Culturable fungi associated with the marine shallow-water hydrothermal vent crab Xenograpsus testudinatus at Kueishan Island, Taiwan

: Reports on fungi occurring on marine crabs have been mostly related to those causing infections/diseases. To better understand the potential role(s) of fungi associated with marine crabs, this study investigated the culturable diversity of fungi on carapace of the marine shallow-water hydrothermal vent crab Xenograpsus testudinatus collected at Kueishan Island, Taiwan. By sequencing the internal transcribed spacer regions (ITS), 18S and 28S of the rDNA for identi ﬁ cation, 12 species of fungi were isolated from 46 individuals of X. testudinatus : Aspergillus penicillioides, Aspergillus versicolor, Candida parapsilosis, Cladosporium cladosporioides, Mycosphaerella sp ., Parengyodontium album, Penicillium citrinum, Penicillium paxili, Stachylidium bicolor, Zasmidium sp. (Ascomycota), Cystobasidium calyp-togenae and Earliella scabrosa (Basidiomycota). With additional data from other published reports, a total of 26 species of fungi (23 Ascomycota, three Basidiomycota) have been recorded from X. testudinatus . Aspergillus is the most spe-ciosegenusonthecrab,followedby Penicillium and Candida . All but one species ( Xylaria arbuscula ) had been previously isolated from substrates in the marine environment, although many are typical terrestrial taxa. None of the recorded fungi on X. testudinatus are reported pathogens of crabs, but some have caused diseases of other marine animals. Whether the crab X. testudinatus is a vehicle of marine fungal diseases requires further study.


Introduction
Kueishan Island (also known as Turtle Island with reference to its shape) is an active volcano situated at the northeastern end of the main Taiwan island.At one end of the island, a shallowwater hydrothermal vent system is present with roughly 50 hydrothermal vents, where hydrothermal fluids (between 48 and 116 °C) and volcanic gases (carbon dioxide and hydrogen sulfide) are constantly being emitted (Chen et al. 2005).The hydrothermal vent system of Kueishan Island is considered to be a mixed photosynthetic-chemosynthetic ecosystem in contrast to the obligate chemoautotrophic primary production in deep-sea hydrothermal vents (Chang et al. 2018).
With the possibly extreme environmental conditions (high temperature, low pH), macro-and microorganisms have been reported at/near the hydrothermal vent system of Kueishan Island, including fishes, crabs, mussels, sea anemones, snails, sipunculid worms, algae and zooplankton (Chen et al. 2005;Kâ and Hwang 2011;von Corsel 2008).In particular, the crab Xenograpsus testudinatus is the dominant animal species at the vents with a high population density (Tseng et al. 2020).The crab mainly consumes zooplankton killed by the plumes from the vents which eventually settle to the seafloor (Jeng et al. 2004).Algae, fishes, bivalves and anthozoans were also reported to be possible food sources for the crab (Ho et al. 2015), as well as vent particulate organic matter (Chang et al. 2018).X. testudinatus occupies the highest trophic level in the hydrothermal ecosystem of Kueishan Island (Wang et al. 2014).
Concerning microorganisms, the ε-Proteobacteria including the genera Sulfurovum and Sulfurimonas dominated the bacterial community in sediment samples collected near the hydrothermal vents at Kueishan Island using metabarcoding analysis of the 16S rRNA gene sequences (Wang et al. 2016).In the same study, chemoautotrophic carbon fixation genes of the ε-Proteobacteria were also detected, suggestive of their possible role in the primary production at the hydrothermal vent area of Kueishan Island.For fungi, species of the genus Aspergillus were isolated near the hydrothermal vent area of Kueishan Island (Ding et al. 2016;Jiang et al. 2013;Pan et al. 2016), but these studies mainly explored the secondary metabolites of these fungi.Pang et al. (2019) was the first comprehensive study to look into the culturable and nonculturable diversities of fungi associated with sediment, seawater, and animal samples including the crab X. testudinatus at/near the hydrothermal vent area of Kueishan Island.The main results of Pang et al. (2019) were: (1) a higher diversity of fungi was observed in sediment than in seawater and animal samples, (2) Ascomycota was dominant over Basidiomycota, (3) metabarcoding analysis revealed a more diverse fungal community than the culture method, and (4) both marine and terrestrial Ascomycota were recovered.
Fungi of the marine environment have been mainly reported from plant-based substrata (Jones et al. 2019).Little is known on the mycota associated with marine animals, except those that cause diseases (Pang et al. 2021).For crustaceans, fungi are known to cause diseases of aquacultured animals, such as the black gill disease caused by Fusarium spp., leading to significant mortalities of the giant tiger prawn (Penaeus monodon; Khoa et al. 2004) and the Kuruma prawn (Marsupenaeus japonicus; Khoa andHatai 2005, Khoa et al. 2005).For crabs, the best known is the lethargic crab disease of the mangrove land crab Ucides cordatus in Brazil, caused by Exophiala cancerae and Fonsecaea brasiliensis (Boeger et al. 2007;Vicente et al. 2012).The Microsporidia, instead of the Ascomycota, is the main fungal group causing diseases of marine crabs (Pang et al. 2021).Pang et al. (2019) isolated 13 species of fungi from 10 individuals of X. testudinatus: Hortaea werneckii, Aspergillus sydowii, Aspergillus terreus, Aspergillus unguis, Penicillium citreosulfuratum, Hypocreales sp., Parengyodontium album, Microascus brevicaulis, Candida oceani, Hypoxylon monticulosum, Peroneutypa scoparia, Xylaria sp. and Chondrostereum sp.While some of these fungi are common in the marine environment as saprobes and symbionts, some are potential pathogens of marine animals (Pang et al. 2021).In order to have a better understanding of the potential role(s) of fungi associated with X. testudinatus, a larger number of specimens of this species was collected in the hydrothermal vent area of Kueishan Island, and this study investigated the culturable diversity of fungi on the carapace of this crab based on molecular identification using three nuclear ribosomal genes (18S, ITS, 28S).

Sample collection
The crab X. testudinatus Ng, Huang et Ho, 2000 was collected from the shallow hydrothermal vents of Kueishan Island, Yilan, Taiwan (Figure 1) on 13 August 2018 and 2 September 2018.The crabs were collected by a rectangular cage trap and brought up to the fishing boat by SCUBA divers.Crabs were immediately put into sterile ziplock plastic bags on board, placed in a cool box during transportation to the laboratory, and kept at 4 °C before isolation (time from collection to isolation < 12 h).

Results
A total of 23 colony morphotypes were isolated on the two media from the carapace of the crab X. testudinatus collected at the shallow hydrothermal vents of Kueishan Island, Yilan, Taiwan.These colony morphotypes were identified based on the nucleotide BLAST search of the 18S, ITS and 28S rDNA in NCBI with the highest sequence coverage and similarity (Table 1).Twelve different fungal species were identified: Aspergillus penicillioides, Aspergillus versicolor, Candida parapsilosis, Cladosporium cladosporioides, Mycosphaerella sp., Parengyodontium album, Penicillium citrinum, Penicillium paxili, Stachylidium bicolor, Zasmidium sp.(Ascomycota), Cystobasidium calyptogenae and Earliella scabrosa (Basidiomycota).For most species, the top matches for all three gene regions agreed on one fungal name; for A. penicillioides and Pa.album, two gene regions agreed on one name.For Mycosphaerella sp. and Zasmidium sp., the BLAST results of the three gene regions could refer these two species only to the genus level.For P. paxili NTOU5880, the 28S rDNA phylogenetic tree grouped this isolate with P. paxili MH876591 downloaded from GenBank (results not shown).Based on richness, 10 species of the Ascomycota were isolated (83.3%), in comparison to only two species belonging to the Basidiomycota (16.7%) (Figure 2).The species mostly belonged to the Eurotiales of the Eurotiomycetes (33.3%), and the Capnodiales of the Dothideomycetes (25.0%).These 12 species could be referred to seven different families: Aspergillaceae (33.3%),Mycosphaerellaceae (16.7%),Saccharomycetidae, Cladosporiaceae, Cystobasidiaceae, Polyporaceae, Cordycipitaceae, and incertae sedis (8.3% for the latter five families).Two species of Aspergillus and Penicillium were isolated from the crab while the rest were only represented by one species, including Candida, Cladosporium, Cystobasidium, Mycosphaerella, Parengyodontium, Stachylidium and Zasmidium.Figure 3 shows the taxonomic classification of the 26 species of fungi reported from X. testudinatus based on the results of this study and others (Ding et al. 2016;Jiang et al. 2013;Pan et al. 2017;Pang et al. 2019).Twenty-three species belong to the Ascomycota while there are only three basidiomycetes.Aspergillus is the most speciose genus on the crab, followed by Penicillium (both Eurotiomycetes) and Candida (Saccharomycetes); other genera are only represented by one species.Among the 26 species documented for X. testudi natus, eight species (Ascomycota: Aspergillus sp., A. terreus, H. werneckii, Parengyodondium album, P. citrinum, P. scoparia; Basidiomycota: Chondrostereum sp., E. scabrosa) were also isolated from the sediment samples (either from black or yellow sediment types or both) collected at the hydrothermal areas of Kueishan Island (Figure 4).

Discussion
Twelve species of fungi were isolated from 46 individuals of the hydrothermal vent crab X. testudinatus collected at Kueishan Island, Taiwan with 10 ascomycetes and two basidiomycetes.In our previous study (Pang et al. 2019), 13 species were isolated from 10 individuals of the same crab species at the same area.The low fungal species richness isolated in this study may be related to the fact that the crabs were collected in different months/years in these two studies, i.e.Pang et al. (2019): October 2015, March 2017, June 2017; this study: August 2018, September 2018.Also different methods of isolation were used between the two studies; the biofilm on carapaces was inoculated in this study but whole crabs were crushed and inoculated in Pang et al. (2019).
Twenty-six fungi have been recorded from X. testudinatus based on the results of this study and others; the majority of the fungi belong to the Ascomycota (23 species), with Aspergillus, Penicillium and Candida being the most speciose genera, and these are also three of the most speciose genera in the marine environment (Jones et al. 2015).All but one ascomycete, Xylaria arbuscula, were previously reported from substrates of the marine environment (Jones et al. 2015).In fact, some species appear to be very common, for example, H. werneckii from seawater, sediment and sponges (Anteneh et al. 2019;Leo et al. 2018;Singh et al. 2012), and Parengyodontium album from seawater, sediment, sponges and corals (Huang et al. 2018;Khusnullina et al. 2018;Leo et al. 2018;Proksch et al. 2008).It is worth noting that some of these species are regarded as terrestrial species, including H. monticulosum and X. arbuscula, and their ecological association with X. testudinatus is unknown.Among the three species of the Basidiomycota, Cystobasidium was previously reported from the marine environment (Jones et al. 2015).The polypore E. scabrosa and the gilled mushroom Chondrostereum sp.(both Agaricomycetes) were isolated not only from X. testudinatus, but also from the sediment samples at the hydrothermal vent area of Kueishan Island (Figure 4).E. scabrosa was previously isolated from the sea fan coral Pacifigorgia cf.eximia (Barrero-Canosa et al. 2013).Chondrostereum spp.have been commonly isolated from various substrata in the marine environment.For example, one Chondrostereum sp. was isolated from the soft coral Sarcophyton tortuosum (Li et al. 2020), and another from the red alga Pterocladiella capillacea (Hsiao et al. 2017).Agaricomycetes is the dominant class of Basidiomycota in air over coasts and seas (Fröhlich-Nowoisky et al. 2012), which provides a constant supply of terrestrial fungal propagules for inoculation of marine substrates, including seawater, sediment, marine animals and macroalgae.Moreover, many typical terrestrial ascomycetes isolated from substrates of the hydrothermal area of Kueishan Island were able to grow in seawater (Pang et al. 2020).Further research is required to investigate if Chondrostereum spp., E. scabrosa and other filamentous basidiomycetes can grow and play a role in the marine environment.
The ecological relationships between X. testudinatus and the fungi associated with it are not known.In the hydrothermal ecosystem of Kueishan Island, green and red algae, epibiotic biofilms on crustacean surfaces, and zooplankton form the base of the trophic system based on a stable isotope analysis (Wang et al. 2014), but Chang et al. (2018), using the same technique, discovered that biofilms on biotic surfaces made little contribution to other benthic consumers.None of the recorded fungi on the crab are reported pathogens of crabs, but some of them are known pathogens of other marine animals (Pang et al. 2021).A. terreus was reported to cause pneumonia and severe otitis media in the harbor porpoise Phocoena phocoena (Jepson et al. 2000;Prahl et al. 2011).A. sydowii causes diseases of corals (Smith et al. 1996).A. versicolor formed white masses on the sea fan Annella sp. and caused tissue disintegration (Phongpaichit et al. 2006).Granulomas in the swim bladder of the humpback grouper Cromileptes altivelis were reported to be caused by C. cladosporioides  (Bowater et al. 2003).Whether the crab X. testudinatus is a vehicle of fungal diseases requires further study.

Figure 1 :
Figure 1: (A) Location of Kueishan Island (box) at the northeastern end of the main Taiwan Island.(B) Kueishan Island showing the vent and sampling locations.(C) Xenograpsus testudinatus, the hydrothermal vent crab.

Figure 2 :
Figure 2: Taxonomic classification of the fungi isolated from the carapace of the crab Xenograpsus testudinatus collected in the hydrothermal vent area of Kueishan Island, Taiwan.

Figure 3 :
Figure 3: Taxonomic classification of all fungi reported (by this study and previously published work) on the crab Xenograpsus testudinatus.

Figure 4 :
Figure 4: Comparison of fungal diversity on the vent crab Xenograpsus testudinatus, in yellow sediment (with sulfur granules) and in black sediment (with no sulfur granules) collected in the hydrothermal vent area of Kueishan Island, Taiwan.

Table  :
BLAST search results of the S, ITS (internal transcribed spacer regions) and S rDNA of the fungi isolated from the carapace of Xenograpsus testudinatus collected at the hydrothermal vent area of Kueishan Island, Taiwan in National Center for Biotechnology Information (NCBI).