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Gregarious settlement in barnacles has been related to the settlement-inducing compounds from adult conspecifics, bacteria in the biofilms, and their interaction. Elucidation of larval settlement cues from these sources is limited. The effectiveness of larval settlement cues under different environmental conditions (salinity, temperature) needs evaluation. Pseudomonas aeruginosa, a bacterium isolated from the shell surface of Balanus amphitrite Darwin, was used as a candidate. The influence of bacterial film, culture supernatant and its molecular-weight fractions, and bacterial extract was investigated along with the conspecific adult extract (AE). The influence of culture supernatants and exopolysaccharides obtained from the bacterium cultivated in different nutrient media, effectiveness of leachants and adsorbed (surface-bound) compounds on the metamorphosis of cyprids of B. amphitrite was also assessed. The influence of P. aeruginosa on cyprid metamorphosis varied with salinity and temperature. The differences were not significant as the film and the cyprids aged. When the bacterial film was examined in the presence of an active substance (agonist) such as AE, metamorphosis was facilitated, suggesting the role of competitive antagonism in cue perception. The higher molecular-weight fraction of the bacterial-culture supernatant was inductive at higher salinity. Conversely, the lower molecular-weight fraction of the culture supernatant showed maximum inhibition when the adsorbed (surface-bound) compounds were assessed along with the leachants. Bacterial extract showed the presence of ketonic compounds, and its influence differed with salinity. The inhibitory effect of the extract was nullified in the presence of AE. When the extract was examined in the presence of leachants, a 2-fold increase in the metamorphosis rates was evident where only surface-bound components were inhibitory. Fourier transformed infrared spectroscopy analysis revealed that bacteria grown in different nutrient media yielded culture supernatants with different chemical composition, thus altering their ability to induce metamorphosis of cyprids. Maximum inducement was provoked by the culture supernatant obtained from semi-solid culture, and this positive effect was protein concentration dependent. The exopolysaccharides obtained from bacteria grown in basal salt solution facilitated metamorphosis similar to that of the bacterial film and AE. The response of the cyprids to bacteria and its products seems to be regulated by both contact chemoreception and olfaction, depending on the properties of the settlement-inducing compounds. The need to characterize and distinguish the receptors, which act via different signaling systems on a particular settlement cue, may be a step ahead to resolve the complexities of invertebrate larval recruitment.

Factors regulating the production of different inducers in Pseudomonas aeruginosa with reference to larval metamorphosis in Balanus amphitrite