This is a repository copy of Cucumispora ornata n. sp. (Fungi: Microsporidia) infecting invasive 1 ‘demon shrimp’. White Rose Research Online URL for this paper: http://eprints.whiterose.ac.uk/85921/ Version: Accepted Version Article: Bojko, J, Dunn, A, Stebbing, PD et al. (3 more authors) (2015) Cucumispora ornata n. sp. (Fungi: Microsporidia) infecting invasive 1 ‘demon shrimp’. Journal of Invertebrate Pathology, 128. 22 - 30. ISSN 0022-2011 https://doi.org/10.1016/j.jip.2015.04.005 © 2015, Elsevier. Licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International http://creativecommons.org/licenses/by-nc-nd/4.0/ Reuse Unless indicated otherwise, fulltext items are protected by copyright with all rights reserved. The copyright exception in section 29 of the Copyright, Designs and Patents Act 1988 allows the making of a single copy solely for the purpose of non-commercial research or private study within the limits of fair dealing. 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Stentiford 3,4 5 1Faculty of Biological Sciences, University of Leeds, Leeds, UK, LS2 9JT 6 2Epidemiology and Risk Team, Centre for Environment, Fisheries and Aquaculture Science 7 (Cefas), Weymouth Laboratory, Weymouth, Dorset, UK, DT4 8UB 8 3Pathology and Molecular Systematics Team, Centre for Environment, Fisheries and 9 Aquaculture Science (Cefas), Weymouth Laboratory, Weymouth, Dorset, UK, DT4 8UB 10 4European Union Reference Laboratory for Crustacean Diseases, Centre for Environment, 11 Fisheries and Aquaculture Science (Cefas), Weymouth Laboratory, Weymouth, Dorset, UK, 12 DT4 8UB 13 14 Author e-mails: 15 Jamie Bojko (*Correspondence) - [email protected] 16 Phone Number: N/A 17 18 Alison M. Dunn – [email protected] 19 Paul D. Stebbing – [email protected] 20 Stuart H. Ross – [email protected] 21 Rose C. Kerr – [email protected] 22 Grant D. Stentiford – [email protected] 23 24 Keywords: Invasive Non-native Species, Amphipod, Ponto-Caspian Region, Biological 25 Control, Microsporidia, Phylogeny 26 Abbreviations: Invasive non-native species = INNS 1 27 Abstract 28 Dikerogammarus haemobaphes, the ‘demon shrimp’, is an amphipod native to the Ponto- 29 Caspian region. This species invaded the UK in 2012 and has become widely established. 30 Dikerogammarus haemobaphes has the potential to introduce non-native pathogens into the 31 UK, creating a potential threat to native fauna. This study describes a novel species of 32 microsporidian parasite infecting 72.8% of invasive D. haemobaphes located in the River 33 Trent, UK. The microsporidium infection was systemic throughout the host; mainly targeting 34 the sarcolemma of muscle tissues. Electron microscopy revealed this parasite to be 35 diplokaryotic and have 7-9 turns of the polar filament. The microsporidium is placed into the 36 ‘Cucumispora’ genus based on host histopathology, fine detail parasite ultrastructure, a 37 highly similar life-cycle and SSU rDNA sequence phylogeny. Using this data this novel 38 microsporidian species is named Cucumispora ornata, where ‘ornata’ refers to the external 39 beading present on the mature spore stage of this organism. Alongside a taxonomic 40 discussion, the presence of a novel Cucumispora sp. in the United Kingdom is discussed 41 and related to the potential control of invasive Dikerogammarus spp. in the UK and the 42 health of native species which may come into contact with this parasite. 43 44 45 46 47 48 49 50 2 51 1. Introduction 52 The Microsporidia are a diverse group of obligate parasites within the Kingdom Fungi 53 (Capella-Guitiérrez et al. 2012; Haag et al. 2014). They infect hosts from all animal phyla 54 and from all habitats; are genetically diverse; use a variety of transmission methods; can 55 infect a range of different tissue and organ types; and exhibit high developmental and 56 morphological plasticity (Dunn et al. 2001; Stentiford et al. 2013a; Stentiford et al. 2013b). 57 Plasticity in parasite morphology has led to the formation of polyphyletic taxa whose inter- 58 relationships are now being clarified by application of molecular phylogenetic approaches 59 (e.g. Vossbrinck and Debrunner-Vossbrinck, 2005; Stentiford et al. 2013b). Furthermore, 60 similar approaches are being applied to increase the confidence in placement of the 61 Microsporidia at the base of the Fungi (Capella-Guitiérrez et al. 2012). The discovery and 62 description of novel taxa, such as Mitosporidium daphniae, emphasise this positioning by 63 essentially bridging the gap between true Fungi, the Cryptomycota (e.g. Rozella spp.) and 64 the Microsporidia (Haag et al. 2014). Novel taxonomic descriptions now combine data 65 pertaining to ultrastructural features, lifecycle characteristics, host type and habitat type, and 66 conclusively, phylogenetics (Stentiford et al. 2013b). 67 68 Microsporidia were first identified infecting members of the Gammaridae (a family of 69 omnivorous amphipods found across the world in freshwater and marine habitats), 70 specifically Gammarus pulex, by Pfeiffer (1895). Since this initial discovery, gammarids have 71 been shown to play host to a wide diversity of Microsporidia (Bulnheim, 1975; Terry et al. 72 2003). Ten microsporidium genera are currently known to infect gammarid hosts including: 73 Dictyocoela (unofficially presented by Terry et al. 2004); Nosema (Nägeli, 1857); 74 Fibrillanosema (Johanna et al. 2004); Thelohania (Henneguy and Thélohan, 1892); 75 Stempillia (Pfeiffer, 1895); Pleistophora (Canning and Hazard, 1893); Octosporea (Chatton 76 and Krempf, 1911); Bacillidium (Janda, 1928); Gurleya (Hesse, 1903); Glugea (Thélohan, 77 1891); Amblyospora (Hazard & Oldacre, 1975) and Cucumispora (Ovcharenko and 78 Kurandina, 1987). Based on phylogenetic analysis and tree construction, these gammarid- 3 79 infecting microsporidia appear alongside those infecting fish, insects and other crustacean 80 hosts from marine and freshwater environments (Stentiford et al. 2013b). Members of these 81 genera utilise either horizontal or vertical transmission pathways, or a combination of the 82 two, to maintain infections within populations of target hosts (Smith, 2009). Dictyocoela 83 berillonum (vertical transmission), Pleistophora mulleri (vertical and horizontal transmission) 84 and Gurleya polonica (horizontal transmission solely) provide examples of these 85 transmission methods (Czaplinska et al. 1999; Terry et al. 2003; Terry et al. 2004; Wattier et 86 al. 2007). 87 88 Most organs and tissues of gammarids can become infected by microsporidia. Whilst some 89 taxa cause systemic infections (e.g. Cucumispora dikerogammari), others target specific 90 tissue types such as muscle fibres (e.g. G. polonica in Orchestia sp.). In general, vertically 91 transmitted microsporidia infect gonadal tissues and often elicit only minor pathologies 92 unless they are also capable of horizontal transmission (Terry et al. 2003). Horizontally 93 transmitted microsporidia on the other hand can elicit negative effects on feeding and 94 locomotion and often result in host mortality (Bacela-Spychalska et al. 2014). For these 95 reasons, horizontally transmitted microsporidia are considered a useful target for biological 96 control strategies against agriculturally-important insect pests (Hajek and Delalibera Jr, 97 2010). 98 99 Members of the genus Dikerogammarus are a group of freshwater amphipods, native to the 100 Ponto-Caspian region. Within the genus, two taxa have received considerable attention as 101 invasive non-native species (INNS) within Europe: the ‘killer shrimp’ D. villosus (Rewicz et al. 102 2014) and the ‘demon shrimp’ Dikerogammarus haemobaphes (Bovy et al. 2014). 103 Dikerogammarus villosus is listed in the ‘top 100 worst invasive species in Europe’ (DAISIE, 104 2014) due to its widely documented detrimental impact on native invertebrate fauna and its 105 ability to spread parasites to novel locations (Wattier et al. 2007). In 2010, populations of D. 106 villosus were discovered in several locations within the UK where they have subsequently 4 107 caused significant issues to both native fauna and the environment (MacNeil et al. 2013). 108 Subsequent to the invasion by D. villosus, in 2012, a second invader, D. haemobaphes was 109 also detected in UK freshwater habitats and has since been detected at numerous sites 110 across a wide geographic space (Bovy et al. 2014; Green-Etxabe et al. 2014). 111 112 An extensive survey of D. villosus using histopathology revealed a distinct lack of pathogens 113 and parasites in populations of D. villosus in UK sites (Bojko et al. 2013). These data were 114 reinforced in a subsequent study by Arundell et al, (2014) which demonstrated an absence