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First published online January 19, 2006
Journal of Experimental Biology 209, 558-566 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02037
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The identification and role of a novel eicosanoid in the reproductive behaviour of barnacles (Balanus balanus)

Ben H. Maskrey1, Graham W. Taylor2,* and Andrew F. Rowley1,{dagger}

1 Department of Biological Sciences and Institute of Environmental Sustainability, University of Wales Swansea, Swansea, SA2 8PP, UK
2 Division of Medicine, Imperial College, Hammersmith Campus, London, W12 0NN, UK


Figure 1
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  		Fig. 1. RP-HPLC chromatograms of supernatants from A23187 ionophore-challenged barnacle seminal vesicles/testis separated at pH 7.0. (A) Biologically active BMSF (peak 1) elutes at 9 min. (B) Peak 2 (12 min); (C) Peak 3 (25 min). Insets show characteristic UV profiles of peaks indicated. Asterisks indicate peaks with a similar UV profile to that shown in insets.

 

Figure 2
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  		Fig. 2. Effect of pH prior to solid phase extraction, on the products produced by barnacle seminal vesicles/testis. Chromatograms shown are from RP-HPLC separation at pH 7.0 of material extracted at either pH 3.5 (red trace) or pH 7.0 (black trace). Note the significant reduction in peak 1 (BMSF; A) and the appearance of triene- and pentaene-containing compounds including peaks 2 (B) and 3 (C) following this brief period of acidification. Asterisks indicate peaks with similar UV profiles.

 

Figure 3
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  		Fig. 3. Effect of acidification on BMSF. Product was collected from initial RP-HPLC separation at pH 7.0 (A), the pH was adjusted to 3.5 using acetic acid and the changes in UV absorbtion monitored spectrophotometrically at RT (B).

 

Figure 4
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  		Fig. 4. Effect of acidification on post-HPLC separated products generated from ionophore-challenged barnacle seminal vesicles/testes. (A) The original RP-HPLC separation at pH 7.0 and the fractions collected (green, blue and yellow regions) that were subsequently acidified to pH 3.5, re-extracted and separated again by RP-HPLC at pH 7.0 (B-D).

 

Figure 5
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  		Fig. 5. (A) Exogenous EPA causes a dose-dependent increase in the generation of BMSF by disrupted B. balanus seminal vesicles/testis incubated at 12°C for 20 min. Products were separated by RP-HPLC at pH 7.0 and quantified as detailed in the Materials and methods. Values are means ± s.d. (B) BMSF production is inhibited by lipoxygenase inhibitors NDGA (20 µmol l-1) and esculetin (50 µmol l-1), and by the cytochrome P450 inhibitor, metyrapone (100 µmol l-1). Representative data are shown from an experiment repeated several times.

 

Figure 6
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  		Fig. 6. (A) Spontaneous muscle contractions of an intact B. balanus following its removal from the shell. Note the contraction of the cirri (C) and generation reduction in size of the body mass following contraction. The whitish area highlighted (unlabelled arrow) is the region of the seminal vesicles/testis. (B) Effect of BMSF and breakdown product 2 on muscular contractions of B. balanus over time. Values are means ± s.e.m., N=3, *P<0.05 compared with control (paired t-test).

 

Figure 7
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  		Fig. 7. (A) Collisionally induced dissociation of the deprotonated molecular ion BMSF. The ion at m/z 333 fragments to lose water and CO2. Ions at m/z155 (OHC.C6H10.COO-) and 193 ([C4H3O].C6H10.COO-), are characteristic; both lose CO2 under further MS3 dissociation. (B) Electron impact mass spectrum of the methyl ester O-trimethylsilyl ether of BMSF showing ions at m/z 492 (M+.), 351 and 243 are characteristic of an 8,X-diHEPE. (C) Catalytic hydrogenation of the A240 material results in the formation of 8,13-dihydroxy eicosanoic acid, with characteristic ions at m/z 502 (M+.), 201, 245, 322, 359 and 403.

 

Figure 8
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  		Fig. 8. Proposed pathway for the generation of BMSF (8,13-diHEPE). Note that the stereochemical assignments shown are tentative.

 

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© The Company of Biologists Ltd 2006