Settling barnacle larvae avoid substrata previously occupied by a mobile predator

J. Exp. Mar. Biol. Ecol., 1989, Vol. 128, pp. 87-103

87

Elsevier

JEMBE 01250

Settling barnacle larvae avoid substrata previously occupied by a mobile predator Ladd E. Johnson

and Richard

R. Strathmann

Department of Zoology NJ-IS, University of Washington, Seattle, Washington, U.S.A.; Frida)j Harbor Laboratories, University of Washington, Friday Harbor, Washington, U.S.A.

(Received 12 May 1988; revision received 15 February 1989; accepted 2 March 1989) Abstract: Settlement by the larvae of the barnacle 3a~un~ glandula (Darwin) in the field is decreased on slate tiles that have been previously occupied by a predator of postmetamorphic stages, the whelk Nucella lamellosa (Gmelin). Settlement by another barnacle species, Semibalanus curiosus (Pallas), was not as greatly influenced by the same treatment. The effect on B. glandula also occurs when the substratum is treated with

traces of the whelk by either forcing the foot over the plate or rubbing the plate with crushed individuals. This reduction in settlement can persist up to 3 wk after treatment of the substratum. A similar but lesser effect occurs after treatments with traces of a potential biological disturber of settling larvae, the limpet Tecrura se&urn (Rathke), but also occurs with mucus from the brown alga Focus djstich~ (L.), which is not a demonstrated hazard to the barnacles. Mucus is naturally released by all of the above organisms and may be the cause of the reduced settlement. Control manipulations using a nonmucus-producing animal tissue, the pectoral muscle of the chicken Gallus gallus (L.), produced only a slight decrease in barnacle settlement, Mucus could reduce settlement either by acting as a cue for avoidance of high-risk areas, altering other settlement cues, or physically inhibiting attachment. However, not all mucus inhibits settlement: another investigator demonstrated that prior occupation by another predatory whelk induced greater settlement by another barnacle species; and we found that mucus from the dorid nu~ibr~ch Archidoris montereyensis (Cooper) increased settlement by B. glandula. Thus, our results suggest that the mucus might possibly act as a cue to the larvae, and we conclude that traces of mobile animals can influence the settlement of sessile animals. Since the threat of whelks to barnacles is delayed until some time after settlement, we hypothesize that the barnacle may be taking into account the long-term future risks (i.e., “the ghost of predation future”) when making the decision to settle. The avoidance response of the barnacle to the traces of limpets and macroalgae offers an alternative explanation (differential settlement) to patterns often attributed to biological disturbance (differenti~ ~stsettlement mortality). Key

words: Balanus glandula; Cue;

Larva; Nucella lamellosa; Predator avoidance; Settlement

INTRODUCTION

Many larvae of sessile invertebrates use environmental cues to assess potential settlement sites. While many studies have found responses to cues associated with favorable sites (e.g., Connell, 1961a; Scheltema, 1961; Wilson, 1968; Knight-Jones et al., 1971; Stebbing, 1972; Doyle, 1975; Strathmann et al., 1981; Highsmith, 1982; Had~eld & Scheuer, 1985; R~mondi, 1988), only a few have examined potential cues Correspondence address: L.E. Johnson, Biological Sciences Department, Stanford University, Hopkins Marine Station, Pacific Grove, CA 93950, U.S.A. 0022-0981/89/$03.50 D 1989 Elscvier Science Publishers B.V. (Biomedical Division)

88

L. E. JOHNSON AND R. R. STRATHMANN

used to avoid particular

habitats

1981; Woodin, sedentary

(Grosberg,

1985); all observed

species present

1981; Moyse & Hui, 1981; Young &, Chia,

avoidance

has been

at the time of settlement.

in response

In these situations,

to sessile

or

the stimulus

preventing settlement (the presence of the other organism) is probably highly correlated with future interactions. Risks involving interactions with motile organisms may be more difficult for larvae to assess detectable at the time of settlement is delayed until some later time.

since they may be poorly correlated (Strathmann

with any cue

et al., 1981), especially ifthe interaction

We examined the settlement response of two intertidal barnacle species, Balunus ,&mdula (Darwin) and Semibalanus cariosus (Pallas), to traces of a variety of organisms, some expected to be hazards and others not. These were the whelk Nucella lamellosa (Gmelin), the limpet Tectura scutum (Rathke), the brown alga Fucus distichus (L.), and the dorid nudibranch Archidoris montereyensis (Cooper). All of these organisms are capable of leaving mucus on the substratum; the gastropods use mucus for locomotion, and the alga releases mucus from its thallus. We presume that the traces left on the substratum after occupation by these organisms are either the mucus itself or some substances associated with the mucus. Henceforth, we use the term “mucus” generally to refer to these traces. The whelk is an important predator on the postmetamorphic stages of both barnacle species (Connell, 1970a; Dayton, 1971; Palmer, 1984) although S. cariosus can have a refuge in large size (Connell, 1970b, 1972; Dayton, 1971; Palmer, 1983). Adult whelks will eat recently settled barnacles when nothing else is available (Denley & Underwood, 1979; A. R. Palmer, pers. comm.) but they usually eat larger barnacles first when given a range of barnacle sizes from which to choose (Connell, 1961a, 1970a; Denley & Underwood, 1979; Palmer, 1984; but see Moran, 1985). Thus, adult whelks are probably a greater threat some time after settlement unless other prey are unavailable. Juvenile whelks may pose a greater threat to a recently settled barnacle but their ecology is poorly known. Typically they are difficult even to find (A. R. Palmer, pers. comm.) which may indicate low abundances but might also be due to their small size or use of cryptic microhabitats. Barnacle recruitment can be reduced in the presence of limpets (e.g., Lewis, 1954; Connell, 196 la; Dayton, 1971; Branch, 1975 ; Denley & Underwood, 1979). Although this relationship is attributed to biological disturbance by the limpets (i.e., “bulldozing”), this direct effect of limpets on barnacles has only recently been well documented (Miller, 1986). In that study, the mortality due to limpets decreased with age and was negligible after 4-5 wk. Therefore, limpets are mostly likely to be a more immediate short-term danger to barnacles after they settle. The potential effect of fucoid algae on barnacle settlement is less clear. Along Atlantic shores, the recruitment of Semibalanus balanoides (L.) can be reduced in the presence of Focus spp., presumably through the removal of settling barnacles by the action of the alga’s fronds whipping the substrata (Menge, 1976; Grant, 1977; Hawkins, 1983). However, in the northeastern Pacific, F. distichus has only been shown to have either

LARVAL

AVOIDANCE

OF A PREDATOR

89

a benficial or neutral effect on the recruitment of B. glun~uZ~(Dayton, 1971; Farrell, 1987). Thus, we selected it as a control for any directly inhibitory effect of mucus in general. Likewise, the mucus of A. montereyensis would be expected to have little effect beyond any direct effect of the mucus since this nudibranch eats mostly sponges. We performed four experiments to elucidate the influence of the prior occupation of an area (and thus any traces left behind) on the subsequent settlement of barnacles. In the first, we examined the effect of the natural occupation of a surface by whelks on barnacle settlement; we expected that prior occupation by the whelk, a future predator, might reduce barnacle settlement. Since any negative effects observed might be caused by general properties of mucus itself, we attempted a further experiment to see if mucus from a presumed nonh~~dous source (a fucoid alga) would reduce settlement to the extent of the whelk mucus. Another experiment was performed to confirm the unexpected results of J. Davies & B. Holthuis (unpubl. data) in which they found the mucus of A. montereyensis actually enhanced the settlement of B. glandula. Finally, we contrasted the effects of the whelk relative to the limpet with the expectation that the prior presence of the limpet might have a greater negative effect since it is a more immediate danger to recently settled barnacles. Our results show that the prior occupancy of the substratum by the whelk can greatly decrease the settlement ofB. glandula. Because the other species of barnacle, S. cariosus, was not as greatly influenced by the presence of the whelk, we suggest that the prior occupancy does not merely make the substratum physically unsuitable for barnacle settlement. Instead, it may either leave a cue or change existing cues. The different responses of the two barnacle species may reflect differing aspects of their ecology (see Discussion). The algal mucus also inhibited B. glandala settlement but this response may be due to either the effects of mucus itself or to an unsuspected negative cue associated with the algal mucus. As predicted from previous work (J. Davies & B. Holthuis, unpubl. data), nudibranch mucus increased settlement of B. glundzda but possibly by altering the composition of the epibenthic microflora. Unexpectedly, the inhibition of B. gland&z settlement was greater in response to the whelk than to the limpet. An adaptationist interpretation of these results suggests the hypothesis that the long-term and thus future risks of predation by whelks are greater than the immediate and short-term risk of biological disturbance from limpets. The adaptive consequences, if they exist, of the effects of the alga and the nudibranch are not as readily apparent.

METHODS

Although the distinction between settlement and recruitment has been justly made (Keough & Downes, 1982; Caffey, 1985; Connell, 1985), most of our observations were made within 3 wk after settlement and were consistent with those made within a day after settlement. Moreover, the pattern of settlement by cyprids (the terminal planktonic stage that selects the settlement site) matched patterns seen in the recruitment of

90

L. E. JOHNSON

postmetamorphic reflected

the

postsettlement

juveniles. actual

Thus, we believe that any recruitment

settlement

mortality

AND R. R. STRATHMANN

patterns,

especially

since

patterns no

we observed,

obvious

causes

of

were noticed.

All experiments involved barnacle settlement onto slate plates that were either suspended from the piers at the Friday Harbor Laboratories (San Juan Island, Washington,

48”33’N,

123”Ol’W)

or attached

14.5 x 14.5 x 0.8 cm, and each had

12-15

to the nearby

horizontal

and

shore. The plates were 12-15

vertical

grooves,

z 1 mm wide by 1 mm deep, ground into the surface to enhance barnacle settlement (Crisp & Barnes, 1954); the number of grooves among plates within any replicate was the same. Except for the experiment involving nudibranchs, plates were set out in late winter in order to develop a natural microflora to facilitate settlement (Strathmann et al., 1981) i.e., to season. Plates were sterilized between years using sodium hypochlorite (5 %). Once larvae began to settle, the plates were collected from the field, any barnacles on the plates were removed, and then the specified manipulations were performed. Except where noted, barnacles were removed along with an underlying layer of the slate by scraping all the grooves with a knife point. The flat areas between grooves were not disturbed by this process. The species of barnacles were identified by the size and carapace sculpturing of the cyprids and the setation of the metamorphosed juveniles (Strathmann & Branscomb, 1979; Standing, 198 1). EFFECTS

OF PRIOR OCCUPANCY

BY WHELKS

To determine if the prior occupation of the substratum decreased subsequent settlement by barnacles, we caged whelks on experimental plates and then compared the subsequent settlement of barnacles after the removal of the whelks to that of control plates. Six pairs of plates were set out in March 1986 near the Cantilever Pier and were distributed throughout the intertidal zone dominated by F. distichus (z 1.3 m above MLLW). All plates were initially enclosed by cages of Vexar (a plastic mesh, mesh diameter = 5 mm), and together with the cages were attached to the shore by screws driven into plastic anchors that were set into holes drilled in the rock. On 23 April 1986, 23 whelks were placed in one cage of each pair (alternately left or right along the shore). The whelks and cages were removed 2 days later at which time all settled barnacles

were

removed. The plates were then placed back on the shore. After 4 more days, the plates were collected, taken to the laboratory, and censused for attached cyprids and metamorphosed juveniles. EFFECTS

OF MUCUS

To determine if any inhibition of barnacle settlement was due to mucus in general and not to the specific source, mucus from the whelk was compared to that of F. distichus. In addition, the duration of the effects were also examined. Methods differed from those above in that plates were either entirely rubbed with crushed whelks (without shells), entirely rubbed with crushed receptacles of the alga, or left untouched as controls.

LARVAL

Whelks produce

only minimal

AVOIDANCE

amounts

tarily across a surface in contrast animals.

Since the primary

of mucus when moving voluntarily

to the copious amounts

purpose

91

OF A PREDATOR

of this experiment

or involun-

of mucus produced was to determine

by injured

if mucus was

a nonspecific inhibitor of barnacle settlement, the change in application protocol was made to ensure approximately equal application of mucus from both sources. While the mucus produced

from injury may be different from the pedal mucus used in locomotion,

we hoped that any species-specific cues would still be associated with it. Fresh whelks or fronds were used for each plate. The plates within a replicate were randomly arranged side by side on a fiberglass panel with up to four replicates per panel (two on each side, one directly above the other; the maximum tidal height difference among replicates was 25 cm). The panels were hung off the main laboratory pier and centered at the tidal height where the maximal densities of adult barnacles occurred on nearby pilings (z 1.7 m above MLLW). For eight of the 12 replicates, treatments were applied weekly three times prior to initiation of the experiment. The panel with the other four replicates was lost in a storm and was recovered only in time for the final application of the treatment. The experiment began on 16 May 1985 when the final application was made and all previously settled cyprids and juveniles were removed. The plates were then censused for newly settled barnacles 11, 25 and 39 days after the last treatment. All individuals were removed after each census. EFFECTS

OF NUDIBRANCHS

The effect of the prior presence of A. montereyensis on the settlement of B. glandula was tested as in the previous experiment except for the following differences: (1) the mucus was applied by sliding the nudibranch (foot-side down) over the entire plate; (2) plates were not allowed to season in seawater before initiation of the experiment; (3) only five replicates were used; and (4) the plates were treated only once on 5 May 1987. Plates were examined on 8 May 1987 but settlement was not high enough to make censusing worthwhile; thus those settlers present were removed, and new settlers were recorded censused RELATIVE

and removed after the following 17-day period (8 May-25 May). Plates were again after an additional 21-day period (25 May-15 June). EFFECTS

The influence

OF LIMPETS

of the prior

AND

presence

WHELKS

of the limpet

relative

to the whelk

on the

subsequent settlement of barnacles was tested by rubbing the feet of these gastropods over the surface of the plates. The general methods were similar to those described in the preceding two experiments. Each of the plates within a replicate received one of the following treatments, each coupled with a control for the rubbing (Fig. 2). (1) Whelk effect: half (left or right side, randomly chosen) of a plate was rubbed with the foot of one whelk while the other side was rubbed with a portion of the pectoral muscle of the chicken Gallus gallus (L.). The rubbing with chicken was used to control for the disruption of the microflora that might occur when the gastropod foot was moved across

92

L. E. JOHNSON AND R. R. STRATHMANN

the plate. (2) Limpet e&t: half of a plate was similarly rubbed with the foot of a limpet and the other half with the chicken muscle. (3) Wetted: half of the plate was wetted with seawater to control for the moisture added to the plate when rubbed while the other half was rubbed with the chicken muscle. For all applications of the chicken, the muscle was soaked overnight in seawater. For both gastropods, the shell was broken back around the foot to fully expose the foot and avoid scraping the plate with the shell. The body cavity was not disrupted in this process so that traces left on the plate were derived primarily from the foot. A new animal was used for each plate, and after the initial treatment, the plates were promptly returned to the water and then censused 24 h later for attached cyprids. As an a priori decision, cyprids that settled in the central three vertical grooves were not included in the census to exclude any potential tr~sition zone between sides of the plate. After censusing, the cyprids were removed individually with forceps, the treatments were reapplied, and the plates returned to the water for another 24 h. This procedure was repeated until after the third census when it became visually obvious that the rubbing had removed much of the microflora. Traces of the settlers removed on previous days might have influenced subsequent settlement especially if B. glundulu exhibits the gregarious settlement behavior seen in some other barnacle species (e.g., Knight-Jones, 1953; reviewed by Crisp, 1976). However, because of the low settlement rates, the numbers of cyprids within each half-plate treatment over the three censuses were pooled before statistically analyzed; thus any lack of independence among days does not affect the analysis. Nine replicates were used although three of the replicates were moved to the nearby Cantilever Pier on the 3rd day in hopes of obtaining higher settlement rates. The experiment began on 11 April 1983.

STATISTICAL

COMPARISONS

Because settlement rates were spatially variable and at some times and for some treatments very low, we have relied exclusively on nonparametric statistics for the statistical analysis of data. Treatments were always spatially paired or grouped making the Wilcoxon signed-rank test for matched-pairs almost always the most appropriate test, and we refer to this test simply as “Wilcoxon” from this point on. Although a m~tiple range test would be preferable to analyze experiments with more than two treatments, no nonparametric versions exist that retain the information of the spatial grouping of the treatments. Thus, we have used a pair-wise approach to compare them. We recognize the dangers of making multiple comparisons in a pair-wise manner (Sokal & Rohlf, 198 1, p. 232) but feel justified since the comparisons were planned in advance. Moreover, since the P < 0.05 level is only a convention, we merely report the probability levels at each comparison and let the reader judge the validity of our conclusions.

LARVALAVOIDANCEOF A PREDATOR

93

RESULTS EFFECTS OF PRIOR OCCUPATIONBY WHELKS As predicted, fewer barnacle larvae were attached to plates previously occupied by whelks, and fewer had metamorphosed on such substrata (Fig. 1). Although this pattern was evident when both species of barnacles were pooled together, when statistically examined for each individual species, differences in B. glundula settlement were greater than for S. cariosus (P = 0.02 vs. P = 0.08 (cyprids) and P = 0.02 vs. P = 0.16 (juveniles), respectively; Wilcoxon, one-tailed). To directly compare the two species, we 120 ;

h

90

z 3 Ij

60 30 0

whelk

control

whelk

whelk

control

whelk

control

80 ii

z

2

._)

is

.

g

60

40 20 0

control

S. cariosus

Fig. 1. Settlementand recruitment of B. glanduia and S. cariosus onto slate plates previously occupied or not occupied by whelk. Cyprids: P = 0.02 (B.g~affd~~a), P = 0.08 (S. curiosus); juveniles: P = 0.02 (8. g~a~du~a), P = 0.16 (S. ca~~~~~~).Wilcoxon matched-pairs signed-rank test (one-tailed); N = 6 for each treatment. Bars represent 1 SE. used the ratio of the number of cyprids and juveniles settled on the control plates to those settled on the experimental plate. The ratio was greater for B. glundulu than for S. curiosus (P = 0.05; Wilcoxon, two-tailed) thus indicating that the effect of the treatment was greater for B. glandulu. The remains of the barnacles after removal could influence subsequent settlement patterns if barnacle larvae settled gregariously on the remains of removed barnacles (e.g., Knight-Jones, 1953). If the physical presence of the whelks during their 2-day occupation of the plates blocked access to the plates for barnacle larvae and produced

94

L. E. JOHNSON

lower settlement

AND R.R. STRATHMANN

on the plates during the occupation,

been left after the removal

of settled barnacles;

then fewer remains

perhaps

fewer barnacles

would have would then

settle on the previously occupied plates after the removal of the whelks. However, this alternative hypothesis is difficult to reconcile with the patterns we observed. First, in this experiment barnacles

the grooves

and the underlying

were scraped

sufficiently

hard

slate surface which presumably

enough removed

to remove

the

most if not all

of the remains. Second, a similar effect of the whelk was seen in experiments in which settlement could not occur during the simulated “occupation” of the plates (see below). Thus, the negative effect of whelks previous settlers did not exist.

still occurred

when the possibility

of a bias by

However, one complication exists in a possible interaction between the whelks and the microflora on the plate surface. The settling plates had become fouled by small tilamentous and encrusting algae during the seasoning prior to the initiation of the experiment. Whether by coincidence or as an effect of the 2-day occupation by the whelks, the plates that had been occupied by whelks appeared less fouled by algae. The effect of visible differences in algal fouling was compared to the effect of occupation by the snails by subjectively ranking all plates by degree of algal cover. (This procedure could be done without knowledge of their prior treatment because plates were numbered on their backs.) The plates were then divided into two groups: the six most fouled and the six least fouled. Numbers that had settled on plates of each group were then compared using the Mann-Whitney U test. The same test was applied to the original treatments of the plates (prior occupation by whelks and controls). Although both analyses demonstrated nonrandom patterns, the level of significance for the comparison using the original treatment of whelk occupancy was considerably lower than the post hoc comparison using the degree of fouling [P = 0.002 (U = 1) and P =: 0.02 (U = 5), respectively]. Thus, the prior occupation by whelks was a better predictor of the number of settled barnacles than was visible algal fouling. EFFECT

OF MUCUS

Settlement

of B. glandula

was lowest

on plates

smeared

with crushed

whelks,

intermediate on plates smeared with crushed algal receptacles, and highest on untreated control plates (Table I). The difference between plates decreased over time, but settlement was still lower on both the whelk and the alga treatments > 3 wk after the last application. Persistence of the material from the whelks was quite obvious since the royal purple from their precious body fluids stained the plates until they were bleached with sodium hypochlorite a year later. The whelk treatment was more effective than the alga treatment for at least the first 25 days (Table I). EFFECT

OF NUDIBRANCH

More B. glandula settled on plates treated with A. montereyensis than on control plates (Table II). The settlement decreased during the second interval either from

LARVAL

AVOIDANCE

95

OF A PREDATOR

reduced effects of the treatment or from fewer barnacle larvae encountering the plates. The enhanced settlement on surfaces rubbed with nudibranch feet confirms results from a similar experiment (J. Davies & B. Holthuis, unpubl. data), in which more B. glandulu settled on plates rubbed with A. montereyensis. However, they also found similar results with another dorid nudibranch, Onchidoris bilamellata (L.), which is a barnacle predator, and thus would be expected to inhibit barnacle settlement.

TABLE I Recruitment of B. glandula juveniles onto slate plates smeared with mucus from N. lamellosa, crushed receptacles of F. distichus, and on control plates during three consecutive periods in spring of 1985 (X and SE, n = 12). Juvenile B. glandala plate

Treatments 16-27 May Whelk mucus

4.8

(SE) Fucus mucus

(0.9) 8.7

(SE) Control

(2.3) 19.8

(SE)

(2.6) Wilcoxon

matched-pairs

27May-lOJune

’ lo-24

51.1 (10.7) 68.8

80.5 (13.3) 99.7 (13.7) 137.6 (12.2)

(9.2) 111.3 (15.3) signed-ranks

test (one-tailed)

by dates.

P = 0.001 P = 0.001 P = 0.01

P = 0.001 P = 0.002 P = 0.03

Whelk mucus vs. control Fucus mucus vs. control Whelk mucus vs. Fucus mucus

June

P = 0.001 P = 0.01 P = 0.14

TABLE II Effect of mucus

from A. montereyensis on settlement

of B. glandula in spring of 1987 (X and SD, n = 6).

Treatment

Ind

plate

’

8-25 May Cyprids

25May-15June

Juveniles __~

Cyprids

Juveniles

Dorid mucus

21.2

7.0

(8.6) 0.0

29.2 (10.0) 0.4

3.6

(SD) Control

(2.6) 0.2

(4.8) 0.2

(SD)

(0.0)

(0.9)

(0.4)

(0.4)

Wilcoxon Dorid mucus control

vs.

matched-pairs

signed-ranks

P = 0.03

test (one-tailed)

P = 0.03

by dates.

P = 0.06

P = 0.03

96

L. E. JOHNSON AND R. R. STRATHMANN

RELATIVE EFFECTS OF LIMPETS AND WHELKS

As predicted, the prior presence of both the limpet and the whelk decreased the settlement of B. glandulu. Fewer barnacles settled on either the whelk-treated or limpet-treated plates than on the wetted plates (P = 0.004 and P = 0.01, respectively; Wilcoxon, one-tailed; Fig. 2). However, contrary to expectations, fewer barnacles settled on the whelk-treated than the limpet-treated plates (P = 0.02; Wilcoxon, twotailed).

I

Experimental Plate 1

Design Plate 3

Plate 2

I

30

7

8 ._

l-

b!o

e ;

10

C -1

0 whelk chicken

limpet chicken

wetted chicken

treatments Fig. 2. Settlement of B. g~u~~~Z~on halves of slate plates rubbed with whelk feet, limpet feet, and chicken muscle, and half pIate that was only wetted. Whelk vs. chicken (1A vs. 1B): P < 0.002; limpet vs. chicken (2A vs. 2B); P -=z 0.02; wetted vs. chicken (3A vs. 3B): P < 0.014; whelk vs. wetted (1A vs. 3A): P < 0.004; limpet vs. wetted (2A vs. 3A): P i 0.006; whelk vs. limpet (1A vs. 2A): P c:0.016. Wilcoxon matched-pairs signed-rank test (one-tailed, except for “whelk vs. limpet”); n = 9 for each treatment. Bars represent 1 SE.

As anticipated, the rubbing with chicken muscle also decreased settlement relative to the wetted sides (P = 0.014; Wilcoxon, one-tailed), presumably by disrupting the microflora although the effect on barnacle settlement did not appear until after the second rubbing (Day 2, Fig. 3). However, the decreases in settlement in the whelk and limpet treatments were not due entirely to this disturbance; both treatments had lower settlement on the side rubbed with the gastropod when compared to the other side of the plate which had been rubbed with chicken (P = 0.002 and P = 0.02 for the whelk and limpet treatments, respectively; Wilcoxon, one-tailed).

91

LARVALAVOIDANCEOFAPREDATOR

In summary, the settlement of one species of barnacle, B. gland&z, is less on substrata previously occupied by N. l~~~~~~~~,a major predator on this barnacle. A second species of barnacle, S. caviosus, is much less affected by this prior occupation. The effect of mucus from crushed whelks on the settlement of B. gkzndula is long lasting, > 3 wk.

0

day 1

day 2

day 3

Fig. 3. Daily settlement of B. g~~~~~~u onto halves of slate plates rubbed with pectoral muscle of chicken (“rubbed”) and control halves (“wetted”) after 3 consecutive days of treatments (plate 3; Fig. 2). Day 1: P = 0.52; Day 2: P = 0.08; Day 3: P = 0.05; Wilcoxon matched-pairs signed-rank test (one-tailed); X = 9 for each treatment. Bars represent 1 SE.

Treatment with mucus from an alga that has no known deleterious effect on B. glandufa also decreased settlement but to a lesser extent than the whelk. However, mucus from another source, a dorid nudibr~ch, with no known effect on B. g~a~duZu,enh~ced settlement by B. ghdula. Treatment of the substratum with the mucus of a limpet, which is a potential source of death only shortly after settlement, had a less inhibitory effect on B. glandula settlement than did mucus from the whelk. DISCUSSION

Prior occupation of a substratum might change the substratum in three ways: first, it could remove substances; second, it could rearrange or disrupt existing structures; or last, it could add materials to the substratum. We hypothesize that some of the species we tested left behind some substance that affected the settlement of barnacle larvae. The added substance may be the mucus (or something associated with the mucus) left behind by these organisms. Although some of the techniques employed (e.g., crushing individuals) might not have mimicked the natural release of mucus, the effect still occurred when whelks attached or moved naturally over the substratum (Fig. I). Although barnacle larvae are sensitive to differences in microflora (Strathmann et al., 1981; Hudon et al., 1983; Maki et al., 1988), the physical disruption or removal of the microflora can not entirely account for the decrease in settlement since the results from the experiment comparing limpets and whelks demonstrate that disruption or removal

98

L. E. JOHNSON AND R. R. STRATHMANN

of the microflora by novel materials (chicken muscle) did not produce as great of a decrease (Fig. 2). In addition, settlement patterns were better predicted by the history of occupation than by the count of algal microflora present on the plates in the experiment examining the effect of the prior occupancy of whelks. If it is indeed mucus that produces these results, then several explanations can account for the decreased settlement. One explanation is that mucus inhibits settlement of barnacles by affecting the adhesion of the cyprids or through directly offensive chemicals. Although mucus is associated with predators or biological disturbers, inhibition of settlement by the mucus would only fortuitously decrease settlement in areas of higher risk. Under this interpretation, the low settlement on mucus coated surfaces would not be a result of natural selection to avoid areas of higher risk. Another expl~ation is that the mucus alters existing cues present on the substratum. Mucus can alter the microflora (Connor & Quinn, 1984), and difTerences in microflora can change settlement patterns in barnacles (Strathmann et al., 1981; Hudon et al., 1983; Maki et al., 1988). Again, a decline in settlement in response to mucus does not imply that the larvae are avoiding areas of higher risk unless they are indirectly using the altered conditions as cues for areas of higher risk. An adaptationist explanation is that the mucus acts as a cue to the presence of a hazard and that the patterns of reduced settlement reflect behavior that avoids areas of higher risk. This hypothesis prompted our experiments. Several lines of evidence support this idea. First are the different responses of the two barnacle species (Fig. 1) which would not have been expected if mucus acted merely as a physical barrier to attachment. Differences in the natural history of the barnacles may explain this result. On San Juan Island, B. glandula rarely survives to reproduce in the zone where foraging Nucelfa species are abundant and instead persists in a refuge above this zone (Connell, 1970a, 1972). In contrast, very few S. cariosus that attach above this zone even complete metamorphosis (Strathmann & Branscomb, 1979). Since they have a refuge in size from predation by the whelk (Connell, 1970b, 1972; Dayton, 1971; Palmer, 1983) and are better defended against drilling by whelks (Palmer, 1982, 1983), S. cariosus are better able to coexist with the whelks (Palmer, 1983). Thus avoidance of substratum previously occupied by whelks might be beneficial to B. glandula while perhaps even deleterious to S. caiiosus. Secondly, the response of the barnacles depended on the source of the mucus. The mucus from the whelk had a greater effect than that of the alga. However, to conclude that this difference was due only to cues associated with the mucus would require assuming that the two kinds of mucus had equal persistence in the environment and no important biochemical differences. The reduction of barnacle settlement in response to the algal mucus may indicate that mucus can act as a direct barrier to settlement (but see results for nudibranch mucus) or that there are negative settlement cues also associated with the alga. Nevertheless, the greater negative effect of the whelk mucus does lend further support to the idea that its inhibitory effect is at least partly caused by cues that act beyond any direct effects of mucus.

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Thirdly, adult B. ghndula withdraw their cirri for a longer period of time after contact with a ~ucel~a species than after contact with the herbivorous turban snail Te~la~ui~jgo or with F. d~~~c~u~(Palmer et al., 1982). Thus, adult B, gZu~dulucan discriminate among different kinds of organisms implying that the larval stages might also have this ability. Lastly, traces of some gastropods increase barnacle settlement. The mucus from nudibranchs increased the settlement of B. glundulu. However, the higher settlement on nudibranch-treated plates relative to control plates may be partially due to the change in protocol for that experiment. By not seasoning the plates in seawater several months before the initiation of experiments, we did not allow a microflora to develop. Mucus can enhance microalgal colonization of a surface (Connor & Quinn, 1984), and thus may alter the microflora affecting settling larvae (Strathmann et al., 1981; Hudon et al., 1983; Maki et al., 1988), especially on surfaces that are almost devoid of a microalgal flora. In fact, several of the plates rubbed with nudibranchs became visibly more fouled than any of the control plates during the course of the experiment (pers. obs.). Thus, the influence on settlement may be mediated by the effects of the mucus on the microflora. Fresh unseasoned surfaces were used in the study of Davies & Holthuis (unpubl. data), and thus the mucus again may have been acting indirectly by affecting the microflora and not as a direct cue. Nevertheless, the results still demonstrate that the larvae of B. giu~duZado not avoid traces of all gastropods and that mucus does not appear to act as a barrier to settlement. Further evidence against a directly inhibitory effect of mucus is the work of Raimondi (1988) in which he demonstrated that prior occupation by the predaceous gastropod Acanfhina angelica could influence settlement of the barnacle C~r~u~~ius anisopoma. However, in that system it inc~effsed the settlement rate which indicates the mucus was not acting as a barrier to settlement or at least that the inducement it provided overwhelmed any effect it had as a barrier. One explanation for the difference between his and our results is that B. glundula has a refuge above the zone of Nucella species while C. anisopoma does not survive above the zone occupied by A. aplgelica. Thus, the risk of predation by A. angefica at lower levels is less than the risk of death from desiccation at higher levels, and the larvae may be using the presence ofA. angelica as a cue to induce settlement in the more favorable habitat (Raimondi, 1988). However, he did not use seasoned plates, and thus the possibility exists that the effects he demonstrated were the indirect effects of the mucus influencing the microflora. All of the above cases of enhanced settlement involved unseasoned (no initial microflora) substrata while all of the cases of reduced settlement involved seasoned (established ~croflora~ substrata. Thus, the movement of organisms over the substratum (or the subsequent traces left behind) may interact with the microflora in a complex way to affect barnacle settlement. This subject is likely to be a fruitful area for future research. The differential response of B. glanduia to whelk and limpet treatments is difficult to interpret. Both are potential sources of mortality for barnacles, and we predicted that the limpets would have a greater effect since they present a more immediate danger to barnacles after settlement. However, our results demonstrated that whelks reduced

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settlement to a greater extent (Fig. 2). One explanation is that the cue from the whelk is either more detectable by the larvae, adheres better to the substratum, or was released in greater amounts by the treatment methods. Another interpretation is that the strength of the response to a cue reflects either the degree of risk from its source or the reliability of that cue. If this were true, then our results suggest that the risk from the whelk predation exceeds that from disturbance by the limpet or that the whelk’s cue is more reliable. The latter possibility seems unlikely given that the whelk is motile and may not pose a threat for some time while disturbance by limpets presents an immediate danger. Indeed, the patchiness, mobility, and perhaps delayed effect of the predatory A. angelica is such that C. a~~opo~a preferentially settles on rocks that have been previously occupied by the snail, apparently using the presence of its predator as a cue to the appropriate intertidal height (Raimondi, 1988). Thus, we hypothesize that, although the instantaneous risk of predation by whelks might be less than that of disturbance from limpets shortly alter settlement, the overall risk is greater due to the long-term nature of the interaction. If so, the importance of bulldozing may be overestimated, especially since some of the best demonstrations of this effect (e.g., Lewis, 1954; Connell, 1961a; Dayton, 197 1; Branch, 1975) have often been done on cleared or flat substrata on which little of the natural three-dimensional topography exists that would normally offer refuge from such disturbance (Miller, 1986). The negative effect of the alga could be interpreted as the adaptive use of cues but there is no supporting evidence that this alga reduces the survival of settling B. g~anduZa. On other shores, fucoid algae can reduce recruitment of barnacles, presumably by whipping settling cyprids off the substratum (Menge, 1976; Grant, 1977; Hawkins, 1983), but experiments on the northwest shores of the U.S.A. have not shown a negative effect of F. distichus on the recruitment of B. glandufa (Dayton, 1971; Farrell, 1987) though the settlement of barnacles was not directly examined in these studies. A complication in a priori ranking of hazards is the possibility that there are benefits as well as costs for barnacles associating with other organisms. Limpets might remove epiphytic or competing algae (e.g., Hawkins et al., 1983; Jernakoff, 1985 ; Dungan, 1986; Van Tamelen, 1987); whelks might selectively remove competing invertebrates (e.g., Connell 1961b; Dayton, 1971); algae might provide cover which might reduce thermal stresses. For example, on San Juan Island, initial recruitment of B. gla~d~la was lower in areas where the canopy of F. distic~~s was removed (Dayton, 1971). Thus, the presence of the alga was beneficial for at least a part of the life of B. glandula. A longer-term study that included initial settlement patterns would be needed to critically evaluate whether the beneficial effects of the presence of F. distichus outweighed any deleterious ones. However, even then the balance might shift both temporally and spatially; Dayton (1971) could not detect an effect of removing F. distichus in a similar experiment on an exposed coast. Likewise, Hawkins (1983) found that a cover of fucoid algae could have either beneficial or deleterious effects on barnacle settlement and recruitment depending on factors such as tidal height, wave exposure, and the species involved. Such variation in costs and

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benefits combined with the long dispersal distances of barnacles might erase any movement towards local adaptation. Although we have followed an adaptationist approach in discussing our results, care must be used to avoid doing so blindly. While some results can be easily explained, others can not. For example, the prior presence of another potential barnacle predator, the nudibranch Onchidoris bilamellata, enhanced settlement of B. glandula (J. Davies & B. Holthuis, unpubl. data) which is contrary to expectations but might also be explained by the absence of an initial microflora. Likewise, our initial expectations for the relative effects of whelks and limpets were not supported. Raimondi (1988) discusses the circumstances that would select for responses to potential cues, but further speculation on our part is unwarranted until more is known about this system. Nevertheless, to our knowledge, this study is the first demonstration that settling larvae avoid areas previously occupied by a mobile predator. Since this predator is dangerous throughout the prey’s benthic life, we hypothesize that the larvae may be taking into account long-term future risks (i.e., “the ghost of predation future”) when making the decision to settle. The influence on the prey’s distribution may be substantial since some results suggest that the effects can persist long after the actual occupation of the area. These results and the responses to other potential hazards call for a more careful examination of recruitment patterns and their inferred causes. Patterns attributed to bulldozing by gastropods or whipping by algal fronds may instead be caused by selective larval settlement to avoid such hazards. By focusing on the process of settlement and the early survival of juveniles, the actual causes of such patterns can begin to be unequivocally understood. Research on settlement cues has emphasized stimuli that induce settlement and often deals with only single factors. However, the number of stimuli potentially detectable by larvae is great, and they may be correlated with either costs or benefits to varying degrees (Hadfield, 1987). Larvae may use many sources of information when deciding whether or not to settle and must integrate the sometimes conflicting information. Thus the relative importance of various stimuli must be determined, not only in terms of larval responses but also in terms of the correlated benefits or risks.

ACKNOWLEDGEMENTS

NSF Grants OCE8415258 and OCE8606850 to R. R. Strathmann, 0CE8415707 to R.T. Paine, a NSF Predoctoral Fellowship to L. E. Johnson, the Friday Harbor Laboratories and Zoology Department of the University of Washington supported this project. We thank S. Cohen, J. Davies, M. Hadfield, B. Holthuis, R. Kvitek, J. Marks, R. T. Paine, A. R. Palmer and T. Williams. The manuscript was improved by comments from R. K. Grosberg and an anonymous reviewer.

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