Small Polar Molecules: A Challenge in Marine Chemical Ecology

Abstract

Due to increasing evidence of key chemically mediated interactions in marine ecosystems, a real interest in the characterization of the metabolites involved in such intra and interspecific interactions has emerged over the past decade. Nevertheless, only a small number of studies have succeeded in identifying the chemical structure of compounds of interest. One reason for this low success rate is the small size and extremely polar features of many of these chemical compounds. Indeed, a major challenge in the search for active metabolites is the extraction of small polar compounds from seawater. Yet, a full characterization of those metabolites is necessary to understand the interactions they mediate. In this context, the study presented here aims to provide a methodology for the characterization of highly polar, low molecular weight compounds in a seawater matrix that could provide guidance for marine ecologists in their efforts to identify active metabolites. This methodology was applied to the investigation of the chemical structure of an algicidal compound secreted by the bacteria Shewanella sp. IRI-160 that was previously shown to induce programmed cell death in dinoflagellates. The results suggest that the algicidal effects may be attributed to synergistic effects of small amines (ammonium, 4-aminobutanal) derived from the catabolization of putrescine produced in large quantities (0.05⁻6.5 fmol/cell) by Shewanella sp. IRI- 160.

Keywords: algicide; allelochemicals; characterization; extraction; polyamines.

Figure 1

Activity of the algicide IRI-160 in % after being submitted to various treatments: heat (A), dialysis bags (B), and pH modification (C). All bioassays were done using G. instriatum and Rhodomonas sp. as positive and negative control species, respectively. Statistical significance is indicated by **, *, and · for p < 0.005, <0.05 and <0.1, respectively.

Figure 2

Reaction scheme involving dansyl chloride (1) and dansylated ammonium (2), and the chemical structures of the amino compounds identified in this study (3 to 6).

Figure 3

(A) HPLC-fluorescence chromatogram comparing f/2 blank (black line) and IRI-160AA (red line): 1 = DNS-OH, 2 = DNS-NH₃, 3 = DNS-glycine, and 4 =DNS-n-butylamine. Lower panels: Fragmentation patterns (MSMS spectra) of peaks 2 and 4 confirming the presence of DNS-NH₃ (B) and DNS-n-butylamine (C).

Figure 4

(A) UHPLC-HRMS chromatogram of the Shewanella 160 cells previously derivatized: 1 = DNS-NH₃, 2 = DNS-glycine, and 3 = DNS-putrescine. Lower panels: Fragmentation patterns (MSMS spectra) of peaks 2 and 3 confirming the presence of DNS-4-aminobutanal (B) and DNS-putrescine (C).

Figure 5

Relative fluorescence of Gyrodinium instriatum (a), Prorocentrum minimum (b), and Rhodomonas sp. (c) after 24 h exposure to 20 µM n-butylamine, 200 µM n-butylamine, 20 µM n-butylamine + 100 µM NH₄⁺, and 200 µM n-butylamine + 100 µM NH₄⁺. Asterisks indicate significant differences in relative fluorescence between n-butylamine alone and n-butylamine + NH₄⁺ treatments.

Figure 6

UHPLC-HRMS chromatograms of a standard of 4-aminobutanal at a concentration of 180 µM, as well as extracted chromatogram showing ions in the range m/z 361.15-361.16 in f/2 media blank and the algicide. A comparison between the fragmentation patterns (MSMS spectra) of 4-aminobutanal retrieved in the standard and in the algicide is also given for confirmation.