Cultivable Fungi from Amazon River Dolphins Engaged in Wildlife Ecotourism in the Anavilhanas National Park, Brazil

Amazon River dolphins are an important flagship species in the Anavilhanas National Park, Brazil, where they interact with visitors. This study aimed to quantify and identify fungi isolated from dolphin skin and oral samples and their surrounding environment in this unique ecosystem. Samples were collected from three dolphins and water samples from Flutuante dos Botos and the Novo Airão city harbor. Fungi were isolated using culture media and identified through micromorphology assays and ITS region sequencing. Oral swab samples resulted in culture of Trichosporon montevideense and Exophiala dermatitidis. Skin samples from one dolphin revealed Toxicocladosporium irritans and Diaporthe lithocarpus. Water samples exhibited higher fungal counts and diversity, with Rhodotorula mucilaginosa, Exophiala dermatitidis, Penicillium citrinum, Fomitopsis meliae, and Nectria pseudotrichia identified at the collection site and Candida spencermartinsiae and Penicillium chermesinum at the city harbor. This study provides important insights into the fungal diversity associated with Amazon River dolphins and their environment, enhancing our understanding of the public health and ecological dynamics in the Anavilhanas National Park.


Introduction
Advancements in microbiology have improved the understanding of the microbiota of living animals.Tis enhanced understanding is important to study the complex relation animal-biota and for identifying potential zoonoses with potential risk to society [1].
Recent works have been describing the biota associated to animals, including cetaceans [1].However, research specifcally focusing on the Amazon River dolphins (Inia geofrensis) is scarce.Among these limited studies, some have explored diseases afecting these dolphins.For example, cases of Streptococcus iniae infection, typically seen in captive dolphins, have been documented [2][3][4].Furthermore, the occurrence of fungal pathogens, including those responsible for Jorge Lôbo's disease (JLD) or Lobomycosis, a chronic skin disease prevalent in humans in Central and South America, is of interest [5].Te causative agent of JLD is the fungus Lacazia loboi [6][7][8][9].
Conducting research on the skin microbiota and fungal pathogens of wild dolphins presents unique challenges.Gaining access to these animals and implementing suitable methodologies to describe the organisms present in their biota can be particularly daunting [10].However, a unique opportunity for studying Amazon River dolphins has emerged at the Flutuante dos Botos, a private foating house situated in the Anavilhanas National Park in Novo Airão city, Amazonas state, Brazil [11,12].Since 1998, these dolphins have interacted with visitors and local residents at this location.Tis proximity ofers a relatively easy access to conditioned Amazon River dolphin individuals and facilitates the collection of invaluable biological samples.
Tis study aims to fll the existing knowledge gaps concerning the fungal microbiota found in freshwater dolphins and leverage the potential for sample collection due to the distinctive interaction between these animals and humans in the city of Novo Airão-AM (Brazil).Specifcally, our objectives are to collect skin and oral cavity samples from Amazon River dolphins, analyze the isolated fungi, and address the following research questions: (a) "what are the number of aerobic fungal colony-forming units (CFUs) present on the skin and in the oral cavity of the Inia geofrensis?";(b) "which fungal species are identifed in the samples obtained from the skin and oral cavity?;" and (c) "are these fungal isolates previously documented in relation to infections in humans or these cetaceans?."By providing answers to these questions, we aim to initiate a discussion about Inia geofrensis microbiota and its potential implications.

Materials and Methods
2.1.Collection Site."Sample collections were carried out in the "Flutuante dos Botos" (a private foating house), where dolphins are provisioned on a daily basis and interact with visitors, and in the Novo Airão city harbor (Figure 1), both located in the south-central region of the Anavilhanas National Park, Amazonas state, Brazil.Novo Airão is a small town situated 183 km by land from Manaus and represents one of the main destinations for local inhabitants and tourists alike.Te Anavilhanas National Park has approximately 350,000 hectares of area and about 400 islands, making it the second largest river archipelago in the world [10,13,14].Its aquatic environment is formed by black waters of the Negro River, which are poor in nutrients and have high concentrations of organic compounds, resulting an acidic pH 4.6-4.8while the dissolved oxygen level was 5.0-5.5 mg/L [15,16].It is important to note that compared to the Flutuante dos Botos, the city harbor of Novo Airão exhibited a higher degree and nature of pollution, infuenced by boats pollutants infux and city sewage."

Animals.
Te study involved the participation of three adult dolphins named Curumim, Chico, and Priscila.Tese dolphins (Inia geofrensis) were visually identifed with the assistance of the employees at Flutuante dos Botos, who possess expertise in recognizing individual dolphins based on their scars, pigmentation patterns, and behaviors [15,16].It should be noted that since these are free-ranging animals, estimating factors such as approximate age and sex becomes inherently challenging.Te dolphins regularly partake in provisioning sessions at Flutuante dos Botos and willingly engaged in sample collection during the sessions conducted from June 6th to June 8th, 2022.During our visit to the facility, we observed that these animals did not exhibit any visible skin wounds; however, they did bear several scars, likely resulting from aggressive intraspecifc interactions, previous trauma, and infections.
Te research was carried out within the scope of the Chico Mendes Institute for Biodiversity Conservation (ICMBio), being authorized by the research committee of this institution and registered in the System of Authorization and Information on Biodiversity (SISBIO) under the numbers 37309-1 and 45110-1.

Sample Collection.
To investigate the presence of fungi in both the skin and oral samples of dolphins (I.geofrensis), we collected specimens and inoculated them immediately into culture media specifcally designed for fungal isolation (Table 1).Te dolphins voluntarily approached the foating deck surface, providing an opportunity for us to collect samples during their natural ascent.Figure 2 displays images depicting the process of sample collection.For the collection of oral cavity and skin swabs, we utilized sterile disposable swabs PurFlock Ultra-6″ Sterile Standard Flock Swab w/Polystyrene Handle (Puritan Medical Products, Guilford, ME, USA).In our experimental conditions, these swabs keep about 100 ± 20 μL of the biological specimens.
Swab from the oral cavity sample was collected from the interior region of the mouth, particularly from the space adjacent to and between the teeth, to accurately represent the oral microbiota.Te skin samples were obtained from the dolphin's melon and rostrum.
In addition to the skin swab collection, we also collected a second sample from the dolphin's skin using a saw commonly employed in civil construction (Blade Single 75 × 3 mm, Bu38 Athol, Starrett Company, Massachusetts, United States) (Figure 1).During the dolphin's ascent, the saw was gently rubbed against the skin in the head region.Te material obtained from the skin scraping was frst transferred to the swab (PurFlock Ultra-6″ Sterile Standard Flock Swab w/Polystyrene Handle) and subsequently inoculated into the culture medium for further analysis.
With the purpose of investigating the fungi present in the water, we collected samples from the same collection site as the dolphins, approximately 30 cm deep, using sterile 25 mL conical tubes.Tese samples were transferred to the swab (PurFlock Ultra-6″ Sterile Standard Flock Swab w/Polystyrene Handle) and fnally transferred to the culture media.Te choice of these culture media was deliberate to isolate various fungi species, where Sabouraud agar supported medically relevant fungi, Brain heart infusion facilitated growth of dimorphic fungi, and CHROMagar ™ Candida aided in the identifcation of Candida yeast species.In addition, the in-house prepared niger seed agar was specifcally formulated for cultivating Cryptococcus genus agents.Following inoculation onto these media, plates were incubated at 25 °C for 120 hours to optimize growth conditions.Te composition of niger seed agar comprised Guizotia abyssinica (niger) seeds at 70 g/L, chloramphenicol at 0.05 g/L, agar at 20.0 g/L, and distilled water to achieve a fnal volume of 1000 mL, maintaining a pH of 6.7 ± 0.2 at 25 °C [17,18].

Fungi Identifcation.
Te fungi that developed were quantifed and grouped into morphotypes and identifed using conventional taxonomic keys [18,19].Te morphological identifcation was associated with DNA sequencing analysis.Identifcation by DNA sequencing initiated with culture DNA was extracted with the kit QIAamp Tissue and Blood (Qiagen, Hilden, Germany) according to manufacturer.Te PCR was performed in accordance with [20] from the amplifcation of the regions ITS (ITS1-5′ TCC GTA GGT GAA CCT GCGG 3′ and ITS4-5′ TCC TCC GCT TAT TGA TAT GC 3′).Te following were used: 40 ng of DNA in a fnal volume of 30 µL, with 1X reaction bufer (200 mM TrisHCl (pH 8.4), 500 mM KCl); 0.2 mM of each nucleotide; 2 mM of magnesium chloride (MgCl 2 ); 50 ng of each primer; and 2.5U of Platinum ® Taq polymerase enzyme (Invitrogen, Brazil).
Te amplicons were sequenced according to the instructions from the manufacturer of the kit "BigDye ® Terminator v3.1 Cycle Sequencing Kit" (Applied Biosystems) and capillary electrophoresis was performed in an ABI 3130 Genetic Analyzer sequencer.Te sequences were analyzed in the Sequencher 5.4.6 program and then compared with existing sequences in the NCBI database (https://blast.ncbi.nlm.nih.gov/Blast.cgi)and the Wester Dijk Fungal Biodiversity Institute (https://wi.knaw.nl/page/Pairwisealignment).  4 Veterinary Medicine International
In Curumim, a single CFU of Exophiala dermatitidis was isolated from the oral swab using agar niger, with no fungal growth observed on skin swabs or scrapings.Priscila showed no fungal growth in any of the samples.However, in Chico, 3 CFUs of Trichosporon montevi-deense were identifed in the oral swab, and 6 CFUs of Toxicocladosporium irritans along with an unidentifed fungal colony were detected in skin scrapings.
Brain heart infusion (BHI) agar was employed to detect dimorphic fungi.Although BHI agar is not selective for fungi, it revealed signifcant bacterial growth.In Curumim's samples, over 10 colony-forming units (CFUs) were recorded in the oral swab, skin swab, and skin scraping.A similar pattern was observed in Priscila's samples.Notably, Chico's samples and water samples exhibited an exceedingly high bacterial presence, with over 100 CFUs in all types of samples.

Discussion
In the Anavilhanas National Park, Brazil, we quantitatively and qualitatively assessed fungal species on Amazon River dolphins and in their aquatic environment.Trough morphological analysis and sequencing of the ITS region, we identifed diverse fungi, including Trichosporon montevideense and Exophiala dermatitidis from dolphin oral samples and Toxicocladosporium irritans from skin.Water samples revealed a higher fungal diversity, with signifcant fndings such as Rhodotorula mucilaginosa, Exophiala, Fomitopsis, and Penicillium citrinum.Tis investigation enriches our understanding of the microbial landscape associated with these cetaceans and highlights potential implications for public health and ecosystem dynamics.
Te lower fungal colony count in dolphins, as compared to their environment, points towards a robust immune system and a unique microbiota composition, potentially skewed towards bacterial dominance previous described [21,22].Te fungi isolated from the water included Penicillium, Rhodotorula, and others belong to genera previously identifed in water samples from the Rio Negro [23][24][25].Predominantly from the phylum Ascomycota, these fungi are mostly transient in the environment, seeking suitable substrates for their growth and development [25].Te isolation of fungi from freshwater dolphins is scantily documented in literature [3,22].In our study, we detected Trichosporon montevideense and Exophiala dermatitidis in the oral cavity and Toxicocladosporium irritans from a skin sample.Tough these genera are not known primary pathogens in dolphins, they hold signifcance in other contexts.Trichosporon, prevalent on mammalian skin, can cause skin infections such as onychomycosis and invasive infections in humans [18,26].Exophiala dermatitidis, an opportunistic fungus, can cause fatal brain infections and pheohyphomycosis in humans [27].Toxicocladosporium irritans, isolated from dolphin "Chico," have been found in patients with atopic dermatitis in Japan and in human blood and fngernails [28].Tis investigation enriches our understanding of the skin microbiology of these cetaceans.
In the evaluation of zoonotic potential from cetacean oral microfora, it is crucial to consider the natural occurrence of these microorganisms within their habitat.Te presence of diverse microbes in the oral cavities/skin of cetaceans, as observed through culture growth, does not inherently signify a pathogenic state.Te low quantity of colony-forming units (CFUs) cultured suggests low potential pathological implications.Interestingly, according to our fnds exposure to river water could pose a greater risk to fungal infection than the cetaceans themselves [18].
Te present work has certain limitations that need to be acknowledged.Tese include the small number of animals and samples that were investigated.Due to these limitations, it was not possible to quantitatively evaluate the collection methods.To obtain a more comprehensive description of the microbiota, it is recommended that future experiments be conducted with a larger number of animals.However, it is important to consider the challenges involved in handling these animals.Moreover, the inability to collect samples beyond the designated ecotourism areas limited the comparative analysis of microbiota between dolphins engaged in ecotourism and those existing in non-tourism-associated habitats, signifying a potential constraint in drawing comprehensive conclusions regarding microbiota variations infuenced by ecotourism activities.In addition, we believe that future work focusing on metagenomic studies will allow us to directly analyze the genetic material present in environmental samples.Unlike cultivable studies which rely on growing microorganisms in laboratory conditions, the metagenomic approach enables us to uncover the vast diversity of microorganisms, including non-cultivable species, and understand their functional potential and ecological roles.Nonetheless, this current work serves as a pioneering efort in describing the methodologies and presenting preliminary results.While providing foundational knowledge, it also opens avenues for further exploration into the complex interplay of environmental factors, microbial communities, and dolphin health.

Conclusions
In conclusion, our study has successfully addressed the revised research questions by providing a comprehensive analysis of the aerobic fungal colony-forming units (CFUs) present on the skin and in the oral cavity of Amazon River dolphins, identifying specifc fungal species and assessing their potential implications for both dolphin health and human interaction.Our fndings reveal the presence of Trichosporon montevideense, Exophiala dermatitidis, and Toxicocladosporium irritans, among others, highlighting a complex interplay between these dolphins and their microbial environment.Trough these insights, we contribute to animal microbiology, emphasizing the need for integrated approaches that consider both animal and human health within ecologically sensitive habitats.

2 Veterinary Medicine International 2 . 4 .
Fungi Isolation.In our experimental conditions, we assumed that each swab (PurFlock Ultra-6″ Sterile Standard Flock Swab w/Polystyrene Handle) transferred an approximate volume of 100 μL or 100 mg of biological or environmental samples.Tese samples, one swab per sample and one swab for each culture media, were directly applied onto 9 cm plates containing the following fve distinct culture media: Sabouraud agar, Mycosel, brain heart infusion, CHROMagar ™ Candida (Becton, Dickinson and Company, USA), and niger seed agar.

Figure 2 :
Figure 2: Images of sample collections (Inia geofrensis).Collection of swabs from oral cavity from the boto "Curumim" (a); foating facility where the interaction with Amazon River dolphins occurred (b); collection of a skin sample from "Chico" (c); and small saw used to generate greater friction with the skin of dolphins (d).