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Comparative attachment of barnacle cyprids (Balanus amphitrite Darwin, 1854; B. improvisus Darwin, 1854; & B. ebumeus Gould, 1841) to polystyrene and glass substrata
Journal
of Experimental Marine Biology and Ecology 183 (1994) 213-225
JOURNAL OF EXPERIMENTAL MARINE BIOLOQY AND ECOLOGY
Comparative attachment of barnacle cyprids (Balanus amphitrite Darwin, 1854; B. improvisus Darwin, 1854; & B. eburneus Gould, 1841) to polystyrene and glass substrata Nancy J. O’Connor *, Donnia L. Richardson South Carolina Marine Resources Research Institute, 217 Fort Johnson Road, Box 12559, Charleston. SC 29422-2559, USA Received
21 December
1993, revision
received
20 June 1994, accepted
7 July 1994
Abstract The cyprid larval stage of the barnacle Balanus amphitrite Darwin is often used in assays of larval attachment to substrata, yet its settlement response may not be representative of other barnacles. We compared attachment of cyprids of three balanomorph species to polystyrene and glass substrata. Larvae released by adults in the laboratory were reared to the cyprid stage, and attachment was monitored at daily intervals to determine whether it varied with larval age, substratum type, salinity, or storage at low temperatures. Attachment of B. amphitrite and B. improvisus Darwin after a one-day period was low in almost all assays, but increased over a few days to a high level of attachment. Balunus amphitrite tended to attach in higher numbers to glass than to polystyrene whereas B. improvisus attached more to polystyrene. Older cyprids did not attach in higher numbers than younger cyprids. Cooling cyprids prior to experimentation sometimes temporarily retarded attachment of B. improvisus and B. amphitrite. Balanus ebumeus Gould cyprids attached in low numbers to both substrata in all assays. All three species attached in salinities from 10 to 30 ppt despite more restricted distributions of adults along salinity gradients in nature. Keywords: Attachment; ment
Balanus amphitrite; Balanus ebumeus; Balanus improvisus; Cyprid; Settle-
1. Introduction Barnacles have proven useful as a model organism for studies of larval settlement, both in the laboratory (e.g. Knight-Jones, 1953; Crisp, 1974; Branscomb & Rittschof, * Correspondence North Dartmouth,
and present address: MA 02747, USA.
Biology
Department,
University
0022-0981/94/$7.00 0 1994 Elsevier Science B.V. All rights reserved SSDI 0022-0981(94)00107-3
of Massachusetts
Dartmouth,
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N.J. O’Connor. D.L. Richardson
; J. E-up. Mar. Biol. Ecol. 183 (15~~1 ,713-Z’
1984; Rittschof et al., 1984; Dineen & Hines, 1992), and in the field (e.g. Connell, 1961a,b; Strathmann et al., 1981; Wethey, 1984; Gaines et al., 1985; Chabot & Bourget, 1988; Raimondi, 1988; Sutherland, 1990). Assays examining the attachment ofbarnac1e larvae to substrata are routinely used in biofouling research and the testing of natural antifoulants (e.g. Standing et al., 1984; Rittschof et al., 1985, 1986, 1992: Maki et al., 1988, 1990, 1992; Szewzyk et al., 1991; Holmstrom et al,, 1992). Laboratory assays in biofouling research have been most thoroughly developed for monitoring settlement (defined as permanent attachment to a surface) of cyprid larvae of Balanus amphitrite Darwin, using polystyrene and glass as the experimental substrata (Branscomb & Rittschof, 1984; Rittschof et al., 1984). However, assay results obtained for B. amphitrite might not be generalizable to settlement behavior of other balanomorph species. Species-specific settlement behaviors may exist that would limit the predictive value of assay results in natural systems. Two other barnacle species present in intertidal and shallow subtidal habitats along the eastern coast of North America are B. eburneus Gould and B. improvisus Darwin. Both species are euryhahne and common on hard substrata in estuarine habitats (Zullo, 1979). We performed settlement assays to determine whether larvae of B. eburneus and B. improvisus attach to polystyrene and glass substrata in a manner similar to B. amphitrite. If they do, then these species also could be used in assays of responses of cyprid larvae to environmental factors. The primary questions we asked were: (1) What are temporal attachment patterns of cyprid larvae of the three species to polystyrene and glass substrata, and do they vary with larval age or substratum type? (2) Does letting the cyprids age by storing them at low temperatures prior to use, a common practice in biofouling research to increase settlement response, affect the subsequent attachment of cyprids? and (3) Does the salinity of seawater used in the assays affect the number of cyprids attaching to the substrata?
2. Materials and methods 2.1. Adult broodstocks At least 100 adults of each species were collected every 2-3 months year-round and placed into laboratory cultures. Balanus eburnew and B. improvisus attached to rocks and oyster shells were collected from polyhaline and mesohaline habitats in Charleston County, South Carolina, USA. Adult B. amphitrite were collected from pilings and oysters in euhaline habitats of Georgetown County, South Carolina. In the laboratory, adults were maintained in buckets of % 5 1 of aerated, filtered (5 pm) seawater following methods similar to those used by Rittschof et al. (1984). Seawater used for B. amphitrite and B. ebumeus adults was ~30 ppt, and that for B. improvisus was % 20 ppt. Adults were fed a diet of brine shrimp (Artemiu sp.) nauplii and phytoplankton (the diatom Chaetoceros gracilis Schutt and the flagellate Isochrysis galbana Parke (Tahitian strain = T-lso) in a 3:l ratio). Phytoplankton were cultured following the method of Guillard (1983) and used when cultures had reached densities of at least 1 x lo6 cells . ml-‘. Each day adult barnacles were placed in fresh sea-
N.J. O’Connor, D.L. Richardson /J. Exp. Mar. Biol. Ecol. 183 (1994) 213-225
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water and given fresh food. Adults were maintained at room temperature (x 25 ‘C) and a 14 h light:10 h dark photoperiod. 2.2. Larval cultures Balanus amphitrite and B. improvisus adults spontaneously released larvae when adults were placed in fresh seawater without aeration, and the water surface was illuminated. However, B. eburneus adults did not release larvae by this method, and it was necessary to sieve newly-released (within w 24 h) nauplii from seawater in which adults had been maintained. Approximately 500-2000 larvae were placed in 4 1 polypropylene containers with aged, filtered (0.8 pm) seawater (30 ppt), to which was added a 3:l mixture of Chaetoceros gracilis and T-Zso. Initial larval density was = 1 per 2 ml, in a solution of three parts filtered seawater:one part algae. The antibiotics sodium penicillin G and streptomycin sulfate were added to give a final concentration of 21.9 mg . 1-t and 36.5 mg . l-‘, respectively. Cultures were aerated gently. Each day 100-300 ml of the algal mixture were added to each culture. Cultures were maintained in an incubator at 27 “C ( _+1 “C). After 3 days of culturing, the nauplii were sieved (150 pm mesh) from the culture, inspected for assessment of feeding, activity, mortality, and development, and placed into fresh filtered seawater with phytoplankton and antibiotics. When cyprids appeared in the cultures (after 4-5 days for B. amphitrite, 5-6 days for B. eburneus, and 6-7 days for B. improvisus), the cultures were sieved (200 pm mesh), and cyprids were removed for experimentation. The day of molting to the cypris stage was termed day 0, following the convention of Rittschof et al. (1984). Cyprids were used immediately in assays, or stored at 6 “C (k 1 “C) in covered glass culture dishes until used. Only batches of cyprids that were active and had numerous oil cells, representing energy reserves (Lucas et al., 1979), were saved for experimentation.
2.3. General attachment assay procedures Assay procedures were similar to those used by Rittschof et al. (1984, 1992). Experimental containers consisted of sterile polystyrene petri dishes (50 x 9 mm, Falcon no. 1006), and borosilicate glass vials (25 x 57 mm) that had been placed in an oven for 4 h at 500 “C to remove organic material. Containers were filled with 5 ml of sterile-filtered (0.2 pm) seawater (FSW), and a group of ~25 cyprids (range 15-50) was added to each container. Varying the number of cyprids within this range did not affect the number attaching (unpubl. data). Containers were covered and placed in an incubator at 27 “C ( +_1 “C) with a 14 h light:10 h dark photoperiod ( = constant conditions). At %24-h intervals, each container was examined microscopically, and the locations of all settled animals (attached cyprids and metamorphosed juveniles) were marked on the outside of the container. Attached cyprids could be readily recognized with practice by their “firm attachment and obtuse anterior end” (Knight-Jones, 1953, p. 585). The number of dead unattached cyprids was also noted. At the end of the assay period, assays were terminated by rinsing unattached cyprids
from containers with distilled water onto filter paper in a vacuum filtration unit. The number of living and dead cyprids and attached animals was determined for each container, and the proportion settled on each day of the assay was calculated, correcting for cyprid mortality. Containers with mortality > 30”, were omitted from further analysis. Generally, mortality of cyprids was very low (< IO’,,,). 2.4. Specific attachmenr assuys 2.4.1. Attachment vs. qvprid age Cyprids were harvested from Iarval cultures on day 0. Approximately 25 cyprids were placed into an experimental container (dish or vial) containing 5 ml of FSW of 30 ppt (B. ~?~phitr~teand B. eburneus) or FSW diluted with distilled water to 20 ppt (B. improvisu.s).The remaining cyprids were stored at 6 ‘C ( + I ‘C). Each day for the next
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Exp. Mar. Biol. Ecol. 183 (1994) 213-225
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Fig. 1. Cumulative attachment of B. amphitrite, B. improvisus, and B. ebumeus cyprids of different ages over time. A group of -25 cyprids was placed in each polystyrene Petri dish (p) or glass vial (g). Assays were initiated both when cyprids first appeared in culture (0, day 0 cyprids) and on successive days with older larvae (day 1 cyprids, 0; day 2, 0; day 3, n ; day 4, A; day 5, A; day 6, +). Numbers denote particular batches of larvae used in the assays.
5 or 6 days, a group of cyprids from the same batch of larvae was slowly warmed to ambient temperature and placed in a container at constant conditions. Attachment of cyprids to the experimental containers was monitored over time as described above. 2.4.2. Attachment vs. storage temperature Day 0 cyprids from a batch of larvae were divided into two groups and stored in FSW with antibiotics in glass culture dishes at 6 “C and 27 “C for 1 and 3 days. To begin an assay, day 1 or day 3 cyprids from culture dishes stored at each temperature were placed into experimental containers at 27 “C. Four replicate containers were used for each treatment group. Assays with B. amphitrite cyprids were run at 30 ppt, those for B. eburneus at 20 ppt, and those for B. improvisus at 15 ppt. 2.4.3. Attachment vs. salinity Larvae were placed in experimental containers with 5 ml of FSW (30 ppt) or FSW diluted with distilled water to 25, 20, 15, or 10 ppt. Three or four replicate containers were used for each salinity. For assays with B. amphitrite and B. improvisus, day 0 cyprids were used; day 0 and day 1 cyprids were used in assays with B. ebumeus. Containers were placed at constant conditions and monitored as described above. 2.5.
Statistical analyses
All data were examined Rohlf, 198 1) and normality
for homogeneity of variances using the F,,, test (Sokal & using stem-and-leaf and normal probability plots. Data were
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arcsin transformed when necessary to meet these assumptions of analysis of variance (ANOVA). Data from assays examining the effect of salinity on attachment were analyzed using multivariate ANOVA and repeated measures ANOVA techniques, to examine differences among treatment means on each day of the assay and differences among treatments over the duration of the assay, respectively. When significant differences existed (p 50.05) among treatments on any day of the assay, differences among means were examined with t-tests (Fisher’s least-significant-difference test). Data from assays examining the effects of storage temperature were analyzed using t-tests. Separate tests were used to compare day 1 cyprids and day 3 cyprids (stored at 6 “C and 27 “C) on each day of an assay. All statistical analyses were performed using SAS version 6.06.02 (SAS Institute Inc., Cary, North Carolina) installed on a Data General MVl9500.
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Fig. 2. Mean attachment ( f SEM) of B. amphitrite, B. improvisus, and B. ebumeus cyprids over time in polystyrene (p) and glass (g) containers. Cyprids were stored at 6 and 27 “C for 1 and 3 days before use in attachment assays. Stars indicate significant differences existed among treatment means.
N.J. O’Connor, D.L. Richardson /J. Exp. Mar. Biol. Ecol. 183 (1994) 213-225
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3. Results 3.1. Attachment
vs. cyprid age
Attachment of B. amphitrite cyprids to polystyrene after a one-day period was generally low (t40%), regardless of cyprid age (Fig. 1). Attachment of B. amphitrite cyprids to glass after one day was variable; one batch of larvae attached in high numbers whereas another attached in numbers similar to those seen on polystyrene. In other assays not reported here the same variable responses were seen. However, the pattern of attachment over the duration of the assay was similar on both polystyrene and glass: attachment increased rapidly during the first few days until most of the larvae had attached, then leveled off for the remainder of the assay. B. improvisus cyprids attached to polystyrene in a manner similar to B. amphitrite: attachment was relatively low after one day, regardless of age, then increased rapidly so that a high proportion of the cyprids had attached within 5-6 days (Fig. 1). As seen for B. amphitrite, aging the cyprids did not increase subsequent attachment rate. Attachment of B. improvkus cyprids to glass was very low after one day, increasing after 1 or more days to relatively high levels similar to those seen on polystyrene. Balanus eburneus cyprids attached in variable numbers to both polystyrene and glass substrata (Fig. 1). Attachment was lower for this species than for either of the other two species. Younger cyprids tended to settle in higher numbers than those that had been aged at 6 “C for longer periods, on both polystyrene and glass. 3.2. Attachment
vs. storage temperature
Storing cyprids at 6 or 27 ‘C prior to use in the assay had little effect on subsequent attachment of B. amphitrite cyprids (Fig. 2). In only one instance were any statistically significant differences observed. For day 2 of the assay with batch no. 55 on glass, attachment was higher for cyprids stored at 27 than 6 “C. However, this difference disappeared with time. Cooling somewhat delayed attachment of B. improvisus cyprids (Fig. 2). On 2 days of the assay with polystyrene substrata, larvae stored for 3 days at 27 “C attached in greater numbers than larvae stored for 3 days at 6 “C. Attachment on glass substrata tended to be higher for cyprids stored for one day at 27 “C than at 6 “C, although differences were not statistically significant. In both assays, however, differences in attachment decreased as time passed. In general, attachment of B. improvisus cyprids was lower on glass than on polystyrene substrata. Attachment of B. eburneus cyprids was not signihcantly affected by storage temperature, in assays with either polystyrene or glass substrata (Fig. 2). However, attachment was less than that of either B. amphitrite or B. improvisus. 3.3. Attachment
vs. salinity
Salinity had little effect on either initial attachment or the temporal tachment of B. amphitrite cyprids (Fig. 3). No statistically signiIkant
pattern of atdifferences in
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3.J. 0 ‘Connor. D. L. Richmdson 1 J. Erp. Mur. Bid. Ed.
attachment due to salinity were observed except for batch no. 120 on glass, where on one day (day 5) of the assay attachment was greater at 20 and 25 ppt than at 10 and 30 ppt. The magnitude of the differences, however, was very small and probably is not biologically significant. Initial attachment was higher on glass than polystyrene. Salinity affected attachment of B. improvisus cyprids to polystyrene and glass substrata, in terms of both numbers and rate of attachment (Fig. 3). Attachment of batch no. 35 cyprids to both polystyrene and glass was signifkantly higher at 10 and 20 ppt than at 30 ppt on 2 or 3 days of the assays. Throughout most of the assay with batch no. 11, attachment to polystyrene was significantly lower at 30 ppt than at 10, 15, 20, or 25 ppt. On days 4 and 5 of the assay with batch no. 70 in glass containers, attachment at 10 and 15 ppt was greater than that at 25 and 30 ppt, and on day 6 attachment was greater at 10, 15, and 20 ppt than at 30 ppt. Attachment of B. ~mprov~s~~ cyprids was
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N.J. O’Connor, D.L. Richardson 1 J. Exp. Mar. Biol. Ecol. 183 (1994) 213-225
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Fig. 3. Mean attachment ( f SEM) of B. amphitrite, B. improvisus, and B. eburneus cyprids over time in polystyrene (p) and glass (g) containers with filtered seawater of various salinities. For figures in the left-hand column: 0, 10 ppt; n , 20 ppt; A, 30 ppt. For figures in the right-hand column: 0, 10 ppt; l , 15 ppt; 0, 20 ppt; n , 25 ppt; A, 30 ppt). Three or four replicate containers were used for each treatment of each assay. Numbers denote particular batches of larvae used in the assays. Stars indicate days where significant differences existed among treatment means.
generally more rapid and greater on polystyrene than on glass, and differences due to salinity were more pronounced on glass than on polystyrene. Attachment of B. eburneus cyprids to both polystyrene and glass was relatively low at all salinities (Fig. 3). There were no statistically significant differences in attachment at any salinity on any day of the assays. However, attachment tended to be lowest at 30 ppt in all assays. 4. Discussion Our study demonstrates several differences in the response of larvae of three balanomorph species during attachment to polystyrene and glass substrata. BaZunus amphitrite cyprids initially attached in greater numbers to glass than to polystyrene in almost all assays, substantiating the apparent preference of B. amphitrite for glass substrata noted by Rittschof et al. (1984) and Roberts et al. (1991). However, attachment of cyprids after a one-day period did not increase with cyprid age. In addition, storing cyprids at cool temperatures did not increase their attachment, as was found by Rittschof et al. (1984) and Pechenik et al. (1993). Cyprids stored at either 6 or 27 “C attached similarly in assays at 27 “C (Fig. 2). However, some cyprids settled during storage at 27 “C, so results of those assays may have been biased by using cyprids that were slower to settle. Contrary to the preference for glass substrata displayed by B. amphitrite cyprids, B. improvisus cyprids attached in greater numbers to polystyrene than to glass surfaces. As seen for B. amphitrite, attachment increased during the duration of the assays, so
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that most cyprids had attached within l-2 wk. Aging cyprids did not increase subsequent attachment. In fact, storing cyprids at 6 “C caused a temporary decrease in attachment. This result was unexpected, given that the range of B. improvisus in North America extends to higher latitudes than that of B. ~~~~~~~~~e (Bous~eld, 1954). and B. improvisuscyprids probably experience cooler temperatures in nature. We observed that cyprids of both species stored for extended periods (3-4 wk) at 6 “C began attaching to and metamorphosing on the bottom and sides of the storage containers. ~fflu~~ ebumeus cyprids attached to polyst~ene and glass surfaces in relatively low numbers in all assays performed. This could have been because we were not providing cyprids with conditions favorable for attachment, or that WCwere not producing larvae that were competent to settle. All assays were performed without water movement within containers. We tested the hypothesis that water motion would stimulate attachment of B. eburneus cyprids by placing some experimental containers containing cyprids and FSW (20 ppt) on shakers that rotated (1.1 revolutions s ‘) or moved back and forth (1.4 cycles + s-l). Cyprids in containers experiencing water movement did not attach in significantly higher numbers than cyprids not subjected to water motion (p > 0.05 in ANOVA or Kruskal-Wallis tests, Fig. 4). We then determined whether B. eburneus cyprids reared using our procedures were competent to settle by placing a variety of potential settlement substrata (a polystyrene dish and a glass vial; small pieces of slate, tile, formica, and wood) in the larval culture containers of three different batches of larvae once cyprids began to appear. Five days later, we examined the substrata and found that most of the cyprids had settled, primarily on the wooden and unglazed tile surfaces. Many also set on the inside of the polypropylene culture container. Several attached on the inside of the glass vial (none on the outside) and very
Fig. 4. Attachment of B. ehurneus cyprids in polystyrene (p) and giass (g) containers that were stationary. rotated. or moved back and forth. Wues represent mean attachment ( _+SEM) of three replicate containcrs.
N.J. O’Connor.D.L. Richardson f J. Exp. Mar. Bid. Ecoi. 183 (1994) 213-225
223
few attached to the polystyrene dish. Like other barnacle species (Crisp, 1974), B. ebumeus apparently prefers to attach to textured or porous surfaces (see also Norris, 1991; Weis & Weis, 1992). The culture containers presumably contained large amounts of metabolites from the larvae and ph~opl~kton, which also might have stimulated attachment. Differences in settlement responses of cyprids of the three species to the two substratum types that emerged during the assays were tested for statistical significance using nonp~~et~c tests. Data used for the analysis were from the storage temperature assays where cyprids were not exposed to low temperatures and were placed in assay containers one day after molting to the cyprid stage ( = day 1, 27 “C treatment, Fig. 2). The proportion attached in each of the replicate containers on the seventh day of the assay was used in the analysis, to determine whether any differences in attachment that occurred early in the assay persisted over time. The statistical analyses indicated that significantly more B. amp&rite cyprids attached to glass than polystyrene, whereas more B. ~mprovisus cyprids attached to polystyrene compared to glass (Table 1). In among-species comparisons (Kruskal-Wallis test followed by Wilcoxon rank-sum tests between species), attachment of B. amphitrite and B. improvises to polystyrene was higher than that of B. ebumeus (p = 0.03), and attachment to glass was greater for B. am~hitri~~ than either B. imFrovisus or B. eburneus (PI 0.03) (Table 1). Cyprids of the three species settled at a wide range of salinities. This result was surprising, as the species generally occur within narrower salinity ranges in nature. Balanus improvises typically occurs in lower salinities than either B. ebu~eus or B. amphitrite (Kennedy & DiCosimo, 1983), and usually does not occur in areas with salinities > 25 ppt (Bousfield, 1954). It is possible that B. improvisus embryos and larvae became acclimated to higher salinities used during culturing, which could explain their settlement at salinities higher than expected (Bacon, 1971; Dineen & Hines, 1992). Bafanus amphitrite usually occurs in salinities higher than those of habitats for either B. eburneus or B. improvisus (Bousfield, 1954; pers. obs. in South Carolina). Perhaps B. amphitrite settled at relatively low salinities in our assays because the substrata became more attractive due to modi~cation by proteins deposited by cyprids as they explored surfaces with their antennules (Yule & Walker, 1985). The present study demonstrates that both B. amphitrite and B. improvisus will settle Table t Mean percent attachment of cyprids of three barnacle species to polystyrene and glass substrata Species
B. amphitrite * B. improvisus* B. eburneus
Polystyrene
Glass
Mean
SD
Mean
SD
66.7
16.7 6.4 19.7
94.3 38.1 3.8
1.6 26.1 5.2
78.0
32.3
Data are from the storage temperature assays (Fig. 3, “day 1, 27 “c” treatment, day 7 of the assay). * Mean % attachment values to polystyrene and glass differ (p = 0.03, Wilcoxon rank-sum test).
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predictably and in relatively high numbers in the assay designed by Rittschof and coworkers, although there are differences between the species in their response to polystyrene and glass substrata. However, the assay system must be modified to include other substrata when assays using B. ebumeus as the test organism are desired. In addition, assays should be conducted using more natural surfaces likely encountered by cyprids in the wild, to increase the ecological relevancy of the assay system.
Acknowledgements We thank D. Rittschof and J. Dineen for advice during initiation of the project, and the shellfish mariculture group at the Marine Resources Research Institute for providing the phytoplankton used in larval culturing. This research was supported by U.S. Office of Naval Research Grant NOOO14-90-J-4048 to South Carolina State University (J.B. Stukes, P.I.). Contribution number 346 from the South Carolina Marine Resources Center.
References Bacon. P.R., 1971. The maintenance of a resident population of Balmus ehurwus (Gould) in relation to salinity fluctuations in a Trinidad mangrove swamp. J. Eqr. Mar. Biol. Ecol.. Vol. 6. pp. 187-198. Bousfield. E.L.. 1954. The distribution and spawning seasons of barnacles on the Atlantic coast of Canada. &ill. Ricrrl. Mus. Can., Vol. 132, pp. 113-l 54. Branscomb. ES. & D. Rittschof, 1984. An investigation of low frequency sound waves as a means of inhibiting barnacle settlement. J. E.up. Mar. Biol. Ecol.. Vol. 79, pp. 149-154. Chabot. R. & E. Bourget. 1988. Influence of substratum heterogeneity and settled barnacle density on the settlement of cypris larvae. Mur. Eiol.. Vol. 97, pp. 45-56. Connell. J.H., 1961a. Effects of competition, predation by Thuis lapillus, and other factors on natural populations of the barnacle Balanus bulanoides. Ecol. Monogr.. Vol. 31. pp. 61-104. Connell, J.H., 1961b. The influence of interspecific competition and other factors on the distribution of the barnacle Chthamalus siellatus. Ecology, Vol. 42, pp. 710-723. Crisp. D.J.. 1974. Factors influencing the settlement of marine invertebrate larvae. In, Chemoreception in nlurine organisms. edited by P.T. Grant & A.M. Mackic, Academic Press, New York, pp. 177-265. Dineen. J.F., Jr. & A.H. Hines, 1992. Interactive effects of salinity and adult extract upon settlement of the cstuarine barnacle Balunu.~ improvisus (Darwin. 1854). J. Exp. Mar. Biol. Ecol.. Vol. 156. pp. 239-252. Gaines, S.. S. Brown & J. Roughgarden, 1985. Spatial variation in larval concentrations as a cause of spatial variation in settlement for the barnacle. Balanus glundtdu. Oecokogiu, Vol. 67, pp. 367-271. Guillard. R.R.L., 1983. Culture of phytoplankton for feeding marine invertebrates. in. Culture oj’murine inwrtebrrrtest selected readings. edited by C.J. Berg, Jr., Hutchinson Ross Publishing Company. Stroudsburg. Pennsylvania, pp. 108-132. Holmstrom, C., D. Rittschof & S. Kjelleberg. 1992. Inhibition of settlement by larvae of Boiunus umphitrite and Cirjilu intestinalis by a surface-colonizing marine bacterium. Appl. Environ. 44icrobiol., Vol. 58. pp. 2111-2115. Kennedy. V.S. & J. DiCosimo. 1983. Subtidal distribution of barnacles (Cirrtpedia: Balamdae) in Chesapeake Bay. Maryland. Estuuries. Vol. 6, pp. 95- 10 1. Knight-Jones. E.W.. 1953. Laboratory experiments on gregartousncss during scttmg in Bulunu.~ hafunoider dnd other barnacles. J. Exy. Biol.. Vol. 30. pp. 584-598. L.ucas. M.I., G. Walker, D.L. Holland & D.J. Crisp. 1979. An energy budget for the free-swimming and metamorphosing larvae of Balanus balanoides (Crustacea: Cirripedia). Mrrr. Biol.. Vol. 55. pp. 221-29-Y.
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Maki, J.S., D. Rittschof, J.D. Costlow & R. Mitchell, 1988. Inhibition of attachment of larval barnacles, Balanus amphitrite, by bacterial surface films. Mar. Biol., Vol. 97, pp. 199-206. Maki, J.S., D. Rittschof, M.-O. Samuelsson, U. Szewzyk, A.B. Yule, S. Kjelleberg, J.D. Costlow & R. Mitchell, 1990. Effect of marine bacteria and their exopolymers on the attachment of barnacle cypris larvae. Bull. Mar. Ski., Vol. 46, pp. 499-511. Maki, J.S., D. Rittschof & R. Mitchell, 1992. Inhibition of larval barnacle attachment to bacterial films: an investigation of physical properties. Microb. Ecol., Vol. 23, pp. 97- 106. Norris, D.R., 1991. Recruitment, growth, and survivorship of Balanus eburneus with respect to settlement, surface, and season. J. Elisha Mitchell Sci. Sot., Vol. 107, pp. 123-130. Pechenik, J.A., D. Rittschof & A.R. Schmidt, 1993. Influence of delayed metamorphosis on survival and growth of juvenile barnacles Balanus amphitrite. Mar. Biol., Vol. 115, pp. 287-294. Raimondi, P.T., 1988. Settlement cues and determination of the vertical limit of an intertidal barnacle. Ecology, Vol. 69, pp. 400-407. Rittschof, D., E.S. Branscomb & J.D. Costlow, 1984. Settlement and behavior in relation to flow and surface in larval barnacles, Balanus amphitrite Darwin. J. Exp. Mar. Biol. Ecol., Vol. 82, pp. 131-146. Rittschof, D., I.R. Hooper, E.S. Branscomb & J.D. Costlow, 1985. Inhibition of barnacle settlement and behavior by natural products from whip corals, Leptogorgia virgulata (LaMarck, 18 15). J. Chem. Ecol., Vol. 11, pp. 551-563. Rittschof, D., I.R. Hooper & J.D. Costlow, 1986. Barnacle settlement inhibitors from sea pansies, Renilla reniformis. Bull. Mar. Sri., Vol. 39, pp. 376-382. Rittschof, D., A.S. Clare, D.J. Gerhart, Sister Avelin Mary & J. Bonaventura, 1992. Barnacle in vitro assays for biologically active substances: toxicity and settlement inhibition assays using mass cultured Balanus amphitrice amphitrite Darwin. Biofouling, Vol. 6, pp. 115-122. Roberts, D., D. Rittschof, E. Holm & A.R. Schmidt, 1991. Factors influencing initial larval settlement: temporal, spatial and surface molecular components. J. Exp. Mar. Biol. Ecol., Vol. 150, pp. 203-221. Sokal, R.R. & F.J. Rohlf, 1981. Biometry. W.H. Freeman & Co., San Francisco, California, second edition, 859 pp. Standing, J.D., I.R. Hooper & J.D. Costlow, 1984. Inhibition and induction of barnacle settlement by natural products present in octocorals. J. Chem. Ecol., Vol. 10, pp. 823-834. Strathmann, R.R., E.S. Branscomb & K. Vedder, 1981. Fatal errors in set as a cost of dispersal and the influence of intertidal flora on set of barnacles. Oecologia, Vol. 48, pp. 13-18. Sutherland, J.P., 1990. Recruitment regulates demographic variation in a tropical intertidal barnacle. Ecology. Vol. 71, pp. 955-972. Szewzyk, U., C. Holmstrom, M. Wrangstadh, M.-O. Samuelsson, J.S. Maki & S. Kjelleberg, 1991. Relevance of the exopolysaccharide of marine Pseudomonas sp. strain S9 for the attachment of Ciona intestinalis larvae. Mar. Ecol. Prog. Ser., Vol. 75, pp. 259-265. Weis, J.S. & P. Weis, 1992. Construction materials in estuaries: reduction in the epibiotic community on chromated copper arsenate (CCA) treated wood. Mar. Ecol. Prog. Ser., Vol. 83, pp. 45-53. Wethey. D.S., 1984. Spatial pattern in barnacle settlement: day to day changes during the settlement season. J. Mar. Biol. Assoc. U.K., Vol. 64, pp. 687-698. Yule, A.B. & G. Walker, 1985. Settlement of Balanus balanoides: the effect of cyprid antennular secretion. J. Mar. Biol. Assoc. U.K., Vol. 65, pp. 707-712. Zullo, V.A., 1979. Marine flora and fauna of the northeastern United States. Arthropoda: Cirripedia. NOAA Tech. Rep. NMFS. Circ., No. 425, 29 pp.
of Experimental Marine Biology and Ecology 183 (1994) 213-225
JOURNAL OF EXPERIMENTAL MARINE BIOLOQY AND ECOLOGY
Comparative attachment of barnacle cyprids (Balanus amphitrite Darwin, 1854; B. improvisus Darwin, 1854; & B. eburneus Gould, 1841) to polystyrene and glass substrata Nancy J. O’Connor *, Donnia L. Richardson South Carolina Marine Resources Research Institute, 217 Fort Johnson Road, Box 12559, Charleston. SC 29422-2559, USA Received
21 December
1993, revision
received
20 June 1994, accepted
7 July 1994
Abstract The cyprid larval stage of the barnacle Balanus amphitrite Darwin is often used in assays of larval attachment to substrata, yet its settlement response may not be representative of other barnacles. We compared attachment of cyprids of three balanomorph species to polystyrene and glass substrata. Larvae released by adults in the laboratory were reared to the cyprid stage, and attachment was monitored at daily intervals to determine whether it varied with larval age, substratum type, salinity, or storage at low temperatures. Attachment of B. amphitrite and B. improvisus Darwin after a one-day period was low in almost all assays, but increased over a few days to a high level of attachment. Balunus amphitrite tended to attach in higher numbers to glass than to polystyrene whereas B. improvisus attached more to polystyrene. Older cyprids did not attach in higher numbers than younger cyprids. Cooling cyprids prior to experimentation sometimes temporarily retarded attachment of B. improvisus and B. amphitrite. Balanus ebumeus Gould cyprids attached in low numbers to both substrata in all assays. All three species attached in salinities from 10 to 30 ppt despite more restricted distributions of adults along salinity gradients in nature. Keywords: Attachment; ment
Balanus amphitrite; Balanus ebumeus; Balanus improvisus; Cyprid; Settle-
1. Introduction Barnacles have proven useful as a model organism for studies of larval settlement, both in the laboratory (e.g. Knight-Jones, 1953; Crisp, 1974; Branscomb & Rittschof, * Correspondence North Dartmouth,
and present address: MA 02747, USA.
Biology
Department,
University
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of Massachusetts
Dartmouth,
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; J. E-up. Mar. Biol. Ecol. 183 (15~~1 ,713-Z’
1984; Rittschof et al., 1984; Dineen & Hines, 1992), and in the field (e.g. Connell, 1961a,b; Strathmann et al., 1981; Wethey, 1984; Gaines et al., 1985; Chabot & Bourget, 1988; Raimondi, 1988; Sutherland, 1990). Assays examining the attachment ofbarnac1e larvae to substrata are routinely used in biofouling research and the testing of natural antifoulants (e.g. Standing et al., 1984; Rittschof et al., 1985, 1986, 1992: Maki et al., 1988, 1990, 1992; Szewzyk et al., 1991; Holmstrom et al,, 1992). Laboratory assays in biofouling research have been most thoroughly developed for monitoring settlement (defined as permanent attachment to a surface) of cyprid larvae of Balanus amphitrite Darwin, using polystyrene and glass as the experimental substrata (Branscomb & Rittschof, 1984; Rittschof et al., 1984). However, assay results obtained for B. amphitrite might not be generalizable to settlement behavior of other balanomorph species. Species-specific settlement behaviors may exist that would limit the predictive value of assay results in natural systems. Two other barnacle species present in intertidal and shallow subtidal habitats along the eastern coast of North America are B. eburneus Gould and B. improvisus Darwin. Both species are euryhahne and common on hard substrata in estuarine habitats (Zullo, 1979). We performed settlement assays to determine whether larvae of B. eburneus and B. improvisus attach to polystyrene and glass substrata in a manner similar to B. amphitrite. If they do, then these species also could be used in assays of responses of cyprid larvae to environmental factors. The primary questions we asked were: (1) What are temporal attachment patterns of cyprid larvae of the three species to polystyrene and glass substrata, and do they vary with larval age or substratum type? (2) Does letting the cyprids age by storing them at low temperatures prior to use, a common practice in biofouling research to increase settlement response, affect the subsequent attachment of cyprids? and (3) Does the salinity of seawater used in the assays affect the number of cyprids attaching to the substrata?
2. Materials and methods 2.1. Adult broodstocks At least 100 adults of each species were collected every 2-3 months year-round and placed into laboratory cultures. Balanus eburnew and B. improvisus attached to rocks and oyster shells were collected from polyhaline and mesohaline habitats in Charleston County, South Carolina, USA. Adult B. amphitrite were collected from pilings and oysters in euhaline habitats of Georgetown County, South Carolina. In the laboratory, adults were maintained in buckets of % 5 1 of aerated, filtered (5 pm) seawater following methods similar to those used by Rittschof et al. (1984). Seawater used for B. amphitrite and B. ebumeus adults was ~30 ppt, and that for B. improvisus was % 20 ppt. Adults were fed a diet of brine shrimp (Artemiu sp.) nauplii and phytoplankton (the diatom Chaetoceros gracilis Schutt and the flagellate Isochrysis galbana Parke (Tahitian strain = T-lso) in a 3:l ratio). Phytoplankton were cultured following the method of Guillard (1983) and used when cultures had reached densities of at least 1 x lo6 cells . ml-‘. Each day adult barnacles were placed in fresh sea-
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water and given fresh food. Adults were maintained at room temperature (x 25 ‘C) and a 14 h light:10 h dark photoperiod. 2.2. Larval cultures Balanus amphitrite and B. improvisus adults spontaneously released larvae when adults were placed in fresh seawater without aeration, and the water surface was illuminated. However, B. eburneus adults did not release larvae by this method, and it was necessary to sieve newly-released (within w 24 h) nauplii from seawater in which adults had been maintained. Approximately 500-2000 larvae were placed in 4 1 polypropylene containers with aged, filtered (0.8 pm) seawater (30 ppt), to which was added a 3:l mixture of Chaetoceros gracilis and T-Zso. Initial larval density was = 1 per 2 ml, in a solution of three parts filtered seawater:one part algae. The antibiotics sodium penicillin G and streptomycin sulfate were added to give a final concentration of 21.9 mg . 1-t and 36.5 mg . l-‘, respectively. Cultures were aerated gently. Each day 100-300 ml of the algal mixture were added to each culture. Cultures were maintained in an incubator at 27 “C ( _+1 “C). After 3 days of culturing, the nauplii were sieved (150 pm mesh) from the culture, inspected for assessment of feeding, activity, mortality, and development, and placed into fresh filtered seawater with phytoplankton and antibiotics. When cyprids appeared in the cultures (after 4-5 days for B. amphitrite, 5-6 days for B. eburneus, and 6-7 days for B. improvisus), the cultures were sieved (200 pm mesh), and cyprids were removed for experimentation. The day of molting to the cypris stage was termed day 0, following the convention of Rittschof et al. (1984). Cyprids were used immediately in assays, or stored at 6 “C (k 1 “C) in covered glass culture dishes until used. Only batches of cyprids that were active and had numerous oil cells, representing energy reserves (Lucas et al., 1979), were saved for experimentation.
2.3. General attachment assay procedures Assay procedures were similar to those used by Rittschof et al. (1984, 1992). Experimental containers consisted of sterile polystyrene petri dishes (50 x 9 mm, Falcon no. 1006), and borosilicate glass vials (25 x 57 mm) that had been placed in an oven for 4 h at 500 “C to remove organic material. Containers were filled with 5 ml of sterile-filtered (0.2 pm) seawater (FSW), and a group of ~25 cyprids (range 15-50) was added to each container. Varying the number of cyprids within this range did not affect the number attaching (unpubl. data). Containers were covered and placed in an incubator at 27 “C ( +_1 “C) with a 14 h light:10 h dark photoperiod ( = constant conditions). At %24-h intervals, each container was examined microscopically, and the locations of all settled animals (attached cyprids and metamorphosed juveniles) were marked on the outside of the container. Attached cyprids could be readily recognized with practice by their “firm attachment and obtuse anterior end” (Knight-Jones, 1953, p. 585). The number of dead unattached cyprids was also noted. At the end of the assay period, assays were terminated by rinsing unattached cyprids
from containers with distilled water onto filter paper in a vacuum filtration unit. The number of living and dead cyprids and attached animals was determined for each container, and the proportion settled on each day of the assay was calculated, correcting for cyprid mortality. Containers with mortality > 30”, were omitted from further analysis. Generally, mortality of cyprids was very low (< IO’,,,). 2.4. Specific attachmenr assuys 2.4.1. Attachment vs. qvprid age Cyprids were harvested from Iarval cultures on day 0. Approximately 25 cyprids were placed into an experimental container (dish or vial) containing 5 ml of FSW of 30 ppt (B. ~?~phitr~teand B. eburneus) or FSW diluted with distilled water to 20 ppt (B. improvisu.s).The remaining cyprids were stored at 6 ‘C ( + I ‘C). Each day for the next
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5 or 6 days, a group of cyprids from the same batch of larvae was slowly warmed to ambient temperature and placed in a container at constant conditions. Attachment of cyprids to the experimental containers was monitored over time as described above. 2.4.2. Attachment vs. storage temperature Day 0 cyprids from a batch of larvae were divided into two groups and stored in FSW with antibiotics in glass culture dishes at 6 “C and 27 “C for 1 and 3 days. To begin an assay, day 1 or day 3 cyprids from culture dishes stored at each temperature were placed into experimental containers at 27 “C. Four replicate containers were used for each treatment group. Assays with B. amphitrite cyprids were run at 30 ppt, those for B. eburneus at 20 ppt, and those for B. improvisus at 15 ppt. 2.4.3. Attachment vs. salinity Larvae were placed in experimental containers with 5 ml of FSW (30 ppt) or FSW diluted with distilled water to 25, 20, 15, or 10 ppt. Three or four replicate containers were used for each salinity. For assays with B. amphitrite and B. improvisus, day 0 cyprids were used; day 0 and day 1 cyprids were used in assays with B. ebumeus. Containers were placed at constant conditions and monitored as described above. 2.5.
Statistical analyses
All data were examined Rohlf, 198 1) and normality
for homogeneity of variances using the F,,, test (Sokal & using stem-and-leaf and normal probability plots. Data were
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arcsin transformed when necessary to meet these assumptions of analysis of variance (ANOVA). Data from assays examining the effect of salinity on attachment were analyzed using multivariate ANOVA and repeated measures ANOVA techniques, to examine differences among treatment means on each day of the assay and differences among treatments over the duration of the assay, respectively. When significant differences existed (p 50.05) among treatments on any day of the assay, differences among means were examined with t-tests (Fisher’s least-significant-difference test). Data from assays examining the effects of storage temperature were analyzed using t-tests. Separate tests were used to compare day 1 cyprids and day 3 cyprids (stored at 6 “C and 27 “C) on each day of an assay. All statistical analyses were performed using SAS version 6.06.02 (SAS Institute Inc., Cary, North Carolina) installed on a Data General MVl9500.
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3. Results 3.1. Attachment
vs. cyprid age
Attachment of B. amphitrite cyprids to polystyrene after a one-day period was generally low (t40%), regardless of cyprid age (Fig. 1). Attachment of B. amphitrite cyprids to glass after one day was variable; one batch of larvae attached in high numbers whereas another attached in numbers similar to those seen on polystyrene. In other assays not reported here the same variable responses were seen. However, the pattern of attachment over the duration of the assay was similar on both polystyrene and glass: attachment increased rapidly during the first few days until most of the larvae had attached, then leveled off for the remainder of the assay. B. improvisus cyprids attached to polystyrene in a manner similar to B. amphitrite: attachment was relatively low after one day, regardless of age, then increased rapidly so that a high proportion of the cyprids had attached within 5-6 days (Fig. 1). As seen for B. amphitrite, aging the cyprids did not increase subsequent attachment rate. Attachment of B. improvkus cyprids to glass was very low after one day, increasing after 1 or more days to relatively high levels similar to those seen on polystyrene. Balanus eburneus cyprids attached in variable numbers to both polystyrene and glass substrata (Fig. 1). Attachment was lower for this species than for either of the other two species. Younger cyprids tended to settle in higher numbers than those that had been aged at 6 “C for longer periods, on both polystyrene and glass. 3.2. Attachment
vs. storage temperature
Storing cyprids at 6 or 27 ‘C prior to use in the assay had little effect on subsequent attachment of B. amphitrite cyprids (Fig. 2). In only one instance were any statistically significant differences observed. For day 2 of the assay with batch no. 55 on glass, attachment was higher for cyprids stored at 27 than 6 “C. However, this difference disappeared with time. Cooling somewhat delayed attachment of B. improvisus cyprids (Fig. 2). On 2 days of the assay with polystyrene substrata, larvae stored for 3 days at 27 “C attached in greater numbers than larvae stored for 3 days at 6 “C. Attachment on glass substrata tended to be higher for cyprids stored for one day at 27 “C than at 6 “C, although differences were not statistically significant. In both assays, however, differences in attachment decreased as time passed. In general, attachment of B. improvisus cyprids was lower on glass than on polystyrene substrata. Attachment of B. eburneus cyprids was not signihcantly affected by storage temperature, in assays with either polystyrene or glass substrata (Fig. 2). However, attachment was less than that of either B. amphitrite or B. improvisus. 3.3. Attachment
vs. salinity
Salinity had little effect on either initial attachment or the temporal tachment of B. amphitrite cyprids (Fig. 3). No statistically signiIkant
pattern of atdifferences in
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attachment due to salinity were observed except for batch no. 120 on glass, where on one day (day 5) of the assay attachment was greater at 20 and 25 ppt than at 10 and 30 ppt. The magnitude of the differences, however, was very small and probably is not biologically significant. Initial attachment was higher on glass than polystyrene. Salinity affected attachment of B. improvisus cyprids to polystyrene and glass substrata, in terms of both numbers and rate of attachment (Fig. 3). Attachment of batch no. 35 cyprids to both polystyrene and glass was signifkantly higher at 10 and 20 ppt than at 30 ppt on 2 or 3 days of the assays. Throughout most of the assay with batch no. 11, attachment to polystyrene was significantly lower at 30 ppt than at 10, 15, 20, or 25 ppt. On days 4 and 5 of the assay with batch no. 70 in glass containers, attachment at 10 and 15 ppt was greater than that at 25 and 30 ppt, and on day 6 attachment was greater at 10, 15, and 20 ppt than at 30 ppt. Attachment of B. ~mprov~s~~ cyprids was
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Fig. 3. Mean attachment ( f SEM) of B. amphitrite, B. improvisus, and B. eburneus cyprids over time in polystyrene (p) and glass (g) containers with filtered seawater of various salinities. For figures in the left-hand column: 0, 10 ppt; n , 20 ppt; A, 30 ppt. For figures in the right-hand column: 0, 10 ppt; l , 15 ppt; 0, 20 ppt; n , 25 ppt; A, 30 ppt). Three or four replicate containers were used for each treatment of each assay. Numbers denote particular batches of larvae used in the assays. Stars indicate days where significant differences existed among treatment means.
generally more rapid and greater on polystyrene than on glass, and differences due to salinity were more pronounced on glass than on polystyrene. Attachment of B. eburneus cyprids to both polystyrene and glass was relatively low at all salinities (Fig. 3). There were no statistically significant differences in attachment at any salinity on any day of the assays. However, attachment tended to be lowest at 30 ppt in all assays. 4. Discussion Our study demonstrates several differences in the response of larvae of three balanomorph species during attachment to polystyrene and glass substrata. BaZunus amphitrite cyprids initially attached in greater numbers to glass than to polystyrene in almost all assays, substantiating the apparent preference of B. amphitrite for glass substrata noted by Rittschof et al. (1984) and Roberts et al. (1991). However, attachment of cyprids after a one-day period did not increase with cyprid age. In addition, storing cyprids at cool temperatures did not increase their attachment, as was found by Rittschof et al. (1984) and Pechenik et al. (1993). Cyprids stored at either 6 or 27 “C attached similarly in assays at 27 “C (Fig. 2). However, some cyprids settled during storage at 27 “C, so results of those assays may have been biased by using cyprids that were slower to settle. Contrary to the preference for glass substrata displayed by B. amphitrite cyprids, B. improvisus cyprids attached in greater numbers to polystyrene than to glass surfaces. As seen for B. amphitrite, attachment increased during the duration of the assays, so
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183 (19941 213-225
that most cyprids had attached within l-2 wk. Aging cyprids did not increase subsequent attachment. In fact, storing cyprids at 6 “C caused a temporary decrease in attachment. This result was unexpected, given that the range of B. improvisus in North America extends to higher latitudes than that of B. ~~~~~~~~~e (Bous~eld, 1954). and B. improvisuscyprids probably experience cooler temperatures in nature. We observed that cyprids of both species stored for extended periods (3-4 wk) at 6 “C began attaching to and metamorphosing on the bottom and sides of the storage containers. ~fflu~~ ebumeus cyprids attached to polyst~ene and glass surfaces in relatively low numbers in all assays performed. This could have been because we were not providing cyprids with conditions favorable for attachment, or that WCwere not producing larvae that were competent to settle. All assays were performed without water movement within containers. We tested the hypothesis that water motion would stimulate attachment of B. eburneus cyprids by placing some experimental containers containing cyprids and FSW (20 ppt) on shakers that rotated (1.1 revolutions s ‘) or moved back and forth (1.4 cycles + s-l). Cyprids in containers experiencing water movement did not attach in significantly higher numbers than cyprids not subjected to water motion (p > 0.05 in ANOVA or Kruskal-Wallis tests, Fig. 4). We then determined whether B. eburneus cyprids reared using our procedures were competent to settle by placing a variety of potential settlement substrata (a polystyrene dish and a glass vial; small pieces of slate, tile, formica, and wood) in the larval culture containers of three different batches of larvae once cyprids began to appear. Five days later, we examined the substrata and found that most of the cyprids had settled, primarily on the wooden and unglazed tile surfaces. Many also set on the inside of the polypropylene culture container. Several attached on the inside of the glass vial (none on the outside) and very
Fig. 4. Attachment of B. ehurneus cyprids in polystyrene (p) and giass (g) containers that were stationary. rotated. or moved back and forth. Wues represent mean attachment ( _+SEM) of three replicate containcrs.
N.J. O’Connor.D.L. Richardson f J. Exp. Mar. Bid. Ecoi. 183 (1994) 213-225
223
few attached to the polystyrene dish. Like other barnacle species (Crisp, 1974), B. ebumeus apparently prefers to attach to textured or porous surfaces (see also Norris, 1991; Weis & Weis, 1992). The culture containers presumably contained large amounts of metabolites from the larvae and ph~opl~kton, which also might have stimulated attachment. Differences in settlement responses of cyprids of the three species to the two substratum types that emerged during the assays were tested for statistical significance using nonp~~et~c tests. Data used for the analysis were from the storage temperature assays where cyprids were not exposed to low temperatures and were placed in assay containers one day after molting to the cyprid stage ( = day 1, 27 “C treatment, Fig. 2). The proportion attached in each of the replicate containers on the seventh day of the assay was used in the analysis, to determine whether any differences in attachment that occurred early in the assay persisted over time. The statistical analyses indicated that significantly more B. amp&rite cyprids attached to glass than polystyrene, whereas more B. ~mprovisus cyprids attached to polystyrene compared to glass (Table 1). In among-species comparisons (Kruskal-Wallis test followed by Wilcoxon rank-sum tests between species), attachment of B. amphitrite and B. improvises to polystyrene was higher than that of B. ebumeus (p = 0.03), and attachment to glass was greater for B. am~hitri~~ than either B. imFrovisus or B. eburneus (PI 0.03) (Table 1). Cyprids of the three species settled at a wide range of salinities. This result was surprising, as the species generally occur within narrower salinity ranges in nature. Balanus improvises typically occurs in lower salinities than either B. ebu~eus or B. amphitrite (Kennedy & DiCosimo, 1983), and usually does not occur in areas with salinities > 25 ppt (Bousfield, 1954). It is possible that B. improvisus embryos and larvae became acclimated to higher salinities used during culturing, which could explain their settlement at salinities higher than expected (Bacon, 1971; Dineen & Hines, 1992). Bafanus amphitrite usually occurs in salinities higher than those of habitats for either B. eburneus or B. improvisus (Bousfield, 1954; pers. obs. in South Carolina). Perhaps B. amphitrite settled at relatively low salinities in our assays because the substrata became more attractive due to modi~cation by proteins deposited by cyprids as they explored surfaces with their antennules (Yule & Walker, 1985). The present study demonstrates that both B. amphitrite and B. improvisus will settle Table t Mean percent attachment of cyprids of three barnacle species to polystyrene and glass substrata Species
B. amphitrite * B. improvisus* B. eburneus
Polystyrene
Glass
Mean
SD
Mean
SD
66.7
16.7 6.4 19.7
94.3 38.1 3.8
1.6 26.1 5.2
78.0
32.3
Data are from the storage temperature assays (Fig. 3, “day 1, 27 “c” treatment, day 7 of the assay). * Mean % attachment values to polystyrene and glass differ (p = 0.03, Wilcoxon rank-sum test).
224
N.J. O’Connor,
D.L.
Richurdson
/ J. E.yp. Mrtr. Biol. Ecol. 183 il Y541 713-1725
predictably and in relatively high numbers in the assay designed by Rittschof and coworkers, although there are differences between the species in their response to polystyrene and glass substrata. However, the assay system must be modified to include other substrata when assays using B. ebumeus as the test organism are desired. In addition, assays should be conducted using more natural surfaces likely encountered by cyprids in the wild, to increase the ecological relevancy of the assay system.
Acknowledgements We thank D. Rittschof and J. Dineen for advice during initiation of the project, and the shellfish mariculture group at the Marine Resources Research Institute for providing the phytoplankton used in larval culturing. This research was supported by U.S. Office of Naval Research Grant NOOO14-90-J-4048 to South Carolina State University (J.B. Stukes, P.I.). Contribution number 346 from the South Carolina Marine Resources Center.
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