Adult Extracts Research Articles

Role of conspecific cues and sugars in the settlement of cyprids of the barnacle, Balanus amphitrite

Gregarious settlement in barnacle is attributed to the settlement-inducing protein complex of cuticular glycoprotein, arthropodin. In this study, we characterized arthropodin protein complex (APC) from crude protein extracts of whole barnacle (AE), and also from soft body (SbE) and shell (ShE). The settlement of cyprids exposed to surfaces coated with different crude protein extracts and APC components was evaluated. In the natural environment, larvae are also exposed to different dissolved sugars. Therefore, the cyprids were tagged with different sugars and exposed to AE, SbE and ShE in order to elucidate their specific role in determining the way barnacle cyprids identify conspecifics. A previously undescribed 66-kDa subunit was observed in shell and soft body APC, and a 98-kDa subunit was observed in shell APC. Both the subunits were shown to inhibit settlement when alone but promoted settlement in combination (ShE), suggesting that these proteins interact in a coordinated manner. The crude adult extract (AE) facilitated higher settlement compared with shell or soft body extract. However, when cyprids were tagged with different sugars and exposed to the surfaces coated with different crude protein extracts, settlement response differed and was jointly determined by the type and concentration of sugars. Such interactions could play an important role in nature as larvae encounter surfaces covered with different glycoproteins and also experience different dissolved cues.

Field-based video observations of wild barnacle cyprid behaviour in response to textural and chemical settlement cues

Many marine invertebrate larvae respond behaviourally to environmental settlement cues, yet behaviours are often only inferred from settlement patterns or are limited to laboratory studies. The behaviour of wild cypris larvae of Semibalanus balanoides L. was filmed on settlement tiles in the field. Tiles were of five different textures with a nested treatment of crude conspecific adult extract (AE). The effects of texture and AE on eleven defined behaviours were analysed. Texture affected the gross and net exploratory distances, velocity, acceleration and time spent exploring. AE attracted more cyprids during the first minute of immersion and increased the time spent on surfaces. Relatively few arrivals that either travel far and fast, or exit the surface rapidly, may indicate a lower chance of settlement. An increase in time spent on a surface may increase the probability of being in contact with the surface when the sign stimulus to settle occurs.

Exploration and metamorphosis in Balanus amphitrite Darwin (Cirripedia; Thoracica) cyprids: significance of sugars and adult extract

Earlier observations have shown that sugars in solution adsorb electrostatically through –OH groups to polar groups associated with the cypris larva temporary adhesive (CTA). Lens culinaris agglutinin (LCA)-binding sugar chains of the adult extract (AE) have been suggested to be involved in the settlement of Balanus amphitrite. In the present study experiments were carried out to assess how cypris larvae would explore and metamorphose when treated with LCA-specific sugars (i.e. d-glucose and d-mannose). The influence of d-galactose was also assessed similarly. Evaluation of sugar-treated as well as nontreated cyprids was carried out with AE-coated and non-coated multiwells containing filtered sea water (FSW). d-mannose-treated cyprids resulted in higher metamorphosis than the nontreated cyprids at 10 −8 M, whereas cyprids treated with the other LCA-specific sugar ( d-glucose) showed an inhibitory effect in the absence of AE. This result implies the involvement of d-mannose moieties of AE in the settlement promotion of B. amphitrite. The barnacle cyprid, while exploring some surfaces, leaves behind ‘footprints’ of temporary adhesive. In the absence of AE, sugar-treated cyprids did not deposit footprints. Concurrently, the sugar-treated cyprids deposited footprints when exposed to multiwells coated with AE. A comparison of observations between single cyprid and multiple cyprid assays showed a similar trend, thus indicating that larva–larva interaction may not play an important role, when such pretreated larvae are subjected to assays. The third antennular segment with its attachment disc is the most obvious point of contact between the cyprid and the substratum during exploration. The detection of AE, even after blockage of polar groups of CTA on the third antennular segment, suggests availability of alternate sites for pheromone reception.

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

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.