Sharks breathe through various mechanisms. Regardless of the mechanisms by which they breathe, all sharks must maintain water passage through their gills to receive oxygen(Meng et al., 1987; De Maddalena, 2007). The water flow through sharks’ gills is striking; for Cetorhinus maximus, up to 2 000 tons of water per hour passes through the gills(Knickle et al., 2011). A sponge-like structure facilitates the accumulation of a diverse mixture of marine microorganisms, including fungi, from their diverse living environments.

Here, we isolated gill fungi from a Carcharodon carcharias shark. Furthermore, we screened this population for their antimicrobial, larvicidal, and cytotoxic bioactivities. Finally, we investigated the active isolates’ taxonomy, and their bioactive metabolite production with the aim of finding useful biopharmaceutical fungal strains.

The gill samples were obtained as a donation, in March of 2010, from a shark, which was accidentally captured and killed by punching its head promptly to terminate suff ering in a legal fishery activity in the East China Sea near Wenzhou City, Zhejiang Province, China, not for the purpose of this study. The fishing was licensed by the local Administration of Ocean and Fisheries. The shark was identified as a Carcharodon carcharias, commonly known as a great white shark, by Prof. ZHAO Yongbo, an ichthyologist with the Dalian Museum of Natural History. The gill rakers were removed using sterile scissors, washed with sterile sea water, sealed in containers under sterile conditions, and stored on ice during air transport to our facility.

In a sterile environment, the largest gill raker was washed with sterile sea water and dissected into small pieces. Next, the pieces were transferred onto agar plates, cultured at 28°C for 3-7 days, and monitored every 12 h. When fungal colonies formed on the gill tissues, they were transferred onto new agar plates. To avoid repeated isolation from the same piece, only the colonies with diff erent morphologies were isolated. The colonies were purified using a repeated hyphal tipping method until pure cultures were obtained(Strobel et al., 1996). The pure isolates were then inoculated onto marine potato dextrose agar(PDA)slants and immersed in sterile liquid paraffin for preservation at 4°C. Four basic types(3 sub-types containing diff erent additives for each type)of culture media were used for strain isolation, including the GPY(glucose-peptone-yeast extract), PSP(potatosucrose- peptone, based on the common PDA medium), M(malt extract), and CY(Czapek’s-yeast extract)media(see supplementary Table S1 online).

As an additive for some culture media(Table S1), the sterile shark gill extract was prepared as described here. The gill pieces were mixed with 2% sterile seawater(v/v=1:1) and ground with a sterilized mortar and pestle, and the homogenate was then diluted 10- fold and filtrated through sterile gauze. The filtrate was centrifuged at 4°C at 5 000 r/min for 30 min, and finally, the upper liquid layer was micro-filtrated(0.45 and 0.22 μm)to yield the sterile gill extract. Marine PDA slants: normal PDA culture medium contains 2% sea salt(Pinkerton and Strobel, 1976).

All the isolates were inoculated in Petri dishes containing approximately 15 mL of the isolation agar medium without antibiotics(see the details in Table S1 online). After 7 day of static fermentation at 28°C, the agar media containing the fungal cultures were sectioned and extracted together with one volume of methanol for 24 h at room temperature. Afterwards, the supernatants were filtered and evaporated to dryness in a rotary evaporator at 45°C. Finally, these crude extracts were dissolved in 3 mL of absolute methanol. Therefore, 1 μL of the crude extract sample was equal to the metabolites produced in 5 μL of the solid culture medium. For the following biassays, methanol and blank culture media extracts(going through incubation without inoculum)were used as negative blank and medium controls, respectively.

Samples(30 μL)were seeded into 96-microwell plates. Ampicillin was used as a positive control. Each sample was tested in triplicate. The plates were dried in a vacuum oven overnight at room temperature. Next, 100 μL of Muller-Hinton broth, containing 10 ⁵ CFU/mL of MRSA(strain number A7983, donated by the Dalian Friendship Hospital)was added into the samples and incubated at 35°C for 24 h. Finally, 20 μL of MTT(3-(4, 5-dimethylthiazol-2-yl)- 2, 5-diphenyltetrazolium bromide, 1 mg/mL)was added and the plates were incubated for an additional 30 min. Absorbency measurements were taken at 540 nm using a microplate reader. The inhibition ratios were calculated using the following formula: Inhibition ratio(%)=100 (OD _blank -OD _sample)/OD _blank.

The DDRT experiment is an antimicrobial and antitumor related model for detecting potential DNA damaging agents using a pair of E. coli indicator strains(purchased from Yale University): AB1157(+)(wild type), which is able to repair DNA damage, and AB3027(−)(deficient), which is deficient in this function(Bartus et al., 1984; Li and Lin, 2004; Alam et al., 2009).

Preliminary antimicrobial screening using the deficient strain was performed using the paper disk method. Briefly, 30 μL of a sample was placed on a 6 mm-paper disk and air dried. The disks were then placed on nutrient agar plates coated with 100 μL of a bacteria suspension(10 5 CFU/mL) and then cultivated at 37°C for 16 h. The plates were then examined for inhibition zones, and their diameters were measured. The isolates that yielded inhibition zones larger than 7 mm were characterized as active and subjected to further testing, in which the DDRT was used to evaluate the samples’ selective inhibition ability, and the wild type and deficient E. coli strains were used as indicators. To determine the samples that were capable of targeting DNA, the samples were incubated with both E. coli strains at the same concentration. The experiment was performed in triplicate using a similar method as the above described anti-MRSA test. A st and ard nutrient broth medium was used to cultivate the E. coli, and adriamycin was used as the positive control. When a given sample exhibited a ratio(selectivity index, SI)of inhibition against the deficient strain to inhibition against the wild type strain that was larger than 1.5, that sample was characterized as a potentially DNA damaging active sample. If a sample showed 100% inhibition to both E. coli strains at a dose of 30 μL per well, the dose was sequentially decreased(10, 5, 2, or 1 μL per well), until inhibitory diff erences no less than 1.5 times were observed between the two strains or inhibition was lost entirely.

The inhibition of P. oryzae mycelium growth is a modified antifungal and antitumor related model aimed at detecting anti-mitosis agents that are able to interfere with microtubule formation(Kobayashi et al., 1996). The experiments were performed using a similar methodology to the anti-MRSA test. The samples were again loaded into 96-well microwell plates. Sabouraud’s broth(100 μL)containing 3%(v/v)Pyricularia oryzae mycelium suspension was added and incubated at 30°C for 36 h. Next, MTT dye was applied to the samples and absorbance readings were taken at 540 nm. Taxol was used as a positive control.

The above-mentioned P. oryzae mycelium suspension was prepared using the following method. Sterile water(20 mL)was added to P. oryzae cultured in 9-cm Petri dishes for 10 days. Then, the mycelium was minced into fragments using a sterile blade. Next, a suspension containing mycelial fragments was collected and separated into small mycelium pellets using a supersonic cleaner(250 W, 40 kHz)for 5 min. Finally, the suspension was filtrated using two layers of sterile gauze.

The brine shrimp larva lethality test is commonly used as a cytotoxicity model in antitumor and pesticidal assays(Hu et al., 2000; Carballo et al., 2002; Xiong et al., 2004). The test was performed as previously described(Zhang et al., 2012). The samples were tested for their lethality in 96-well microwell plates. Approximately 20 to 30 vivid instar II−III Artemia parthenogenetica larva in 200 μL of seawater were incubated per well. The dosage for each sample was 30 μL/well(the solvent, methanol, was similarly dried in vacuum before the test). Taxol, adriamycin, and trichlorphon were used as positive controls. The test was performed in triplicate, and the corrected average lethality rate(16 h)of each sample was calculated according to the Abbott formula(Abbott, 1925).

Those isolates showing strong bioactivities in the preliminary bioassays were statically fermented for re-screening in 500-mL Erlenmeyer flasks containing 100 mL of liquid culture broths at 28°C for 30 days. Here, pairs of culture broths were used(Table S1 online). If a given fungus was initially isolated from the isolation agar medium without the host gill extract, it was fermented both in its previous medium(without agar) and the liquid medium with the host gill extract as an additive. If the fungus was initially from the isolation agar medium containing the host gill extract, it was fermented both in its previous medium(without agar) and the liquid medium without the host gill extract. After fermentation, the broth and mycelia were extracted using ethyl acetate and methanol under room temperature, respectively. The two extracts were finally combined and rotary evaporated to dryness and then resuspended in 3 mL of MeOH for the use in the following bioassays.

These fungal extracts were firstly checked for their reproducibility of anti- E. coli DDRT and brine shrimp larvicidal bioactivities using the same methods as described in the above 2.2.

Then, out of those extracts reproducing their activity, some samples were r and omly selected and screened in a MTT cytotoxic assay against a human breast cancer cell line(MCF-7) and a cervical cancer cell line(Hela)(Mueller et al., 2004). The results were reported as hemi-inhibitory concentrations(IC ₅₀)after a 48 h treatment. The morphology of the MCF-7 cells treated with 200 μg/mL for 48 h was observed using an Olympus CX41-32RFL fluorescence microscope.

The taxonomy evaluations were performed using ITS-LSU(ITS1-5.8 S-ITS2 and partial 28 S)rDNA analysis and supported by the morphology. Before the molecular experiments, the bioactive isolates were preliminarily classified into diff erent groups according to their colonial and microscopic characteristics on the isolation culture media, marine PDA and CA(normal Czapek’s agar medium containing 1.6% sea salts)plates. Then, after one week of cultivation on the marine PDA plates, representative active isolates’ mycelia were harvested for DNA isolation. The DNA was extracted and purified using the Plant Genomic DNA Kit DP305(Tiangen, Beijing, China)according to the manufacturer’s protocol.

DNA amplification was performed via PCR using TaKaRa Ex Taq polymerase(TaKaRa, Dalian, P. R. China) and the primer pair ITS1(5'-TCCGTAGGTGAACCTGCGG- 3') and ITS4(5'-TCCTCCGCTTATTGATATGC- 3')in an TaKaRa Thermal cycler Dice TP600. The following protocol was used: 1)initial denaturation 94°C/5 min; 2)denaturation 94°C/0.5 min; 3)annealing 55°C/0.5 min; 4)extension 72°C/1 min; 5)final extension 72°C/5 min; steps 2-4 were repeated 30 times(White et al., 1990). The PCR-product mixtures were analyzed using electrophoresis and purified using the TaKaRa Agarose Gel DNA Purification Kit DV805A according to the manufacturer’s protocol. The purified PCR products were sequenced using an ABI PRISMTM 3730XL sequencer with the ITS1 primer. The sequence data obtained during this project have been submitted to GenBank.

The ITS-LSU rDNA sequences were then used to search the GenBank database with the BlastN 2.2.19+ algorithm to determine the genus taxonomy of the samples using the closest matches to the corresponding rDNA sequences of known fungi. Thirteen isolates were identified in the same genera as molecularly identified isolates using colonial and microscopic characteristics on the isolation culture media, as well as on marine PDA and CA plates.

The sequences of the bioactive strains in rescreening were aligned with the representative fungal ITS-LSU rDNA sequences using Clustal X 1.81, and a phylogenetic tree was constructed using MEGA 4.0. The bootstrap values were calculated using 1 000 bootstrap samples, and the maximum parsimony(MP)method was used to infer the tree topologies(Thompson et al., 1997; Tamura et al., 2007).

The extracts of the strains with representative bioactivity and taxonomy were analyzed using HPLCDAD- HRMS. For this analysis, a 1-mg/mL methanol solution was prepared. The analysis was performed on an Ultimate 3000 HPLC connected to a DAD detector and a Bruker Daltonics maXis 4G UHR-TOF mass spectrometer equipped with an electrospray ionization source. Stationary phase: Nucleosil 100 C18, 3 μm, 100  2 mm ID, equipped with a guard column 10  2 mm ID. Mobile phases: A=water/formic acid(999.4:0.6), B=methanol/formic acid(999.9:0.1); gradient was from⁰% B to 100% B over 20 min and 100% B for another 10 min; flow rate: 300 μL/min; column temperature: 40°C. MS condition: mode: ESI-TOF, positive and negative, alternating; capillary voltage: 3.5 kV; temperature: 350°C; mass scanning range: m/z 50 to m/z 1 000. The injection volume was 3.0 μL. The Bruker Daltonics DataAnalysis software 4.0 was used for the data analysis.

For clear peak recognition, the base peak chromatography(BPC)results were extracted from the positive and negative full-scan total ion chromatography. For each peak, the retention time, maximum UV-visible absorption, and an accurate mass were recorded. The software’s SmartFormulae function was used to generate molecular formulae.

During this process, the default settings were adopted, such as the minimum carbon content, upper, and lower limits for elements, number of rings and double bonds, C/H ratios. Halogens were added to elements when their typical isotopic patterns were observed. Mass error tolerances were set to be ≤3 parts per million. Furthermore, to confirm the correct molecular formulae, the mass errors and true isotopic patterns(mSigma values)were taken into consideration for more than one pseudo-molecular peak, e.g., [M+H] +, [M+Na] +, [2M+H] +, [2M+Na] +, [M+H−H ₂ O] +, [M−H] −, [M+Cl] −, and [M+HCOO] −, etc., in the mass spectra of each chromatographic peak under both positive and negative MS modes. If necessary, the MS/MS fragment information was also used to exclude improper c and idate formulae. Then, the molecular formulae were used to search microbial(mostly fungal)metabolites in the Dictionary of Natural Products 2011(DNP2011)database(Buckingham, 2011). The resulting list was shortened using UV spectral comparison if the UV data were also available for the hits. The c and idates with very diff erent UV characteristics were eliminated.

Fig. 1 Representative fungal isolate colonies growing on common cultural media a. BP2T4 Arthrinium sp. on Czapek’s agar(CA), 7 day; b. TBG3-5 Mucor sp. on potato dextrose agar(PDA), 7 day; c. TBG2-10 Chaetomium sp. on PDA, 7 day; d. TBG3-12 Penicillium sp. on PDA, 7 day; e. TBG1-12 Penicillium sp. on CA, 7 day; f. BP2T2 Penicillium sp. on CA, 7 day; g. TBG1-1 Penicillium sp. on CA, 7 day; h. TBG3-10 Penicillium sp. on CA, 7 day; i. TBG3-3 Aspergillus sp. on PDA, 7 day; j. BCT1-4 Aspergillus sp. on PDA, 7 day; k. BCT2-3 Aspergillus sp. on PDA, 7 day; l. BP321 Aspergillus sp. on CA, 7 day. It is worth noting that the Penicillium and Aspergillus in these isolates showed quite diverse morphological diff erence.

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In total, we isolated 115 fungal strains with diverse colonial characteristics from gill samples from a shark(Carcharodon carcharias), which was obtained by accident during a fishing activity in the East China Sea near Wenzhou City, Zhejiang Province, China(Figs.1 and 2). Among the four types of isolation culture media employed, the GPY type(glucosepeptone- yeast extract)yielded the most fungal isolates, followed by the PSP type(potato-sucrosepeptone) and the M type(malt extract). In other reported studies, GPY/GP(glucose-peptone)media and malt extract media were also used very often and were recommended as the most suitable media for isolating diverse marine hosts-associated fungi(Baker et al., 2009; Menezes et al., 2010; Ding et al., 2011; Zhou et al., 2011). The reason may be the presence of the rich and easily utilizable carbon and nitrogen sources in these media, as well as the growth factors. Although PSP type media were not reported as often as the former two, they performed even better than M type in our study, which should be due to the same reason. The CY medium(Czapek’s agar-yeast extract), which was the most oligotrophic medium used in this study, yielded the smallest number of fungal isolates.

Fig. 2 The number of fungal isolates from the gills of C. carcharias acquired through cultivation in four types(12 kinds)of culture medium For GPY medium(glucose, peptone, and yeast extract as the main ingredients), GPY-1 represents the enriched medium without the host extract, GPY- 2 represents the enriched medium with the host extract, and GPY-3 represents the oligotrophic medium with the host extract; for the PSP medium(potato, sucrose, and peptone as the main ingredients), M medium(malt extract as the main ingredient), and CY medium(Czapek’s agar with yeast extract as the main ingredient), a similar naming strategy was followed.

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In previous reports, marine organisms like sponges and seaweeds have been commonly used as host materials for investigation of culturable fungi(Baker et al., 2009; Menezes et al., 2010; Ding et al., 2011; Wiese et al., 2011; Zhou et al., 2011; Passarini et al., 2013). As reported, usually 20-80 isolates can be obtained from each host. Therefore, preliminary comparison with these cases showed that shark’s gills are possibly a comparable source of marine-derived fungi if merely from the point of pure microbiological research.

The isolates were screened for their bioactivities, including anti-MRSA activity, activity against E. coli and selectivity in the DNA damage repair test(DDRT), inhibition to the mycelium growth of Pyricularia oryzae, and lethality to brine shrimp larva. These assays act to determine the potential for these organisms to be used as antimicrobial, pesticidal, and possible antitumor agents.

Out of the 115 isolates, 58 demonstrated meaningful activity in one or more tests(see the details in supplementary Table S2 online). Among these active isolates, 33 exhibited activity against MRSA(G+; proportion: 28.7%), 23 against E. coli(G-; 20.0%; 16 of these 23 also had selectivity in DDRT, 13.9%), 7 against P. oryzae(6.1%), and 25 against brine shrimp larva(21.7%). Furthermore, a subset of these isolates(20 in the anti-MRSA test, 6 in the DDRT, 3 in the anti- P. oryzae test, and 17 in the anti-brine shrimp test)exhibited strong inhibitory or lethality rates higher than 85% or with a SI value higher than 5. Twenty of the isolates exhibited broad activity spectra towards at least two models, including 3 isolates that presented 3 types of activities. These results suggested that a significant portion of these shark gill-derived fungi possess the ability to produce antimicrobial(G+, G-, fungi) and larvicidal substances.

After the above pre-screening, 24 strongly active isolates were statically fermented(Table S2). Each isolate was fermented in two kinds of liquid culture media(with and without host extract), and then extracted. These extracts were re-screened for bioactivity using two rapid antitumor related models, i.e., DDRT and brine shrimp larva. In this experiment, the bioactivity of 22 extracts from¹⁴ strains was reproduced(Table 1). The fermentation conditions used here, such as static cultivation in liquid and 30-day’s cultural cycle, were possibly not suitable for all the strains to produce active substances, which may be the reason why not all of them reproduced activity.

Table 1 The list of 22 liquid fermentation extracts showing strong bioactivity in the re-screening

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To further investigate their inhibition against cancer cells, 5 isolates’ extracts were r and omly selected from the above mentioned 22 extracts, and the cytotoxic activity was assayed. In this set of experiments, 4 extracts showed remarkable antiproliferative activity against human cancer cell lines MCF-7 and /or Hela, with the IC ₅₀ values ranging from⁷.8 μg/mL to 61.7 μg/mL(Table 1). Microscopy of the MCF-7 cells treated with these fungal extracts also displayed remarkable morphological change, i.e. from large polygonous cells to microspheres, particularly for extracts BP2T2(PSP-2) and BP3T3(PSP-4), when compared to the negative controls(Fig. 3). Although not all active isolates were tested for their cytotoxic activity for practical reasons, the performance of these five r and om samples is thought to be representative of the group and their potential usage as source of cytotoxins is promising. Of course, more detailed and exp and ed bioassays using both cancerous and normal human cell lines are necessary in further study on anti-cancer drug c and idates’ discovery.

Fig. 3 Microscopy of breast cancer cells (MCF-7) treated using the fungal extracts a. negative control; b. TBG3-14 (fermented in GPY-3 culture broth); c. BP2T2 (PSP-2); d. TBG2-2 (GPY-1); e. BP2T1 (PSP-1); f. BP3T3 (PSP-4). The cells were treated with 200 μg/mL for 48 h. The images were magnifi ed 100 times.

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Given the high percentage of the shark gill-derived fungal isolates exhibited multiple bioactivities, a potentially important source of biopharmaceutical fungi has been identified. To underst and the natural role of these bioactivity in shark gills, we suppose that an important motive for the producers may be the need for competition with other microorganisms, fouling organisms, or parasites just as some endophytic microorganisms in other marine hosts do(Xu and Yan, 2006). It’s not excludable that they may also be involved in their host’s chemical defense against pathogens or canceration. However, further chemecological investigation is still needed for answering these questions.

In total, 45 of the 58 active isolates, as the representatives from diff erent morphological groups, were sequenced for their internal transcribed spacerlarge subunit(ITS-LSU)rDNA and identified at the genus level through comparison with known nucleotide sequences in GenBank using BLASTN. These genera included Penicillium(31), Aspergillus(6), Neosartorya(1), Chaetomium(2), Arthrinium(1), and Mucor(4)(see details in supplementary Table S3 online). In this comparison, the maximum identities(MI)to corresponding reference isolates(mainly with marine or coastal origins)ranged from⁹⁸% to 100%. Although ITS-LSU rDNA is widely used to identify fungi at the species level, it was incapable of disseminating the highly similar species in some genera such as Penicillium and Aspergillus(Peterson, 2012). Therefore, we cautiously characterized these isolates on the genus level in this report. Additionally, we classified 13 isolates(the rest in 4 morphological groups)to the same genera as their molecularly identified representatives due to their identical colonial morphology and microscopic characteristics, including typical Penicillium(11) and Mucor(2).

The producers’ taxonomical distribution was used to assess the relationship between the fungal isolates’ bioactivities and their taxonomy. There were four dominate taxa, Penicillium(42 active strains), Aspergillus(6), Mucor(6), and Chaetomium(2).

Furthermore, in all the four basic bioassays, Penicillium was the predominant active genus(Fig. 4). The genera, Aspergillus, Mucor, and Chaetomium were the mutual active genera in anti-MRSA, anti- E. coli(DDRT), and anti-brine shrimp assays. The Arthrinium(1) and Neosartorya(1)isolates only exhibited anti-brine shrimp bioactivity. As is well known, the members of Genera Penicillium, Aspergillus, and Chaetomium are important antibiotics’ producers. The other three taxa also gave bioactive metabolites’ records in the Dictionary of Natural Products 2011(DNP2011)(Buckingham, 2011). The presence of these fungi in shark gill and their bioactivity indicated that shark gill could be used as a new source of biopharmaceutical fungi.

Fig. 4 Group distribution of bioactive isolatesThe bars showed the bioactive isolates’ taxonomical distribution for eachset of bioassay. For anti- E. coli active strains, a further DDRT test wasperformed. The bars showed the bioactive isolates’ taxonomical distribution for each set of bioassay. For anti- E . coli active strains, a further DDRT test was performed.

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In the re-screening after static liquid fermentation, the 14 producers that displayed anti- E. coli(DDRT) and /or anti-brine shrimp activity were mostly Penicillium strains, as well as one Chaetomium(BP3T10) and three Mucor strains(TBG1-21, TBG2- 1, and TBG3-4). Their taxonomy was further investigated using the maximum parsimony(MP)phylogenic tree construction, which was able to show the diff erences between highly similar ITS sequences, especially those of the Penicillium species. In the MP tree(Fig. 5), the 10 Penicillium strains clearly fell into 5 clades based on their genetic affiliation, displaying their intragenera diversity. These clades showed close lineage with P. polonicum, P. crustosum, P. commune, P. chrysogenum, and P. atramentosum. These genetic clades also corresponded to the main morphological groups for the Penicillium strains. For the Chaetomium and Mucor strains, the MP tree also revealed their close relationship to C. globosum and M. circinelloides rather than with other species in their own genera.

Fig. 5 A phylogenic tree of the reference sequences from GenBank(not underlined) and the ITS1-5.8S-ITS2 rDNA sequencesof the 14 bioactive strains(underlined)that displayed activity in the re-screeningSoftware: Mega 4.0; method: maximum parsimony; bootstrap value: calculated using 1 000 replications

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As mentioned above, in the liquid fermentation and re-screening experiments, 22 extracts from¹⁴ strains showed obvious anti- E. coli AB3027 and /or larvicidal activities. To investigate the chemical properties of their bioactive molecules, the extracts of 9 strongly active strains were selected for HPLCDAD- HRMS constituent analysis. These strains were chosen because they represented diff erent phylogenic clades/subclades, including 6 Penicillium strains(BP2T1(extract in medium PSP-2), BP2T2(in PSP- 2), BP2T3(in PSP-2), TBG1-4(in GPY-2), TBG2-2(in GPY-1), and TBG1-21(in GPY-1)), 2 Mucor strains(TBG2-1(in GPY-2) and BP1T2(in PSP-1)), and one Chaetomium strain(BP3T10(in PSP-3)). The HPLC-DAD-HRMS analysis demonstrated the richness of their secondary metabolites. In total, 119 recognizable peaks(17-34 for each sample)under positive and /or negative ESI modes were described(Fig. 6 and Table S4). Because of the similarity in the basic peak chromatography(BPC)profiles, these strains were classified into 5 groups: group 1(extracts 2 and 3), group 2(extract 4), group 3(extracts 7, 10, and 21), group 4(extracts 9 and 18), and group 5(extract 15). These groups were consistent with the result of the phylogenic analysis.

Fig. 6 Basic peak chromatography(BPC)analysis using HPLC-DAD-MS of nine representative strains For each sample, the upper spectrum was detected using positive ESI-MS mode, and the lower spectrum was detected using negative ESI-MS mode. Theunified peak numbers were assigned for the spectra in order of retention time. The peaks marked with bold numbers were possible new metabolites.

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By use of the accurate mass information from TOF-MS and the SmartFormulae function of the DataAnalysis software 4.0, totally, 109 molecular formulae were deduced for the peaks, with the exception of 10 peaks that had no credible c and idate formulae with acceptable mass errors(≤3 parts per million). The results were surprising, as 75 of the 109 identified peaks were alkaloids or nitrogen-containing compounds. In addition, 9 peaks with C ₂₈ carbon backbones were most likely ergosteroids, nortriterpenoids or others natural products, 4 peaks were possibly anthraquinones or xanthones(C _{14- 16} H _{10 - 12} O_{4 - 6}), and the other peaks that did not contain nitrogen had diverse carbon skeletons(i.e., C ₅, C ₆, C ₁₂, C ₁₃, C ₁₈, C ₂₁, C ₂₂, C ₂₄, and C ₂₅) and belonged to diff erent structural types.

By searching the Dictionary of Natural Products 2011(DNP2011)database with the molecular formulae of the peaks(Buckingham, 2011) and further comparing the UV characteristics(if UV data were available for the hits), some fungal or microbial metabolites from the database were chosen as putative hits for those peaks with identified molecular formulae.

As a result, 40 hits had reports of antimicrobial, cytotoxic, antitumor, or pesticidal activity or were able to inhibit target enzymes(see details in supplementary Table S4 online). Moreover, some peaks also showed clear UV spectra that were similar to the UV reports for these hits.

For example, peak 39(RT=8.7 min, C ₁₈ H ²⁰ N ⁴ O₃)in extract 10 displayed similar UV maxima, i.e., 225 and 300 nm to its hit, aurantiomide C, a cytotoxic quinazoline alkaloid from a marine sponge-derived Penicillium strain SP0-19(Xin et al., 2007). Peak 44(RT=9.0 min, C ₁₅ H ₁₁ NO₃)in extract 10 showed UV maxima at 225, 195, 240, 285, and 320 nm, which were close to its hit, viridicatol, an antitumor quinolinone alkaloid produced by a Penicillium strain and a marine-derived Aspergillus strain. Furthermore, a series of possible viridicatol precursors or derivatives were also detected in extracts 7, 10, and 21, with peak numbers of 29(cyclopenol), 42(cyclopenin), and 69(O - methylviridicatin), respectively(Mohammed and Luckner, 1963).

Peak 54(RT=9.7 min, C ₁₅ H ₁₀ O₅)in extracts 9 and 18 showed UV maxima at 230, 260, and 330 nm, similar to several xanthone or anthraquinone hits(toxic, antimicrobial, or antineoplastic)produced by Penicillium and other fungal species.

Peak 57(RT=10.1 min, C ₂₃ H ₂₃ N ₅ O₄)in extracts 2 and 3 displayed UV maxima at 230 and 330 nm, agreeing with its hit, meleagrin, a cytotoxic alkaloid from two Penicillium species(Du et al., 2010). Additionally, possible derivatives of meleagrin, including oxaline(also cytotoxic), neoxaline, and 9-epineoxaline, were found in extracts 4 and 2 with peak numbers of 67, 46 or 49, and 49 or 46, respectively; besides, several possible roquefortine(oxaline’s precursor)analogues were also detected in extracts 4, 7, and 21, with peak numbers of 62(N ₆ - formylroquefortine C), 68(roquefortine C), and 81(roquefortine F), respectively(Pieter and Robert, 1983).

Peak 85(RT=12.4 min, C ₂₇ H ₂₉ N ₃ O₃)in extract 10 displayed UV maxima at 225, 280, and 320 nm, which is similar to fructigenine A, a cytotoxic diketopiperazine alkaloid produced by many Penicillium species(Buckingham, 2011).

Although this DNP searching can not definitively link these peaks to the putative hits, it off ers some possible explanation for the bioactivity of the strains. Further investigation is needed to determine if these bioactive peaks are indeed these metabolite hits or if they are uncharacterized natural products.

Another interesting finding is that these strains can also produce some possible novel metabolites that were not recorded in the DNP2011 database. In detail, 22 peaks had no formula or UV-matching formula hits in the database, and 12 peaks only had plausible hits from bacteria, streptomycetes, higher plants or animals but not from fungi. With their high proportion of novel peaks(34 out of 119), these shark gill-derived fungi should be valuable as producers of new bioactive compounds. Based on the molecular formulae, UV characteristics, and co-existence with identified metabolites in extracts, some peaks may be new derivatives of known bioactive compounds. For example, peak 36(RT=8.4 min, C ₁₆ H ₁₄ N ₂ O₃)may be a derivative of O -methylviridicatin(peak 69(C ₁₆ H ₁₃ NO₂)also in extract 10); peak 79(RT=11.9 min, C ₂₂ H ₂₁ N ₅ O₂)may be a derivative of roquefortine C(peak 68(C ₂₂ H ₂₃ N ₅ O₂)also in extract 21); and peaks 61(RT=10.2 min, C ₃₂ H ₃₈ N ₂ O₇), 66(RT=10.8 min, C ₃₂ H ₃₈ N ₂ O₇), 70(RT=11.2 min, C ₃₂ H ₃₈ N ₂ O₇), 77(RT=11.8 min, C ₃₂ H ₃₆ N ₂ O₆), and 78(RT=11.8 min, C ₃₁ H ₃₈ N ₂ O₆)were possibly new cytotoxic chaetoglobosin or penochalasin derivatives of peak 65(C ₃₂ H ₃₈ N ₂ O₅, also in extract 15). Given these results, we may find partial clues of these shark gill-fungi’s meaningful products and more new bioactive products may be expectable. Overall, the HPLC-DAD-MS analysis and the database mining reflect these strains’ potential for producing diverse bioactive metabolites, including novel natural products.

In the field of marine natural product chemistry, sponges are viewed as important host source of pharmaceutical microorganisms due to their water filtering behavior(Bugni and Irel and , 2004; Proksch et al., 2010; Hentschel et al., 2012; Swathi et al., 2013). To date, sharks have not drawn similar attention even though they also possess the ability to collect cultivable fungi that produce diverse bioactive metabolites by their gills’ filtration. Here, we report that the fungi from the gills of Carcharodon carcharias can produce remarkable antimicrobial, larvicidal and cytotoxic bioactivities in high yield. The molecular and morphological taxonomy determined that the bioactive substances were produced mainly by organisms from the genera Penicillium, Aspergillus, Mucor and Chaetomium. The HPLC-DAD-HRMS analysis clearly showed the high diversity and novelty of the bioactive secondary metabolites produced by these fungi. This research revealed the potential of shark gill-derived fungi as a novel source of antimicrobial, pesticidal and antitumor agents.

The authors express their thanks to Prof. ZHAO Yongbo from Dalian Museum of Natural History for shark specimen’s identification and to Dr. ZHANG Hong from Dalian University of Technology for her help on cytotoxic assay using cancer cell lines.