Aquaculture 334-337 (2012) 111

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Aquaculture
journal homepage: www.elsevier.com/locate/aqua-online

Review

Vaccines and crustacean aquacultureA mechanistic exploration

Andrew F. Rowley , Edward C. Pope
Department of Biosciences & Centre for Sustainable Aquatic Research, College of Science, Swansea University, Singleton Park, Swansea, SA2 8PP, UK

a r t i c l e

i n f o

s u m m a r y
One of the dogmas of comparative (evolutionary) immunology for the last few decades has been that all invertebrates lack any form of immunological memory similar to that found in jawed vertebrates. Vaccinating invertebrates, such as shrimp, should therefore be an ineffective management strategy resulting in no more than short-lived, non-specic immune stimulation. Recent studies in crustaceans and insects, however, suggest a form of immune memory in these animals described variously as immune priming, specic immune priming or line specic immune memory. Workers have injected various arthropod species with inactivated pathogens and then later challenged them with a dose of the same pathogen capable of killing the host, reporting enhanced survival or increased reproductive fecundity compared with animals vaccinated with an unrelated organism. Interestingly, in some studies this enhanced immunity is trans-generational (i.e. passed on to progeny). This brief review focusses on a mechanistic exploration of specic immune priming with particular reference to shrimp culture. Humoral antimicrobial factors, such as antimicrobial peptides and lysozyme, do not possess the required specicity to explain this phenomenon. Instead, recent studies have demonstrated elevated phagocytic activity after vaccination that does not result from general (nonspecic) stimulation in phagocytosis. This enhanced phagocytic activity is likely linked to specic recognition of determinants on the outside of microbes by the haemocytes of these vaccinated animals. In some cases, at least, the mechanism for recognising these determinants appears to rely upon an invertebrate homologue of the Down syndrome cellular adhesion molecule (Dscam) and the ability of the Dscam gene to produce variants of this molecule with specic binding capabilities. Pioneering studies have demonstrated that Dscam is intricately involved in phagocytosis in insects and able to produce pathogen-specic splice form variants after immune challenge or infection. This would enable an animal to tailor an immune response specically to a pathogen. It should be noted, however, that the phenomenon of immune priming is certainly not universal, possibly because of the much lower numbers of different immune receptors that arthropods are able to produce compared with jawed vertebrates. The review concludes that immune priming in invertebrates should be re-evaluated on an animal to animal and pathogen to pathogen basis. It also notes that whilst there is evidence that vaccinating shrimp against viral and bacterial infections shows promise, the practicality of such processes and their benet to shrimp requires further evaluation. 2011 Elsevier B.V. All rights reserved.

Article history: Received 25 October 2011 Received in revised form 5 December 2011 Accepted 7 December 2011 Available online 14 December 2011 Keywords: Shrimp WSSV Vaccine development Down syndrome cellular adhesion molecule Specic immune priming dsRNA

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The invertebrate immune system with particular reference to shrimp and other crustaceans . . . . . . . . . . . . . . Immune priming, specic immune priming and trans-generational immune primingthe phenomenon (see . ) . . . . . . . . . Immune priming, specic immune priming and trans-generational immune primingmechanistic explanations . . . . . . 4.1. Changes in humoral antimicrobial activity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Changes in phagocytic activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Limitations of specic immune priminga cautionary note. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Vaccines for crustaceans such as shrimpare they feasible in light of recent reports of specic immune priming and the development of RNA interference techniques? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. 2. 3. 4. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 2 2 3 3 6 8 8 9 9 9

Corresponding author at: Department of Biosciences, College of Science, Swansea University, Singleton Park, Swansea, SA2 8PP, Wales, UK. Tel.: + 44 1792 295455. E-mail address: a.f.rowley@swansea.ac.uk (A.F. Rowley). 0044-8486/$ see front matter 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2011.12.011

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1. Introduction One of the central dogmas of comparative (evolutionary) immunology for the last 3040 years has been that invertebrates do not possess a specic immune system similar to that of jawed vertebrates. Invertebrates lack the lymphocytes and functional antibody (immunoglobulin) of jawed vertebrates and hence their immune system is normally described as non-specic or innate. The specic (acquired) immunity found in mammals and other vertebrates is thought to have evolved over 500 million years ago with the advent of the early jawed vertebrates. The hallmarks of this specic immune system, specicity and memory, are realized through the recognition of antigens by immune receptors on lymphocytes via somatic gene rearrangement and the subsequent production of effector (e.g. plasma) and memory cells through clonal expansion (Male et al., 2006). Vaccination depends upon these mechanisms so that a second encounter between the host and the pathogen in the vaccine results in heightened immunity that is specic for that particular pathogen. With this in mind, the logical conclusion is that invertebrates, apparently lacking a specic immune system, hence cannot be vaccinated to yield animals that show specic protection at some later stage. In this case, the vaccine would behave as a non-specic immune stimulant rather than a true vaccine. There are several examples of such immunostimulants developed for use in aquaculture, including complex polysaccharides derived from a number of sources such as other marine animals (e.g. chitin; Powell and Rowley, 2007), macroalgae (e.g. fucoidan; Chotigeat et al., 2004; Ergosan, Montero-Rocha et al., 2006), microalgae and yeast (e.g. 1, 3 glucans; Burgents et al., 2004) and vascular plants (Blasubramanian et al., 2008). The health benet of continuous prophylactic application of such immunostimulants has been questioned, however, as constant stimulation of the immune system could be detrimental rather than protective (Smith et al., 2003). Indeed, from the perspective of ecological immunology, the cost of heightened immune defence could reduce tness in other key life history events such as growth and reproductive fecundity (Sadd and Schmid-Hempel, 2009; Schmid-Hempel, 2005, 2011) that would, in principle, be detrimental to animal production under aquaculture conditions. It should be noted, however, that although there are many examples of this phenomenon in animals ranging from invertebrates to birds and mammals, such events may not be universal in all animal parasite/pathogen interactions (Labb et al., 2010). Several recent reports using mainly insectan or crustacean models have suggested that the invertebrate innate immune system may be capable of some form of immune memory, described as immune priming (Moret, 2006), specic immune priming (Pham et al., 2007; Roth and Kurtz, 2009) or line specic immune memory (Kurtz and Franz, 2003). As some of these reports are largely reporting phenomena, such as increased survival or increased fecundity, with little or no mechanistic explanations, conclusions in favour of the existence of some form of specic immunity have attracted criticism (Hauton and Smith, 2007). However, increasingly evidencebased explanations of at least some of the phenomena observed are appearing, suggesting that these warrant further research at a molecular and cellular level. This brief review focusses on an exploration of the phenomenon of specic immune priming with particular reference to shrimp culture. It also examines other strategies to vaccinate shrimp centred on the inhibition of viral replication in its host. Overall, it evaluates the potential implications of these two research areas to improve the health of these economically important animals. 2. The invertebrate immune system with particular reference to shrimp and other crustaceans The nature of the immune system of invertebrates is now well established as a result of initial cell-based studies (see review by

Ratcliffe et al., 1985) and more recent biochemical, proteomic and genomic approaches applied in the last couple of decades (see Aoki et al., 2010; Hirono et al., 2011; Robalino et al., 2009; for reviews). Although it is potentially dangerous to over compartmentalise the mechanisms, the invertebrate immune system comprises both cellular and humoral arms that are intimately linked. The main effector cells of this immune system in crustaceans are the blood cells or haemocytes whilst the hepatopancreas is responsible for the biosynthesis of some humoral factors (Fig. 1). In shrimp, as in other crustaceans, there appears to be three main types of haemocytes (blood cells), the hyaline cells, semi-granular cells and granular cells (termed granulocytes by some authors), all of which appear to participate in immune defences (Jiravanichpaisal et al., 2006; Martin and Graves, 1985). Once potential pathogens invade through the gastrointestinal (GI) tract or cuticle, they immediately come into contact with haemocytes in the underlying haemocoel (see Fig. 1). Haemocytes recognise pathogen-associated molecular patterns (PAMPs) associated with these invaders and respond with either phagocytosis or an encapsulation reaction that segregates the pathogen into nodules or capsules. At a later time, either these blood cells or cells in the hepatopancreatic tubules synthesise and release a host of factors including antimicrobial peptides (AMPs), such as penaeidins (in shrimp) and crustins, lysozyme and lectins (Table 1). In shrimp, lectins have recently been recognised as bi-functional molecules that are not only involved in the recognition of sugar residues in some PAMPs, leading to their opsonisation and destruction by phagocytic haemocytes (Luo et al., 2006), but also possess potent antibacterial, antifungal (J. Sun et al., 2008; Y.D. Sun et al., 2008) and antiviral (Zhao et al., 2009) activities. Similarly, the penaeidins synthesised and stored in the granular haemocytes of shrimp (Destoumieux et al., 2000) have potent activity against Gram positive bacteria, bind vibrios (Gram negative bacteria) and contain a chitin-binding domain (Destoumieux et al., 2000) that presumably allows these molecules to be immobilised in the cuticle as part of the external defences against invasion. Fragments of the respiratory pigment, haemocyanin, may act as an alternative phenoloxidase (Adachi et al., 2003) and have both antifungal (Destoumieux-Garzon et al., 2001) and antiviral (Lei et al., 2008) activity. Finally, the prophenoloxidase activating system not only results in the formation of melanin (a hallmark of many cellular events such as encapsulationsee Fig. 1) but also generates a series of cytotoxic factors and is linked to wound healing and blood clotting (Cerenius et al., 2010). The potential importance of the prophenoloxidase activating system has been recently shown by RNA interference (RNAi)-mediated depletion of prophenoloxidase in craysh which resulted in greater susceptibility to infection, probably as a result of a reduced ability to carry out the cellular defence reactions of phagocytosis and encapsulation-type responses (Liu et al., 2007). Similar studies using shrimp (Penaeus monodon) have revealed that RNAi silencing of a gene associated with phenoloxidase activation resulted in a signicant increase in susceptibility of the animals to challenge with Vibrio harveyi (Charoensapsri et al., 2009). 3. Immune priming, specic immune priming and trans-generational immune primingthe phenomenon (see Table 2) Because a signicant number of reports of these phenomena have been made in non-crustacean models (Table 2), these next sections also review our understanding of these in a wide range of invertebrates. Although there are differences in the experimental design and the nature of the animals used in the initial studies on immune priming, in essence these studies all involve a primary exposure (1 vaccination) and usually a second exposure (2 vaccination) followed by either a nal assessment of the animals' ability to survive a challenge with live pathogens (i.e. LD50 type determinations) or measurement of other events such as fecundity. What these studies often demonstrate is that animals initially vaccinated with inactivated pathogen

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Fig. 1. Diagrammatic representation of the cellular and humoral defence reactions of crustaceans including shrimp.

and later challenged with a dose of the same pathogen capable of killing the host, show enhanced survival or increased reproductive fecundity compared with those animals vaccinated with an unrelated organism. In the case of some of the experiments, this enhanced immunity is trans-generational in nature. For example, Little et al. (2003) found the clonal progeny of female water eas, Daphnia magna, that had previously been exposed to one of two strains of the pathogenic bacterium, Pasteuria ramose, showed straindependent enhanced fecundity and survival upon subsequent challenge with homologous (i.e. the same strain their mother had experienced) compared with heterologous (i.e. mother and offspring exposed to different strains) bacterial strains. The authors' interpretation was that progeny from mothers previously exposed to homologous challenge agent showed specic elevation in their tness. To date, this work lacks a mechanistic explanation of how this transfer of specic protection could occur between mothers and offspring. 4. Immune priming, specic immune priming and trans-generational immune primingmechanistic explanations 4.1. Changes in humoral antimicrobial activity In an effort to nd a mechanism for trans-generational immune priming, Moret (2006) challenged mealworms (Tenebrio molitor)

with bacterial lipopolysaccharide (LPS) and subsequently analysed the phenoloxidase and antimicrobial activity in their offspring 3 days after further challenge with LPS. He found that the antibacterial activity of haemolymph was greater in the progeny of parents that themselves were also exposed to LPS and hence concluded that this enhanced immunity was probably a result of enhanced production of antimicrobial peptides. A similar experimental approach was employed by Sadd et al. (2005) using the social insect, Bombus terrestris. During maternal challenge, queens of B. terrestris were injected with either saline or heat-inactivated bacteria (Arthrobacter globiformisa pathogen of bees) and the antibacterial activity in the haemolymph of subsequent offspring was measured 24 h after stimulation with LPS. They found evidence of heightened antibacterial activity in the haemolymph of workers which were the progeny of queens exposed to A. globiformis compared to those that were injected with saline. A more recent and detailed study again using an insect model (the red our beetle, Tribolium castaneum) has further explored trans-generational immune priming (Roth et al., 2010). The authors used a series of parental exposures to heat-killed Bacillus thuringiensis (an insect pathogen), Escherichia coli (an opportunistic pathogen), saline or no treatment followed by the formation of breeding pairs. The progeny of these was subsequently challenged with live B. thuringiensis or E. coli. These experiments showed trans-generational immune priming with specicity to the nature of the original immunogen

Table 1 Examples of key immune factors in crustaceans with particular reference to shrimp.
Name Anti-lipopopolysaccharide factors Crustins Category Antimicrobial peptides Activities Active against Vibrio harveyi, fungi and viruses (WSSV) Mainly active against Gram +ve bacteria but also Vibrio penaecida (and other vibrios?) based on RNAi inhibition of crustin expression studies of Shockey et al. (2008) Recognition of foreignness (pathogens) and self (during brain development) Antimicrobial against fungi Species described in Various, including Litopenaeus vannamei and Penaeus monodon L. vannamei and L. setiferus Location Expression Constitutive but upregulated by various agents (including WSSV) Haemocyteassociated Mainly constitutive but variable changes following microbial challenge including down regulation (see Smith et al., 2008 for details) Key references Liu et al., 2005; de la Vega et al., 2008; Ponprateep et al., 2009; Tharntada et al., 2009 Gross et al., 2001; Bartlett et al., 2002; Vargas-Albores et al., 2004; Shockey et al., 2008 Key reviews Liu et al., 2009; Hirono et al., 2011 Smith et al., 2008

Antimicrobial peptides (related to carcinin in crabs)

Down syndrome cell adhesion molecule (Dscam) homologue Haemocyanin fragments Haemocyanin fragments

Recognition

Daphnia magna, P. monodon, L. vannamei

Dscam homologue in insects

Dscam found free in haemolymph (in insects)

Antimicrobial peptides

L. vannamei and P. stylirostris P. japonicus

Plasma

Antiviral peptide

Antiviral activity against WSSV

Plasma

Haemocyanin fragments Lectins

Alternative phenoloxidase Recognition, antimicrobial 1. Recognition of foreigness by crosslinking foreign cells and effector cells of immune system (pattern recognition proteins) 2. Antiviral activity 3. Antibacterial activity 4. Antifungal activity Active against both Gram +ve and G ve bacteria (including pathogenic vibrios) Many, including Fennero-penaeus chinensis, L. vannamei, P. monodon

Plasma Synthesised by hepatopancreas

Bacterial challenge causes increase in concentration in plasma Expression of one gene associated with fragment generation induced by WSSV infection Constitutive (?) Constitutive. Expression up-regulated following challenge with bacteria (Vibrio anguillarum) and viruses (WSSV)

Watson et al., 2005; Dong et al., 2006; Brites et al., 2008; Chou et al., 2009, 2011; Watthanasurorot et al., 2011 Destoumieux-Garzon et al., 2001 Lei et al., 2008

Du Pasquier, 2005; Gross, 2006; Boehm, 2007; Ghosh et al., 2011 No recent reviews

A.F. Rowley, E.C. Pope / Aquaculture 334-337 (2012) 111

No recent reviews

Adachi et al., 2003 Luo et al., 2006; J. Sun et al., 2008; Y.D. Sun et al., 2008; Zhang et al., 2009; Zhao et al., 2009

No recent reviews Ghosh et al., 2011

Lysozymes

Antimicrobial (enzymatic)

L. vannamei, Marsupenaeus japonicus, P. monodon

Haemocyteassociated

Constitutive

Gross et al., 2001; Hikima et al., 2003; de-la-Re-Vega et al., 2006; Burge et al., 2007; Tyagi et al., 2007; Bu et al., 2008;

No recent reviews

Penaeidins (PEN2, PEN3, PEN 4)

Antimicrobial peptides (57 kDa)

Peroxinectin

Recognition and antimicrobial Activation of phagocytic uptake

Phagocytosis activating protein (Ribosomal protein L26 homologue)

1. Antimicrobial: mainly Gram + ve bacteria and fungi (at high concentrations) 2. Vibrio-binding 1. Cell adhesion/opsonisation 2. Peroxidase activity (microbial killing?) Enhanced phagocytosis

Widespread including L. vannamei

Synthesised and stored in haemocytes within granules

Up-regulated in a range of tissues following bacterial challenge Constitutive (transcription is not induced by bacterial infection)

Muoz et al., 2002, 2003, 2004; de Lorgeril et al., 2005; Jiravanichpaisal et al., 2007 Liu et al., 2004

Bachre et al., 2004; Tincu and Taylor, 2004; Cuthbertson et al., 2008; Jiravanichpaisal et al., 2006; Zamocky et al., 2008 No recent review

L. vannamei

Constitutive

P. monodon

Haemocytes

Phenoloxidase (prophenoloxidase cascade) Toll receptors

Antimicrobial (cytotoxic factors) Recognition (enhanced encapsulation, etc.) Recognition of PAMPs (?)

Key enzyme in prophenoloxidase cascade

In insects at least, key receptor in activation of AMP expression

Various but most key studies in craysh (Pacifastacus leniusculus) L. vannamei

Haemocyte associated and released upon activation Haemocytes and several other cell types?

Injection of inactivated V. harveyi, WSSV or fucoidan causes increased gene expression > 4 week post-challenge. Potential immunostimulant Mechanism of stimulation of phagocytosis unknown Constitutive

Deachamag et al., 2006; Chotigeat et al., 2007

Liu et al., 2007; Charoensapsri et al., 2009, 2011

Sritunyalucksana and Sderhll, 2000; Jiravanichpaisal et al., 2006; Cerenius et al., 2008, 2010

Constitutive

Yang et al., 2007

Table 2 Recent examples of reported acquired (specic) immunity in arthropods and its mechanistic explanation (adapted and updated from Rowley and Powell, 2007).
Event Trans-generational enhanced immunity Animals Nature of immunogen Main ndings A form of enhanced immunity is present in the offspring. In some cases this shows specicity towards the original immunogen Mechanistic explanation Enhanced antibacterial (humoral defences) in progeny Lectin binding Comments Nature of antibacterial factors unknown; level of specicity in relation to challenge unknown Short timescale between primary and secondary exposure may invalidate conclusions; antigenic relatedness of different strains of parasites not revealed Specicity of the reaction untested (i.e. is this general immune stimulation?) and overall duration limited to maximum tested of 25 days Strong evidence for specic and relatively long-term memory as evidenced by cumulative survival after pathogen challenge References Little et al., 2003; Sadd et al., 2005; Moret, 2006 Kurtz and Franz, 2003

Line-specic memory

Insects, including Various bumblebees and mealworms, and Daphnia magna Copepods Tapeworm larvae (Macrocyclops albidus) (Schistocephalus solidus)

Vaccination resulting Shrimp in enhanced survival following subsequent challenge Specic immune priming Bumblebees

Envelope proteins (VP19, VP28) of white spot syndrome virus Gram + ve and Gram ve bacteria

Drosophila

N/A

Shrimp can be partially protected against WSSV by exposure to some viral envelope proteins (VP28) Bumble bees were injected with one of three bacterial pathogens and later challenged with heterologous, related (same genus but different species) or homologous forms. Differences in survival were dependent upon the nature of the primary vaccination Flies unable to express Dscam show reduction in haemocyte-mediated phagocytosis

None presented

Witteveldt et al., 2004a, 2004b Sadd and SchmidHempel, 2006

Dscam homologue found on haemocytes

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Dscam mediated immune specicity

Mosquitoes (Anopheles gambia)

Various bacteria, and strains of parasites (Plasmodium berghei and P. falciparum)

Dscam acts as a pattern recognition protein (PRP) involved in the recognition of foreign cells by phagocytic cells This work shows convincing experimental Exposure of the immune proof that Dscam homologue is a key molecule system of mosquitoes to in the immune system of mosquitoes various foreign agents results and that the gene (Dscam) produces in production of Dscam challenge-specic splice form variants variants with enhanced capability to recognise PAMPs

This work implies that Dscam acts as a PRP with the capability of producing a wide range of isoforms of different pathogen binding abilities 1. Challenge with a range of bacteria or parasites triggers alternative splicing of Dscam 2. This results in Dscam molecules with differing interaction specicity 3. Dscam gene silencing results in a signicant reduction in anti-bacterial defence 4. Phagocytic activity is linked to Dscam and this molecule can attach to bacteria 5. Hence Dscam acts as a PRP Study failed to assess the phagocytic activity of haemocytes following immune priming Not all bacteria were capable of inducing specic immune priming 1. Study shows high level of specicity where enhanced survival is related to nature of bacteria used as vaccine 2. Study also shows that immune priming does not occur in response to all immunogens 1. Study shows that trans-generational immune priming can occur via both male and female parents 2. Enhanced survival of progeny cannot be totally explained by either changes in phenoloxidase activity or haemolymph antibacterial activity Study shows high level of discriminatory abilitythe immune system appears to be able to differentiate between two strains of B. thuringiensis Study shows that elevated phagocytosis observed following exposure to vibrios has a level of selectivity in that unrelated Gram positive bacteria fail to show enhanced uptake by haemocytes from vibrioinjected shrimp This study may have commercial implications for the development of vaccines to protect shrimp from viral diseases

Watson et al., 2005

Dong et al., 2006

Specic immune priming

Drosophila

Streptococcus pneumoniae and the entomopathogenic fungus, Beauveria bassiana Bacteria: Bacillus thuringiensis and Escherichia coli

Experiments suggest the importance of phagocytic haemocytes and elements of the Toll pathway Beetles show a high level of specicity in terms of survival following challenge with heterologous or homologous pathogens

Dscam and Toll

Pham et al., 2007

Strain-specic priming

Beetle (Tribolium castaneum)

Dscam

Roth et al., 2009

Trans-generational immune priming

Beetle (T. castaneum)

Heated killed B. thurigiensis and E. coli

Paternal and maternal trans-generational immune priming that appears to be specic in nature in that progeny exposed to the same species of bacteria as their parents survived longer after challenge.

Phenoloxidase and humoral antibacterial activity

Roth et al., 2010

Specic immune priming

Woodlouse (Porcellio scaber) Shrimp (Litopenaeus vannamei)

Elevated phagocytosis following vaccination

Two strains of the insect pathogen, Bacillus thuringiensis and E. coli Vibrios (constituents of Vibromax vaccine) and V. harveyi

Bacterial strain-specic elevation in haemocyte phagocytic responses Bacterial strain specic/selective elevation in haemocyte phagocytic responses

Toll pathway and Dscam

Roth and Kurtz, 2009 Powell et al., 2011; Pope et al., 2011

Dscam?

Elevated phagocytosis following vaccination

Shrimp (L. vannamei)

VP28 protein from WSSV expressed in Bacillus subtilis

Haemocytes from VP28 immunised shrimp show selective elevation in their uptake of WSSV

Fu et al., 2011

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(i.e. B. thuringiensis or E. coli). Attempts to explain this heightened resistance to bacteria in the progeny of vaccinated insects that used phenoloxidase and antibacterial activity as markers of humoral immunity largely failed to explain the effectsperhaps suggesting other changes in defence could provide an answer to the effect observed. Overall, these three papers using insects as model organisms show changes in antibacterial activity that are trans-generational in nature but whether these are responsible for any level of specicity (pathogen-specic elevation) is not clear. There is also still a lack of mechanistic explanation for some of the original trans-generational immune priming studies that show a specic action (e.g. Little et al., 2003). Two studies have examined if changes in antibacterial activity are associated with the phenomenon of specic immune priming in crustaceans (Pope et al., 2011; Powell et al., 2011). Powell et al. (2011) used post-larvae of Litopenaeus vannamei exposed to a commercial anti-vibrio vaccine (Vibromax) whilst Pope et al. (2011) used the larger juveniles of L. vannamei and a bacterin composed of formalininactivated V. harveyi. In the former experiments, post-larvae were exposed to the commercial vaccine using the manufacturer's recommended method (via food, Artemia salina). As this vaccine includes formalin-inactivated strains of Vibrio anguillarum, V. harveyi, V. parahaemolyticus and V. vulnicus, the workers were able to look for changes in antibacterial activity in post-larvae both related and unrelated to the bacteria in the original vaccination to determine if any specicity was displayed. They observed that post-larvae exposed to vaccine showed elevated levels of antibacterial activity against V. anguillarum (in the vaccine) up to 14 days post-exposure. However, in the case of V. harveyi (also in the vaccine) they found no antibacterial activity either in the nave or vaccine-exposed animals. Finally, antibacterial activity against Vibrio alginolyticus (not in the vaccine) was found in post-larvae after exposure to vaccine or a vehicle (no bacteria but other components of the full commercial vaccine). The main conclusion that can be drawn from this study is that exposure to Vibromax can cause an elevation in antibacterial activity against some vibrios (e.g. V. anguillarum) but this does not appear to be the case for all bacteria tested and this change may not be related to the bacteria in the vaccine (i.e. may not show specicity). Although these experiments used an established quantitative antibacterial assay, the small size of the post-larvae necessitated the use of whole body homogenates rather than whole haemolymph (blood), which may explain why antibacterial activity was not found against some vibrios, such a V. harveyi. The later studies of Pope et al. (2011) used juvenile L. vannamei and were therefore able to monitor changes in antibacterial activity of whole blood following exposure to a virulent strain of V. harveyi. Unlike the earlier study with post-larval shrimp, they found evidence of natural antibacterial activity against V. harveyi in the blood but this was not signicantly altered by previous vaccination with that organism. Whilst the experiments described previously showed changes in antibacterial activity in both insects and crustaceans following exposure to various foreign agents, there is no clear evidence to suggest that this event shows specicityone of the key hallmarks of vertebrate specic acquired immunity and the observations made in whole animal studies of invertebrate specic immune priming. Indeed, it is worth questioning whether such specicity could exist based on our existing knowledge of the antimicrobial factors found in insects and crustaceans. By reference to Table 1, it can be seen that none of the antibacterial factors found in crustaceans is specic in terms of the microorganism that they kill at the species or genus level. For example, penaeidins and other AMPs tend to have broadspectrum activity against a wide range of bacteria, even fungi and perhaps viruses. If vaccination results in higher levels of these molecules in the haemolymph it is therefore very difcult, if not impossible, to use this as a basis to create a model for specic immune priming with the key hallmarks of specicity. It could be argued

that changes in the prole of the wide range of humoral antimicrobial factors described in Table 1 could show some selectivity towards particular groups of pathogens but only at the major division of bacteria vs. fungi or potentially Gram positive vs. Gram negative bacteria. Certainly, pioneering studies in insects by the 2011 Nobel Laureate, Jules Hoffmann and colleagues, have shown that challenge with a Gram positive bacterium results in the elevation of AMP biosynthesis via the Toll pathway appropriate for the destruction of such organisms, whilst the equivalent experiment with Gram negative bacteria results in increased levels of such molecules to eliminate these bacteria via the immune deciency (Imd) pathway (see Hoffmann, 2003 for review). Hence the insectan immune system, at least, can vary its response to pathogens to improve the chance of the hosts' survival. Whether this same level of selectivity exists in crustaceans is unclear but there is evidence for the existence of both Toll (Yang et al., 2007) and Imd (Wang et al., 2009) pathways in these animals. A series of genomic and proteomic studies with shrimp may shed some light on the changes that occur following infection and may be of value in the search for a mechanism of specic immune priming. Genes that appear to be regulated following infection include those for lysozyme, anti-LPS factor, and C-lectins (Robalino et al., 2007, 2009). Some of these studies looked at relatively immediate changes of up to 24 h post-infection (e.g. de Lorgeril et al., 2008), which may be too short-term to give any insight into immune priming (a longer term event). Whilst these approaches give us a detailed insight into changes in the immune system of shrimp following infection they may not help us in a search for a mechanism of specic immune priming based on antimicrobial factors detailed in Table 1. Overall, there is little or no evidence to suggest that the phenomenon of specic immune priming or trans-generational immune priming can be explained by changes in humoral antimicrobial factors such as AMPs and lysozyme alone. These factors may play a limited role in a concept of selective but not specic elevated immunity, however. 4.2. Changes in phagocytic activity Phagocytosis is the universal, non-specic, defensive phenomenon in all multicellular animals from simple invertebrates through to highly advanced mammals. Whilst the cell types involved in this process may differ from one group of animals to another, the process is largely identical in its basic biochemical pathways, resulting in the generation of killing factors through oxygen-dependent and oxygenindependent mechanisms. It has been over 30 years since a series of studies reported that the phagocytic activity of invertebrate haemocytes could be enhanced by vaccination, although these studies are now largely forgotten. For instance, craysh (Parachaeraps bicarinatus) vaccinated with a strain of killed Pseudomonas showed enhanced in vivo clearance of injected particles (McKay and Jenkin, 1970a) that was demonstrated in subsequent experiments to be linked to elevated phagocytic activity of the haemocytes (McKay and Jenkin, 1970b). These experiments failed to test whether this elevated phagocytosis was specic as the test particles were sheep erythrocytes and not the original immunogen in the vaccine. A similar phenomenon was observed in American lobsters (Homarus americanus) where injection of a range of vaccines including those derived from Pseudomonas petrolens and Aerococcus viridians var. homari (the causative agent of gaffkaemia) caused signicant elevation in the rates of phagocytosis of sheep erythrocytes, especially when these test particles were pre-sensitized (opsonised) in lobster serum (Paterson et al., 1976). These early studies show enhanced phagocytosis that does not seem to be linked to the nature of the agent in the vaccine suggesting non-specic elevation (activation) unlike the phenomena seen in specic immune priming. Thirty years later, the seminal experiments by these two groups of workers have been revisited using similar assays but with a view to

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determining if elevated phagocytosis shows any specicity in its action. Five studies merit detailed discussion. The rst by Mori and Stewart (2006) again studied phagocytosis in lobsters. They reported that animals injected with LPS showed elevated non-specic levels of phagocytosis in their in vitro assay. Animals that had been injected over 30 days previously with A. viridans var. homari, however, showed an increase in phagocytosis that was specic to this immunogen. More impressive levels of specicity were found in a second study by Roth and Kurtz (2009) where woodlice (Porcellio scuber) were primed with either two different strains of B. thuringiensis or E. coli and changes in the phagocytic activity of haemocytes from these animals examined against the same bank of bacteria two weeks later. They found that elevated phagocytosis was linked to the nature of the original challenge and that priming with B. thuringiensis strain A resulted in elevated phagocytosis for this bacterium only and not the second strain of this species. The third study comes from work with juvenile shrimp (L. vannamei) that were injected with a mix of formalin-inactivated vibrios as found in the commercial Vibromax vaccine or saline (Powell et al., 2011). The animals were sacriced 14 days later and the haemocytes from each shrimp were incubated with a 1:1 ratio of vibrios found in the vaccine: Bacillus subtilis (an unrelated Gram positive bacterium). Selective elevation of phagocytosis was found in haemocytes taken from vibrio-injected shrimp (i.e. only the vibrios were ingested by haemocytes at a higher rate). In related experiments, Pope et al. (2011) used a similar experimental design but with a vaccine formed by formalin-inactivation of a virulent strain of V. harveyi. They also showed that vaccination caused a selective increase in the phagocytic activity (as determined by measuring the number of internalised bacteria/100 haemocytes) and the % of haemocytes showing phagocytosis. The nal work for discussion is that of Fu et al. (2011) who used a novel method to deliver a recombinant form of the viral protein, VP28, from white spot syndrome virus (WSSV) within the spores of the Gram positive bacterium, B. subtilis. They vaccinated adult shrimp (L. vannamei) via feed using a recombinant strain of B. subtilis where their vegetative cells secrete the recombinant viral protein, VP28. Control animals were fed the equivalent number of normal B. subtilis spores without the ability to secrete the VP28 protein. Twenty days later the phagocytic activity of the haemocytes from both groups of shrimp was assessed against WSSV or an unrelated virus (Taura syndrome virus; TSV). The authors reported that the haemocytes from VP28-B. subtilis immunized animals showed significantly higher phagocytic activity against WSSV than the same cells taken from control shrimp. Furthermore, the same cells taken from these animals but incubated with TSV showed no elevation in phagocytic activity showing that the enhanced uptake of WSSV was selective or specic in nature. Taken together these ve studies imply that specic immune priming or vaccination may be explained by elevated phagocytosis that does not appear to be a result of some general (non-specic) stimulation of such activity. The mechanism of this event is likely to be linked to specic recognition of determinants on the outside of the test microbes by the haemocytes of previously vaccinated animals. Our knowledge of how phagocytic cells of the invertebrate immune system recognise foreignness is still rather limited. It is thought that pattern recognition proteins (PRPs) are able to interact with PAMPs on the surface of foreign cells so that they bind to and are internalised by the phagocytes. Known examples of PRPs in invertebrates include lectins that interact with particular carbohydrate residues associated with the surface structures of microbes. Several different types of lectins have been reported in crustaceans, including shrimp and presumably they all have slightly different carbohydrate binding capacities. For example, a C-type lectin has been found in Fenneropenaeus chinensis that contains two carbohydrate recognition domains that are predicted to bind to galactose and mannose residues (Zhang et al., 2009). This lectin also agglutinates a wide variety of

Gram positive and Gram negative bacteria, binds to microbial products, LPS, peptidoglycan and lipoteichoic acid, and has antimicrobial activity against bacteria and fungi (J. Sun et al., 2008; Y.D. Sun et al., 2008). A similar C type lectin from P. monodon also acts as an opsonin for the haemocyte-derived engulfment of E. coli (Luo et al., 2006) and a further mannose-specic lectin from L. vannamei has apparent direct antiviral activity against WSSV as a result of its ability to bind viral envelope proteins (Zhao et al., 2009). Indeed, challenge of shrimp with WSSV causes an increase in the expression of genes for such lectins, suggesting their key role in anti-viral responses (Zhao et al., 2009). Overall it can be seen that lectins are important PRPs that also have antibacterial and antiviral activity. The question remains, however, whether they may be key elements responsible for the phenomenon of specic immune priming? There is no evidence from any studies with either crustaceans or arthropods in general, that lectins have the diversity and specicity of binding sufcient for them to differentiate between closely related pathogens in a highly discriminatory way. However, a series of studies with molluscs provides compelling evidence of such a lectin family that shows somatic diversication (Harington and Zhang, 2011; Harington et al., 2010; Mon et al., 2010; Zhang et al., 2004) presumably resulting in interaction with pathogens and parasites that have a degree of specicity or selectivity. Recent work has focussed on the potential of an invertebrate homologue of the Down syndrome cellular adhesion molecule (Dscam) as a PRP and the ability of the Dscam gene to produce variants of this molecule with specic binding capabilities. In mammals and insects this molecule acts a neural adhesion molecule that plays a role in neuronal development (see Schmucker and Chen, 2009 for review). The groundbreaking study of Watson et al. (2005) was the rst to show that the Drosophila homologue of Dscam is probably also involved in immunity, specically acting as a PRP during phagocytosis. These authors made two major discoveries: (i) that interference of Dscam expression in ies dramatically reduced the ability of haemocyte-mediated phagocytosis, suggesting its role as a PRP and (ii) that alternative splicing of the Dscam gene generates many thousand potential combinations. They calculated that over 18,000 Dscam isoforms in circulation could be formed, each with varying binding capability (c.f. the somatic diversication of immune receptors in the vertebrate specic immune system). From their data, they suggested that Dscam was a receptor involved in phagocytosis through its ability to bind foreign cells. A further signicant development followed with the discovery of another homologue of Dscam in the mosquito (Anopheles gambiae; Table 2) (Dong et al., 2006). Essentially, these authors proved that Dscam is a PRP in mosquitoes with the ability to produce pathogen-specic splice form variants after immune challenge or infection. This would give these animals the ability to tailor an immune response specically to the pathogen such that this foreign agent would be actively recognised and ingested by the phagocytic haemocytesessentially the key hallmarks of specic immune priming. Hence Dscam together with the lectins in molluscs, is a likely candidate to provide a mechanistic explanation of the phenomenon of specic immune priming. Currently we do not know how universal Dscam is in invertebrates in relation to its putative importance in specic immune priming. Similarly, all descriptions of this event linked to Dscam have been found within the arthropods alone. Therefore, whether deuterostomate invertebrates, such as echinoderms and urochordates, display evidence of specic immune priming is currently not known. As has been suggested, they may have alternative PRPs with functional similarities to arthropodan Dscam but without homology (Litman and Cooper, 2007; Loker et al., 2004). The recent papers by Brites et al. (2008) and Chou et al. (2009, 2011) at least show that Dscam is also found in shrimp. Of signicance is a recent report on the biological activity of Dscam in the freshwater craysh, Pacifastacus leniusculus (Watthanasurorot et al., 2010). These authors were able

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to produce recombinant isoforms of Dscam following exposure of craysh to either E. coli or Staphylococcus aureus which had differential binding activities to these two distinct types of bacteria. Changes in the binding activities in turn caused changes in the rate of in vivo bacterial clearance and phagocytic activity leading to the conclusion that the isoforms of Dscam act as bacteria-specic PRPs. 5. Limitations of specic immune priminga cautionary note As already reviewed, the reports on immune priming and specific immune priming are largely limited to arthropods with an emphasis on insects. Whether such events occur across all invertebrates with a similar mechanistic explanation is unknown. Although we have cited two examples of enhanced phagocytosis published in the 1970s (McKay and Jenkin, 1970a, 1970b; Paterson et al., 1976) that may be linked to the more recent description of specic immune priming, it must be pointed out that these studies are in the minority to those of other workers from that time that failed to nd any such elevation in terms of cellular or humoral immunity. Indeed, in the review by Hauton and Smith (2007) that is critical of some of the work on immune priming, it is also pointed out that the prevailing view at that time (i.e. 1970s1980s) was that invertebrates simply did not show any form of specic immunity based on personal experience of the active workers in the eld and as a result this became the central dogma within evolutionary immunology for the ensuing decades. Furthermore as negative ndings tend to either not be published in journals or often simply reside in dormant theses and reports, there are likely to be examples from other workers who explored the potential for specic immunity in invertebrates and failed to show any evidence for this. An example of this comes from the unpublished doctoral thesis of Thomas (1984), where he reported trying unsuccessfully to emulate the earlier experiments already discussed that show elevated phagocytosis. He carried out a wide range of experiments using an insect model (a cockroach, Blaberus craniifer) and an insect pathogen, Bacillus cereus, as both immunogen and test particle in the phagocytosis assay only to nd no evidence of any change in the phagocytic potential. Worryingly, there is some evidence from the recent reports to suggest that specic immune priming may not occur in response to all foreign agents. For example, Pham et al. (2007) concluded that specic protection (i.e. specic immune priming) occurred in Drosophila in response to challenge with Streptococcus pneumoniae (a human pathogen) and the fungus Beauveria bassiana (a natural insect pathogen) but not to Salmonella typhimurium (Salmonella enterica serovar Typhimurium), Listeria monocytogenes and Mycobacterium marinum. These latter three organisms, although pathogenic to Drosophila, may not naturally come into contact with this host. Roth et al. (2009) also reported that whilst specic immune priming could be observed with B. thuringiensis and B. subtilis, this did not hold for E. coli. Finally, in our recent work with shrimp we (Pope et al., 2011) found that whilst injection of vibrios into shrimp resulted in the selective elevation of phagocytosis of such organisms, injection of B. subtilis (a marine isolate of what is usually considered to be a terrestrial bacterium) was without such effect. In their discussion of the unexpected ndings, Roth et al. (2009) suggested that one reason for this lack of specic response to some microorganisms may be related to whether the host would normally come into contact with such agents. This would imply that immune priming may not be able to emulate the vertebrate specic immune system's ability to deal with all comers regardless of their natural distribution. This may represent the much lower number of different immune receptors that arthropods are predicted to be able to produce (currently the maximum is around 31,000 potential Dscam isoforms in A. gambiae, Dong et al., 2006) compared with jawed vertebrates (many millions of different antibody molecules in terms of sequence variation). Certainly it suggests that invertebrate specic immune priming is not a universal phenomenon either in terms of the evolutionary status of the host or the natural habitat of their pathogens.

6. Vaccines for crustaceans such as shrimpare they feasible in light of recent reports of specic immune priming and the development of RNA interference techniques? As has been discussed in this review, some invertebrates appear to be able to mount an immune response that demonstrates selective enhancement such that pathogens can be managed in a more efcient manner upon secondary exposure. This may have implications for the strategy of how to combat bacterial diseases such as vibriosis and viral diseases such as that caused by white spot syndrome virus (WSSV) in shrimp. Several independent studies suggest that it is possible to immunise shrimp against WSSV using inactivated virus (e.g. Singh et al., 2005), viral envelope proteins such as VP28 (Fu et al., 2010, 2011; Wei and Xu, 2009; Witteveldt et al., 2004a, 2004b) DNA vaccines (Li et al., 2010; Rout et al., 2007) or with dsRNA vaccines (Kim et al., 2007; Liu et al., 2009; Meja-Ruz et al., 2011; Ongvarrasopone et al., 2008; Robalino et al., 2007; Sarathi et al., 2010). As recently reviewed by Hirono et al. (2011), injection of shrimp with dsRNAs or short interfering RNAs (siRNAs) targeted against particular viral genes can reduce the severity of such diseases as a result of inhibition of viral multiplication within host cells. For instance, as VP28 is of importance in the internalisation of WSSV into host cells, silencing of the VP28 gene with sequence-specic dsRNA protects P. monodon (Sarathi et al., 2008) and L. vannamei (Meja-Ruz et al., 2011) against this disease. Of importance, Sarathi et al. (2008) were able to achieve this by oral administration of VP28dsRNA which could have obvious commercial implications in terms of ease of delivery. However, similar studies aimed to determine if oral application of dsRNA targeted to gill-associated virus failed to provide any protection to P. monodon from later challenge whilst delivery by injection was effective (Sellars et al., 2011). In the case of dsRNA vaccines, Robalino et al. (2004, 2007) make a reasoned argument of how delivery does not simply block viral multiplication by gene silencing alone but may also prime the immune system to deal more effectively with the virus. Further evidence of this comes from subtractive suppression hybridization studies comparing nave with virus-resistant shrimp, where a number of immune genes coding for products with antiviral activity were differentially expressed (Garca et al., 2009; Zhao et al., 2007). Whilst all of these aforementioned reports show clear evidence of enhanced survival following vaccination it should be noted that this approach only appears to give limited protection (i.e. some vaccinated animals still die of the disease after challenge) and the longevity of protection is not always fully elucidated. For example, oral delivery of various formulations of VP28dsRNA to shrimp over a 5 day period only resulted in a maximum of 68% survival 30 days after exposure to WSSV that killed 100% of the control animals (Sarathi et al., 2008). Similarly, L. vannamei vaccinated by injection with a recombinant clone of the VP28 gene were challenged with a lethal dose of WSSV at 7 or 14 days post-vaccination and cumulative mortality measured (Li et al., 2010). They found that shrimp challenged 7 days post-vaccine delivery showed ca. 50% mortality 5 days later (i.e. 12 days post vaccination) whilst those shrimp challenged 14 days post-vaccination had ca. 80% mortality in the same timescale (total of 14 + 5 days post vaccination)suggesting a loss of protection in just a week. A further recent report using proteins from the envelope and nucleocapsid of the gill-associated virus that infects P. monodon failed to show any protective responses following earlier vaccination (Underwood et al., 2010). These authors concluded the efcacy of protection following protein-based vaccination may vary amongst different shrimp viruses. Hence, both traditional (e.g. protein based) and dsRNA or DNA vaccines often appear to at best give only some protection against various viral diseases that may also be short term in nature. Of more promise is the novel approach taken by Fu et al. (2010, 2011) in which shrimp were immunised by oral application of

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spores of B. subtilis harbouring the ability to secrete the recombinant form of the viral coat protein, VP28. They fed shrimp the B. subtilis VP28 complex for 20 days and challenged with live WSSV 7 days later. Shrimp immunised with this material showed a highly signicant improvement in the relative percent survival over a further 24 day period compared to controls. The advantages of this approach are that the putative vaccine can be delivered orally and with an apparent high level of protection. As the spores of B. subtilis are resistant to environmental factors, it may also be possible to store the vaccine for signicant periods without the need to refrigerate. Vibriosis is a serious disease to early developmental stages of shrimp as well as the juveniles and adults (Saulnier et al., 2000a, 2000b; Vandenberghe et al., 1999; Walling et al., 2010) and vaccines may therefore offer some assistance in its control. In the case of postlarvae of P. monodon, bath vaccination with formalin killed vibrios results in elevated survival up to 30 days post-delivery (Teunissen et al., 1998). Similarly, a commercial product (Vibromax) has been shown in eld trials to improve the growth and survival of shrimp (L. vannamei) post-larvae (Wongtavatchai et al., 2010). The study of Powell et al. (2011) already discussed in this review, may give a mechanistic explanation for such ndings, based upon elevated antibacterial activity and selectively enhanced phagocytosis. It would be interesting to know how long this protective response lasts as a short-term protective response would be of limited value. Also, does vaccine delivery result in enhanced long term survival under both eld and laboratory conditions? Overall there are some promising results from the work reviewed in this section but whether these various vaccines can be routinely delivered under eld conditions in a cost effective manner needs to be evaluated (see Johnson et al., 2008 for review) as does the longevity of the protective response. 7. Concluding remarks The invertebrate immune system was once thought to be incapable of exhibiting any specicity or immune memory in its repertoire. Since the initial reports of immune priming and the more recent discovery of Dscam, however, this view should now be re-evaluated on an animal to animal and pathogen to pathogen basis. If invertebrates, such as shrimp, do show such phenomena then this may present a basis for the development of disease control strategies by means of vaccination. It would seem unlikely, however, that putative vaccines will totally control potential disease outbreaks alone without the assistance of other measures such as improved sanitation, diets formulated with pre- and pro-biotics, and heightened biosecurity. Acknowledgements The nancial support of Intervet/Schering-Plough in preparing this review is gratefully acknowledged. We also thank our colleagues in CSAR in Swansea University for their continued support. Finally, we thank Dr Miranda Walker for her review of the text. References
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