For Peer Review Only

Journal of Aquatic Animal Health

Exophiala angulospora causes systemic mycosis in Atlantic halibut (Hippoglossus hippoglossus) −−−−−− A Case Report

Journal: Journal of Aquatic Animal Health

Manuscript ID: UAAH-2014-0011

Manuscript Type: Communication

Keywords: Pathology < Culture, Disease and Parasites, Coldwater < Culture

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1 Exophiala angulospora causes systemic mycosis in Atlantic halibut ( Hippoglossus 2 hippoglossus ) −−−−−− A Case Report 3 4 5 David P. Overy 6 Department of Pathology and Microbiology, University of Prince Edward Island, 550 7 University ForAvenue Charlottetown, Peer PEI, Review Canada, C1A 4P3, andOnly Nautilus Biosciences 8 Canada, Duffy Research Center (NRC-INH), 550 University Ave., Charlottetown, Prince 9 Edward Island, Canada, C1A 4P3 10 11 David Groman and Jan Giles 12 Aquatic Diagnostic Services, University of Prince Edward Island, 550 University Ave., 13 Charlottetown, Prince Edward Island, Canada, C1A 4P3 14 15 Stephanie Duffy 16 Nautilus Biosciences Canada, Duffy Research Center (NRC-INH), 550 University Ave., 17 Charlottetown, Prince Edward Island, Canada, C1A 4P3 18 19 Mellisa Rommens 20 Aquaculture Solutions, 1475 Peter Rd., Emyvale, Prince Edward Island, Canada, 21 C0A1Y0 22 23 Gerald Johnson 24 Department of Pathology & Microbiology, University of Prince Edward Island, 550 25 University of Prince Edward Island, Prince Edward Island, Canada, C1A 4P3 26 27 28 29 ∗ Corresponding author: [email protected] 30

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31 Abstract 32 33 Filamentous black yeasts from the genus Exophiala are ubiquitous, opportunistic 34 pathogens causing both superficial and systemic mycoses in warm- and cold-blooded 35 animals. Infections by black yeasts have been reported relatively frequently in a variety 36 of captive and farmed freshwater and marine fish. In November 2012, moribund and 37 recently deadFor farm raised Peer Atlantic halibut Review (Hippoglossus hippoglossus Only ) were necropsied 38 to determine the cause of death. Histopathology revealed that three of five fish were 39 affected by a combination of an ascending trans-ureter granulomatous mycotic nephritis, 40 necrotizing histiocytic encephalitis, and in one fish the addition of a fibrogranulomatous 41 submucosal branchitis. Microbial cultures of kidney using selective mycotic media 42 revealed pure growth of a black pigmenting septated agent. Application of molecular and 43 phenotypic taxonomy methodologies determined that all three isolates were genetically 44 consistent with Exophiala angulospora . This is the first report of Exophiala 45 angulospora as the causal agent of systemic mycosis in Atlantic halibut. 46

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47 Introduction and Clinical History 48 49 Exophiala spp. are ubiquitous fungi, historically isolated from a variety of environmental 50 substrates such as soil and sediment, decaying wood and plant material, human hair and 51 nails and drinking water (Iwatsu et al. 1991; Domsch et al. 2007; de Hoog et al. 2011). 52 These filamentous black yeasts are also opportunistic pathogens causing both superficial 53 and systemicFor mycoses Peer in warm- and Reviewcold-blooded animals (UijthofOnly et al. 1997; de Hoog 54 et al. 2011; Gjessing et al. 2011). De Hoog and collaborators suggest that animals with 55 moist skin are more susceptible to infection by filamentous black yeasts, where the ability 56 of the pathogen to assimilate alkylbenzenes and accumulate melanin within the hyphae 57 and conidia are purported to be general virulence factors for these agents (de Hoog et al., 58 2011). Black yeast infections in aquarium and farmed fish as well as amphibians are 59 relatively frequent. An accurate estimation of the magnitude of the disease outbreaks has 60 been difficult to ascertain due to the infrequent and happenstance nature of disease 61 reports, in spite of the fact that outbreaks of this mycotic disease have been reported to 62 result in severe losses. (de Hoog et al. 2011). 63 64 Although first isolated and described from drinking water (Iwatsu et al. 1991), Exophiala 65 angulospora is also an opportunistic pathogen of both freshwater and marine fish. 66 Exophiala angulospora has been reported to cause mycosis in aquaria-maintained weedy 67 sea dragons (Phyllopteryx taeniolatus ) in the United States (Nyaoke et al. 2009). This 68 pathogen has also been isolated from the freshwater whitefish Stenodus leucichtys in 69 southern Russia (de Hoog et al. 2011) and was determined to be the cause of systemic 70 mycosis in farmed Atlantic cod (Gadus morhua ) from Norway (Gjessing et al. 2011). 71 Necropsy of moribund cod revealed chronic multifocal inflammation in the internal 72 organs, consisting of dematiaceous fungal hyphae surrounded by distinct layers of 73 inflammatory cells. Gjessing and co-workers noted that the fungal infection was not 74 eliminated by this inflammatory response in the cod, resulting in a systemic infection and 75 mortality. 76

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77 In this Case Report we describe the diagnostic evaluation of Atlantic halibut 78 (Hippoglossus hippoglossus ) which were submitted on multiple occasions from a grow- 79 out population of approximately 4000 fish, during November of 2012. Necropsies and 80 diagnostic testing on both fresh dead and moribund fish harvested from the slowest 81 growing group in the population were undertaken at the Aquatic Diagnostic Services Unit 82 of the Atlantic Veterinary College, University of Prince Edward Island, Canada. Fish

83 had been rearedFor in 5 'Peer by 12' fiberglass Review tanks ( water temperature Only 12.1- 12.2 ° C, O 2 =

84 105-119 % saturation, N 2 < 96% saturation ) and were being fed dry commercial fish feed 85 ( 6mm Europa 18 – Skretting Feeds). The mean weight of the three mortalities was 227 86 grams. Clinical observations of affected fish, as reported by the on-site clinician, were 87 limited to reduced appetite and loss of equilibrium. Cumulative mortality during the 88 previous two months was five to 10 times higher than normally expected, ranging from 89 0.8 to 1.5 % per month as compared to the normal rate of < 0.1 % per month. Diagnostic 90 investigation into the cause of death revealed that the halibut were suffering from a 91 systemic mycotic infection caused by Exophiala angulospora . To the authors’ 92 knowledge this is the first report of a clinical myosis due to Exophiala angulospora in 93 farm reared Atlantic halibut. 94 95 96 Methods 97 Necropsy 98 Live fish were euthanized using an overdose of a saturated solution of benzocaine 99 dissolved in 95% ethanol. All fish were subsequently examined for gross external and 100 internal lesions, assessed for sex determinations and content of the alimentary tract. The 101 gills of all live fish were examined by wet mounts by phase contrast microscopy. 102 103 Histopathology: 104 The following tissues were selected from each of five fish at the time of necropsy, 105 trimmed approximately 1 cm in diameter and placed into 10% Neutral Buffered 106 Formalin (1 : 10 ratio of tissue to formalin): gill, brain, eye, kidney, spleen, heart, liver, 107 nares, gonad, esophagus, stomach, intestine, pyloric caeca, pancreas and body wall.

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108 Fixation was completed by holding all samples on a shaker for a minimum of 48 hours 109 prior to routine histology processing. For tissues which contained bone and/or cartilage 110 (cranium, eye, body wall, gill), decalcification was undertaken by immersion for a further 111 24 hours in a commercially available decalcification fluid ( CAL-EX II ® Fisher 112 Chemical ). Sections from all tissues were subsequently trimmed into cassettes, 113 processed to wax blocks, sectioned to 5 µm, mounted/dried onto glass slides and stained 114 by the HematoxylinFor andPeer Eosin (H&E) Review method (Luna, L.G. 1968).Only Selected tissues were 115 recut and stained by the Periodic Acid-Schiff (PAS) technique (Luna, L.G. 1968). 116 Stained slides were examined using a Lieca DM2500 light microscope and, where 117 appropriate, digital images were captured with a top mounted PixeLink PL-B625 digital 118 microscope camera using the PixeLINK® µScope Standard v3.6 software. 119 120 Bacteriology 121 Phenotypic Identification. − A sterile swab of the kidney was provided at necropsy for 122 routine bacteriology. Swabs were streaked onto Blood Agar and Blood Agar containing 123 2 % NaCl, and held at both 22°C and 15°C for a minimum of seven days. Any bacterial 124 growth deemed significant was sub-cultured onto new media and subsequently 125 indentified using routing biochemical and immunologic methodologies (American 126 Fisheries Society-Fish Health Section 2010) 127 128 Mycology 129 Phenotypic Identification .− A sterile swab of the kidney was provided at necropsy for 130 routine mycology. Swabs were streaked out onto Sabouraud’s agar and incubated at 131 room temperature ( ~22°C ) and monitored daily for fungal outgrowth. Obtained axenic 132 fungal isolates were maintained on potato dextrose and Sabouraud’s agar at 22 °C for a 133 two month period. Micromorphology was visualized from cellotape slide mounts 134 prepared in lactic acid observed under 40x and 100x magnification using Phase Contrast 135 microscopy on a Leica DME microscope. Digital photomicrographs were obtained using 136 a Leica EC3 camera (Leica Microsystems, Switzerland) and micromorphological 137 measurements were made from digital photomicrographs using the Leica LAS EZ 138 software (v2.1.0).

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139 140 DNA Extraction and PCR Amplification. − Genomic DNA was obtained from isolated 141 strains using the fast DNA extraction kit (FASTDNA SPIN KIT FOR SOIL®, MP 142 Biomedicals) according to the manufacturer’s protocols. The ITS rRNA gene was 143 amplified by PCR using 50 µL of reaction mixture consisting of 25 µL of Econo Taq ®

144 PLUS GREEN 2× Master Mix (Lucigen), 17 µL of sterile ddH 2O, 2 µL of each primer 145 (ITS-1 andFor ITS-4 (White Peer et al. 1990)) Review and 4 µL of genomic OnlyDNA. Reactions were run in 146 a Biometra thermocycler using: an initial denaturation step at 96 °C for 3 min; 35 cycles 147 of 45s for denaturation at 96 °C, primer annealing at 54.5 °C for 45 s and extension at 72 148 °C for 1 min; and the reaction was completed with a final extension step of 10 min at 72 149 °C. PCR amplicons were checked for correct length and concentration by electrophoresis 150 in 1 % agarose gel in 1× TAE buffer (Tris Base 2.42 g, glacial acetic acid 0.572 mL, 0.5

151 M EDTA 1 mL; add ddH 2O to 500 mL). 152 153 DNA Sequencing and Phylogenetic Analysis. − The ITS amplicons were sent to a 154 commercial sequencing facility (Eurofins MWG Biotech) and sequenced on a 3730xl 155 DNA Analyzer coupled with BigDye Terminator ver. 3.1 Cycle Sequencing reagents, 156 Applied Biosystems (ABI). The generated sequences were compared with other fungal 157 ITS sequences from NCBI’s GenBank sequence database using a Blastn search 158 algorithm. Using the software Molecular Evolutionary Genetics Analysis ver. 5 159 (MEGA5) (Tamura et al. 2011), a dataset was compiled of 42 ITS nucleotide sequences 160 of Exophiala spp. (39 obtained from GenBank) and a sequence alignment was 161 subsequently performed using the ClustalW algorithm with a DNA Gap Open Penalty = 162 15.0, DNA Gap Extension Penalty = 6.66 and a delay divergent cutoff of 30 %. To infer 163 the evolutionary history of the dataset, the Neighbor-Joining method was used to 164 construct a bootstrap consensus tree from 2000 replicates. Evolutionary distances were 165 computed using the Maximum Composite Likelihood method and all ambiguous 166 positions were removed for each sequence pair during analysis. 167 168 169

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170 Results 171 172 Necropsy Findings 173 A total of seven fish were examined: three mortalities and four live moribund specimens. 174 None of the fish showed evidence of significant external lesions to the body proper. Wet 175 mount evaluations of the gills from the four live fish were found to be negative for both 176 bacteria andFor parasites. Peer Lenticular opacity Review was noted in three Only of seven fish and digesta 177 was found in the lumen of the intestine in three of seven fish. Three of seven fish showed 178 significant internal changes as specified below, including two fish with melanized 179 discoloration of the mid-brain including regions of the cerebellum and tegumentum 180 (Figure 1A,C). U30818_F3 was a fresh mortality, which presented on gross examination 181 with flared gills. U29736_F4 was a live female which presented on gross examination 182 with both splenomegaly and serosanguineous ascites. And, U29736_F1 was a female 183 mortality which presented on gross examination with an enlarged kidney that on cut- 184 section oozed out a chalky white fluid and the urinary bladder was filled with a soft 185 mineral-like substance. 186 187 Histopathology Findings 188 U30818_F3. − The gills of this fish showed low frequency, random distribution of 189 respiratory epithelial cells containing a chlamydial-like organism with no associated 190 inflammatory infiltrate. Both kidney and spleen sections contained regions of histiocytic 191 / granulomatous infiltration containing septate, brown-colored dematiaceous hyphae 192 (golden-brown in H&E staining). Brain sections revealed locally extensive multifocal 193 fungal colonization with moderate diffuse histiocytic infiltrate and intracerebral 194 hemorrhage (Figure 1B). No additional significant morphological changes were noted. 195 196 U29736_F4. − This fish presented with a unilateral liquefactive lenticular cataract. No 197 significant morphological changes were noted in tissue sections of the body wall and 198 nares. Brain sections revealed multifocal fungal colonization of the forebrain with 199 evidence of necrosis and granulomatous infiltration (Figure 1B,D), especially prominent 200 in association with the meningeal vasculature (Figure 1E). Tissue sections of the gill

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201 showed marked necrosis, granulomatous infiltrate and hemorrhage in the gill arch and 202 extending into an adjacent base of filaments. The lesion was colonized by a brown- 203 colored dematiaceous septated fungus (Figure 2A,B). Kidney sections contained a 204 locally extensive, necrotizing granulomatous infiltration of the renal interstitium and 205 ureters in the rostral trunk kidney, with a marked colonization by a brown-colored 206 dematiaceous, septate fungus. Fungal hyphae were present in high frequency in both the 207 ureter lumenFor and adjacent Peer peritubular Reviewinterstitial tissue (Figure Only 2D). 208 209 U29736_F1 . − Kidney sections from this fish showed locally extensive necrotizing 210 granulomatous infiltration of the renal interstitium and ureters of the rostral trunk kidney, 211 with a marked fungal colonization of both the ureter lumen and adjacent peritubular 212 interstitial tissue (Figure 2C,D). Fungal hyphae were brown-colored dematiaceous and 213 septate (Figure 2B) . Application of the PAS stain highlighted the morphology of the 214 fungal hyphae (Figure 2D). No significant morphological changes were noted in tissue 215 sections of the ovary, liver, esophagus, stomach, intestine, spleen, liver, heart, nares, 216 brain and eye. 217 218 Bacteriology Findings 219 Moderate to heavy growth of Lactobacillus sp. was recovered on blood agar from the 220 kidney of two fish ( U29736_F4 and U30818_F1 ), all other fish were found to be free of 221 significant bacterial agents. 222 223 Mycology Findings 224 Molecular Identification and Phylogenetic Analysis . − Three dematiaceous fungal 225 isolates were obtained from the kidney samples in each of the three fish submitted 226 (denoted as isolates U29736_F1, U29736_F4, and U30818_F3). Blastn ITS sequence 227 searches against GenBank returned a 100% sequence homology of the three isolates to 228 various isolates of Exophiala angulospora . A dataset of the ITS rDNA gene was 229 compiled and analyzed to infer the relative evolutionary history of isolates U29736_F1, 230 U29736_F4, and U30818_F3 with other representative Exophiala spp (Figure 3). The 231 analysis involved 42 sequences and included 604 positions in the final dataset with an

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232 overall mean distance calculated as 0.095 with a standard error of 0.035. Isolates 233 U29736_F1, U29736_F4, and U30818_F3 formed a well supported clade (100% 234 bootstrap support) with other isolates of Exophiala angulospora , including the type 235 strain, which was phylogenetically distinct from other representative taxa of the genus. 236 Alignment of the ITS sequences revealed a 100% agreement between isolates 237 U29736_F1 and U29736_F4 and the Exophiala angulospora isolates VI05436 and 238 VI03759 (isolatedFor from Peer Atlantic Cod) Review and CBS 11911 (isolated Only from weedy seadragon). 239 Isolate U30818_F3 aligned in 100% agreement with Exophiala angulospora CBS 121503 240 (isolated from the freshwater whitefish Stenodus leucichtys ) and these strains differed 241 from the aforementioned isolates by a single nucleotide basepair substitution. 242 243 Phenotypic Taxonomy . − Morphological phenotypes of the isolates were examined and 244 compared with the published description of Exophiala angulospora (Iwatsu et al. 1991). 245 Colonies were slow growing, olivaceous black and velvety in appearance with a black 246 reverse (Figure 4A) . Conidiogenesis was annellidic with conidia accumulating in slimy 247 masses from intercalary (Figure 4B) and branched (Figure 4D) or unbranched flask- 248 shaped or cylindrical conidiogneous cells that tapered to a short beak at the apex and 249 were septate at the base (Figure 4E). Conidia were single celled and variable in shape, 250 ranging from obovoid and oblong to angular (Figure 4C). Mature hyphae, conidiophores 251 and conidia were dematiaceous. 252 253

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254 Discussion 255 256 Systemic infection caused by a dematiaceous fungus was evident in tissue samples from 257 three of the seven halibut that were submitted for necropsy. From tissue outgrowths, a 258 dematiaceous fungus was isolated and confirmed by both phenotypic examination and 259 ITS sequence homology to be Exophiala angulospora . The lack of significant external 260 lesions on Forthe three fish, Peer as well as the Review consistent occurrence Only of fungal hyphae within the 261 ureter and renal interstitium of the kidney, suggest that the urinary tract was the site of 262 infection ingress prior to the ultimate dissemination of the infection to the brain. In one 263 of the three fish, evidence of fungal infection of the gills may suggest a second locus of 264 fungal ingress, however, it is more likely that the fungus was disseminated to other 265 tissues from the kidney via the circulatory system. Systemic mycosis caused by 266 Exophiala angulospora has been previously described from two marine fish: weedy 267 seadragons ( Phyllopteryx taeniolatus ) and Atlantic cod ( Gadus morhua ). Similar to our 268 observations, in the case of Atlantic cod, ingress was suggested to occur via the urinary 269 tract to the kidney, as the kidney was found to be infected in all of the diseased fish 270 examined (Gjessing et al. 2011). Fungal ingress in weedy seadragons was believed to 271 occur via the gill or skin through preexisting lesions such as abrasions (Nyaoke et al. 272 2009). 273 274 Exophiala angulospora is a ubiquitous fungus, with reports of isolations made from 275 water, decorticated wood, human skin and nails, soils and from diseased fish (de Hoog et 276 al. 2011; Gjessing et al. 2011). Due to the ubiquitous nature of the fungus, it is unclear as 277 to origin of the Exophiala angulospora inoculum found in the diseased Altantic halibut. 278 Determination of the source of the Exophiala angulospora inoculum was outside the 279 scope of this investigation, however, multiple sources are possible: from the stocked 280 juvenile halibut, commercial feed, sea water supply, or surfaces of the rearing facility. In 281 this case the water supply is filtered to remove fungal propagules so that incoming water 282 supply is likely not the source of the agent. The absence of intestinal mucosal lesions, as 283 would be expected from infection via feed, futher suggests that the feed was not the 284 primary source. Since the stocked juveniles were not originally assessed for fungal

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285 agents, it is possible these fish were carriers of the agent and that over time disseminated 286 the fungus throughout the facility and colonizing tank walls and piping. As the kidney 287 and ureter pathology supported initial infection via the urinary tract, it is likely that the 288 benthic distribution of the fish within the rearing tanks contributed to this route of 289 infection. Thus it could be hypothesized the the agent most likely gained access to the 290 halibut via the urinary pore from the biofilm of the tank surfaces. To confirm of disprove 291 this hypothesisFor additional Peer epidemiological Review investigation would Only be useful, as infections by 292 Exophiala angulospora clearly pose a pathological threat to onshore rearing of Atlantic 293 halibut. 294 295 Species specific primers have been successfully applied in the molecular diagnosis of 296 fungal infections from a variety of symptomatic tissues (Atkins and Clark 2004). 297 Phylogenetic analysis of the ITS gene demonstrated that this region is suitable for the 298 identification of Exophiala spp. and distinguishes Exophiala angulospora from all other 299 taxa of the genus. Alignment of the Exophiala ITS dataset indicated that sufficient 300 sequence variance between taxa is present in loci of both the ITS1 and ITS2 regions, 301 while these regions remained conserved within species, and therefore are ideal for 302 forward and reverse, species specific primer development. Extraction of gDNA from 303 urinary tracts routinely collected from culled fish during harvest and PCR amplification 304 using Exophiala angulospora specific primers would allow for adequate surveillance and 305 rapid diagnosis of disease outbreak. 306

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307 References 308

309 American Fisheries Society-Fish Health Section. 2010. FHS blue book: suggested 310 procedures for the detection and identification of certain finfish and shellfish pathogens, 311 2010 edition. AFS-FHS, Bethesda, Maryland.

312 Atkins, S. ForD., and I. PeerM. Clark. 2004. Review Fungal molecular Only diagnostics: a mini review. 313 Journal of Applied Genetics 45:3–15. 314 315 de Hoog, G. S., V. A. Vicente, M. J. Najafzadeh, M. J. Harrak, H. Badali, and S. 316 Seyedmousavi. 2011. Waterborne Exophiala species causing disease in cold-blooded 317 animals. Persoonia 27:46–72. 318 319 Domsch, K. H., W. Gams, and T. H. Anderson. 2007. Compendium of soil fungi, 2 nd edn. 320 IHW-Verlag, Eching. 321 322 Gjessing, M. C., M. Davey, A. Kvellestad, and T. Vralstad. 2011. Exophiala angulospora 323 causes systemic inflammation in Atlantic cod Gadus morhua . Diseases of Aquatic 324 Organisms 96:209–219. 325 326 Iwatsu, T., S. Udagawa, and T. Takase. 1991. A new species of Exophiala recovered 327 from drinking water. Mycotaxon 41:321–328. 328 329 Luna, L.G. 1968. Manual of histologic staining methods of the Armed Forces Institute of 330 Pathology. Third Edition. McGraw Hill Book Corp. Toronto. Pages 38-39 and 158. 331 332 Nyaoke, A., E. S. Weber, C. Innis, D. Stremme, C. Dowd, L. Hinckley, T. Gorton, B. 333 Wickes, D. Sutton, S. de Hoog, and S. Frasca. 2009. Disseminated phaeohyphomycosis 334 in weedy seadragons ( Phyllopteryx taeniolatus ) and leafy seadragons ( Phycodurus eques ) 335 caused by species of Exophiala , including novel species. Journal of Veterinary 336 Diagnostic Investigation 21:69–79.

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337 338 Tamura, K., D. Peterson, N. Peterson, G. Stecher, M. Nei, and S. Kumar. 2011. MEGA5: 339 molecular evolutionary genetics analysis using maximum likelihood, evolutionary 340 distance, and maximum parsimony methods. Molecular Biology and Evolution 28:2731– 341 2739. 342 343 Uijthof, J.For M. J., M. Peer J. Figge, G. Review S. de Hoog. 1997. MolecularOnly and physiological 344 investigations of Exophiala species described from fish. Systematic and Applied 345 Microbiology 20:585–594. 346 347 White, T. J., T. Bruns, S. Lee, and J. Taylor. 1990. Amplification and direct sequencing 348 of fungal ribosomal RNA genes for phylogenetics. Pages 315–322 in M. A. Innis, D. H. 349 Gelfand, J. J. Sninsky, and T. J. White, editors. PCR protocols: A guide to methods and 350 applications. Academic Press, New York, USA.

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1 Figure 1:

2 Gross and microscopic images of brain with encephalitis due to infection with Exophiala

3 angulospora in Atlantic halibut. (A) and (C) gross mid-sagittal section through formalin fixed

4 infected brain ( b) from 2 different fish, with circled area showing melanization of affected 5 neuropile; (B) Forinflammation Peer of neuropile Reviewwith perivascular hemorrh Onlyage (arrow) ; (D) higher 6 magnification of neuropile with fungal hyphae (arrows) ; (E) PAS staining of fungal hyphae

7 (arrow) in affected neuropile.

8 Figure 2:

9 Microscopic image of the gill with granulomatous and hemorrhagic branchitis (arrows) due to

10 infection with Exophiala angulospora in Atlantic halibut (A) , and (B) higher magnification of

11 affected gill showing melanized intralesional fungal hyphae (arrow) . (C) Microscopic image of

12 the trunk kidney with necrotizing granulomatous interstitial nephritis and transmural intratubular

13 nephrocystitis due to infection with Exophiala angulospora in Atlantic halibut. (Ur) indicates

14 central region of the affected urinal duct, containing mineral deposits or calculi as well as fungal

15 hyphae. (D) higher magnification of an affected ureter with PAS stained fungal hyphae (arrow) .

16 Figure 3:

17 Bootstrap consensus tree inferred from 2000 replicates using the neighbor-joining method based

18 on Exophiala spp. ITS rDNA sequences. The percentage of replicate trees (>50 %) in which the

19 associated taxa clustered together in the bootstrap tests of 2000 replicates are shown next to the

20 branches. Evolutionary distances were computed using the maximum composite likelihood

21 method and are in the units of the number of base substitutions per site. The tree was rooted with

22 Ceramothyrium melastoma (CBS 133576).

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24 Figure 4:

25 Morphology of Exophiala angulospora isolated from Atlantic halibut (isolate U29736_F1).

26 Gross colony morphology (A) on PDA after 14 days incubation at 22 °C (scale=1mm).

27 ConidiogenesisFor was annellidic Peer from intercalary Review (B) , branched (D) Only, or unbranched (E) flask-

28 shaped or cylindrical conidiogenous cells (scale=10µm). Conidia were single celled and variable

29 in shape ranging from obovoid and oblong to angular (C) (arrows) .

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A B

C D

E Figure 1

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C D

Figure 2

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Figure 4

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