Bioassay-guided fractionation of antifouling compounds using computer-assisted motion analysis of brown algal spore swimming

Abstract

Antifouling extracts from the sea stars Astropecten articulatus and Luidia clathrata and from the brittle star Astrocyclus caecilia were fractionated by solid phase extraction and high performance liquid chromatography. Bioactive fractions were identified with the use of computer-assisted motion analysis-based bioassays utilising previously described Hincksia irregularis spore swimming behaviour parameters. Quantified parameters of spore movement were rate of change of direction (RCD) and speed (SPEE). The methods used initially required only 10 μg equivalent amounts of total crude extract and each resultant resolving step (normalised to 1 mg ml⁻¹ of crude, unfractionated extract) required far less material. Statistical analyses of RCD and ratios of RCD:SPEE values in experiments comparing swimming in the presence of extract fractions to controls revealed that both parameters were useful individually and in combination for efficiently following compound bioactivity throughout the fractionation procedure. This technique was also able to detect synergistic or additive interactions between compounds.

Keywords:

• Algal spores
• antifouling
• behaviour
• brown algae
• echinoderm
• Hincksia irregularis

Acknowledgements

We thank Drs Robert Angus and Scott Brande for their help and critical insight regarding statistical analyses and Drs Daniel Jones, Bill Baker, Donald Muccio, and two anonymous reviewers for constructive comments on earlier versions of this manuscript. This work is a result of research sponsored by the National Oceanic and Atmospheric Administration (NOAA), the US Department of Commerce under grant no. R/MT-40 to CDA and JBM, the Mississippi-Alabama Sea Grant Consortium, and the University of Alabama at Birmingham. SPG also received salary support from a National Science Foundation GK-12 Fellowship and KI was partially funded by a grant from the German Science Foundation (IK 4/2-1). The US Government is authorised to produce and distribute reprints notwithstanding any copyright notation that may appear hereon. The views expressed herein are those of the authors and do not necessarily reflect the views of NOAA or its subagencies.