- Email: [email protected]
Histological analysis of an outbreak of red gill disease in cultured oriental river prawn Macrobrachium nipponense
Aquaculture 507 (2019) 370–376
Contents lists available at ScienceDirect
Aquaculture journal homepage: www.elsevier.com/locate/aquaculture
Short communication
Histological analysis of an outbreak of red gill disease in cultured oriental river prawn Macrobrachium nipponense
T
Zhengfeng Dinga, , Yuwei Yana, Yan Wua, Yinbin Xub, Qingguo Mengb, Gongcheng Jianga, ⁎
⁎
a
Institute of Aquatic Biology and Jiangsu Key Laboratory for Biofunctional Molecules, College of Life Science and Chemistry, Jiangsu Second Normal University, 77 West Beijing Road, Nanjing, China, 210013 b College of Life Sciences, Nanjing Normal University, 1 Wenyuan Road, Nanjing 210046, China
ARTICLE INFO
ABSTRACT
Keywords: Macrobrachium nipponense Red gill disease Histology Ultrastructure
An epidemic of red gill disease with a high mortality rate recently occurred in the cultured oriental river prawn Macrobrachium nipponense in China. The prawns had flaccid bodies and gills were red in color with numerous black spots. By wet smear microscopy and negative staining, numerous membrane-bound plasmodia or degenerated spores were observed. Histological sections indicated the infection was limited to the gills. The gill filaments were filled with masses of mononuclear parasites, multinucleate and multicellular plasmodia and became enormously hypertrophic. Host encapsulation response and inflammatory foci with phagocytized parasite spores were evident. Transmission electron microscopy (TEM) analysis confirmed the gross plasmodia almost filling the entire space of gill epithelial cell. The intracellular parasite was a multinucleate plasmodium enclosed in a vacuole within the cytoplasm of the host cell. Large numbers of trophonts were visible in each plasmodium. High degree of vesiculation of early sporonts was evident. Scanning electron microscopy (SEM) and TEM observations all revealed characteristic tubular extension from the surface of a late sporont. We recognized the risk of associating the presence of a parasite with a particular pathology in the absence of total evidence that demonstrates an unequivocal link between the parasite, the perceived pathology and a clear disease manifestation that would be demonstrated by e.g. experimental studies. However, given the widespread importance of the host in aquaculture, it is necessary to provide this short report to highlight to other researchers and to the industry the potential for a new and emerging disease. It is hoped that this will lead to additional sightings of the condition and additional research to confirm the link between parasite and the pathology seen. It is our intention to complete life-cycle studies with to satisfy Koch's postulate.
1. Introduction The oriental river prawn Macrobrachium nipponense is widely distributed in Myanmar, Vietnam, Japan, and Korea. It is also a significant aquaculture species in China with an annual cultured production of 265,061 tons worth 20 billion RMB (Fu et al., 2012). The frequency of disease outbreaks for the prawn has previously been very low. However, during the 2015 to 2016 winter stocking seasons, mortalities of cultured M. nipponensis occurred in several grow-out ponds in Nanjing, China. The prawns had flaccid bodies and gills were red in color with numerous black spots (named as “red gill disease” by us). Coupled with the recent report of a new microsporidian infection in M. nipponensis (Ding et al., 2016), it is de-
⁎
monstrated that disease outbreaks limit the successful production of this valuable prawn. Using a histological approach as a frontline tool to assess the health of prawns is useful as it provides information on localization in the host and an assessment of the pathology associated with any infection (Longshaw, 2011). In this study, histological analysis of the prawn M. nipponensis with red gill disease was accomplished with both light and electron microscopy methods. The results may be fundamentally important for assessing the nature and host effects of a potentially novel pathogen infecting the freshwater prawn sampled from Nanjing city, Jiangsu Province of China. This is an area where crustacean fisheries are generating significant economic impacts and hence knowledge of the newly emerging disease is necessary for effective management.
Corresponding authors. E-mail addresses: [email protected] (Z. Ding), [email protected] (G. Jiang).
https://doi.org/10.1016/j.aquaculture.2019.04.050 Received 10 October 2018; Received in revised form 15 April 2019; Accepted 16 April 2019 Available online 17 April 2019 0044-8486/ © 2019 Elsevier B.V. All rights reserved.
Aquaculture 507 (2019) 370–376
Z. Ding, et al.
Fig. 1. External appearance of the cultured oriental river prawn Macrobrachium nipponense suffering mortalities. Major developmental phases of the infection could be characterized, from light to heavy infection. A, light infection with a few black spots; BeC, moderate infection with more spots and light red gill; D, heavy infection with the gill turning almost red and large areas of melanistic spots. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
2. Materials and methods
From November 2015 to December 2016, 130 prawns (a carapace length of 17–38 mm) with clinical symptoms out of approximately 900 prawns (apparent prevalence 14.4%), were sampled in 12 culture ponds (from Changzhou and Liyang city of Jiangsu Province, China). Tissues were then dissected and frozen immediately in liquid nitrogen for total RNA extraction, fixed in 4% paraformaldehyde solution (pH 7.4) for hematoxylin and eosin (H&E) staining analysis, and in 2.5% glutaraldehyde (pH 7.3) for EM (electron microscopy) examinations. Each tissue was prepared in vials and numbered separately.
were embedded in Epon 812. Ultrathin sections were then double stained with uranyl acetate and lead citrate, and examined using a Hitachi H-7650 TEM. For the negative stain, small fragments (1 mm3) of gill tissues were fixed in 4% glutaraldehyde in 0.3 M sodium phosphate buffer (pH 7.4) for 4 h at 4 °C, from which 10 μl of the supernatant was then collected on formvar-coated copper grids for 1 min. The wet residues were immediately covered with sodium phosphotungstate (2%) for 30s. The grid was air-dried before observation with the TEM (Hitachi H-7650). For SEM (scanning electron microscopy), the infected gills were fixed in 2.5% glutaraldehyde and smeared on glass slides. After airdrying, the slides were coated with gold at 10 mA for 3 min and examined using JSM5610LV SEM.
2.2. Histological analysis
3. Results
For histological analysis, representative pieces of tissue from the gills, hepatopancreas, muscle, and intestine were fixed in 4% paraformaldehyde solution for 24 h and then transferred to 70% ethanol. After dehydration in a graded ethanol series, samples were embedded in paraffin. Six 4–5 μm thick sections were obtained from each piece using a manual rotary microtome. Sections were then stained with H&E and observed under the light microscope (Olympus, BX53). Wet smears of infected gills were also observed for signs of haplosporidian infection.
3.1. Gross pathology
2.1. Samples
Prawns were screened for common pathogens including IHHNV (Tang and Lightner, 2006), WSSV, HPV, MBV (Umesha et al., 2006), spiroplasma (Xiu et al., 2015) and microsporidium (Ding et al., 2016). They were negative for all these pathogens. The prawns had flaccid bodies and gills were red in color with numerous black spots. Major developmental phases of the infection could be characterized, from light to heavy infection (Fig. 1). In addition to their reduced viability during holding and transportation, the severe pathology (black spots and reddish gill) was likely to cause considerable reduction in the yield and appearance of the individuals, which also had potential commercial impacts. Unstained wet smears of gill filaments from diseased M. nipponense were examined. In areas where the filament was fractured and
2.3. EM (electron microscopy) examinations For TEM (transmission electron microscopy), the gill samples fixed in 2.5% glutaraldehyde were post-fixed in 1% (w/v) osmium tetroxide for 2 h. After dehydration through a graded series of acetone, samples 371
Aquaculture 507 (2019) 370–376
Z. Ding, et al.
Fig. 2. Unstained wet smears and negative staining of gill filaments from diseased prawns Macrobrachium nipponense. A, by wet smear microscopy, membrane-bound plasmodia were evident in areas where the filament was fractured and disrupted during the preparation of squash. B, large numbers of spherical, uninucleate trophonts were readily apparent in plasmodia that had ruptured. C, by negative staining, numerous degenerated spores were observed. D, characteristic tubular extension emerged haphazardly around the spore surface. Inserts, higher-power images. Scale bar A, B = 20 μm (insert 10 μm), C = 10 μm, D = 2 μm.
disrupted during the preparation of squash, membrane-bound plasmodia were evident (Fig. 2A). Mature plasmodia were characterized as containing large numbers of spherical, uninucleate trophonts, which were readily apparent in plasmodia that had ruptured (Fig. 2B). Negative staining using an electron microscope showed numerous degenerated spores (Fig. 2C). Characteristic tubular extension emerged haphazardly around the spore surface (Fig. 2D).
tissues, including hepatopancreas, muscle, and intestine (data not shown). 3.3. Electron microscopy observations In accordance with the H& E results, gross plasmodia almost filled the entire space of the gill epithelial cell (Fig. 4A). A higher magnification view indicated that the intracellular parasite was a multinucleate plasmodium enclosed in a vacuole within the cytoplasm of the host cell. The vacuolar membranes were closely applied to the plasmalemma of the parasite. Large numbers of trophonts were visible in each plasmodium. There were amounts of vesicles, spherical and elongated, containing more or less electron opaque substance (Fig. 4B). A characteristic tubular extension from the surface of a late sporont was observed (Fig. 4C). Clear margins between the membrane of uninucleate parasite and the plasma membrane; and profiles of smooth endoplasmic reticulum were evident (Fig. 4D). Mitochondria with well-developed tubular cristae arrayed tightly around the nuclei, which had a central mass of chromatin with a prominent nucleolus (Fig. 4E). High degree of vesiculation in the cytoplasm of early sporonts was evidently distributed (Fig. 4F). Spore ornamentation observed by SEM (Fig. 5) consisted of spore wall-derived, thin and flat ribbons that emerged haphazardly around the spore. Ribbons were often twisted along their length. The number of
3.2. Histopathology The gill filaments of healthy prawns were orderly arranged and no pathological alterations could be observed (Fig. 3A). But in diseased prawns, the gill filaments were filled with masses of mononuclear parasites, multinucleate and multicellular plasmodial forms and became enormously hypertrophic (Fig. 3B), The intracellular parasite was usually rounded (about 4.3 μm in diameter) and enclosed in a vacuole within the host cell. In some specimens, the affected area approached 80 to 90% of the section, and nearly all epithelial cells were infected (Fig. 3C). The resulting destruction of gill epithelium was usually associated with a granulomatous inflammatory host reaction (Fig. 3D). The overall damage to the gill was heavy enough to preclude any possibility of restoration of the normal function. Although masses of discharged bodies were widely distributed in the gill epithelium, no spores or plasmodia were observed in other 372
Aquaculture 507 (2019) 370–376
Z. Ding, et al.
Fig. 3. H&E staining of gill tissues from diseased prawn Macrobrachium nipponense. A, in healthy prawns, the gill filaments were orderly arranged and no pathological changes could be observed; B, in diseased prawns, the gill filaments became enormously hypertrophic and filled with parasitic pathogens (arrows); C, in heavily infected prawns, nearly all epithelial cells were infected. The intracellular parasite was usually rounded (4.565 ± 0.688 μm in diameter) and enclosed in a vacuole within the host cell; D, granulomatous inflammatory of host reactions were evidently distributed (arrows). Inserts of A, B & D, higher-power images; C, size statistics in diameter. Scale bar A = 100 μm, B = 100 μm (insert 50 μm), C, D = 20 μm.
internationally notifiable diseases. In contrast, knowledge of haplosporidian infections in crustaceans is limited, with even fewer reports from freshwater invertebrates. The haplosporidian targeted infection towards the digestive gland and hepatopancreas of shrimp Penaeus vannamei, where all epithelial cell types appeared to be susceptible (Nunan et al., 2007). In the shore crab Carcinus maenas, systemic infection was established through the haemal sinuses and connective tissues (Stentiford et al., 2004). In prawns Pandalus platyceros, the haplosporidian-like parasites were observed in the haemal sinuses of all tissues (Bower and Meyer, 2002). In heavily infected edible crabs Cancer pagurus, a haplosporidian-like parasite spread from the antennal gland to the gills, suggesting that the parasite was released into the hemolymph (Bateman et al., 2011; Thrupp et al., 2013). In this study, the haplosporidian-like parasite was limited to the gill of M. nipponensis, while the other tissues including hepatopancreas, intestine, and muscle showed no sign of infection. All these results indicated the diverse characteristics of tissue tropisms of these economically important pathogens. Additionally, high intensity of the haplosporidian-like parasite was likely to have led to hypoxia in infected prawns and was probably the primary cause of mortalities in M. nipponensis. Few of the haplosporidia have been shown to directly transmit between hosts. For instance, attempts to demonstrate direct transmission of H. nelsoni between oysters have consistently failed (Malek and Byers, 2017). Speculation has persisted that other intermediate hosts may be involved or other developmental stages may occur in the life cycle. In the crab Cancer pagurusthe, production and liberation of uninucleate and plasmodial stages of haplosporidian into the urine provides the most likely transmission route for the haplosporidian-like parasite and explains its high prevalence (Bateman et al., 2011). In the current study, the haplosporidian-like organism was limited to the gill of M. nipponense and the intestines were free of infection, hence the
ribbons on each spore varied considerably from about 1 to 10 ribbons per spore. All this light and electron microscopy evidence suggested a possible parasite infection in the diseased prawns. 4. Discussion The frequency of disease outbreaks for M. nipponense has been thought to be very low in the past. However, an increasing amount of data has been accumulated on pathogens affecting the cultured prawn. M. nipponense are susceptible to the viruses Macrobrachium nipponense reovirus (MnRV) (Bateman and Stentiford, 2017), and WSSV (Yun et al., 2014; Zhao et al., 2017) and have been recorded as hosts for Spiroplasma eriocheiris (Srisala et al., 2018; Xiu et al., 2015), Aeromonas hydrophlia (Ding et al., 2015), the ciliates Gymnodinioides (=Hyalophysa) caridinae on the gills and exoskeleton (Morado and Small, 1995), Lagenophrys branchium and Lagenophrys eupagurus (Clamp, 1989; Felgenhauer, 1982; Mayén-Estrada and Clamp, 2016), the microsporidian Potaspora macrobrachium (Ding et al., 2016), and the digeneans Pseudophyllodistomum lesteri (Goodchild, 1943; Long and Wai, 1958; Cribb, 1987), P. macrobranchicola (Cribb, 1987), Paragonimus westermani (Toledo and Fried, 2014), Pseudolevinseniella cheni (Kudlai et al., 2015) and Microphallus minus (Kudlai et al., 2015). It should be noted that Sokolova and Overstreet (2018) suggest that the position of Potaspora macrobrachium requires reevaluation and may need to be moved to the genus Apotaspora. The current study presents for the first time the detailed description of a potentially new parasite in M. nipponense with red gill disease. Most of the studies on the haplosporidian group are focused on infections of molluscs because species such as Bonamia spp. and Haplosporidium nelsoni are causing epizootics and listed by the OIE as 373
Aquaculture 507 (2019) 370–376
Z. Ding, et al.
Fig. 4. Ultrastructure of the gill tissues from diseased prawn Macrobrachium nipponense. A, in accordance with the H&E results (Fig. 3B), gross plasmodia almost filled the entire space of the gill epithelial cell; B, the intracellular parasite was a multinucleate plasmodium enclosed in a vacuole within cytoplasm of the host cell. Numbers of trophonts were visible in each plasmodium. There was a variety of vesicles, spherical and elongated, containing more or less electron opaque substance; C, characteristic tubular extension from the surface of a late sporont was revealed; D, clear margins between membrane of uninucleate parasite and the plasma membrane and profiles of smooth endoplasmic reticulum (arrows); E, mitochondria (arrows) arrayed tightly around the nuclei, which had a central mass of chromatin with a prominent nucleolus; F, high degree of vesiculation in cytoplasm of early sporonts (★). N, host nuclei; Insert of A, higher-power image. Scale bar A = 20 μm, B = 5 μm, C, E = 1 μm, D, F = 2 μm.
transmission was probably not by the oral route. Even though the route of entry of the parasite into target cells is still unknown, an enzootic presence or introduction via transfer of infected prawn stocks are 2 plausible explanations for the emergence of the disease (Nunan et al., 2007). The disease showed a seasonal epizootiology, with peak infections occurring during the winter (November and December) and spring
(March and April), and with a latent infection or absence during the summer and early autumn. This was of significance because usually the diseases and parasites caused greater losses at higher temperatures in aquaculture seasons. This epizootiology was mostly similar with the prevalence of Hematodinium parasite in some crab species, which also suggested highly seasonal disease outbreaks, with peak infections occurring over a relatively narrow time period, followed by a longer 374
Aquaculture 507 (2019) 370–376
Z. Ding, et al.
Fig. 5. Spore ornamentation observed by scanning electron microscopy (SEM). A, spore wall-derived, thin, flat ribbons that emerged haphazardly around the spore; B, ribbons were often twisted along their length.
period of undetectable or low-level prevalence (Stentiford et al., 2002). Additionally, in the M. nipponensis rearing ponds, the virulence of the parasite could increase due to environmental stresses such as pollution, a scarcity of food and increase in population density. Therefore, adequate measures should also be taken in the susceptible months to avoid massive mortality outbreaks. The haplosporidian from blue crabs, Callinectes sapidus was about 3.4–7.3 μm in diameter (Burreson and Ford, 2004). In the spot prawn P. platyceros, the haplosporidian was 12.3 μm in diameter for round cells and 14.4 μm in length for ovoid cells (Bower and Meyer, 2002). The haplosporidian reported from shrimp P. vannamei was 2 μm in diameter (Nunan et al., 2007). While in the current study, the haplosporidian-like parasite infecting M. nipponense was around 4 μm in diameter (Fig. 3C). In addition, unique spore ornamentation was observed by TEM (Fig. 2D, 4C) and SEM (Fig. 5). The ribbons projecting from the spore wall and terminating in a definite capped structure were most similar to that of Bonamia perspora and H. edule (Carnegie et al., 2010; Rf and Montes, 2003). These features might provide classification criterions for this potentially new haplosporidian pathogen. Some of the infected gills showed granuloma-like foci that may have been similar to granulomatous lesions in shrimp P. vannamei infected by a similar haplosporidian (Nunan et al., 2007). Such foci have been described as aggregations of hemocytes encapsulating pathogens or foreign particles and which lead to the deposition of melanin either on the object or within the hemocyte matrix (Stentiford et al., 2002). In the case of parasite infection, the parasite is destroyed as the inner layers of these foci became necrotic. Such encapsulating lesions have also been thought to indicate a previous microsporidian infection in this prawn M. nipponense (Ding et al., 2016). It needs to be recognized that experimental studies will not always provide equivocal evidence of cause / effect (lack of SPF hosts, lack of stressors that exacerbate disease, failed challenge because parasite needs additional hosts or to be at a particular stage to be infective, etc.). While it is critical that challenge infections are run to name an agent, these are not always quick to conduct and there may be challenges to this (availability of SPF hosts, seasonal components to the infection, proximity of the lab to the study site etc.).
sightings of the condition and additional research to confirm the link between parasite and the pathology seen. It is our intention to complete life-cycle studies with to satisfy Koch's postulate. Acknowledgments We have received valuable advice from Jeffery D. Shields (Virginia Institute of Marine Science, College of William & Mary, USA). We thank Dr. Roland Pevsner from the University of Exeter, UK for reviewing the manuscript and the anonymous reviewers for valuable comments. This work was supported by grants from Natural Sciences Foundation of Jiangsu Province (BK20171406), Natural Science Fund of Colleges and Universities in Jiangsu Province (16KJA180005, 16KJB180006), innovation of agricultural science (CX (17) 2008), project for aquaculture of Jiangsu Province (Y2016-29, Y2018-22) and China Scholarship Council (2018). Conflict of interest The authors declare that they have no conflict of interests. References Bateman, K.S., Stentiford, G.D., 2017. A taxonomic review of viruses infecting crustaceans with an emphasis on wild hosts. J. Invertebr. Pathol. 147, 86–110. Bateman, K.S., Hicks, R.J., Stentiford, G.D., 2011. Disease profiles differ between nonfished and fished populations of edible crab (Cancer pagurus) from a major commercial fishery. ICES J. Mar. Sci. 68, 2044–2052. Bower, S.M., Meyer, G.R., 2002. Morphology and ultrastructure of a protistan pathogen in the haemolymph of shrimp (Pandalus spp.) in the northeastern Pacific Ocean. Can. J. Zool. 80, 1055–1068. Burreson, Eugene M., Ford, Susan E., 2004. A review of recent information on the Haplosporidia, with special reference to Haplosporidium nelsoni (MSX disease). Aquat. Living Resour. 17, 499–517. Carnegie, R.B., Burreson, E.M., Hine, P.M., Stokes, N.A., Audemard, C., Bishop, M.J., Peterson, C.H., 2010. Bonamia perspora n. sp. (Haplosporidia), a parasite of the oyster Ostreola equestris, is the first Bonamia species known to produce spores. J. Eukaryot. Microbiol. 53, 232–245. Clamp, J.C., 1989. Redescription of Lagenophrys eupagurus Kellicott (Ciliophora, Peritricha, Lagenophryidae) and a comparison of it with three similar species. J. Protozool. 36, 596–607. Cribb, T.H., 1987. Studies on gorgoderid digeneans from Australian and Asian freshwater fishes. J. Nat. Hist. 21, 1129–1153. Ding, Z., Zhang, Y., Ye, J., Du, Z., Kong, Y., 2015. An evaluation of replacing fish meal with fermented soybean meal in the diet of Macrobrachium nipponense: growth, nonspecific immunity, and resistance to Aeromonas hydrophila. Fish Shellfish Immun. 44, 295–301. Ding, Z., Sun, M., Liu, H., Zhao, Y., Pan, J., Hui, X., 2016. A new microsporidium, Potaspora macrobrachium n.sp. infecting the musculature of pond-reared oriental river prawn Macrobrachium nipponense (Decapoda: Palaemonidae). J. Invertebr. Pathol. 136, 57–64. Felgenhauer, B.E., 1982. A new species of Lagenophrys (Ciliophora: Peritrichida) from the fresh-water shrimp Palaemonetes kadiakensis. Trans. Am. Microsc. Soc. 101, 142–150. Fu, H., Jiang, S., Xiong, Y., 2012. Current status and prospects of farming the giant river prawn (Macrobrachium rosenbergii) and the oriental river prawn (Macrobrachium
5. Conclusion We recognize the risk of associating the presence of a parasite with a particular pathology in the absence of total evidence that demonstrates an unequivocal link between the parasite, the perceived pathology and a clear disease manifestation that would be demonstrated by e.g. experimental studies. However, given the widespread importance of the host in aquaculture, it is necessary to provide this short report to highlight to other researchers and to the industry the potential for a new and emerging disease. It is hoped that this will lead to additional 375
Aquaculture 507 (2019) 370–376
Z. Ding, et al. nipponense) in China. Aquac. Res. 43, 993–998. Goodchild, G.C., 1943. The life-history of Phyllodistomum solidum Rankin, 1937, with observations on the morphology, development and taxonomy of the Gorgoderinae (Trematoda). Biol. Bull. 84, 59–86. Kudlai, O., Cutmore, S.C., Cribb, T.H., 2015. Morphological and molecular data for three species of the Microphallidae (Trematoda: Digenea) in Australia, including the first descriptions of the cercariae of Maritrema brevisacciferum Shimazu et Pearson, 1991 and Microphallus minutus Johnston, 1948. Folia. Parasit. 62, 053. Long, S., Wai, M.T., 1958. Parasitic worms from Tai Hu fishes: digenetic trematodes.I. The genus Phyllodistomum Braun, 1899 (Gorgoderidae), with descriptions of four new species. Acta Zool. Sin. 10, 348–368. Longshaw, M., 2011. Diseases of crayfish: a review. J. Invertebr. Pathol. 106, 54–70. Malek, J.C., Byers, J.E., 2017. The effects of tidal elevation on parasite heterogeneity and co-infection in the eastern oyster, Crassostrea virginica. J. Exp. Mar. Biol. Ecol. 494, 32–37. Mayén-Estrada, R., Clamp, J.C., 2016. An annotated checklist of species in the family Lagenophryidae (Ciliophora, Oligohymenophorea, Peritrichia), with a brief review of their taxonomy, morphology, and biogeography. Zootaxa 4132, 451–492. Morado, J.F., Small, E.B., 1995. Ciliate parasites and related diseases of Crustacea: a review. Rev. Fish. Sci. 3, 275–354. Nunan, L.M., Lightner, D.V., Pantoja, C.R., Stokes, N.A., Reece, K.S., 2007. Characterization of a rediscovered haplosporidian parasite from cultured Penaeus vannamei. Dis. Aquat. Org. 74, 67–75. Rf, A.C.C., Montes, J., 2003. Description of Haplosporidium edule n. sp (Phylum Haplosporidia), a parasite of Cerastoderma edule (Mollusca, Bivalvia) with complex spore ornamentation. Eur. J. Protistol. 39, 161–167. Sokolova, Y.Y., Overstreet, R M.A., 2018. A new microsporidium, Apotaspora heleios ng, n. sp., from the Riverine grass shrimp Palaemonetes paludosus (Decapoda: Caridea: Palaemonidae)[J]. J. invertebr. pathol. 157, 125–135. Srisala, J., Pukmee, R., McIntosh, R., Choosuk, S., Itsathitphaisarn, O., Flegel, T.W.,
Sritunyalucksana, K., Vanichviriyakit, R., 2018. Distinctive histopathology of Spiroplasma eriocheiris infection in the giant river prawn Macrobrachium rosenbergii. Aquaculture 493, 93–99. Stentiford, G.D., Green, M., Bateman, K., Small, H.J., Neil, D.M., Feist, S.W., 2002. Infection by a hematodinium-like parasitic dinoflagellate causes pink crab disease (PCD) in the edible crab Cancer pagurus. J. Invertebr. Pathol. 79, 179–191. Stentiford, G.D., Feist, S.W., Bateman, K.S., Hine, P.M., 2004. Haemolymph parasite of the shore crab Carcinus maenas: pathology, ultrastructure and observations on crustacean haplosporidians. Dis. Aquat. Org. 59, 57–68. Tang, K.F., Lightner, D.V., 2006. Infectious hypodermal and hematopoietic necrosis virus (IHHNV)-related sequences in the genome of the black tiger prawn Penaeus monodon from Africa and Australia. Virus Res. 118, 185–191. Thrupp, T.J., Lynch, S.A., Wootton, E.C., Malham, S.K., Vogan, C.L., Culloty, S.C., Rowley, A.F., 2013. Infection of juvenile edible crabs, Cancer pagurus by a haplosporidian-like parasite. J. Invertebr. Pathol. 114, 92–99. Toledo, R., Fried, B., 2014. Digenetic Trematodes. Springer, New York. Umesha, K.R., Dass, B.K.M., Naik, B.M., Venugopal, M.N., Karunasagar, I., Karunasagar, I., 2006. High prevalence of dual and triple viral infections in black tiger shrimp ponds in India. Aquaculture 258, 91–96. Xiu, Y., Wu, T., Meng, X., Meng, Q., Wang, W., 2015. Identification and isolation of a spiroplasma pathogen from diseased oriental river prawn, Macrobrachium nipponense, in China: a new freshwater crustacean host. Aquaculture 437, 270–274. Yun, J.M., Kim, B.S., Hwang, S.M., Kim, Y.B., Choi, W.B., Choi, T.J., 2014. Artificial infection of the native Korean freshwater prawn Macrobrachium nipponense (De Haan, 1849) (Decapoda, Palaemonidae) with white spot syndrome virus (WSSV). Crustaceana 87, 866–880. Zhao, C., Fu, H., Sun, S., Qiao, H., Zhang, W., Jin, S., Jiang, S., Xiong, Y., Gong, Y., 2017. Experimental inoculation of oriental river prawn Macrobrachium nipponense with white spot syndrome virus (WSSV). Dis. Aquat. Org. 126, 125–134.
376
Contents lists available at ScienceDirect
Aquaculture journal homepage: www.elsevier.com/locate/aquaculture
Short communication
Histological analysis of an outbreak of red gill disease in cultured oriental river prawn Macrobrachium nipponense
T
Zhengfeng Dinga, , Yuwei Yana, Yan Wua, Yinbin Xub, Qingguo Mengb, Gongcheng Jianga, ⁎
⁎
a
Institute of Aquatic Biology and Jiangsu Key Laboratory for Biofunctional Molecules, College of Life Science and Chemistry, Jiangsu Second Normal University, 77 West Beijing Road, Nanjing, China, 210013 b College of Life Sciences, Nanjing Normal University, 1 Wenyuan Road, Nanjing 210046, China
ARTICLE INFO
ABSTRACT
Keywords: Macrobrachium nipponense Red gill disease Histology Ultrastructure
An epidemic of red gill disease with a high mortality rate recently occurred in the cultured oriental river prawn Macrobrachium nipponense in China. The prawns had flaccid bodies and gills were red in color with numerous black spots. By wet smear microscopy and negative staining, numerous membrane-bound plasmodia or degenerated spores were observed. Histological sections indicated the infection was limited to the gills. The gill filaments were filled with masses of mononuclear parasites, multinucleate and multicellular plasmodia and became enormously hypertrophic. Host encapsulation response and inflammatory foci with phagocytized parasite spores were evident. Transmission electron microscopy (TEM) analysis confirmed the gross plasmodia almost filling the entire space of gill epithelial cell. The intracellular parasite was a multinucleate plasmodium enclosed in a vacuole within the cytoplasm of the host cell. Large numbers of trophonts were visible in each plasmodium. High degree of vesiculation of early sporonts was evident. Scanning electron microscopy (SEM) and TEM observations all revealed characteristic tubular extension from the surface of a late sporont. We recognized the risk of associating the presence of a parasite with a particular pathology in the absence of total evidence that demonstrates an unequivocal link between the parasite, the perceived pathology and a clear disease manifestation that would be demonstrated by e.g. experimental studies. However, given the widespread importance of the host in aquaculture, it is necessary to provide this short report to highlight to other researchers and to the industry the potential for a new and emerging disease. It is hoped that this will lead to additional sightings of the condition and additional research to confirm the link between parasite and the pathology seen. It is our intention to complete life-cycle studies with to satisfy Koch's postulate.
1. Introduction The oriental river prawn Macrobrachium nipponense is widely distributed in Myanmar, Vietnam, Japan, and Korea. It is also a significant aquaculture species in China with an annual cultured production of 265,061 tons worth 20 billion RMB (Fu et al., 2012). The frequency of disease outbreaks for the prawn has previously been very low. However, during the 2015 to 2016 winter stocking seasons, mortalities of cultured M. nipponensis occurred in several grow-out ponds in Nanjing, China. The prawns had flaccid bodies and gills were red in color with numerous black spots (named as “red gill disease” by us). Coupled with the recent report of a new microsporidian infection in M. nipponensis (Ding et al., 2016), it is de-
⁎
monstrated that disease outbreaks limit the successful production of this valuable prawn. Using a histological approach as a frontline tool to assess the health of prawns is useful as it provides information on localization in the host and an assessment of the pathology associated with any infection (Longshaw, 2011). In this study, histological analysis of the prawn M. nipponensis with red gill disease was accomplished with both light and electron microscopy methods. The results may be fundamentally important for assessing the nature and host effects of a potentially novel pathogen infecting the freshwater prawn sampled from Nanjing city, Jiangsu Province of China. This is an area where crustacean fisheries are generating significant economic impacts and hence knowledge of the newly emerging disease is necessary for effective management.
Corresponding authors. E-mail addresses: [email protected] (Z. Ding), [email protected] (G. Jiang).
https://doi.org/10.1016/j.aquaculture.2019.04.050 Received 10 October 2018; Received in revised form 15 April 2019; Accepted 16 April 2019 Available online 17 April 2019 0044-8486/ © 2019 Elsevier B.V. All rights reserved.
Aquaculture 507 (2019) 370–376
Z. Ding, et al.
Fig. 1. External appearance of the cultured oriental river prawn Macrobrachium nipponense suffering mortalities. Major developmental phases of the infection could be characterized, from light to heavy infection. A, light infection with a few black spots; BeC, moderate infection with more spots and light red gill; D, heavy infection with the gill turning almost red and large areas of melanistic spots. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
2. Materials and methods
From November 2015 to December 2016, 130 prawns (a carapace length of 17–38 mm) with clinical symptoms out of approximately 900 prawns (apparent prevalence 14.4%), were sampled in 12 culture ponds (from Changzhou and Liyang city of Jiangsu Province, China). Tissues were then dissected and frozen immediately in liquid nitrogen for total RNA extraction, fixed in 4% paraformaldehyde solution (pH 7.4) for hematoxylin and eosin (H&E) staining analysis, and in 2.5% glutaraldehyde (pH 7.3) for EM (electron microscopy) examinations. Each tissue was prepared in vials and numbered separately.
were embedded in Epon 812. Ultrathin sections were then double stained with uranyl acetate and lead citrate, and examined using a Hitachi H-7650 TEM. For the negative stain, small fragments (1 mm3) of gill tissues were fixed in 4% glutaraldehyde in 0.3 M sodium phosphate buffer (pH 7.4) for 4 h at 4 °C, from which 10 μl of the supernatant was then collected on formvar-coated copper grids for 1 min. The wet residues were immediately covered with sodium phosphotungstate (2%) for 30s. The grid was air-dried before observation with the TEM (Hitachi H-7650). For SEM (scanning electron microscopy), the infected gills were fixed in 2.5% glutaraldehyde and smeared on glass slides. After airdrying, the slides were coated with gold at 10 mA for 3 min and examined using JSM5610LV SEM.
2.2. Histological analysis
3. Results
For histological analysis, representative pieces of tissue from the gills, hepatopancreas, muscle, and intestine were fixed in 4% paraformaldehyde solution for 24 h and then transferred to 70% ethanol. After dehydration in a graded ethanol series, samples were embedded in paraffin. Six 4–5 μm thick sections were obtained from each piece using a manual rotary microtome. Sections were then stained with H&E and observed under the light microscope (Olympus, BX53). Wet smears of infected gills were also observed for signs of haplosporidian infection.
3.1. Gross pathology
2.1. Samples
Prawns were screened for common pathogens including IHHNV (Tang and Lightner, 2006), WSSV, HPV, MBV (Umesha et al., 2006), spiroplasma (Xiu et al., 2015) and microsporidium (Ding et al., 2016). They were negative for all these pathogens. The prawns had flaccid bodies and gills were red in color with numerous black spots. Major developmental phases of the infection could be characterized, from light to heavy infection (Fig. 1). In addition to their reduced viability during holding and transportation, the severe pathology (black spots and reddish gill) was likely to cause considerable reduction in the yield and appearance of the individuals, which also had potential commercial impacts. Unstained wet smears of gill filaments from diseased M. nipponense were examined. In areas where the filament was fractured and
2.3. EM (electron microscopy) examinations For TEM (transmission electron microscopy), the gill samples fixed in 2.5% glutaraldehyde were post-fixed in 1% (w/v) osmium tetroxide for 2 h. After dehydration through a graded series of acetone, samples 371
Aquaculture 507 (2019) 370–376
Z. Ding, et al.
Fig. 2. Unstained wet smears and negative staining of gill filaments from diseased prawns Macrobrachium nipponense. A, by wet smear microscopy, membrane-bound plasmodia were evident in areas where the filament was fractured and disrupted during the preparation of squash. B, large numbers of spherical, uninucleate trophonts were readily apparent in plasmodia that had ruptured. C, by negative staining, numerous degenerated spores were observed. D, characteristic tubular extension emerged haphazardly around the spore surface. Inserts, higher-power images. Scale bar A, B = 20 μm (insert 10 μm), C = 10 μm, D = 2 μm.
disrupted during the preparation of squash, membrane-bound plasmodia were evident (Fig. 2A). Mature plasmodia were characterized as containing large numbers of spherical, uninucleate trophonts, which were readily apparent in plasmodia that had ruptured (Fig. 2B). Negative staining using an electron microscope showed numerous degenerated spores (Fig. 2C). Characteristic tubular extension emerged haphazardly around the spore surface (Fig. 2D).
tissues, including hepatopancreas, muscle, and intestine (data not shown). 3.3. Electron microscopy observations In accordance with the H& E results, gross plasmodia almost filled the entire space of the gill epithelial cell (Fig. 4A). A higher magnification view indicated that the intracellular parasite was a multinucleate plasmodium enclosed in a vacuole within the cytoplasm of the host cell. The vacuolar membranes were closely applied to the plasmalemma of the parasite. Large numbers of trophonts were visible in each plasmodium. There were amounts of vesicles, spherical and elongated, containing more or less electron opaque substance (Fig. 4B). A characteristic tubular extension from the surface of a late sporont was observed (Fig. 4C). Clear margins between the membrane of uninucleate parasite and the plasma membrane; and profiles of smooth endoplasmic reticulum were evident (Fig. 4D). Mitochondria with well-developed tubular cristae arrayed tightly around the nuclei, which had a central mass of chromatin with a prominent nucleolus (Fig. 4E). High degree of vesiculation in the cytoplasm of early sporonts was evidently distributed (Fig. 4F). Spore ornamentation observed by SEM (Fig. 5) consisted of spore wall-derived, thin and flat ribbons that emerged haphazardly around the spore. Ribbons were often twisted along their length. The number of
3.2. Histopathology The gill filaments of healthy prawns were orderly arranged and no pathological alterations could be observed (Fig. 3A). But in diseased prawns, the gill filaments were filled with masses of mononuclear parasites, multinucleate and multicellular plasmodial forms and became enormously hypertrophic (Fig. 3B), The intracellular parasite was usually rounded (about 4.3 μm in diameter) and enclosed in a vacuole within the host cell. In some specimens, the affected area approached 80 to 90% of the section, and nearly all epithelial cells were infected (Fig. 3C). The resulting destruction of gill epithelium was usually associated with a granulomatous inflammatory host reaction (Fig. 3D). The overall damage to the gill was heavy enough to preclude any possibility of restoration of the normal function. Although masses of discharged bodies were widely distributed in the gill epithelium, no spores or plasmodia were observed in other 372
Aquaculture 507 (2019) 370–376
Z. Ding, et al.
Fig. 3. H&E staining of gill tissues from diseased prawn Macrobrachium nipponense. A, in healthy prawns, the gill filaments were orderly arranged and no pathological changes could be observed; B, in diseased prawns, the gill filaments became enormously hypertrophic and filled with parasitic pathogens (arrows); C, in heavily infected prawns, nearly all epithelial cells were infected. The intracellular parasite was usually rounded (4.565 ± 0.688 μm in diameter) and enclosed in a vacuole within the host cell; D, granulomatous inflammatory of host reactions were evidently distributed (arrows). Inserts of A, B & D, higher-power images; C, size statistics in diameter. Scale bar A = 100 μm, B = 100 μm (insert 50 μm), C, D = 20 μm.
internationally notifiable diseases. In contrast, knowledge of haplosporidian infections in crustaceans is limited, with even fewer reports from freshwater invertebrates. The haplosporidian targeted infection towards the digestive gland and hepatopancreas of shrimp Penaeus vannamei, where all epithelial cell types appeared to be susceptible (Nunan et al., 2007). In the shore crab Carcinus maenas, systemic infection was established through the haemal sinuses and connective tissues (Stentiford et al., 2004). In prawns Pandalus platyceros, the haplosporidian-like parasites were observed in the haemal sinuses of all tissues (Bower and Meyer, 2002). In heavily infected edible crabs Cancer pagurus, a haplosporidian-like parasite spread from the antennal gland to the gills, suggesting that the parasite was released into the hemolymph (Bateman et al., 2011; Thrupp et al., 2013). In this study, the haplosporidian-like parasite was limited to the gill of M. nipponensis, while the other tissues including hepatopancreas, intestine, and muscle showed no sign of infection. All these results indicated the diverse characteristics of tissue tropisms of these economically important pathogens. Additionally, high intensity of the haplosporidian-like parasite was likely to have led to hypoxia in infected prawns and was probably the primary cause of mortalities in M. nipponensis. Few of the haplosporidia have been shown to directly transmit between hosts. For instance, attempts to demonstrate direct transmission of H. nelsoni between oysters have consistently failed (Malek and Byers, 2017). Speculation has persisted that other intermediate hosts may be involved or other developmental stages may occur in the life cycle. In the crab Cancer pagurusthe, production and liberation of uninucleate and plasmodial stages of haplosporidian into the urine provides the most likely transmission route for the haplosporidian-like parasite and explains its high prevalence (Bateman et al., 2011). In the current study, the haplosporidian-like organism was limited to the gill of M. nipponense and the intestines were free of infection, hence the
ribbons on each spore varied considerably from about 1 to 10 ribbons per spore. All this light and electron microscopy evidence suggested a possible parasite infection in the diseased prawns. 4. Discussion The frequency of disease outbreaks for M. nipponense has been thought to be very low in the past. However, an increasing amount of data has been accumulated on pathogens affecting the cultured prawn. M. nipponense are susceptible to the viruses Macrobrachium nipponense reovirus (MnRV) (Bateman and Stentiford, 2017), and WSSV (Yun et al., 2014; Zhao et al., 2017) and have been recorded as hosts for Spiroplasma eriocheiris (Srisala et al., 2018; Xiu et al., 2015), Aeromonas hydrophlia (Ding et al., 2015), the ciliates Gymnodinioides (=Hyalophysa) caridinae on the gills and exoskeleton (Morado and Small, 1995), Lagenophrys branchium and Lagenophrys eupagurus (Clamp, 1989; Felgenhauer, 1982; Mayén-Estrada and Clamp, 2016), the microsporidian Potaspora macrobrachium (Ding et al., 2016), and the digeneans Pseudophyllodistomum lesteri (Goodchild, 1943; Long and Wai, 1958; Cribb, 1987), P. macrobranchicola (Cribb, 1987), Paragonimus westermani (Toledo and Fried, 2014), Pseudolevinseniella cheni (Kudlai et al., 2015) and Microphallus minus (Kudlai et al., 2015). It should be noted that Sokolova and Overstreet (2018) suggest that the position of Potaspora macrobrachium requires reevaluation and may need to be moved to the genus Apotaspora. The current study presents for the first time the detailed description of a potentially new parasite in M. nipponense with red gill disease. Most of the studies on the haplosporidian group are focused on infections of molluscs because species such as Bonamia spp. and Haplosporidium nelsoni are causing epizootics and listed by the OIE as 373
Aquaculture 507 (2019) 370–376
Z. Ding, et al.
Fig. 4. Ultrastructure of the gill tissues from diseased prawn Macrobrachium nipponense. A, in accordance with the H&E results (Fig. 3B), gross plasmodia almost filled the entire space of the gill epithelial cell; B, the intracellular parasite was a multinucleate plasmodium enclosed in a vacuole within cytoplasm of the host cell. Numbers of trophonts were visible in each plasmodium. There was a variety of vesicles, spherical and elongated, containing more or less electron opaque substance; C, characteristic tubular extension from the surface of a late sporont was revealed; D, clear margins between membrane of uninucleate parasite and the plasma membrane and profiles of smooth endoplasmic reticulum (arrows); E, mitochondria (arrows) arrayed tightly around the nuclei, which had a central mass of chromatin with a prominent nucleolus; F, high degree of vesiculation in cytoplasm of early sporonts (★). N, host nuclei; Insert of A, higher-power image. Scale bar A = 20 μm, B = 5 μm, C, E = 1 μm, D, F = 2 μm.
transmission was probably not by the oral route. Even though the route of entry of the parasite into target cells is still unknown, an enzootic presence or introduction via transfer of infected prawn stocks are 2 plausible explanations for the emergence of the disease (Nunan et al., 2007). The disease showed a seasonal epizootiology, with peak infections occurring during the winter (November and December) and spring
(March and April), and with a latent infection or absence during the summer and early autumn. This was of significance because usually the diseases and parasites caused greater losses at higher temperatures in aquaculture seasons. This epizootiology was mostly similar with the prevalence of Hematodinium parasite in some crab species, which also suggested highly seasonal disease outbreaks, with peak infections occurring over a relatively narrow time period, followed by a longer 374
Aquaculture 507 (2019) 370–376
Z. Ding, et al.
Fig. 5. Spore ornamentation observed by scanning electron microscopy (SEM). A, spore wall-derived, thin, flat ribbons that emerged haphazardly around the spore; B, ribbons were often twisted along their length.
period of undetectable or low-level prevalence (Stentiford et al., 2002). Additionally, in the M. nipponensis rearing ponds, the virulence of the parasite could increase due to environmental stresses such as pollution, a scarcity of food and increase in population density. Therefore, adequate measures should also be taken in the susceptible months to avoid massive mortality outbreaks. The haplosporidian from blue crabs, Callinectes sapidus was about 3.4–7.3 μm in diameter (Burreson and Ford, 2004). In the spot prawn P. platyceros, the haplosporidian was 12.3 μm in diameter for round cells and 14.4 μm in length for ovoid cells (Bower and Meyer, 2002). The haplosporidian reported from shrimp P. vannamei was 2 μm in diameter (Nunan et al., 2007). While in the current study, the haplosporidian-like parasite infecting M. nipponense was around 4 μm in diameter (Fig. 3C). In addition, unique spore ornamentation was observed by TEM (Fig. 2D, 4C) and SEM (Fig. 5). The ribbons projecting from the spore wall and terminating in a definite capped structure were most similar to that of Bonamia perspora and H. edule (Carnegie et al., 2010; Rf and Montes, 2003). These features might provide classification criterions for this potentially new haplosporidian pathogen. Some of the infected gills showed granuloma-like foci that may have been similar to granulomatous lesions in shrimp P. vannamei infected by a similar haplosporidian (Nunan et al., 2007). Such foci have been described as aggregations of hemocytes encapsulating pathogens or foreign particles and which lead to the deposition of melanin either on the object or within the hemocyte matrix (Stentiford et al., 2002). In the case of parasite infection, the parasite is destroyed as the inner layers of these foci became necrotic. Such encapsulating lesions have also been thought to indicate a previous microsporidian infection in this prawn M. nipponense (Ding et al., 2016). It needs to be recognized that experimental studies will not always provide equivocal evidence of cause / effect (lack of SPF hosts, lack of stressors that exacerbate disease, failed challenge because parasite needs additional hosts or to be at a particular stage to be infective, etc.). While it is critical that challenge infections are run to name an agent, these are not always quick to conduct and there may be challenges to this (availability of SPF hosts, seasonal components to the infection, proximity of the lab to the study site etc.).
sightings of the condition and additional research to confirm the link between parasite and the pathology seen. It is our intention to complete life-cycle studies with to satisfy Koch's postulate. Acknowledgments We have received valuable advice from Jeffery D. Shields (Virginia Institute of Marine Science, College of William & Mary, USA). We thank Dr. Roland Pevsner from the University of Exeter, UK for reviewing the manuscript and the anonymous reviewers for valuable comments. This work was supported by grants from Natural Sciences Foundation of Jiangsu Province (BK20171406), Natural Science Fund of Colleges and Universities in Jiangsu Province (16KJA180005, 16KJB180006), innovation of agricultural science (CX (17) 2008), project for aquaculture of Jiangsu Province (Y2016-29, Y2018-22) and China Scholarship Council (2018). Conflict of interest The authors declare that they have no conflict of interests. References Bateman, K.S., Stentiford, G.D., 2017. A taxonomic review of viruses infecting crustaceans with an emphasis on wild hosts. J. Invertebr. Pathol. 147, 86–110. Bateman, K.S., Hicks, R.J., Stentiford, G.D., 2011. Disease profiles differ between nonfished and fished populations of edible crab (Cancer pagurus) from a major commercial fishery. ICES J. Mar. Sci. 68, 2044–2052. Bower, S.M., Meyer, G.R., 2002. Morphology and ultrastructure of a protistan pathogen in the haemolymph of shrimp (Pandalus spp.) in the northeastern Pacific Ocean. Can. J. Zool. 80, 1055–1068. Burreson, Eugene M., Ford, Susan E., 2004. A review of recent information on the Haplosporidia, with special reference to Haplosporidium nelsoni (MSX disease). Aquat. Living Resour. 17, 499–517. Carnegie, R.B., Burreson, E.M., Hine, P.M., Stokes, N.A., Audemard, C., Bishop, M.J., Peterson, C.H., 2010. Bonamia perspora n. sp. (Haplosporidia), a parasite of the oyster Ostreola equestris, is the first Bonamia species known to produce spores. J. Eukaryot. Microbiol. 53, 232–245. Clamp, J.C., 1989. Redescription of Lagenophrys eupagurus Kellicott (Ciliophora, Peritricha, Lagenophryidae) and a comparison of it with three similar species. J. Protozool. 36, 596–607. Cribb, T.H., 1987. Studies on gorgoderid digeneans from Australian and Asian freshwater fishes. J. Nat. Hist. 21, 1129–1153. Ding, Z., Zhang, Y., Ye, J., Du, Z., Kong, Y., 2015. An evaluation of replacing fish meal with fermented soybean meal in the diet of Macrobrachium nipponense: growth, nonspecific immunity, and resistance to Aeromonas hydrophila. Fish Shellfish Immun. 44, 295–301. Ding, Z., Sun, M., Liu, H., Zhao, Y., Pan, J., Hui, X., 2016. A new microsporidium, Potaspora macrobrachium n.sp. infecting the musculature of pond-reared oriental river prawn Macrobrachium nipponense (Decapoda: Palaemonidae). J. Invertebr. Pathol. 136, 57–64. Felgenhauer, B.E., 1982. A new species of Lagenophrys (Ciliophora: Peritrichida) from the fresh-water shrimp Palaemonetes kadiakensis. Trans. Am. Microsc. Soc. 101, 142–150. Fu, H., Jiang, S., Xiong, Y., 2012. Current status and prospects of farming the giant river prawn (Macrobrachium rosenbergii) and the oriental river prawn (Macrobrachium
5. Conclusion We recognize the risk of associating the presence of a parasite with a particular pathology in the absence of total evidence that demonstrates an unequivocal link between the parasite, the perceived pathology and a clear disease manifestation that would be demonstrated by e.g. experimental studies. However, given the widespread importance of the host in aquaculture, it is necessary to provide this short report to highlight to other researchers and to the industry the potential for a new and emerging disease. It is hoped that this will lead to additional 375
Aquaculture 507 (2019) 370–376
Z. Ding, et al. nipponense) in China. Aquac. Res. 43, 993–998. Goodchild, G.C., 1943. The life-history of Phyllodistomum solidum Rankin, 1937, with observations on the morphology, development and taxonomy of the Gorgoderinae (Trematoda). Biol. Bull. 84, 59–86. Kudlai, O., Cutmore, S.C., Cribb, T.H., 2015. Morphological and molecular data for three species of the Microphallidae (Trematoda: Digenea) in Australia, including the first descriptions of the cercariae of Maritrema brevisacciferum Shimazu et Pearson, 1991 and Microphallus minutus Johnston, 1948. Folia. Parasit. 62, 053. Long, S., Wai, M.T., 1958. Parasitic worms from Tai Hu fishes: digenetic trematodes.I. The genus Phyllodistomum Braun, 1899 (Gorgoderidae), with descriptions of four new species. Acta Zool. Sin. 10, 348–368. Longshaw, M., 2011. Diseases of crayfish: a review. J. Invertebr. Pathol. 106, 54–70. Malek, J.C., Byers, J.E., 2017. The effects of tidal elevation on parasite heterogeneity and co-infection in the eastern oyster, Crassostrea virginica. J. Exp. Mar. Biol. Ecol. 494, 32–37. Mayén-Estrada, R., Clamp, J.C., 2016. An annotated checklist of species in the family Lagenophryidae (Ciliophora, Oligohymenophorea, Peritrichia), with a brief review of their taxonomy, morphology, and biogeography. Zootaxa 4132, 451–492. Morado, J.F., Small, E.B., 1995. Ciliate parasites and related diseases of Crustacea: a review. Rev. Fish. Sci. 3, 275–354. Nunan, L.M., Lightner, D.V., Pantoja, C.R., Stokes, N.A., Reece, K.S., 2007. Characterization of a rediscovered haplosporidian parasite from cultured Penaeus vannamei. Dis. Aquat. Org. 74, 67–75. Rf, A.C.C., Montes, J., 2003. Description of Haplosporidium edule n. sp (Phylum Haplosporidia), a parasite of Cerastoderma edule (Mollusca, Bivalvia) with complex spore ornamentation. Eur. J. Protistol. 39, 161–167. Sokolova, Y.Y., Overstreet, R M.A., 2018. A new microsporidium, Apotaspora heleios ng, n. sp., from the Riverine grass shrimp Palaemonetes paludosus (Decapoda: Caridea: Palaemonidae)[J]. J. invertebr. pathol. 157, 125–135. Srisala, J., Pukmee, R., McIntosh, R., Choosuk, S., Itsathitphaisarn, O., Flegel, T.W.,
Sritunyalucksana, K., Vanichviriyakit, R., 2018. Distinctive histopathology of Spiroplasma eriocheiris infection in the giant river prawn Macrobrachium rosenbergii. Aquaculture 493, 93–99. Stentiford, G.D., Green, M., Bateman, K., Small, H.J., Neil, D.M., Feist, S.W., 2002. Infection by a hematodinium-like parasitic dinoflagellate causes pink crab disease (PCD) in the edible crab Cancer pagurus. J. Invertebr. Pathol. 79, 179–191. Stentiford, G.D., Feist, S.W., Bateman, K.S., Hine, P.M., 2004. Haemolymph parasite of the shore crab Carcinus maenas: pathology, ultrastructure and observations on crustacean haplosporidians. Dis. Aquat. Org. 59, 57–68. Tang, K.F., Lightner, D.V., 2006. Infectious hypodermal and hematopoietic necrosis virus (IHHNV)-related sequences in the genome of the black tiger prawn Penaeus monodon from Africa and Australia. Virus Res. 118, 185–191. Thrupp, T.J., Lynch, S.A., Wootton, E.C., Malham, S.K., Vogan, C.L., Culloty, S.C., Rowley, A.F., 2013. Infection of juvenile edible crabs, Cancer pagurus by a haplosporidian-like parasite. J. Invertebr. Pathol. 114, 92–99. Toledo, R., Fried, B., 2014. Digenetic Trematodes. Springer, New York. Umesha, K.R., Dass, B.K.M., Naik, B.M., Venugopal, M.N., Karunasagar, I., Karunasagar, I., 2006. High prevalence of dual and triple viral infections in black tiger shrimp ponds in India. Aquaculture 258, 91–96. Xiu, Y., Wu, T., Meng, X., Meng, Q., Wang, W., 2015. Identification and isolation of a spiroplasma pathogen from diseased oriental river prawn, Macrobrachium nipponense, in China: a new freshwater crustacean host. Aquaculture 437, 270–274. Yun, J.M., Kim, B.S., Hwang, S.M., Kim, Y.B., Choi, W.B., Choi, T.J., 2014. Artificial infection of the native Korean freshwater prawn Macrobrachium nipponense (De Haan, 1849) (Decapoda, Palaemonidae) with white spot syndrome virus (WSSV). Crustaceana 87, 866–880. Zhao, C., Fu, H., Sun, S., Qiao, H., Zhang, W., Jin, S., Jiang, S., Xiong, Y., Gong, Y., 2017. Experimental inoculation of oriental river prawn Macrobrachium nipponense with white spot syndrome virus (WSSV). Dis. Aquat. Org. 126, 125–134.
376