Article

A haplosporidian parasite associated with high mortality and slow growth in Penaeus (Litopenaeus) vannamei cultured in Indonesia

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Abstract

High mortality of juvenile Penaeus (Litopenaeus) vannamei less than one month old in Indonesia has been observed in shrimp farms and hatcheries since 2007. The gross signs of disease included shrinkage of the hepatopancreas, flaccid bodies, cuticular melanization and retarded growth. Overall loss in the severely-infected farms was 60 to 90% due to progressive mortality. The loss since 2007 is estimated to be more than 5 million US dollars. PCR testing of shrimp samples for major viral pathogens gave negative results, but histopathological analysis of hepatopancreatic (HP) tissue showed evidence of a haplosporidian para-site indicated by cytoplasmic multinucleate plasmodia and trophonts in tubule epithelial cells. Plasmodia caused disruption of HP tubule epithelial cells when they discharged trophonts into the tubular lumen. To confirm the presence of haplosporidia, PCR was performed to amplify a small subunit rRNA gene (SSU rDNA) fragment and a cloned 1.5 kb amplicon showed 96% sequence identity to that of a haplosporidian previously described in P. vannamei from Belize, Central America suggesting that the Indonesian haplosporidian was either conspecific or very closely related. In situ hybridization assays using a probe based on this amplicon gave positive results spe-cifically with the putative haplosporidian lesions. Prevalence of infections in broodstock and ponds from 2004 to 2010 suggested that the haplosporidian disease outbreaks had resulted from stocking of infected post-larvae.

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... Dykova et al. (1988) described multinucleate plasmodia developing within the hepatocytes of the whiteleg shrimp, Penaeus vannamei, imported from Nicaragua to Cuba. Similar infections were also reported in hepatopancreas tissues from P. vannamei in Belize (Nunan et al., 2007) and Indonesia (Utari et al. 2012). In both cases pathology was similar to the infection previously described by Dykova et al. (1988) and it was suggested that they may be the same pathogen due to the structural similarities, high sequence identity and similar geographical isolations, and the fact that shrimp were initially imported to Indonesia from America (Nunan et al., 2007;Utari et al., 2012). ...
... Similar infections were also reported in hepatopancreas tissues from P. vannamei in Belize (Nunan et al., 2007) and Indonesia (Utari et al. 2012). In both cases pathology was similar to the infection previously described by Dykova et al. (1988) and it was suggested that they may be the same pathogen due to the structural similarities, high sequence identity and similar geographical isolations, and the fact that shrimp were initially imported to Indonesia from America (Nunan et al., 2007;Utari et al., 2012). Dykova et al. (1988) reported that the infected hepatopancreas cells became hypertrophied as the nucleus was displaced and the cytoplasm was replaced by the developing plasmodia. ...
... No host immune response was observed, even in heavily infected tissues. Severity of infection in most jelly prawns suggested that the normal functions of the hepatopancreas tissue would be affected, and as highlighted by Nunan et al. (2007), could result in clinical signs such as slow growth, possibly resulting in increased mortality levels as reported by Utari et al. (2012) in Penaeus vannamei cultured in Indonesia. Phylogenetic analyses show that Haplosporidium acetes is most closely related to the haplosporidian found in P. vannamei, from the USA and Indonesia, which is supported by morphological characteristics including its similar tissue trophism and pathological effect on hepatopancreas tissues. ...
Article
Wild Acetes sibogae australis from northern Moreton Bay, Australia displaying opacity of the hepatopancreas were sampled and examined histologically, revealing infection by multinucleate plasmodia of a haplosporidian-like parasite in the epithelial cells of the hepatopancreas. A morphological and phylogenetic investigation identified the parasite as a novel species of the order Haplosporida, and the parasite is described as Haplosporidium acetes n. sp. This is the first report of disease caused by a haplosporidian in wild Australian decapod crustaceans, and the first record of haplosporidiosis in sergestid shrimp. Infections of H. acetes were observed in all cell types (R, B, F and E) within the hepatopancreas. Infected epithelial cells became hypertrophied as they filled with haplosporidian parasites and, in heavy infections, caused almost complete displacement of normal hepatopancreas tissue. Although sporulation was not observed, infected jelly prawns appeared terminally diseased. Infections became grossly evident in around 5% of wild prawns during early autumn at a time of year when jelly prawn populations decline rapidly with decreasing water temperatures, however histopathology indicated at least 13% of apparently normal jelly prawns were also infected. Further studies are required in order to determine if this parasite influences jelly prawn population dynamics. In addition, we report co-infection of a novel microsporidian parasite in the Enterocytozoon Group Microsporidia (EGM) infecting nuclei of hepatopancreatic epithelial cells. The microsporidian was phylogenetically distinct from Enterocytozoon hepatopenaei (EHP) known to infect penaeid shrimp in Asia.
... Both Bonamia species are pathogens listed by and notifiable to the World Organization for Animal Health (OIE) and the European Union (EU). In addition, unnamed haplosporidan parasites were responsible for high mortality of cultured shrimps Penaeus vannamei in the Caribbean Sea (Nunan et al., 2007) and Indonesia (Utari et al., 2012). ...
... Both H. nelsoni and H. tuxtlensis sporulate in the digestive gland tubules of their hosts, the eastern oyster Crassostrea virginica and the striped false limpet Siphonaria pectinata, respectively (Couch et al., 1966;Vea and Siddall, 2011). Additionally, heavy infection of the epithelium of digestive gland tubules of the shrimp P. vannamei with Haplosporidium sp., causing disruption of host epithelial cells, has been reported from Caribbean countries and Indonesia (Dykova et al., 1988;Nunan et al., 2007;Utari et al., 2012). Similar to H. pinnae, H. nelsoni and the haplosporidan parasite of P. vannamei have been responsible for mass mortalities of their hosts, eastern oysters (Ford and Tripp, 1996) and penaeid shrimps (Utari et al., 2012). ...
... Additionally, heavy infection of the epithelium of digestive gland tubules of the shrimp P. vannamei with Haplosporidium sp., causing disruption of host epithelial cells, has been reported from Caribbean countries and Indonesia (Dykova et al., 1988;Nunan et al., 2007;Utari et al., 2012). Similar to H. pinnae, H. nelsoni and the haplosporidan parasite of P. vannamei have been responsible for mass mortalities of their hosts, eastern oysters (Ford and Tripp, 1996) and penaeid shrimps (Utari et al., 2012). The low prevalence of H. tuxtlensis could explain that no abnormal mortality of false-limpets was mentioned when this parasite was described (Vea and Siddall, 2011). ...
... Indonesia (Utari et al., 2012). ...
... vannamei with Haplosporidium sp., causing disruption of host epithelial cells, has been reported from Caribbean countries and Indonesia (Dykova et al., 1988;Nunan et al., 2007;Utari et al., 2012). Similar to H. pinnae, H. nelsoni and the haplosporidan parasite of P. vanamei have been responsible for mass mortalities of their hosts, eastern oysters (Ford and Tripp, 1996) and penaeid shrimps (Utari et al., 2012). ...
... vannamei with Haplosporidium sp., causing disruption of host epithelial cells, has been reported from Caribbean countries and Indonesia (Dykova et al., 1988;Nunan et al., 2007;Utari et al., 2012). Similar to H. pinnae, H. nelsoni and the haplosporidan parasite of P. vanamei have been responsible for mass mortalities of their hosts, eastern oysters (Ford and Tripp, 1996) and penaeid shrimps (Utari et al., 2012). The low prevalence of H. tuxtlensis could explain that no abnormal mortality of false-limpets was mentioned when this parasite was described (Vea and Siddall, 2011). ...
Article
This study provides morphological and molecular characterization of a new species, Haplosporidium pinnae, very likely responsible for mass mortality of fan mussels, Pinna nobilis, in the Western Mediterranean Sea. The parasite was found in dead or moribund P. nobilis but did not occur in healthy fan mussels from locations that were not affected by abnormal mortality. Histological examination of infected fan mussels showed uninucleate cells of a haplosporidan parasite throughout the connective tissue and hemolymph sinuses of the visceral mass and binucleate cells and, rarely, multinucleate plasmodia were also detected in the connective tissue. Additionally, stages of sporulation occurred in the epithelium of the host digestive gland tubules. Spores were slightly ellipsoidal with a hinged operculum in one pole. Typical haplosporosomes were not found with TEM but vesicles with two concentric membranes resembling haplosporosomes were abundant in the cytoplasm of the multinucleate plasmodia occurring in host digestive gland tubules. SEM analysis showed multiple structures on the spore surface; some spores had two or four long tape-like filaments attached to the spore wall. Phylogenetic analysis based on the SSU rDNA sequence placed this parasite within a large clade including species of the order Haplosporida, not in the Bonamia/Minchinia subclade or the subclade containing most Haplosporidium species, but within a subclade of Haplosporidium sp. from Penaeus vannamei. Our results suggested that H. pinnae and the parasite of P. vannamei may represent a distinct new genus within the order Haplosporida.
... Between 2007 and 2009 in Indonesia, severe outbreaks of hepatopancreatic haplosporidiosis (HPH) were recorded in cultivated P. vannamei (Utari et al., 2012). The rearing pond outbreaks were linked to infected PL arising from infected, imported broodstock purportedly free of the pathogen. ...
... By histological examination, the causative agent morphologically resembled an unnamed haplosporidian previously reported from Central America (Dyková et al., 1988;Nunan et al., 2007) (Fig. 9). A portion of the ssu rRNA sequence was amplified by PCR and subjected to BLAST analysis and phylogenetic comparison using a 1582 bp consensus sequence (GenBank HQ285783) (Utari et al., 2012). The gene fragment had 96% sequence identity to that of the haplosporidian previously described from Central America (GenBank DQ653412) (Nunan et al., 2007), suggesting that they were either conspecific or very closely related. ...
... Based on the amplified sequence (HQ285783), primers were designed for nested PCR that would work for both the Indonesian and Central American isolates (Fig. 9) and to prepare a DIG-labeled probe that was used to confirm the infections by in situ hybridization (Utari et al., 2012). Since 2010, close monitoring of broodstock and PL using histology and the PCR method developed has resulted in no further reports of disease outbreaks by this pathogen in Indonesia. ...
... Asian shrimp cultivation is an important industry that generates billions of US dollars in export income annually (Flegel 2012). The main cultivated species are Penaeus monodon and Penaeus (Litopenaeus) vannamei, with the latter currently dominating the world market (FAO 2010). ...
... In China and southeast Asia, shrimp cultivation is a multi-billion dollar industry that contributed 3.9 million tons of production to the global shrimp market in 2010 (FAO 2010). However, widespread epidemic diseases caused by viruses, such as white spot syndrome virus (WSSV), yellow head virus (YHV), hepatopancreatic parvovirus (HPV), monodon baculovirus (MBV), Taura syndrome virus (TSV), and infectious hypodermal and hematopoietic virus (IHHNV), have caused serious losses for shrimps farmers, estimated at nearly US$15 billion over the last 15 years, with about 80 % of this loss occurring in Asia (Flegel 2012). ...
... The determined DNA sequence was novel, and phylogenetic analysis placed the P. vannamei parasite within the phylum Haplosporidia as Aquacult Int a sister taxon to a clade that included Bonamia and Minchinia species (Nunan et al. 2007). A similar pathogen caused high mortality of juvenile P. vannamei (\1 month old) in Indonesia, which showed 96 % sequence identity to the haplosporidian from Central America (Utari et al. 2012). Enterocytozoon hepatopenaei causes white feces syndrome and has no causal relationship with AHPND; it seems likely that the increased prevalence of E. hepatopenaei and AHPND bacteria has resulted in the contamination of brood stock or post-larvae (Sriurairatana et al. 2014). ...
Article
Full-text available
Acute hepatopancreatic necrosis disease (AHPND) of shrimps is an important disease, first appearing in China in 2009. Since then, AHPND has caused serious drops in shrimp production (up to 20% worldwide). Although AHPND (originally termed acute hepatopancreatic necrosis syndrome (AHPNS)) first appeared in 2009, it was not until 2013 that a laboratory infection model was devised and the causative agent identified as certain strains of Vibrio parahaemolyticus. AHPND has caused mortality from 40 to 100% which usually occurs early (within approximately 35 days) after stocking shrimp fry in shrimp ponds; therefore, it was initially referred to as early mortality syndrome (EMS). Confusingly, other pathogens and environmental factors also cause EMS, and are often attributed to AHPND by shrimp farmers. Frequently, farmers do not send samples for confirmatory tests requiring detection of the unique histopathology at the acute stage of disease (massive sloughing of hepatopancreatic epithelial cells without any accompanying signs of a pathogen). The gross signs presumptive of AHPND (lethargy, slow growth, empty stomach and midgut, and a pale to white, atrophied hepatopancreas) are insufficient for confirmatory diagnosis. Recently, molecular detection of AHPND bacteria using PCR has been developed, which has sped up diagnosis and increased research on the causative agent, alternative detection methods and possible therapies. We hope that this review of research progress on AHPND will serve as a useful introduction for researchers who are currently unfamiliar with AHPND, but have backgrounds in bacterial virulence, detection, and epidemiology, and may be encouraged to participate in the research effort to reduce AHPND's impact on shrimp cultivation.
... Haplosporidia have also been reported from penaeid and caridean shrimp but none have been adequately described (Dyková et al. 1988;Bower and Meyer 2002;Nunan et al. 2007;Utari et al. 2012). Localised outbreaks with prevalence as high as 30% and low host survival have occurred sporadically in penaeid cultures (Utari et al. 2012). ...
... Haplosporidia have also been reported from penaeid and caridean shrimp but none have been adequately described (Dyková et al. 1988;Bower and Meyer 2002;Nunan et al. 2007;Utari et al. 2012). Localised outbreaks with prevalence as high as 30% and low host survival have occurred sporadically in penaeid cultures (Utari et al. 2012). An outbreak in Indonesia was thought to arise from infected postlarval shrimp. ...
Chapter
As with all animals, crustaceans serve as hosts to a very diverse taxa of parasites. These parasites range from unusual dinoflagellates that parasitise the hemocoels or eggs of their hosts, to classical helminths that use crustaceans as intermediate hosts, and to the bizarrely adapted tantulocarid and rhizocephalan crustaceans with their highly derived life styles. Here, I review the major parasitic taxa that use Crustacea as hosts. The parasites of decapods, particularly those in shrimps, crabs, and lobsters are the best known, primarily because of their impact on populations of their commercially important hosts. Several of these parasites are outright pathogens that cause widespread mortality, feminisation, and stunting in their host populations. Other parasites, particularly those in copepods, cladocerans, and amphipods have also received attention because of the ecological importance of these hosts in food webs. They have received notable studies on vertical transmission, the influence of cryptic species complexes (both host and parasite), as well as the emergence of new pathogens in these hosts. A few parasites are also known from brine shrimp (Anostraca) and barnacles (Cirripedia) which have served as laboratory or ecological models, respectively, but few of these parasites have received much study other than their initial taxonomic descriptions and systematic placement. Although molecular tools have revealed the systematics of many of the parasitic taxa, their biology and ecology remain poorly known.
... A haplosporidian disease has caused high mortalities in juvenile P. vannamei in Indonesian shrimp farms and hatcheries since 2007 (Utari et al. 2012). Loss in the severely-infected farms was 60−90%. ...
... The nuclei of the infected host cells appeared normal. The plasmodia caused disruption of the epithelial cells and the trophonts were discharged into the tubular lumen (Utari et al. 2012). ...
Article
The hepatopancreas of decapod crustaceans is used as an example to illustrate the range of cytopathologies, detoxification mechanisms, and immune responses that environmental toxicants and pathogens can induce in a single organ. The hepatopancreas is the central metabolic organ of decapods and consists of hundreds of blindly-ending tubules and intertubular spaces. The tubular epithelium contains 5 structurally and functionally different cell types, and the interstitium contains haemolymph, haemocytes, connective tissue, and fixed phagocytes. Some physiological conditions such as moulting and starvation cause marked but reversible ultrastructural alterations of the epithelial cells. Environmental toxicants induce either detoxification mechanisms or structural damage in cells, depending on toxicant and concentration. The hepatopancreas is also a main target organ for pathogens, mainly viruses, bacteria, and protists that enter the body via the digestive tract and gills and replicate in the hepatopancreatocytes. The cytopathologies caused by toxicants and pathogens affect single cell types specifically or, more often, several cell types simultaneously. Pathogenesis often begins in a certain cell organelle such as the nucleus, mitochondrion, or endoplasmic reticulum, spreads to other organelles, and ends with death of the infected cell. Fixed phagocytes in the interstitium capture and degrade pathogens that move from the infected tubules into the intertubular spaces or enter the hepatopancreas via circulation. Relatively few disease agents elicit the melanisation and encapsulation reaction that encloses infected tubules by a rigid melanised capsule and kills the entrapped pathogens.
... Although molluscs are the best-known hosts of haplosporidian parasites, awareness of the role played by crustacean hosts has increased in recent decades (Hine et al. 2009). Some of these relationships potentially have ecological and commercial importance, as haplosporidians infect several species of crabs (Stentiford et al. 2013) and shrimps (Bower & Meyer 2002, Utari et al. 2012. However, there is very limited information on Haplosporida infecting amphi pods. ...
... While molluscs may be the most diverse hosts for haplosporidian parasites, crustaceans are increas-ingly reported as hosts (Stentiford et al. 2013), including infections affecting commercial species (Bower & Meyer 2002, Utari et al. 2012). However, crustacean-derived haplosporidians have so far appeared to branch without discernible pattern among mollusc-infecting lineages. ...
Article
Full-text available
This study provides morphological, ultrastructural and phylogenetic characterization of 2 novel species of Haplosporidia (Haplosporidium echinogammari n. sp. and H. orchestiae n. sp.) infecting amphipods of the genera Echinogammarus and Orchestia collected in southwestern England. Both parasites infect the connective tissues associated with the digestive gland and the tegument, and eventually infect other organs causing disruption of host tissues with associated motor impairment and fitness reduction. Prevalence of infection varied with host species, provenance and season, being as high as 75% for individuals of E. marinus infected with H. echinogammari in June (n = 50). Although no spores were found in any of the infected amphipods examined (n = 82), the morphology of monokaryotic and dikaryotic unicellular stages of the parasites enabled differentiation between the 2 new species. Phylogenetic analysis of the new species based on the small subunit (SSU) rDNA gene placed H. echinogammari close to H. diporeiae in haplosporidian lineage C, and H. orchestiae in a novel branch within Haplosporidium. Genetic diversity of the haplosporidians infecting these and other amphipod species was evaluated and compared to morphological and ultrastructural changes to host tissues. The phylogenetic relationship of haplosporidian infections in other crustacean hosts is discussed after inclusion into the analysis of 25 novel SSU rDNA sequences obtained from crabs, isopods and crayfish.
... Other eukaryotic pathogens, such as members of the Ascetosporea class of parasites, also cause problems in crustacean culture, such as the haplosporidian parasite that has been associated with high mortality and slow growth in P. vannamei. 173 In addition to a gap in our understanding of eukaryotic pathogens of M. rosenbergii, there is also information missing from the literature surrounding the life-stages that pathogens affect, especially whether viruses that are lethal in postlarvae/juveniles can also infect larvae and adults. This is especially important at the stages in culture when animals are moved to new locations e.g. from hatchery to grow-out pond, where pathogens could be transferred from one life stage to another. ...
Article
Full-text available
The giant river prawn, Macrobrachium rosenbergii, is a major focus of aquaculture in tropical and sub‐tropical regions around the globe. Over the last 30 years, culture of M. rosenbergii has increased exponentially as demand has risen both for domestic consumption and for international export trade. As with many aquaculture species increases in production have been accompanied by the emergence of diseases affecting yield, profit and trading potential. Disease‐causing agents include pathogens infecting other crustaceans, such as Decapod Iridescent Virus (DIV1), as well as pathogens only known from M. rosenbergii such as White Tail Disease caused by Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV). Here, we review the pathogenic agents associated with the culture of M. rosenbergii since commercial culture began in earnest during the 1970s. Particular emphasis is given to pathogens first identified in other aquaculture host species, but which have subsequently been shown to infect and cause disease in M. rosenbergii. As polyculture of M. rosenbergii with other aquaculture species is common practice, including culture with other decapods, crabs and fish, increased pathogen transfer among these farmed species may occur as M. rosenbergii aquaculture increases in the future.
... Tourtip et al., 2009;Chaijarasphong et al., 2021;Geetha et al. (2022) Intracytoplasmic -connective tissue (A. penaei) and tubules (E. hepatopenaei) of the hepatopancreas.Hepatopancreatic haplosporidiosisHaplosporidium sp.-Melanisation of the cuticle, hepatopancreas shrinkage (pale and atrophied), reduced growth, and flaccid bodies.(Utari et al., 2012;Thitamadee et al., 2016) ...
Article
Full-text available
The farming of decapod crustaceans is a key economic driver in many countries with production reaching around 9.4 million tonnes (USD 69.3 billion) in 2018. These efforts are currently dominated by the farming of Pacific whiteleg shrimp, Penaeus vannamei, which translates into approximately 167 billion farmed P. vannamei being harvested annually. Further production growth is expected in the future and hence the need for more research into its health and welfare is required. Herein, from an extensive survey of the available literature, we scrutinise farming practices and the challenges associated with the production of P. vannamei from an animal-centric welfare perspective (1), we propose potential welfare indicators (2) and we critically review current scientific evidence of sentience in penaeid shrimp among other commercially important decapods (3), since it is plausible that in the near future not only the largest, but in fact all decapod crustaceans will receive welfare protection. This review highlights that despite the wide knowledge on crustacean stress physiology and immunology as well as disease control, still little is known about some key parameters related to the five welfare dimensions. We recommend that further research should focus on developing a systematic integrated welfare assessment encompassing all the different aspects of the crustaceans farming and life cycle up to slaughter. Furthermore, direct and indirect species-specific operational welfare indicators should be developed for all decapod crustaceans currently farmed, similar to the ones suggested in this review for P. vannamei.
... Further, these authors attributed the slow growth observed to hepatopancreatic dysfunction due to infection with a haplosporidian pathogen. Similarly, since 2007, high mortality along with the gross signs of shrunken hepatopancreas, flaccid bodies, cuticular melanisation and retarded growth have been reported in juvenile P. vannamei farmed in Indonesia (Utari et al. 2012). Similar to the observation made by Nunan et al. (2007), the surviving animals showed retarded growth compared to animals of the same age from the uninfected ponds. ...
Article
Full-text available
The phenomenon of slow growth in farmed shrimp was observed for the first time in 1989 in Penaeus vannamei as a clinical manifestation of runt deformity syndrome caused by infectious hypodermal and hematopoietic necrosis virus. Subsequently, it was recorded in Penaeus monodon during 2001–2002. Since then, the condition described as monodon slow growth syndrome (MSGS) has been reported from many countries. Though not leading to mortality, retarded growth at the pond level results in significant economic losses. Several potential pathogens have been identified from affected shrimp; however, no confirmed causal relationship has yet been established. Initially, P. vannamei farmed alongside slow growth‐affected P. monodon were unaffected by the condition. Further, experimental studies suggested that P. vannamei was not susceptible. However, in recent years, increasing incidence of severe growth retardation has been reported in farmed P. vannamei. Currently, slow growth is considered as one of the most impactful production‐limiting conditions affecting shrimp farming across Asia. Similar to MSGS, many infectious agents have been detected in slow growth‐affected P. vannamei, especially the microsporidian Enterocytozoon hepatopenaei. Further, genetic and environmental factors have been suggested to play a role. However, the exact cause of slow growth remains elusive, proposing that it may be a multi‐factor syndrome. This review tracks the trajectory of the condition over the period of significant expansion of shrimp farming and postulates that conventional single pathogen/single disease paradigms are insufficient to deal with chronic yield‐limiting syndromes. We suggest more realistic, multi‐dimensional aetiological consideration of syndromic and emerging diseases in shrimp aquaculture.
... The molecular and phylogenetic analysis conducted by Catanese et al. (2018) revealed that the parasite infecting P. nobilis is clearly included in the Haplosporida group and is closely related to another haplosporidan parasite causing high mortality in the cultured shrimp Penaeus vannamei in the Caribbean Sea (Nunan et al. 2007) and Indonesia (Utari et al. 2012). Nevertheless, they identified many differences between the two parasites, which appeared to be a separate group from other Haplosporidium, Minchinia and Bonamia species, and suggested that these two species may represent a distinct new genus within the order Haplosporida. ...
... The molecular and phylogenetic analysis conducted by Catanese et al. (2018) revealed that the parasite infecting P. nobilis is clearly included in the Haplosporida group and is closely related to another haplosporidan parasite causing high mortality in the cultured shrimp Penaeus vannamei in the Caribbean Sea (Nunan et al. 2007) and Indonesia (Utari et al. 2012). Nevertheless, they identified many differences between the two parasites, which appeared to be a separate group from other Haplosporidium, Minchinia and Bonamia species, and suggested that these two species may represent a distinct new genus within the order Haplosporida. ...
... The molecular and phylogenetic analysis conducted by Catanese et al. (2018) revealed that the parasite infecting P. nobilis is clearly included in the Haplosporida group and is closely related to another haplosporidan parasite causing high mortality in the cultured shrimp Penaeus vannamei in the Caribbean Sea (Nunan et al. 2007) and Indonesia (Utari et al. 2012). Nevertheless, they identified many differences between the two parasites, which appeared to be a separate group from other Haplosporidium, Minchinia and Bonamia species, and suggested that these two species may represent a distinct new genus within the order Haplosporida. ...
Article
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The cryptogenic parasite Haplosporidium pinnae has caused mass mortality of the protected endemic Mediterranean bivalve Pinna nobilis in the western Mediterranean, since the autumn of 2016. Herein, we confirm the spread of the parasite in the eastern Mediterranean, and report a mass mortality event, with > 93% average mortalities, in the coastal waters of Lesvos Island (Greece, Aegean Sea). Histopathological study of collected specimens revealed the presence of a haplosporian-like protozoon in different life cycle stages, mainly within the digestive gland of the infected Pinna nobilis, with many uni- and bi-nucleate parasite cells, plasmodia and sporocysts in the wide lumen of digestive tubules causing the collapse of epithelial cells, and apparently low host haemocyte reaction. The parasite was identified as H. pinnae by molecular methods (PCR amplification and sequencing of a part of small subunit ribosomal DNA gene, and comparison with available records in Genbank). In many sites, 100% mortality was recorded, whereas in a single site (among 13 surveyed sites) mortality was relatively low (36%), successful recruitment was observed and the parasite was not detected. The latter observation stresses the importance of possible parasite-free refugia sites. We call for continuous monitoring of the spread of the parasite and its impacts, and for urgent targeted research and actions to identify the factors affecting the parasite’s virulence, investigate biotic and abiotic conditions that characterize refugia sites, and strictly protect the remaining P. nobilis populations to increase the chances for the survival of the species.
... The molecular and phylogenetic analysis conducted by Catanese et al. (2018) revealed that the parasite infecting P. nobilis is clearly included in the Haplosporida group and is closely related to another haplosporidan parasite causing high mortality in the cultured shrimp Penaeus vannamei in the Caribbean Sea (Nunan et al. 2007) and Indonesia (Utari et al. 2012). Nevertheless, they identified many differences between the two parasites, which appeared to be a separate group from other Haplosporidium, Minchinia and Bonamia species, and suggested that these two species may represent a distinct new genus within the order Haplosporida. ...
... Recombinant plasmid containing a 442-bp DNA fragment derived from 16S rDNA of H. chejuensis HN01 was used as template in the labeling reaction. Saccharomyces cerevisiae TRP1 gene encoding phosphoribosylanthranilate isomerase was employed as unrelated negative control probe (Utari et al., 2012). ...
Article
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The present study is the first report of a novel disease called Hahellosis or “red egg disease” that has been affecting tilapia (Oreochromis spp.) hatcheries in Thailand. Hahella chejuensis, a red pigmented Gram negative bacterium, was recovered from samples of the red egg and identified to species level based on 99.5% - 99.7% nucleotide homology to 16S rDNA of the type strain H. chejuensis KCTC 2396. Experimental infection of eggs indicated that H. chejuensis was able to cause red egg disease and also reduce their hatching. PCR protocols were developed for detection of H. chejuensis in tilapia samples including eggs and ovaries and testes of tilapia broodstocks. Positive signals were obtained in the ovaries and testes of tilapia broodstocks with in situ DNA hybridization using probes specific for H. chejuensis, suggesting possible vertical transmission of the red pigmented bacteria from broodstock to eggs.
... Ituarte et al. (2014), describing the limpet parasite Haplosporidium patagon, found strong (100% bootstrap) support for a sister relationship to the S. nipponica parasite, but for little else besides a close relationship among H. littoralis, H. montforti, H. lusitanicum, H. pickfordi, and H. tuxtlensis, which were recovered on a monophyletic clade in both parsimony and maximum likelihood analyses. In general, with regard to the Haplosporidium region of the haplosporidian phylogeny, we can conclude based on presently available data that H. littoralis, H. montforti, H. lusitanicum, H. pickfordi, and H. tuxtlensis do comprise a clade of closely related species; that H. raabei and H. edule, and H. costale and the Haplosporidium sp. from O. edulis, are pairs of sister species that may (see Fig. 1) or may not (Ituarte et al., 2014) represent another monophyletic lineage; that the S. nipponica parasite and H. patagon, the parasites from the shrimp Litopenaeus vannamei in Belize (Nunan et al., 2007) and from Indonesia (Utari et al., 2012), and H. nelsoni and the amphipod parasite Haplosporidium diporeiae (Winters and Faisal, 2014) are three additional pairs of sister species; but that the relationships among these lineages are also not well resolved (Fig. 1). We note also a growing uncertainty about the position of H. louisiana, which has reliably been positioned as basal to H. nelsoni and all the other Haplosporidium, Bonamia, and Minchinia spp. ...
Article
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The protist phylum Haplosporidia comprises over 40 described species with representatives infecting a range of mollusc hosts, including several ecologically and economically significant pathogens. Continuing exploration of haplosporidian diversity has added ten new species in recent years and brought the phylogenetics of the group into somewhat clearer focus, with monophyletic Bonamia and Minchinia lineages continuing to be supported. However, the addition of new sequences to phylogenetic analyses has left the paraphyletic genus Haplosporidium's picture less resolved. It is not clear that even two genera will be enough to accommodate the species presently drawn to the Haplosporidium regions of the haplosporidian tree. In this review, we summarize recent findings in haplosporidian diversity and phylogenetics, and provide a synthesis of our understanding of the life cycles and environmental influences on haplosporidians, with particular emphasis on the important pathogens Haplosporidium nelsoni and Bonamia ostreae. Additionally, we consider the evolution of the "microcell haplosporidian" lifestyle of Bonamia parasites, and suggest that colonization of high-density oyster host populations in relatively stable euhaline marine environments may have been an important development favoring the evolution of the microcell haplosporidian life strategy. Copyright © 2015 Elsevier Inc. All rights reserved.
... Manajemen kesehatan ikan hias seharusnya mulai diterapkan untuk mencegah kerugian yang lebih besar bagi pedagang dan konsumen dan tersebarnya berbagai spesies parasit ke lokasi baru, seperti yang terjadi pada penyebaran Cyrptocaryon iritans pada hampir semua lokasi budidaya di Irlandia Barat (Jackson et al., 2012). Implikasi lain masuknya parasit dari luar akan menjadi karir penyebaran patogen lain seperti bakteri dan virus yang lebih mematikan seperti yang terjadi pada udang vannamei (Litopenaeus vannamei) yang terinfeksi parasit haplosporidian yang menyebabkan kematian tinggi dan sindrom pertumbuhan yang terhambat (Utari et al., 2012). ...
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Infeksi parasit dapat menyebabkan kerugian pada usaha budidaya dan konsumen penggemar ikan hias. Kota Bandar Lampung merupakan salah satu pusat penjualan ikan hias air tawar yang potensial secara ekonomi. Penelitian dilakukan untuk mengidentifikasi spesies parasit Trichodina dari ikan komet (Carassius auratus) yang berasal dari 5 toko ikan hias yang berada di Kota Bandar Lampung. Penelitian ini menggunakan sampel ikan komet sebanyak 250 ekor. Identifikasi spesies dilakukan dengan mempelajari perbedaan karakter morfologi dari setiap Trichodina yang ditemukan pada ikan komet. Hasil penelitian menunjukkan terdapat dua spesies yang teridentifikasi sebagai Trichodina nobilis dan Trichodina reticulata. Keragaman karakter morfologi yang membedakan kedua spesies Trichodina adalah variasi bentuk, jumlah dan ukuran beberapa parameter tubuh. Teridentifikasinya dua spesies Trichodina yang berbeda menunjukkan potensi penyebaran spesies parasit yang tinggi bagi ikan hias air tawar yang diperjualbelikan di Kota Bandar Lampung karena sebagian besar kebutuhan ikan hias air tawarnya berasal dari Bogor atau Jakarta.
... Haplosporidia include the causative agents of the commercially significant oyster diseases MSX (caused by Haplosporidium nelsoni (Burreson and Ford, 2004)), bonamiosis (caused by various Bonamia spp. (Pichot et al., 1980; Carnegie et al., 2006)) and agents of several decapod crustacean diseases (Stentiford et al., 2004; Utari et al., 2012). The reticulate amoebae, Filoreta and Gromia, are the closest known free-living relatives; phytomyxids, vampyrellids and other Cercozoa are more distantly related (Smith and Chao, 2003; Bass et al., 2009 ). ...
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Haplosporidians are rhizarian parasites of mostly marine invertebrates. They include the causative agents of diseases of commercially important molluscs, including MSX disease in oysters. Despite their importance for food security, their diversity and distributions are poorly known. We used a combination of group-specific PCR primers to probe environmental DNA samples from planktonic and benthic environments in Europe, South Africa and Panama. This revealed several highly distinct novel clades, novel lineages within known clades and seasonal (spring vs autumn) and habitat-related (brackish vs littoral) variation in assemblage composition. High frequencies of haplosporidian lineages in the water column provide the first evidence for life cycles involving planktonic hosts, host-free stages or both. The general absence of haplosporidian lineages from all large online sequence data sets emphasises the importance of lineage-specific approaches for studying these highly divergent and diverse lineages. Combined with host-based field surveys, environmental sampling for pathogens will enhance future detection of known and novel pathogens and the assessment of disease risk.
Chapter
Haplosporidiosis occurs in cultured and wild shrimp, as for examples, Penaeus vannamei from Cuba and Nicaragua, P. stylirostris from Mexico, and P. monodon from Indonesia and the Philippines. Severe outbreaks of hepatopancreatic haplosporidiosis (HPH) of cultured P. vannamei occurred in Indonesia during 2007–09. In the Indonesian outbreak of HPH, affected P. vannamei showed shrinkage of the hepatopancreas, flaccid shrimp bodies, cuticular melanization, and retarded growth. The cumulative mortality in farms ranged by 60%–90%. An outbreak of HPH in Belize, Central America, P. vannamei showed inappetence, reduced growth rate, flaccid bodies with red tails, melanization, and atrophy of the hepatopancreatic tubules. Surveillance of broodstocks and postlarvae using histology and polymerase chain reaction are tools useful to prevent the recurrence of HPH.
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This paper thoroughly approaches fish parasites its prevalence and economical loss mainly in aquaculture leading hubs of the world, aiming to point out the adverse effects of fish parasites and their economic loss. Fishes are one of the most significant group of vertebrates that assist humans in a variety of ways. So, they are more widespread and common in practically every part of the globe nearly all fish freshwater and marine are edible and have been a significant source of protein with the ever-increasing demand for inexpensive protein sources, more and more attention has been given to fish. But icthyo fauna is at great risk to parasitic infestation and truly is a serious matter of concern. As these parasites are harmful for both aquatic fauna as well as human being so for prevention from such noxious organisms, this matter must be brought on the table of scientific discussion to ensure the hygienic production of fish. Our reservoirs, rivers, and lakes are precious root for mass production of fishes. In spite of the fact that parasites are causing serious damage to fish fauna, resulted in huge economic loss as well as disturb the natural taste of fish meat, attention to this matter and to find their control measures is still at the beginnings. To prevent mortality of fish fauna, surge in economic loss and unwanted taste of fish meat, serious attention is needed.
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Shrimp farming is a main aquaculture industry in Indonesia. Over the past two years, Indonesia shrimp production increase gradually and reach a peak in 2017. One of the challenges facing the shrimp industry is disease issue and healthy broodstock. Since 2009, the emerging disease of Early Mortality Syndrome in Asia has become a threat to shrimp Industry in Indonesia. The disease has spread in China, Vietnam, Malaysia, Thailand, Mexico and lastly in Philippines between 2009-2014. A study found that the toxin produced by Vibrio parahaemolyticus damages the hepatopancreas of the shrimp causing mass mortality. Understanding the aetiology of the disease, method to observing the disease and measures to manage the disease might decrease the risk of disease outbreak in some area. To date, Ministry of Marine and Fishery Affairs claims that Indonesian shrimp farming is free from AHPND/EMS. This paper will discuss the strategies and opportunities to prevent the spread of AHPND/EMS in Indonesian shrimp farming. Abstrak Tambak udang merupakan salah satu industri perikanan terbesar di Indonesia. Selama dua tahun terakhir, produksi udang Indonesia meningkat secara bertahap dan mencapai puncaknya pada tahun 2017. Salah satu tantangan yang dihadapi industri budidaya udang adalah masalah penyakit dan ketersediaan induk udang sehat. Sejak tahun 2009, kemunculan penyakit Early Mortality Syndrome (EMS) di Asia telah menjadi ancaman bagi industri udang di Indonesia. Penyakit ini telah menyebar di Cina, Vietnam, Malaysia, Thailand, Meksiko and terakhir di Filipina antara tahun 2009-2014. Sebuah penelitian menemukan bahwa racun yang dihasil oleh Vibrio parahaemolyticus merusak hepatoprankreas udang dan menyebabkan kematian massal. Pemahaman etiologi penyakit, metode pemeriksaan, dan langkah-langkah Pengendalian penyakit akan mampu mengurangi risiko wabah di beberapa daerah. Sampai saat ini, Kementrian Kelautan dan Perikanan mengumumkan bahwa tambak udang Indonesia bebas dari AHPND/EMS. Makalah ini akan membahas langkah strategi untuk mencegah penyebaran AHPND/EMS pada tambak udang di Indonesia yaitu penerapan sistem karantina di pintu pemasukan dan pengeluaran, program pengawasan penyakit AHPND, penggunaan teknologi dan biosecurity.
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The host:parasite interactions of the 3 serious haplosporidian pathogens of oysters, on which most information exists, are reviewed. They are Bonamia ostreae in Ostrea spp. and Crassostrea gigas; Bonamia exitiosa in Ostrea spp.; and Haplosporidium nelsoni in Crassostrea spp. Understanding the haemocytic response to pathogens is constrained by lack of information on haematopoiesis, haemocyte identity and development. Basal haplospridians in spot prawns are probably facultative parasites. H. nelsoni and a species infecting Haliotis iris in New Zealand (NZAP), which have large extracellular plasmodia that eject haplosporosomes or their contents, lyse surrounding cells and are essentially extracellular parasites. Bonamia spp. have small plasmodia that are phagocytosed, haplosporosomes are not ejected and they are intracellular obligate parasites. Phagocytosis by haemocytes is followed by formation of a parasitophorous vacuole, blocking of haemocyte lysosomal enzymes and the endolysosomal pathway. Reactive oxygen species (ROS) are blocked by antioxidants, and host cell apoptosis may occur. Unlike susceptible O. edulis, the destruction of B. ostreae by C. gigas may be due to higher haemolymph proteins, higher rates of granulocyte binding and phagocytosis, production of ROS, the presence of plasma β-glucosidase, antimicrobial peptides and higher levels of haemolymph and haemocyte enzymes. In B.exitiosa infection of Ostrea chilensis, cytoplasmic lipid bodies (LBs) containing lysosomal enzymes accumulate in host granulocytes and in B. exitiosa following phagocytosis. Their genesis and role in innate immunity and inflammation appears to be the same as in vertebrate granulocytes and macrophages, and other invertebrates. If so, they are probably the site of eicosanoid synthesis from arachidonic acid, and elevated numbers of LBs are probably indicative of haemocyte activation. It is probable that the molecular interaction, and role of LBs in the synthesis and storage of eicosanoids from arachidonic acid, is conserved in innate immunity in vertebrates and invertebrates. However, it seems likely that haplosporidians are more diverse than realized, and that there are many variations in host parasite interactions and life cycles.
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Giant freshwater prawn, Macrobrachium rosenbergii is an important freshwater aquaculture species worldwide, and China contributes the most to its global production. However, in recent years in China, many prawns have shown serious growth retardation, which is referred to as “iron prawn.” To explore the mechanism behind this phenomenon, we compared the difference between these “iron prawns” and normal prawns in three aspects—changes in genetic diversity, DNA methylation, and transcriptomes—as well as comparing differences in their molt performance. The results are as follows: first, compared with normal prawns, “iron prawns” showed no significant decrease in genetic diversity, but they did show obvious genetic differentiation, and different DNA methylation levels were observed. The genetic and epigenetic variations that existed between “iron prawn” and normal prawn indicated the influence of germplasm on growth performance. Second, transcriptome analysis revealed 1813 differentially expressed genes (DEGs) between the “iron prawn” and normal prawn, and the DEGs mainly enriched the glucose metabolism- and immune-related pathways, such as in glycolysis/gluconeogenesis metabolism, insulin secretion, glucagon signaling pathway, antigen processing and presentation, as well as in complement and coagulation cascades. Enrichment analysis indicated the importance of the glucose level and pathogen attacks to growth performance in the “iron prawn.” Finally, a comparison of the molt performance showed that the length of the molt cycle in the “iron prawn” was comparable to normal prawns with the same size, but the specific growth was much lower in the “iron prawn.” This result suggested that lower body weight gain per molt cycle should be responsible for growth retardation in the “iron prawn,” but not in the longer molt cycle. The results in this study provided fundamental information about the mechanism behind growth retardation in M. rosenbergii.
Article
Shrimp aquaculture in India has an extensive history of successes and challenges reflecting both the potential and the problems of developing this industry. The industry initially grew rapidly during the 1990s, largely through the efforts of individual farmers, but operated in an environment where there was often a lack of adequate regulatory guidance. During this time the total area occupied by farms engaging in shrimp aquaculture increased dramatically, but over 85% of farms were of small size (< 2 Ha). On these farms the average shrimp production was ~ 0.73 tons per hectare, representing yields that were low compared to other shrimp producing countries. To increase productivity, and without adequate regulatory oversight, farmers imported seed stock and feed materials from countries like Taiwan and the Philippines (among others) which claimed to have better growth rates and productivity. At this time the species most often used in culturing was Penaeus monodon. In 1994, a disease affecting this shrimp known as WSSV became widespread and negatively impacted the entire industry in India, resulting severe losses to several research and regulatory control become a major national issue. Partly in response to the negative assessments that were available at the time, in 1996 the Supreme Court of India passed a ban on coastal aquaculture. Soon after that, an emergency session of the Indian Parliament passed a bill to form an Aquaculture Authority. Though, due to continuous failures, at the same time farmers started looking for alternate candidate species for culture. In 2003, a major shift in this industry in India took place with the introduction of Litopenaeus vannamei as a candidate species for aquaculture. By the end of the year 2017, the area under culture had increased 50%, production had increased by almost 83% and India became the second highest shrimp producer in the world. Nevertheless, questions about these production statistics and the extent to which unregulated and/or unregistered farming was being practiced remained. Partly in response to this, in 2005 the Coastal Aquaculture Authority Act was enacted by the Parliament to regulate activities connected with aquaculture in coastal areas. Given the scope and potential for future expansion of this industry, the need for continuing regulation for safeguarding and improving shrimp aquaculture in India as well as to maximize export potential, is apparent.
Chapter
Haplosporidian species (phylum Haplosporidia Caullery & Mesnil, 1899) are a small group of four genera of sometimes pathogenic protozoan parasites usually with uninucleated spores. They are widely distributed in marine and freshwater invertebrates, although their status in Africa, much of Eurasia and Central and South America, is largely unknown. They infect molluscs including commercially important bivalves and other molluscs, annelids, crustaceans, ascidians, trematodes, turbellarians, and probably many invertebrate groups. The morphology, development, and ultrastructure of the four haplosporidian genera (Haplosporidium, Minchinia, Urosporidium, and Bonamia) are described using light, scanning, and transmission electron microscopy. Several new haplosporidian species have recently been described based on their genetic sequences, spore morphology, and ornamentation. Ultrastructural organization of the spores and the origin of the spore wall ornamentation are discussed. This phylum contains 52 described species and several unnamed species reported in the four genera. Life cycle stages involve exosporulation of the endosporoplasm to form multinucleate plasmodia and sporoblasts giving rise to the spores that are described from some species. The phylogenetic analysis based on SSU rRNA strongly supports that the genera Minchinia, Urosporidium, and Bonamia are monophyletic, while the genus Haplosporidium is paraphyletic. The taxonomic positions and affinities between these genera within phylum Haplosporidia are discussed. The negative economic impact on the commercially important infected hosts is reported.
Chapter
Haplosporidian species (phylum Haplosporidia Caullery & Mesnil, 1899) are a small group of four genera of sometimes pathogenic protozoan parasites usually with uninucleated spores. They are widely distributed in marine and freshwater invertebrates, although their status in Africa, much of Eurasia and Central and South America, is largely unknown. They infect molluscs including commercially important bivalves and other molluscs, annelids, crustaceans, ascidians, trematodes, turbellarians, and probably many invertebrate groups. The morphology, development, and ultrastructure of the four haplosporidian genera (Haplosporidium, Minchinia, Urosporidium, and Bonamia) are described using light, scanning, and transmission electron microscopy. Several new haplosporidian species have recently been described based on their genetic sequences, spore morphology, and ornamentation. Ultrastructural organization of the spores and the origin of the spore wall ornamentation are discussed. This phylum contains 52 described species and several unnamed species reported in the four genera. Life cycle stages involve exosporulation of the endosporoplasm to form multinucleate plasmodia and sporoblasts giving rise to the spores that are described from some species. The phylogenetic analysis based on SSU rRNA strongly supports that the genera Minchinia, Urosporidium, and Bonamia are monophyletic, while the genus Haplosporidium is paraphyletic. The taxonomic positions and affinities between these genera within phylum Haplosporidia are discussed. The negative economic impact on the commercially important infected hosts is reported.
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SUMMARY Parasites have a major impact on global finfish and shellfish aquaculture, having significant effects on farm production, sustainability and economic viability. Parasite infections and impacts can, according to pathogen and context, be considered to be either unpredictable/sporadic or predictable/regular. Although both types of infection may result in the loss of stock and incur costs associated with the control and management of infection, predictable infections can also lead to costs associated with prophylaxis and related activities. The estimation of the economic cost of a parasite event is frequently complicated by the complex interplay of numerous factors associated with a specific incident, which may range from direct production losses to downstream socio-economic impacts on livelihoods and satellite industries associated with the primary producer. In this study, we examine the world's major marine and brackish water aquaculture production industries and provide estimates of the potential economic costs attributable to a range of key parasite pathogens using 498 specific events for the purposes of illustration and estimation of costs. This study provides a baseline resource for risk assessment and the development of more robust biosecurity practices, which can in turn help mitigate against and/or minimise the potential impacts of parasite-mediated disease in aquaculture.
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Previously, we described the pathology and ultrastructure of an apparently asporous haplosporidian-like parasite infecting the common shore crab Carcinus maenas from the European shoreline. In the current study, extraction of genomic DNA from the haemolymph, gill or hepatopancreas of infected C. maenas was carried out and the small subunit ribosomal DNA (SSU rDNA) of the pathogen was amplified by PCR before cloning and sequencing. All 4 crabs yielded an identical 1736 bp parasite sequence. BLAST analysis against the NCBI GenBank database identified the sequence as most similar to the protistan pathogen group comprising the order Haplosporida within the class Ascetosporea of the phylum Cercozoa Cavalier-Smith, 1998. Parsimony analysis placed the crab pathogen within the genus Haplosporidium, sister to the molluscan parasites H. montforti, H. pickfordi and H. lusitanicum. The parasite infecting C. maenas is hereby named as Haplosporidium littoralis sp. nov. The presence of a haplosporidian parasite infecting decapod crustaceans from the European shoreline with close phylogenetic affinity to previously described haplosporidians infecting molluscs is intriguing. The study provides important phylogenetic data for this relatively understudied, but commercially significant, pathogen group.
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Shrimp farming has a long and colorful history whose development can be divided into three distinct periods: Start-Up Era, Hatchery Era, and Breeding Era. Key characteristics of these periods are summarized in Table 1 and Figure 1. Start-Up Era During the Start-up Era of the 1980s, nearly all stocking material was wild postlarvae gathered from the sea. In Asia, farms raised primarily black tiger shrimp, Pe-naeus monodon, while in the Western Hemisphere the industry used Pacific white shrimp, P. vannamei. During this era, annual production increased over 100%/year. Growth was driven by the combination of very strong market acceptance and demand for farmed shrimp, and a relative absence of disease that allowed simple pond culture methods to succeed. Hatchery Era During the 1988-1996 Hatchery Era, postlarvae (PL) were produced in land-based hatcheries. While cultured, these PL were genetically wild animals because the parents were collected in the sea. Shrimp farming in the West was dominated by P. vannamei production, while Asian farming was dominated by P. monodon. Asian shrimp production was at least five times greater than Western production throughout this era, so production statistics were dominated by P. monodon. Total world production rose from 604,000 to 693,000 mt, with an average annual gain of only 2%. The main obstacle to growth was widespread shrimp disease that moved through the industry with the hatch-ery-produced PL, because the hatcheries paid little attention to animal biosecurity. Diseases carried by wild broodstock were passed to the PL offspring in the hatch-eries, then introduced to farms when the PL were stocked. The other obstacle to growth was the continued reliance on wild animals. During this period, shrimp farming production reached an effective " carrying capacity " for the use of wild shrimp. While farmers tried to use higher stocking densities to increase yields and profits, their wild animals – which often carried disease – were not well suited to intensive culture systems.
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Mortalities of Penaeus vannamei, cultured in ponds in Belize, Central America, began during the last part of the grow-out cycle during the cold weather months from September 2004 through February 2005. Tissue squashes of infected hepatopancreata and histological examination of infected shrimp revealed that the mortalities might have been caused by an endoparasite. To confirm the diagnosis, DNA was extracted from ethanol preserved hepatopancreata and the small-subunit rRNA gene was sequenced. The 1838 bp sequence was novel and phylogenetic analysis placed the P. vannamei parasite within the phylum Haplosporidia as a sister taxon to a clade that includes Bonamia and Minchinia species. In situ hybridization was performed using anti-sense DNA probes that were designed to hybridize specifically with the parasite's nucleic acid. This organism presents similar characteristics to those of a haplosporidian that infected cultured P. vannamei imported from Nicaragua into Cuba, as described by Dyková et al. (1988; Fish Dis 11:15-22).
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Polymerase chain reaction conditions were established for the in vitro amplification of eukaryotic small subunit ribosomal (16S-like) rRNA genes. Coding regions from algae, fungi, and protozoa were amplified from nanogram quantities of genomic DNA or recombinant plasmids containing rDNA genes. Oligodeoxynucleotides that are complementary to conserved regions at the 5' and 3' termini of eukaryotic 16S-like rRNAs were used to prime DNA synthesis in repetitive cycles of denaturation, reannealing, and DNA synthesis. The fidelity of synthesis for the amplification products was evaluated by comparisons with sequences of previously reported rRNA genes or with primer extension analyses of rRNAs. Fewer than one error per 2000 positions were observed in the amplified rRNA coding region sequences. The primary structure of the 16S-like rRNA from the marine diatom, Skeletonema costatum, was inferred from the sequence of its in vitro amplified coding region.
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As the size and intensity of aquaculture enterprises increase in this millennium, so will the scale for potential loss from disease. Economies dependent on them for export earnings will be vulnerable to disastrous epizootics that must be avoided by implementation of appropriate safeguards. These should be based on a scientific determination of disease transfer risks and be designed to have minimum impact on international trade. The risk assessment process outlined by the WTO SPS agreement exemplifies this approach. We will illustrate the issues on preventing the spread of fish and shellfish diseases based on our experience with two shrimp viruses (IHHNV and WSSV). The greatest risk for disease spread lies with careless movement of living animals for aquaculture. Other risks will also be reviewed. Ultimately, intensive aquaculture will move towards closed recycle systems based on domesticated and genetically improved stocks. The impetus for this will come mostly as a result of pressure from national trade requirements concerning human and veterinary health risks, and from consumer groups concerned about the environment and animal welfare.
Article
Outbreaks of disease due to infectious myonecrosis virus (IMNV) were first reported from Brazil in 2004 in Pacific white shrimp Penaeus (Litopenaeus) vannamei, an exotic species to Brazil that was relatively recently imported for cultivation in earthen ponds. In Indonesia, this non-native species has also been imported and successfully cultivated on a large scale since 2003. Starting in early 2006, there were anecdotal reports from Indonesian shrimp farmers of high mortality in cultivated P. vannamei with gross signs of white muscle, similar to those reported for IMNV outbreaks in Brazil. We obtained a sample of shrimp from one of these outbreak ponds and it gave a positive result for the presence of IMNV using a standard commercial detection kit. After sequencing the PCR fragment to confirm the presence of IMNV, additional primers were designed for cloning and sequencing the full 7.5 kb IMNV genome. Subsequent analysis (GenBank accession no.EF061744) revealed that the Indonesian IMNV sample had 99.6% nucleic acid sequence identity (a total of 29 differences in 7.5 kb) to that of Brazilian IMNV reported at GenBank. It is interesting that one of these differences was a single base insertion at nucleotide 7431 leading to the creation of a delayed termination (stop) codon that led to 13 additional amino acid residues in the deduced RdRp (RNA-dependent RNA polymerase) protein product. Due to some difficulty with very weak false positive results obtained using the commercial detection kit with some samples, we designed an alternative, nested RT-PCR detection method with specific primers to target the viral RdRp region instead of the capsid gene targeted by the commercial kit. In our hands, this protocol gave more consistent results and higher sensitivity that did the kit.
Article
A hitherto unrecorded haplosporean was found in the hepatopancreas of Penaeus vannamei. Multinucleate plasmodia developing within the hepatocytes fragment into unimicleate stages. Eventually the host cells are destroyed and the uninuclear stages are released into the lumen of the secretory tubules. The haplosporosomes are oval, have a thick cortex and contain a small spherical medulla at the broader end. No spores were found and thus the parasite, which is highly pathogenic, could not be assigned any precise taxonomic position.
Article
In Thailand, several PCR-based methods are used by private and public service laboratories for the detection of white spot syndrome virus (WSSV) infection in penaeid shrimp post larvae (PL) before they are stocked in rearing ponds. Conflicting test results for similar samples sent to two service laboratories has decreased confidence in PCR testing. Thus, we compared the sensitivity of several PCR methods commonly employed in Thailand using Taqman real-time PCR as the gold standard with a purified WSSV template stock. Using this stock for assays, we found no significant inhibitory effect by WSSV-free host shrimp DNA over the range 0 to 300 ng per reaction or by added DNA from WSSV-infected shrimp. Real-time PCR could detect WSSV with certainty at dilutions of approximately 5 copies per reaction while 1000 copies were needed for a common one-step PCR method and 50 for a common single-tube nested PCR (1N-PCR) method. Of 2 two-tube nested PCR protocols tested, one required 100 and the other 1000 copies. In addition to these sensitivity tests, a triple-blind ring test was carried out employing sets of 10 WSSV-infected DNA extracts sent to 12 commercial and public laboratories in Thailand, without specifying the PCR method to be used. Returned results included no false positives and two false negatives, the latter both from light infection vials. This translated into a test sensitivity of 97.3% and a specificity of 100%. Overall, the results confirmed the validity of PCR-based methods in Thailand for detection of WSSV in shrimp DNA extracts.
Article
The sensitivity of the commonly used progressive multiple sequence alignment method has been greatly improved for the alignment of divergent protein sequences. Firstly, individual weights are assigned to each sequence in a partial alignment in order to downweight near-duplicate sequences and up-weight the most divergent ones. Secondly, amino acid substitution matrices are varied at different alignment stages according to the divergence of the sequences to be aligned. Thirdly, residue-specific gap penalties and locally reduced gap penalties in hydrophilic regions encourage new gaps in potential loop regions rather than regular secondary structure. Fourthly, positions in early alignments where gaps have been opened receive locally reduced gap penalties to encourage the opening up of new gaps at these positions. These modifications are incorporated into a new program, CLUSTAL W which is freely available.
Molecular Cloning: A Laboratory Manual. Cold Spring Harbor
  • J Sambrook
  • D W Russell
Sambrook, J., Russell, D.W., 2001. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, New York.
Aquaculture development. Ecosystem approach to aquaculture. Food and Agricultural Organization of The United State of America
FAO, 2010. Aquaculture development. Ecosystem approach to aquaculture. Food and Agricultural Organization of The United State of America, Rome.