Analysis of gut-associated fungi from Chinese mitten crab Eriocheir sinensis

Abstract

Chinese mitten crab, Eriocheir sinensis (Varunidae), is one of the most popular and widely cultivated freshwater crab species in the Chinese food industry, with high commercial importance and nutritional value. We analyzed the diversity of culturable fungi in the gut of E. sinensis collected from a rice-crab co-culture system in Tianjin, China. We isolated 41 fungal strains from the gut of analyzed male and female crabs, using the dilution plate method. Morphological and molecular identification based on nuclear ribosomal internal transcribed spacer (ITS) sequencing suggested that these isolates belonged to 16 genera in Ascomycota, Basidiomycota, and Mucoromycota. Aspergillus was the dominant identified genus followed by Penicillium and Talaromyces. Yeasts, including Candida, Clavispora, Meyerozyma, and Trichosporon genera, accounted for a significant portion (12.2%) of the isolated strains. Statistical analysis showed significant differences in gut-associated fungal communities between female and male crabs, with female individuals showing a higher species diversity. Our study represents the first report on intestinal fungal communities of Chinese mitten crab, providing valuable microbiological information that could be essential for supporting the effective management and conservation of this crab species, and for the improvement of the economic performance of the crab industry.

KEYWORDS:

• Chinese mitten crab
• Eriocheir sinensis
• gut-associated fungi
• fungal diversity
• ITS sequencing
• morphology

Introduction

Rice-aquatic animal co-culture systems, which combine animal production (e.g. fish, shellfish, crab, shrimp, or duck) with rice plantation, are a sustainable strategy to fulfill the increasing demand for rice yield and aquaculture (Bashir et al. 2020). As a novel rice-aquatic animal system, the rice-crab co-culture system has been vigorously promoted by the Chinese government. In this co-culture system, rice and Chinese mitten crab (Eriocheir sinensis) are in a beneficial symbiotic relationship. Rice fields provide a suitable environment for crab growth, with sufficient water level, good light conditions, and enough dissolved oxygen (Zhang et al. 2017), while the foraging activities of crabs can reduce weed emergence, avoid pest propagation, and improve soil fertility in rice fields (Fernando 1993; Xu et al. 2014). Compared to traditional rice monoculture, this efficient rice-aquatic animal co-culture model is characterized by complementary resource use, reduction of chemical fertilizers and pesticides, improvement of rice yield and biodiversity enhancement (Halwart 2008; Xu et al. 2014; Hu et al. 2016).

Chinese mitten crab, Eriocheir sinensis H. Milne-Edwards, is a crustacean belonging to the family Varunidae, order Decapoda. It is one of the most popular freshwater crab species in the Chinese food industry, with high commercial importance and nutritional value, widely cultivated in the Liaoning, Jiangsu, and Anhui provinces (Sui et al. 2009; Ding et al. 2016). As a crucial component of the Chinese freshwater aquaculture industry, the annual output of Chinese mitten crab has increased rapidly from 200,000 tons in 2000 to over 756,000 tons in 2018 (Yuan 2005; People's Republic of China Ministry of Agriculture 2019). However, frequent outbreaks of Hepatopancreatic Necrosis Disease (HPND) with a high mortality rate (40–50%) have resulted in a significant loss in production and economic value of Chinese mitten crab. HPND is an infection within the cytoplasm of the epithelial cells of the hepatopancreas by microsporidia, which were considered Protozoa for a long time. Microsporidia were reclassified by Cavalier-Smith (1998) into the kingdom Fungi in 1998, and described as Cellular Organisms, Eukaryote, Fungi/Metazoa group, Fungi, Microsporidia in 2002 by NCBI, although there is no universal agreement on their classification yet (Dong et al. 2010). Crabs with HPND exhibit multiple clinical symptoms, including colour change of the hepatopancreas (from golden yellow to almost white), soft shells that are darker than usual, loss of appetite, slow action, muscle atrophy and edema (Shen et al. 2017). After the stomachs and intestines become empty, diseased crabs eventually die (Ding et al. 2016). In spite of the crucial influence of microbes in Chinese mitten crab health, there is a lack of knowledge on microbial diversity associated with E. sinensis, especially at gut level. The gut microbiome is a diverse and complex ecosystem consisting of bacteria, archaea, viruses, fungi, protists, and (sometimes) helminths (Enaud et al. 2018). The gut-associated microbiome plays a crucial role in the host health by supplying essential nutrients, maintaining normal gut functions (Zhang et al. 2015), modulating immune response, and contributing to host defense against pathogens (Enaud et al. 2018). Recently, the intestine microbial diversity has been studied in a wide range of animals, particularly in mammals (Hermann-Bank et al. 2013). Crustaceans, which have received the highest attention among aquatic invertebrates, have been found to harbor various genera of bacteria including Vibrio, Pseudomonas, Flavobacterium, Micrococcus, and Aeromonas in their gut (Harris 1993). In addition, the presence of intestinal fungi in aquatic crustaceans, such as shrimps, has been reported. For instance, the intestinal mycobiota of Litopenaeus vannamei (Pacific white shrimp) was dominated by the genera Alternaria, Tuber, Hortaea, Sarocladium, and Stagonospora (Li et al. 2019). Nevertheless, up to now, studies of microbial communities in crab gut have mainly focused on bacteria. Four bacterial phyla, Proteobacteria, Firmicutes, Tenericutes, and Bacteroidetes, have been found to be dominant in the gut of mud crab (Scylla paramamosain) and E. sinensis (Li et al. 2012; Chen et al. 2015). Firmicutes and Tenericutes have been described as the most abundant phyla in the foregut, Tenericutes and Proteobacteria in the midgut, and Proteobacteria, Tenericutes, and Bacteroidetes in the hindgut of E. sinensis (Dong et al. 2018). Only a few studies have focused on the composition of fungal communities in crab species. Fonsecaea brasiliensis and Exophiala cancerae, two black yeast-like fungal species, were identified in the mangrove-land crab (Ucides cordatus) infected with Lethargic Crab Disease (LCD) (Vicente et al. 2012). Thirteen fungal species have been found from the vent crab Xenograpsus testudinatus including Aspergillus terreus, Hortaea werneckii, Parengyodontium album, and Peroneutypa scoparia, using both culture-based and metabarcoding methods (Pang et al. 2019).

Thus far, gut bacteria have been the most common object of gut microbiome studies due to the abundant presence of bacteria in the gut (Chin et al. 2020), whereas little attention has been given to gut fungi because of their comparatively much lower presence (Chin et al. 2020). While gut bacterial communities of E. sinensis have been previously studied, there is no information related to the gut fungal diversity of Chinese mitten crab. In this study, we provided the first assessment of culturable fungal diversity associated with the gut of E. sinensis growing in a rice-crab co-culture area in Tianjin, China, in order to increase our knowledge of gut microbial communities in this economically important crab species and provide useful information for the management of E. sinensis aquaculture.

Materials and methods

Study site and sampling

This study was conducted in a rice-crab co-culture farm located in Qilihai Town, Ninghe District, Tianjin, China (39°19′19″ N, 117°39′44″ E). The investigated farm included two rice paddy fields that were connected with two adjacent ponds by means of canals located along two opposite sides of the fields. Three female and three male Chinese mitten crab (Eriocheir sinensis) individuals, with a weight of approximately 100 g each, were captured alive in the studied environment. All the sampled individuals appeared healthy. The freshly collected crabs, labeled by sex (F and M) and individual (1–3), were placed in sterilized bags and kept on ice in sampling box. They were immediately transported to the laboratory where they were put at 4°C to slow their activity and induce anesthesia within 15–20 min.

Isolation of crab gut-associated fungi

The collected crab individuals were dissected and their intestines were transferred into 15 ml sterilized centrifuge tubes (Eppendorf, Germany) with 3 ml sterilized water. The tubes were shaken thoroughly on an Analog Vortex Mixer (OHAUS, USA). About 100 μl dilutions from each sample were spread onto Petri dishes with Potato Dextrose Agar (PDA; Solarbio, China) supplemented with Streptomycin Sulfate (100 mg/l) to inhibit bacterial growth. The isolation procedure was repeated three times for each diluted sample. All the plates (9 cm diameter) were incubated at 25_°C in darkness and monitored daily to assess the growth of fungal hyphae. Colonies appearing on the agar medium were picked up and isolated onto new PDA plates every two days to obtain pure cultures. All isolated fungal strains were stored at 4°C for further analysis. All strains were deposited in the LP Culture Collection (personal culture collection held in the laboratory of Prof. Lorenzo Pecoraro) at the School of Pharmaceutical Science and Technology, Tianjin University, Tianjin, China.

Molecular and morphological identification of isolated fungi

Total genomic DNA was extracted from fresh fungal mycelia isolated from the gut of Eriocheir sinensis using a modified cetyltrimethyl ammonium bromide (CTAB) method (Doyle 1987). The fungal nuclear ribosomal internal transcribed spacer (ITS) region of the rDNA gene were amplified by polymerase chain reaction using the universal primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) (White et al. 1990). The PCR reactions were performed in 50 μl volume containing 25 μl 2× Rapid Taq Master Mix (Vanzyme, China), 2 μl of each forward and reverse primer (10 mM), 2 μl of template DNA, and 19 μl of double-distilled water. The amplification program consisted of an initial denaturation at 95_°C for 3 min, followed by 35 cycles of 95_°C for 15 s, 55_°C for 15 s, 72_°C for 15 s and a final elongation step at 72_°C for 5 min using a Mastercycler^® thermal cycler (Eppendorf, Germany). PCR products, along with a reference 2000 bp ladder marker (TsingKe, China), were analyzed by gel electrophoresis in 1% agarose gels stained with 3× Gel Red (TsingKe, China) and visualized under Gel imager (TianNeng, China). Sequencing of the PCR products was performed by Tsingke Biological Technology Company (Beijing, China) using the same PCR primers. All obtained sequences were submitted to the National Center for Biotechnology Information (NCBI) for a nucleotide BLAST search (GenBank; http://www.ncbi.nlm.nih.gov/BLAST/index.html) to determine the closest sequence matches that enabled taxonomic identification. DNA sequences from the fungal isolates were deposited in GenBank under the accession numbers MW958029–MW958069. Fungal morphology (branched septate hyphae, pseudohyphae, conidiophores, conidia, poroconidia, arthroconidia and sporangiosphores, etc.) was examined under a Nikon ECLIPSE Ci microscope for identification of isolates following the taxonomic keys for different taxa (Gilman 1957; Alexopoulos and Mims 1979).

Statistical analysis

Krona Charts illustrating the composition of fungal communities identified from gut of Chinese mitten crab were prepared using Krona Tools (https://github.com/marbl/Krona/wiki/KronaTools) (Ondov et al. 2011). In order to quantify differences in the gut fungal communities between female and male crabs, alpha diversity indexes were calculated in Multi-Variate v. 3.22 (MVSP 3.22). The fungal diversity was evaluated using Shannon index (H′), which has two main components: evenness and the number of species (Spellerberg and Fedor 2003). The Simpson index estimates the probability that two randomly selected fungal individuals from a community belong to different species (Simpson 1949). The Margalef diversity index (Margalef 1957) and Pielou index (Pielou 1966) were used to assess the species richness and evenness, respectively.

Results

Culturable fungal diversity associated with Eriocheir sinensis gut

A total of 41 strains were isolated from 6 gut samples of Chinese mitten crab growing naturally in the investigated rice-crab co-culture system. The isolated strains belonged to 3 fungal phyla, 6 classes, 7 orders, 15 families, 16 genera, and 30 species as assessed through BLAST searches and morphological identification (Figure 1). Among the identified species, 95.12% (28 species and 39 strains) belonged to 14 genera of Ascomycota, while Basidiomycota and Mucoromycota were represented by a single stain each (2.4%) of Trichosporon coremiiforme and Mucor circinelloides, respectively (Table 1). Aspergillus was the dominant genus with 10 strains followed by Penicillium (7 strains), Talaromyces (6 strains), Pyrenochaetopsis (3 strains), Acremonium (2 strains), Clavispora (2 strains), and Fusarium (2 strains), while Alternaria, Candida, Cladosporium, Meyerozyma, Mucor, Paraconiothyrium, Phoma, Trichoderma, and Trichosporon were represented by a single strain each (Figure 2). The most common species were Aspergillus niger (6 strains) and Talaromyces verruculosus (4 strains), followed by A. tubingensis, T. funiculosus, and Clavispora lusitaniae, which yielded 2 strains each (Table 1).

Figure 1. Krona chart indicating the taxonomic identification and relative abundance of fungi isolated from gut samples of Chinese mitten crab.

Figure 2. Fungal genera obtained from gut samples of Chinese mitten crab.

Table 1. Fungal diversity molecularly detected in the gut of Chinese mitten crab.

Strain no.	Host	GenBank code	Best BLAST matches	Accession code	Max score	Overlap length	Query cover	E value	% match
1	M 1	MW958029.1	Aspergillus niger	MT729934.1	1027	1027	97%	0	99.30
2	M 1	MW958030.1	Aspergillus awamori	HM037957.1	1014	1014	97%	0	99.46
3	M 1	MW958031.1	Aspergillus niger	MT218381.1	1009	1093	97%	0	99.82
4	M 1	MW958032.1	Aspergillus tubingensis	MG551279.1	1020	1348	98%	0	99.47
5	M 1	MW958033.1	Aspergillus niger	MG759551.1	1035	1035	97%	0	99.47
6	M 1	MW958034.1	Penicillium sp.	KX008632.1	981	981	98%	0	99.45
7	M 1	MW958035.1	Aspergillus tubingensis	MH237653.1	1013	1013	98%	0	99.82
8	M 1	MW958036.1	Pyrenochaetopsis indica	MH410656.1	867	867	93%	0	99.38
9	M 1	MW958037.1	Aspergillus niger	MT597435.1	1038	1038	99%	0	98.80
10	M 1	MW958038.1	Talaromyces verruculosus	MN429191.1	987	987	97%	0	99.09
11	M 1	MW958039.1	Cladosporium perangustum	KF706664.1	915	915	99%	0	100.00
12	M 1	MW958040.1	Talaromyces verruculosus	MN429191.1	994	994	96%	0	99.28
13	M 1	MW958041.1	Aspergillus niger	KX099623.1	1018	1018	99%	0	99.29
14	M 2	MW958042.1	Penicillium sp.	KX008632.1	965	965	98%	0	99.81
15	M 2	MW958043.1	Talaromyces verruculosus	MN429191.1	994	994	97%	0	99.28
16	M 2	MW958044.1	Candida intermedia	DQ665263.1	623	623	99%	2e–174	99.42
17	M 2	MW958045.1	Fusarium incarnatum	MN853391.1	894	894	99%	0	100.00
18	M 2	MW958046.1	Talaromyces verruculosus	MN429191.1	1003	1003	96%	0	99.82
19	M 2	MW958047.1	Talaromyces funiculosus	MN429201.1	987	987	94%	0	100.00
20	M 2	MW958048.1	Talaromyces funiculosus	MH590622.1	977	977	96%	0	99.08
21	M 2	MW958049.1	Penicillium sp.	MN093874.1	970	970	99%	0	99.26
22	M 3	MW958050.1	Phoma eupyrena	KJ686363.1	870	870	97%	0	98.97
23	F 1	MW958051.1	Paraconiothyrium sp.	MF185997.1	987	987	92%	0	99.45
24	F 1	MW958052.1	Trichosporon coremiiforme	KY105733.1	911	911	98%	0	99.02
25	F 1	MW958053.1	Clavispora lusitaniae	MT731390.1	645	645	98%	0	99.72
26	F 1	MW958054.1	Aspergillus niger	MT446087.1	1031	1031	99%	0	99.47
27	F 1	MW958055.1	Penicillium sp.	MN093874.1	974	974	97%	0	99.44
28	F 1	MW958056.1	Acremonium sp.	MK541538.1	869	869	98%	0	99.79
29	F 1	MW958057.1	Mucor circinelloides	KX349461.1	1081	1081	99%	0	99.01
30	F 2	MW958058.1	Acremonium furcatum	LR026793.1	872	872	92%	0	98.58
31	F 2	MW958059.1	Aspergillus flavus	MT584825.1	712	712	88%	0	92.59
32	F 2	MW958060.1	Meyerozyma caribbica	KU200440.1	1042	1042	99%	0	99.65
33	F 2	MW958061.1	Trichoderma harzianum	KU375456.1	1044	1044	96%	0	99.65
34	F 2	MW958062.1	Pyrenochaetopsis sp.	LT592946.1	876	876	97%	0	98.79
35	F 2	MW958063.1	Penicillium sp.	MN093874.1	981	981	97%	0	99.63
36	F 2	MW958064.1	Pyrenochaetopsis sp.	LT592946.1	872	872	97%	0	99.58
37	F 2	MW958065.1	Alternaria alternata	MT446076.1	893	893	96%	0	96.65
38	F 2	MW958066.1	Clavispora lusitaniae	MT731390.1	625	625	96%	6e–175	99.42
39	F 3	MW958067.1	Penicillium sp.	KM222497.1	974	1239	98%	0	99.08
40	F 3	MW958068.1	Penicillium terrigenum	MH864538.1	974	974	95%	0	98.73
41	F 3	MW958069.1	Fusarium verticillioides	MK392032.1	952	952	96%	0	99.62

Notes: BLAST search closest matches of fungal internal transcribed spacer DNA sequences amplified from E. sinensis. Sample GenBank accession codes, accession codes for the closest GenBank matches, sequence identity, and overlap of each match are reported.

Comparison of gut fungal communities in female and male crabs

There were significant differences in gut fungal diversity between female and male Chinese mitten crabs. The 22 strains isolated from the gut of male crabs were found to represent 13 species and 8 genera of Ascomycota, whereas the 19 strains isolated from the gut of female crabs belonged to 16 species and 10 genera of Ascomycota (17 strains), 1 species of Mucoromycota (1 strain) and 1 species of Basidiomycota (1 strain) (Figure 3). Female gut samples showed a higher genus richness than male: 12 genera from female and 8 genera from male crabs. Four genera including Aspergillus, Fusarium, Penicillium and Pyrenochaetopsis were found in common for both female and male crab individuals (Figure 4). The dominant fungi were different in the two analyzed groups of samples, with Aspergillus being the most abundant genus in male gut, with 8 strains, and Penicillium in female gut, with 4 strains. The four genera Candida, Cladosporium, Phoma, and Talaromyces were only present in male crabs, while 8 genera (Acremonium, Alternaria, Clavispora, Meyerozyma, Mucor, Paraconiothyrium, Trichoderma, and Trichosporon) were exclusively found in female crabs. The Shannon (H′) and Simpson (D) indexes for fungal diversity from female crabs were 2.871 and 0.005847, respectively, whereas for male crabs the two measured indexes were 2.347 and 0.07792, respectively. The Shannon index of female crab gut was higher than male, while in contrast, the Simpson index of female crab gut was lower than males. Pielou index and Margalef index of female samples (0.9934 and 5.774, respectively) was significantly higher than that of male samples (0.9150 and 3.882, respectively) (Table 2). The results indicated that the taxonomic diversity of the fungal community in the gut of the female crab was higher than in males.

Figure 3. Taxonomic profiles of gut fungal communities at different taxonomic levels. (a) phylum; (b) class; (c) family; and (d) genus.

Figure 4. Venn diagram of the culturable fungal genera from female and male crabs.

Table 2. Indexes of alpha diversity of fungal communities associated with the gut of Chinese mitten crab.

	Shannon (H′)	Pielou (E)	Simpson (D)	Margalef (DMG)
Female	2.871	0.9934	0.005847	5.774
Male	2.347	0.9150	0.07792	3.882

Discussion

This is the first study exploring the fungal community in the gut of Chinese mitten crab E. sinensis using molecular and morphological identification of isolated strains. The analysis of gut microbial diversity could be essential for understanding the ecological functions of microbes in E. sinensis, and their influence on crab health, given that the interactions between gut microorganisms and hosts have been found to be an important factor influencing aquatic animal health (Chaiyapechara et al. 2012; Yan et al. 2012; Dong et al. 2018). The results obtained in this study indicated the presence of various filamentous fungi and yeasts inhabiting the gut of E. sinensis. These results contribute to filling the gap in our knowledge of gut microorganisms in the studied crab species from a mycological point of view and complement previous information on bacterial diversity associated with E. sinensis, which revealed the dominant presence of the phyla Proteobacteria, Firmicutes, Tenericutes, and Bacteroidetes (Chen et al. 2015).

Ascomycota (95.12%), Basidiomycota (2.4%), and Mucoromycota (2.4%) were identified from the intestine of E. sinensis using ITS sequencing. Ascomycota and Basidiomycota have been frequently isolated from various crustaceans, including Xenograpsus testudinatus (shallow water hydrothermal vent crab), Litopenaeus vannamei (Pacific white shrimp), and Cherax quadricarinatus (red claw crayfish) (Li et al. 2019; Pang et al. 2019; Victor and Pahirulzaman 2020). On the contrary, the presence of Mucoromycota represents a peculiar aspect of the Chinese mitten crab gut mycobiota, since previous records of zygomycetous fungi in animals gut have been mainly from mammals, including human and panda (Hamad et al. 2012; Yang et al. 2018). The sole previous study reporting on the presence of Mucoromycota in the gut of crabs was performed by Pitts and Cowley (1974) on fiddler crab (Uca pugilator) individuals collected from South Carolina. However, in the latter study, Mucor sp. was only found in the midgut of a group of crabs that was maintained in a covered pan at 20°C for 87 days, supplied with sea water, and fed pellets of Purina rabbit chow (Pitts and Cowley 1974). It seems likely that the presence of Mucor sp. in this group of Uca pugilator crabs was induced by the experimental procedure used by the authors, given that this fungal taxon was not found in the other two groups of crab individuals that were directly analyzed, without maintenance period, within 3 h after collection (Pitts and Cowley 1974).

We found a total of 16 fungal genera colonizing the gut of the studied crab species (Table 1). Among them, Aspergillus, Candida, Cladosporium, Fusarium, Penicillium, Phoma, and Trichosporon have been previously isolated from aquatic micro- and macroinvertebrates, and vertebrates from freshwater, estuarine, and marine environments (Rand 2004). Aspergillus, a taxon including ubiquitous saprotrophic fungi that play an important role in recycling carbon and nitrogen on Earth (Fang and Latgé 2018) was found to have the highest relative abundance (24.39%) among the fungal genera recorded in E. sinensis. The dominant Aspergillus species was A. niger, which was isolated from both female and male gut samples, while A. awamori, A. flavus, and A. tubingensis, were only isolated from male individuals. Previous studies have demonstrated that Aspergillus species are essential components of crab fungal communities. For instance, Aspergillus has been described as the dominant genus retrieved from the crushed dilutions of the vent crab Xenograpsus testudinatus (Pang et al. 2019). More specifically, A. niger has been previously isolated from midgut of the fiddler crab (Uca pugilator) (Pitts and Cowley 1974), whereas A. flavus has been found from dried powder of fresh water crab (Sesarma boulengeri) living in the Shatt-Al-Arab River in southern Iraq (Al-Duboon and Farhan 2007). Although the presence of Aspergillus fungi has been documented in several crab species, the role of these fungi in the crab host is still unclear and further physiological studies are needed to understand the real function of this fungus-crab association. Phoma, a ubiquitous fungal genus reported in various natural habitats, including aquatic environments, water distribution systems, soil and air (Bennett et al. 2018), was only recorded once from the gut of the male crab (M3) in the present study. This fungal taxon has been previously described as the dominant genus isolated from cuticle of the invasive crayfish Procambarus clarkii (Dörr et al. 2012). In addition, Phoma has been isolated from the exoskeleton of snow crab (Chionoecetes bairdi) collected in the USA by Hyning and Scarborough (1973) hypothesis.

According to our BLAST results, yeasts, represented by Candida intermedia, Clavispora lusitaniae, Meyerozyma caribbica, and Trichosporon coremiiforme, accounted for a high portion (12.2%) of the 41 isolated plates. Trichosporon, a genus of anamorphic basidiomycetous yeasts, is widely distributed in rotting wood, sludge, soil, plants, leaf-cutting ants, birds, and mammals (Obana et al. 2010). Trichosporon species, represented by T. caseorum, T. asahii, and T. cutaneum, have been found in gastrointestinal microbiota from a healthy African man using culture-independent molecular identification methods based on various primer sets (Hamad et al. 2012), whereas T. coremiiforme has never been described as a gut-associated fungus. Meyerozyma caribbica (anamorph Candida fermentati) recorded with a single strain in this study has been previously isolated from the gut of four different Diptera species, namely Drosophila suzukii, Culex quinquefasciatus, Anopheles stephensi, and Aedes aegypti (De Marco et al. 2018). Two strains isolated from two E. sinensis female individuals (F1 and F2) were molecularly found to share a high similarity with Clavispora lusitaniae (Synonymy: Candida lusitaniae) previously isolated from the gastrointestinal tracts of warm-blooded animals (Lodder 1970). The Candida strain isolated from male individual M2 highly matched (>99% similarity) with Candida intermedia (accession number DQ665263.1), which was isolated from the marine environment in China by Gao et al. in an unpublished study. Candida species have been frequently documented to colonize the gut of numerous different hosts. For instance, Candida species, such as C. albicans, have been often detected in human stool (Truss 1981), and sometimes associated with diseases, such as diabetes and inflammatory bowel disease (IBD) (Sonoyama et al. 2011; Gosiewski et al. 2014). Other studies have demonstrated that species of the genus Candida could colonize the gut of birds (Larus michahellis) and insects (Dendroctonus adjunctus, D. approximatus, D. pseudotsugae, and Megaplatypus mutatus) (Rivera et al. 2009; Al-Yasiri et al. 2016; Ceriani-Nakamurakare et al. 2020). To our knowledge, there is no previous information about the presence of Candida intermedia in crab intestine. Our culture-dependent approach to assess the gut microbial diversity in E. sinensis showed the presence of a rich variety of yeasts in the studied crab species gut. Further studies based on metabarcoding culture-independent analyses are needed to provide a comprehensive description of the intestinal yeasts associated with E. sinensis, in particular for those species that are difficult or impossible to isolate in axenic culture.

With the prevalence of foodborne diseases, close attention has been paid to the food safety problems originated from microbial infections since 1992 in China (Liu et al. 2004; Xu and Zhang 2012). A total of 5021 foodborne incidents were reported in China during 2001–2012, where microbial infections were the most common cause, accounting for 40.9% of total outbreaks (Xu and Zhang 2012). It has been reported that 260,000 people get sick from contaminated fish every year in the United States (Barrett et al. 2017). In China, freshwater food production and consumption have increased rapidly during the past few years, and the yield of freshwater products in 2018 was over 3,1562,000 tons (Republic of China Ministry of Agriculture 2019). However, little is known about the fungal pathogens harbored by freshwater food in China. Our results showed that E. sinensis harbored a variety of human fungal pathogens. Among them, species from the genera Aspergillus and Candida are primarily responsible for the lethal fungal infections (Li et al. 2019). White piedra is defined as the superficial infection of hair shafts caused by Trichosporon (Erer et al. 2000). Trichoderma species, comprising T. harzianum, T. koningii, T. longibrachiatum, T. pseudokoningii, and T. viride, have been regarded as important human pathogenic fungi (Loeppky et al. 1983; Ragnaud et al. 1984; Tanis et al. 1995; Guiserix et al. 1996; Rota et al. 2000). People who wear contact lenses are likely to suffer from keratomycosis caused by Acremonium species (Fincher et al. 1991). Fusarium has emerged as a pathogenic factor of disseminated infection in patients who are undertaking intensive chemotherapy or bone marrow transplantation (Anaissie et al. 1988). Some species of the genus Penicillium are known to cause pneumonia and disseminated disease in cancer patients (Anaissie et al. 1989). A variety of fungi previously recognized as plant pathogen such as Fusarium (Burdon and Silk 1997) have been recently found to represent potential pathogens for humans (Fleming et al. 2002). Therefore, a more comprehensive risk assessment for fungal pathogens derived from Chinese mitten crab is required to effectively protect public health.

It has been shown that human males and females have gender-specific differences in their gut microbial composition (Haro et al. 2016). Additionally, a study conducted on the diversity of hand surface bacteria showed that women had significantly higher diversity than men (Fierer et al. 2008). In our study, we hypothesized that fungal communities associated with the gut of males and females were different. The results confirmed our hypothesis, showing that there were statistically significant differences in fungal species diversity between analyzed female and male crab individuals. The Shannon and Simpson indexes were 2.871 and 0.005847, respectively, for gut from female crabs, while the two calculated values were 2.347 and 0.07792 for male crabs. This result revealed that the fungal population observed in E. sinensis female individuals was more diverse and species rich than that in males. A previous study investigating the gut mycobiota of a cohort of healthy human subjects revealed that female subjects possessed a higher number of fungal isolates and species compared to male subjects (Strati et al. 2016 ). It has been described that sex hormones may play a key role in modulating microbiota composition in mice (Markle et al. 2013). Moreover, sex-diet interaction may also influence the composition of vertebrate microbiota (Bolnick et al. 2014). Our findings showed, for the first time, gender-related differences in the Chinese mitten crab gut fungi. Due to the limited knowledge on sex-dependent microbiota in crabs, it is difficult to assess the mechanisms that trigger this special phenomenon. Further investigations are necessary to understand which host sexual differences influence the gut fungal diversity in Chinese mitten crabs and how.

Conclusion

Our study represents the first report on intestinal fungal communities of Chinese mitten crab. We observed that the gut of E. sinensis harbored a considerable number of filamentous fungi in combination with ascomycetous and basidiomycetous yeasts. Significant difference of intestinal fungal diversity was found between female and male crabs, with female individuals showing a higher number of fungal species. Our findings provide valuable information on microbes associated with one of the most popular fresh water crab species in the Chinese food industry, and represent a starting point in the analysis of Chinese mitten crab microbiology to support the effective management and conservation of this crab species. Knowledge of gut-associated microbial communities is essential for the improvement of economic performance of the crab industry. Further studies are necessary to describe the interaction mechanisms between intestinal fungal communities and Chinese mitten crab hosts, and to understand the exact role that gut-associated microbes play in the analyzed crab species health.

Data availability statement

The Fungal DNA sequences amplified during this study are available in GenBank (https://blast.ncbi.nlm.nih.gov/Blast.cgi) under accessions MW958029–MW958069.

Disclosure statement

No potential conflict of interest was reported by the author(s).