Future prospects for prophylactic immune stimulation in crustacean aquaculture – the need for improved metadata to address immune system complexity

Developmental and Comparative Immunology xxx (2014) xxx–xxx

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Developmental and Comparative Immunology

journal homepage: www.elsevier .com/locate /dci

Future prospects for prophylactic immune stimulation in crustaceanaquaculture – the need for improved metadata to address immunesystem complexity

http://dx.doi.org/10.1016/j.dci.2014.04.0170145-305X/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding author. Tel.: +44 (0)2380 595784; fax: +44 (0)2380 593059.E-mail address: [email protected] (C. Hauton).

Please cite this article in press as: Hauton, C., et al. Future prospects for prophylactic immune stimulation in crustacean aquaculture – the nimproved metadata to address immune system complexity. Dev. Comp. Immunol. (2014), http://dx.doi.org/10.1016/j.dci.2014.04.017

Chris Hauton ⇑, Meggie Hudspith, Laetitia GuntonOcean and Earth Science, University of Southampton, National Oceanography Centre, Southampton, European Way, Southampton, Hants SO14 3ZH, UK

a r t i c l e i n f o

Article history:Available online xxxx

Keywords:Immune stimulationPattern Recognition ReceptorDigestive tract microbiomeCrustaceanMetadata

a b s t r a c t

Future expansion of the crustacean aquaculture industry will be required to ensure global food security.However, this expansion must ensure: (a) that natural resources (including habitat use and fish meal) aresustainably exploited, (b) that the socio-economic development of producing nations is safeguarded, and(c) that the challenge presented by crustacean diseases is adequately met. Conventionally, the problem ofdisease in crustacean aquaculture has been addressed through prophylactic administration of stimulants,additives or probiotics. However, these approaches have been questioned both experimentally andphilosophically. In this review, we argue that real progress in the field of crustacean immune stimulantshas now slowed, with only incremental advances now being made. We further contend that an overtfocus on the immune effector response has been misguided. In light of the wealth of new data reportingimmune system complexity, a more refined approach is necessary – one that must consider theimportant role played by pattern recognition proteins. In support of this more refined approach, thereis now a much greater requirement for the reporting of essential metadata. We propose a broad seriesof recommendations regarding the ‘Minimum Information required to support a Stimulant Assessmentexperiment’ (MISA guidelines) to foster new progression within the field.

� 2014 Elsevier Ltd. All rights reserved.

The fundamental contribution that finfish and shellfish aqua-culture will have in safeguarding food security for an estimatedglobal population of 9 billion people by 2050 is well established(Stentiford et al., 2012). The UN Food and Agriculture Organisation(FAO) define food security in terms of (a) Availability, (b) Access,(c) Utilization and (d) Stability (FAO, 2006). Without question,however, the incidence of disease in aquaculture and particularlyshellfish aquaculture, represents a key threat to the availabilityand stability of this commodity for global consumption(Subasinghe and Phillips, 1999; Stentiford et al., 2012). It seemsclear now that no single strategy will meet the challenge ofinfectious outbreaks in farmed stocks. As such, efforts to limitthe incidence of disease will need to rely on a holistic approach,based on the principle of the disease triad first proposed bySnieszko (1974). Under Sineszko’s model, a pathogen, host andthe environment interact to produce a situation in which aninfectious disease can first break out and then spread. Underthis model, the administration of treatments or additives by

prophylaxis that significantly improve host health or immuneperformance, even on a short-term basis, does have merit.Consequently there has been considerable long standing interest,both scientific and commercial, in the identification of novelcomponents or extracts to use as additives in the feed or culturesystem to promote the health, growth and survival of the crop toharvest (e.g. Karunasagar and Karunasagar, 1999; Meena et al.,2013).

In the vast majority of published cases the success of potentialstimulants has been assessed using measures of immune effectoractivation, either in terms of gene transcription or protein titre(Fig. 1). Often, such treatments have provided evidence for a sus-tained period, of hours to days, of heightened immune reactivityin certain immune indices measured in a proportion of the treatedcrop (Smith et al., 2003). Such data have used to support the devel-opment of prophylactic treatments in aquaculture on a globalscale. The published literature continues to report enhancedimmune effector responses following treatment with extracts ofnatural products, subsequently advocating their application at afarm scale as a solution to disease. However, over the last decadeprogress in this field has slowed to only incremental advances

eed for

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Fig. 1. Summary of conventional approaches to immune stimulation applied to crustaceans highlighting the application of stimulants to induce systemic degranulation ofcirculating granulocytes and the release of potent non-specific antimicrobial effector molecules (+/� indicates that some proteins and gene isoforms have been reported thatare not regulated after infection, depending on the pathogen used). Stimulation of effector pathways and proteins is conventionally followed by an interval of increased genetranscription during haemopoesis to regenerate the immune system for the next challenge. The duration of stimulated protein expression, gene transcription andhaemopoesis is variable, as is the density of the new population of haemocytes. Timings of changes in protein and gene expression are only indicative.

2 C. Hauton et al. / Developmental and Comparative Immunology xxx (2014) xxx–xxx

which have not made significant inroads into the continuingproblem of bacterial, viral and protozoan diseases.

1. Limitations of existing prophylactic approaches to diseasecontrol

The concept of stimulating or priming an immune response as aprotective solution in crustacean aquaculture has been questionedpreviously on both experimental and philosophical grounds (for

Please cite this article in press as: Hauton, C., et al. Future prospects for proimproved metadata to address immune system complexity. Dev. Comp. Immu

example: Smith et al., 2003; Kounatidis and Ligoxygakis, 2012;Rowley and Pope, 2012). These challenges remain a significantimpediment to the successful contribution of crustacean aquacul-ture to global food security.

Experimental challenges to this field often stem from the insuf-ficient detail being included within the published literature. Impli-cit in the publication of scientific research is the presumption thatresults can be independently corroborated or validated by otherresearch groups with the aim that the field of research can collec-tively advance. A fundamental impediment to this, however, starts

phylactic immune stimulation in crustacean aquaculture – the need fornol. (2014), http://dx.doi.org/10.1016/j.dci.2014.04.017


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with the often very poor description of what a test stimulant oradditive actually is. There can be little gained by the publicationof data from studies that make use of ‘alkali-extracts’, ‘ethanolextracts’ or ‘hot water extracts’ of organic materials, for example.Such bulk prepared and undefined additives cannot easily beindependently replicated: will the same plant/algal species andstrain be used, will the growth conditions of that species in differ-ent locations produce the same biochemical profile in the sourcematerial, will the same part of a source species be used (e.g. root,leaves, stem?), does the biochemical profile change with the differ-ent tissues used from the source material? Ultimately, it is impos-sible to definitively corroborate the outcomes of one study withsubsequent work on the same or closely related species by otherresearch groups. In some reviewed cases colour or odour has beenused to compare the identity of one putative stimulant with thosein published reports from earlier experiments. These concerns,loosely identified as issues of ‘origin’ or ‘purity’ of the additive, alsoapply to studies of pre- and probiotics. As has been report, anumber of commercial products that have been claimed to beBacillus-based probiotics, have been shown to contain eitherseveral strains of bacteria (Lata et al., 2006) or even not to containstrains of Bacillus at all (Hoa et al., 2000). These uncertaintiesrender host response data almost meaningless and certainlyunverifiable.

Many of the publications reporting the validation of immuno-stimulants, additives or treatments are based on experimentswhere both the stimulant and the pathogen challenge are pre-sented by injection directly into the body cavity. Whilst in experi-mental studies this methodological convenience ensures that anexact dose of either stimulant or treatment is produced, this isentirely unrepresentative of what will take place at farm scale(Rowley and Pope, 2012). The complex interactions of the digestivetract (DT) microbiome (discussed below), combined with the harshdigestive environment, means that the prophylactic treatment andthe test pathogen will be likely subject to considerable amendmentor interaction before entering the haemocoel. Whilst it is inevitablethat any study employing oral exposures to stimulants andpathogens is likely to produce more variable outcomes, it is surelyessential that any treatment recommended for farm use is provenfor administration by this route (see: Azad et al., 2005).

However, whilst presenting additives orally mixed with feed isa necessary validation step, it does present additional challengesfor interpretation. Often it is impossible to state what the effectivedose is in each treated animal; too often no data are provided onthe feeding rate, feed conversion efficiency of the treated livestock,or an assessment of the uneaten food removed from the culturesystem. In some reports it is clear that failure to remove uneatenfood and faeces from the experimental system has led to issuesof poor water quality that might have been the prime cause of dif-ference in performance of animals on a control versus a test diet.

Determination of feeding rates would permit more accurateestimates of the maximum quantity of additive that might be takenup by an individual. Studies have repeatedly shown that therealised dose of an adjuvant, stimulant or probiotic can havefundamental consequences on the outcome of the treatment(Kirubakaran et al., 2010; Harikrishnan et al., 2012). Very oftenthe dose response relationship has proved to be non-linear, withlow doses consistently having no effects but too high doses causingnegative impacts on the physiology (e.g. Sung et al., 1994; Caipanget al., 2011) or immune function (Figueras et al., 1997; Lopez et al.,2003; Bai et al., 2010). To complicate this, often the optimum dosefor one particular response, for example: an immune response suchas respiratory burst, is quite different to the optimum responseassessed using another indicator, such as haemocyte counts. Thesedifferences complicate the prescription for each treatment within afarm setting (see for example: Peraza-Gómez et al., 2014). The


resolution of optimal dosing of feed additives to achieve desiredoutcomes (e.g. growth versus immune function and disease resis-tance) is a non-trivial problem which has not received sufficientattention.

Further experimental difficulties remain with the selectiveinterpretation of response variables as indications of positiveeffects. For example: increases in the activity of superoxidedismutase (SOD), and other antioxidants have been used as indica-tors for a positive influence on immune response. Researchers haveinterpreted these results by arguing that additives or stimulantspositively increase SOD activity to regulate the effects of respira-tory burst that accompanies phagocytosis. However, antioxidantenzymes are produced to detoxify excess superoxide anions whichare produced through metabolic processes, often in times of stress(e.g. Brouwer and Brouwer, 1998; Tavares-Sanchez et al., 2004). Toconclude that an increase in antioxidant activity represents apositive outcome after stimulation is not necessarily correct. Ifthe additive or treatment promotes or stimulates an immuneresponse, which would involve the generation of toxic oxygenradicals, then a more parsimonious explanation is that the treatedorganism is required to up-regulate its antioxidant pathways tocounteract the negative effects of excess superoxides in theabsence of any actual infection (see Fig. 1). The increase in activitycan, at best, be seen as an indirect consequence of exposure to theadditive but more likely represents a negative impact of additiveon the physiology of the treated crop species.

An increasing number of studies have quantified the change intranscription of genes coding for key effector proteins within thehost immune system, such as antimicrobial peptides (AMPs) orenzymes of the phenoloxidase system in crustaceans. Increasinglythese assessments are making use of the exquisitely sensitive tech-nique of quantitative real time PCR (qPCR). However, compared toother fields within the biological and medical sciences, there tendsto be minimal or no reporting of essential metadata on the designand validation of robust qPCR assays using RNA of proven quality.The significant pitfalls of poor RNA quality or lack of qPCR valida-tion have been comprehensively presented as justification for therecently established MIQE guidelines (Bustin et al., 2009, 2013).These essential metadata include: (1) the validation of RNA purityand integrity by gel electrophoresis or RQI/RIN determination, and(2) details of an optimized qPCR assay including optimized primerconcentrations, amplification efficiency, detection limit, qPCR cycledetails, and specificity of assay by melt analysis or by directsequencing of amplicons. Vandesompele et al. (2002) have alsoadvocated strongly for the use of the geometric mean of multipleendogenous reference genes for normalizing gene expression andthese concepts are also incorporated into the MIQE-guidelines(Bustin et al., 2009).

A final weakness of some published work is that sometreatments have been presented as cost-effective solutions to theproblem of disease in aquaculture, but with no supporting detailedcost benefit analysis. For such recommendations to be takenseriously it is absolutely essential that data are provided on thefinancial costs of preparation or labour costs associated withadministering a particular stimulant at farm scale.

Beyond experimental and interpretational issues substantiveprogress in this field has been hampered by: (1) the often inconsis-tent response of individuals within a treatment group, or betweenreplicate treatment groups or repeat studies; (2) the variability induration of protective effect; and (3) the energetic penalty sufferedby over stimulation of the immune system through repeat treat-ments (summarised in Fig. 1). It has been argued that progress inthe field of disease prevention through prophylaxis has reachedan impasse and that current techniques represent a blunt approachto a very complex problem (Hauton, 2012). This lack of recent pro-gress in the identification of effective treatments is at odds with




the really significant, rapid, and exciting insights that have be gen-erated in arthropod (crustacean and insect) research labs aroundthe world. These rapid developments have broken new ground inour mechanistic understanding of the invertebrate immunemechanism, from recognition to effector response, which shouldsurely support new approaches to the challenge of disease preven-tion within aquaculture.

2. Recent advances in invertebrate mechanistic immunology –realising the complexity

Within the last decade there have been significant advances inthe field of comparative invertebrate immunology that, for arthro-pods in particular, has been driven by advances made with insectmodels. Many studies have identified previously unknown levelsof complexity and diversity in the immune system, data whichnow provide a possible mechanistic basis for concepts such asmemory and specificity. Indeed, these advances are forcing thecommunity to reconsider the merits of the classic paradigm ofinnate versus adaptive immunity (Rodrigues et al., 2010). Whatis clear is that the invertebrates comprise very successful phylathat have developed diverse solutions to the problems of survival.Available data indicate that invertebrates have evolved multiplesolutions to same problems across diverse phylogenetic groups(Ghosh et al., 2011; Cerenius and Söderhäll, 2013). In particular,two areas of significant progress that are relevant to the problemsof disease in crustacean aquaculture merit brief review. Significantdevelopments have been made: (1) in the field of Pattern Recogni-tion Receptors (PRRs); and (2) our knowledge of the complexity ofthe digestive tract (DT) microbiome. These two areas of activeresearch may foster new insights into tackling the problems of dis-ease within crustacean aquaculture.

2.1. Novel and variant putative Pattern Recognition Receptors (PRRs)

Ghosh et al. (2011), Cerenius and Söderhäll (2013) have pre-sented extensive overviews of recently characterised transcription-ally variant PRRs in invertebrates. These include examples ofarthropod highly variable Down Syndrome Cell Adhesion Mole-cules (hv-Dscam gene; Armitage et al., 2012), Fibrinogen-RelatedProteins (FREPs) in molluscs and arthropods and Toll-Like Recep-tors (TLRs) with variable numbers of leucine-rich repeats reportedacross invertebrate and vertebrate phyla. Whilst some of thesePRRs have been reported from diverse phyla (e.g. TLRs), other fam-ilies show different patterns of evolution. For example, whilstalternatively-spliced FREPs from a single gene have been identifiedin molluscs, in arthropods this family has seen gene expansionwithin the genome, such that 59 separate FREP genes have been

Fig. 2. Summary of SMART (Letunic et al., 2012) domain predictions of the putative: (a) 10tail-less hv-DSCAM (Accession number HG964670) receptors isolated from the haemocyincludes Leucine Rich Repeats (LRRs), a transmembrane domain (blue), a cytoplasmicPreliminary sequence analysis of the hv-Dscam gene shows splice variation in the Ig2,transmembrane domain and only a short 30 region. In both figures regions of low seque


identified within the mosquito (Anopheles gambiae) genome(reported in Cerenius and Söderhäll, 2013). Similarly, whilst withinthe arthropods the hv-Dscam gene family exhibits extreme splicevariation, potentially producing tens of thousands of potentialisoforms, Dscam transcripts have also been reported within somemolluscs (including the mussel Mytilus edulis and the oyster Cras-sostrea gigas) although these do not exhibit splice variation anddo not appear universally with the Mollusca (e.g. absent from thegastropod Littorina littorea) (Gorbushin and Iakovleva, 2013). How-ever, more work on a wider range of taxa is necessary to developour understanding of the evolution and diversity of receptorswithin the invertebrates and, to that end, work conducted in thislaboratory has preliminarily identified at least one Toll-like and atail-less hv-Dscam isoform expressed by the haemocytes of theshore crab Carcinus maenas (Fig. 2, Accession numbers HG964671and HG964670, respectively), adding to the recorded incidencesof these receptors from the Pleocyemata.

The diversity of putative receptor families found within theinvertebrates is compelling, as is the potential for generating splicevariants. It would seem that whilst the immune effector arm offerslittle scope for specificity in response to infection, there is consid-erable scope for generating specific repertoires of PRRs which maycontrol downstream activation. Using conventional and ultra-deepsequencing approaches, teams have already presented evidence forimmunoglobulin domain (Ig) repertoire selection in hv-Dscam afterinfection with bacteria, viruses and protozoa (Dong et al., 2006;Chou et al., 2009; Watthanasurorot et al., 2011; Hung et al.,2013; Sun et al., 2013). Additional support for these ideas has beenthe recent identification of pathways and molecules that mightcreate splice variation post transcriptionally. Chiang et al. (2013)have reported that the expression of the splicing activator serine/arginine (SR)-rich protein B52 in the white shrimp Litopenaeus van-namei is up-regulated with hv-Dscam expression in response toinfection with White Spot Syndrome Virus (WSSV). Schwarz andEvans (2013) have shown also that the transcription of hv-Dscam,MyD88 and Imd in the honey bee Apis mellifera are consistently cor-related both systemically and locally following infection with dif-ferent protozoans (trypanosomes and microsporidians), whilstDong et al. (2012) have shown that activation of the immune defi-ciency pathway (Imd) or the Toll pathway can lead to the tran-scription of different splice variants of hv-Dscam in the mosquitoAnopheles gambiae. Confirmation of these pathways in the widerArthopoda will be essential to build a consistent model for theretention and transfer of antigenic information, supporting a the-ory of specificity in invertebrate immune responses. Future work,making greater use of deep sequencing technologies, will also berequired to test the realised repertoire of expression of hv-Dscamtranscripts, to determine if the theoretical maximum diversity cal-

10 amino acid Toll-like (Accession number HG964671), and the (b) 1690 amino acidtes of the Pleocyemata decapod Carcinus maenas. The Toll-like receptor predictionToll – interleukin 1 – resistance (TIR) domain and a 50 signal sequence in red.

Ig3 and Ig7 domains (data not shown), additional Fibronectin type 3 domains, nonce complexity are shown in pink.




culated from the product of all available alternately spliced exonsis, in reality, seen in vivo (see discussion in Smith et al., 2011).

The identification of transcriptional variation in PRR sequencestructure within invertebrates provides a mechanism forspecificity. A second goal of researchers has been to identify amechanism to support the concept of memory within these samesystems. Recent data from the mosquito Anopheles gambiae hasidentified the necessary differentiation of granulocytes to conferpriming after oral challenge with malarial Plasmodium (Rodrigueset al., 2010). By combining these recent breakthroughs from withinthe Arthropoda it is now possible to postulate a mechanism forspecific memory, involving the differentiation of new granulocyteswith a selected cell-surface expressed PRR repertoire after

Fig. 3. Overview of a mechanistic concept for memory and specificity within the arthrointeractions within the host digestive tract which can cause systemic degranulation odifferentiation after degranulation can produce a new repertoire of hv-Dscam transcriptslife span of those new cells, potentially providing a memory of previous pathogen expo


non-lethal challenge by a pathogen (Fig. 3). This model relies onthe local to systemic degranulation of granulocytes in responseto the primary infection, followed by the release and differentia-tion of new granulocytes during haemopoesis. This new generationof granulocytes might then exhibit a selected PRR repertoire, onethat is specific to the pathogens seen in the first infection (Donget al., 2006; Chou et al., 2009; Watthanasurorot et al., 2011;Hung et al., 2013; Sun et al., 2013). So long as these granulocytesremain in circulation then a memory (or information) of that initialinfection might be retained. This memory would vary in duration,depending on the life span of the granulocytes or the incidence ofrepeat infections causing further degranulation. Without question,this model requires considerable validation and further studies are

pod immune system. Local or systemic immunity is under the control of complexr haemocyte migration to sites of infection. Data suggests that haemopoesis and, some of which are expressed at the cell surface. These will then remain during thesure. See text for further explanation.




required to address obvious gaps, specifically to identify how infor-mation from the first infection is retained and used to regulate thesubsequent PRR repertoire expressed by the transcriptionallyvariant receptors. Nevertheless, if the gaps can be addressed thenthe community will be able to describe a complete pathway frominitial exposure to a pathogen to subsequent specific memory.

Nevertheless, continuing research on transcriptionally variantreceptors, particularly those of the hv-Dscam family, should notdetract from the valid pursuit of other receptor classes that mighthave specific roles in the detection of particular microbial associ-ated molecular patterns (MAMPs). Despite the growing work sug-gesting that splice variation of hv-Dscam, and other, transcripts canbe influenced by infection, it would be naïve conclude that theseisoforms represent the only mediators of specificity within crusta-cean immune pathways. In time, we might find that hv-Dscamproves to be a dead end. Viral pathogens of crustaceans presentthe most serious economic diseases globally (Stentiford et al.,2012) and in recent years key advances have been made also inidentifying the mechanisms of viral entry into host cells (reviewedby Flegel and Sritunyalucksana, 2011). This work is essential. If thehost immune system does not recognise or respond to a viral infec-tion under normal conditions then it is unlikely that stimulationvia current approaches will prove to be of significant benefit. Forviral pathogens, direct intervention at the point of viral entrymight be more successful in restricting the ability of the virus toreplicate. The role of viral coat proteins in mediating host cell entryis now established and here again potential farm-scale treatmentshave been advocated (e.g. Satoh et al., 2009; Yang et al., 2012). Thecontinued identification of host receptors and activating proteinsthat mediate viral entry, such as the PmRab7 receptor and theGTP-ase activating protein PmTBC1D20 identified in Penaeus mon-odon (Sritunyalucksana et al., 2006; Yingvilasprasert et al., 2014,respectively), will be essential to further a comprehensive viewof host/viral interactions that might also present alternative strat-egies for intervention in the case of these challenging diseases.

2.2. The role of the digestive tract (DT) and gut microbiome inmediating local and systemic immunity

The inconsistent response to stimulation in invertebrates hasproved challenging for the industry (see Section 1 and Fig. 1).Often, when presented with a particular stimulant, a proportionof the treated stock respond as anticipated whilst the rest fail torespond appropriately. This variability in response to stimulationis set against a widely-reported background of variable susceptibil-ity to infection with host life stage (e.g. Sano et al., 1985;Overstreet et al., 1988; Eleftherianos et al., 2008; Wua et al.,2010; Wang et al., 2011). As mentioned, others have identified thatoptimal dose for immune stimulation is also highly variable whenassessed using different proxies for immune performance.

As recently reviewed extensively by Garcia-Garcia et al. (2013),in large part this complexity in the invertebrate immune responseto stimulation is attributable to role of the DT microbial commu-nity in mediating both the host immune response and in vivo path-ogen virulence. The DT represents a dynamic environment inwhich Pathogen Associated Molecular Patterns (PAMPs) and otherstimulants can be detected, but also moderated or degraded, toproduce positive and negative (Zaidman-Rémy et al., 2006) feed-backs on local and systemic immunity. This complexity ofresponses to the DT microbiome has also been more broadlyreviewed by Nyholm and Graf (2012), who have made clear a def-inition of conserved PAMPs as a subset of conserved Microbe Asso-ciated Molecular Patterns (MAMPs) (Fig. 3).

In short, we now know that pathogen interactions with thecommensal microbiome can be key determinants of the local orsystemic immune outcome, whilst the presentation of an immune


stimulant via the DT might adversely affect the natural microfloraof the DT and so confound valid interpretation of cause and effect.For example, Dong et al. (2009) demonstrated that the absence ofbacterial microflora in the gut of the mosquito Anopheles gambiaemade them more susceptible to infection with the malarial para-site Plasmodium falciparum. Mosquitoes grown in aseptic condi-tions had increased susceptibility to the protozoan whilst thosefed on a mixed diet of P. falciparum and bacteria had reduced levelsof infection. Conversely Ryu et al. (2010) reviewed evidence thatthe commensal microflora within the gut of the fruit fly Drosophilaplayed a pivotal role in mediating the activation or suppression ofkey immune pathways such as Toll and Imd. Ryu et al. (2010)reported that in flies over-expressing gut antimicrobial peptides,such as might be the case in organisms being repeatedly stimu-lated by prophylaxis, there was a loss of the commensal microflorawithin the DT that could be replaced by opportunistic disease-causing microbial pathogens. (see also Ryu et al., 2008). A thirdtype of interaction has been reported in the case of the mothLymantria dispar by Broderick et al. (2009). Antibiotic reared mothlarvae, which had increased susceptibility to infection with theGram-positive bacteria Bacillus thuringiensis, could be rescued byfeeding with Enterobacter sp. NAB3, a normal gut bacterium of L.dispar.

In light of the microbiome complexity and crosstalk betweenthe microbiome, host PRRs and downstream signalling pathways,the variability in immune response and disease resistance withstimulation has to be expected. (see: Wua et al., 2010 and thereview of Chu and Mazmanian, 2013). This complexity alsoextends to consideration of different strains of the same pathogen,which might exhibit antigenic variation or differences in virulencebetween replicate studies (Frank and Barbour, 2006). It is clear thatthe outcome of any stimulation experiment will be a function ofthe life stage, DT microbiome and the dietary history of the host.Indeed, Smith et al. (2011) have argued that as genetic backgroundof both hosts and pathogens plays a critical role in determining theprobability of infection and the severity of any outcome (see Tangand Lightner, 2006), there is also need to constrain these identitiesfirst using molecular approaches. Future validation of putativestimulants will need to consider these details so that differentexperiments can be meaningfully compared and that any resultscan be unequivocally and independently verified.

3. Moving from laboratory advances to farm scale validation

From the preceding overview it is apparent that the field ofinvertebrate immunity has made very significant and rapidadvances in identifying complexity, both in terms of immune sig-nalling pathways and also the interaction of the host immune sys-tem with the commensal DT microbiome and with pathogens.Whilst we are a very long way from resolving the full detail, andindeed some large gaps remain in theories for memory and specific-ity, we contend that a shift in focus to the receptor arm of theimmune system will likely offer new approaches to disease control.These new approaches to priming immune surveillance throughaction on the receptor arm of the immune system might circumventthe problem of short-lived activation of the immune effector arm,which at the very least represents an energetic cost to the host.

Hauton (2012) and Rowley and Pope (2012) have already pre-sented recommendations on technical laboratory developmentsthat are necessary to support advances in this field. These includethe wider development of annotated genomic and transcriptomicresources (e.g. Clark et al., 2013; O’Rorke et al., 2014) and thedevelopment of specific crustacean cell lines. In particular, theapplication of deep and ultra-deep high throughput sequencingapproaches will be necessary in order to provide real detail on



Table 1Summary of proposed Minimum Information required to support a Stimulant Assessment experiment (MISA guidelines).

Recommendations for the Minimum Information required to support a Stimulant Assessment experiment (MISA guidelines)Host organismFully identified host species, including data on:

(1) Life stage/age(2) Origin of test organisms (geographic location, hatchery, wild collected)(3) Rearing history of test organism, including stock density during acclimation, stimulation and challenge(4) Molecular characterization of host strain(5) Basal diet composition, feeding regime and feed conversion efficiency(6) Categorization of existing gut flora, including molecular characterization(7) Tank environmental conditions, including: temperature, salinity, light regime, water change regime, water filtration or sterilization, microbial characterization,

nutrient load, oxygen concentration(8) Assessment of the existing pathogen load of the test organisms using molecular methods or histopathology to corroborate external visual assessments of organ-

ism healthStimulantFull characterization of test stimulant including:

(1) Full biochemical characterization(2) Source (e.g. plant or algal tissue, harvest season, growth location, growth conditions)(3) Detailed extraction method(4) Dose administered by injection or immersion (if used) or by diet(5) Interval of repeat exposure (if any), number of repeat exposures, doses(6) Some assessment of uptake (detection in faeces?)?(7) Mortalities in treated hosts during stimulation regime

Test pathogenAn infection study to confirm a protective effect is essential post stimulation. This will confirm an improvement in disease resistance over control treated animals. This should

follow from the characterization of test pathogen including:(1) Species and strain, including molecular characterization(2) Origin of pathogen culture(3) Dose administered by injection or immersion (if used) and by oral exposure(4) Mortalities in hosts exposed to pathogen and in treatment controls with appropriate survivorship analysis

Data analysisRaw data should available upon request or as supplementary informationFull descriptions of statistical analysis should be provided, including tests for normality and homoscedacity of variance, power of any statistical analysis and anticipated

effect sizeIf gene expression is quantified as part of the assessment, qPCR assays should be MIQE compliant


the variation in transcript profile of variant PRRs that might conferspecificity to a particular stimulant or situation (Sun et al., 2013).The more widespread use of unbiased sequencing approaches isalso likely to generate greater insight of downstream signallingpathways and also of cross talk between different pathways, andso also must be seen as a more informative initial approach thanthe a priori selection of target genes (Lee and Brey, 2013).

Nonetheless, it is clear that the transition from well-constrainedhost/pathogen model systems studied in a laboratory to the valida-tion of effective treatments for use in crustacean aquaculture atfarm scale will not be straightforward, even with the technicaladvances summarised above and by others. The emerging com-plexity variant PRR transcription, and of the DT microbiome andits interactions with host and pathogens, require a step change inthe detailed metadata that are provided to support the interpreta-tion of each large scale challenge experiment. This is necessary inorder that informed comparisons can be made between replicateor comparative experiments and to foster genuine progress withinthe field. Indeed, the reporting of metadata has become the normin other scientific fields, including those of microarray experiments(MIAME – Minimum Information Required for a Microarray Exper-iment; Brazma et al., 2001), quantitative PCR (MIQE – MinimumInformation for publication of Quantitative real-time PCR Experi-ments; Bustin et al., 2009) and, more recently, in the field of OceanAcidification (Riebesell et al., 2010). As the contribution of crusta-cean aquaculture to food security grows in the future we see thatthe inclusion of detailed metadata will become as essential as itis in the fields of human health and climate change.

To foster this development and to promote debate we haveidentified a list of key data that would contribute toward the Min-imum Information required to support a Stimulant Assessmentexperiment (MISA guidelines) (see Table 1) and would argue thatresearchers should move toward providing as much of this infor-


mation as possible in the future. Metadata should include consid-eration of the host and pathogen as well as a full description ofthe stimulant, its purification and how it was administered in theexperiment. Response data should be included whenever this ispossible to ensure that the full spectrum of individual variationis recorded. Often the extreme outlying individuals will prove tobe more informative than the treatment mean. As an extensionof this, tissue samples should be collected for routine pathologyso that the response of individual outliers can be explored in termsof their existing pathogen burden (Table 1).

It is acknowledged that the cost and technical feasibility of pro-viding all details will prove challenging but argue strongly that thisfield needs to move in this direction to ensure progress continues.This level of detail will also require the collaboration of wider dis-ciplines in the conduct of stimulant experiments, but again we seethis as essential to the maturation of this field of science. The openand detailed reporting of all data and metadata, including non-sig-nificant results, will provide the strongest foundation from whichwe can build solutions to the problems of disease within crusta-cean aquaculture.

4. Summary

Crustacean aquaculture can make a very significant contribu-tion to the provision of meat protein as a part solution to the prob-lem of food security. However, pathogenic diseases represent aconsiderable threat to the development of this industry. To addressthis threat the industry has sought stimulants and other treat-ments that will enhance the disease resistance of the cultured crop.This review has presented a summary of limitations of using pro-phylactic treatment to stimulate immune effector responses, argu-ing that progress using current approaches has slowed and is now




only incremental. This slow progression is in complete contrast tothe exciting detailed descriptions of diverse and variable immunereceptors in arthropods, which begin to provide us with a mecha-nistic basis of selective and sustained immune responses to infec-tion. At the same time, very detailed descriptions of the arthropodDT tract and microbiome have emphasized the complexity in thiskey system which mediates host local and systemic immuneresponses. This step change in our mechanistic understanding ofthe receptor arm of arthropod immune system has the potentialto identify significant new prospects for disease control withincrustacean aquaculture. However, in order to capitalize on thisnew knowledge, future studies of prophylactic stimulation mustaddress the issue of complete metadata for all experiments toensure that future work can be replicated and validated. Hereinwe propose the introduction of a series of guidelines for metadatareporting entitled Minimum Information required to support aStimulant Assessment experiment (MISA guidelines) as a structurethat will foster new progression within the field.

Acknowledgements

Isolation and sequencing of the PRRs from Carcinus maenasreported herein was funded by the University of Southamptonand the NERC National Oceanography Centre, Southampton (NOCS)under the NOCS-School of Medicine Interface Fund.

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Future prospects for prophylactic immune stimulation in crustacean aquaculture – the need for improved metadata to address immune system complexity