Longitudinal Study of Winter Mortality Disease in Sydney Rock Oysters Saccostrea Glomerata

Vol. 110: 151–164, 2014 DISEASES OF AQUATIC ORGANISMS Published July 24 doi: 10.3354/dao02629 Dis Aquat Org Contribution to DAO Special 7 'Microcell parasites of molluscs' Longitudinal study of winter mortality disease in Sydney rock oysters Saccostrea glomerata Zoe B. Spiers1, Melinda Gabor1, Shayne A. Fell1, Ryan B. Carnegie2, Michael Dove3, Wayne O’Connor3, Jane Frances3, Jeffrey Go1, Ian B. Marsh1, Cheryl Jenkins1,* 1NSW Department of Primary Industries, Elizabeth Macarthur Agricultural Institute, Menangle, New South Wales 2568, Australia 2Virginia Institute of Marine Science, College of William and Mary, Gloucester Point, Virginia 23062–1346, USA 3NSW Department of Primary Industries, Port Stephens Fisheries Institute, Port Stephens, New South Wales 2315, Australia ABSTRACT: Winter mortality (WM) is a poorly studied disease affecting Sydney rock oysters Sac- costrea glomerata in estuaries in New South Wales, Australia, where it can cause significant losses. WM is more severe in oysters cultured deeper in the water column and appears linked to higher salinities. Current dogma is that WM is caused by the microcell parasite Bonamia roughleyi, but evidence linking clinical signs and histopathology to molecular data identifying bonami- asis is lacking. We conducted a longitudinal study between February and November 2010 in 2 estuaries where WM has occurred (Georges and Shoalhaven Rivers). Results from molecular testing of experimental oysters for Bonamia spp. were compared to clinical disease signs and histopathology. Available environmental data from the study sites were also collated and compared. Oyster condition declined over the study period, coinciding with decreasing water temperatures, and was inversely correlated with the presence of histological lesions. While mortalities occurred in both estuaries, only oysters from the Georges River study site showed gross clinical signs and histological changes characteristic of WM (lesions were prevalent and intralesional microcell-like structures were sometimes noted). PCR testing for Bonamia spp. revealed the presence of an organism belonging to the B. exitiosa−B. roughleyi clade in some samples; however, the very low prevalence of this organism relative to histological changes and the lack of reactivity of affected oysters in subsequent in situ hybridisation experiments led us to conclude that this Bonamia sp. is not responsible for WM. Another aetiological agent and a confluence of environmental factors are a more likely explanation for the disease. KEY WORDS: Bonamia roughleyi · Microcell-like structure · Lesion · Haemocyte Resale or republication not permitted without written consent of the publisher INTRODUCTION caused by Marteilia sydneyi, is considered among the key contributing factors for S. glomerata produc- Winter mortality is a poorly studied disease affect- tion declines since the mid-1970s (Nell 2007). ing Sydney rock oysters Saccostrea glomerata (Gould The first investigation into the potential causes of 1850) in the southern half of its cultivation range in winter mortality was carried out between 1924 and New South Wales (NSW), Australia. The disease was 1925 (Roughley 1926), where it was determined that first observed in 1923 in the Georges River and regu- cold temperatures were a contributing but indirect larly causes economically significant oyster losses cause of mortality. Subtidal oysters were found to be during winter and early spring. Winter mortality dis- more prone to winter mortality than intertidal oys- ease, along with Queensland unknown (QX) disease ters, suggesting that prolonged exposure to an agent *Corresponding author: [email protected] © Inter-Research 2014 · www.int-res.com 152 Dis Aquat Org 110: 151–164, 2014 within the water column was important in inducing first assumption has also been challenged via the clinical disease (Roughley 1926). More recent obser- assertion that lesion morphology cannot be used to vations (Wolf 1967), and controlled studies in which differentiate between microcell pathogens occurring raising the growing height of Saccostrea glomerata in different host species due to variations in tissue had a significant positive effect on oyster survival structure (Cochennec-Laureau et al. 2003). (Smith et al. 2000), corroborated the earlier findings. Current dogma is that winter mortality is caused by Winter mortality occurs primarily in oysters 2 to 3 yr Bonamia roughleyi, a member of the phylum Hap- of age in areas of high salinity (Wolf 1967, Nell & losporidia (Carnegie & Cochennec-Laureau 2004, Perkins 2006). Oyster growers currently move oyster López-Flores et al. 2007). Reclassification of Mikro- stocks to upstream leases prior to the winter season cytos roughleyi into the genus Bonamia was based on to avoid winter mortality losses (Smith et al. 2000, ultrastructural observations of a parasite within Sac- Nell 2007). costrea glomerata gill and digestive gland tissue Winter mortality is characterised by the presence (Cochennec-Laureau et al. 2003). In that study, mito- of pustules, abscesses and ulcerations, and these chondria and haplosporosomes were observed with - lesions have been reported in various tissues includ- in parasites, while M. mackini is amitochondriate ing the mantle, palps, gills, adductor muscle, gonad, and lacks haplosporosomes (Hine et al. 2001). These digestive diverticulum and alimentary tract (Rough- observations, combined with molecular testing, which ley 1926, Farley et al. 1988, Bower 2012). Micro- revealed the presence of Bonamia sp. DNA within S. scopic observations of haemocyte accumulations glomerata oysters, led to the conclusion that M. within these lesions led Roughley (1926) to infer roughleyi belonged in the genus Bonamia. Nonethe- that the cause of disease was an unidentified bacte- less, the organism M. roughleyi, first described by rial agent; however, a histological investigation of Farley et al. (1988), appears to be somewhat different winter mortality conducted some decades later indi- from that described by Cochennec-Laureau et al. cated that the causative agent was a protistan (2003), in that M. roughleyi is 1 to 2 µm in size and ‘microcell’ parasite (Farley et al. 1988). This conclu- spherical, rather than the 3 to 5 µm, ovoid and occa- sion was based on observations of small (1−2 µm sionally multinucleate organism observed in the lat- diameter), spherical, single-celled organisms with ter study. The ultrastructure of the organism in the eccentric nuclei that were both intracellular (pri- study by Cochennec-Laureau et al. (2003) could be marily within haemocytes) and associated with considered consistent with a number of different lesions (Farley et al. 1988). organisms. Indeed, these morphological and ultra- The identity of the microcells associated with win- structural descriptions combined with the fact that ter mortality has been the subject of some confusion. the parasites resided primarily in the digestive Farley et al. (1988) classified the Saccostrea glo - tubules and gill, could be considered consistent merata parasite as Mikrocytos roughleyi based on with Marteilia sydneyi daughter cells (Kleeman et comparisons with other microcell organisms of the al. 2002), a haplosporosome-containing paramyxean genera Mikrocytos and Bonamia. Two major obser- parasite that is prevalent in S. glomerata in a number vations that led to this classification were that (1) of NSW estuaries. A further question regarding the both M. mackini, the causative agent of Denman study by Cochennec-Laureau et al. (2003) is whether Island disease in Crassostrea gigas, and M. roughleyi molecular testing and ultrastructural investigations of S. glomerata cause focal abscesses, whereas B. were conducted on tissue derived from the same oys- ostreae infections in the European flat oyster Ostrea ters. Thus, while a Bonamia sp. has been detected edulis are more disseminated; and (2) Mikrocytos within S. glomerata oysters using molecular meth- spp. only infect crassostreid oysters while Bonamia ods, its connection with winter mortality and the orig- spp. only infect ostreid oysters. Indeed, Brock (1990) inal disease descriptions (Roughley 1926, Farley et al. demonstrated that the genus Ostrea is more distant 1988) have not been fully established. Molecular from Crassostrea and Saccostrea than the latter 2 identification of the aetiological agent of winter mor- genera are from each other. Therefore, the second tality has been further confounded by the fact that B. assumption made by Farley et al. (1988) has since roughleyi found in NSW oysters does not appear to been violated given that Mikrocytos spp. have been be easily distinguishable from B. exitiosa, the cause found to infect O. edulis (Bower et al. 1997, Gagné et of bonamiosis in Ostrea chilensis based on 18S rRNA al. 2008), and conversely, 1 or more Bonamia spp. gene sequences (Corbeil et al. 2006, Hill et al. 2010). have been shown to infect C. ariakensis (C. rivularis) The State Veterinary Diagnostic Laboratory at the (Cochennec et al. 1998, Audemard et al. 2008). The Elizabeth Macarthur Agricultural Institute (NSW) Spiers et al.: Winter mortality in Saccostrea glomerata 153 has received numerous Saccostrea glomerata samples for winter mortality diagnosis from estuaries prone to the disease over a period of approximately 5 yr. Diagnostic testing, consisting of a Bonamia genus-specific PCR, failed to return positive results in all but a very small proportion of clinical cases during this period. In light of these findings

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