Effects of the “all-fish” growth hormone transgene expression on non-specific immune functions of common carp, Cyprinus carpio L.

Aquaculture 259 (2006) 81 – 87 www.elsevier.com/locate/aqua-online

Effects of the “all-fish” growth hormone transgene expression on non-specific immune functions of common carp, Cyprinus carpio L. Wen-Bo Wang a,b,c , Ya-Ping Wang a , Wei Hu a , Ai-Hua Li a , Tao-Zhen Cai a , Zuo-Yan Zhu a , Jian-Guo Wang a,⁎ a

State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, Hubei Province, P.R. China b Institute of Hydrobiology, Jinan University, Guangzhou 510632, Guangdong Province, P.R. China c Graduate School of the Chinese Academy of Sciences, China Received 2 December 2004; received in revised form 14 May 2006; accepted 15 May 2006

Abstract This study investigated non-specific immune functions of the F2 generation of “all-fish” growth hormone transgenic carp, Cyprinus carpio L. Lysozyme activity was 145.0 (±30.7) U ml− 1 in the transgenic fish serum and 105.0 (±38.7) U ml− 1 in agematched non-transgenic control fish serum, a significant difference (P < 0.01). The serum bactericidal activity in the transgenics was significantly higher than that in the controls (P < 0.05), with the percentage serum killing of 59.5% (± 6.83%) and 50.8% (± 8.67%), respectively. Values for leukocrit and phagocytic percent of macrophages in head kidney were higher in transgenics than controls (P < 0.05). However, the phagocytic indices in the transgenics and the controls were not different. In addition, the mean body weight of the transgenics was 63.4 (± 6.65) g, much higher than that of the controls [39.2 (±3.30) g, P < 0.01]. The absolute weight of spleen of the transgenics [0.13 (± 0.03) g] was higher than that of the controls [0.08 (±0.02) g, P < 0.01]. However, there was no difference in the relative weight of spleen between the transgenics and the controls, with the spleen mass index being 0.21% (± 0.02%) and 0.20% (± 0.03%), respectively. This study suggests that the “all-fish” growth hormone transgene expression could stimulate not only the growth but also the non-specific immune functions of carp. © 2006 Published by Elsevier B.V. Keywords: “All-fish” growth hormone gene; Transgenic fish; Growth hormone; Non-specific immune functions

1. Introduction In 1984, the first batch of growth hormone (GH) transgenic fish was produced in China (Zhu et al., 1985). Since then, many laboratories all over the world have turned to the study of transgenic fish. In the early ⁎ Corresponding author. Tel.: +86 27 68780720; fax: +86 27 68780123. E-mail address: [email protected] (J.-G. Wang). 0044-8486/$ - see front matter © 2006 Published by Elsevier B.V. doi:10.1016/j.aquaculture.2006.05.016

studies, the recombinant gene was MThGH, i.e., the human GH gene (hGH) under the transcriptional control of the mouse metallothionein-1 (MT-1) promoter (Pavlakis and Hammer, 1983). Due to biosafety and bioethical issues, the MT-1 promoter that needs heavy metal ion induction and the hGH that encodes a human protein are not appropriate for the purpose of human food production. For this reason, Zhu et al. (1989) proposed the idea of constructing an “all-fish” transgene which contains only piscine sequences. Transgenic fish

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have been studied now for 20 years and the research results are very extensive, including growth effects, mechanism of transgene integration, and energy, body composition, cultivation, and ecological safety of transgenic fish (Zhu and Zeng, 2000). However, the immunology of the GH transgenic common carp remains unknown. What pleiotropic effects have been induced by the exogenous GH gene expression on the immune functions of carp? Are these effects beneficial or harmful to host fish? Is resistance to disease of the host fish altered by GH transgene expression? Investigation of these questions will enrich basic research on GH transgenics and supply information relating to the prevention and control of fish disease. GH, produced by the hypothalamus-pituitary-adrenal (HPA) axis, is a pleiotropic hormone regulating many aspects of fish physiology, including growth (Donaldson et al., 1979), osmoregulation (Sakamoto et al., 1997), and reproduction (Trudeau, 1997). Recently, the relationship between the HPA axis and the immune system in fish has been given considerable attention by investigators. There is increasing evidence that GH also exerts an immunostimulatory function in fish (Calduch-Giner et al., 1997; Harris and Bird, 2000; Yada and Nakanishi, 2002). For example, in some teleost fishes, GH can promote lymphopoiesis, leukocyte mitogenesis, natural killer cell activity, antibody production, serum haemolytic activity and resistance to disease (Sakai et al., 1996; Harris and Bird, 2000; Yada and Nakanishi, 2002; Johansson et al., 2004). However, Jhingan et al. (2003) found that GH suppresses immune function in GH transgenic coho salmon. Teleost fishes possess a variety of specific and nonspecific defence mechanisms against invading organisms. When a pathogen penetrates the physical barriers of the animal, the first lines of defence are those of the non-specific immune system. Chemical defences, such as serum lysozyme, complement components, lectins and C-reactive protein, attack the pathogen, or may opsonise it for further destruction by the cellular components of the non-specific immune system (Secombes, 1996; Yano, 1996). The present work studies the effects of “all-fish” GH transgene expression on the non-specific immune functions of common carp.

carp (Cyprinus carpio L.), which was modified from the construct pCAgcGH (Zhu, 1992; Wang et al., 2001). The production of P0 and F1 “all-fish” GH transgenics was described in detail by Wang et al. (2001). In brief, P0 transgenics were produced by microinjection of pCAgcGHc into the fertilized eggs of Yellow River carp (C. carpio L.) (for details of gene transfer, see Zhu et al., 1989). (1) F1 transgenics were produced as follows: P0 transgenic ♂ (sperm revealed to be pCAgcGHc positive) × non-transgenic ♀. (2) F2 transgenics were produced by a similar method in 2004: F1 transgenic ♂ (sperm revealed to be pCAgcGHc positive) × non-transgenic ♀. On the same day, a batch of non-transgenic control carp (Yellow River carp) was also produced. The fry of F2 transgenics and controls were reared in separate ponds at the Institute of Hydrobiology, Chinese Academy of Sciences (Wuhan, Hubei province, China). Polymerese chain reaction (PCR) was performed on F2 transgenics according to Wang et al. (2001) to detect the pCAgcGHctransgene positive fish. PCR-positive fish and controls were randomly taken to use as the experimental fish in this study (n = 10 for each group). 2.2. Rearing of the experimental fish After PCR detection, the 10 transgenics and 10 controls were transferred to two identical tanks (1800 l each) in a recirculation system, supplied with a constant flow of water (flow rate 2.5 l min− 1). The temperature range of the water during the experimental period was 22– 26 °C. They were fed twice daily with a carp feed without growth additive until use. The feed was produced by the Institute of Hydrobiology, Chinese Academy of Sciences. 2.3. Bacteria Micrococcus lysodeikticus, purchased from Sigma Chemical Co. (St Louis, MO, USA), was stored at − 20 °C and used for determining the lysozyme activity. Vibrio fluvialis (XS 91-24-3) and Aeromonas hydrophila (XS 91-4-1) were originally isolated from a virulent outbreak of bacterial hemorrhagic septicemia in a carp farm in China and stored in the laboratory at − 20 °C, with the former used for the bactericidal assay and the latter for the phagocytic assay.

2. Materials and methods 2.4. Sampling 2.1. Production of transgenic fish The “all-fish” gene construct pCAgcGHc included the grass carp (Ctenopharyngodon idellus) GH cDNA (gcGHc) driven by the β-actin gene promoter of common

The 10 transgenics and 10 controls were sampled after 3.5 months of rearing. The fish were rapidly placed in water containing a concentration of anaesthetic (0.2 g l− 1; MS 222; Sigma) for about 10 s, then weighed and

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blood was sampled from the caudal vessel. An aliquot of each blood sample was stored on ice for 30 min, centrifuged at 400×g for 10 min at 4 °C, and the serum collected was stored at − 80 °C for analyses. The remainder of the blood sample was used to determine leukocrit values. The head kidney and spleen of each fish were aseptically dissected after blood sampling, with the former placed in Leibovitz-15 medium (Sigma) and stored at 4 °C for the phagocytic assay, and the latter examined for spleen mass index directly. 2.5. Lysozyme assay Lysozyme activity was measured according to Parry et al. (1965), using a turbidity assay in which 0.2 mg ml− 1 lyophilized M. lysodeikticus in 0.04 M sodium phosphate buffer (pH 5.75) was used as substrate. Five microlitres of fish serum was added to 3 ml of the bacterial suspension, and the reduction in absorbance at 540 nm determined after 0.5 and 4.5 min incubations at 22 °C. One unit of lysozyme activity was defined as a reduction in absorbance of 0.001 per min. 2.6. Serum bactericidal activity Serum bactericidal activity was assessed as described by Ainsworth et al. (1995) with some modifications. V. fluvialis (XS 91-24-3) was grown in 100 ml of tryptone soy broth (TSB) in a rotary shaker (200 rpm) at 28 °C for 24 h. The bacterium was washed with 0.5% saline and the bacterial density adjusted to 106 bacteria ml− 1 in saline by spectrophotometer (OD 620), then 50 μl of serum was mixed with 50 μl of suspension of V. fluvialis (XS 91-24-3). After 5 h of incubation at 22 °C, 10-fold and 100-fold dilutions of the mixture (10 μl per dilution) were plated on tryptone soy agar (TSA) plates. After 24 h of incubation at 28 °C, colony-forming units (CFUs) on the TSA plates were counted. The results were expressed as the percentage serum killing using the formula: percentage serum killing ¼100  ðCFU number  100Þ  ðCFU number in controlsÞ1 : 2.7. Leukocrit value Blood was collected in heparinized microleukocrit tubes and centrifuged in a swinging-bucket rotor at 400×g for 20 min. The packed leukocyte layer height was measured using a vernier caliper, and the leukocrit value [white blood cell (WBC) volume] was defined as the percentage of the leukocyte layer height to total blood height inside the tube.

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2.8. Phagocytic assay Macrophage cells and suspensions of A. hydrophila (XS 91-4-1) were prepared according to the methods of Secombes (1990) and Thompson et al. (1996), respectively. The phagocytic assay was performed using the method of Sarder et al. (2001). In brief, the head kidney tissue was dissected from the fish and pushed through a 100 μl nylon mesh into L-15 medium containing 20 I.U. ml− 1 of heparin. Cell suspensions were layered onto a 34%/51% Percoll (Sino-American Biotechnology Co., Shanghai, China) gradient and centrifuged at 400×g for 25 min at 4 °C. The macrophage-enriched band was collected and washed with L-15 medium and the cell numbers were adjusted to 5 × 106 cells ml− 1 L-15 medium containing 1% penicillin/streptomycin. Two hundred microlitre aliquots of the suspension were added to circles of clean glass slides (6 wells per slide, 15 mm diameter per well) and incubated for 3 h at 22 °C, after which the glass slides were washed with L-15 medium to remove the nonadherent cells. Each of the resultant macrophage monolayer was incubated together with 200 μl of A. hydrophila (XS 91-4-1) bacterium suspension (5 × 107 cells ml− 1 sterile saline) for 1 h at 22 °C. The macrophage monolayers then were washed with L-15 medium, fixed with formaldehyde and stained with Giemsa. Finally, 200 macrophages were examined microscopically and the number of macrophages containing phagocytosed bacteria, as well as the number of phagocytosed bacteria, were counted. The results were expressed as phagocytic percent and phagocytic index, where: phagocytic percent ¼ the number of the macrophages containing phagocytosed bacteria  2001  100

and phagocytic index ¼ the number of phagocytosed bacteria  2001 : 2.9. Spleen mass index Spleens of fish were excised, washed with saline, dried using filter paper and then weighed. The spleen mass index was calculated using the following formula: spleen mass index ¼ ðspleen weightÞ ðbody weightÞ1  100: 2.10. Statistical analysis All results are expressed as means ± standard errors (S.E.), and the effects of the “all-fish” GH transgene

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the controls (P < 0.01), but the spleen mass indices were not different between the transgenics and the controls, with values of 0.21% (± 0.02%) and 0.20% (± 0.03%), respectively.

expression were analysed using a Student's t-test (P < 0.05). All analyses were carried out on 10 fish per group. 3. Results

4. Discussion 3.1. Lysozyme activity Lysozyme is an important innate humoral factor of the fish immune system, having an antibacterial effect both by attacking the bacterial cell wall, thereby causing lysis, and by stimulating phagocytosis of bacteria. It is produced by neutrophils and macrophages, of which binding sites for GH have been found on both (CalduchGiner et al., 1995). In the present study, we observed higher lysozyme activity in transgenic fish than in agematched control fish (Fig. 1). Was this the result of stimulation of neutrophils and macrophages induced by transgene expression? We presume that in the F2 transgenic fish, the expressive production of the foreign GH gene may possess the same biological functions as that of the endogenous GH gene. A similar conclusion can be found in other articles (Cui, 1998; Dialynas et al., 1999; Wang et al., 2001). In aquaculture, GH usually acts as an immunostimulant to enhance the non-specific immunity of fish. When flounder (Paralichthys olivaceus) was fed with recombinant yeast (Saccharomyces cerevisiae) containing salmon (Oncorhynchus keta) GH, the serum lysozyme activity in fish was greatly improved, and this effect was dose-dependent (Wang and Zhang, 2000). In addition, endogenous GH can also regulate lysozyme activity. After brown trout (Salmo trutta) was transferred from freshwater to seawater, the GH level in fish blood rose, accompanied with an increase in serum lysozyme activity (Marc et al., 1995). Although both endogenous GH and exogenous GH transgene expression can stimulate lysozyme activity, whether their mechanisms are the same requires further study. There are many aspects exerting stimulative or suppressive effects on the immune system in fish, one of which is growth rate or size. It can influence not only innate immune functions in fish, but also their disease

The range of lysozyme activity in the transgenics was between 100 and 200 U ml− 1, with a mean of 145.0 (±30.7) U ml− 1; while the range in the controls was between 50 and 175 U ml− 1, with a mean of 105.0 (±38.7) U ml− 1. Lysozyme activity in the transgenics was significantly higher than that in the controls (P < 0.01). 3.2. Bactericidal activity The average number of CFUs on the TSA plates was 9.50 (± 1.82) in the transgenics and 11.5 (± 2.16) in the controls (P < 0.01). The percentage serum killing in the transgenics was 59.5 (± 6.83), higher than that in the controls [50.8 (± 8.67); P < 0.05]. 3.3. Leukocrit The leukocrit value in the transgenics was significantly higher than that in the controls (P < 0.05), with values of 4.22% (± 0.37%) and 3.87% (± 0.39%), respectively. 3.4. Phagocytic activity The phagocytic percent of macrophages in head kidney was 45.73 (± 5.25) in the transgenics and 39.20 (± 7.21) in the controls (Table 1; P < 0.05). With respect to the phagocytic index, there was no statistical difference between the transgenics and the controls. 3.5. Spleen mass index As shown in Table 2, both the spleen weight and the body weight of the transgenics were higher than those of

Table 1 Comparison of phagocytic activity of macrophages in head kidney between the F2 “all fish” GH transgenic carp (C. carpio L.) and control carp No.

Phagocytic percent (%) Phagocytic index

transgenic carps control carps transgenic carps control carps

1

2

3

4

5

6

7

8

9

10

47.25 43.75 2.80 3.13

51.75 39.75 2.53 1.94

41.50 38.50 3.27 2.33

45.00 29.75 2.78 3.57

54.50 47.00 2.74 2.78

47.50 33.75 3.78 2.57

41.00 49.75 3.36 3.53

49.50 44.00 2.84 2.26

39.50 38.00 2.78 3.61

39.75 27.75 2.15 3.03

Means ± S.E.

t-test

45.73 ± 5.25 39.20 ± 7.21 2.90 ± 0.46 2.87 ± 0.60

P < 0.05 P > 0.05

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Table 2 Comparison of spleen mass index between the F2 “all fish” GH transgenic carp (C. carpio L.) and control carp No.

Spleen weight (g) Body weight (g) Spleen mass index (%)

transgenic carps control carps transgenic carps control carps transgenic carps control carps

1

2

3

4

5

6

7

8

9

10

0.12 0.07 56.9 36.9 0.21 0.19

0.15 0.09 65.7 41.2 0.23 0.22

0.11 0.08 58.4 37.5 0.19 0.21

0.12 0.09 58.3 39.8 0.21 0.23

0.19 0.11 75.7 44.5 0.25 0.25

0.15 0.09 70.2 41.5 0.21 0.22

0.13 0.07 60.5 38.6 0.21 0.18

0.12 0.06 63.2 39.5 0.19 0.15

0.09 0.08 55.8 40.4 0.16 0.20

0.13 0.06 69.4 32.1 0.19 0.19

Means ± S.E.

t-test

0.13 ± 0.03 0.08 ± 0.02 63.4 ± 6.65 39.2 ± 3.30 0.21 ± 0.02 0.20±0.03

P < 0.01 P < 0.01 P > 0.05

susceptibility (Magnadóttir, 2006). Owing to lack of size-matched controls in our experimental design, the increased non-specific immune response presented in this study may have been caused directly by transgene expression or indirectly through the effect of transgene expression on size of individual fish. It is well known that lysozyme performs a bacteriolytic function only on Gram-positive bacteria, whereas the V. fluvialis (XS91-24-3) used in the bactericidal assay was a Gram-negative bacterium, which would not be dissolved by lysozyme. Therefore, the elevation of the serum bactericidal activity in the transgenics (Fig. 2) was unlikely to have been caused by the increased lysozyme activity. Then, what was responsible for this? Fish size may be taken into account and, on the other hand, besides lysozyme, the complement components in fish serum also play an important role in killing pathogens. It was reported by Doong (2000) that after injection with black porgy (Acanthopagrus schlegeli) GH, both the lysozyme activity and the alternative complement activity in black porgy were improved. But in the present study, whether GH transgene expression has caused the activation of the complement system and thereby promoted the bactericidal activity in the transgenics requires further investigation.

There are two main issues when it comes to the cellular immunity: one is the number of immune cells involved in the immune response, and the other is the activity of these cells. Leukocrit value and phagocytic activity of macrophages in head kidney are two typical indices to assess the amount and activity of the immune cells. The leukocrit value in the transgenics was higher than that in the controls (Fig. 3; P < 0.05), indicating that GH transgene expression accelerated the leukocyte proliferation. Among the leukocytes, lymphocytes and monocytes are important immune cells mediating specific and non-specific immune response in fish, respectively. Thus, the enhanced leukocytosis in the transgenics supports the finding of Yada et al. (2004), who suggested that GH showed stimulatory effects not only on lymphopoiesis, but also on proliferation of other leukocytes in trout (O. mykiss) in vitro, resulting in an increase in the total blood cell number. Under common status, the amount of GH in a fish is not fully saturated, so that the expressive GH production by the transgene has a chance to exert its biological effects on the fish (Donaldson et al., 1979). Perhaps this is a reason why GH transgene expression could stimulate the leukocyte proliferation. It is reported that as an in vitro phagocyte-

Fig. 1. Comparison of serum lysozyme activity between the F2 “all fish” GH transgenic carp (C. carpio L.) and control carp. Bar graphs indicate means ± S.E. (n = 10). ⁎⁎P < 0.01.

Fig. 2. Comparison of percentage serum killing between the F2 “all fish” GH transgenic carp (C. carpio L.) and the control carp. Bar graphs indicate means ± S.E. (n = 10). ⁎P < 0.05.

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expected to become the first transgenic animal to come into the market in China (Zhu and Zeng, 2000; Wu et al., 2003). Acknowledgements The authors would like to thank Liu Weiyi and Cao Ting for their help in collecting the experimental fish. This work was supported by the National Natural Science Foundation of China (Grant No. 30130050), the National Basic Research Program of China (Grant No. 2001CB109006) and the National High Technology Research and Development Program of China (Grant No. 2004AA213120). Fig. 3. Comparison of leukocrit value between the F2 “all fish” GH transgenic carp (C. carpio L.) and the control carp. Bar graphs indicate means ± S.E. (n = 10). ⁎P < 0.05.

activating factor, GH can enhance the phagocytic activity of macrophages (Calduch-Giner et al., 1997). Increased phagocytic activity and phagocytic index have been observed after rainbow trout (O. mykiss) was treated with chum salmon (O. keta) GH in vitro (Sakai et al., 1996). Similarly, GH transgene expression also stimulated the phagocytosis of macrophages in head kidney (Table 1). As was mentioned previously, macrophages can produce lysozyme to kill pathogens, including the A. hydrophila used in the phagocytic assay (Grinde, 1989). Therefore, it can be inferred that the phagocytic activity promoted in the transgenics may correlate with their increased lysozyme activity. In mammals, it has been documented that bovine GH transgenic mice showed significant increases in spleen weight and mitogenesis of splenic cells after a mitogenic stimulation (Dialynas et al., 1999). In the present study, GH transgene expression increased the absolute weight of fish body and spleen, but did not increase the relative weight of spleen, as shown by spleen mass index (Table 2), which closely match the findings derived from the human GH transgenic carp (C. carpio L.) (Cui, 1998). As one of the important immune organs in fish, the spleen contains a variety of immune cells, such as lymphocytes, macrophages and granulocytes. Hence, the accelerated development of spleen may be beneficial to the immunity of fish. In conclusion, the non-specific immune functions of common carp were significantly enhanced after “allfish” GH transgene expression, but the underlying molecular mechanism requires more study. With growth and immunity enhancement, “all-fish” GH transgenic carp is a good breed of fish for aquaculture, and is

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