Marine Biology (1996) 127:125-130 ~ Springer-Verlag 1996 C. G. Satuito • K. Shimizu • K. Natoyama M. Yamazaki • N. Fusetani Age-related settlement success by cyprids of the barnacle Balanus amphitrite, with special reference to consumption of cyprid storage protein Received: 13 May 1996/Accepted: 12 June 1996 Abstract Newly molted (0-d-old) cyprids of the bar- nacle Balanus amphitrite Darwin were prevented from settling for 0 to 14 d at four different temperatures (25, 20, 15 and 5°C treatments). The effect on settlement success of prolonging the cyprid lifetime was evaluated using a nitrilocellulose membrane assay. In addition, protein extract prepared from these cyprids was ana- lyzed using gel electrophoresis to characterize the effect of age on protein content and composition. Settlement success was significantly affected for larvae aged at 25 (P < 0.001), 20 (P < 0.001) and 15°C (P < 0.05), while differences in settlement success between age groups was negligible at 5~C (P = 0.09). Settlement success of cyprids increased with time for up to 3 d (P < 0.001, Phase 1), following which settlement success signifi- cantly declined (P < 0.001, Phase II). Temperature had no significant effect on settlement in Phase I (P = 0.17), but did enhance the decline in settlement success with age during Phase II (P < 0.001). Gel electrophoresis revealed a significant decline in the quantity of the cyprid storage protein CMP (Cyprid Major Protein) with increasing age at 25, 20 and 15°C, but CMP levels remained constant at 5°C, These results suggest that, upon molting to the cyprid stage, larvae may still require a settlement-competence attainment period. This may be achieved by CMP utilization during Phase I, depletion of which during Phase II may be responsible for reduction in settlement success with cyprid age such that remaining CMP stores can no longer support the production of adult structures following settlement. Communicated by T. Ikeda, Hakodate C. G. Satuito ((2N)' K. Shimizu. K. Natoyama M. Yamazaki. N. Fusetani Fusetani Biofouling Project, ERATO, Research Development Corporation of Japan, c/o Yokohama R&D Center, Niigata Eng. Co. Ltd., 27 Shin isogo-cho, Isogo-ku, Yokohama 235, Japan Introduction Larval development in barnacles consists of six nau- pliar stages followed by metamorphosis to a nonfeed- ing cyprid stage. As has already been described by several researchers, the sole function of the cyprid is to find a suitable site for attachment where metamorpho- sis to the adult phase can proceed. Settlement behavior of the larval barnacle has been studied from various perspectives, including stimulation by exogenous fac- tors, e.g., adult extracts of conspecies (Crisp and Meadows 1963; Dineen and Hines 1994), cyprid foot- prints (Yule and Walker 1985; Clare et al. 1994), inhibi- tion by natural products (Rittschof et al. 1985) and bacterial films (Maki et al. 1992), and the influence of various environmental factors including water flow (Rittschof et al. 1984) and salinity (Dineen and Hines 1992, 1994; O'Connor and Richardson 1994). Larval age is also known to affect settlement. In the laboratory, older cyprids were observed to respond more readily to external cues than were newly molted ones, which was explained as a decrease in response threshold with larval age (Rittschof et al. 1984). Prolonging the swimming period of the cyprid may also have detrimental effects once energy reserves fall below a critical level required for successful meta- morphic and postmetamorphic consumption (Holland and Walker 1975; Lucas et al. 1979). Lucas et al. (1979) demonstrated that BaIanus balanoides could be main- tained at the cyprid stage for 28 d at 10°C before the energy supply was consumed, resulting in the loss of metamorphic ability. Pechenik et al. (1993) reported that the prolongation of the cyprid stage in Balanus amphitrite for 3 to 5 d dramatically depressed the post- metamorphic growth rate, but in this case meta- morphic success and survival were not affected. It is apparent, therefore, that with the exception of studies which have prolonged larval-lifetime artificially by storage at low temperatures, the influence of cyprid age on settlement has received little attention.