Fusarium species isolated from post-hatchling loggerhead sea turtles (Caretta caretta) in South Africa

Species in the Fusarium solani species complex are fast growing, environmental saprophytic fungi. Members of this genus are filamentous fungi with a wide geographical distribution. Fusarium keratoplasticum and F. falciforme have previously been isolated from sea turtle nests and have been associated with high egg mortality rates. Skin lesions were observed in a number of stranded, post-hatchling loggerhead sea turtles (Caretta caretta) in a rehabilitation facility in South Africa. Fungal hyphae were observed in epidermal scrapes of affected turtles and were isolated. The aim of this study was to characterise the Fusarium species that were isolated from these post-hatchling loggerhead sea turtles (Caretta caretta) that washed up on beaches along the South African coastline. Three gene regions were amplified and sequenced, namely the internal transcribed spacer region (ITS), a part of the nuclear large subunit (LSU), and part of the translation elongation factor 1 α (tef1) gene region. Molecular characteristics of strains isolated during this study showed high similarity with Fusarium isolates, which have previously been associated with high egg mortality rates in loggerhead sea turtles. This is the first record of F. keratoplasticum, F. falciforme and F. crassum isolated from stranded post-hatchling loggerhead sea turtles in South Africa.

www.nature.com/scientificreports/ falciforme and F. keratoplasticum of clade III, have been described as emerging animal pathogens, causing both localised and systemic infections 6,16,17,23 . These infections can result in mortality rates as high as 80-90% in animal populations 7,17 . Cafarchia and colleagues (2019) suggested that fusariosis should be included in differential diagnosis of shell and skin lesions in sea turtles and that species level identification is required to administer appropriate treatment and infection control 12 . Loggerhead sea turtles nest on the beaches of Southern Africa between November and January 24,25 . Hatchlings that find their way into the ocean are carried south in the Aghulas current, with some turtles stranding on the South African coast, mainly between the months of March and May each year. Between 2015 and 2016, a total of 222 post-hatchling (turtles that have absorbed the yolk-sac and are feeding in open ocean but have yet to return to coastal waters to enter the juvenile stage) loggerhead sea turtles were admitted to a rehabilitation centre after stranding along the Indian and Atlantic Ocean coastline of South Africa, between Mossel Bay and False Bay (Fig. 1). During their time at the rehabilitation centre a number of these turtles developed skin lesions. Fungal dermatitis was diagnosed based on skin scrape cytology findings. Fungal strains resembling Fusarium were isolated from the affected areas.
The aim of this study was to characterise the strains isolated from skin lesions of post-hatchling loggerhead Sea turtles that washed up on beaches along the South African coastline, and to determine the molecular relationships between these isolates and those strains reported from literature that pose significant conservation risks to sea turtles from other geographic localities.

Materials and methods
Gross observations and Fungal isolations. Post-hatchling turtles with skin lesions were isolated from unaffected turtles. Clinical signs observed were as follows; excessive epidermal sloughing on the limbs, head and neck, where scales on the skin lifted easily and were frequently lost. A softening and sloughing of the carapace and plastron were observed, where scutes of the carapace and plastron became crumbly, soft and were frequently shed. Turtles were diagnosed with fungal skin infection if they had clinical signs of epidermal sloughing and a positive epidermal scrape. Epidermal scrapes taken from lesions of affected turtles were examined by light microscopy (20 to 50 × objective) and deemed positive if significant numbers of hyphae were observed. For fungal isolation, samples (scrapings) were taken from affected areas of skin in a sterile manner and placed onto culture media. During 2015 and 2016, 10 fungal isolates were isolated from 10 clinically affected loggerhead sea turtles (Caretta caretta) onto marine phycomycetes isolation agar (12.0 g Agar, 1.0 g Glucose, 1.0 g Gelatin hydrolysate, 0.01 g Liver extract, 0.1 g Yeast extract, 1 000 mL Sea water) supplemented with streptomycin sul-    Table 1 for specific annealing temperatures) and 72 °C for 1 min, followed by a final extension at 72 °C for 7 min. Purified PCR products were sequenced by using BigDye Terminator Cycle Sequencing Kit (Applied Biosystems) and an ABI PRISM 310 genetic analyser. Sequencing was done in one direction. Each sequence was edited in BioEdit sequence alignment editor v7.2.5. Phylogenetic analyses were conducted using the dataset from Sandoval-Denis et al. (2019) combining sequences of three loci (LSU, ITS and tef1) to identify species [28][29][30][31][32] (Table 2 lists all the sequences included in the phylogenetic analyses). Alignments were done in ClustalX using the L-INS-I option.
Phylogenetic analysis was performed using Maximum likehood (ML) analysis, with GTR + I + G. The partitioning scheme and substitution models were selected using Partitionfinder v 2.1.1 33 . The software package PAUP was used to construct the phylogenetic trees and confidence was calculated using bootstrap analysis of 1 000 replicates. Geejayessia atrofusca was used as an outgroup. A Bayesian analysis was run using MrBayes v. 3.2.6 34 . The analysis included four parallel runs of 500 000 generations, with a sampling frequency of 200 generations. The posterior probability values were calculated after the initial 25% of trees were discarded.
Morphological observation. Agar plugs (6 mm diameter) of the selected isolates were transferred onto fresh PDA and Carnation leaf agar (CLA) plates and incubated at 26 °C ± 1 °C for 7 and 21 days, respectively for further morphological characterisation. Morphological characterisation was based on the taxonomic keys of Leslie and Summerell, 2006 35 . Gross macro-morphology of all isolates was examined on PDA after 7 days, this comprised (i) colony colour on top of the plate, (ii) colony colour on the reverse side (iii) colony size and (iv) texture of the hyphal growth. With a primary focus on 3 strains namely ML16006, ML16011 and ML16012. Micro-morphological evaluation of the respective isolates was achieved by examining CLA plates in situ under the 20X or 40X objective, using a Nikon eclipse Ni compound microscope. The following characteristics were noted: (i) microconidia; shape, size, number of septa and their arrangement on phialide cells (ii) macroconidia; shape, size, number of septa and the shape of the apical and basal cells (iii) sporodachia; when present colour was noted and (iv) chlamydospores; texture of cell walls, position on hyphae and the arrangements. The length and the width of 30 micro-and macroconidia were measured for each isolate (Online Resource 1). The oval shape of the microconidia was measured by drawing a straight line from top to the bottom for the length and the width was measured across the septa or when no septa was observed, at the widest part of the cell. The length of the macroconidia was measured by drawing a straight line from the apical side of the cell to the basal side of the cell. The width was measured at the apical side of the middle septa. Conidia and chlamydospores were mounted on glass slides using water as mounting medium from fungal structures grown on carnation leaf agar 36 and photographed.
All methods were carried out in accordance with relevant guidelines and regulations. All experimental protocols were approved by a named institutional and/or licensing committee.  Chlamydospores were first observed after 14 days of incubation on CLA plates, and were globose in shape with rough walls, positioned terminally, sometimes single but mostly in pairs. Distinct hyphal coils were observed in all three strains (Fig. 4). The morphology is consistent with that described for N. crassum 28 (Fig. 4).

Discussion
Fusarium infections, specifically F. keratoplasticum and F. falciforme have been reported from infected eggs and embryos of turtle species, including endangered species, at major nesting sites along the Atlantic, Pacific and Indian Oceans, as well as the Mediterranean and Caribbean Sea 15,16,18,20,21,37 . Management strategies to mitigate emerging fungal diseases, like Fusarium infections in turtle eggs, are influenced by identifying whether a pathogen is novel or endemic and the understanding of its ecology and distribution. A novel pathogen gains access to and infects naïve hosts as a result of migration of the pathogen or the development of novel pathogenic genotypes, in contrast endemic pathogens occur naturally in the host's environment, but shifts in environmental conditions and/or host susceptibility influence pathogenicity 37 . Thus, effective management strategies to mitigate novel pathogens should aim at preventing pathogen introduction and expansion, while disease caused by endemic pathogens relies on an understanding of environmental and host factors that influence disease emergence and severity 37 . Phylogenetic analysis provides important information to assist in understanding the ecology, introduction and distribution of infectious agents 37,38 . The first aim of this study was to use multigene phylogenetic analyses to identify Fusarium strains isolated from the carapace, flippers, head, and neck area of post-hatchling loggerhead sea turtles (Caretta caretta) with fungal skin infections that stranded along the South African coastline and kept at a rehabilitation centre. The genus Fusarium was recently revised, with an attempt to standardise the taxonomy and nomenclature after a lack of formal species descriptions, Latin names and nomenclatural type  Table 2. Fusarium strains included in the phylogenetic analyses. www.nature.com/scientificreports/ specimens were identified 39 . Strains from this study grouped with three Fusarium species of which two species, F. keratoplasticum and F. falciforme, were previously reported to occur on animal hosts, including turtles. The third species, F. crassum is rather surprising as this species is only known from a human toenail and nematode eggs, while the origin of the type strain is unknown. Three strains (ML16011, ML16012 and ML16006) grouped with two F. crassum strains. Strain identifications were confirmed with the morphological characteristics that agreed with species descriptions published in 2019 28 , with the one exception of chlamydospore wall texture for F. crassum. Chlamydospore walls in this study for all three F. crassum strains were smooth, while previously it has been documented with a rough texture. Turtle egg fusariosis (STEF) is a disease that has increasingly been reported over the last decade and is considered a potential conservation threat to six out of seven species of marine turtles 16,37 . Skin disease and systemic infections caused by Fusarium species has been reported in adult and subadult turtles and in captive reared hatchlings 5,[11][12][13][14][15]19 , but has not been reported in post-hatchling loggerhead sea turtles (C. caretta) undergoing rehabilitation. Clinical signs reported in juvenile, subadult and adult loggerhead sea turtles (C. caretta) with Fusarium infections were localised and generalised lesions of the skin and carapace, consisting of areas of discolouration and loss of shell 12 . Clinical signs observed in post-hatchling loggerhead sea turtles (C. caretta) in this study were similar, but generalised sloughing of scales on the limbs and head, and a soft, crumbly carapace and plastron were more common than focal lesions. Histopathology was not performed in this study to confirm the association of fungal hyphae with pathological changes in the skin, and, therefore, the role of the Fusarium isolates in the skin lesions cannot definitively be identified (as isolation of fungus could be from normal skin flora or the environment), however, fungal hyphae, often in dense mats, were seen in epidermal scrapes from affected turtles (Online Resource 2). Although Fusarium isolates (and other fungi) have been identified in the skin of healthy adult C. caretta 12 , a finding of numerous hyphae (hyphal mats) in skin scrapings would not be considered a normal finding in healthy turtle skin and thus it is considered likely that the fungal elements observed, and therefore the isolates identified, were associated with the observed pathology. The epidemiology of turtle pathogenic isolates F. keratoplasticum and F. falciforme in sea turtle nesting sites are not fully understood 37 , however, it has been suggested that tank substrates and/or biofilms forming in the water supply infrastructure or filtering systems may act as a source of infection, to traumatised and immunocompromised sea turtles 11,12,40 .
Investigations into the source of infection were not undertaken in this study, so it is not clear if the fungal isolates originated in the rehabilitation environment or were present in the skin on admission. Cafarchia and colleagues (2019) found increased length of stay to be a risk factor for fungal colonisation, where turtles staying in a rehabilitation centre for over 20 days were more frequently colonised with Fusarium 12 . Loggerhead sea turtles (C. caretta) in this study exhibited clinical signs around 20-30 days after admission and it is likely that most individuals experienced some degree of immunocompromise in the initial stages of rehabilitation. This, combined with physical skin trauma that may be present on admission may have provided a suitable environment for fungal colonisation. The second aim of study was to establish the phylogenetic relationship between F. keratoplasticum and F. falciforme strains isolated during this study and strains that were previously associated www.nature.com/scientificreports/ with brood failure and high mortality rates 17,18 . Combined sequence data of the ITS and LSU regions revealed that seven of the strains formed part of the monophyletic F. keratoplasticum clade. Strains isolated during this study showed a close phylogenetic relation with other species in this clade, consisting of species that were previously isolated from Hawksbill (E. imbricata) and green sea turtle (C. mydas) eggs shells from nesting beaches along the Pacific Ocean in Ecuador 7,16 . Furthermore, phylogenetic analyses of the F. falciforme group showed close resemblance to strains that were previously isolated from olive ridley sea turtle (L. olivacea), green sea turtle (C. mydas), flatback sea turtle (N. depressus) and loggerhead sea turtle (C. caretta) egg shells and C. caretta embryos on nesting beaches in Australia, Cape Verde and Ecuador, Turkey, along the Pacific, Atlantic and Indian Ocean 7,[15][16][17]20,21 . In addition, these strains showed a close resemblance to a strain that was previously isolated from a lesion in an adult turtle nare from the USA 29 . Based on the ITS and LSU gene regions, a genetic relationship exists between Fusarium species associated with turtle egg infections (also known as STEF) and Fusarium species isolated from post-hatchling loggerhead sea turtles (C. caretta) that stranded on beaches in South Africa along the Indian ocean.
Infections caused by members of this genus have been reported in numerous other aquatic animals in the past 6,9,10 , but for many of these, identification has been limited and mostly based on morphological characteristics. Many reports based on morphology only identified causative agents as Fusarium (F. solani), lacking further identification. Accurate identification of pathogenic Fusarium members is essential for epidemiological purposes and for assisting in management programs, however, more research is required to complete the puzzle and fully understand the ecology and distribution of these pathogens, especially amongst reptiles and aquatic animals. This is the first confirmed record of F. keratoplasticum and F. falciforme strains isolated from post-hatchling loggerhead sea turtles (Caretta caretta) from the South African coastline that were not associated with nesting sites. This is also the first record of F. crassum to be associated with loggerhead sea turtles.