Antifouling effects of the periostracum on algal spore settlement in the mussel Mytilus edulis
© Kang et al. 2016
Received: 9 March 2016
Accepted: 12 March 2016
Published: 18 March 2016
In nature, marine mussels (Mytilus edulis) suffer less fouling colonization on the newly formed sides of their shells. Using settlement assays with algal spores of Porphyra suborbiculata, we determined that spore attachment and germination on the periostracum decreased to 36.8 and 3.3 %, respectively. Additionally, the spore settlement was considerably diminished by periostracum dichloromethane extracts containing 19 % oleamide, a major antifouling compound. A scanning electron micrograph of the surface revealed a regular ripple structure with approximately 1.4 μm between ripples. Based on these results, mussel periostraca or their associated biomimetic materials may become environmentally friendly, antifouling agents for preventing the settlement of soft foulants.
Biofouling is a natural marine ecosystem process caused by the surface colonization and development of micro- and macrofoulers on submerged natural or artificial marine structures, and can lead to economic and environmental losses worldwide. The fouling of ship hulls and fishing nets results in major costs for the marine industry through increased maintenance and fuel requirements due to greater levels of hull drag, lost productivity due to an increased frequency of dry-docking for the removal of fouling organisms, and compliance with environmental regulations (Yebra et al. 2004). Previously utilized antifouling agents, such as the common vessel tributyltin biocidal coatings, although effective against fouling, are also toxic (Minchin et al. 1996; Atanasov et al. 2005; Sonak 2009). As a result of the negative environmental impacts associated with its toxicity, tributyltin has become the subject of a relatively recent worldwide ban by the International Maritime Organization (IMO). Due to the limitations of conventional coatings, research on biomimetic surfaces and compounds inspired by natural systems has become important (Scardino and de Nys 2011). Researchers have observed that some marine species, such as mussels, can resist fouling when in good physiological condition (Scardino and de Nys 2004; Bers et al. 2006). Mussels have a tough, yet pliable, proteinaceous shell covering secreted by the mantle, known as the periostracum (Harper and Skelton 1993; Scardino et al. 2003). Wahl et al. (1998) found that when the periostracum was physically removed in Mytilus edulis, they observed an increase in the settlement of barnacles and algae on the shell. Conversely, mussels with an intact periostracum showed a greater resistance to fouling pressure (Scardino et al. 2003). Several studies have reported the benefits of microtopography on mussel shells as a physical fouling deterrent (Bers and Wahl 2004; Scardino and de Nys 2004). However, the general antifouling role of the periostracum in deterring settlement of fouling organisms and its underlying mechanisms remain unclear. Therefore, we investigated chemical elements from periostracum extracts and the physical surface of the periostracum responsible for defense against algal spore settlement. Monospores of Porphyra suborbiculata, one of common wild seaweed and easily obtainable throughout the year in a laboratory scale, were conveniently used as an assay organism for spore attachent and germination.
Aquacultured (6.5 ± 0.4 cm) and wild (4.4 ± 0.3 cm) Mytilus edulis mussels were purchased from the Namcheon fish market and collected from the rocky intertidal area at Eegidae (35°11′97″ N, 129°12′74″ E), on the east coast of Busan, Korea. For solvent extraction, shells of aquacultured mussels were gently cleaned to remove associated detritus and epibionts prior to submersion in vinegar solution.
Whole shells were submerged in a vinegar and seawater mixture (1:2 vinegar: seawater, approximately 2 % acetic acid) to aid in removing the periostracum (Grandison et al. 2011). Shells were retained in this mixture for 1 d, after which the periostracum was peeled from the shell with forceps and stored in seawater. After rinsing with distilled water, the peeled periostraca were freeze-dried and ground to a powder by hand for 5 min using a mortar and pestle. Twenty mg of periostracum powder was extracted with each one mL solvent of dichloromethane, ethyl acetate, and methanol. Extraction with each solvent was repeated three times for 1 h using pulses of an ultrasonic water bath (low-intensity frequency of 40 kHz), and the extracts were then dried with nitrogen. A stock solution of each extraction was prepared by adding 1 mL dimethyl sulfoxide (DMSO) to each 40 mg of dried extract. The prepared stocks were filtered through a 0.45-μm syringe filter before use.
The dichloromethane extracts of non-treated (i.e., no vinegar) periostraca were analyzed by gas chromatography–mass spectrometry (GC-MS) using a QP5050A instrument (Shimadzu, Kyoto, Japan) equipped with a flame-ionization detector and compared with spectral data from the database. Analysis was performed on an HP-5 column (30 m × 0.25 mm, 0.25 μm; Agilent Technologies, Santa Clara, CA, USA). The temperature was initially held at 50 °C for 2 min and raised to 150 °C at 4 °C/min and to 250 °C at 7 °C/min. Helium carrier gas was controlled at 0.6 mL/min with a split ratio of 1:50. The mass spectrometer was operated in electron-ionization mode at 70 eV.
Scanning electron microscopy
The periostracum, peeled from the shell, was rinsed with distilled water and freeze-dried under vacuum before scanning electron microscopy (SEM) analysis. For SEM images, periostracum was mounted on conductive carbon tabs of a SEM post (Ted Pella, Inc., Redding, CA), sputter-coated using a Desk-II coater equipped with a gold target (Alfa Aesar, Ward Hill, MA), and imaged in a scanning-electron microscope (JSM-6700F; JEOL, Tokyo, Japan).
Juvenile blades of P. suborbiculata were collected from the rocky intertidal area at the mussel collection site. The fresh blades were rinsed, sonicated (40 kHz) twice for 1 min in autoclaved seawater, and immersed in 1 % Betadine solution with 2 % Triton X-100 for 1 min to eliminate epiphytes (Choi et al. 2005). To liberate the monospores, blades were cultured in Provasoli-enriched seawater (PES) medium (Provasoli 1968) under a 40 μmol/m2/s light intensity (10 L:14D) at 18 °C. Monospores were then used for attachment and germination assays under the same conditions.
Attachment and germination assays
For assays of algal spore attachment, aliquots of 100 μL seawater were first distributed into a 96-well plate. We added 1 μL of periostracum extract (40 mg/ml), 4 μL PES stock, and approximately 100–200 spores to each, with the final volume being 200 μL. The resulting spore suspensions were placed in the dark for 1 d at 18 °C to allow for even settlement on the bottom. At the end of this period, nonattached spores were removed from the bottom by centrifugation in an inverted position at 1500 × g for 15 min. The number of attached spores was counted under a microscope after replacing the PES solution. Relative attachment (%) was expressed as a percentage of the attached spores against total spores added. The reference for each test was prepared using the same procedure but with no extract. The minimum detectable inhibition of spore attachment by DMSO occurred at 0.5 %. Thus, the solvent extracts and reference were always added to the assay medium to provide a DMSO concentration of less than 0.5 %. For spore germination assays, approximately 100–200 spores were added to a 200-μL aliquot of PES in a 96-well plate and placed in the dark for 1 d at 18 °C to allow spores to settle on the bottom. After removing the nonattached spores by centrifugation in an inverted position, a fresh 200 μL of PES was added. Then, 1 μL of each extract (40 mg/ml) was immediately added to each 200 μL culture. The resulting germination cultures were placed at 18 °C and 80 μmol/m2/s light intensity on a 12 L:12D cycle for 1 week to permit development. The number of germlings was counted under a microscope and expressed as relative germination (%), i.e., the percentage of germinated spores to the total number of spores attached. The minimum DMSO concentration leading to detectable inhibition was 0.5 %. Thus the final concentration of DMSO was kept below 0.5 % in all assays.
The experiments were repeated at least three times with each independent assay. The means of each indicator were compared to the controls using Student’s t-tests.
Results and discussion
Attachment and germination of algal spores on periostracum peels of the mussel Mytilus edulis
Relative attachmentc (%)
Relative germinationc (%)
232/629 (36.8 %)*
10/299 (3.3 %)**
1532/1823 (84.0 %)
1042/1288 (80.9 %)
Effects of periostracum extracts (200 μg/mL each) on the attachment and germination of Porphyra suborbiculata monospores
Relative attachmenta (%)
Relative germinationa (%)
35 ± 17**
5.1 ± 1.8**
41 ± 7**
8.9 ± 2.4**
55 ± 9*
10.6 ± 5.0**
31 ± 7**
3.5 ± 3.2**
32 ± 1**
5.2 ± 2.6**
34 ± 6**
10.6 ± 2.4**
35 ± 5**
3.4 ± 5.4**
37 ± 9**
4.5 ± 4.1**
47 ± 8*
16.5 ± 3.6**
81 ± 7
83.2 ± 5.9
Profile of the major compounds in the dichloromethane extract of periostraca using GC-MS
Pelargonic acid (C7:0)
Myristic acid (C14:0)
Palmitic acid (C16:0)
Stearic acid (C18:0)
Our research has shown that periostracum extracts display some antifouling effects, while the periostracum also physically deters the settlement of spores. These findings strengthen mimetic application claims holding that the components and surface microtopographies of M. edulis periostraca can be used as models for antifouling materials. Our results, periostracum composition and structure results, suggest that mussel periostraca or their associated biomimetic materials may become environmentally friendly antifouling materials preventing the attachment of diverse fouling organisms.
This work was supported by a National Research Foundation of Korea grant funded by the Korean government (MEST) (NRF-M1A5A1-2011-0029963).
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