Veterinary Microbiology 158 (2012) 109–122 Contents lists available at SciVerse ScienceDirect Veterinary Microbiology jo urnal homepage: www.elsevier.com/locate/vetmic Black yeast-like fungi associated with Lethargic Crab Disease (LCD) in the mangrove-land crab, Ucides cordatus (Ocypodidae) a,b b,c,d a,e f Vania A. Vicente , R. Ore´lis-Ribeiro , M.J. Najafzadeh , Jiufeng Sun , b,d a d g Raquel Schier Guerra , Stephanie Miesch , Antonio Ostrensky , Jacques F. Meis , g a,h,i,j, c,d, Corne´ H. Klaassen , G.S. de Hoog *, Walter A. Boeger ** a CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands b Department of Basic Pathology, Federal University of Parana´, Curitiba, PR, Brazil c Department of Zoology and Molecular Ecology and Parasitology, Federal University of Parana´, Curitiba, PR, Brazil d Laborato´rio de Ecologia Molecular e Parasitologia Evolutiva, Grupo Integrado de Aqu¨icultura e Estudos Ambientais, Federal University of Parana´, Curitiba, PR, Brazil e Department of Parasitology and Mycology, and Cancer Molecular Pathology Research Center, Ghaem Hospital, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran f Key Laboratory of Tropical Disease Control, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, China g Department of Medical Microbiology and Infectious Diseases, Canisius-Wilhelmina Hospital, Nijmegen, The Netherlands h Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, Amsterdam, The Netherlands i Peking University Health Science Center, Research Center for Medical Mycology, Beijing, China j Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China A R T I C L E I N F O A B S T R A C T Lethargic Crab Disease (LCD) caused extensive epizootic mortality of the mangrove land Article history: Received 29 July 2011 crab Ucides cordatus (Brachyura: Ocypodidae) along the Brazilian coast, mainly in the Received in revised form 26 January 2012 Northeastern region. The disease was named after the symptoms of slow movement of Accepted 31 January 2012 infected crabs. Causative agents were suspected to be two black yeast-like fungi of the family Herpotrichiellaceae (ascomycete order Chaetothyriales), judged by infected tissue Keywords: biopsies from moribund U. cordatus. The aim of the present study is to prove that two Ucides cordatus species are involved in the disease: the recently described black yeast Exophiala cancerae, Mangrove crab but also a less virulent, hitherto undescribed fonsecaea-like species, introduced here as the Lethargic Crab Disease (LCD) novel species Fonsecaea brasiliensis. Strains were identified by ITS rDNA sequencing, and Exophiala cancerae species borderlines were established by multilocus sequencing and AFLP analysis. Fonsecaea brasiliensis Virulence Fonsecaea brasiliensis proved to be closely related to the pathogenic species Cladophia- lophora devriesii which originally was isolated from a systemic infection in a human patient. The virulence of F. brasiliensis is lower than that of E. cancerae, as established by artificial inoculation of mangrove crabs. ß 2012 Elsevier B.V. All rights reserved. 1. Introduction Since the year 1997 an extensive epizootic causes high * Corresponding author at: CBS-KNAW Fungal Biodiversity Centre, P.O. mortality of the mangrove-land crab Ucides cordatus Box 85167, NL-3508 AD, Utrecht, The Netherlands. Tel.: +31 30 2122 663; (Brachyura: Ocypodidae) along the Brazilian coast. The fax: +31 30 251 2097. ** Corresponding author at: Department of Zoology, Federal University disease was termed Lethargic Crab Disease (LCD) by Boeger of Parana´, Curitiba, PR, CEP 81531–980, Brazil. Tel.: +55 41 33611765; et al. (2005) because of clinical signs of ataxia observed in fax: +55 41 33611765. moribund organisms. The histopathology of crabs in E-mail addresses: [email protected], [email protected] diverse stages of development of the disease shows that (G.S. de Hoog), [email protected] (W.A. Boeger). 0378-1135/$ – see front matter ß 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2012.01.031 110 V.A. Vicente et al. / Veterinary Microbiology 158 (2012) 109–122 the most affected tissues are the epidermis, connective tissues, heart, and hepatopancreas, and that the fungus disseminates hematogenously (Boeger et al., 2007). The ) main signs of disease include increasingly weak motor control, particularly of pereiopods and chelae, causing lethargy and poor balance, followed by the death of the BT2, TEF1 affected crab. Tetany of the claws is also observed in many crabs with other signs of the disease. Despite the importance of the crab species for low- income populations in the coastal regions of Brazil, only JF747063, JN112443, – JF747064, JN112444, – JN173784, JN368478, JN187943 JN173785, JN368479, JN187944 JN173787, JN368481, – JN173788, JN368480, JN187945 few scientific studies addressing LCD are available (e.g. Pie et al., 2011; Ore´lis-Ribeiro et al., 2011; Boeger et al., 2005). All evidence from a variety of sources (light and electron microscopy, behavioral and experimental tests, and molecular phylogenetics) indicate that LCD signs were associated with the presence of a black yeast-like fungus, Exophiala cancerae (de Hoog et al., 2011). However, occasional co-infection may take place by another black o state, Icatu city JN173791, –, – o state, Icatu city JN173792, –, – ˜ ˜ yeast-like fungus, an as yet unknown species of Fonsecaea. Exophiala and Fonsecaea are members of the ascomy- cete order Chaetothyriales, comprising the black yeasts and allies, which are regularly encountered as causative agents Grande city, mangrove Goiana city, mangrove Grande city, mangrove Grande city, mangrove Grande city, mangrove Grande city, mangrove of disorders in humans and animals (Nyaoke et al., 2009; de Hoog et al., 2011; Kano et al., 2000). In humans, infections by chaetothyrialean fungi range from commensalism or mild cutaneous infection to fatal neurotropism with severe mutilation. All infections are preponderantly found in patients without immune disorder or underlying metabolic disease. Outside humans, particularly cold-blooded water- borne vertebrates are susceptible to a diversity of Exophiala species (de Hoog et al., 2011). Some of these are host-specific to particular fish taxa (Nyaoke et al., 2009). As virulence factors the ability to assimilate alkylbenzenes, present in sweat and nervous tissues of mammals and in the toxic skin of amphibians, has been suggested (Prenafeta-Boldu´ et al., Toad liver Israel JF747065, JN112445, – 2006; de Hoog et al., 2011). Many reports of epizootics from the older literature clearly demonstrate that black yeast infection is a relatively common phenomenon in cold-blood vertebrates (e.g. Fijan, 1969; Richards et al., 1978; Reuter et al., 2003). Recent studies have shown that different pathogenic species are involved (e.g. Manharth et al., 2005; Nyaoke et al., 2009; de Hoog et al., 2011) which are morpholo- gically very similar. Vakili (1993) was the first to isolate an Cunningham 179/99 Exophiala species from invertebrate hosts (earthworms); recently Dover et al. (2007) reported on an epizootic in mussels by a black yeast. The mangrove crab epizootic species involved in Lethargic Crab Disease (LCD), with reference isolates. (Boeger et al., 2005, 2007) underlines the ability of black yeasts to infect a larger spectrum of animals. The present study clarifies the etiology of the dis- CBS 120532CBS 120420 (T) dH 17408 = HFIICBS 119920 dH 17409 = HFCBS 119710 (T) IMI 380731 = CBS 119718 dH 16818 = Crab isolate 3/2 HCBS 120419 Crab Crab dH 16816 = isolateCBS 1/2H 120418 dH 17407 = 2ECBS Crab 16/08 127898CBS 127905 dH 17406 = 1GCBS 16/08 127896CBS 127904 Crab dH 21422 = R32A1CBS 127819 dH 21423 = R43B1 Crab dH 21421 = R27A1 dH21431 = R42B1 dH 21198 Mangrove Brazil, Branch Sergipe in state, mangrove Brejo Branch in Brazil, mangrove Pernambuco state, Branch Brazil, in Sergipe mangrove state, Brejo Brazil, Sergipe state, Soil Brejo in rotten stem of palm Brazil, Sergipe state, Brazil, Brejo Bahia state, Acupi Brazil, Bahia Brazil, state, Sergipe Canavieiras state, Brazil, Brazil, Brejo Bahia Bahia state, state, Acupi Canavieiras Brazil, Maranha CBS 126872 dH 20498 Leaf of Babassu palm tree Brazil, Maranha Exophiala seminated LCD in mangrove-land crabs, describes the epidemiology of the disease and establishes the relative and pathogenicity of the agents. The undescribed fonsecaea- like species is introduced here as a novel taxon. Fonsecaea 2. Materials and methods 2.1. Fungal strains A total of 84 strains (Table 1) were analyzed, the set Name Strain number Cross ref. number Source Geography GenBank (ITS, Exophiala cancerae E. cancerae E. cancerae Fonsecaea brasiliensis F. brasiliensis F. brasiliensis F. brasiliensis F. brasiliensis F. brasiliensis F. brasiliensis F. brasiliensis F. brasiliensis F. brasiliensis including three isolates of E. cancerae and four of an Table 1 Analyzed strains of F. brasiliensis CBS 126729 dH 20490 Leaf of Babassu palm tree Brazil, Maranha˜o state, Pinheiros, JN173795, –, – village of Fortaleza F. brasiliensis CBS 126723 dH 20497 Leaf of Babassu palm tree Brazil, Maranha˜o state, JN173794, –, – Icatu city F. brasiliensis CBS 126732 dH 21169 Leaf of Babassu palm tree Brazil, Maranha˜o state, JN173793, –, – Icatu city F. brasiliensis CBS 127813 dH 20515 = VM20 Bark of Babassu coconut palm tree Brazil, Maranha˜o state, Icatu city F. brasiliensis CBS 126741 dH 20482 = VM35A5 Leaf of Babassu palm tree Brazil, Maranha˜o state, Bacabera city F. brasiliensis