NORTH-WESTERN JOURNAL OF ZOOLOGY 16 (2): 216-219 ©NWJZ, Oradea, Romania, 2020 Article No.: e202501 http://biozoojournals.ro/nwjz/index.html

First report of water mold (Aphanomyces sp.) documented on skin of pool frog (Pelophylax lessonae) in Serbia

Miloš STUPAR*, Katarina BREKA, Imre KRIZMANIĆ, Srđan STAMENKOVIĆ and Milica LJALJEVIĆ GRBIĆ

University of Belgrade, Faculty of Biology, Studentski trg 16, 11000 Belgrade, Serbia. * Corresponding author, M. Stupar, E-mail: [email protected]

Received: 25. October 2019 / Accepted: 27. January 2020 / Available online: 25. January 2020 / Printed: December 2020

Abstract. In the last several decades, numerous factors contributing to amphibian populations decline have been recorded, including emerging infectious diseases. The aim of this study was to investigate integument-associated microbiota of the P. esculentus complex from the locality Jaruga in South Banat (Serbia) using non-aggressive adhesive tape method. Morphological structures (hyphae, zoosporangia, primary and secondary zoospores) belonging to a water mold from the genus Aphanomyces which is a causative agent of aphanomycosis in amphibians, were detected on the skin of Pelophylax lessonae, the only member of the P. esculentus complex included in the National red book and supposedly the most susceptible to environmental threats in Serbia.

Key words: Anura, aphanomycosis, pathogen, skin infection.

The members of genus Aphanomyces de Bary (formerly Oo- chochytrium dendrobatidis (Bd) Longcore, Pessier & D.K. mycota, order: Saprolegniales), are eukaryotic microorgan- Nichols and ranavirus. Kolenda et al., (2017) reported that isms, morphologically similar to fungi, and hence known as Bd infection was associated with reduced fitness in wild “water molds”, but genetically related to brown algae populations of P. lessonae in Poland. According to Vörös et (Gleason et al. 2018) or straminipilous fungi (Dick 2001) in al. (2018), Bd was documented on the members of P. esculen- Kingdom Straminipila (Beakes 2012, 2014). Aphanomyces wa- tus complex in Hungary. Baláž et al. (2014) reported Bd on ter molds are frequently isolated from freshwater, as sap- members of P. esculentus complex in Czech Republic and robes and colonizers of dead plant and animal remains sub- showed that Pelophylax was one of the most commonly in- merged in water. However, Aphanomyces spp. could be op- fected genera. According to Mali et al. (2017), Bd is also pre- portunistic pathogens associated with cutaneous injuries of sent in green frog populations in Serbia with the highest in- aquatic and semi-aquatic animals, i.e. crustaceans, fishes and fection rate in P. lessonae but not from this region. The causa- amphibians (Scott 1961, Takuma et al. 2010). Among, the tive agent of amphibian chromomycoses, Fonsecaea Negroni best-described animal pathogen Aphanomyces species are A. sp., was detected on P. esculentus in Serbia (Stupar et al. astacci Schikora, the causative agent of crayfish plague 2017). To our knowledge, other fungal and fungal-like path- (Strand et al. 2012), as well as A. laevis de Bary and A. in- ogens, including causative agents of amphibian aphano- vadans Willoughby, R.J. Roberts & Chinabut, both fish path- mycosis or saprolegniosis on P. esculentus complex have not ogens (Buller 2015). Sinmuk et al. (1996) described Aphano- been reported. myces infection of Chinese softshell turtle, Pelodiscus sinensis The main goal of this research was to investigate the Wiegmann. Interactions of Aphanomyces spp. as a pathogen “hidden dwellers” of the P. esculentus complex skin via ap- with amphibians are seldom reported. While saprolegniasis plication of the non-harmful adhesive tape method, which is the general term used to describe amphibian infections provides minimal stress to the studied animal, and could caused by the Aphanomyces spp. and related genera, Sapro- lead to recognition and registration of novel potential patho- legnia Nees, Achlya Nees and Leptolegnia de Bary (Pessier gens of amphibians in this region. 2002, Paré 2003), the term aphanomycosis, proposed by Baldock et al. (2005), is used exclusively for Aphanomyces in- A total of 24 individuals belonging to Pelophylax esculentus complex fections. Berger et al. (2001) reported that Aphanomyces sp. were collected in 2017 on the locality Jaruga, South Banat, Serbia was the pathogen responsible for the mucocutaneous fungal (near the village Kusić ,44°52'30.8"N 21°28'16.0"E). Jaruga is a canal formed to regulate floodwaters of the Nera river and to meliorate ag- disease of the cane toad Rhinela marina L. tadpoles in north ricultural surrounding areas. Losing its primary purpose, the flow of Queensland (Australia), and this finding is regarded as the the canal was reduced and overgrown with macrophyte vegetation. first report of aphanomycosis in anurans. Also, cutaneous Frogs were collected from the canal as well as from flood–meadows ulcers described on the skin of the spotted salamander along the canal, individually stored in denim sacks and transported Ambystoma maculatum Shaw were caused by Aphanomyces sp. to a field laboratory. The identification of frogs was based on their (Pessier 2002). To our knowledge, there are no other reports quantitative and qualitative attributes following Krizmanić (2008). regarding the presence of members of genus Aphanomyces on Integument-associated microbiota was sampled superficially the skin of adult anurans. from five different skin areas (dorsal and ventral side, head, fore- and hind limbs), using the adhesive-tape method, specially modified The Pelophylax esculentus complex consists of two species for amphibian skin investigations by Stupar et al. (2017). Before Pelophylax lessonae Camerano and Pelophylax ridibundus Pal- sampling captured specimens were rinsed with sterile distilled wa- las and a hybridogenetic form between these two species – ter. Standard mycological dye LactophenolCottonBlue (LCB) was Pelophylax kl. esculentus Linnaeus. Although P. lessonae and used for mounting the adhesive tape samples. Optical microscopy P. ridibundus are widespread in Europe and Serbia, data on (Nikon Eclipse E200, equipped with camera BresserMikroCam PRO their skin microbiota are scarce. Recent studies were mainly HDMI, Japan) was used for detailed analyses of samples. All ob- focused on the detection of amphibian chytrid fungus, Batra- served constituents of frog-skin biofilm were photographed and for Water mold found on pool frog skin 217 the proper identification of frog skin dwellers, the following keys mented. Detailed microscopical analyses of the integument were used: Scott 1961, Khulbe 2001, Buller 2015. Adhesive-tape slides of a single specimen identified as female Pelophylax lessonae are preserved and kept in the slide collection of the Department for revealed the presence of coenocytic mycelium, with morpho- Algology, Mycology and Lychenology, Faculty of Biology, Universi- logical features of water molds. Moderately branched, deli- ty of Belgrade. After sampling, all captured specimens were released within 12 cate hyaline aseptate hyphae (8-10 µm Ø) were found on the hours at the capture sites. skin. Isodiametric zoosporangia, with the same diameter as the hyphae were also observed. Two types of zoospores Microscopical analyses of samples taken directly from cap- were recorded: the primary, barrel-shaped zoospores (10 x tured P. esculentus complex specimens revealed the presence 20 µm) in a single row and linked with cytoplasm strands of organic and inorganic material, soil particles, plant frag- (Fig 1c) within the zoosporangia, encysted within the ments originating from submerged vegetation, and attached achlyoid-type clusters formed at the apical tips, and the glo- to amphibian skin, but also, various microorganisms, mostly bose or subglobose secondary zoospores (8 µm Ø) (Fig 1a,b). fungi, fungal-like organisms and algae. Although the majori- The sexual stages (oogonium and antheridia) were not found ty of observed microorganisms could be regarded as transi- in examined samples, suggesting that frog skins were sam- ents, autochthonous frog skin microbiota and potential pled prior to the molds undergoing sexual reproduction, or pathogens were detected in investigated samples. Fungal that sexual reproduction of water molds does not occur on structures recorded in this research were, predominantly, amphibian skin. Based on documented morphological fea- spores of air-borne and soil-borne fungi, and in some rare tures of the asexual stage (morphology and formation of cases, spore germination and mycelia formation were docu- primary zoospores), the water mold was identified as repre-

Figure 1. Water mold structures documented directly on the Pelophylax lessonae integument a) Achlyoid clusters with secondary zoospores b) Secondary zoosporangium c) Primary zoospores linked with cytoplasm strands within prima- ry zoosporangium. 218 M. Stupar et al.

sentative of genus Aphanomyces de Barry. The identification ever, Vujičić et al. (2013) pointed out, that according to litera- of Aphanomyces spp. to the species level based solely on ture data presented by Alderman (1996), the A. astacii, was morphological criteria is rather difficult, and requires isola- for the first time introduced through the Danube River to the tion and further molecular analyses. Analyses of other frogs territory of Banat and Bačka (today Republic of Serbia) and captured on Jaruga revealed no presence of water molds as- in 1880 spread through the Sava River to northern Bosnia. sociated with frog integument. Also, Čomić et al. (2010) reported the presence of A. leavis, Although hyphal invasion into dermal layers of exam- the causative agent of cotton-wool disease in fish, in Lake ined P. lessonae specimens was not documented, and no visi- Ohrid (North Macedonia). The distribution of Aphanomyces ble symptoms (such as skin lesions, ulcers, cottony appear- spp. in aquatic habitats in Serbia and the surrounding Bal- ance) were present on the specimen’s skin, the obtained evi- kan countries is presumably wider and interactions of dence that water mold species, a potential causative agent of Aphanomyces spp. with crustaceans, fishes, amphibians, and aphanomycosis, could grow directly on amphibian skin, turtles in this region require further studies. must not be neglected. Also, for the members of the genus This study confirms the adhesive tape method as a non- Aphanomyces the process called repeated zoospore emer- aggressive and readily available sampling technique, proven gence (RZE) as an adaptation to parasitism has been de- to be adequate for studying skin microbiota of green frogs scribed (Iberahim et al. 2018). Encysted secondary zoospores and identifying novel pathogens. In the presented case, a formed in achlyoid clusters are capable of releasing a new water mold from the genus Aphanomyces, the causative agent zoospore generation instead of germinating and developing of aphanomycosis, is recorded for the first time on the skin mycelium. The RZE continues until the next suitable host or of the Pool frog (Pelophylax lessonae). This result is significant substrate is found. It might be assumed that, if the studied since P. lessonae is the only species of P. esculentus complex specimen of P. lessonae is not affected by the species causing listed in The Red Book of Fauna of Serbia and potentially the aphanomysocis pathogenic to frogs, it could probably be re- most vulnerable. garded as a natural reservoir of infection for other aquatic and semi-aquatic animals. It is important to emphasize that

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