Gregarious behaviour of marine larvae is perhaps most clearly associated with finding a suitable habitat in a changeable or restricted environment, or with finding other conspecifics with which to mate. Prior work has shown that in settlement assays using cypris larvae of the barnacle Balanus amphitrite, gregarious interactions significantly affected the interpretation of experiments testing the activity of organic settlement promoters and inhibitors. Other studies have also shown effects of cyprid age and pheromone concentration on settlement behaviour. However, the effects of interactions between gregariousness and these two factors are not known. The aim of this study was to test the hypotheses that i) as cyprids age the effects of gregariousness become less apparent, and ii) as the duration of the experiment increases gregarious effects become more apparent, using cypris larvae of B. amphitrite and Balanus improvisus. Three age classes of cyprids were used at six densities in a fully factorial design. For B. improvisus cyprids significant gregarious effects occurred between 3 or more larvae, and although larval age and experiment duration had significant main effects, there were no interactions between these important factors and gregariousness. For B. amphitrite cyprids significant gregarious effects also occurred with 3 larvae per well, though this effect was strongly dependent upon experiment duration. B. amphitrite cyprid sensitivity to conspecific cues does not change with age, although increasing experiment duration and age interact to increase settlement. Differences between species may be due to different thresholds to conspecific larval cues, or B. improvisus cyprids release much more larval temporary adhesive during exploration.

Repetitive testing of TBTO, sea-nine 211 and farnesol using Balanus amphitrite (Darwin) cypris larvae: Variability in larval sensitivity, Biofouling, 12:1-3, 133-147, Settlement inhibition assays with mass cultured cypris larvae of the barnacle Balanus amphitrite are widely used in (anti)fouling research. In this study, TBTO, Sea-Nine 211 and farnesol were tested repeatedly using multiple batches of larvae to study variability in larval sensitivity. There were significant differences in EC 50 values among batches of cyprids of the same age and a maximum three-fold difference between EC 50 values. Larval sensitivity did not change significantly when culture duration was longer than normal (5 or 6d instead of 4d). The effect of the 3 compounds on settlement was independent of cyprid age. Notwithstanding the variability encountered, the assay is suitable for screening purposes, but for a more precise comparison of antisettlement activity improved reproducibility is desirable, which might be achieved using artificial seawater, sibling barnacles and including an internal reference in the assay.

The in vitro settlement assay using cyprids of Balanus. amphitrite is an important tool in basic and applied research. The aim of this study was to quantify the effect of gregariousness within these assays, and to determine the interaction between gregariousness and container size, and settlement promoters and inhibitors. Assays with 1, 5, 10, 15, 20, and 40 cyprids in containers with a log range of surface area to volume ratios were conducted in a fully factorial design. Assays with the same range of cyprid numbers and six concentrations of the settlement promoter 3-isobutyl-1-methylxanthine (IBMX) or the inhibitor phloroglucinol were also conducted in a fully factorial design. Percentage settlement was analysed by GLM ANOVA. Significant (p < 0.05) gregarious effects were detected at > or = 5 cyprids in a well. Surface area:volume ratio had a strong effect on cyprid settlement, but this effect could be masked by overcrowding in very small wells. Gregarious interactions between only five cyprids magnified the effect of IBMX by a factor of 10, whereas phloroglucinol had no effect without gregarious interactions. The cyprid settlement bioassay is a valuable tool for basic and applied research but must be used with care.