Viral-like particles associated with cuticular epithelium necrosis in cultured Litopenaeus vannamei (Decapoda: Crustacea) in Ecuador

Aquaculture Research, 2000, 31, 519±528

Viral-like particles associated with cuticular epithelium necrosis in cultured Litopenaeus vannamei (Decapoda: Crustacea) in Ecuador R JimeÂnez1, R Barniol1, L de Barniol1, M Machuca1 & X Romero2 1

Acuatecnos, PO Box 09-01-5738, Guayaquil, Ecuador

2

PO Box 09-01-5554, Guayaquil, Ecuador

Correspondence: Dr Roberto JimeÂnez, Acuatecnos, PO Box 09-01-5738, Guayaquil, Ecuador

Abstract

Introduction

A new viral agent was found associated with the endoplasmic reticulum of epithelial cells of the Paci®c white shrimp Litopenaeus vannamei (Boone) sampled during mass mortalities. A 40% mortality rate affected nursery and grow-out ponds during the ®rst 50±60 days of culture, and peak mortality in ponds occurred when shrimp reached 2±4 g. Histopathological changes of affected shrimp showed different grades of necrosis in epithelial cells and, in some cases, other tissues were affected. Transmission electron microscopy (TEM) of columnar cells of the cuticular epithelium showed the accumulation of viral particles, either dispersed in the cytoplasm or in a string-like or paracrystalline array. These arrays of virions were within membrane-bound vesicles formed from the endoplasmic reticulum (ER), in orderly arrays on the outer nuclear membrane or along the ER. The virus particles had apparently proliferated in the ER. The virions had an opaque area with an approximate diameter of 20 nm and an electron-lucent surface layer. The approximate diameter of the nonenveloped virions was 25 nm. The cytological changes observed are similar to those associated with the Picornaviridae and Nodaviridae families. The histopathology and ultrastructure of a new disease in L. vannamei is associated with the presence of a putative new virus. Until further isolation and characterization is performed, it is recommended to refer to the agent as Litopenaeus vannamei viral-like particles (LvVLPs).

Increased production of cultured shrimp in different areas of the world has also brought attention to the role of bacterial and viral diseases and their negative impact on this industry (Sindermann & Lightner 1988; Lin 1989; Frelier, Sis, Bell & Lewis 1992; Shariff & Subasinghe 1992; Jiravanichpaisal, Miyasaki & Limsuwan 1994; Chen 1995; Flegel, Boonyaratpalin & Withyachumnarnkul 1997; JimeÂnez, Barniol & Machuca 1997; Lucien-Brun 1997). To date, more than a dozen viruses have been reported to cause diseases in penaeid shrimp (Bonami & Lightner 1991; Lightner 1996). The new taxonomy will be used when referring to Litopenaeus vannamei and L. stylirostris, which were formerly placed in the genus Penaeus (Perez-Farfante & Kensley 1997). Since the beginning of the shrimp farming industry in Ecuador, viral diseases have been observed in cultured Litopenaeus vannamei (Boone) and L. stylirostris (Stimpson) (Lightner 1983, 1988a,b), but they caused no serious epizootics in commercial ponds during the 1980s. In June 1992, a new disease affecting L. vannamei cultured in farms near the Taura River in the inner estuary of the Gulf of Guayaquil was described by JimeÂnez (1992) and named Taura syndrome (TS). Regarding the aetiology of TS and Taura syndrome virus (TSV), research groups have offered different opinions (Lightner, Redman, Hasson & Pantoja 1995; Intriago, JimeÂnez, Machuca, Barniol, Krauss & Salvador 1997; Brock, Gose, Lightner & Hasson 1997).

# 2000 Blackwell Science Ltd,

519

Cuticular epithelium necrosis in cultured Litopenaeus vannamei R JimeÂnez et al. Aquaculture Research, 2000, 31, 519±528

A major effort was made to study Taura syndrome virus ®rst reported by Brock, Gose, Lightner & Hasson (1995). Further research included isolation of a viral agent, characterization, developing sensitive and speci®c techniques for its detection and reporting its geographic extension (Brock et al. 1995; Hasson, Lightner, Poulos, Redman, White, Brock & Bonami 1995; Lightner 1996; Bonami, Hasson, Mari, Poulos, & Lightner 1997; Mari, Bonami & Lightner 1998; Nunan, Poulos & Lightner 1998; Hasson, Lightner, Mari, Bonami, Poulos, Mohney, Redman & Brock 1999a). The viral disease named TSV by Brock et al. (1995) has been reported in different locations in the Americas (Lightner 1996; Lightner & Redman 1998; Hasson et al. 1999a). This viral disease also affects other species of penaeids with varying degrees of virulence (Lightner 1996; Overstreet, Lightner, Hasson, McIlwain & Lotz 1997). Size does not seem to protect L. vannamei, but there is a reported difference in survival depending on the route of infection (Lotz 1997). The onset of clinical signs and mortality is also related to the dose of infected tissue in per os experiments (Hasson, Lightner, Mohney, Redman, Poulos & White 1999b). Intriago et al. (1997) used the name infectious cuticular epithelium necrosis virus (ICENV) to refer to the disease named TSV by Brock et al. (1995). No new viruses have been reported in L. vannamei commercial aquaculture in Ecuador since these publications. However, in May 1999, using light microscopy, white spot syndrome virus (WSSV) was diagnosed in shrimp ponds in Ecuador (JimeÂnez, personal observation). Although two species of penaeid shrimp are cultured in Ecuador, the Paci®c white shrimp L. vannamei and the blue shrimp L. stylirostris, of these, L. vannamei production (» 80%) dominates. Pond-reared shrimp in Ecuador reached a record year in 1997 with a total production of 109 000 metric tons (MeleÂndez 1998). The intention of this paper is: (1) to report the presence of viral-like particles (VLPs) that have a different arrangement and size from previously reported viral diseases affecting and causing necrosis in the cuticular epithelium of moribund L. vannamei; and (2) to discuss the role of this putative new virus in penaeid aquaculture. Until the viral agent is isolated and characterized, it should be referred to as L. vannamei viral-like particles (LvVLPs). 520

Materials and methods As part of an animal health survey and control programme in Ecuador, Paci®c white shrimp L. vannamei were collected from ponds experiencing mortalities during August and September 1996 using a cast net. The shrimp were being fed with 28% crude protein feed pellets. A total of 496 shrimp were taken as a sample for histological examination by light microscopy and for electron microscopy. Owing to the nature of this new disease and its association with an outbreak in commercial farms, the sample size was above the number usually taken during an epizootic in farms. Davidson's AFA ®xative was used to preserve all samples taken for light microscopy observations during the histopathological study. Shrimp tissues were processed according to the procedures outlined by Bell & Lightner (1988). Histological stains used included Mayer Bennet haematoxylin and eosin (H&E), Brown and Brenn tissue Gram stain (Luna 1968), Steiner and Steiner stain (Steiner & Steiner 1944), Macchiavello (Luna 1968) and Giemsa (Luna 1968). Specimens selected for electron microscopy were ®xed with Karnovsky's ®xative (Karnovsky 1965) and, from those specimens that presented necrosis in the cuticular epithelium, only sections of this tissue were processed. Tissues were post-®xed in 1% osmium tetroxide in 0.1 sodium cacodylate buffer for 1 h, processed further and embedded in Spurr's resin, sectioned, stained with lead citrate and uranyl acetate and examined with a JEOL/JEM 1200 EX-II transmission electron microscope (JEM, Japan). In situ hybridization Tissue sections from specimens exhibiting necrosis in the cuticular epithelium in paraf®n-embedded tissues were also tested by cDNA in situ hybridization with a probe for TSV marketed by DiagXotics. The protocols for using the in situ probe for TSV have been outlined by Lightner (1996). Specimens tested were those transferred from Davidson's ®xative after 24±48 h of ®xation; this avoided samples in which degradation of RNA could have occurred (Hasson, Hasson, Aubert, Redman & Lightner 1997). The in situ probe for TSV has been used by our laboratory on numerous occasions and, according to the manufacturer's guidelines, positive and negative controls were present to verify the results. # 2000 Blackwell Science Ltd,, Aquaculture Research, 31, 519±528

Aquaculture Research, 2000, 31, 519±528 Cuticular epithelium necrosis in cultured Litopenaeus vannamei R JimeÂnez et al.

Results Gross signs Affected ponds had salinity ranging from 33 to 35 p.p.t. and water temperature of 23±25 °C. Mortality in both nursery and grow-out ponds gave an average of 40% during the ®rst 50± 70 days of culture. Mortalities occurred with shrimp from 1 to 10 g, and peak mortality occurred in shrimp of 2±4 g. Survivors of the initial acute attack were lethargic, and chronic low mortality persisted thereafter over a 2-month period. The surviving shrimp exhibited a noticeable pink and pale brown discoloration of the cuticular epithelium. Af¯icted shrimp, surviving and moribund, showed soft cuticles, and such shrimp may behave and feed normally.

Figure 1 Photomicrograph of histological section of healthy shrimp showing columnar cells in the cuticular epithelium (E) under the cuticle (C). H&E (bar = 14 mm).

Histopathology The cuticular epithelium of healthy shrimp consists of well-organized columnar cells under the cuticle (Fig. 1). The histopathology of acute and subacute LvVLPs disease was dominated by the presence of degeneration of columnar cells of the cuticular epithelium with hypertrophied nuclei (Fig. 2), karyorrhexis and pyknosis. Degeneration was also present in the cells of the subcuticular connective tissue (Fig. 2). In some cases, the cuticular epithelial cells and subcuticular connective tissue cells did not show hypertrophied nuclei, but a prominent basophilic nucleus and general deformation of nuclear morphology was apparent, with nucleoplasm devoid of the normal structure of chromatin and nucleoli, exhibiting a homogeneous appearance. Vacuolization and alteration of the nucleus/ cytoplasm ratio were observed. The cytoplasm in columnar cells appeared ®brillar in texture and commonly basophilic (Fig. 3). Subacute stages of infection showed in®ltration of haemocytes from the subcuticular tissue to the cuticular epithelium. The acute form of the disease showed multifocal areas of necrosis, with hyperplasia of the large epithelial cells and in®ltration of haemocytes with posterior accumulation of haemocytes in a circular arrangement in the areas of the epithelial tissue and cells. Fifty per cent of shrimp examined presented histological changes indicative of LvVLPs. # 2000 Blackwell Science Ltd,, Aquaculture Research, 31, 519±528

Figure 2 Photomicrograph of histological section of subacute LvVLPs disease in the cuticular epithelial cells. Note the dissolution of columnar cell membranes of the epithelium (E), hypertrophied nuclei (arrow) and in®ltration of haemocytes (arrowhead) in a whirling formation under the epithelium. H&E (bar = 14 mm).

Figure 3 Photomicrograph of histological section of acute LvVLPs disease in the cuticular epithelial cells (E). Note degeneration and necrosis of columnar cells with deformation of nuclear morphology, exhibiting a homogeneous appearance and karyorrhexis (arrow). Vacuolization and loss of cytoplasmic density, in®ltration of haemocytes (arrowhead) in cuticular and subcuticular epithelial tissues are shown. H&E (bar = 14 mm).

521

Cuticular epithelium necrosis in cultured Litopenaeus vannamei R JimeÂnez et al. Aquaculture Research, 2000, 31, 519±528

Ultrastructure Ultrastructure of subacute and acute forms of LvVLPs lesions in juvenile and adult shrimp using transmission electron microscopy (TEM) showed prominent nuclear deformations and speci®c cytoplasmic changes. Small virus particles were observed also individually, but commonly in paracrystalline arrays in the cytoplasm of necrotic epithelial and subepithelial cells and within intercellular spaces. In®ltration of haemocytes in response to cellular degeneration and necrosis was common (Fig. 4). Viral-like particles could also be observed in the cytoplasm of a ®broblast (Fig. 5). Viral-like particles in the columnar cells were arranged in string-like arrangements of one or more ranks between unit membranes. This may have constituted the ®rst step in the formation of the membrane-bound vesicles containing viral particles (Fig. 6). The arrays of virions can be within membrane-bound vesicles and within the endoplasmic reticulum (ER); the virus particles had apparently proliferated in the ER, and viral particles were seen in an orderly array on the surfaces of the outer nuclear membrane and along the ER (Fig. 7). Rupture of the membranes surrounding the masses of virions caused viral particles to look more widely separated than in paracrystalline arrays. Occasionally, they could be observed interspersed

Figure 4 TEM showing degeneration of columnar cells of the cuticular epithelium (E), deformation of nuclear morphology (arrows), subcuticular connective tissue (C) with in®ltration of ®broblast (F) and viral particles (arrowhead). Uranyl acetate and lead citrate (bar = 4 mm).

522

with viroplasm needle-like crystals (Fig. 10). These needle-like crystals are considered to be a ®xation artifact (Lightner 1996). ER membranes, lined with viral particles and enclosing a dense matrix, were

Figure 5 Higher magni®cation of Fig. 3 showing ®broblast (F) containing LvVLP virions in an early stage of replication in the cytoplasm (arrow) and free in the subcuticular connective tissue (arrowhead). Uranyl acetate and lead citrate (bar = 0.8 mm).

Figure 6 TEM of cuticular epithelium with viral particles in string-like arrangement between two unit membranes. This arrangement can contain one or several rows of virions (arrow) in the cytoplasm. Note electron-dense reticular strands in the deformed nucleus (N) and fragments of the outer membrane of the nuclear envelope (arrowhead). Uranyl acetate and lead citrate (bar = 1 mm).

# 2000 Blackwell Science Ltd,, Aquaculture Research, 31, 519±528

Aquaculture Research, 2000, 31, 519±528 Cuticular epithelium necrosis in cultured Litopenaeus vannamei R JimeÂnez et al.

arranged in reticulate con®guration. The number of viral particles keeps building up (Fig. 8) and, probably after cell lysis, viral particles reach other cells. Differences in thickness of the sections and in the plane of section caused bizarre variations in the appearance of the particles: in thicker sections, virus particles in linear arrays blended together into `®lamentous' forms, whereas in thinner sections, individual virus particles were resolved. At this stage, the nuclei seem to exhibit particular changes or ultrastructural modi®cations as irregular electron-dense reticular or granular strands within the nucleus and alterations in the outer nuclear envelope (Figs 6±8). Later, the degeneration of the cytoplasm causes the vacuolization of the ER (Fig. 9), swelling and loss of the internal structure of the mitochondria and the membrane-bound vesicles, leading to cytolysis. Masses of virions discharged into the extracellular space (Fig. 10) by disruption of the plasma membrane, where they could then make their way to the haemolymph and become distributed. Paracrystalline arrays of this virus could be observed close to the cuticle, in the columnar epithelial cells and, in some cases, in the interspace of muscle tissue under the cuticular epithelium. In the case of myocytes, disorganization of muscular ®bres and ®brils was present. The muscles lose their

Figure 7 TEM of cuticular epithelium containing membrane-bound masses of virions accumulated within distended cisternae of endoplasmic reticulum in epithelial cells. Note decondensed nucleus (N) of granular component. Uranyl acetate and lead citrate (bar = 1 mm).

# 2000 Blackwell Science Ltd,, Aquaculture Research, 31, 519±528

usual cross-striations. In the advanced stage of infection, the cytoplasm or sarcoplasm of infected cells is invaded by large viral inclusions. The non-enveloped particles had an opaque area

Figure 8 Higher magni®cation of Fig. 6 with large masses of viral particles in paracrystalline arrays. The degree of virus packing and thickness of the section altered the appearance of such masses (arrowhead). Uranyl acetate and lead citrate (bar = 0.5 mm).

Figure 9 Low-magni®cation TEM of the cuticular epithelium. The cytoplasm shows the accumulation of viral particles inside membrane-bound vesicles. In these vesicles, the virions are surrounded by endoplasmic reticulum (arrow). Dense particles surrounding the endoplasmic reticulum appear to represent ribosomes (arrowhead). Uranyl acetate and lead citrate (bar = 0.6 mm)

523

Cuticular epithelium necrosis in cultured Litopenaeus vannamei R JimeÂnez et al. Aquaculture Research, 2000, 31, 519±528

In situ hybridization Tests done with the in situ hybridization probe for TSV showed a negative reaction in the cuticular epithelium and the rest of the infected tissues. Positive and negative controls con®rmed that the appropriate protocols were followed.

Discussion

Figure 10 Higher magni®cation with masses of virions within distended endoplasmic reticulum, interspersed with virioplasm and needle-like crystals (arrow). Virallike particles are more widely separated than in paracrystalline arrays as a result of rupture of the membrane (arrowhead). Uranyl acetate and lead citrate (bar = 0.4 mm).

Figure 11 Higher magni®cation TEM of virions with possible icosahedral pro®les, showing an opaque area and lucent surface layer morphology. Note the regular interspace of viral-like particles. Uranyl acetate and lead citrate (bar = 100 nm).

with an approximate diameter of 20 nm and an electron-lucent surface layer. The approximate diameter of the virions was 25 nm (Fig. 11). The virus shows periodical arrangement and regular interspace of the particles. 524

The presence of these viral-like particles in the cytoplasm of L. vannamei, either dispersed or in a string-like or paracrystalline array, and arrays of virions within membrane-bound vesicles (Figs 6 and 8±10) is very similar to the TEM photographs published from a Nodamura virus affecting the larvae of the Lepidopteran Galleria mellonella (Garzon, Charpentier & Kurstak 1978). Since the early description of Nodamuravirus, authors have mentioned that this virus was morphologically indistinguishable from Picornaviruses (Murphy, Scherer, Harrison, Dunne, & William 1970). The Nodaviridae icosahedral viral particles have a diameter of 29±30 nm (Garzon & Charpentier 1991), and this might cause confusion with other RNA viruses, especially the reported apparent diameter of 27 nm of the Picornaviridae (Moore & Eley 1991). It is the bipartite singlestranded RNA genome that differentiates the Nodaviridae family of viruses from other small RNA viruses (Newman & Brown 1973; Garzon & Charpentier 1991). The morphological similarities between LvVLPs and other small viruses are interesting; in our investigation, Fig. 11 is similar to Fig. 7c of an experimental viral infection in G. mellonella presented by Garzon et al. (1978). Arrangement of viral-like particles, surrounded by a `membrane' (Longworth, Payne & Macleod 1973), `membrane-bound vacuoles' (Hess, Summers & Falcon 1978) or `virus-induced vesicles' (Chao, Young & Kim 1985), have been reported in insects and present similarity to the membranebound virus particles described in this investigation. Virus particles free in the cytoplasm, and within the same size range (20±25 nm), have also been reported in insects (Longworth et al. 1973; Binnington, Lockie, Hines & van Gerwen 1987). Small virus-like particles (20±30 nm), present in the cytoplasm, have been reported associated with infections in the eye and brain tissues of different # 2000 Blackwell Science Ltd,, Aquaculture Research, 31, 519±528

Aquaculture Research, 2000, 31, 519±528 Cuticular epithelium necrosis in cultured Litopenaeus vannamei R JimeÂnez et al.

species of ®sh (Glazebrook, Heasman & de Beer 1990; Yoshikoshi & Inove 1990; Bloch, Gravmingen & Larsen 1991; Breuil, Bonami & Pichot 1991; Mori, Nakai, Nagahara, Muroga, Mekuchi & Kanno 1991; Glazebrook & Heasman 1992; Munday, Langdon, Hyatt & Humprey 1992; Hetrick & Hedrick 1993; Danayadol, Direkbusarakom & Supamattaya 1995; Boonyaratpalin, Supamattaya, Kasorchandra & Hoffman 1996). The agent of a similar disease affecting larval striped jack Pseudocaranx dentex has been identi®ed as a Nodavirus (Mori, Nakai, Muroga, Arimoto, Musiake & Firusawa 1992). It is probable that many of the picorna-like viruses reported in ®sh actually belong to the Nodavirus family (Comps, Pepin & Bonami 1994; Nguyen, Mekuchi, Imura, Nakai, Nishikawa & Muroga 1994; Nakai, Nguyen, Nishizawa, Muroga, Arimoto & Ootsuki 1994; Grotmol, Totland & Kryvi 1997a; Grotmol, Totland, Thorud & Hjeltnes 1997b; LeBreton, Grisez, Sweetman & Ollevier 1997; Tanaka, Aoki & Nakai 1998). The dif®culty of dependency upon ®nding crystalline arrays in the cytoplasm of infected cells is mentioned in reviews of small RNA viruses in insects, the group of invertebrates in which they have mostly been studied (Moore & Tinsley 1982). As observed by other investigators working with small viruses, the early phases of replication and the different stages of the replicative stage cannot be described, mainly because of the dif®culty of differentiating between viral particles and ribosomes (Garzon et al. 1978; Gildow & D'Arcy 1990) or other cytoplasmic components (Grotmol, Bergh & Totland 1999). Small viruses can be confused with transverse sections of collagen ®bres, collagen ®bres in extracellular matrix and myo®brils. When these small RNA viruses infect muscle cells, the interpretation of results is even more dif®cult; nevertheless, some authors have differentiated them from myo®brils (Murphy et al. 1970; Garzon et al. 1978; Binnington et al. 1987; Gildow & D'Arcy 1990; Grotmol et al. 1997b). One important observation is that myo®brils present ®ne ®laments under TEM, and these structures have not been observed in our TEM photographs, in which the nucleus is also pyknotic (Figs 7 and 8). We can conclude that this TEM photograph is not a cross-section of a muscle; it is important to remark that all the sections processed by us for TEM only included the cuticular epithelium. # 2000 Blackwell Science Ltd,, Aquaculture Research, 31, 519±528

In the early description of Nodamuravirus, Murphy et al. (1970) mentioned: `in thicker sections virus particles in linear arrays blended together into `®lamentous' forms¼.', a presentation under TEM very similar to our observations. The word `®lamentous' might cause confusion, as readers unfamiliar with this description in the presentation of these viruses may suppose that the observed TEM photograph shows collagen ®brils in transverse section. The af®nity of LvVLPs to the cuticular epithelium is not unique in crustaceans, because a picorna-like virus described by Johnson (1978) in the blue crab Callinectes sapidus affects this tissue and others. Other viruses within the same size range and location reported to date in other commercial penaeid shrimp species do not present a similar viral arrangement to LvVLPs (Figs 6, 8± 10). The infection of these epithelial tissues could affect the normal formation of the new cuticle and moulting in L. vannamei, eventually leading to high mortality rates in the affected population. This effect of interfering with the normal formation of the cuticle is a similar process to a Picornavirus infecting the cuticular epithelium of L. vannamei, referred to as TSV by Brock et al. (1995) and as ICENV by Intriago et al. (1997). To the naked eye, affected animals present a similar appearance. The literature of viral diseases affecting penaeids has many descriptions of viruses causing extensive cell and tissue damage but, as the aquaculture industry has expanded, some of these were not associated with major epizootics in culture ponds (Lightner 1996). One may assume, however, that, given the right conditions, such as a host whose immune system is negatively affected, all viruses that infect crustaceans will cause major cell and tissue necrosis. Although in the case of LvVLPs, there is extensive tissue necrosis and an association with mortalities in aquaculture ponds, it should be remarked that, until controlled infection experiments are done, it would be speculative to predict the effects of this viral agent on the whole industry. This applies especially to L. vannamei from different stocks and genetic origins, cultured under different environmental conditions and stocking densities. Authors should also have caution when extrapolating laboratory results to ®eld conditions and must note the route of infection in their experiments and the levels and frequency of doses given during per os 525

Cuticular epithelium necrosis in cultured Litopenaeus vannamei R JimeÂnez et al. Aquaculture Research, 2000, 31, 519±528

infections. All these variables may give signi®cant differences in survival rates. One of the main limits in working with disease outbreaks in naturally infected populations is describing the progressive changes that affect infected and necrotic cells at TEM level, and it is a constraint in our study. This is a situation that has also been faced by other investigators because, although at the light microscopy level, nuclear pyknosis and karyorrhexis are observed, all these changes are not presented in published TEM photographs (Brock et al. 1995, 1997). Nuclear pyknosis was observed in cells infected by LvVLPs during our investigation (Figs 9 and 10). After the ®rst incidents of LvVLPs in 1996, the prevalence of this disease in commercial shrimp ponds has been moderate without serious mortalities until early 1998. The presence and association of this virus with events of high mortalities (above 40% in the next 50±70 days after stocking commercial ponds) stresses the need for a continuous research programme to identify and determine the presence of new viral agents in penaeid shrimp aquaculture. The criteria for virus classi®cation cannot be based merely on an electron microscopic examination and demonstration of correct macromolecular complement (Murphy, Fauquet, Mayo, Jarvis, Ghabrial, Summers, Martelli & Bishop 1995). Until further research can be done to purify and characterize it, the described viral-like particles can belong to a virus in either the Nodaviridae or the Picornaviridae families. Considering this last statement, the agent causing this disease should temporarily be referred to as Litopenaeus vannamei viral-like particles (LvVLPs). Acknowledgments Gratitude is extended to Dr Frederick Murphy for comments and suggestions after observing copies of the original TEM photographs. References Bell T.A. & Lightner D.V. (1988) A Handbook of Normal Penaeid Shrimp Histology. The World Aquaculture Society, Baton Rouge, LA. Binnington K.C., Lockie E., Hines E. & van Gerwen A.C.M. (1987) Fine structure and distribution of three types of virus-like particles in the sheep blow¯y, Lucilia cuprina and associated cytopathic effects. Journal of Invertebrate Pathology 49, 175±187.

526

Bloch B., Gravmingen K. & Larsen J.L. (1991) Encephalomyelitis among turbot associated with a picorna-like agent. Diseases of Aquatic Organisms 10, 65±70. Bonami J.R. & Lightner D.V. (1991) Unclassi®ed viruses of crustacea. In: Atlas of Invertebrate Viruses (ed. by J.R. Adams & J.R. Bonami), pp. 597±622. CRC Press, Boca Raton, FL. Bonami J.R., Hasson K.W., Mari J., Poulos B.T. & Lightner D.V. (1997) Taura syndrome of marine penaeid shrimp: characterization of the viral agent. Journal of General Virology 78, 313±319. Boonyaratpalin S., Supamattaya K., Kasorchandra J. & Hoffman R.W. (1996) Picorna-like virus associated with mortality and spongious encephalopathy in grouper Epinephelus malabaricus. Diseases of Aquatic Organisms 26, 75±80. Breuil G., Bonami J.R. & Pichot Y. (1991) Viral infection (picorna-like virus) associated with mass mortalities in hatchery reared seabass (Dicentrarchus labrax) larvae and juveniles. Aquaculture 97, 109±116. Brock J.A., Gose R., Lightner D.V. & Hasson K.W. (1995) An overview of Taura syndrome, an important disease of farmed Penaeus vannamei. In: Swimming Through Troubled Water, Proceedings of the Special Session on Shrimp Farming Aquaculture `95 (ed. by C.L. Browdy & J.S. Hopkins), pp. 84±94. The World Aquaculture Society, Baton Rouge, LA. Brock J.A., Gose R., Lightner D.V. & Hasson K. (1997) Recent developments and an overview of Taura Syndrome of farmed shrimp in the Americas. In: Diseases in Asian Aquaculture III (ed. by T.W. Flegel & I.H. MacRae), pp. 275±283. Fish Health Section, Asian Fisheries Society, Manila, Philippines. Chao Y.C., Young S.Y. & Kim K.S. (1985) Cytopathology of the soybean looper Pseudoplusia includens infected with the Pseudoplusia includens icosahedral virus. Journal of Invertebrate Pathology 45, 16±23. Chen S. (1995) Current status of shrimp aquaculture in Taiwan: 1995. In: Swimming Through Troubled Water, Proceedings of the Special Session on Shrimp Farming Aquaculture `95 (ed. by C.L. Browdy & J.S. Hopkins), pp. 29±34. The World Aquaculture Society, Baton Rouge, LA. Comps M., Pepin J.F. & Bonami J.R. (1994) Puri®cation and characterization of two ®sh encephalitis virus (FEV) infecting Lates calcarifer and Dicentrarchus labrax. Aquaculture 123, 1±10. Danayadol Y., Direkbusarakom S. & Supamattaya K. (1995) Viral nervous necrosis in brownspotted grouper, Epinephelus malabaricus, cultured in Thailand. In: Diseases in Asian Aquaculture II (ed. by M. Shariff, J.R. Arthur & R.P. Subasinghe), pp. 227±233. Fish Health Section, Asian Fisheries Society, Manila, Philippines. Flegel T.W., Boonyaratpalin S. & Withyachumnarnkul B. (1997) Progress in research on Yellow-head virus and

# 2000 Blackwell Science Ltd,, Aquaculture Research, 31, 519±528

Aquaculture Research, 2000, 31, 519±528 Cuticular epithelium necrosis in cultured Litopenaeus vannamei R JimeÂnez et al. White-spot virus in Thailand. In: Diseases in Asian Aquaculture III (ed. by T.W. Flegel & I.H. MacRae), pp. 285±295. Fish Health Section, Asian Fisheries Society, Manila, Philippines. Frelier P.F., Sis R.S., Bell T.A. & Lewis D.H. (1992) Microscopic and ultrastructural studies of necrotizing hepatopancreatitis in Texas cultured shrimp (Penaeus vannamei). Veterinary Pathology 29, 269±277. Garzon S. & Charpentier G. (1991) Nodaviridae. In: Atlas of Invertebrate Viruses (ed. by J.R. Adams & J.R. Bonami), pp. 351±370. CRC Press, Boca Raton, FL. Garzon S., Charpentier G. & Kurstak E. (1978) Morphogenesis of the Nodamura virus in the larvae of the Lepidopteran Galleria mellonella (L.). Archives of Virology 56, 61±76. Gildow F.E. & D'Arcy C.J. (1990) Cytopathology and experimental host range of Rhopalosiphum padi virus, a small isometric RNA virus infecting cereal aphids. Journal of Invertebrate Pathology 55, 245±257. Glazebrook J.S. & Heasman M.P. (1992) Diagnosis and control of picorna-like virus infections in larval barramundi, Lates calcarifer Bloch. In: Diseases in Asian Aquaculture I (ed. by M. Sharifff, R.P. Subasinghe & J.R. Arthur), pp. 267±272. Fish Health Section, Asian Fisheries Society, Manila, Philippines. Glazebrook J.S., Heasman M.P. & de Beer S.W. (1990) Picorna-like viral particles associated with mass mortalities in larval barramundi, Lates calcarifer (Bloch). Journal of Fish Diseases 13, 245±249. Grotmol S., Totland G.K. & Kryvi H. (1997a) Detection of nodavirus-like agent in heart tissue from reared Atlantic salmon Salmo salar suffering from cardiac myopathy syndrome (CMS). Diseases of Aquatic Organisms 29, 79± 84. Grotmol S., Totland G.K., Thorud K. & Hjeltnes B.K. (1997b) Vacuolating encephalopathy and retinopathy associated with a nodavirus-like agent: a probable cause of mass mortality of cultured larval and juvenile Atlantic halibut Hippoglossus hippoglossus. Diseases of Aquatic Organisms 29, 85±97. Grotmol S., Bergh O. & Totland G.K. (1999) Transmission of viral encephalopathy and retinopathy (VER) to yolksac larvae of Atlantic halibut Hippoglossus hippoglossus: occurrence of nodavirus in various organs and a possible route of infection. Diseases of Aquatic Organisms 36, 95±106. Hasson K.W., Lightner D.V., Poulos B.T., Redman R.M., White B.L., Brock J.A. & Bonami J.R. (1995) Taura syndrome in Penaeus vannamei: demonstration of a viral etiology. Diseases of Aquatic Organisms 23, 115±126. Hasson K.W., Hasson J., Aubert H., Redman R.M. & Lightner D.V. (1997) A new RNA-friendly ®xative for the preservation of penaeid shrimp samples for virological detection using cDNA genomic probes. Journal of Virological Methods 66, 227±236. Hasson K.W., Lightner D.V., Mari J., Bonami J.R., Poulos

# 2000 Blackwell Science Ltd,, Aquaculture Research, 31, 519±528

B.T., Mohney L.L., Redman R.M. & Brock J.A. (1999a) The geographical distribution of Taura Syndrome Virus in the Americas: determination by histology and in situ hybridization using TSV-speci®c cDNA probes. Aquaculture 171, 13±26. Hasson K.W., Lightner D.V., Mohney L.L., Redman R.M., Poulos B.T. & White B.M. (1999b) Taura syndrome virus (TSV) lesion development and the disease cycle in the Paci®c white shrimp Penaeus vannamei. Diseases of Aquatic Organisms 36, 81±93. Hess R.T., Summers M.D. & Falcon L.A. (1978) A mixed virus infection in midgut cells of Autographa californica and Trichoplusia ni larvae. Journal of Ultrastructure Research 65, 253±265. Hetrick F.M. & Hedrick R.P. (1993) New viruses described in ®n®sh from 1988 to 1992. Annual Review of Fish Diseases 3, 187±207. Intriago P., JimeÂnez R., Machuca M., Barniol R., Krauss E. & Salvador X. (1997). Experiments on toxicosis as the cause of Taura syndrome in Penaeus vannamei in Ecuador. In: Diseases in Asian Aquaculture III (ed. by T.W. Flegel & I.H. MacRae), pp. 365±379. Fish Health Section, Asian Fisheries Society, Manila, Philippines. JimeÂnez R. (1992) Sindrome de Taura (Resumen). Revista Acuacultura Del Ecuador 1, 1±16. CaÂmara Nacional de Acuacultura, Guayaquil, Ecuador. JimeÂnez R., Barniol R. & Machuca M. (1997) An epizootic of an intracellular bacterium in cultured penaeid shrimp (Crustacea: Decapoda) in the Gulf of Guayaquil, Ecuador. In: Diseases in Asian Aquaculture III (ed. by T.W. Flegel & I.H. MacRae), pp. 305±311. Fish Health Section, Asian Fisheries Society, Manila, Philippines. Jiravanichpaisal P., Miyasaki T. & Limsuwan C. (1994) Histopathology, biochemistry and pathogenicity of Vibrio harveyi infecting black tiger prawn Penaeus monodon. Journal of Aquatic Animal Health 6, 27±35. Johnson P.T. (1978) Viral diseases of the blue crab Callinectes sapidus. Marine Fisheries Review of 40 (10), 13±15. Karnovsky M.L. (1965) A formaldehyde±glutaraldehyde ®xative of high osmolarity for use in electron microscopy. Journal of Cell Biology 27, 137A±138A. LeBreton A., Grisez L., Sweetman J. & Ollevier F. (1997) Viral nervous necrosis (VNN) associated with mass mortalities in cage-reared sea bass, Dicentrarchus labrax (L.). Journal of Fish Diseases 20, 145±151. Lightner D.V. (1983) Diseases of penaeid shrimp. In: Handbook of Mariculture Vol. I. Crustacean Aquaculture (ed. by J.P. McVey), pp. 283±320. CRC Press, Boca Raton, FL. Lightner D.V. (1988a) IHHNV virus disease of penaeid shrimp. In: Disease Diagnosis and Control in North American Marine Aquaculture, 2nd edn (ed. by C.L. Sindermann & D.V. Lightner), pp. 11±15. Elsevier, New York. Lightner D.V. (1988b) BP (Baculovirus penaei) virus

527

Cuticular epithelium necrosis in cultured Litopenaeus vannamei R JimeÂnez et al. Aquaculture Research, 2000, 31, 519±528 disease of penaeid shrimp. In: Disease Diagnosis and Control in North American Marine Aquaculture, 2nd edn (ed. by C.L. Sindermann & D.V. Lightner), pp. 16±25. Elsevier, New York. Lightner D.V. (1996) A Handbook of Pathology and Diagnostic Procedures for Diseases of Penaeid Shrimp. The World Aquaculture Society, Baton Rouge, LA. Lightner D.V. & Redman R.M. (1998) Strategies for the control of viral diseases of shrimp in the Americas. Fish Pathology 33, 165±180. Lightner D.V., Redman R.M., Hasson K.W. & Pantoja C.R. (1995) Taura syndrome in Penaeus vannamei (Crustacea: Decapoda): gross signs, histopathology and ultrastructure. Diseases of Aquatic Organisms 21, 53±59. Lin C.K. (1989) Prawn culture in Taiwan ± what went wrong ?. World Aquaculture 20, 19±20. Longworth J.F., Payne C.C. & Macleod R. (1973) Studies on a virus isolated from Gonometa podocarpi (Lepidoptera: Lasicampidae). Journal of General Virology 18, 119±125. Lotz J.M. (1997) Effect of host size on virulence of Taura virus to the marine shrimp Penaeus vannamei (Crustacea: Penaeidae). Diseases of Aquatic Organisms 30, 45±51. Lucien-Brun H. (1997) Evolution of world shrimp production: ®sheries and aquaculture. World Aquaculture 28 (4), 21±33. Luna L.G. (1968) Manual of Histological Staining Methods of the Armed Forces Institute of Pathology, 3rd edn. McGraw-Hill, New York. Mari J., Bonami J.R. & Lightner D.V. (1998) Taura syndrome of penaeid shrimp: cloning of viral genome fragments and development of speci®c gene probes. Diseases of Aquatic Organisms 33, 11±17. MeleÂndez M.L. (1998) Ecuador: exportaciones de camaroÂn 1997, anÄo record. Revista Acuacultura Del Ecuador 23, 22±27. CaÂmara Nacional de Acuacultura, Guayaquil, Ecuador. Moore N.F. & Eley S.M. (1991) Picornaviridae: Picornaviruses of invertebrates. In: Atlas of Invertebrate Viruses (ed. by J.R. Adams & J.R. Bonami), pp. 371±386. CRC Press, Boca Raton, FL. Moore N.F. & Tinsley T.W. (1982) The small RNA viruses of insects. Archives of Virology 72, 229±245. Mori K., Nakai T., Nagahara M., Muroga K., Mekuchi T. & Kanno T. (1991) A viral disease in hatchery-reared larvae and juveniles of redspotted grouper. Fish Pathology 26, 209±210. Mori K., Nakai T., Muroga K., Arimoto M., Musiake K. & Firusawa I. (1992) Properties of a new virus belonging to Nodaviridae found in larval striped jack (Pseudocaranx dentex) with nervous necrosis. Virology 187, 368±371. Munday B.L., Langdon J.S., Hyatt A. & Humprey J.D. (1992) Mass mortality associated with viral-induced vacuolating encephalopathy and retinopathy of larval and juvenile barramundi, Lates calcarifer Bloch. Aquaculture 103, 197±211.

528

Murphy F.A., Scherer W.F., Harrison A.K., Dunne H.W. & William G.G. (1970) Characterization of Nodamura virus, an arthropod transmissible Picornavirus. Virology 40, 1008±1021. Murphy F.A., Fauquet C.M., Mayo M.A., Jarvis A.W., Ghabrial S.A., Summers M.D., Martelli G.P. & Bishop D.H. (1995) The Classi®cation and Nomenclature of Viruses: Sixth Report of the International Committee on Taxonomy of Viruses. Archives of Virology SpringerVerlag, Wien, Austria. Nakai T., Nguyen H.D., Nishizawa T., Muroga K., Arimoto M. & Ootsuki K. (1994) Occurrence of viral nervous necrosis in kelp grouper and tiger puffer. Fish Pathology 29, 211±212. Newman J.F.E. & Brown F. (1973) Evidence for a divided genome in Nodamura virus, an arthropod-born Picornavirus. Journal of General Virology 21, 371±384. Nguyen H.D., Mekuchi T., Imura K., Nakai T., Nishikawa T. & Muroga K. (1994) Occurrence of viral nervous necrosis (VNN) in hatchery-reared juvenile ¯ounder Paralichthys olivaceous. Fisheries Science 60, 551±554. Nunan L.M., Poulos B.T. & Lightner D.V. (1998) Reverse transcription polymerase chain reaction (RT-PCR) used for the detection of Taura Syndrome Virus (TSV) in experimentally infected shrimp. Diseases of Aquatic Organisms 34, 87±91. Overstreet R.M., Lightner D.V., Hasson K.W., McIlwain S. & Lotz J.M. (1997) Susceptibility to Taura Syndrome Virus of some penaeid shrimp species native to the Gulf of Mexico and the Southeastern United States. Journal of Invertebrate Pathology 69, 165±176. Perez-Farfante I. & Kensley B. (1997) Penaeoid and Sergestoid shrimps and prawns of the world. Keys and diagnoses for the families and genera. MeÂmoires du Museum National D'Histoire Naturelle Tome 175. Shariff M. & Subasinghe R.P. (1992) Major diseases of cultured shrimp in Asia: an overview. In: Diseases of Cultured Penaeid Shrimp in Asia and the United States (ed. by W. Fulks & K.L. Manned), pp. 37±46. The Oceanic Institute, Honolulu, Hawaii. Sindermann C.J. & Lightner D. (1988) Disease Diagnosis and Control in North American Marine Aquaculture, 2nd edn. Elsevier, New York. Steiner G. & Steiner G. (1944) New simple silver stain for demonstration of bacteria, spirochetes and fungi in sections from paraf®n embedded tissue blocks. Journal of Laboratory Clinical Medicine 29, 868±871. Tanaka S., Aoki H. & Nakai T. (1998) Pathogenicity of the Nodavirus detected from diseased seven band grouper Epinephelus septemfasciatus. Fish Pathology 33, 31±36. Yoshikoshi N. & Inove K. (1990) Viral nervous necrosis of hatchery-reared larvae and juveniles of Japanese parrot®sh Oplegnathus fasciatus (Temminck & Shlegel). Journal of Fish Diseases 13, 69±77.

# 2000 Blackwell Science Ltd,, Aquaculture Research, 31, 519±528