Diseases in Asian Aquaculture VII

Current trends in the study of molluscan diseases

ABSTRACT
The study of molluscan diseases has a long history. The first publication on the redial stages of a trematode appeared in the 18th century; early papers on molluscan phagocytosis appeared in the last half of the 19th century and yet much work published before about 1975 does not appear in electronic abstract databases and is effectively lost. By contrast, a recent search of a leading abstract database for the terms mollusc and disease shows that the number of publications has exploded in the last eight years and the exponential trend looks set to continue. Much of the increase has been driven by the introduction of molecular technologies, the rediscovery that the immunology of invertebrates generally is a rich hunting ground for new biochemical defence systems and thus potential medical breakthroughs and the desire to publish multiple papers from the same project. As this publication trend continues, it will become increasingly difficult to be knowledgeable on all aspects of molluscan diseases and considerable specialisation is inevitable. It is not only our knowledge about known mollusc diseases that has grown, since new diseases continue to be reported as: aquaculture becomes more intensive; the Asia/Pacific regional skills base develops; and international reporting becomes more accurate. Transfer of disease between jurisdictions is also becoming more rapid as products are sent live around the world both as broodstock and for human consumption. Thus, the work of the Network of Aquaculture Centres in the Asia and the Pacific and the Food and Agriculture Organization of the United Nations in awareness raising and skills development will continue to make an impact. It is inevitable that, as the initial work on mollusc diseases developed around shellfish growing areas in Europe and America, the next generation of molluscan disease experts will be based in the Asia and the Pacific region. Key words: diagnosis, taxonomy, physiology, parasite-host relationships

Jones, J.B. 2011. Current trends in the study of molluscan diseases, pp. 75-92. In BondadReantaso, M.G., Jones, J.B., Corsin, F. and Aoki, T. (eds.). Diseases in Asian Aquaculture VII. Fish Health Section, Asian Fisheries Society, Selangor, Malaysia. 385 pp. Corresponding author: Brian Jones, brian.jones@agric.wa.gov.au 75

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INTRODUCTION
Global demand for seafood, including molluscs, continues to grow (FAO, 2009). However, disease continues to be a major financial constraint to growth of mollusc culture. Losses due to Marteilia refringens and Bonamia ostreae in French oyster farms over the period 19801983 were estimated at US$31 million dollars (Grizel and Hral, 1991). Abalone mortalities of unknown aetiology in Taiwan cost US$11 million (Bondad-Reantaso and Subasinghe, 2005). Ongoing mortalities in abalone in Australia had, by February 2007, resulted in a loss of US$4.5 million in exports (Lannen, 2007). The study of molluscan diseases has a long history. The first publication on the redial stages of a trematode appeared in the 18th century (Swammerdam, 1737), based on work he completed during the 17th century. Early papers on molluscan phagocytosis appeared in the last half of the 19th century (Metchnikoff, 1893; De Bruyne, 1893, 1896) as well as early papers on the histology of molluscan hosts (Grey, 1853; Peck, 1877). According to Yonge (1926), in his excellent description of the histology of the digestive diverticula in lamellibranchs, it was in 1880 that the name hepatopancreas for the digestive gland of crustacea was first used, a term still sometimes used for the digestive diverticula in molluscs. Unfortunately, much work published before about 1975 does not appear in electronic abstract databases and is effectively lost. By contrast, a recent search of CABI abstract database for the terms mollusc and disease showed that the total number of publications in the database for the period 19001950 was only 16, all of which were human or veterinary health references, and clearly missed all of the pertinent aquatic mollusc disease papers. In the next 25 years (1951-1975) there were 55 references, only three of which were on aquatic molluscs and none of which included those cited above. The next 25-year period (1976-2000) revealed 1 815 references, yet in the next eight years there were 10 814 references, of which 1 174 were of aquatic relevance. A similar trend is evident when using Aquatic Sciences and Fisheries Abstracts (ASFA) or other such databases. While clearly, much work (including foreign language papers) is not being captured by such databases, it is clear that this exponential trend is set to continue. It also means that, where it was possible to have read all of the literature on the subject up to about 1990, it is now no longer the case. As this publication trend continues, it will become increasingly difficult to be knowledgeable on all aspects of molluscan diseases and considerable specialisation in a team environment is inevitable (Sparks, 2005; Whitfield, 2008). The increasing specialisation has been driven by a number of factors including the: introduction of molecular, genomic and proteomic technologies; microfluidics and the development of microfluidic biochips capable of continuous sampling and real-time (and remote) testing of air/water samples for pathogens and toxins; rediscovery that the immunology of invertebrates generally is a rich hunting ground for new biochemical defence systems and thus potential medical breakthroughs;

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science-wide moves to publish frequently, which forces scientists to publish their research in parts, rather than waiting until the research has been completed. In this regard there is a clear trend towards papers with a multitude of authors; and increase in number of scientists in general. For the purpose of this review, the current trends in the study of mollusc diseases can be divided into three categories: (i) diagnostic testing for known mollusc diseases; (ii) taxonomic and phylogenetic studies on pathogens and their host molluscs; and (iii) investigations of known diseases, their impact on the host and the environment. Of necessity, these categories are artificial the boundaries between them tend to merge. (i) Diagnostic testing of molluscs for disease. Diagnostic tests fall into three broad categories: Screening apparently healthy animals for specific pathogens of concern. This is most commonly applied to stocks destined for live transfer or as part of a surveillance program. Determining the cause of poor health/mortality. This can often be a very complex process in determining the relationships between the host, pathogens (there may be more than one) and the environment (Snieszko, 1974; Berthe, 2002; Garnier et al., 2007). Development of new test methodologies or procedures, or improvement of existing procedures. This is becoming more important as issues of sensitivity, specificity and fitness for purpose become more important. Unfortunately, the results derived from molecular methods are sometimes at odds with more conventional methods, but too often the assumption is made that a positive polymerase chain reaction (PCR) result verifies an infection in a tested host, or that a negative PCR means that infection is not present. This assumption is valid only if the assay has been properly validated for the geographic area and for the hosts examined (Burreson, 2008). For example, based on histology, epidemiology and visualised by Transmission Electron Microscopy (TEM), Hine, Wesney and Hay (1992) and Hine, Wesney and Besant (1998) reported the presence of a herpes virus in oysters (Ostrea chiliensis and Crassostrea gigas) in New Zealand, which is certainly there (Jones, unpublished TEM obs.). A more recent study using histology and confirmatory PCR (Webb, Fidler and Renault, 2007) failed to amplify ostreid herpesvirus (OsHV-1) from New Zealand shellfish, leading the authors to discount the previous observations and question whether OsHV-1 is present in New Zealand at all. Ulrich et al. (2007) claim, based only on PCR results, the presence of Haplosporidium nelsoni infections in the Gulf of Mexico despite the results of thousands of oysters having been examined by histology from the same area for over 20 years which failed to observe a single infection (Burreson, 2008). PCR may show that parasite DNA1 is apparently present in a sample, but in the absence of independent verification it does not confirm or refute infection in the environment (see also Kanagawa, 2003).

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(ii) Taxonomic and phylogenetic studies on pathogens and their host molluscs Work to classify the large numbers of pathogenic organisms associated with molluscs has suffered from the ongoing decline in numbers of trained taxonomists. It is becoming difficult to find people who can identify and describe new metazoan parasites, especially since all of the classical taxonomic literature is based on morphology, and relatively few species are represented in Genbank. Nevertheless, accurate identification of both the host and the parasite is of importance. For example, Nakano and Spencer (2007) used phylogenetic analysis of DNA sequences to show that a species of small intertidal limpet Notoacmea helmsi from New Zealand was in fact a taxon comprising 5 morphologically cryptic species. Any study of the parasitology of the group would have been confounded by this discovery. The current taxonomic difficulties are well illustrated by studies of the molluscan parasites informally grouped as microcells because of their small size (about 2 microns). The genera Marteilia, Mikrocytos, Bonamia and Haplosporidium spp. are relatively easily detected by routine histology, but species determination is much more problematic. Marteilia refringens and Marteilia maurini are morphologically indistinguishable (Longshaw et al., 2001) but can be separated by molecular means (Le Roux et al., 2001) and both infect oysters and mussels. Lpez-Flores et al. (2004) suggested that the Marteilia from oysters and from mussels may be two different strains of the same species that appear to readily infect both hosts while other authors accept that they are closely related species - thus their taxonomic status is still open to debate (Berthe et al., 2004). A similar situation occurs with the occurrence of Bonamia sp. in Australia that has molecular and histological differences to B. exitiosa (J. Handlinger, pers. comm.). The distinction is important, for some species are internationally reportable, while other species of the genus are not. While the use of sequence data from the small subunit ribosomal RNA (SSU r RNA2) gene is now commonly referred to in the description of new haplosporidians (Azevedo et al., 2006), sequence data alone is generally not sufficient to separate a new species. The distance between Bonamia sp. from New South Wales (Australia) and B. exitiosa from New Zealand over the 1 586 base pair sequence from the 18S gene is only 0.9% (Corbeil et al., 2006), yet there are differences in geography, morphology, ultrastructure and histopathology between the two microcells (B. Jones pers. obs., J. Handlinger, pers. comm.) (iii) Investigations of known diseases of molluscs, their interaction with the host and the environment There is a rich and growing body of literature on molluscan diseases and their associated pathogens, their interactions with each other and with the wider environment. Comparative genomics is also a source for major advances in our understanding of the regulatory systems not only in molluscs but also in their parasites.

1 2

Deoxyribonucleic acid Ribonucleic acid 78

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In order to make some sense of the voluminous literature, it is useful to adapt Figure 1 below, developed by Snieszko (1974), to show the interrelationships of the host, parasite and the environment and to categorise the types of studies undertaken, as follows: Host-parasite interactions fall into two groups: firstly, the investigation of molluscan host impact on the pathogen (including host defence mechanisms involving the detection of and subsequent neutralising of pathogen); and secondly, studies on the impact of the parasites on molluscan host (including mechanisms by which the parasite overcomes the host defences and appropriates the host to its own purposes). Parasite-environment interactions can also be divided into two groups: the investigation of the pathogens impact on environment and the investigation of the environments effect on the pathogen (as opposed to the environmental impact on the host). Environment-host interactions include investigation of the environment on the host mollusc (including the effects of stress, pollution, tumours- environmental diseases); and investigations of how the host mollusc alters or affects the environment. Host-parasite interactions a) Impact of host on pathogen Host defense mechanisms now constitute a rich area of research. By the end of last century, it was thought that molluscs did not have an acquired immune system and lacked immunoglobulin antibodies (Chu, 1988). Defense was ascribed to both cellular and humoral factors with phagocytosis as the primary response to foreign matter (Feng, 1988).

Figure 1. Interrelationship of the host, parasite and the environment (Snieszko, 1974)

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Phagocytosis, in particular was studied in the 1890s and that early work was built on by Stauber (1950) who studied the effect of injected India ink particles in Crassostrea virginica. There is clearly variation in haemocytes among molluscs; for example, scallops and abalone do not have granulocytes yet other gastropods do (Hine, 1999; Travers et al., 2008; Mahilini and Rajendran, 2008). Despite over 100 years of research and numerous papers describing the various morphological forms that haemocytes display, Allam, Ashton-Alcox and Ford (2002) were still able to write the origin, life cycle and life span of bivalve haemocytes are still largely unknown and the role of each cell type has not been completely elucidated. Harris, Lambkin and OByrne-Ring (2006) and others have been using immunohistochemistry to identify structural and functional proteins in abalone leading Travers et al. (2007) to recommend that resolving the controversies over the classification of haemocytes in molluscs would require the obligatory use of mollusc-specific antibodies and gene probes. Whether the application of these new technologies will settle the controversy remains to be seen. Part of the problem is probably an assumption that haemocyte form and function will be the same across all molluscan groups, and a review, such as that by Hine, Wain and Boustead (1987) for teleost leucocytes, is well overdue. It could have been added that both the origin and fate of haemocytes is also unknown. Haemocytes can be seen to pass through the intact columnar epithelial layers, especially those of the gut, in a process known as diapedesis (from the Greek diapedan = to ooze through). Though in human pathology the term is confined to the passage of blood cells through unruptured vessel walls, in invertebrates, especially molluscan pathology, the term is also used for the passage of haemocytes, which may or may not contain phagocytosed material, across epithelial borders to the exterior of the body (Onstad et al., 2006). Cheng (1967) noted that it was unknown if haemolymph was lost during diapedesis and what factors influenced the rate of diapedesis. That is still unknown and it is also still unclear what role diapedesis plays in the response to infectious diseases. What we do know is that haemocytes are both mediated by and also produce humoral factors. For example, killing mechanisms associated with haemocytes involve reactive oxygen molecules, such as the phenoloxidase cascade, that are now known to be important for phagocytosis, melanisation and encapsulation. Inducible serum antimicrobial factors including lysozyme (a bacteriolytic protein) are released by degranulation when phagocytosis occurs (Cheng et al., 1975; Mohandas, Cheng and Cheng, 1985; Xu-Tao Hong, Li-Xin Xiang and Jian-Zhong Shao, 2006). However, the distinction between immunomediators, hormones and neurotransmitters, has become blurred by the finding that haemocytes can synthesis neuroendocrine peptide hormones and also have receptors for these peptides (Ottaviani and Franceschi, 1998a, 1998b). Though most work has concentrated on the haemocyte-mediated response, other defense mechanisms have been studied. Wright (1959) demonstrated species specific substances in the mucous of a number of snail species and Cheng, Shuster and Anderson (1966a, 1966b) showed that haemolymph of C. virginica and C. gigas will stimulate cercariae of Himasthla quitessetensis to encyst, immobilising them and preventing infection.
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There has been increasing activity looking for bioactive compounds in molluscs. glucuronidase, phosphatases, lipases, aminopeptidase amylase and antimicrobial factors have been described from molluscs (Chu, 1988; Montes, Durfort and Garcia-Valero, 1996; Montes et al., 1997; Roch et al., 2008). Agglutinins including haemaglutinins have also been widely reported and these may increase phagocytosis by acting as opsonins (Olafsen et al., 1992). Faisal, Oliver and Kaattari (1999) also showed that resistance to Perkinsus infections by Crassostrea species is effected by protease inhibitors to the extracellular serine proteases secreted by the parasite. The finding of virus particles in molluscs, when looking for causes of disease, is complicated by the ability of molluscs to sequester live viruses (such as norwalk and hepatitis viruses) from their environment. The reason why this occurs and the relationship of these sequestered viruses to the host mollusc immune systems are unknown. The virus is not simply bioaccumulated since recent work (Le Guyader et al., 2006; Tian et al., 2007) suggests that clams, mussels and oysters trap the norwalk virus (or the virus actively binds) through an intestinal type A-like histo-blood group antigen. Flegel proposed in 1998 that crustaceans accommodate live virus as a means of continually challenging the immune system in the absence of an acquired immune system (Flegel, 2006). Berthe (2002) also suggested that the invertebrate immune system response is complex and suggests that an ecological, or whole system approach to immunology should be considered, rather than relying on the mechanistic cause-effect interpretation of host-pathogen relationships that have, to date, dominated studies on infectious diseases of invertebrates. b) Impact of pathogen on host It has long been hypothesised that pathogens may be able to secrete extracellular products to inhibit the host response. For example, there is a strong negative association between the presence of Marteilia sydneyi and phenoloxidase activity in Saccostrea glomerata, possibly through the release of serum proteases, as happens with Perkinsus marinus (Faisal et al., 1999; Peters and Raftos, 2003). Variations in phenoloxidase also affect the resistance of oysters to QX disease3 (Peters and Raftos, 2003; Bezemer et al., 2006; Aladaileh, Nair and Raftos, 2007). Bezemer et al., (2006) used native-PAGE4 to identify five discrete forms of phenoloxidase in wild oysters, of which one was associated with disease susceptibility. This raises the question, is it the lack of a specific phenoloxidase type that permits disease or can the pathogen knock out a specific phenoloxidase molecule? Some pathogens are clearly able to inhibit or modify the host response. For example, Bonamia roughleyi microcells stimulate phagocytosis by suitable haemocytes but are not killed and instead proliferate within the host cell, eventually lysing the host to release more microcells (Da Silva et al., 2008). The cycle results in massive destruction of haemocytes leading to death of the host oyster.
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QX stands for Queensland Unknown the title given to this disease prior to the discovery of the organism that is now known to cause it. Native-Page stands for native polyacrylamide gel electrophoresis 81

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Parasites may also affect biochemical processes other than those involved in defence. Cheng, Sullivan and Harris (1973) reported that the marine gastropod Nassarius obsoletus was castrated by chemicals secreted by the sporocysts of Zoogonius rubellus and that were specific for germinal epithelium and gametes. Studies on the freshwater snail Lymnaea stagnalis infected with Trichobilharzia ocellata have shown that substances secreted by the trematode induce changes in host gene expression to directly inhibit mitotic division in the male copulatory organ and also stimulate development of the female endocrine dorsal bodies (De Jong-Brink, Bergamin-Sassen and Solis-Soto, 2001). Likewise Rice et al. (2006) showed that infection of the mollusc Haliotis asinina by the trematode Allopodocotyle sp. results in parasitic castration and is accompanied by differential expression of a number of regulatory genes. Manger, Christensen and Yoshino (1996) found that Schistosoma mansoni appropriated, for its own use, the hosts neurotransmitters serotonin and dopamine leading to changes in the host Biomphalaria glabra endocrine system. Perkinsus marinus, P. olseni and Haplosporidium nelsoni have all been shown to affect the growth and condition of their host molluscs which led Flye-Sainte-Marie et al. (2007) to demonstrate that brown ring disease of clams (Ruditapes philippinarum) caused by the bacteria Vibrio tapetis affected the energy budget of the clams and resulted in a reduction of the clearance and respiration rate, possibly due to the energy requirements associated with the immune response and tissue repair. How many published studies of the physiology of molluscs have been compromised because the disease status of the animals was not considered a factor? Parasite-environment interactions a) Impact of pathogen on the environment of the host It is known that parasite mortality events can alter the host density leading to major changes in the ecology of an area. Marteilia sp. infections lead to the commercial extinction of Ostrea edulis from the Gulf of Thessalonaiki (Virvilis and Angelides, 2006). Also, Miura et al. (2006) showed that the mud snail Batillaria cumingi, when infected by the trematode Cercaria batillariae, develop a different morphological form, move to the lower intertidal zone and consume different resources from uninfected snails. The parasites are, thus, indirectly altering the food web of many marsh species. b) Impact of environment on pathogen Studies on the effect of the environment (including pollution) on the susceptibility of the host to the pathogen have been done, particularly for MSX (Haplosporidium nelsoni) in oysters in the United States of America (USA), but studies on other pathogens and their hosts are more limited. Hgaret et al. (2007) showed that infection of clams (Rudetapes philippinarum) with Perkinsus olseni had no measurable effect on haemocyte parameters measured, but when the clams were exposed to toxic algal blooms there was a measurable change in haemocyte parameters monitored, and thus immunomodulation, in heavily infected clams exposed to toxic algae when compared to heavily infected clams that were not exposed. While we know a lot about the effects of the environment on the mollusc immune system, we know very little

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about the effect of the environment on the biochemistry of the pathogen when it is in the host. The optimal conditions for the host may not be those of the pathogen, such that changes in the environment (salinity, temperature) may place the pathogen at a disadvantage. Research has tended not to disengage the effects on the external infectious stages from the impact of the environment on established infections. For example, the probability of a severe kill due to winter mortality (B. roughleyi) is higher after dry autumns and early winters. Growers minimise losses by relaying oysters to low salinity upstream locations during periods of potential infection. Whether these actions are mitigating the effects of subclinical infections, perhaps by reducing parasite replication in the host, or are avoiding new infections by breaking the life cycle has apparently not been studied. Environmental pollution affects both host communities and parasite populations. Again, there has been much recent work done on the effect of pollutants on the host, and recognition in the literature that parasite communities are a potential indicator of environmental disturbance but there has been little study on the effect of pollutants on the parasite itself. Changes in parasite abundance and prevalence have also been used as an environmental monitor. Ectoparasites tend to increase and endoparasites decrease in prevalence and abundance in fish after chronic exposure to xenobiotics and aromatic hydrocarbons (MacKenzie, 1999; Kahn, 2004), and the impact on invertebrate hosts is likely to be similar, leading to establishment of monitoring programs such as the USAs mussel watch programme (Kim et al., 2008). Contaminants may favour the propagation of parasites by excluding predators, reducing host resistance, improving living conditions of host, or may interfere with parasite biochemistry thus reducing parasite burden or pathogenicity. Environment-host interactions a) Non-pathogenic diseases directly induced by environmental changes and pollution. Examples include tumours, toxins, endocrine disruptors and other non-infectious diseases. Describing tumours in molluscs has a long history starting with Ryder (1883), see also subsequent reviews by Pauley (1969), Farley and Sparks (1970), Elston, Moore and Brooks (1992), and Sparks (2005). More recently there has been a growing body of literature on the tissue effects of pollutants, particularly heavy metals, on molluscs following on from the work on tributyl tin antifouling and the imposex5 that it causes (Smith, 1981; Tallmon and Hoferkamp, 2009). Work has also focussed on oil pollution and endocrine disruption associated with veterinary and medical drug residues (Marigomez et al., 2006; Matthiessen, 2008; Morley, 2008). There has been a parallel growth in papers researching the use of mollusc diseases as a bioindicator of environmental pollution (see review by Au, 2004).
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Imposex is a descriptive term applied to some seasnails, marine gastropod molluscs which, under the toxic effects of pollutants, develop sex organs that are in contrast to their actual sex. It is a pathological condition where male sex characteristics, such as the development of male sex organs, (for example the penis and the vas deferens) form in female gastropods (http://en.wikipedia.org/ wiki/Imposex) 83

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b) Changes to host susceptibility driven by the environment, leading to disease Environmental changes do not have to damage tissues and interfere with biochemical processes to affect oyster immune status. More subtle environmental changes are often classified as stress and include, but are not limited to: mechanical disturbance (Lacoste et al., 2002; Ballarin, Pampanin and Marin, 2003); salinity changes (Fisher, Auffret and Ballouet, 1987; Butt, Shaddick and Raftos, 2006); temperature (Soudant et al., 2004; Cheng et al., 1975; Cheng et al., 2004; Zhang et al., 2006); chemical pollution (Pipe and Coles, 1995; Oliver et al., 2001; Cheng, Hsiao and Chen, 2004; Cheng, Juang and Chen, 2004) and diet including starvation (Butt et al., 2007). For example, Hong Chen et al. (2005) found that both phagocytosis and phenoloxidase activity in Haliotis discus hannai were affected by lack of dietary pyridoxine (vitamin B6). It is also certain that the enzyme systems of molluscs will be adapted to optimally perform at the normal temperature range. Changes in temperature may change host biochemistry thus favouring pathogens. The 1986 Bonamia epizootic in Foveaux Strait, New Zealand, began in areas that had been fished intensively for years and where benthic habitat was highly modified (Cranfield, Michael and Doonan, 1999). Similar escalating disease mortality in oysters in Chesapeake Bay, USA, has been attributed to modification of oyster habitat by fishing (Rothschild et al., 1994), and the environmental stress caused by this modification has been directly implicated in increasing acetosporan disease levels of oysters in experiments (Lenihan et al., 1999).

CONCLUSIONS
It is not only our knowledge about known mollusc diseases that has grown. New diseases continue to be regularly reported as aquaculture becomes more intensive; as the Asia and the Pacific regional skills base develops and more diseases are reported; as international reporting also becomes more accurate; and as transfer of disease between jurisdictions becomes more rapid as products are sent live - both as broodstock and as products for human consumption (such as the spread of Bonamia spp. infected oysters throughout Europe). There is a need for an increase in the numbers of trained diagnosticians as well as those investigating the ecology of diseased molluscs at a local level. It has been recognised by agencies such as the Network of Aquaculture Centres in Asia/Pacific and the Food and Agriculture Organization of the United Nations that the skills base needs to be developed at three levels: Level I (farm/production site observations, record-keeping and health management) is strongly emphasized throughout the Asian Diagnostic Guide (BondadReantaso et al., 2000) as this forms the basis for triggering the other diagnostic levels (II and III). Level II includes specialisations such as parasitology, histopathology and bacteriology that, generally speaking, cannot be conducted at the farm or culture site. Level III comprises advanced diagnostic specialisation that requires significant capital and training investment, such as TEM. Immunology and biomolecular techniques are included in Level III, although field kits are now being developed for farm or pond-side use (Level I) as well as use in microbiology or histology laboratories (Level II). These efforts are good indication that technology transfer is now enhancing diagnostics and, with solid quality control and field
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validation, it is certain that more Level III technology will become field accessible in the near future (Walker and Subasinghe, 2000). It is inevitable that, as the initial work on mollusc diseases developed around shellfish growing areas in Europe and America, the next generation of molluscan disease experts will be based in the Asia Pacific region.

REFERENCES
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