The redbelly toads, genus Melanophryniscus (Bufonidae), comprises 31 species of small anurans that are geographically restricted to South America, occurring in Brazil, Paraguay, Bolivia, Uruguay, and Argentina [1]. Currently, several species of redbelly toads are listed as threatened in regional or global red lists [2, 3]. Melanophryniscus admirabilis is listed as Critically Endangered and is included as a priority species in the Action Plan for the Conservation of Amphibians and Reptiles in southern Brazil [4, 5]. This is a small toad with a microendemic distribution, presented on a single-site of the Atlantic Forest of southern Brazil [6]. This forest fragment has been suffering anthropogenic impacts, such as hydroelectric power generation, deforestation, pesticides contamination, intensive agricultural and livestock activities [7]. Recently, our research group evaluated the association of the oral microbiota with M. admirabilis conservation [9].
The knowledge about host-associated bacterial communities can help with conservation actions for endangered species, as well as play a role like bioindicator of a pathogen-free population [9-11]. Amphibian skin microbiomes are considered components of the amphibian immune system, acting as a barrier to pathogen infection, such as chytridiomycosis [12-14]. This is an infectious disease caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd) and has been linked to dramatic declines and extinctions of amphibian populations around the world [15, 16]. In this context, this study aimed to identify the cultivable bacteria present in the skin of wild M. admirabilis, and to address the ability of these bacterial populations to protect the toads against pathogens, such as Bd fungus.
Skin swabs were collected from 15 wild M. admirabilis in September 2019 (Figure 1). The toads were captured as described by Mann et al. [8]. This study was carried out following the recommendations of the Chico Mendes Institute for Biodiversity Conservation and was approved by the Research and Ethics Committees at the Federal University of Rio Grande do Sul (Projects 19541, 25526, and 25528). The protocol was approved by the Information and Authorization System in Biodiversity (SISBIO), numbers 40004-5 and 10341-1 (for M. Borges-Martins). All possible measures were taken to reduce the impact of our sampling protocol, which is part of a larger program intended for the study, monitoring, and conservation of the only known population of admirable redbelly toad. Samples were inoculated immediately in peptone water and incubated overnight. Subcultures were performed on Brain Heart Infusion (BHI) agar, MacConkey agar, and Mannitol-salt agar plates [17]. Plates were incubated aerobically at 37 °C for 24–48 h. Identification of the isolates was performed by standard microbiological techniques such as colony morphology, and microscopic features. In total, 62 isolates were obtained, which were subcultured and preserved in BHI-15% glycerol (v/v) at −80 ◦C. Species were identified by MALDI-TOF MS or 16S rRNA gene sequencing. The identification of B. dendrobatidis was performed as previously described by Coutinho et al. [18]. Significant differences were determined by using Student’s t-test. All statistical analyses were performed in the Past [19] software and the diversity graphics were elaborated through GraphPad Prism 9. To analyze alpha-diversity, the taxon diversity study (richness and evenness) within the samples was performed by employing the Chao1, Shannon diversity, and the Simpson diversity index.
The bacterial strains isolated from skin samples of wild M. admirabilis belong to different families, such as Enterobacteriaceae (n=28; 45.16%); Bacillaceae (n=22; 35.48%), Moraxellaceae (n=5; 8.06%), Aeromonadaceae (n=4; 6.45%), and Hafniaceae (n=3; 4.84%) (Table 1). Enterobacteriaceae was the most abundant cultivable family identified in all skin samples of wild M. admirabilis. This result is in concordance with Proença et al., [20] that also showed Enterobacteriaceae was the most dominant cultivable family in skin samples of Perez’s frogs (Pelophylax perezi) living in non-contaminated and contaminated rivers from Portugal. Besides, Bacillaceae, Moraxellaceae, and Aeromonadaceae were also observed in Perez's and Panamanian frogs [20, 21].
Among the 62 bacteria collected, 19 species were identified: Acinetobacter calcoaceticus (n=1; 1.62%), Aeromonas hydrophila (n=3; 4.83%), Aeromonas jandaei (n=1; 1.62%), Bacillus cereus (n=11; 17.74%), Bacillus sp. (n=11; 17.74%), Citrobacter braakii (n=2; 3.22%), , Citrobacter freundii (n=7; 11.29%), Citrobacter sp. (n=1; 1.62%), Enterobacter asburiae (n=1; 1.62%), Enterobacter cloacae (n=2; 3.22%), Enterobacter sp. (n=2; 3.22%), Hafnia alvei (n=3; 4.83%), Providencia rettgeri (n=1; 1.62%), Serratia marcescens (n=6; 10.34%), Serratia fonticola (n=3; 4.83%), Serratia liquefaciens (n=3; 4.83%), Yersinia enterocolitica (n=1; 1.62%), Yersinia nurmii (n=2; 3.45%), and Yersinia sp. (n=1; 1.62%) (Table 1). Based on the Kruskal-Wallis analysis there is no significant difference in bacterial composition between males and females individuals (H(chi²) = 6.872; Hc = 16.27; p = 0.574). The same result was observed through alpha diversity metrics (Shannon, Simpson, Chao1 index) (p > 0.05). The occurrence of the genera Acinetobacter, Serratia, Citrobacter, and Bacillus in skin samples of M. admirabilis is in concordance with previous studies, which have also found these genera in skin samples of different anurans species [22-25]. On the other hand, the occurrence of Providencia rettgeri, Enterobacter spp., Yersinia spp., and Hafnia alvei might be associated with the external environment since these species are commonly found in fecal and urine samples of animals, and in many natural environments, such as surface waters, soil, sewage, and vegetation [26-28]. The establishment of the microbial skin community in amphibians occurs throughout host generations by vertical or horizontal transmission (both aquatic and terrestrial environment) forming coevolutionary symbiotic relationships [29].
All the skin samples were tested using nested PCR and found Bd negative. Despite the absence of Bd fungus in our samples, we cannot confirm that this pathogen still has not affected the M. admirabilis population. However, the methodology used to identify the fungus in our samples is very sensitive. In general, the detection limit of this technique is at least ten fungus zoospores, in both asymptomatic as well as symptomatic individuals [18]. Furthermore, some bacterial genera identified in our study might be acting in a synergic relationship and protecting them against the Bd fungus. Previous studies have demonstrated that the skin microbiota of adult anurans can produce secondary metabolites with antimicrobial, antifungal, and antiparasitic properties [23, 30-32]. Acinetobacter sp. Bacillus sp., Citrobacter freundii, Janthinobacterium lividum, Pseudomonas sp., Serratia sp. found on amphibian’s skin already showed inhibitory activity against Bd fungus [20, 24, 33-38]. In addition, several bacteria of the amphibian skin act as component of the innate immune system of amphibians, acting as a barrier to pathogen infection and maintaining the species in an environment modulated by anthropic action [20, 38].
The main limitation of our study is the low number of sampled animals. However, considering the critically endangered status of M. admirabilis and the need to reduce sampling impact, the results obtained in this study are very important to numerous fields, including species conservation, the detection and quantification of environmental changes and stressors.
In conclusion, our results shows that bacteria present in the skin M. admirabilis might play an important role in defenses the host against the Bd fungus, as well as in maintaining this microendemic toads species living in an anthropogenic environment, including herbicides and antimicrobials use. This first report of skin cultivable bacteria from M. admirabilis natural population improves our knowledge of skin amphibian microbiomes and contributes to a better understanding of the ecology of this threatened species.
Table 1. Cultivable bacteria isolated from skin swab samples of wild Melanophryniscus admirabilis (admirable redbelly toads)
Family/genus/species
|
N of bacterial isolated (%)
|
Bacillaceae
|
|
22 (35.48)
|
|
Bacillus cereus
|
11 (17.74)
|
|
Bacillus sp.
|
11 (17.74)
|
Aeromonadaceae
|
|
4 (6.45)
|
|
Aeromonas hydrophila
|
3 (4.83)
|
|
Aeromonas jandaei
|
1 (1.62)
|
Enterobacteriaceae
|
|
28 (45.16)
|
|
Citrobacter braakii
|
2 (3.22)
|
|
Citrobacter freundii
|
7 (11.29)
|
|
Citrobacter sp.
|
1 (1.62)
|
|
Enterobacter asburiae
|
1 (1.62)
|
|
Enterobacter cloacae
|
2 (3.22)
|
|
Enterobacter sp.
|
2 (3.22)
|
|
Providencia rettgeri
|
1 (1.62)
|
|
Serratia liquefaciens
|
3 (4.83)
|
|
Serratia marcescens
|
6 (9.6)
|
|
Serratia fonticola
|
3 (4.83)
|
Hafniaceae
|
|
3 (4.83)
|
|
Hafnia alvei
|
3 (4.83)
|
Moraxellaceae
|
|
5 (8.06)
|
|
Acinetobacter calcoaceticus
|
1 (1.62)
|
|
Yersinia enterocolitica
|
1 (1.62)
|
|
Yersinia nurmii
|
2 (3.22)
|
|
Yersinia sp.
|
1 (1.62)
|
Total
|
|
|
62 (100)
|