Inhibition of barnacle larval settlement and crustacean toxicity of some hoplonemertine pyridyl alkaloids William R. Kem a, *, Ferenc Soti a , Dan Rittschof b a Department of Pharmacology and Therapeutics, University of Florida College of Medicine, Gainesville, FL 32610-0267, USA b Department of Zoology and Nicholas School of the Environment, Duke University Marine Laboratory, 135 Duke Marine Lab Road, Beaufort, NC 28516-9721, USA Abstract Hoplonemertines are carnivorous marine worms, which prey upon crustaceans and annelids. They paralyze their prey by injecting alkaloids with a stylet-bearing proboscis. The dermis of these animals also secretes alkaloids to repel predators. Besides affecting central and peripheral nervous system nicotinic receptors, some pyridyl alkaloids also activate certain chemoreceptor neurons in crustacean walking legs, which sense environmental chemicals. Anabaseine (2-[3-pyridyl]-3,4,5,6-tetrahydropyridyl) and 2,3?- bipyridyl (2,3?-BP) are two nemertine alkaloids, which potently paralyze crustaceans. Anabaseine is an agonist of vertebrate as well as invertebrate nicotinic receptors. While 2,3?-BP is non-toxic to mice, it is toxic to crustaceans. We tested a variety of nemertine pyridyl alkaloids for inhibition of barnacle (Balanus amphitrite ) larval settlement and for crustacean toxicity in order to determine whether toxicity could be dissociated from inhibition of larval settlement. We prepared eight C-methylated 2,3?-BP isomers to determine where substitution is permitted without loss of activity. Anti-settlement and toxicity activities were not always related. For instance, 4?-methyl-2,3?-BP displayed only 3% of the crayfish paralytic activity of 2,3?-BP, but inhibited settlement almost 2-fold more effectively. Two other isomers displaying exceptional anti-settlement activity were the 4- and 5-methyl-2,3?-BPs; these also displayed high crustacean toxicity. Nemertelline inhibited barnacle settlement at concentrations similar to 2,3?-BP but was 136-fold less toxic when injected into crayfish. Thus, certain bipyridyls and tetrapyridyls may be useful anti-fouling additives. # 2003 Elsevier Science B.V. All rights reserved. Keywords: Alkaloid; Balanus ; Biofouling; Bipyridyl; Crustacean; Nemertine 1. Introduction A variety of organisms settle and colonize marine surfaces. These include bacteria, algae, and sedentary invertebrates such as bryozoans, sponges, mollusks and barnacles. Such fouling organisms can colonize the hulls of ships to a degree that fuel efficiency is greatly reduced. Thus, the exposed surface must be scrapped and repainted at frequent intervals. Organotin additives to marine paints are effective anti-fouling agents, but unfortunately also damage the marine environment, killing and sterilizing intoxicating many free-living organisms where vessels are docked. The world-wide ban on the use of these toxic additives is to be initiated in 2003. Thus, there is much interest in finding new methods to inhibit the colonization of man-made marine surfaces by these organisms [1  /4]. Future anti-fouling additives should be more selective for the targeted fouling organisms and less generally toxic to non-fouling organisms. One approach might be to target the relatively unique mechanisms associated with the process of settlement. Thus, we need to understand the process by which the encrusting organ- ism senses the surface, attaches to it, and then grows upon it. Little is known about the chemoreceptive capabilities of barnacle larvae compared with knowl- edge on chemoreceptors of other crustaceans [5]. Besides being receptive to amino acids, some decapod crustacean neuronal chemoreceptors are sensitive to certain pyridine compounds, especially 3-substituted pyridines [6  /8]. Some of the most potent 3-pyridyls are natural toxins found in certain nemertines, a phylum of nearly 1000 recorded species of carnivorous flat- worms [9]. Bacq [10] first demonstrated that nemertines * Corresponding author. Tel.:  /1-352-392-0669; fax:  /1-352-392- 9696. E-mail address: kem@pharmacology.ufl.edu (W.R. Kem). Biomolecular Engineering 20 (2003) 355  /361 www.elsevier.com/locate/geneanabioeng 1389-0344/03/$ - see front matter # 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S1389-0344(03)00049-2