Maternal transmission of immunity to white spot syndrome associated virus (WSSV) in shrimp (Penaeus monodon)

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Maternal transmission of immunity to white spot syndrome

associated virus (WSSV) in shrimp (Penaeus monodon )

Chih-Cheng Huang, Yen-Ling Song*

Department of Zoology, National Taiwan University, Taipei, Taiwan

Received 26 August 1998; received in revised form 12 January 1999; accepted 14 January 1999

Abstract

b-1,3-1,6-glucan, derived from bakers' yeast Saccharomyces cerevisiae, was used in the present study to investigate the extent to which glucan is able to protect spawners from white spot syndrome associated virus (WSSV), and whether this protection (if any) can be passed on to hatchlings via maternal transmission of immunity. Results showed that fewer spawners in the glucan-injected groups showed the clinical symptoms of red body coloration and white spots on the shell during the 15 days between eyestalk ablation and the end of repeated spawning. This suggests that the application of glucan might lead to a slight enhancement of disease resistance in spawners, although the dierences were not statistically signi®cant within the con®dence limit chosen. Challenge results showed a signi®cant increase in relative percent survival for larvae derived from groups of glucan-injected spawners compared to those derived from groups of untreated spawners. It therefore seems that a maternally transmitted disease resistance induced by glucan, protected the larvae against a WSSV infection. Glucan immersion was not only shown to be eective for nauplii derived from spawners that were not injected with glucan, it also provided additional, cumulative protection for nauplii which already had a maternally transmitted resistance to WSSV. This is the ®rst documented demonstration of a maternal transmission of immunity in invertebrates. # 1999 Elsevier Science Ltd. All rights reserved.

Keywords: Maternal transmission; Immunity; White spot syndrome associated virus (WSSV); Shrimp

1. Introduction

The high mortality of spawners and hatchlings is a common problem in shrimp nurseries. The vulnerability of the spawners is increased by the

stress induced by eyestalk ablation, wound infec-tion and/or brooding, all of which are believed to generally depress the animals' immune status and make them more susceptible to infection by baculovirus and Vibrio [1±5]. For hatchlings, on the other hand, the most critical periods seem to be the four metamorphic stages from nauplius, zoea and mysis to postlarvae. The transition from zoea to mysis is especially dicult and it is

* Corresponding author. Tel.: +886-2-23630231, Extn 3355; fax: +886-2-23660243.

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typically only survived by about 30±50% of the hatchlings. Various strategies against these high mortalities have been investigated. Thus, for example, since bacterial infections are often as-sociated with the deaths of these hatchlings, Liu [6] investigated the eciency of a sulfa drug (fur-azolidone) administered both orally and via an immersion route. In fact, the drug never reached bactericidal concentrations in the shrimp hemo-lymph [6], but in any case the problem with this approach is that drug residues not only induce drug-resistant strains but also result in environ-mental pollution. An alternative approach, in which an insoluble b-1,3-1,6-linkaged glucan functions as an immunostimulant, has however, been demonstrated to enhance resistance to vibriosis and WSSV disease in shrimp [7,8]. The yeast glucan enhances disease resistance by acti-vating the prophenoloxidase (proPO) system, releasing highly reactive oxygen species from hemocytes and inducing antimicrobial activity in plasma [7,9,10].

Maternal transmission of immunity can occur in several dierent ways. In mice and chicken it is known to take place mainly via the egg sac [11±14] and a positive correlation has recently been found between the Ig level in the plasma of a mature female salmon and the Ig content in her eggs [15]. In Drosophila, embryonic pattern-ing appears to be controlled by the bindpattern-ing of extracellular ligands that initiate and activate the Toll-Dorsal signaling pathway, but maternal eect genes in the signaling pathway have been found to be related to Drosophila's innate immune response at later developmental stages [16,17]. In the present study we investigate the extent to which glucan is able to protect spaw-ners from white spot syndrome associated virus (WSSV), and whether this protection (if any) can be passed on to hatchlings via maternal trans-mission of immunity.

2. Materials and methods

Glucan suspension (supplied as an adjuvant ingredient by Biotech-Mackzymal AS, Norway) was processed as described in a previous report

[8] in preparation for both injecting spawners and bathing nauplii. Glucan was administered to spawners and nauplii during times when other cultural procedures already required the shrimp to be handled, so that the additional stress of multiple handling could be avoided.

In two separate trials, spawners of sucient sexual maturity were selected based on body size (10.5±11.5 in. in body length) [18,19] and reared in concrete ponds (6 mL 4 mW 2 mH) con-taining re-circulated brackish water. Salinity was adjusted to 3.0% and the temperature was kept at 288C. One hundred ml shrimp sterile saline containing 100 mg glucan was injected into the pericardial cavity of each experimental spawner using a 30 ga needle. Control spawners were similarly injected with saline alone. After being injected, spawners were disinfected in oxolinic acid solution for 3 min and then returned to the concrete ponds. The next day one eyestalk was ablated from each spawner to promote egg matu-ration. Spawners were fed with live feed 4±6 times per day until spawning at the ®fth day. Diseased and dead spawners were counted during each of the 15 days from post ablation to the end of repeated spawning. The percentage of dead and diseased spawners in the glucan-injected and control groups were statistically ana-lyzed with a t-test.

Eggs were produced either by natural crosses or by in vitro fertilization, and no attempt was made to distinguish between these two groups. Hatched nauplii were pooled 13±14 h post-spawning, with nauplii derived from the glucan-injected spawners being kept separate from those derived from the spawner controls. Half of each group were then bathed in aerated brackish water containing glucan at 0.5 mg/ml (i.e. group A: +/+ and group C: ÿ/+). The other nauplii were bathed in the brackish water only (i.e. group B: +/ÿ and group D: ÿ/ÿ; control group). Bath density was adjusted to 5.5 million nauplii per 500 liters of water in a round plastic container. The temperature was held at 328C. After immersion for 3 h, each group of 5.5 million nauplii was transferred to separate indoor ponds which each contained 40 metric tons of brackish water.

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To determine whether glucan gave the larvae immunity to WSSV infection, 250 larvae were randomly taken from each of the four groups at the mysis and postlarva 0 stages (about 7 and 10 days post-hatching, respectively) and subjected to a challenge test. These viral infection experiments broadly followed the method of Momoyama and Sano [20] with modi®cations which have already been described in detail in a previous paper [8]. After exposure to WSSV by immersion, the lar-vae were returned to a beaker and maintained for another 5±6 days. They were fed with both artemia nauplii and arti®cial feed. Larvae were checked for viability every 4 h and bodies were removed immediately after death to keep the water clean. Because deaths that occurred within 24 h post-challenge probably resulted from mech-anical injury or handling stress rather than

infec-tion, these `non-infectious deaths' were

subtracted from the calculations of larvae mor-tality. Larvae mortality (LM) was thus de®ned as follows: LM 0 B B @

Total number of deaths ÿnon-infectious deaths Total number of shrimp tested

ÿnon-infectious deaths 100% 1 C C A The challenge tests were terminated until two days had passed without an infectious death. The cumulative mortalities of larvae at the stationary phase were expressed in terms of relative percent survival (RPS):

RPS

1 ÿMortality in experimental group_{Mortality in control group} 100%

Dierences between the paired groups (A and B, B and D, C and D) were examined using a Duncan's multiple range test of the cumulative mortalities of each group. Dierences between the two sets of challenge groups (i.e. challenged at mysis vs challenged at PL0) were examined using a variance analysis (ANOVA) of the RPSs of each group. Dierences between the four pro-tocols (i.e. groups A, B, C and D) were also com-pared using Duncan's multiple range test.

3. Results

During the 15 days between ablation and the end of repeated spawning, spawner mortality was slightly higher in the glucan-injected groups than in the control groups (Table 1), although there were no statistical dierences when the results were analyzed using a t-test. On the other hand, fewer spawners in the glucan-injected groups showed the clinical symptoms of red body color-ation and white spots on the shell (Table 1), which suggests that application of glucan might lead to a slight enhancement of disease resistance in spawners, although again the dierences were not statistically signi®cant within the con®dence limit chosen.

Table 1

Eects of b-1,3-1,6-glucan on the health status of Penaeus monodon spawners during their spawning period Trial Number of

spawners Glucan-injected Deaths afterinjectiona Deaths during_spawningb No. (%) of survivors_{with clinical symptoms}c

I 56 + 0 4 0/52 (0)

235 ÿ 0 4 10/231 (4)

II 49 + 1 2 1/46 (2)

50 ÿ 12 1 7/37 (19)

a_{Deaths occurring in the 24 h between injection and eyestalk ablation.}

b_{Deaths occurring during the 15 days of repeated spawning that followed eyestalk ablation. No signi®cant dierence was shown}

between injected and control spawners (t-test; P = 0.1982 > 0.05).

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The cumulative mortalities for the ospring groups that were challenged at mysis (Fig. 1A)

and PL0 (Fig. 1B) showed that more of the hatchlings derived from the glucan-injected spaw-ners (i.e. group B) survived than those in the cor-responding untreated spawner group (i.e. group D). Mortality rates were nevertheless quite simi-lar, and Duncan's multiple range test showed that there was in fact no signi®cant dierence between any of the pairs of corresponding curves. However, because an ANOVA analysis of the RPS values also showed that there were no sig-ni®cant dierences between the mysis and PL0 challenge groups, the RPS results for these two groups can be combined. When mean RPS values are considered, the dierence between groups D and B (23%; see Table 2) is signi®cant. This suggests that a maternally transmitted disease re-sistance induced by glucan has protected the lar-vae against a WSSV infection. A similar maternal transmission of immunity was also inferred from the dierences between groups C (23.5%) and A (59.5%). Glucan immersion also increased survival rates, as shown by the 23.5% RPS increase between groups D and C and the 36.5% increase between groups B and A. Glucan immersion was thus shown to be eective for nauplii derived from spawners that were not injected with glucan, while it also provided ad-ditional, cumulative protection for nauplii which already had a maternally transmitted resistance to WSSV.

3. Discussion

During the 15 days between eyestalk ablation and the end of repeated spawning, the mortality of the spawners was slightly higher in the injected groups than in the control groups. However, there were fewer spawners showing clinical symp-toms in the glucan-injected groups than in the corresponding controls. Even though neither of these dierences were statistically signi®cant, these data nonetheless suggest that induction of disease resistance by 100 mg glucan does not occur immediately in spawners. At high concen-trations, glucan has been reported to show no, or even adverse, eects in shrimp in terms of tissue damage and resistance to Vibrio infection [7].

Fig. 1. Cumulative mortality in WSSV-challenged ospring. (A) Ospring challenged at mysis (i.e. approx. 7 days after hatching). (B) Ospring challenged at PL0 (10 days post-hatching). None of the nauplii shown here were immersed in the glucan suspension. W=larvae from untreated spawners (ÿ/ÿ), group D); ÐwÐ=larvae from glucan injected spaw-ners (+/ÿ, group B).

Table 2

Relative percent survival (RPS) of larvae challenged by immersion in WSSV ®ltrates prepared from infected P. mono-don

A B C D

Treatmenta_{Spawners injected with glucan +} _{+ ÿ} _ÿ

Nauplii immersed in glucan + ÿ + ÿ RPS (%)b _{Challenge at mysis stage}c ₆₈ _{25 27} ₀

Challenge at postlarva 0 stagec₅₁ _{21 20} ₀

Meand _59.5A₂₃B_23.5B₀C a_{Symbol `+' represents glucan-treated and `ÿ' non-treated.} b_{RPS1 ÿ} Mortality in exp: group

Mortality in control group 100%.

c_{ANOVA showed no signi®cant dierences between these}

two challenges.

d_{Duncan's multiple range test at a 5% con®dence limit}

showed that the means diered signi®cantly. Signi®cantly dierent means are indicated by dierent letters.

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Similarly, a high dose of levamisole suppresses the defense mechanisms of rainbow trout [21]. Robertsen et al. [22] and Jùrgensen [23] also found that the onset timing of disease-resistance and/or stress-tolerance was glucan-dose depen-dent in salmonids, and suggested that while a low dose of immunostimulant (within an optimal range) will induce a quick-on and quick-o pro-tection, a higher dose requires a lag period during which the glucan is metabolized, and pro-tection is only initialized once the glucan concen-tration has fallen back to an optimal range. This in turn suggests that the injection dosage (100 mg glucan per spawner) used in the present study might have been too high. Nevertheless, inspi-ration comes from Su et al. who reported that

Schizophyllum commune-derived b-1,3-glucan

could enhance survival of P. monodon brooders during a 30-day feeding trial. They concluded that this daily oral dose (0.2% glucan in the feed) stimulated hemocytes by signi®cantly increasing phagocytic activity, cell adhesion and superoxide anion production [24].

Jarp and Tverdal [25] suggest that if the cumu-lative cases in immunostimulated and unimmu-nostimulated groups are registered at the end of the observation period, the immunostimulant eect can be measured with the risk ratio, risk dierence or the RPS. RPS is the same as the preventive fraction or the immunostimulant eect [26,27]. RPS represents the fraction of the mor-tality that was prevented by the immunostimu-lant up to the end of the observation period, so RPS is dependent on the experimental conditions and the length of the observation period. A point estimate of RPS is very often used to present the results from ®sh vaccination trials, because a single value is clear, precise and easy to under-stand. Unfortunately, the very precision of this point estimate gives sometimes the misleading im-pression that the value presented is the correct answer [25].

In this study when the mysis and PL0 chal-lenge groups are compared during the obser-vation period, the dierence in cumulative mortalities between any of the pairs of corre-sponding curves in group B and the control group (group D) are not statistically signi®cant.

However, when the mysis and PL0 challenge data are compared at the end of the observation period, the dierence between the mean RPSs does become signi®cant. We have therefore demonstrated here that larvae derived from glu-can-injected mothers had signi®cantly higher sur-vival rates than the control groups against an arti®cial infection with WSSV. To the best of our knowledge, this is the ®rst documented demon-stration of a maternal transmission of immunity in invertebrates.

Glucan immersion also improved survival rates. The 23.5% increase in RPS between groups D and C (Table 2) is similar to the 24±32% improvement in RPS previously reported for shrimp that were fed daily with b-glucan [8]. Furthermore, the larvae from glucan-injected mothers had even higher survival rates when they were restimulated at the nauplius stage (59.5% RPS; see Table 2). In other words, stimulation of spawners and restimulation of the nauplii induced an additive protection eect in shrimp against WSSV infection.

Recently some studies on the proPO activating system (proPO-AS) in shrimp were carried out on the penaeid such as P. californiensis, P. japo-nicus, P. monodon, P. paulensis and as freshwater

prawn (Macrobrachium rosenbergii ).

Phen-oloxidase (PO) activity is found mainly in hemo-cytes [7,28,29] and/or serum [30±32]. Enzyme ac-tivity was cation-dependent [28,32,33]. It can be enhanced by several microbial polysaccharides, including b-glucan from fungal cell walls [7,10,28,32], laminarin from seaweed [31], lipopo-lysaccharides (LPS) from bacterial cell walls [10,28,32]. Additional factors found to activate the proPO system include SDS [31], trypsin [28,32] and high temperature [28,30]. An immune factor, similar to the 76 kDa protein from the cray®sh, was found in the P. paulensis hemocytes, capable of inducing cell-adhesion and degranula-tion of the semigranulocytes and granulocytes [32]. A b-glucan-binding protein (b GBP) of 100 kDa molecular mass, similar to the cray®sh b GBPs [34] in terms of amino acid composition in N-terminal sequence, has been identi®ed in plasma of P. californiensis, capable of enhancing the proPO system activation induced by

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lami-narin [35]. In addition, a LPS-binding agglutinin [29] and a hemolytic factor [36] have been detected in brown shrimp (P. californiensis ) serum, although these proteins do not seem to be involved in proPO system stimulation. Although the proPO-AS is unraveled, the associated pro-teins of the penaeid shrimp are highly similar in terms of both structure and function to those observed in crustaceans [37]. We therefore believe that they may play an important role in shrimp defense mechanisms, too.

Although at this point we can only speculate upon the mechanisms that underlie the maternal transmission of immunity in shrimp, current evi-dence suggests that there might be several paral-lels between shrimp and Drosophila. Recently in kuruma shrimp (P. japonicus ) numerous micro-villi were found on the surface of oocytes. Immunohistochemical staining showed that egg yolk protein may be transferred and incorporated into the ooplasma through these microvilli [38]. SoÈderhaÈll and Cerenius [37] have shown that the shrimp protein LP-1, which seems to be identical to the cray®sh b GBP, is involved in lipid trans-port to the ovary showing that reproduction and immunity might be linked in crustaceans. During the oogenesis Drosophila maternal RNA and pro-teins are transported from follicle cells into the oocytes and aect embryo development [39]. In Drosophila, embryonic patterning appears to be controled by the binding of extracellular ligands that initiate and activate the Toll±Dorsol signal-ing pathway, but maternal eect genes in the sig-naling pathway have been found to be related to Drosophila's innate immune response at later development stages [16,17]. It is tempting to speculate that there may also be a link between the proPO-AS and the induction of antimicrobial syntheses in shrimp, since a b GBP has been identi®ed in brown shrimp P. californiensis [35] and a serine proteinase associated with the proPO-AS is likely to induce activation of the Toll-signaling pathway in Drosophila, leading to synthesis of antimicrobial peptides [37]. Besides, structural similarities in C-terminal portions between coagulogen from coagulation cascade of horseshoe crab and spatzle, the ligand of the Toll receptor involving in Drosophila Toll-signaling

pathway, and proposed similarity between at least two of their processing serine proteinase in each cascade (factor B, proclotting enzyme versus Snake and Easter) have been detected [40]. By analogy with the Drosophila Toll-signaling path-way, we hypothesize that a prospatzle-like pro-tein may be present in shrimp hemolymph or hemocyte and that it is processed by a proteinase which may be involved in the proPO-AS or co-agulation cascade and activated by b-glucan. The spatzle-like molecules may act as ligands to induce activation of the Toll-signaling pathway, leading to synthesis of maternal mRNA or anti-microbial peptides that transfer into the ooplasm through the microvilli and enhance disease resist-ance of larvae at later developmental stages. This interesting possibility will need to be investigated in future studies.

Acknowledgements

We thank Biotech-Mackzymal AS Company (Tromso, Norway) for kindly providing b-glucan for this experiment. We thank Professor Ying-Chou Lee, Institute of Fishery Science, National Taiwan University for the statistical analysis. Sincere thanks are also extended to Professor

Masaaki Ashida, the Institute of Low

Temperature Science, Hokkaido University for the many valuable points he raised during discus-sions. This research was ®nancially sponsored by the Council of Agriculture, under Grants No. 85AD-6.3-F-11(6-10) and 87BT-2.1-FID±01(5-1). References

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