Thielavins W–Z7, New Antifouling Thielavins from the Marine-Derived Fungus Thielavia sp. UST030930-004

Eleven new depsides—thielavins W–Z (1–4) and thielavins Z1–Z7 (5–11)—and also four known thielavins—A, H, J, and K (12–15)—were isolated from the ethyl acetate extract of a marine-derived fungal strain Thielavia sp UST030930-004. All of these compounds were evaluated for antifouling activity against cyprids of the barnacle Balanus (=Amphibalanus) amphitrite. The results showed that compounds 1–3 and 6–13 were active, with EC50 values ranging from 2.95 ± 0.59 to 69.19 ± 9.51 μM, respectively. The inhibitive effect of compounds 1–3 and 7 was reversible. This is the first description of the antifouling activity of thielavins against barnacle cyprids.


Identification of the Fungus
The internal transcribed spacer (ITS) sequence of the fungal isolate UST030930-004 (GenBank accession number KJ716558) had a similarity of 97% with Thielavia terrestris NRRL 8126 (CP003011), indicating that isolate UST030930-004 is a Thielavia sp. closely related to this taxon.
Compound 1 was isolated as a white amorphous powder.

Identification of the Fungus
The internal transcribed spacer (ITS) sequence of the fungal isolate UST030930-004 (GenBank accession number KJ716558) had a similarity of 97% with Thielavia terrestris NRRL 8126 (CP003011), indicating that isolate UST030930-004 is a Thielavia sp. closely related to this taxon.

Anti-Larval Settlement and Recovery Activities
The anti-larval settlement activities of compounds 1-15 against cyprid larvae of B. amphitrite are summarized in Table 5. Compounds 1-3 and compounds 6-13 deterred larval settlement (Figure 3a). Due to the poor solubility of this group of compounds, we could not determine the LC 50 values. Compounds 1-3, 7, and 11 also showed narcotic effects against cyprids of B. amphitrite at a concentration of 10 µM. Many organic molecules cause narcosis in barnacle larvae [11,12]. With increasing concentrations of active thielavins, cyprids lost their phototactic response, showed reduced appendage activity and were completely immobilized at a concentration of 10 µM. However, when the cyprids were transferred into 0.22 µM filtered seawater FSW following exposure to thielavins for 24 h, some recovered quickly from the chemical shock and completed their attachment and metamorphosis. The recovery rates of cyprids treated with 10 µM of compounds 1-3, 7 and 11 demonstrated that larvae had the highest recovery rate from treatment with compound 1, while no larvae recovered from treatment with compound 11 for 24 h (Figure 3b). Of all of the compounds, compound 1 showed excellent antifouling activity, and cyprids treated with this compound had the highest recovery rate. Thus, compound 1 is a promising natural antifoulant.  Asterisks indicate significant differences from control using Tukey's test (* p < 0.05, ** p < 0.01, oneway ANOVA).

Isolation and Identification of the Fungus
The fungal strain UST030930-004 was isolated from 12-d biofilms developed at the pier of the Hong Kong University of Science and Technology (Hong Kong, China) in Port Shelter. The biofilms were developed on polystyrene dishes submerged in seawater for 12 days in September 2003 and scraped from the dishes using a sterile glass coverslip. The scraped samples were suspended in 1 mL and 10 mL of autoclaved Ringer solution separately. For each sample, 200 µL of the mixture was spread on a Corn Meal Agar plate (Oxoid Ltd. Hampshire, UK) containing the antibiotics streptomycin and penicillin (final concentrations of 100 and 50 mg·L −1 ) to inhibit bacterial contamination. These plates were incubated at 27 • C for 7 to 14 days. The hyphal tip was transferred to new agar plates, incubated at 27 • C, and replated until a pure culture was obtained.
The total genomic DNA of the fungal isolate UST030930-004 was extracted as described by Lai et al. [13], and the internal transcribed spacer (ITS) gene sequences were amplified by the polymerase chain reaction (PCR) using primers ITS1 (5 -TCCGTAGGTGAACCTGCGG-3 ) and ITS4 (5 -TCCTCCGCTTATTGAT ATGC-3 ). The ITS region was sequenced and compared with reference sequences in GenBank by BLAST search, showing a similarity of 97% with Thielavia terrestris NRRL 8126 (CP003011). The ITS sequence of the fungal isolate UST030930-004 has been submitted to GenBank (Accession no. KJ716558). The fungal strain UST030930-004 was deposited in the China Center for Type Culture Collection (CCTCC) as CCTCC AF 2014015.

Fermentation, Bioassay-Guided Isolation, and Purification
The fungal strain UST030930-004 was cultured in a liquid medium containing 24 g·L −1 of potato-dextrose broth (Difco Laboratories, Detroit, MI, USA) and 20 g·L −1 of sea salts. Seed cultures were prepared in 50-mL Falcon tubes (BD Labware, Bedford, MA, USA), each containing 25 mL of medium, and cultivated at 23 • C for 3 days with shaking at 160 rpms. Afterwards, 2.8 L flasks, each containing 1.0 L of the same medium, were used for large-scale fermentation (16 L) with the following conditions: inoculation volume 5% (v/v), temperature 23 • C, rotation speed 160 rpms and a duration of 10 days on a shaker. The culture was filtered through 8 layers of cheesecloth to separate the filtrate and mycelia, which were treated separately. The filtrate was extracted with an equal volume of ethyl acetate (EA) three times, while the mycelial pellet was suspended in 80% acetone and sonicated using an ultrasonicator (Branson B-12, Danbury, CT, USA). The wet residue obtained after rotary evaporation to remove acetone was partitioned with EA. The filtrate and mycelial EA extracts were combined and evaporated in vacuo at 35 • C to dryness. The EA extract (22.0 g) was subjected to reversed-phase C18 flash chromatography and then eluted with solvents using a step gradient of H 2 O-MeOH to obtain fractions 1-5 (Fr. [1][2][3][4][5]. Fr. 1-5 were tested for anti-larval settlement activity against Balanus amphitrite, and those showing potential activity were further purified. Fr. 3 was purified using semi-preparative HPLC (Phenomenex Luna C18 (2) Table 4.

Anti-Larval Settlement and Larval Recovery Assay
To prepare the larval culture of the barnacle B. amphitrite, adult brood stocks of B. amphitrite were collected from piling at the Pak Sha Wan Pier, Hong Kong (22 • 36 N, 114 • 25 E). Barnacle larvae were obtained and reared to cyprid stage as previously described by Harder et al. [14]. The cyprids were filtered and maintained in (FSW) at 4 • C overnight, before being used in the settlement assay. A stock solution of each extract or purified compound was prepared at 50 mg·mL −1 in dimethyl sulfoxide (DMSO) and further diluted to varying concentrations (25, 12.5, 6.25, 3.125, 1.562, 0.781 mg·mL −1 ) immediately before each bioassay. One milliliter of the test solutions was added to each well of a 24-well plate (Nunc, Thermo Scientific, Rochester, NY, USA), and approximately 15 larvae were gently pipetted into each well. For the negative controls, 1.0 mL of FSW together with 1.0 µL of DMSO, instead of the testing solution, was added to each well prior to the addition of larvae. The plates were incubated at 28 • C for 48 h in the dark. At the end of the incubation, the numbers of settled and swimming larvae were counted directly under a microscope, and where appropriate, potential toxic effects were noted. The number of settled larvae was expressed as a percentage of the total number of larvae per well. Three replicates of each extract concentration or purified compound were evaluated. EC 50 represents the concentration of a compound that inhibits settlement of 50% of the larval population compared with the negative control, while the LC 50 represents the concentration of a compound that is required to kill 50% of the larvae in a tested population. The experiments were performed in triplicate.
In the recovery assay, cyprids used in the negative controls were placed in new polypropylene containers containing FSW for 24 h, as described in the literature [15]. One milliliter of 10 µM of the test solutions was added to each well of a 24-well plate, and approximately 15 larvae were added to each well. After a 24-h incubation at 28 • C in the dark, the tested solutions were removed, and the treated larvae were washed 3 times with FSW prior to being plated in a new 24-well plate. For the negative controls, swimming larvae in a polypropylene container were transferred to 1.0 mL of FSW containing 1.0 µL of DMSO. The plates were maintained at 28 • C for 48 h before determining the percentage of attached cyprids of B. amphitrite. Three replicates of each experimental concentration were assessed.