Effects of salinity and adult extract on settlement of the oligohaline barnacle Balanus subalbidus

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Marine Biology (1994) 119:423-430 9 Springer-Verlag 1994

J. F. Dineen, Jr. 9 A. H. Hines

Effects of salinity and adult extract on settlement of the oligohaline barnacle Balanus subalbidus

Received: 14 December 1993/Accepted: 17 January 1994

A b s t r a c t Balanus subalbidus (Henry) has the most oligohaline distribution of three congeneric barnacles in Chesapeake Bay and tolerates prolonged exposure to fresh water. We studied larval settlement (i.e., permanent attachment and metamorphosis) of B. subalbidus in the laboratory, over a 3 yr period, May 1989 to March 1992, under the following conditions: (1) across an array of salinities at 25~ in the presence and absence of settlement factor consisting of adult B. subaIbidus extract; (2) in the presence of conspecific or congeneric settlement factors; and (3) cyprids which were, and were not, induced to delay metamorphosis were compared in their capacities to settle in a range of salinities. Discrepancies between salinity pro- files of larval settlement in the laboratory and adult oligohaline distribution in the estuary were striking, and there was a significant interaction between salinity and settlement factor. Averaging results of four different batches of larvae, although peak settlement (87_+9%) ofB. subalbidus occurred at 2 ppt salinity in the presence of adult cue, substantial settlement also occurred at higher salinities: >70%

at 5, 10 and 15 ppt; and 47% at 20 and 25 ppt. In addition, settlement in the absence of settlement factor was relatively high (>50%) and peaked at mid-salinity ranges (e.g.

56+10% at 15 ppt). Variation observed in settlement among larval batches reflected detailed differences in settlement between adjacent test salinities. No difference in settlement occurred between replicate aliquots of cyprids within a batch. Cyprids of B. subalbidus settled most abundantly in the presence o f settlement factor extracted from conspecifics, followed in decreasing order by settlement factor extracted from B. improvisus and B. eburneus. De- lay of metamorphosis produced by keeping B. subalbidus

Communicated by J. R Grassle, New Brunswick J. F. Dineen, Jr. ([]) 9 A. H. Hines

Smithsonian Environmental Research Center, RO. Box 28,

Edgewater, Maryland 21037, USA

cyprids for 8 d at 5~ resulted in a decreased level of settlement, but settlement frequency patterns of delayed and non-delayed cyprids were indistinguishable relative to salinity. These results indicate that the oligohaline distribution of adult B. subalbidus is probably not determined by larval behavior at settlement. We suggest that pre-settlement behavior, resulting in larval retention in low saline waters, could be an important factor in determining distribution of this species.

Introduction

The significance of larval behavior in determining spatial distributions of sessile marine invertebrates is receiving increasing attention (Keough and Downes 1982; Caffey 1985, review by Connell 1985; Roughgarden et al. 1985;

Underwood and Fairweather 1989; Minchinton and Schei- bling 1991). For larval barnacles in particular, pre-settlement phenomena such as retention mechanisms ((Bous- field 1955), location in the water column (Grosberg 1982;

Shanks 1986), relative supply (Gaines et al. 1985; Rough- garden et al. 1988) and predation on the larvae (Gaines and Roughgarden 1987) can have direct effects on spatial patterns of settlement. Field studies of barnacle settlement have demonstrated non-random responses to chemical cues (Strathmann et al. 1981; Bushek 1988; Raimondi 1988;

Johnson and Strathmann 1989), physical cues (Crisp and Barnes 1954; Wethey 1986; Chabot and Bourget 1988) or both (Hudon and Bourget 1983; Le Tournex and Bourget

1988; but see Strathmann et al. 1981).

After the fundamental concept of gregarious settlement of cypris larvae in response of proteinaceous "settlement factor" extracted from adults had been established (Knight- Jones 1953; Crisp and Meadows 1962), larval biologists studied barnacle settlement under controlled laboratory conditions from several perspectives. These included:

stimulation by purified extract, cyprid "footprints", bio- chemically fractionated barnacle odor and bacterial films

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( L a r m o n a n d G a b b o t t 1975; R i t t s c h o f 1985; Yule and W a l k e r 1985; M a k i et al. 1990); i n h i b i t i o n b y n a t u r a l p r o d - ucts as w e l l as b a c t e r i a l f i l m s ( S t a n d i n g et al. 1984; R i t t s - c h o f et al. 1985, 1 9 8 6 a ; M a k i et al. 1988, 1990); and s t r e n g t h o f t e m p o r a r y a d h e s i o n ( C r i s p 1955; W a l k e r and Yule 1984; Yule and W a l k e r 1985; E c k m a n et al. 1990). In our l a b o r a t o r y , w e h a v e b e e n a s s e s s i n g l a r v a l s e t t l e m e n t o f t h r e e c o n g e n e r i c b a r n a c l e s (Balanus eburneus, B. improv- isus, and B. subalbidus) d i s t r i b u t e d a l o n g an e s t u a r i n e gra- d i e n t in C h e s a p e a k e B a y b y e x a m i n i n g the c o m b i n e d effects o f a d u l t s e t t l e m e n t f a c t o r and s a l i n i t y (an e n v i r o n - m e n t a l v a r i a b l e that m i g h t a f f e c t s e t t l e m e n t ) . I o n i c s t i m u - l a t i o n has b e e n s h o w n to i n d u c e m e t a m o r p h o s i s in s o m e i n v e r t e b r a t e s , but not in b a r n a c l e s ( R i t t s c h o f et al. 1986 b).

In a r e c e n t s t u d y o f the i n t e r a c t i o n b e t w e e n s a l i n i t y and a d u l t c h e m i c a l cue in the m e s o h a l i n e b a r n a c l e , B. improv- isus, w e d e m o n s t r a t e d that c y p r i d s o f this s p e c i e s s e t t l e d in a s a l i n i t y r e g i m e c o r r e s p o n d i n g to the a d u l t f i e l d d i s t r i - b u t i o n ( D i n e e n and H i n e s 1992). W e also d e m o n s t r a t e d that o t h e r factors, such as s e t t l e m e n t f a c t o r f r o m c o n g e - neric s p e c i e s , and d e l a y o f m e t a m o r p h o s i s c o u l d i n f l u e n c e l a r v a l s e t t l e m e n t o f B . improvisus. Here, w e p r e s e n t results o f a p a r a l l e l l a b o r a t o r y s t u d y o f a n o t h e r e s t u a r i n e b a r n a - cle, B. subalbidus, w h i c h w e u n d e r t o o k to d e t e r m i n e the r e s p o n s e s o f l a r v a l s e t t l e m e n t to t h e s e f a c t o r s in c o r r e s p o n - d e n c e w i t h a d u l t d i s t r i b u t i o n in t h e f i e l d .

T h e i n t e r a c t i v e e f f e c t s o f e n v i r o n m e n t a l cues u p o n l a r - val s e t t l e m e n t m a y b e p a r t i c u l a r l y critical in l o w s a l i n i t y z o n e s o f e s t u a r i e s , w h e r e the cues m a y be d i s t r i b u t e d a l o n g steep h o r i z o n t a l a n d v e r t i c a l g r a d i e n t s , c o n d i t i o n s f l u c t u - ate r a p i d l y , and p r e v a i l i n g s u r f a c e currents are l i k e l y to c a r r y l a r v a e a w a y f r o m s u i t a b l e habitats. T h e r e are few e x - p e r i m e n t a l s t u d i e s o f the l a r v a l b i o l o g y o f i n v e r t e b a t e s w h i c h are r e s t r i c t e d to the l o w s a l i n i t y z o n e s o f e s t u a r i e s (but see B o u s f i e l d 1955; C a i n 1972; H o p k i n s et al. 1973;

C r o n i n 1982). Balanus subalbidus, a m e m b e r o f the Bala- nus-amphitrite c o m p l e x , was p r e v i o u s l y r e f e r r e d to as BaI- anus amphitrite pallidus until d e s i g n a t e d a n e w s p e c i e s ( H e n r y 1973). T h i s b a r n a c l e is c h a r a c t e r i s t i c a l l y f o u n d in o l i g o h a l i n e w a t e r s o f the e a s t e r n c o a s t a l U n i t e d S t a t e s f r o m M a s s a c h u s e t t s to F l o r i d a , the G u l f o f M e x i c o , and has a l s o b e e n r e p o r t e d f r o m T r i n i d a d ( H e n r y and M c l a u g h l i n 1975) and t o l e r a t e s p r o l o n g e d e x p o s u r e to fresh w a t e r ( P o i r r i e r a n d P a r t r i d g e 1979; D i n e e n p e r s o n a l o b s e r v a t i o n ) . W i t h i n its g e o g r a p h i c r a n g e , B. subaIbidus is the o n l y b a r n a c l e o c - c u r r i n g at s a l i n i t i e s o f 0.1 to 3.5 ppt, and at sites o f i n c r e a s - ing s a l i n i t y the size and a b u n d a n c e o f B. subalbidus de- c r e a s e s ( P o i r r i e r a n d P a r t r i d g e 1979; D i n e e n p e r s o n a l o b - s e r v a t i o n ) . O n o y s t e r b e d s in C h e s e p e a k e Bay, B. subal- bidus was f o u n d at s a l i n i t i e s r a n g i n g f r o m 0.8 to 17.9 ppt, but w a s m o s t a b u n d a n t in l o w s a l i n i t y w a t e r s in u p p e r t r i b - u t a r i e s a n d u p p e r b a y l o c a t i o n s ( K e n n e d y a n d D i C o s i m o 1983). T h e l a t t e r two studies also b o t h i n d i c a t e that a d u l t s o f B. subalbidus, B. improvisus and B. eburneus are dis- t r i b u t e d s e q u e n t i a l l y a l o n g a h o r i z o n t a l g r a d i e n t o f i n c r e a s - ing salinity. O t h e r than m o r p h o l o g i c a l d e s c r i p t i o n s o f the n a u p l i a r s t a g e s o f B . subalbidus ( L a n g 1979), the l a r v a l bi- o l o g y o f this l o w - s a l i n i t y s p e c i e s has n o t b e e n s t u d i e d p r e - viously.

Materials and methods Adult barnacles

Adult Balanus subalbidus (Henry) were obtained from submerged tree branches in the Middle River (upper Chesapeake Bay), near Bal- timore, Maryland, and from settlement plates deployed in Muddy Creek, the primary fresh water tributary to the Rhode River, a sub- estuary of central Chesapeake Bay, in Edgewater, Maryland. The lat- ter site has a seasonal salinity range of 0 to 14 ppt, and B. subalbid- us was the only barnacle to settle on plates deployed at this location from 1989 to 1991. If not ovigerous at the time of sampling, adult barnacles 'were induced to breed in the laboratory by supplemental food [Artemia sp. and phytoplankton, Isochrysis galbana (Tahitian strain)] and a warm temperature (25 ~ for approximately 4 to 6 wk, thereby providing larvae out of the normal breeding season.

Experiments commenced in the spring of 1989 and continued through the spring of 1992. B. subalbidus was induced to breed aseasonally in the laboratory on three occasions: February 1990, early April 1991, and March 1992. B. improvisus and B. eburneus, used in the prepar- ation of congeneric settlement factor, were obtained, respectively, from the Rhode River and the York River, Gloucester Point, Vir- ginia.

Larval cultures

Nauplii were either obtained directly by crushing adults that con- tained ripe egg masses, or by removing egg masses and incubating them in 0.1-gm filtered estuarine water (4 to 5 ppt) for a few days until hatching. Nauplii were placed in 4 liters of aerated~ 0.1-gm filtered estuarine water (4 to 5 ppt salinity) at 25 ~ and fed daily on a mixed algal diet of Isochrysis galbana (Tahitian strain) and Phae- odactylum tricronutum. Sodium Penicillin G (21.9 mg 1 -l) and Strep- tomycin sulphate (36.5 mg 1-1) were used as antibiotics. Cyprids de- veloped in 5 to 7 d and were collected on a 200-gm mesh nylon sieve.

Settlement experiments Settlement factor

Conspecific (BaIanus subalbidus) and congeneric (B. improvisus and B. eburneus) settlement factors were prepared by crushing whole adult barnacles in distilled water, removing solids by centrifugation, and boiling the resulting supernate. The crude extract was applied to slate substrata at a concentration of 50 gg protein m1-1 (determined using the BCA TM Protein Assay, Pierce, Inc.) (Rittschof et al. 1984;

Dineen and Hines 1992).

Settlement assay procedures

Depending on the assay, 100 or 200 cyprids were placed in polysty- rene dishes (4 cm deep, 10 cm diameter) containing 0.1-gm filtered estuarine water at a salinity similar to the larval culture (4 to 5 ppt).

Slate substrata (3x6.5 cm) were held in coarsely filtered estuarine water (4 to 5 ppt) for 24 h prior to an experiment and were either adsorbed with settlement factor or untreated, and placed in dishes prior to introducing larvae. All experiments were conducted at 24 to 25 ~ on a 15 h light:9 h dark schedule. Substrata were examined for metamorphosed cyprids at 24-h intervals for 144 h. Three sets of experiments were conducted to assess: (1) the interactive effects of salinity and adult chemical cue; (2) the taxonomic specificity of adult chemical cue; and (3) the effects of delaying metamorphosis.

Salinity and adult chemical cue

After larvae were placed into settlement assay dishes, salinity was adjusted sequentially, over a period of 6 h, with either distilled water or a saline solution (40 ppt) to produce eight salinities (2, 5, 10, 15, 20, 25, 30, and 35 ppt) in combination with and without settlement factor for a total of 16 treatments. (Although this is a rapid adjustment for those larvae going through the extremes of the salinity range, it is an ecologically relevant adjustment for estuarine larvae which may migrate vertically through a pycnocline over the time course of a tidal cycle during spring when low surface salinities over-

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lay a salinity wedge on the bottom.) No settlement or mortality occurred during the salinity adjustment period, and cyprid behavior was normal. Final volume in all settlement culture dishes was approximately 300 ml, and the experimental trials began after the adjustment period. This experiment was conducted on four different batches of larvae. For two of these batches, cyprids were partitioned into two replicate aliquots. To test for variation in settlement within a batch of larvae, we compared effects of salinity and settlement factor treatments among aliquots of cyprids. Each aliquot was then partitioned into the 16 treatments described above, and the frequencies of settled barnacles after 144 h were compared using log-linear models (see below) comparing main effects of replicate and salinity-settlement factor treatment, as well as interaction effects. To test for differences in settlement among batches of larvae, we compared effects of salinity and settlement factor treatments among batches of cyprids. For each of the four separately cultured batches of larvae (including the two batches cultured for the test of replicated aliquots above), we partitioned the larvae into the 16 treatments. As before, frequencies of settled barnacles after 144 h were compared using log- linear models comparing main effects of batch and treatment, as well as interaction effects. Each of the 96 trials (i.e., 16 treatments times six batches/aliquots) received 200 cyprids.

Congeneric conspecific chemical cue

Slate substrata adsorbed with settlement factor extracted from Bal- anus subalbidus, B. improvisus, or B. eburneus, or untreated were placed in assay dishes as above at a salinity of 5 ppt. This resulted in four treatments which were replicated three times, 100 cyprids per trial.

Metamorphic delay

Balanus subalbidus cyprids from the same larval cohort were divid- ed into two groups, one of which was used in settlement assays the following day, while the other was induced to delay metamorphosis by placing larvae in 450 ml of filter-sterilized 5-ppt river water, slow- ly cooling to 5 ~ and storing in the dark for 8 d. Prior to the experiment, larvae were gradually warmed to room temperature over a 2- h period. Settlement assays were then conducted as above, at four salinities (5, 15, 25, and 35 ppt) with substrata adsorbed with conspecific settlement factor, replicated three times, 100 cyprids per trial.

Statistical methods

Data were treated categorically. Logistic regression analysis (PROC CATMOD with maximum likelihood estimation, SAS Institute 1985) was used to analyze settlement frequencies in experiments investi- gating: (1) the effects of salinity, the presence and absence of conspecific settlement factor and their interaction; as well as (2) the effects of salinity, metamorphic delay and their interaction. Settlement frequencies in the presence of conspecific and congeneric settlement factor were compared using two-way contingency tables. Unplanned multiple comparisons controlling for experiment-wise Type I error were subsequently used to distinguish differences among frequencies [Simultaneous Test Procedures (STP), Sokal and Rohlf 1981, p 728].

Results

T h e t i m e course of cypris s e t t l e m e n t f o l l o w e d a s i m i l a r pattern in all of the e x p e r i m e n t a l trials (Fig. 1). Little or no s e t t l e m e n t occurred in the first 24 h; about 50 per cent o f m a x i m u m s e t t l e m e n t occurred w i t h i n 72 h (slightly slower in the a b s e n c e of s e t t l e m e n t factor); an a s y m p t o t i c level of m a x i m a l s e t t l e m e n t was approached at 120 h; a n d no further s e t t l e m e n t occurred after 144 h. M a x i m u m s e t -

200

"~ 150

o

~ 50 2;

0 0

200

"~. 150-

o

~ ~ -

o

~ 5 0 - Z

0 0

A. S e t t l e m e n t F a c t o r P r e s e n t

T T

1 /

T I I I I 1

24 48 72 96 120 144

Time (h)

B, S e t t l e m e n t F a c t o r A b s e n t - - . - n - - 2, ppt

9 15 ppt

T T

24 48 72 96 120 144

T i m e ( h )

Fig. 1 Balanus subalbidus. Time course of cypris settlement in the A presence and B absence of settlement factor at 2 (--o-) and 15 (--I-) ppt. Values plotted are means (_+SE) or four batches of larvae, 200 cyprids per trial. Values at 144 h are extracted from data pre- sented in Fig. 2 C

t l e m e n t levels r a n g e d from 9 5 + 2 % in the c o n g e n e r i c extract e x p e r i m e n t , to 87+10% in the o p t i m a l t r e a t m e n t of the s a l i n i t y x s e t t l e m e n t factor e x p e r i m e n t , and to 4 1 + 5 % in the m e t a m o r p h i c delay e x p e r i m e n t (see below). W h i l e m a x i m u m s e t t l e m e n t levels also varied s i g n i f i c a n t l y a m o n g t r e a t m e n t s w i t h i n e x p e r i m e n t s (Fig. 1 a n d see below), this pattern of a s y m p t o t i c s e t t l e m e n t at 144 h was observed for all t r e a t m e n t s in all three e x p e r i m e n t s .

Mortality of n o n - m e t a m o r p h o s e d cyprids was low in all e x p e r i m e n t s . T y p i c a l l y n o t m o r e than 10 per cent mortality of cyprids occurred after 144 h in each e x p e r i m e n t , e v e n those in which m a x i m a l s e t t l e m e n t was relatively low.

M o r t a l i t y in the s a l i n i t y x s e t t l e m e n t factor e x p e r i m e n t ws higher at the h i g h e s t salinity levels than the o p t i m u m c o n - ditions, b u t v a r i a b i l i t y in c o m b i n a t i o n with low values does not allow us to draw c o n c l u s i o n s . L a r v a l mortality in the other two e x p e r i m e n t s did not exhibit a n y n o t a b l e pattern a m o n g treatments. Thus, the m a j o r i t y o f n o n - s e t t l e d l a r v a e were alive a n d s w i m m i n g at the 144-h o b s e r v a t i o n . A c -

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426 200

1 5 0 ~ 1 ** 13 ]

ooi ii ^o,, ^/

500 ed d b E be bc F e

" ~ 200

"~ 150 ~D r~ O

100

50

Z d 0 2no

2 5 10 15 20 25 30 35 Experimental Salinity (ppt)

Fig. 2 Balanus subalbidus. A and B cypris settlement of two different batches of larvae and C average of four batches after 144 h in the presence (SF) and absence (NSF) of conspecific settlement factor across an array of salinities. Identical letters above bars indicate settlement frequencies that did not differ significantly (upper case letters for slate substrata treated with settlement factor, lower case letters for untreated substrata). Significant differences in settlement frequencies between settlement factor treatments within each salinity level designated as (**), p<0.01. Bars indicate number of settled barnacles out of 200 cyprids per trial

cordingly, we report experimental results as number of cypris larvae which have settled, i.e., permanently attached and metamorphosed to juvenile barnacles.

When Balanus subalbidus cyprids were assayed for settlement in the presence and absence of settlement factor across an array of salinities, settlement frequencies did not differ significantly between replicate aliquots within

batches of cyprids (log-linear model, 22=2.8, 15 df, p>0.09), nor were there significant interaction effects of replicatextreatment (22=0.02, 1 df, p>0.8). Settlement frequencies did differ significantly among the four batches of larvae (22=319.3, 3 df, p<0.001), and there were significant interaction effects of batchxtreatment (2'2=207.0,

15 df, p<0.001). However, when we examined settlement in the presence and absence of settlement factor across the array of salinities, we observed similar patterns among batches (e.g. Fig. 2 A, B). For each batch of larvae, salinity and settlement factor had significant interactive effects upon settlement frequencies (22>15, 7 df, p<0.05), such that settlement frequncies in the presence of settlement factor were highest at low salinities (2 to 10 ppt), while in the absence of settlement factor, frequencies were highest at mid salinities (10 to 20 ppt). The significant settlement differences among batches of larvae were explained by detailed differences in settlement frequencies between test salinities but not in basic pattern across the array of salinities (Fig. 2 A, B). Thus, we averaged settlement frequencies across all four batches (Fig. 2 C). In parallel with the analyses of each individual batch, settlement frequencies of the combined batches showed significant interactive effects of salinity and settlement factor (log-linear model, 22=485.5, 7 df, p<0.001).

Combined settlement frequencies differed significantly amon~ salinities for substrata treated with settlement factor (Z~=2901.0, 7 df, p<0.001) and for substrata without treatment factor (22=1052.0, 7 df, p<0.001). Settlement frequencies in the presence of settlement factor were highest at 2 ppt (unplanned multiple comparisons, STP Tests;

22=18.5, 7 df, p<0.01) and decreased with increasing salinities. Settlement frequencies in the absence of settlement factor were significantly higher at mid-salinities (10 and 15 ppt), and settlement frequencies did not differ significantly among salinities of 2, 5 and 20 ppt in the absence of settlement factor (unplanned multiple comparisons, STP Tests; 22=18.5, 7 df, p<0.01). When settlement was compared within each salinity level, significantly higher settlement frequencies occurred in the presence of settlement factor at salinities of 2, 5, 10, 15 and 25 ppt (pairwise G- tests; p<0.001, experiment-wise error rate=0.01, 7,1 df, 22=10.2). No significant differences in settlement frequencies occurred at salinities of 20, 30 or 35 ppt with and without settlement factor.

Source of settlement factor (conspecific as opposed to congeneric) significantly influenced settlement frequency at 5 ppt salinity (22=420.9, 3 df, p<0.001) (Fig. 3). Settle- ment frequencies of Balanus subalbidus were highest in the presence of conspecific settlement factor, followed in decreasing order by settlement factor extracted from B. im- provisus and B. eburneus. No significant difference in settlement frequency was seen between slate substrata untreated or adsorbed with settlement factor extracted from B. eburneus (unplanned multiple comparisons, STP tests;

22=9.2, 2 dr, p<0.01).

Settlement frequencies of Balanus subalbidus cryprids differed significantly among salinities for both non-delayed cyprids (22=176.0, 3 df, p<0.001) and those which

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427

100

80

20 d Z 0

A

B

B. subalbidus B. i n ~rovisus B. eburneus Control Source of adult extract

Fig. 3 Balanus subalbidus. Cypris settlement after 144 h in the presence of conspecific and congeneric settlement factors adsorbed to slate substrata at an experimental salinity of 5 ppt. Significant differences in settlement frequency among treatments designated by different letters, p<0.01. Bars indicate means (+SE) of three replicate trials with 100 cyprids per trial

45

4o]

3sl

3 0 ,lz

p~ 20

. 10 Z o

5 0

A ~

A

B ~

Cyprid Treatment 9 untreated [ ] "delayed"

5 15 25 35

Experimental Salinity (ppt)

Fig. 4 Balanus subalbidus. Cypris settlement after 144 h of non- delayed cyprids (untreated) and those induced to delay ("delayed") metamorphosis by maintenance at 5 ~ for 8 d, in the presence of conspecific settlement factor at various salinities. Identical letters above bars indicate settlement frequencies that were not significantly different (upper case letters for newly metamorphosed cyprids and lower case letters for cyprids induced to postpone metamorphosis).

Significant difference in settlement frequencies between settlement factor treatments with in each salinity level designated as (**), p<0.01. Bars indicate means of three replicate trials with 100 cyprids per trial

were induced to postpone metamorphosis (;(2=104.3, 3 df, p<0.O01), but there were no significant interactive effects (2"2=4.5, 3 df, p>0.21) (Fig. 4). More untreated cybrids settled than did those which were metamorphically delayed (20.8 compared to 11.8%) when averaged over the salinity treatments. Cyprids settled in greater abundance at 5 and 15 ppt salinity regardless of treatment. In pairwise comparisons, settlement levels of non-delayed cyprids were significantly higher than "delayed cyprids" at 5 and 25 ppt, but not at 15 and 35 ppt salinity (pairwise G-tests;

p<0.01; experiment-wise error rate=0.05, 3.1 df, Z2=8.6) (Fig. 4).

Discussion

Settlement across salinities

The pattern ofBalanus subalbidus settlement across a wide range of salinities in the laboratory contrasts with the nar- rower salinity distribution of adults in the field. In Chesa- peake Bay and other estuaries, B. subalbidus recruits abundantly at salinities of 0.5 to 10 ppt and occurs rarely up to 18 ppt, but has never been documented at higher salinities (Poirrier and Partridge 1979; Kennedy and DiCosimo 1983; Dineen personal observation). When laboratory- reared cyprids were tested in an array of salinities in the presence of conspecific settlement factor, peak settlement occurred at 2 ppt (87+8.9% after 144 h), but substantial settlement occurred over a broad range of salinities up to 25 ppt (48+11% after 144 h) (Fig. 2). In addition, cyprids of B. subalbidus settled abundantly in the absence of settlement factor in a range of mid-salinities, with a peak at 15 ppt (57_+9.9% after 144 h). Differences in settlement between test salinities obtained for the four larval batches in the present study could be due to subtle differences in larval culturing conditions (Holm 1990) and/or due to variation in environmental conditions during embryogenesis (Dineen and Hines 1992). Nevertheless, all four batches of our test larvae consistently showed a broad, relatively in- discriminate pattern of settlement in the laboratoy. In contrast, laboratory settlement of B. improvisus cyprids ex- posed to a similar array of salinities with conspecific settlement factor coincided with its mesohaline adult distribution, and settlement in the absence of settlement factor was low and showed no discernable pattern relative to experimental salinity (Dineen and Hines 1992). Similarly, laboratory settlement of a third estuarine barnacle, B. ebur- neus, coincided with its polyhaline adult distribution in the presence of conspecific settlement factor (Dineen and Hines in press). Thus, in comparison with congeners, B. subalbidus settlement appears less dependent on salinity.

Larval retention in the upper estuary could markedly limit adult distribution independent of specific cues for settlement but spatial distributions of Balanus subalbidus lar- vae in an estuary have never been documented. Although larvae of B. improvisus exhibited a progression of devel- opmental stages while entrained within the Miramichi Es- tuary, with early stages carried away from, and advanced stages eventually returning to parental stocks (Bousfield 1955), other larval species do exhibit distinct retention in upper zones of estuaries. For exampe, larvae of the xan- thid mud crab, Rhithropanopeus harrisii, are retained in low to medium salinity ranges of the upper Newport River Estuary (Cronin 1982), and veligers of the low salinity bi- valve, Rangia cuneata, also appear to be restricted to low salinities where adults occur in estuaries (Cain 1972; Hop- kins et al. 1973).

Despite many studies on the induction of metamorphosis in larvae, few have tested the interactive effects of two or more factors on settlement (Pawlik 1992). In variable

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environments like estuaries, the interaction of factors might serve as a more reliable cue for good settlement habitat than any single factor. Previous studies with Balanus improvisus indicate that this could be the case because larvae were stimulated to settle at a salinity regime indicative of adult distribution only in the presence of conspecific settlement factor. In contrast, present results with B. subalbidus suggesting relative independence from settlement cues tested, both in a qualitative (Fig. 2) and temporal (Fig. 1) sense, could indicate that even interactive cues may not be sufficient to explain the distribution of this species in the highly fluctuating oligohaline zone. Lar- val retention in this zone, on the other hand, might be more likely to produce effective habitat selection than interactive cues. In addition, if larvae ofB. subalbidus are not dis- persing far from adult populations in low saline waters, the adaptive value of utilizing conspecific settlement factor as a settlement cue would not be as critical as it would be for species like B. improvisus with larvae that are initially carried away from parental stocks.

Post-settlement osmotic/ionic stress (possibly resulting in mortality or slower growth rates of newly metamorphosed juveniles) was not investigated in our study and could potentially be a factor in influencing adult salinity distribution of Balanus subalbidus. However, when adult B. subalbidus were transplanted to mesohaline regions, these barnacles survived over a year and reproduced (Di- neen unpublished data). Alternatively, it could be argued that post-settlement mortality from predation and compe- tition could be influencing horizontal distribution of estuarine barnacles. However, in Chesapeake Bay, flatworms and bryozoans, the chief predators and competitors of barnacles in this estuarine system (at least in mesohaline regions) (Branscomb 1976), are unlikely to distinguish pref- erentially among congeneric barnacles thereby eliminat- ing B. subalbidus while allowing B. improvisus to persist at mesohaline salinities.

Conspecific versus congeneric settlement factor

The present study as well as others have shown that stimulation of gregarious cypris settlement is, not surprisingly, greatest in the presence of conspecific settlement factor compared to settlement factor from congeners or more dis- tantly related species (Knight-Jones 1953, 1955; Crisp 1990; Dineen and Hines 1992). In some cases, a correla- tion between degree of settlement stimulation and phylo- genetic affinity of the cue species and receptor species has been implied (Knight-Jones 1955). This relationship has been demonstrated at the family level (Knight-Jones t955, Crisp 1990) but is probably not a good indicator of affinity at or below the generic level. For instance, cyprids of Balanus amphitrite settled in higher numbers in the presence of Conopea galeatus as opposed to B. eburneus settlement factors with equivalent protein concentration (Crisp 1990). Furthermore, we recently demonstrated that B. improvisus settlement is greater in the presence of settlement factor derived from B. subalbidus than from

B. eburneus (Dineen and Hines 1992), although B. improv- isus is more closely allied to B. eburneus within the Bala- nus amphitrite-complex (Henry and McLaughlin 1975).

Similary, the present study shows greater stimulation of B. subalbidus settlement in the presence of B. improvisus settlement factor (Fig. 3), even though B. subalbidus is more closely allied to B. eburnes (Henry and McLaughlin 1975). It is not known whether the differential response of B. subalbidus cyprids to congeneric extracts could have ecological implications in the field. However, it is inter- esting to note that B. subalbidus was stimulated to settle more in the presence of extract obtained from the congener with which it would more likely co-occur in the field (B. improvisus).

Settlement of metamorphically delayed larvae

Delayed metamorphosis of larvae in the absence of appropriate cues or habitat has been documented in several marine invertebrate groups, including barnacles (review by Pechenik 1990). Barnacle larvae retained within an estuary probably often encounter inappropriate habitat. As a consequence of delay, larvae sometimes settle less discrim- inatingly (Rittschof et al. 1984; Crisp 1988). For example, there were fewer differences in settlement in different test salinities when cyprids of Balanus improvisus were induced to postpone metamorphosis by keeping them at low temperature (Dineen and Hines 1992). However, when delayed metamorphosis was imposed on B. subalbidus cyprids by lowering temperature in the present study, subse- quent settlement did not differ among test salinities relative to the untreated group (in the presence of conspecific settlement factor). Although overall settlement levels were lower for metamorphically delayed cyprids, patterns of settlement as a function of salinity were similar for both delayed and non-delayed larvae. This similarity again could reflect relatively low dependency on salinity as a cue for metamorphosis of B. subalbidus cyprids. Because delaying metamorphosis appears detrimental for metamorphic completion without apparently increasing the likelihood of settlement at "appropriate" salinities, postponing metamorphosis may not be a good strategy for B. subalbidus cyprids.

The causes and effects of metamorphic delay are complex (Pawlik 1992; Pechenik et al. 1993). Although delaying metamorphosis by lowering temperature probably does not produce an effect of aging larvae but rather suspends development, we used this method for two reasons. First, temperature is an ecologically relevant factor for delaying development and settlement in the field. At our Chesapeake study site, larvae hatch in late April to early May, when salinities are low and variable, and when water temperatures are warming rapidly from ca. 8 ~ Settlement at our col- lecting site typically begins in early May (14 ~ and peaks in mid to late May (22 ~ In years when cold persisted longer than normal (e.g. 1992), water temperature was only 10 ~ in early May and settlement occurred at least 2 wk later than usual. Although we are not sure whether hatch-

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i n g was late, w h e t h e r larval d e v e l o p m e n t was slowed, or both, "early" larvae will often e n c o u n t e r t e m p e r a t u r e s l o w e r than 14 ~ W h i l e no s e t t l e m e n t occurs in the laboratory or field at t e m p e r a t u r e s as cold as 5 ~ B a l a n u s sub- albidus larvae along the n o r t h e r n p o r t i o n of its geographic r a n g e ( e x t e n d i n g to Massachusetts) are likely to e n c o u n - ter large v a r i a t i o n in t e m p e r a t u r e s d u r i n g the critical spring period o f d e v e l o p m e n t . Second, this low t e m p e r a t u r e treat- m e n t provides an i n t e r e s t i n g c o m p a r i s o n with e x p e r i m e n - tal studies u s i n g the same t e c h n i q u e for other species (Rittschof et al. 1984, 1 9 8 6 b ; M a k i et al. 1988; Crisp 1990;

M a k i et al. 1990; D i n e e n a n d H i n e s 1992; P e c h e n i k et al.

1993). T h e s e studies d e m o n s t r a t e that low t e m p e r a t u r e does n o t appear to have a n y c o n f o u n d i n g deleterious effects o n the larvae that m a n y others factors do (e.g. star- v a t i o n for some types of larvae). Moreover, P e c h e n i k et al.

(1993) s h o w e d that three e x p e r i m e n t a l t e c h n i q u e s o f de- l a y i n g m e t a m o r p h o s i s in B a l a n u s amphitrite (cold temperature, s i l a n i z e d surfaces, a n d o v e r - c r o w d i n g ) p r o d u c e d set- t l e m e n t results that did n o t differ s i g n i f i c a n t l y from each other, b u t all p r o d u c e d s i m i l a r differences from " n o n - d e - layed" larvae.

I n c o n c l u s i o n , the p r e s e n t study has tested the interactive effects of two s e t t l e m e n t cues for larvae. Cyprids of B a l a n u s subalbidus are c a p a b l e of settling, with and without adult s e t t l e m e n t factor, in m e s o - and p o l y h a l i n e salinities where adults rarely, if ever, occur in the estuary. O u r results suggest that p r e - s e t t l e m e n t behavior, l e a d i n g to larval r e t e n t i o n in o l i g o h a l i n e head waters, m a y be the u n d e r - l y i n g m e c h a n i s m that u l t i m a t e l y accounts for the adult dis- t r i b u t i o n of B. subalbidus.

Acknowledgements We thank D. Rittschof and A. Schmidt for larval culturing advice, L. Weichert, M. Haddon, G. Gore and D. Palm- er for field and/or laboratory assistance, and D. Smith for statistical help. Helpful comments by W. Jaeckle, and two anonymous review- ers improved the manuscript.

References

Bousfield EL (1955) Ecological control of the occurrence of barnacles in the Miramichi estuary. Bull natn Mus Can 137:1-69 Branscomb ES (1976) Proximate causes of mortality determining the

distribution and abundance of the barnacle Balanus improvisus Darwin in Chesapeake Bay. Chesapeake Sci 17:281-288 Bushek D (1988) Settlement as a major determinant of intertidal oys-

ter and barnacle distributions along a horizontal gradient. J exp mar Biol Ecol 122:1 - 18

Caffey HM (1985) Spatial and temporal variation in settlement and recruitment of intertidal barnacles. Ecol Monogr 55:313 - 332 Cain TD (1972) The reproductive cycle and larval tolerances of Ran-

gia cuneata in the James River, Virginia. Ph D dissertation, Univ.

Virginia, Charlottesville

Chabot R, Bourget E (1988) Influence of substratum heterogeneity and settled barnacle density on the settlement of cypris larvae.

Mar Biol 97:45-56

Connell JH (1985) The consequences of variation in initial settlement vs. post-settlement mortality in rocky intertidal commu- nities. J exp mar Biol Ecol 93:11-45

Crisp DJ (1955) The behavior of barnacle cyprids in relation to water movement over a surface. J exp Biol 32:569-590

Crisp DJ (1988) Reduced discrimination of laboratory reared cyprids of the barnacle, Balanus amphitrite amphitrite Darwin, Crusta-

cea, Cirripedia, with a description of a common abnormality. In:

Thompson, MF, Serojini, R, Nagebhushanam R (eds) Marine bi- odegradation. Oxford and I.B.H. Publ Co Ltd./New Delhi, pp 409 -432

Crisp DJ (1990) Gregariousness and systematic affinity in some North Carolinian barnacles. Bull mar Sci 47:516-525

Crisp DJ, Barnes H (1954) The orientation and distribution of barnacles at settlement with particular reference to surface contour.

J Anim Ecol 23: 142-162

Crisp D J, Meadows PS (1962) The chemical basis of gregariousness in cirripedes. Proc R Soc Lond (Ser B) 156:500-520

Cronin TW (1982) Estuarine retention of larvae of the crab Rhithrop- anopeus harrisii. Estuar cstl Shelf Sci 15:207-220

Dineen JF Jr, Hines AH (1992) Interactive effects of salinity and adult extract upon settlement of the estuarine barnacle Balanus improvisus (Darwin, 1854). J exp mar Biol Ecol 156:239-252 Dineen JF Jr, Hines AH (in press) Larval settlement of the polyha-

line barnacle BaIanus eburneus (Gould): cue interactions and comparisons with two estuarine congeners. J exp mar Biol Ecol Eckman JE, Savidge WB, Gross TF (1990) Relationship between du- ration of cyprid attachment and drag forces associated with de- tachment of Balanus amphitrite cyprids. Mar Biol 107:111 - 118 Gaines S, Brown S, Roghgarden J (1985) Spatial variation in larval concentration as a cause of spatial variation in settlement for the barnacle, BaIanus glandula. Oecologia 67:267 - 272

Gaines SD, Roughgarden J (1987) Fish in offshore kelp forests af- fect recruitment to intertidal barnacle populations. Science, NY 235:479-481

Grosberg RK (1982) Intertidal zonation of barnacles: the influence of planktonic zonation of larvae on vertical distribution of adults.

Ecology 63:894-899

Henry DP (1973) Descriptions of four new species of the Balanus amphitrite-complex (Cirripedia: Thoracica). Bull mar Sci 23:

964-1001

Henry DR Mclaughlin PA (1975) The barnacles of the Balanus am- phitrite complex (Cirripedia: Thoracica). Zool Verh, Leiden 141:

1-254

Holm ER (1990) Attachment behavior in the barnacle Balanus am- phitrite amphitrite (Darwin): genetic and environmental effects.

J exp mar Biol Ecol 135:85-98

Hopkins SH, Anderson JW, Horvath K (1973) The brackish water clam Rangia cuneata as indicator of ecological effects of salinity changes in coastal waters. US Army Engineer Waterways Ex- periment Station contract report H-73-1, Vicksburg, Mississippi Hudon C, Bourget E (1983) An integrated study of factors influencing the choice of the settling site of Balanus erenatus cyprid larvae. Can J Fish aquat. Science 40:1186-1194

Johnson LE, Strathmann RR (1989) Settling barnacle larvae avoid substrata previously occupied by a mobile predator. J exp mar Biol Ecol 128:87 - 103

Kennedy VS, DiCosimo J (1983) Subtidal distribution of barnacles (Cirripedia: Blanidae) in Chesapeake Bay, Maryland. Estuaries 6: 95-101

Keough M J, Downes BJ (1982) Recruitment of marine invertebrates:

the role of active larval choices and early mortality. Oecologia 54:348 - 352

Knight-Jones EW (1953) Laboratory experiments on gregariousness during settling in Balanus balanoides and other barnacles. J exp Biol 30:584-598

Knight-Jones EW (1955) The gregarious setting reaction of barnacles as a measure of systematic affinity. Nature, Lond 174:266 Lang WH (1979) Larval development of shallow water barnacles of

the Carolinas (Cirripedia: Thoracica) with keys to naupliar stages. NOAA tech Rep US Dep Commerce NMFS Circular 421:

1 - 3 9

Larman VN, Gabbott PA (1975) Settlement of cyprid larvae of Bal- anus balanoids and Elminius modestus indcued by extracts of adult barnacles and other marine animals. J mar biol Ass UK 55:

183 - 190

LeTourneux E Bourget E (1988) Importance of physical and biolog- ical settlement cues used at different spatial scales by the larvae of Semibalanus balanoides. Mar Biol 97:57-66

(8)

Maki JS, Rittschof D, Costlow JD, Mittchell R (1988) Inhibition of attachment of larval barnacles, Balanus amphitrite, by bacterial surface films. Mar Biol 97:199-206

Maki JS, Rittschof D, Samuelsson M-O, Szewzyk U, Yule AB, Kjelleberg S, Costlow JD, Mitchell R (1990) Effect of marine bacteria and their exopolymers on the attachment of barnacle cypris larvae. Bull mar Sci 46:499-511

Minchinton TE, Scheibling RE (1991) The influence of larval supply and settlement on the population structure of barnacles. Ecol- ogy 72: 1867-1879

Pawlik JR (1992) Chemical ecology of the settlement of benthic marine invertebrates. Oceanog mar Biol A Rev 30:273-335 Pechenik JA (1990) Delayed metamorphosis by larvae of benthic

marine invertebrates: Does it occur? Is there a price to pay? Ophe- lia 32:63 - 9 4

Pechenik JA, Rittschof D, Schmidt AR (1993) Influence of delayed metamorphosis on survival and growth of juvenile barnacles Bal- anus amphitrite. Mar Biol 115:287-294

Poirrier MA, Partridge MR (1979) The barnacle, Balanus subalbid- us, as a salinity bioindicator in the oligohaline estuarine zone.

Estuaries 2: 204- 206

Raimondi PT (1988) Settlement cues and determination of the vertical limit of an intertidal barnacle. Ecology 69:400-407 Rittschof D (1985) Oyster drills and the frontiers of chemical ecol-

ogy: unsettling ideas. Bull Am malac Union (special edn) 1:

111-116

Rittschof D, Branscomb ES, Costlow JD (1984) Settlement and behavior in relation to flow and surface in larval barnacles, Bala- nus amphitrite Darwin. J exp mar Biol Ecol 82:131 - 146 Rittschof D, Hooper IR, Branscomb ES, Costlow JD (1985) Inhibi-

tion of barnacle settlement and behavior by natural products from whip corals, Leptogorgia virgulata (Lamarck, 1815). J chem Ec- ol 11:551-563

Rittschof D, Hooper IR, Costlow JD (1986 a) Barnacle settlement in- hibitors from sea pansies, RenilIa reniformis. Bull mar Sci 39:

376-382

Rittschof D, Maki J, Mitchell R, Costlow JD (1986b) Ion and neu- ropharmological studies of barnacle settlement. Neth J Sea Res 20:269-275

Roughgarden J, Gaines S, Possingham H (1988) Recruitment dy- namics in complex life cycles. Science, NY 241: 1460-1466 Roughgarden J, Iwasa Y, Baxter C (1985) Demographic theory for

an open marine population with space-limited recruitment. Ecol- ogy 66:54-67

SAS Institute, Inc (1985) SAS user's guide. Version 5. SAS Insti- tute, Inc., Cary, North Carolina

Shanks AL (1986) Tidal periodicity in the daily settlement of intertidal barnacle larvae and an hypothesized mechanism for the cross-shelf transport of cyprids. Biol Bull mar biol Lab, Woods Hole 170:429 - 4 4 0

Sokal RR, Rohlf FJ (1981) Biometry, 2nd edn. W. H. Freeman and Co., New York

Standing JD, Hooper IR, Costlow JD (1984) Inhibition and induction of barnacle settlement by natural products present in octoc- orals. J chem Ecol 10:823-834

Strathmann RR, Branscomb ES, Vedder K (1981) Fatal errors in set as a cost of dispersal and the influence of intertidal flora on set of barnacles. Oecologia 48:13 - 1 8

Underwood A J, Fairweather PG (1989) Supple-side ecology and benthic marine assemblages. Trends Ecol Evol 4: 16-19

Walker G, Yule AB (1984) Temporary adhesion of the barnacle cyprid: the existence of an antennular adhesive secretion. J mar biol Ass UK 64:679-686

Wethey DS (1986) Ranking of settlement cues by barnacle larvae:

influence of surface contour. Bull mar Sci 39:393-400 Yule AB, Walker G (I985) Settlement ofBaIanus balanoides: the ef-

fect of cyprid antennular secretion. J mar biol Ass UK 65:

707-712

Effects of salinity and adult extract on settlement of the oligohaline barnacle Balanus subalbidus

Effects of salinity and adult extract on settlement of the oligohaline barnacle Balanus subalbidus

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