

96

Control 8.48 × 104

22–34 22

3 × 10 3 × 10

0 0

8.48 × 104

26

3 × 10

0

8.48 × 104

30

3 × 10

0

8.48 × 104

34

3 × 10

0

0 13.3 ± 3.3a (1, 2, 1) 3.3 ± 3.3b (0, 1, 0) 3.3 ± 3.3b (0, 0, 1) 10.0 ± 0a (1, 1, 1)

0 26.7 ± 3.3a (2, 3, 3) 3.3 ± 3.3c (0, 1, 0) 6.7 ± 3.3bc (0, 1, 1) 16.7 ± 3.3ab (2, 1, 2)

0 30.0 ± 5.8a (2, 3, 4) 13.3 ± 3.3b (1, 2, 1) 13.3 ± 3.3b (1, 2, 1) 30.0 ± 5.8a (3, 2, 4)

0 46.7 ± 3.3a (4, 5, 5) 16.7 ± 3.3b (1, 2, 2) 16.7 ± 3.3b (1, 2, 2) 33.3 ± 6.7a (4, 2, 4)

0 46.7 ± 3.3a (4, 5, 5) 16.7 ± 3.3b (1, 2, 2) 16.7 ± 3.3b (1, 2, 2) 36.7 ± 3.3a (4, 3, 4)

Data in the challenge groups in the same column with different letters are significantly different (p b 0.05) among treatments. Values are mean ± S.E. (n = 30 shrimp in each case).

2.4. Effect of temperature change on the immune parameters of P. monodon For hemocyte count and enzyme activity assays, the test was conducted in eight replicates of 20 l PVC tanks (one shrimp per tank). Each tank contained 10 l of water at different temperatures (22, 26, 30 and 34 °C). P. monodon at 25‰ seawater and 26 °C were transferred to 22, 26, 30 (control) and 34 °C for 96 h. There were four temperatures with four sampling times (12, 24, 48 and 96 h). Eight shrimp for each treatment and time were used for the study. In addition, eight shrimp were used as the initial group. Hemolymph was sampled individually at the beginning of the test, and after 12, 24, 48 and 96 h. Hemolymph (100 μl) was withdrawn from the ventral sinus of each shrimp into a 1 ml sterile syringe (25 gauge) containing 0.9 ml anticoagulant (30 mM trisodium citrate, 0.34 M sodium chloride, 10 mM EDTA, 0.115 M glucose, pH 7.55, osmolality 780 mOsm kg− 1). A drop of the anticoagulant–hemolymph mixture was placed on a hemocytometer, and THC and DHC were made under an inverted phase-contrast microscope (Leica DMIL, Leica Microsystems, Wetzlar GmbH, Germany), while the remainder of the mixture was used for subsequent tests. Phenoloxidase activity was measured spectrophotometrically by recording the formation of dopachrome produced from L-dihydroxyphenylalanine (L-DOPA) following the procedures of Hernández-López et al. (1996). The details of the measurements were described previously (Liu and Chen, 2004). The optical density of the shrimp's phenoloxidase activity for all test conditions was expressed as dopachrome formation in 50 μl of hemolymph. Respiratory burst activity of hemocytes was quantified using the reduction of nitroblue tetrazolium (NBT) to formazan as a measure of superoxide anion, as des-

cribed previously (Liu and Chen, 2004). The optical density at 630 nm was measured using a microplate reader (Model VERSAmax, Molecular Devices, Sunnyvale, CA, USA). Respiratory burst was expressed as NBT-reduction in 10 μl of hemolymph. Superoxide dismutase (SOD) activity was measured by its ability to inhibit superoxide radical dependent reactions using the Ransod Kit (Randox, Crumlin, UK). The details of the measurements were described previously (Liu and Chen, 2004). The optical density was measured at 505 nm, 37 °C, and the rate of reaction was estimated from the absorbance readings 30 s and 3 min after adding xanthine oxidase. A reference standard SOD was supplied with the Ransod Kit. One unit of SOD was defined as the amount required to inhibiting the rate of xanthine reduction by 50%. Specific activity was expressed as SOD units ml− 1 (Biagini et al., 1995). For phagocytic activity and bacterial clearance tests, there were four treatments (22, 26, 30 and 34 °C) with four sampling times (12, 24, 48 and 96 h). Eight shrimp for each treatment and time were used for the study. In addition, eight shrimp were used as the initial group. Shrimp held in 25‰ and 26 °C were transferred individually to 22, 26, 30 and 34 °C. After 0, 12, 24, 48 and 96 h of transfer, shrimp were injected in the ventral sinus with 20 μl bacterial suspension (4.70 × 106 cfu ml− 1 in 0.85% NaCl) resulting in 9.40 × 104 cfu shrimp− 1. After injection, each shrimp was held in a separate tank containing one of the test solutions (20, 26, 30 and 34 °C) for 1.5 h at 25‰. Then 100 μl of hemolymph from the ventral sinus was collected, and mixed with 100 and 900 μl of sterile anticoagulant for the measurement of phagocytic activity and clearance efficiency, respectively. Phagocytic activity was measured following the method described by Weeks-Perkins et al. (1995). Briefly,

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PI ¼ 100−fðcfu in test groupÞ=ðcfu in control groupÞg  100 2.5. Statistical analysis A multiple comparison (Tukey) test was conducted to compare the significant differences among treatments using the SAS computer software (SAS Institute Inc., Cary, NC, USA). Percent data (susceptibility test) were normalized using an arcsin transformation before analysis. For statistically significant differences, it was required that p b 0.05. 3. Results 3.1. Effect of temperature change on the susceptibility of P. monodon to P. damselae subsp. damselae All the unchallenged control shrimp survived. By contrast, mortalities began to occur at 12 h in the challenged shrimp. After 12 h, cumulative mortalities for the shrimp transferred to 22 and 34 °C were significantly higher than those for the shrimp held at 26 and 30 °C (Table 1).

Hyaline cell (× ×105 ml-1)

Clearance efficiency was measured following the method of Adams (1991). The 1 ml of volume of diluted hemolymph was further diluted to 100 ml with saline solution. Three 50 μl portions of each diluted hemolymph sample were spread on separate TSA plates and incubated at 26 °C for 24 h before colonies were counted using a colony counter. The number of colony of shrimp kept in 25‰ and 26 °C was expressed as the control group, and the number of colony of shrimp transferred to 22, 30 and 34 °C after 12, 24, 48 and 96 h was expressed as the test group. Clearance efficiency to P. damselae subsp. damselae, defined as percentage inhibition (PI) was calculated as:

For the shrimp transferred to 22 °C, the HC decreased significantly by 45%, 46%, 43% and 46% after 12, 24, 48 and 96 h, respectively. For the shrimp transferred to 30 °C, the HC decreased significantly by 7%, 4% and 4% after 24, 48 and 96 h, respectively. For the shrimp transferred to 34 °C, the HC decreased significantly by 4%, 27%, 22% and 25% after 12, 24, 48 and 96 h, respectively (Fig. 1A). The HC made up 75.0% of THC for the shrimp held at 26 °C, whereas the contribution of HC in THC decreased to 70.7% for the shrimp transferred to 22 °C after 48 h. For the shrimp transferred to 22 °C, the SGC 250

A

200

a a b

150 c

100

Temperature (°C) 22 26 30 34 a b a a b b c c c d d d

50 0 0

Semi-granular cell (×105 ml-1)

Percentage phagocytosis ¼ fðphagocytic hemocytesÞ=ðtotal hemocytesÞg  100

3.2. Effect of temperature change on the immune parameters of P. monodon

12

24

48

96

Time elapsed (h) 50

B

40

a a a

30

Temperature (°C) 22 26 30 34 a a a a a a b b b b b b

b

20 10 0 0

12

24

48

96

Time elapsed (h) Granular cell (×105 ml-1)

200 μl of the diluted hemolymph sample were fixed with 200 μl 0.1% paraformaldehyde for 30 min at 4 °C to fix the hemocytes, and then centrifuged at 800 ×g (Model 5403, Eppendorf, Hamburg, Germany). The details of the measurements were described previously (Liu and Chen, 2004). Two hundred hemocytes were counted. Phagocytic activity, defined as percentage phagocytosis was expressed as:

37

30

Temperature (°C) 22 26 30 34 a a a a

C a a a

20

a ab b

b

b

b

b

b

b

10

0 0

12

24

48

96

Time elapsed (h) Fig. 1. Mean (± S.E.) hyaline cell (A), semi-granular cell (B) and granular cell (C) of P. monodon kept at a salinity of 25‰ and 26 °C at the beginning, and after 12, 24, 48 and 96 h transfer to 22, 26 (control), 30 and 34 °C. Each bar represents the mean value from eight shrimp with standard error. Data (mean ± S.E.) in the same exposure time with different letters are significantly different (p b 0.05) among different temperatures.

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350

A

Temperature (°C)

300

22

250

a

a a a

a

b

200 150

30 34 a a

a b

c c

d

b

26

b c

100 50 0

0

12

24

48

96

0.30

Temperature (°C) 22 26 30 34

B

0.25 a ab b

0.20

a

a a b

0.15

b b

c

c

c

a

a

c d

0.10 0.05 0.00 0

12

24

48

96

Time elapsed (h)

Fig. 2. Mean (±S.E.) total hemocyte count (A) and phenoloxidase activity (B) of P. monodon kept at a salinity of 25‰ and 26 °C at the beginning, and after 12, 24, 48 and 96 h transfer to 22, 26 (control), 30 and 34 °C. See Fig. 1 for statistical information.

decreased significantly by 50%, 31%, 27% and 30% after 12, 24, 48 and 96 h, respectively. For the shrimp transferred to 34 °C, the SGC decreased significantly by 25%, 22% and 25% after 24, 48 and 96 h, respectively (Fig. 1B). The SGC made up 15.7% of THC for the shrimp held at 26 °C, whereas the contribution of SGC in THC increased to 18.7% for the shrimp transferred to 22 °C after 48 h. For the shrimp transferred to 22 °C, the GC decreased significantly by 33%, 25%, 35% and 34% after 12, 24, 48 and 96 h, respectively. For the shrimp transferred to 34 °C, the GC decreased significantly by 23%, 22% and 22% after 24, 48 and 96 h, respectively (Fig. 1C). The GC made up 9.3% of THC for the shrimp held at 26 °C, whereas the contribution of GC in THC increased to 10.0% for the shrimp transferred 22 °C after 48 h. For the shrimp transferred to 22 °C, the THC decreased significantly by 41%, 42%, 39% and 43% after 12, 24, 48 and 96 h, respectively. For the shrimp transferred to 34 °C, the THC decreased by 25%, 22% and 25% after 24, 48 and 96 h, respectively (Fig. 2A). For the shrimp transferred to 22 °C, phenoloxidase activity decreased sig-

Respiratory burst (O.D.630 nm)

Phenoloxidase activity (O.D. 490 nm)

Time elapsed (h)

nificantly by 41%, 42%, 39% and 43% after 12, 24, 48 and 96 h, respectively. For the shrimp transferred to 30 °C, phenoloxidase activity decreased significantly by 14% after 96 h. For the shrimp transferred to 34 °C, phenoloxidase activity decreased significantly by 7%, 18%, 21% and 27% after 12, 24, 48 and 96 h, respectively (Fig. 2B). For the shrimp transferred to 22 °C, respiratory burst decreased significantly by 40%, 37%, 27% and 36% after 12, 24, 48 and 96 h, respectively. For the shrimp transferred to 30 °C, respiratory decreased significantly by 20% after 24 h. For the shrimp transferred to 34 °C, respiratory burst decreased significantly by 21%, 29%, 23% and 13% after 12, 24, 48 and 96 h, respectively (Fig. 3A). For the shrimp transferred to 22 °C, SOD activity decreased significantly by 41%, 30%, 38% and 17% after 12, 24, 48 and 96 h, respectively. For the shrimp transferred to 30 °C, SOD activity decreased significantly by 14% after 96 h. For the shrimp transferred to 34 °C, SOD activity decreased significantly by 19%, 21% and 16% after 24, 48 and 96 h, respectively (Fig. 3B). For the shrimp transferred to 22 °C, phagocytic activity decreased significantly to 15%, 15%%, 18% and 18% 0.25

A

Temperature (°C)

0.20

b

b

0.15

22 26 a a

a

a a

b

bc b

c

c

c

30 34 a a b

0.10 0.05 0.00 0

12

24

48

96

Time elapsed (h) 0.9

SOD activity (units ml-1)

Total hemocyte count (× ×105 ml-1)

38

0.8

Temperature (°C)

B

22 a

a

a a a

0.7

ab

0.6 0.5

30

34

a ab

bc c

26

b

b

b b

c

b

0.4 0.3 0.2 0.1 0.0 0

12

24

48

96

Time elapsed (h) Fig. 3. Mean (±S.E.) respiratory burst (A) and superoxide dismutase (SOD) activity (B) in the hemocytes of P. monodon kept at a salinity of 25‰ and 26 °C at the beginning, and after 12, 24, 48 and 96 h transfer to 22, 26 (control), 30 and 34 °C. See Fig. 1 for statistical information.

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Phagocytlc activity (%)

60

A

Temperature (°C) 22 26 30 34 a a

50 a

a 40

a

a

b 30

c

b 20

c

ab bc

b c

c d

10 0 0

12

24

48

96

48 a

96 a

Time elapsed (h)

Clearance efficiency (%)

100 50

Time elapsed (h)

B 0

0

12 a

24 a

-50 b

b

-100 c

-250

d d Temperature (°C) 22 26 30 34

b

c

c

-150 -200

b

b

d

c

Fig. 4. Mean (± S.E.) phagocytic activity (A) and clearance efficiency (B) in the hemocytes of P. monodon against P. damselae subsp. damselae kept at 25‰ seawater and 26 °C at the beginning, and after 12, 24, 48 and 96 h transfer to 22, 26 (control), 30 and 34 °C. See Fig. 1 for statistical information.

after 12, 24, 48 and 96 h, respectively. For the shrimp transferred to 30 °C, phagocytic activity decreased significantly to 31% after 24 h. For the shrimp transferred to 34 °C, phagocytic activity decreased significantly to 23%, 25%, 25% 23% after 12, 24, 48 and 96 h, respectively (Fig. 4A). A similar trend was observed for the clearance efficiency against P. damselae subsp. damselae. For the shrimp transferred to 22, 30 and 34 °C after 12 h, clearance efficiency decreased by 160%, 60% and 102%, respectively (Fig. 4B). 4. Discussion Giant freshwater prawn M. rosenbergii was more susceptible to L. garvieae when reared in 33 °C than when reared in 27 and 30 °C water (Cheng and Chen, 1998). Crayfish P. leniusculus were exposed to different temperatures after WSSV (white spot syndrome virus) injection, the pathogenicity of the WSSV was significantly higher at 22 °C as compared to temperatures at 4 and 12 °C (Jiravanichpaisal et al., 2004). White shrimp L. vannamei was more susceptible to V. alginolyticus when the shrimp were transferred from 32 to 28 °C in 24 h (Cheng et al.,

39

2005). In the present study, we found that tiger shrimp P. monodon was more susceptible to P. damselae subsp. damselae when the shrimp were transferred from 26 °C to 22 and 34 °C in 24 h. Therefore, change in water temperature can trigger an outbreak of the disease by weakening the immunity of shrimp, prawn and crayfish. Decreases in both THC and phenoloxidase activity occurred when animals reared in an optimal temperature condition were transferred to low temperature. Giant freshwater prawn M. rosenbergii reared in 20 °C had significantly lower THC and phenoloxidase activity as compared to the prawn reared at 27 and 30 °C (Cheng and Chen, 2000). Blue shrimp Litopenaeus stylirostris reared in 18 °C had a significantly lower THC, as compared to the shrimp reared at 27 °C (Le Moullac and Haffner, 2000). White shrimp L. vannamei transferred to 20 and 24 °C had a significantly lower THC and phenoloxidase activity after 24 h, as compared to the shrimp at 28 °C (Cheng et al., 2005). A similar phenomenon was also observed in the present study: P. monodon transferred from 26 to 20 °C had a significantly lower THC and phenoloxidase activity over 12–96 h, as compared to the shrimp at 26 °C. Decreases in both THC and phenoloxidase activity also occurred when animals reared in an optimal temperature condition were transferred to high temperature. For example, giant freshwater prawn M. rosenbergii reared at 33 °C had significantly lower THC and phenoloxidase activity, as compared to the prawn reared at 27 and 30 °C (Cheng and Chen, 2000). A decrease in THC, phenoloxidase activity and respiratory burst were observed in the white shrimp L. vannamei when transferred from 28 to 32 °C over 24–96 h (Cheng et al., 2005). In the present study, a decrease in THC, phenoloxidase activity and respiratory burst was also observed in P. monodon when transferred from 26 °C to 30 and 34 °C over 24–96 h. Phenoloxidase is stored in the secretory granules of semi-granular and granular hemocytes, whereas agranular hemocytes are involved in phagocytosis and the release of superoxide anion and other ROS (Bachère et al., 1995). The present study indicated that decreases of both phenoloxidase activity and respiratory burst are also well correlated with decreases of THC and DHC for P. monodon when transferred to 22 and 34 °C over 24–96 h. This fact indicated that decreases of phenoloxidase activity and respiratory burst are a consequence of decreases in THC, HC, SGC and GC (Cheng et al., 2005). The expression of peroxinectin cDNA obtained from the hemocytes of white shrimp L. vannamei was significantly reduced when the shrimp were transferred to high temperature (34 °C) (Liu et al., 2004). In the present study, phenoloxidase activity decreased when tiger

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shrimp P. monodon were transferred from 26 °C to 22 and 34 °C in 24 h. It is expected that the transcript encoding peroxinectin and prophenoloxidase may also reduce for the P. monodon under low or high temperature stress. We found that tiger shrimp P. monodon when transferred from 26 °C to 22 and 34 °C decreased the respiratory burst and SOD activity in 24 h. This fact indicated that the activity of NADPH-oxidase, responsible for the release of superoxide anion decreased together with a decrease in the activity of SOD responsible for scavenging superoxide anion. Further research is needed to examine the activities of catalase and peroxidase (Holmblad and Söderhäll, 1999) for P. monodon under temperature stress. Elevated temperature has been reported to decrease phagocytic activity of freshwater prawn M. rosenbergii against L. garvieae (Cheng et al., 2003). The phagocytic activity and clearance efficiency of V. alginolyticus decreased for the white shrimp L. vannamei when transferred from 27 to 34 °C in 24 h, which correlated well with increase in susceptibility of L. vannamei to V. alginolyticus (Cheng et al., 2005). In the present study, we found that phagocytic activity and clearance efficiency of P. damselae subsp. damselae decreased for the P. monodon when transferred from 26 °C to 22 and 34 °C in 24 h. This also correlated well with increase in susceptibility of P. monodon to P. damselae subsp. damselae. In conclusion, the present study documented that P. monodon transferred from 26 °C to low temperature (22 °C) or high temperature (34 °C) showed a higher susceptibility to P. damselae subsp. damselae, together with lower THC, DHC, phenoloxidase activity, SOD activity, and decreases in phagocytic activity and clearance efficiency against P. damselae subsp. damselae, indicating a reduction in immune ability. Acknowledgements This research was supported by a grant from the National Science Council (NSC 93-2815-C-019-016B), ROC. We thank Mr. S.T. Yeh and Miss Y. W. Fu for their assistances in the experiment. Reference Adams, A., 1991. Response of penaeid shrimp to exposure to Vibrio species. Fish Shellfish Immunol. 1, 59–70. Bachère, E., Mialhe, E., Noöl, D., Boulo, V., Morvan, A., Rodriguez, J., 1995. Knowledge and research prospects in marine mollusc and crustacean immunology. Aquaculture 132, 17–32. Bayne, C.J., 1990. Phagocytosis and non-self recognition in invertebrates. Phagocytosis appears to be an ancient line of defense. BioScience 40, 723–731.

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