key: cord-0925060-v321m7g9
authors: Jiang, Guojian; Yu, Rencheng; Zhou, Mingjiang
title: Studies on nitric oxide synthase activity in haemocytes of shrimps Fenneropenaeus chinensis and Marsupenaeus japonicus after white spot syndrome virus infection
date: 2006-01-05
journal: Nitric Oxide
DOI: 10.1016/j.niox.2005.11.005
sha: 31a1d28e9d3bf612e830b2d5c3cbd5ec17d729ca
doc_id: 925060
cord_uid: v321m7g9

The nitric oxide synthase (NOS) activity in the haemocytes of shrimps Fenneropenaeus chinensis (Osbeck) and Marsupenaeus japonicus (Bate) was studied after white spot syndrome virus (WSSV) infection to determine its characteristics in response to virus infection. First, the NOS activity in haemocytes of shrimps was determined by the means of NBT reduction and changes in cell conformation. And the variations of NOS activity in shrimps after challenge with WSSV intramuscularly were evaluated through the analysis of l-citrulline and total nitrite/nitrate (both as NO derivates) concentrations. The result showed that NOS activity in the haemocytes of F. chinensis increased slightly from 0 to 12 h postchallenge, indicated by the variations of l-citrulline (from 11.15 ± 0.10 to 12.08 ± 0.64 μM) and total nitrite/nitrate concentrations (from 10.45 ± 0.65 to 12.67 ± 0.52 μM). Then it decreased sharply till the end of the experiment (84 h postchallenge), the concentrations of l-citrulline and total nitrite/nitrate at 84 h were 1.58 ± 0.24 and 2.69 ± 0.70 μM, respectively. The LPS-stimulated NOS activity kept constant during the experiment. However, in M. japonicus, the NOS activity kept increasing during the first 72 h postchallenge, the concentrations of l-citrulline and total nitrite/nitrate increased from 7.82 ± 0.77 at 0 h to 10.79 ± 0.50 μM at 72 h, and from 8.98 ± 0.43 at 0 h to 11.20 ± 0.37 μM at 72 h, respectively. Then it decreased till the end of the experiment (216 h postchallenge), and the concentrations of l-citrulline and total nitrite/nitrate at 216 h were 5.66 ± 0.27 and 4.68 ± 0.16 μM, respectively. More importantly, an apparent increase of LPS-stimulated NOS activity was observed in M. japonicus at 48 h postchallenge, which was about 4 times higher than that in the control group of health shrimps. In correspondence with the difference of NOS activity between the two species of shrimps, the cumulative mortalities of the shrimps were also different. All shrimps of F. chinensis in the mortality experiment died in 66 h, much more quickly than M. japonicus, whose accumulative mortality reached 100% after 240 h. Data here reported let us hypothesize that NOS activity in the haemocytes of shrimps F. chinensis and M. japonicus responses to WSSV infection differently, and this might be one of the reasons for the different susceptibility of F. chinensis and M. japonicus to WSSV infection.

Penaeid shrimp culture is a worldwide economic activity. However, the intensiWcation and environment degradation of shrimp farming have been accompanied by the development of many infectious diseases, especially from viral origin, which cause a dramatic decrease in shrimp production [1, 2] . White spot syndrome virus (WSSV), a circular double-strand DNA virus [3] [4] [5] [6] , has led to heavy crop loss and continues to seriously aVect the cultured penaeids in the shrimp farming systems around the world [7] . In this context, control of disease is very important to insure the long-term survival of shrimp aquaculture. Therefore, it is necessary to study the immune factors of shrimp in response to the invasion of WSSV, which may be helpful in understanding the mechanism of shrimp immunity to WSSV infection and developing strategies for management of the disease.

Belonging to invertebrate, shrimp lack a true adaptive immune response system [8] . However, living in an aquatic environment rich in microorganisms, shrimps have developed eVective systems for detecting and eliminating noxious microorganism depending on non-speciWc innate immune response based on the activity of haemocytes, including phagocytosis, phenoloxidase releasing, bactericidin synthesis, and so on [2] . Non-speciWc molecules like reactive oxygen intermediates (ROIs) and reactive nitrogen intermediates (RNIs) are also involved. Surprisingly, in shrimps, the function and immune status of nitric oxide synthase (NOS) and its catalyzing products-nitric oxide, have been seldom reported. Nitric oxide is a very important component of innate immune system, which has the antiviral, anti-bacterial, and anti-parasite properties by damaging the DNA, enzyme and membrane of pathogen directly or indirectly through the action on the bases and strands of DNA, protein and lipid of membrane [9] [10] [11] [12] . Therefore, the role of NO and NOS in disease resistance of shrimps is worth studying. In this paper, we have tried diVerent methods to determine NOS activity in the haemocytes of Fenneropenaeus chinensis and Marsupenaeus japonicus qualitatively and quantitatively, and the role of the NOS in haemocytes of shrimps is also discussed in response to virus infection.

Marsupenaeus japonicus and F. chinensis were obtained from Ocean Shrimp Farm in Qingdao, China, with average length of 14.43 § 0.86 cm (mean § SD, N D 80) and 16 .70 § 1.40 cm (mean § SD, N D 80), and average weight of 19.84 § 3.71 g (mean § SD, N D 80) and 25.90 § 7.05 g (mean § SD, N D 80), respectively. They were fed with arti-Wcial diet twice a day and acclimated in aerated Xat-bed tanks containing sand Wltrated sea water (20 § 1°C; salinity, 30‰) for 7 days prior to experiment. Every day the unconsumed food and faeces were removed carefully with a siphon. All shrimps used in experiment were intermoult males.

All chemicals used in the experiment were A.R (Analytical reagent) grade or better. The glassware was pyrogenfree to avoid the enzymatic interruption by endotoxin.

Haemolymph of shrimp was collected from the ventralsinus with a 26-gauge needle and 1-ml syringe containing pre-cooled (4°C) anti-coagulant solution [13] (450 mM NaCl, 10 mM KCl, 10 mM EDTA-Na 2 , and 10 mM HEPES, pH 7.3, 850 mOsmol/kg, sterilized by Wltration (0.2 m) before using). The mixture was then injected into a 50 ml centrifuge tube containing anti-coagulant solution. The solution was mixed thoroughly and centrifuged at 600g for 10 min at 4°C. The supernatant was removed and haemocytes were adjusted to a certain cell density and resuspended in L-arginine solution (0.1 M Bis-Tris buVer containing 5 mM L-arginine (Sigma) and 5 mM Ca 2+ ) [14] to make haemocytes suspension for NOS activity analysis.

The NOS activity in haemocytes of shrimps was determined with NBT reduction method, according to Weiske and Wiesner [15] . The procedure was introduced brieXy as below: 500 l LPS solution [0.1 M Bis-Tris buVer containing 200 g/ml LPS (Sigma L2630)] [14] was mixed with 500 l haemocytes suspension with cell density of 10 6 and incubated at 4°C for 4 hours. The mixture was then centrifuged at 600g for 2 min at 4°C, and the supernatant was discarded. The residue haemocytes were Wxed with 4% formaldehyde (dissolved in 0.1 M Tris buVer with Wnal pH of 7.5-8.0) for 20 min, and the cells were washed at least twice with 0.1 M Tris buVer [pH 7.8, containing 0.1% Triton X-100 (Serva 37240)]. The cells were then resuspended in the freshly prepared 0.01% NBT (Sigma N6876) solution [in 0.1 M Tris buVer containing 0.1% Triton X-100 and 0.01% NADPH (Sigma N1630)] and incubated for 10 min at room temperature. Subsequently the mixture was centrifuged at 600g for 5 min at 4°C, and the supernatant was replaced with 0.7 ml of 70% methanol. After intensive mixing and a further centrifugation step at 600g for 10 min at room temperature, the methanol was replaced with 0.6 ml DMSO solution [54% (v/v) dimethyl sulfoxide (Sigma D8779) and 46% (v/v) 2 N KOH] to dissolve the formazan. After a Wnal incubation for 30 min at room temperature with intensive mixing at 10 min intervals, the contents were centrifuged at 10,000g for 5 min at room temperature, and the absorbance of the resulting supernatants was determined at 670 nm (BECKMAN DU 650 spectrophotometer). The NADPH in the 0.01% NBT solution was replaced with NADH (Sigma N8129) was used as control.

The NOS activity in haemocytes of shrimps was also determined by haemocyte conformation approach, according to Ottaviani et al. [16] . BrieXy, 25 l mixture of anticoagulant and haemolymph with cell density of 10 6 was placed on a dichromate cleaned glass slides and incubated in humidiWed chamber for 30 min at room temperature. The slides were then rinsed with 0.1 M Bis-Tris buVer to remove the anti-coagulant and the cells that did not adhere to the slide. Subsequently the slides were divided into four groups (A-D). Group A was added with 25 l Escherichia coli suspension at density of 1 £ 10 8 cells/ml, group B was added with 25 l E. coli suspension and 25 l LPS solution, group C was added with 25 l E. coli suspension, 25 l LPS solution, and 25 l L-NMMA (N -monomethyl-L-arginine, Sigma M7033) solution with Wnal concentration of 800 M, and group D was added with 25 l E. coli suspension and 25 l SNP (sodium nitroprusside, Sigma) solution with Wnal concentration of 10 mM. After incubation in humidiWed chamber for 4 h, samples were stained with toludine blue solution (0.5% toludine blue in 3 mM borate solution) for 5 min, and rinsed with water for 1 min. The samples were then observed under microscope (Zeiss, Jena, Germany) at a magniWcation of 400.

NOS activity was analyzed quantitatively by determination of L-citrulline and total nitrite/nitrate concentrations. BrieXy, 500 l haemocytes suspension with cell density of 10 5 cell/ml was mixed with 500 l LPS solution, and incubated at 4°C for 8 h [14] . Then, the haemocytes were lysed by adding 1% Triton X-100 and agitating on a vortex mixer. After centrifugation at 13,000g for 15 min at room temperature, the supernatants were removed and ultraWltrated. The Wltrate was used to determine the concentration of L-citrulline and total nitrite/nitrate. The haemocytes not incubated with LPS were used as control. The diVerences in NOS activity between the LPS treated haemocytes and the control was considered as LPS stimulated NOS activity.

L-Citrulline was determined according to Marzinzig et al. (1997) [17] with modiWcations described in Weiske and Wiesner (1999) [15] . For each sample, 150 l Wltrate was mixed with 50 l urease (Sigma U2125) solution (165 U/ml) and incubated at 37°C for 30 min. Then, 50 l of 2.45 M trichloroacetic acid was added and mixed thoroughly. After centrifugation at 13,000g for 15 min at room temperature, 150 l supernatant was collected and mixed with 450 l ADMS reagent [18% (v/v) 47.7 mM anti-pyrine (Sigma A5882) in distilled water + 40% (v/v) 79 mM diacetyl monoxime (Sigma B0753) in 83 mM acetic acid + 42% (v/v) 15 N H 2 SO 4 ]. The mixture was incubated for 25 min in a boiling water bath. Samples were cooled to room temperature in the dark and then centrifuged at 13,000g for 5 min, and the absorbance of the supernatant was measured at 450 nm (BECKMAN DU 650 spectrophotometer). The concentration of L-citrulline (Sigma C7629) was calculated according to the standard curve of L-citrulline prepared.

Nitrite/nitrate determination was carried out according to Marzinzig et al. [17] with improvements described in Weiske and Wiesner [15] . For each assay, 300 l of ultraWltrated supernatant was mixed with 20 l Bis-Tris buVer containing FAD [Xavin adenine dinucleotide (Sigma F6625), Wnal concentration 5.5 M], NADPH (Wnal concentration 30.0 M), and nitrate reductase (Sigma N7265, Wnal concentration 0.1 U/ml). The mixture was incubated for 60 min at 37°C under dark condition [18] . Then, 20 l Bis-Tris buVer containing L-lactic dehydrogenase (Sigma L2500, Wnal concentration 0.1 kU/ml) and sodium pyruvate (Sigma P2256, Wnal concentration 0.3 mM) were added and incubated for another 30 min at 37°C. After centrifugation at 10,000g for 15 min at 4°C, 300 l supernatant was mixed thoroughly with 150 l dapsone solution (Sigma D2505, Wnal concentration 4.7 M), and 150 l NEDA [N-(1-naphthyl)ethylenediamine, Sigma N9125] solution (Wnal concentration 1 mM) was added 5 min later. The resulted solution was centrifuged at 10,000g for 5 min at 4°C, and the absorbance of supernatant was measured at 550 nm (BECK-MAN DU 650 spectrophotometer). The concentration of total nitrite/nitrate was calculated according to the standard curve of nitrite prepared.

The carapace, appendages, and hepatopancreas were discarded from frozen F. chinensis with severe WSSV-infections. The residual tissues were then ground into pieces and 1 g tissue was taken to mix with 4 ml of PPB [19] (NaCl, 23.0 g/L; K 2 SO 4 , 1.1 g/L; CaCl 2 , 1.6 g/L; MgSO 4 · 7H 2 O, 1.6 g/L; NaH 2 PO 4 · 2H 2 O, 0.35 g/L; and NaHCO 3 , 0.05 g/L; pH 6.5; osmolarity, 867.9 mOsmol/L, sterilized by Wltration (0.2 m) before using). The mixture was homogenized at 3000 rpm for 5 min and then centrifuged at 5520g for 30 min at 4°C. The supernatant was centrifuged at 9820g for 30 min at 4°C for 2 times. Then, the supernatant was centrifuged at 15,300g for 60 min at 4°C, the precipitate diluted by PPB was used as a virus stock solution.

After acclimation, 80 shrimps were injected intramuscularly with 0.1 ml diluted virus stock solution (100£) in the lateral area of the fourth abdominal segment. The infected shrimps were then subdivided randomly into two groups and placed in two aquaria (200 L) with Xow-through seawater supply. One group of shrimps was used for determination of NOS activity, and the other group was used for mortality observation. Another 80 shrimps injected with 0.1 ml PPB solution were used as control. The shrimps were fed with artiWcial diet twice a day, the unconsumed food and faeces were removed carefully with a siphon once a day.

For both infected and health shrimps, haemolymph sample was collected from three shrimps each time and mixed together, as described in the paragraph of Haemocytes sample preparation. The haemocytes were adjusted to a cell density of 10 5 cells/ml and resuspended in L-arginine solution. The mixture was divided into six Eppendorf micro-centrifuge tubes, three of them were incubated with LPS, the other three were used as control, as described in the paragraph of Analysis of NOS activity in haemocytes of shrimps. For each tube, both L-citrulline and total nitrite/ nitrate were analyzed.

The NOS activity of M. japonicus were assayed at 0, 24, 48, 72, 96, 120, 144, 168, 192 , and 216 h postchallenge, and that of F. chinensis were assayed at 0, 12, 36, 60, and 84 h postchallenge, depending on the surviving time of the shrimps in the experiment.

Diagnosis of WSSV was performed by Digoxigenin Labeled DNA Probe Dot-blot hybridization Kit purchased from the Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences. Following blood sampling for analysis of NOS activity, gills of the same shrimps collected were used for WSSV detection [20] .

Data are reported as means § SE for each treatment, respectively. The signiWcance of variations between diVerent treatments or groups was analyzed by Student's t test or ANOVA in software Excel. The variation was considered to be signiWcant if P < 0.01.

Both NBT method and haemocyte conformation approach conWrmed the NOS activity in haemocytes of the shrimps. The results of NBT method showed that LPS stimulated the activity of NOS in both species of shrimps, after Wxation with 4% formaldehyde, with the absorbance value signiWcantly higher than the control (P < 0.001). For F. chinensis and M. japonicus, the absorbance value increased from 0.091 § 0.006 to 0.160 § 0.008 and from 0.106 § 0.021 to 0.458 § 0.048, respectively.

The results of haemocyte conformation approach also conWrmed the NOS activity in the haemocytes of both species. Most of the haemocytes without any treatment were in amoeboid shape with sporadic bacteria congregated or clumped in their vicinity, as shown in Figs. 1A and 2A. However, most of the haemocytes incubated with LPS became round instead of amoeboid, with a lot of bacteria aggregated around the haemocytes (Figs. 1B and Fig. 2B ). The phenomenon was the same as those haemocytes treated with SNP, a donor of NO, which suggested the production of NO stimulated by addition of LPS (Figs. 1D and Fig. 2D ). This process could be eVectively blocked by the addition of NOS inhibitor L-NMMA, which showed similar results with the haemocytes not treated with LPS (Fig. 1C, Fig. 2C ). These results suggested the existence of NOS in haemocytes of shrimps, and the enzyme activity could be stimulated by the addition of LPS, and blocked by L-NMMA.

The two methods for the analysis of NOS activity are based on the principle that NOS can catalyze L-arginine to generate L-citrulline and NO quantitatively, therefore, concentrations of L-citrulline or total nitrite/nitrate (product of NO) determined could be used to evaluate the activity of NOS. Our results suggested that the two methods coincide well with each other, and either of the methods could be used for the analysis of NOS activity in haemocytes of shrimps.

In the group of F. chinensis infected by WSSV, the results showed that NOS activity in haemocytes increased slightly from 0 to 12 h postchallenge, indicated by the variations of L-citrulline (from 11.15 § 0.10 to 12.08 § 0.64 M) and total nitrite/nitrate concentrations (from 10.45 § 0.65 (Fig. 3) . The LPS-stimulated NOS activity kept constant during the experiment (Table 1 ). In contrast, both the NOS activity and LPS-stimulated NOS activity in the health shrimps used as control was relatively stable during the experiment, and the LPS-stimulated NOS activity in healthy shrimps is generally higher than that in the WSSV-infected shrimps ( Fig. 3 and Table 1 ). In the group of M. japonicus infected by WSSV, the NOS activity in haemocytes kept increasing during the Wrst 72 h postchallenge, the concentrations of L-citrulline and total nitrite/nitrate increased from 7.82 §0.77 at 0 h to 10.79 § 0.50 M at 72h, and from 8.98 §0.43 at 0 h to 11.20 § 0.37 M at 72 h, respectively. The NOS activity in the haemocytes of infected shrimps kept higher than the control from 0 to 120 h postchallenge. Then it decreased till the end of the experiment, and the concentrations of L-citrulline and total nitrite/nitrate at 216 h were 5.66 § 0.27 and 4.68 § 0.16 M, respectively (Fig. 4) . For LPS-stimulated NOS activity, an apparent increase was observed during the Wrst 48 h, and the highest value at 48 h postchallenge was about 4 times higher than that in the control group of health shrimps ( Table 2 ). The LPS-stimulated NOS activity after 48 h then decreased dramatically and became lower than the control. For the health shrimps used as control, the NOS activity and LPS-stimulated NOS activity were relatively stable during the experiment (Fig. 4, Table 2 ). Compared to F. chinensis, the NOS activity in the haemocytes of M. japonicus was more potential to be stimulated by LPS at the early stage of infection (P < 0.01). The value of LPS-stimulated NOS activity was much higher than that of F. chinensis (P <0.01, Figs. 3 and 4) .

The results of Dot-blot hybridization test showed that both species of shrimp were free of WSSV infection prior to experiment, and the shrimps used as control in the experiment were also free of WSSV infection. However, the infection of WSSV in both species after WSSV injection could be conWrmed by the method. The virus was detected at 72 h in M. japonicus and at 36 h in F. chinensis after virus injection, which was in correspondence with the diVerence of NOS activities in the two species.

In correspondence with the diVerence of NOS activity between the two species, the cumulative mortalities were also diVerent. All shrimps of F. chinensis in the mortality experiment died in 66 h, much more quickly than M. japonicus, whose accumulative mortality reached 100% 

In the present study, we examined the NOS activity in the haemocytes of shrimps after LPS stimulation, and the variation of NOS activity after WSSV infection were also studied and compared between two species of shrimps, F. chinensis and M. japonicus.

NOS has been extensively studied among vertebrates and invertebrates. Some of the recent researches have con-Wrmed the presence of NOS activity in haemocytes of invertebrates [15, 17, 21, 22, 29, 30] . Among these researches, techniques of analytical chemistry, immunohistochemistry, and cell and molecular biology have been used to identify NOS [15, 17, [23] [24] [25] [26] [27] [28] . In this experiment, the NBT reduction and cellular conformation approach were employed to identify NOS in the haemocytes of M. japonicus and F. chinensis, under the stimulation of LPS. Both of the methods could conWrm the presence of NOS activity in the two species of shrimps, as reported previously in haemocytes of other invertebrates, like Mytilius edulis and Viviparus ater [15, 22] . However, the expression of NOS in haemocytes still needs to be further conWrmed with techniques like immunochemistry or polymerase chain reaction (PCR).

In vertebrates, three isoforms of NOS, which can be divided into two functional classes, have been found so far. The Ca 2+ /calmodulin-dependent neuronal NOS (nNOS) and endothelial NOS (eNOS) belong to the constitutive class. They produce NO in short bursts in low concentrations for physiological purposes. The other class, which is mainly composed of Ca 2+ -independent inducible NOS (iNOS), is related to the diseases resistance of the organisms. iNOS found in innate immune cells and non-immune cells, such as macrophages and epithelial cells, can produce NO in high concentrations for as long as the enzyme is activated. The NO produced has anti-microbial actions against various intruding microbes, mediated by oxygen radicals, and active oxygen species through the formation of reactive nitrogen oxides like peroxynitrite (ONOO ¡ ), via a variety of mechanisms. However, in invertebrates, only Ca 2+ /calmodulin dependent form was found so far [28, [31] [32] [33] [34] [35] [36] [37] . And in crustaceans, NOS was reported mainly in the nervous system, regulating neuronal activity [38] [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] . Recently, Kim [28] reported that NOS could also be expressed in the Y-organ, gill, testis and ovary of the land crab, which suggested that NO was also involved in other physiological processes except for neuromodulation. It was also suggested by Kim that the localization of NOS in connective tissue and epithelia in Y-organ and gill might function in an immune response to pathogens. The expression of NOS caused by bacterial infection in haemocytes of insects, suggesting the potential role of NOS related to disease resistance, have also been reported previously [15, 50] . Here in our experiment, only a slight increase in NOS activity could be observed in the two species of shrimps shortly after WSSV infection, and then it decreased till the end of the experiment, probably due to the destruction of haemocytes caused by WSSV infection at the late stage of experiment [51] . However, the NOS activity in M. japonicus kept at a high level for a longer time than that of F. chinensis after WSSV infection.More interestingly, there was an apparent increase of LPS-stimulated NOS activity in M. japonicus 48 h after WSSV infection, in contrast to the relatively stable level of LPSstimulated NOS activity in F.chinensis. Referred to the diVerent sensitivity of the two species to WSSV infection, as indicated in the mortality experiment, the results on NOS activity analysis let us hypothesize that the NOS activity in haemocytes of the shrimps might have a relation to the disease resistance ability of the shrimps to WSSV infection.

Although the importance of NO and iNOS in host defense reactions against bacteria have been extensively studied in vertebrates [11] , their roles in resisting virus infections is not very well deWned due to the very complicated situation in virus resistance of organisms. From some previous in vitro studies, it has been demonstrated that NO could inhibit the replication of a diverse range of viruses, especially for DNA viruses, such as murine poxvirus and herpes viruses, and some RNA viruses, such as Coxsackievirus [52] . However, for virus like coronavirus, lymphocytic choriomeningitis virus and murine vaccinia virus, etc., the anti-viral eVect of NO remains unclear or uncertain [52] . Besides, the overproduction of NO will aVect the host defense through its eVects on the balance between the type 1 helper T cell (Th1) and type 2 helper T cell (Th2), and the pathological processes through the oxidative stress it caused. Therefore, its intriguing to understand the role of NO in resistance of virus infection. For iNOS activity, it has been reported that it could be induced by many diVerent virus in vertebrates [53] [54] [55] , through the direct up-regulation of virus or the indirect eVect mediated by pro-inXammatory cytokines such as interferon-. However, the inhibition of iNOS activity and NO production was also reported recently. It's hard to make a straightforward remark for the role of NO and iNOS in virus resistance among diVerent organisms to the infection caused by a variety of virus. And the situation is even worse for invertebrates, although there are some reports about the invertebrate NO synthesis [56] [57] [58] [59] [60] [61] [62] [63] [64] , none of them gave information either on NOS or NO in virus resistance. In our present study, we found that NOS activity in the haemocytes of the two species of shrimp's response diVerently to WSSV infection, which is in correspondence with the diVerent sensitivity of the two species. WSSV, reported as a large dsDNA virus belonging to the virus family Nimaviridae, genus Whispovirus [65] , might be a target of NO. And the observed increase of NOS activity and LPS-induced activity in M. japonicus shortly after WSSV infection might play a role in resistance to virus infection, which lead to a relatively longer surviving time of the species after WSSV infection. However, all these hypotheses need to be further studied to elucidate the role of NO and NOS in host defense and pathological process in WSSV infected shrimps.

LPS have been successfully used in many studies to induce the NOS activity in haemocytes of invertebrates [17, 66, 29, 21] . In the present study, the addition of LPS could also induce the NOS activity in haemocytes of shrimps, and the LPS-stimulated NOS activity in M. japonicus was much higher than that without LPS stimulation after WSSV infection. However, it still needs to be further examined that the induction of NOS activity is good or bad for the mitigation of WSSV infection. The only thing we observed was that M. japonicacus, with higher LPSstimulated NOS activity after WSSV infection, could survive longer than F. chinensis. If the mitigation eVect of increased NOS activity on WSSV infection could be con-Wrmed, it would be helpful for the development of proper immunostimulants, and for the understanding of the mechanisms of certain immunostimulants in host defense ability against the virus infection.

In summary, our results show that the haemocytes of both species of shrimps present NOS activity after LPS stimulation. The comparison of NOS activity in the two species of shrimps after WSSV infection shows that M. japonicus, with higher LPS-stimulated NOS activity after WSSV infection, could survive longer than F. chinensis. The results suggest that NOS in haemocytes of shrimps might be involved in the non-speciWc defense of shrimps to WSSV infection. However, further experiments are still needed to conWrm the expression of NOS in haemocytes of shrimps, and to elucidate the role of NOS in resisting the infection of WSSV virus.

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The mosquito Anopheles stephensi limits malaria parasite development with inducible synthesis of nitric oxide

Time-course and levels of apoptosis in various tissues of black tiger shrimp Penaeus monodon infected with whitespot syndrome virus

Nitric oxide and virus infection

Role of nitric oxide on the replication of haemorrhagic septicemia virus (VHSV), a Wsh rhabdovirus

EVect of nitric oxide on poliovirus infection of two human cell lines

Inducible nitric oxide synthase expression in chronic viral hepatitis. Evidence for a viral induced up-regulation

Nitric oxide synthesis and action in an invertebrate brain

Direct single cell determination of nitric oxide synthase related metabolites in identiWed nitrergic neurons

Immunocytes modulate ganglionic nitric oxide release which later aVects their activity level

Localization of nitric oxide synthaselike immunoreactivity in the developing nervous system of the snail llyanassa obsolete

Complementary distribution of NADPH-diaphorase and L-arginine in the snail nervous system

Seasonal periodicity of enteric nitric oxide synthesis and its regulation in the snail, Helix lucorum

Cannabinoid receptors are coupled to nitric oxide release in invertebrate immunocytes, microglia, and human

Nitric oxide release by hemocytes of the mussel Mytilus galloprovincialis Lmk was provoked by interleukin-2 but not by lipopolysaccharide

Sources and targets of nitric oxide signalling in insect nervous systems

A summary of taxonomic changes recently approved by ICTV

Nitric oxide generation by hemocytes of the mussel Mytilus galloprovincialis

The research is supported by "973" project from Ministry of Science and Technology of PR China (G1999012011). Authors deeply appreciate the advice from Prof. Enzo Ottaviani (Department of Animal Biology University of Modena and Reggio Emilia, Italy) in editing the paper.