Disease of Aquatic Organisms 132:125

Vol. 132: 125–134, 2019 DISEASES OF AQUATIC ORGANISMS Published January 10 https://doi.org/10.3354/dao03316 Dis Aquat Org Morphological and molecular identification of a new Kudoa thyrsites isolate in Mediterranean silver scabbardfish Lepidopus caudatus Lucilla Giulietti1,*, Simonetta Mattiucci2, Michela Paoletti3, Didrik H. Grevskott1, Miguel Bao1, Paolo Cipriani1, Arne Levsen1 1Institute of Marine Research (IMR), Nordnes, Bergen 5009, Norway 2Department of Public Health and Infectious Diseases, Section of Parasitology, Sapienza-University of Rome 00185, Italy 3Department of Ecological and Biological Sciences, Tuscia University, Viterbo 01100, Italy ABSTRACT: Myxozoans of the genus Kudoa (Myxosporea, Multivalvulida) infect marine and estuarine fish species worldwide. Some Kudoa species are of concern to the seafood industry since they may generate macroscopic cysts in the fish host’s musculature, or cause post mortem myo - liquefaction, commonly known as ‘soft flesh’. One of the economically most important species is K. thyrsites, a myoliquefactive myxosporean parasite that occurs in many wild and cultured marine fish species worldwide. Here we identified a K. thyrsites isolate as the causative agent of myoliquefaction in silver scabbardfish Lepidopus caudatus from the Alboran Sea (western Medi- terranean Sea). For comparative and validation purposes, the morphological and molecular char- acteristics of the isolate were compared with fresh spores of a K. thyrsites isolate infecting Atlantic mackerel Scomber scombrus from the Norwegian Sea. Myxospores of both isolates shared a stel- late appearance and contained 4 unequal pyriform polar capsules (1 large, 1 small and 2 intermediate). These morphological traits were consistent with all other previously described K. thyrsites isolates. Moreover, the small subunit rDNA sequences of the Mediterranean and Norwegian Sea isolates revealed 100% similarity, and matched 100% with K. thyrsites isolates previously re - corded in myoliquefactive Atlantic mackerel from the North Sea and off southern England. The findings suggest that K. thyrsites is the primary cause of myoliquefaction in silver scabbardfish from the Alboran Sea. This report represents the first morphological and molecular characteriza- tion of K. thyrsites in the Mediterranean Sea. A set of new allometric characters is proposed as additional descriptors for more accurate and specific description of kudoid myxospores. KEY WORDS: Kudoa thyrsites · Mediterranean Sea · ‘Soft flesh’ · Myoliquefaction · Lepidopus caudatus · Molecular identification · New morphological characters · Fish parasite Resale or republication not permitted without written consent of the publisher 1. INTRODUCTION oligochaetes and polychaetes) as invertebrate defini- tive hosts and fish as vertebrate intermediate hosts Species of the genus Kudoa Meglitsch, 1947 (Wolf & Markiw 1984, Shaw et al. 1997, Young 2002, (Myxo zoa, Multivalvulida) are parasites of marine Lom & Dyková 2006). Actinospores released from the and estuarine fishes with a worldwide geographical annelid host infect fish through various entry sites, distribution (Moran et al. 1999, Whipps & Kent 2006, such as the skin, fins or gills (Yokoyama et al. 2012). Eiras et al. 2014, MacKenzie & Kalavati 2014). The However, the transmission pathways of Kudoa spe- typical life cycle of myxozoans involves annelids (e.g. cies, as well as the life cycle putatively involving an *Corresponding author: [email protected] © Inter-Research 2019 · www.int-res.com 126 Dis Aquat Org 132: 125–134, 2019 alternate invertebrate host and a fish infective stage, range of most Kudoa species, and many myxozoans are still unresolved. The kudoids are defined by their in general (Moran et al. 1999, Whipps & Kent 2006, myxospore life cycle stage that occurs in the verte- Burger et al. 2008, Yokoyama et al. 2012), K. thyrsites brate host (i.e. fish) (Lom 1987). The myxospores are has been recorded from more than 30 phylogeneti- characterized by 4 or more shell valves, each con- cally comparatively distant teleost fish species. taining a single polar capsule (Lom & Dyková 2006). Moreover, infections with K. thyrsites have been In early studies, the identification of kudoids to the reported from commercially important wild fish species level was based on morphology and morpho - stocks such as Northeast Atlantic mackerel (Levsen metry of myxospores, i.e. shape and dimensions of et al. 2008), mahi-mahi Coryphaena hippurus off the spore, polar capsules and polar filaments (Lom & western Australia (Langdon 1991, Whipps et al. 2003) Arthur 1989), and more recently on molecular ana - and snoek Thyrsites atun off South Africa (Henning lyses of the small (SSU) and large (LSU) ribosomal et al. 2013), the latter being the type host species of DNA (rDNA) subunits (Whipps & Kent 2006, Burger K. thyrsites (Gilchrist 1924). K. thyrsites has also been & Adlard 2010, 2011). A more accurate identification documented in several important sea-reared fish of Kudoa species can be achieved by integrating both species such as olive flounder Paralichthys olivaceus morphological and molecular data, together with from Japanese waters (Yokoyama et al. 2004), coho ecological and biological aspects such as tissue tro- salmon Oncorhynchus kisutch and Atlantic salmon pism, host specificity, pathogenicity and geographi- Salmo salar in British Columbia, Canada (Whitaker & cal distribution (Whipps et al. 2004, Adlard et al. Kent 1991, St-Hilaire et al. 1998, Moran et al. 1999, 2005, Shin et al. 2016). Marshall et al. 2016), as well as other areas including To date, more than 100 nominal Kudoa species Ireland, Chile and Australia (Palmer 1994, Munday have been described from many phylogenetically et al. 1998, Lopez & Navarro 2000). For instance, distant fish host species (Moran et al. 1999, Lom & K. thyrsites infections caused significant problems for Dyková 2006, Eiras et al. 2014, Sato & Kasai 2016). the Atlantic salmon aquaculture industry in British Most of these are histozoic parasites infecting the Columbia, where the loss of revenue due to skeletal muscles of fish (Moran et al. 1999, Lom & degraded fish products reached 50 million Canadian Dyková 2006, Eiras et al. 2014), while others occur in dollars in 2002 (Funk et al. 2007, Marshall et al. the brain, heart, gills, kidney, ovary or intestines 2016). (Eiras et al. 2014). Despite its almost cosmopolitan distribution (Moran Kudoa spp. are considered non-pathogenic para- et al. 1999, Whipps & Kent 2006, Henning et al. 2013, sites of fish. However, several species can generate MacKenzie & Kalavati 2014), K. thyrsites has not macroscopic cysts in the muscle and may induce post been previously reported in fish from the Mediter- mortem myoliquefaction (Alvarez-Pellitero & Sitjà- ranean Sea. Only a few reports of Kudoa sp. infec- Bobadilla 1993, Moran et al. 1999, Eiras et al. 2014). tions in fish from the Mediterranean Sea exist so far. The muscle degeneration, commonly known as ‘soft A case of ‘soft flesh’ was observed in 1 swordfish flesh’, ‘milky flesh’ or ‘jelly flesh’, may irreversibly Xiphias gladius caught off Sicily, caused by an reduce the quality of the fish fillet and the mar- unidentified Kudoa sp. (Gaglio et al. 2010). Pam- ketability of the fish product, resulting in economic poulie et al. (1999) described K. camarguensis as the losses to the seafood industry, as well as loss of con- causative agent of myoliquefaction in 2 gobiid fish sumer confidence (Moran et al. 1999, Lom & Dyková caught in the lagoon waters of the River Rhone delta 2006, Levsen 2015). Furthermore, some human in southern France. Moreover, in the same geograph- health issues (e.g. gastrointestinal disorders) associ- ical area, macroscopic pseudocysts of K. lunata were ated with the consumption of raw fish products from found in the muscle of 3 species of scaldfish (Arno - wild and farmed fish infected by Kudoa spp. (e.g. K. glossus spp.) (Lom et al. 1983). Along the Mediter- septempunctata) have recently been reported (Kawai ranean North African coast, spores of K. nova were et al. 2012, Iwashita et al. 2013, Suzuki et al. 2015, detected in whitish macroscopic pseudocysts embed- Yahata et al. 2015). ded in the muscle of Atlantic horse mackerel Trachu- One of the most conspicuous ‘soft flesh’-inducing rus trachurus (Campbell 2005). Some other species species is K. thyrsites (Gilchrist 1924), which infects infecting the ovaries (K. azevedoi, Mansour et al. the somatic and cardiac musculature of several wild 2013), spleen, intestine (K. trifolia, Holzer et al. 2006) and cultured marine fish species throughout temper- and mesenteries (K. unicapsula, Yurakhno et al. 2007) ate seas of the world (Moran et al. 1999, Whipps & of several different Mediterranean fish species have Kent 2006). In contrast to the rather narrow fish host also been reported. However, none of the ‘soft flesh’- Giulietti et al.: Kudoa thyrsites in Mediterranean silver scabbardfish 127 inducing Kudoa species so far reported from the Two subsamples of soft muscle tissue were taken Mediterranean Sea were genetically identified to from each fillet of the affected fish. Following St- species level. Hilaire et al. (1997), the samples were placed on The silver scabbardfish Lepidopus caudatus is a glass slides, moistened with saline water and then benthopelagic trichiurid, widely distributed through- minced with a scalpel blade. The resulting minced out temperate oceans of the world (Parin 1986, De - tissue was collected into plastic tubes and allowed

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