Biofouling The Journal of Bioadhesion and Biofilm Research

ISSN: 0892-7014 (Print) 1029-2454 (Online) Journal homepage: http://www.tandfonline.com/loi/gbif20

Cochliomycin A inhibits the larval settlement of Amphibalanus amphitrite by activating the NO/ cGMP pathway Kai-Ling Wang, Gen Zhang, Jin Sun, Ying Xu, Zhuang Han, Ling-Li Liu, ChangLun Shao, Qing-Ai Liu, Chang-Yun Wang & Pei-Yuan Qian To cite this article: Kai-Ling Wang, Gen Zhang, Jin Sun, Ying Xu, Zhuang Han, Ling-Li Liu, ChangLun Shao, Qing-Ai Liu, Chang-Yun Wang & Pei-Yuan Qian (2016) Cochliomycin A inhibits the larval settlement of Amphibalanus amphitrite by activating the NO/cGMP pathway, Biofouling, 32:1, 35-44, DOI: 10.1080/08927014.2015.1121245 To link to this article: http://dx.doi.org/10.1080/08927014.2015.1121245

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Date: 18 January 2016, At: 01:41

Biofouling, 2016 VOL. 32, NO. 1, 35–44 http://dx.doi.org/10.1080/08927014.2015.1121245

Cochliomycin A inhibits the larval settlement of Amphibalanus amphitrite by activating the NO/cGMP pathway Kai-Ling Wanga,b,e, Gen Zhangc, Jin Sund, Ying Xub,e, Zhuang Hane,f, Ling-Li Liuc, Chang-Lun Shaoa, Qing-Ai Liua, Chang-Yun Wanga and Pei-Yuan Qianc,e a

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Key Laboratory of Marine Drugs, The Ministry of Education of China, School of Medicine and Pharmacy, Ocean University of China, Qingdao, PR China; bCollege of Life Science, Shenzhen University, Shenzhen, PR China; cEnvironmental Science Program, School of Science, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong SAR, PR China; dDepartment of Biology, Hong Kong Baptist University, Hong Kong SAR, PR China; eDivision of Life Science, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong SAR, PR China; f Sanya Institute of Deep-sea Science and Engineering, Chinese Academy of Sciences, Sanya, PR China

ARTICLE HISTORY

ABSTRACT

Cochliomycin A is a compound with anti-barnacle settlement activity and low toxicity, but the molecular mechanism of the compound is unknown. Here, isobaric tags for the relative or absolute quantitation (iTRAQ) labeling proteomic method were applied to analyze changes in the proteome of Amphibalanus(=Balanus) amphitrite cyprids in response to cochliomycin A treatment. Cochliomycin A affected the cytochrome P450, glutathione S-transferase (GST) and NO/cGMP pathways, among which the NO/cGMP pathway was considered to play a key role in barnacle larval settlement, while the cytochrome P450 and the GST pathways are mainly for detoxification. The results of real-time PCR further suggested the NO/cGMP pathway was activated in response to cochliomycin A. Larval settlement assays revealed that S-methylisothiourea sulfate (SMIS) and 1H-(1,2,4)oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) rescued cyprids from cochliomycin A-induced inhibition of larval settlement. The findings supported the hypothesis that cochliomycin A inhibited barnacle larval settlement by stimulating the NO/cGMP pathway.

Introduction Biofouling causes over US $6.5 billion of economic losses to marine industries every year (Bhadury & Wright 2004). Biofoulers include microfoulers such as marine bacteria, algae and protozoa, and macrofoulers such as barnacles, bryozoans and tubeworms (Maki et al. 1992; Hadfield 1998; Yebra et al. 2004; Dobretsov et al. 2006). There have been numerous efforts to develop antifouling (AF) chemicals to prevent the colonization by fouling organisms of man-made surfaces (such as pipes, ships’ hulls and docks) (see reviews by Fusetani 2004, 2011; and Qian et al. 2009, 2015). However, whilst many of the widely used AF compounds, such as tributyltin (TBT), tryphenyltin (TPT), Irgarol 1051 and diuron, are effective following their initial application, they are subsequently highly toxic in field applications (Cresswell et al. 2006; Yamada 2007). These findings demonstrate that the specificity of an AF compound for its targets is no less important than its effectiveness.

KEYWORDS

Cochliomycin A; antifouling; barnacle; nitric oxide; cGMP; mechanism

To avoid the environmental pollution caused by novel AF compounds, additional tests are required prior to their field application. First, a variety of non-target organisms should be included to test the toxicity of antifoulants. For instance, the acute toxicity of butenolide was assessed in microalgae, crustaceans and fish (Zhang et al. 2011). Similarly, the acute toxicities of copper, tributyltin (TBT), and six commonly used booster biocides (Irgarol, diuron, zinc pyrithione, copper pyrithione and chlorothalonil) were tested on the growth and survival of 12 marine species (Bao et al. 2011). Secondly, it is essential to clarify the underlying molecular mechanism of AF compounds against their target biofoulers. Binding assays revealed that butenolide targeted acetyl-CoA acetyltransferase 1 (ACAT1), acyl-CoA dehydrogenase (ACADVL), and succinyl-CoA synthetase β subunit (SCS β) in the barnacle Amphibalanus amphitrite, the bryozoan Bugula neritina, and the marine bacterium Vibrio sp., respectively (Zhang et al. 2012). Proteomic analyses indicated that meleagrin might affect the endocrine

CONTACT  Pei-Yuan Qian  [email protected] The supplemental material for this paper is available online at http://dx.doi.org/10.1080/08927014.2015.1121245 © 2016 Taylor & Francis

Received 17 May 2015 Accepted 12 November 2015

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  K.-L. Wang et al.

Larval culture

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Figure 1. The chemical structure of cochliomycin A.

system and prevent the larval molting cycle (Han et al. 2013). Poly-ether B interfered with the redox-regulatory mechanisms governing the settlement of barnacle larvae (Dash et al. 2012). Compared with the direct toxicity tests, studies investigating the molecular mechanisms of antifoulants not only evaluate specificity but also provide additional information to help improve efficacy through chemical structure modification of these compounds (see reviews by Qian et al. 2013, 2015). Recently, a natural marine product known as cochliomycin A (chemical structure shown in Figure 1) was purified from the culture broth of the fungus Cochliobolus lunatus and showed strong AF activity against cyprids of the barnacle A. amphitrite, with an EC50 of 1.30 μg ml−1 and an LC50/EC50 ratio > 15 (Shao et al. 2011). Cochliomycin A is considered to be a promising AF reagent with potential industrial value (Shao et al. 2011; Liu et al. 2014). However, the molecular mechanism by which cochliomycin A inhibits barnacle larval settlement remains unknown. In the present study, the aim was to identify the pathway(s) that are affected by cochliomycin A using the isobaric tags for the relative or absolute quantitation (iTRAQ) labeling proteomic method. Real-time quantitative PCR and settlement assays were subsequently conducted to further validate some of the pathways found using iTRAQ.

Materials and methods Cochliomycin A preparation Cochliomycin A was isolated from the culture broth of the fungus C. lunatus ZJ-2008002 according to the method described by Shao et al. (2011). The NMR spectra of cochliomycin A were measured using a JEOL Eclips-600 spectrometer (Japan Electronics, Kanagawa Prefecture, Japan) at 600 MHz for 1H and 150 MHz for 13C in CDCl3. High-resolution mass spectra (HREIMS) spectrometry was recorded on a Thermo MAT95XP HREIMS (Thermo Scientific, Bremen, Germany). All of the spectra are provided in the Supporting materials (see Figures S1 and S2).

Adult barnacles of A. amphitrite were collected from the concrete columns of the pier at Pak Sha Wan in Hong Kong (22°21′45′′N, 114°15′35′′E). Nauplius larvae were released from these adults and raised in filtered seawater (0.22 μm pore size, FSW) with a diet of Chaetoceros gracilis Schutt at 1 × 106 cells ml−1. The culture was incubated at 28°C with mild aeration. After 3.5 days, the nauplius larvae transformed into cyprids which were ready to attach and metamorphose. The cyprids were kept in FSW at 4°C overnight before being used in the settlement assays. Larval treatment Pre-tests showed that the LC50 of cochliomycin A was > 50 μg ml−1 against barnacle cyprids. In this experiment, there were four experimental treatments: (1) CON24: the cyprids were cultured in FSW (0.1% DMSO) in the absence of cochliomycin A, and after 24 h, the majority could swim actively at the bottom of the container; (2) INH-L: the cyprids were treated with a low concentration of 10 μg ml−1 cochliomycin A and remained swimming like those in the CON-24 group; (3) INH-M: the cyprids were treated with a moderate concentration of 30 μg ml−1 cochliomycin A but were less active than the INH-L treatment; (4) INH-H: the cyprids were treated with a high concentration of 50 μg ml−1 cochliomycin A, and the majority were alive but inactive. Each treatment contained an equivalent volume of DMSO (0.1%), and was incubated at 28°C in the darkness. After 24 h, the larvae from these four groups were collected by filtration, rinsed three times with FSW, and stored in liquid nitrogen for protein extraction. Each treatment was performed in three independent biological experiments with three replicates each, and 1,000–1,100 cyprids were placed in a new polypropylene container (on the surface of which no larvae had settled within 24 h) for each replicate. Protein extraction and purification Lysis buffer (8  M urea, 40  mM HEPES, pH  =  7.4) was supplemented with protease inhibitor (Roche Applied Science, Mannheim, Germany) just before use. The cyprids were suspended in 200 μl lysis buffer and ultrasonically extracted for 20 s  ×  3 on ice at an amplitude of 20% and pulse of 1 s : 1 s (QSonica Q125 sonicator, Farmingdale, NY, USA). Next, the cell lysates were centrifuged at 15,000 g for 20 min at 4°C, and the supernatants containing the crude protein extracts were transferred to new Eppendorf tubes. The protein concentration was determined using a RC DC protein assay kit (Bio-Rad, Hercules, CA, USA). For

Biofouling 

each sample, 300 μg of total protein were purified using the ReadyPrep 2-D Cleanup Kit (Bio-Rad) according to the manual. Then, the purified proteins were dissolved in 8 M urea, and the protein concentration was determined again as described above.

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Trypsin digestion and iTRAQ labeling Equal amounts of protein (200 μg) from each sample were reduced in 5 mM tris carboxyethyl phosphine hydrochloride (TCEP HCl) at 60°C for 1 h, and then alkylated with methylethanethiosulfonate (MMTS, 10 mM final concentration) at room temperature (~23°C) in the dark for 20 min. Next, the samples were diluted sevenfold with 50  mM triethylammonium bicarbonate (TEAB) to a urea concentration of 

cGMP pathway.

Cochliomycin A is a compound with anti-barnacle settlement activity and low toxicity, but the molecular mechanism of the compound is unknown. Here, is...
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