Orientation of blue crab, Callinectes sapidus (Rathbun), Megalopae: Responses to visual and chemical cues

Journal of Experimental Marine Biology and Ecology, 233 (1999) 25–40

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Orientation of blue crab, Callinectes sapidus (Rathbun), Megalopae: Responses to visual and chemical cues Humberto Diaz a , *, Beatriz Orihuela a , Richard B. Forward, Jr.b , Dan Rittschof b a

Instituto Venezolano de Investigaciones Cientificas, Centro de Ecologia, Apartado 21827, Caracas 1020 A, Venezuela b Duke University, Zoology Department and Nicholas School of the Environment, Marine Laboratory, 135 Duke Marine Lab Road, Beaufort, NC 28516 -9721, USA Received 22 January 1998; received in revised form 4 July 1998; accepted 14 July 1998

Abstract Post-larvae (megalopae) of the blue crab Callinectes sapidus are transported from offshore areas into estuaries where they settle and metamorphose in specific areas, such as seagrass beds. The present study tested the hypothesis that intermolt and premolt megalopae had different behavioral responses to visual and chemical cues that are associated with predator avoidance and settlement. Visually directed movements to solid and striped rectangular targets subtending different visual angles (5–3508) were tested in an arena in the presence of either Offshore Water, Seagrass (Zostera marina) Odor Water or Predator (Fundulus heteroclitus) Odor Water. Intermolt megalopae generally swam away from 108 to 1808 targets in all water types which was interpreted as a predator avoidance response. Premolt megalopae had similar behavior in Offshore and Seagrass Odor Water. However in Predator Odor water, they displayed different predator avoidance behavior. When presented with a solid target, premolt megalopae either remained motionless or swam directly away from the target. If presented with targets resembling stalks of seagrass (vertical stripes), they swam in all directions which was interpreted as a startle response. In a chemical choice chamber, both molt stages were not attracted to Seagrass Odor Water but avoided Predator Odor Water. There was no evidence that megalopae used chemical cues for orientation toward settlement sites. Thus, the hypothesis was supported and the results suggest that behavioral responses to the test chemical and visual cues are involved in predator avoidance.  1999 Elsevier Science B.V. All rights reserved. Keywords: Blue crab; Callinectes sapidus; Megalopa; Post-larva; Orientation; Visual cues; Chemical cues; Settlement

*Corresponding author. 0022-0981 / 99 / $ – see front matter  1999 Elsevier Science B.V. All rights reserved. PII: S0022-0981( 98 )00121-X

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1. Introduction After completing development in offshore areas, post-larvae (megalopae) of the blue crab, Callinectes sapidus (Rathbun), are transported shoreward (Sandifer, 1975; McConaugha et al., 1983; Epifanio, 1988; Epifanio et al., 1989) where they enter estuaries, settle and metamorphose to the first crab stage. Wind-generated surface currents contribute to shoreward transport (Epifanio et al., 1984; Goodrich et al., 1989), and megalopae use selective tidal stream transport (STST) for movement up estuaries to settlement sites. During STST megalopae are abundant in the water column during flood tide at night and in low number at all other times, presumable because they are on or near the bottom (Dittel and Epifanio, 1982; Brookins and Epifanio, 1985; Mense and Wenner, 1989; Little and Epifanio, 1991; De Vries et al., 1994; Olmi, 1994). Areas of submerged vegetation, such as seagrass beds (Orth and van Montfrans, 1987; Olmi et al., 1990), serve as settlement sites, and metamorphosis is accelerated by chemical cues from estuarine and terrestrial vegetation (Wolcott and De Vries, 1994; Forward et al., 1994, 1996, 1997; Brumbaugh and McConaugha, 1995). The present study is based on the postulation that chemical and visual cues mediate orientation responses that facilitate arrival at the settlement sites where blue crab megalopae metamorphose to the first crab stage. Chemical cues are known to affect orientation by a variety of invertebrate larvae including molluscs (Morse et al., 1979; Hadfield and Scheuer, 1985; Zimmer-Faust and Tamburni, 1994), barnacles (Crisp and Meadows, 1962; Rittschof, 1985; Yule and Walker, 1985; Claire et al., 1995), lobsters (Boudreau et al., 1993) and crabs (Forward and Rittschof, 1994; Welch et al., 1997). Frequently chemical cues are involved in habitat selection, as post-larvae of crustaceans are either attracted or repelled by habitat and predation odors (e.g. Boudreau et al., 1993; Rittschof, 1993; Welch et al., 1997. Crustaceans also use visual cues for orientation involved in predator avoidance, prey capture, shelter location, conspecific identification and vertical migration (e.g. Herrnk´ et al., ind, 1968, 1972, 1983; Langdon and Herrnkind, 1985; Forward, 1988; Dıaz ´ et al. (1995a) 1995a,b; McKelvey and Forward, 1995). Among crab post-larvae, Dıaz showed that megalopae of mangrove crabs orient to visual cues and the specific responses vary with species. Cryptic species go toward dark objects, while noncryptic species orient away from dark objects. The present study considered orientation of the blue crab C. sapidus megalopae to visual and chemical cues. Given the movement of megalopae from offshore to estuarine areas as they develop, a logical assumption is that behavior changes with molt stage. Intermolt megalopae occur in offshore and estuarine areas were they mainly participate in horizontal transport. Alternatively, premolt megalopae are more likely to occur at or near estuarine settlement sites, where they will metamorphose. Thus, the study determined the behavior of intermolt and premolt megalopae to visual and current cues in the presence of chemical cues from offshore water, seagrass (Zostera marina) and a planktivorous fish (Fundulus heterclitus). The test hypothesis was that intermolt and premolt megalopae have different behavioral responses to visual and chemical cues that are associated with predator avoidance and settlement. The results indicate that behavior does change with molt stage and is dominated by predator avoidance responses.

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2. Materials and methods Megalopae of the blue crab Callinectes sapidus (Rathbun) were collected in plankton tows (0.67 diameter net; 505 micron mesh) from July to September, 1996 about 1 km inside the entrance to the Newport River Estuary (Beaufort, North Carolina, USA) on flood tides at night. Megalopae were quickly transferred into Offshore Water (see description below) and maintained in the laboratory overnight. The next day, they were sorted according to molt stage into intermolt and premolt megalopae (Aiken, 1973; Anger, 1983; Stevenson, 1985). Intermolt was indicated by body pigmentation and the absence of tissue withdrawal from the rostrum. Megalopae were transferred daily into new Offshore Water and fed brine shrimp nauplii (Artemia fransciscana Kellogg), which had recently hatched in Offshore water. Megalopae were separated according to molt stage before experimentation each day. When not being tested they were maintained in Offshore Water in an environmental chamber (Sherer–Gillet, model CEL 4-4) on the ambient light:dark cycle at a temperature of 258C. There were two general types of test water. ‘Offshore Water’ was collected about 15 km seaward of the Newport River Estuary inlet. This water was beyond the estuarine plume and from an area where blue crab megalopae were collected previously in the neuston (Forward and Rittschof, 1994). It was maintained in acid washed glass carboys throughout the experiments. The salinity of the water was about 35 psu as measured with a refractometer (AO; accuracy 0.5 psu). ‘‘Seagrass Odor Water’’ was prepared by incubating the seagrass Zostera marina at a ratio of 5 g l 21 in Offshore Water for 24 h. ‘Predator Odor Water’ was prepared by similarly incubating the mummichog fish Fundulus heteroclitus at the same ratio in Offshore Water. These ratios were used because this level of odor from Z. marina accelerates metamorphosis of blue crab megalopae (Forward et al., 1996) and odors from fish predators at this concentration affect crustacean photoresponses (McKelvey and Forward, 1995) and time to metamorphosis (R. Forward, unpublished data). Seagrass and mummichogs were removed from the test waters at the beginning of experimentation.

3. Visual experiments Megalopae were tested in a circular Lucite arena (22.5 cm diameter) that had 5 cm high surrounding transparent wall with a white translucent collar. The arena had a translucent white top and a gray Lucite bottom. It rested upon a transparent Lucite table. During tests, it was filled with 500 ml of test water at a temperature of about 248C. In order to avoid the build-up of metabolites during experiments, the water was renewed every 30 min during an experiment. Megalopae were placed individually in a 1.5 cm diameter cylinder in the center of the arena, which was fitted from below with a plunger. The cylinder extended below the arena, and its top was level with the arena floor. For experimentation, the arena was then covered by the translucent plate, and a test initiated by elevating the megalopa to the level of the arena floor with the plunger. Responses of individual megalopa were determined by watching from below through the gray floor. Due to the lighting arrangement, the observer could not be seen from the arena, but

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megalopae were clearly visible through the arena floor. The stimulus light was a circular fluorescent lamp (Phillips model TLE 32W/ 54), which delivered an intensity of 4.15 3 10 15 photons cm 22 s 21 at the center of the arena. The circular lamp was positioned around the arena such that the inner edge was 2 cm from the outside of the arena wall. This apparatus was used previously to test orientation of post-larvae from ´ et al., 1995a). All tests were conducted in a room where the only mangrove crabs (Dıaz light came from the stimulus light and were done during the time of the light phase in the ambient light:dark cycle. A megalopa was considered to respond if it swam from the center of the arena to the arena wall within 60 s. The first point of contact with the wall was recorded as the orientation direction (108 accuracy). Megalopae that did not reach the arena’s wall within 60 s were recorded as not responding. After each trial, the test megalopa was placed in its own finger bowl (8 cm diameter) containing Offshore Water. At least 30 min elapsed between trials, if megalopae were retested in the same water type but with a different visual stimulus on the same day. Each visual target was tested with intermolt and premolt megalopae and each megalopa was only tested once in any condition. A total of 72 orientation tests, each with 50 megalopae, were conducted. Thirty six tests were with intermolt and 36 with premolt megalopae. In each series of 36 tests, 12 each were with Offshore Water, Seagrass Odor Water and Predator Odor Water. Visual targets consisted of eight flat black rectangles and three rectangles with parallel black and white vertical lines. All targets were 2 cm high. For the targets with vertical lines, two rectangles (208 and 908 total width) had narrow black and white lines subtending 28 as viewed from the center of the arena. The third lined target (908 total width) had black lines subtending 28 and wide white lines subtending 48. The solid black rectangles were tested to determine responses to dark objects underwater, and the lined targets were used to mimic different patterns of seagrass stalks and perhaps a school of small fish. Seagrass beds are well documented as settlement sites (Orth and van Montfrans, 1987; Olmi et al., 1990) and the authors have observed schools of small fish in these areas. For each experiment, the transparent arena wall was surrounded by a translucent white collar and targets were attached individually to the inside of the white collar. Each rectangle subtended a different visual angle (5, 7.5, 10, 20, 90, 180 and 3508) as viewed from the center of the arena. With the water volume used, targets extended from the bottom of the arena to above the water level. The white collar alone was tested as a control for orientation to cues in the arena itself. Directional orientation of responding megalopae was determined by circular statistical methods (Zar, 1984). The mean angle and length of the mean vector length (r) were calculated for each experimental condition. The mean vector length varied from 0.0 to 1.0 and was inversely related to the dispersion in a data set. The V test determined whether the distribution was different from uniformity by using both the calculated mean and the expected angle. The expected angle was established a priori as the angle opposite to the center of the test black sector. The one-sample test for the mean angle was used to determine whether the mean angle differed from the expected direction. The 95% confidence interval around each mean angle was determined and then whether the expected orientation direction fell within this interval. For the white collar alone

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(control), there was no black sector that could be used to establish the expected angle. Thus, for the controls, the expected angles were obtained from a random number table. Responsiveness was also analyzed by comparing the proportions of megalopae responding to the different targets. Responsiveness was defined as the proportion of megalopae swimming to the arena wall within 60 s for each test and proportions were compared with the Dunnett test for proportions (Zar, 1984).

4. Chemical choice experiments Megalopae were tested for orientation to chemical cues and current flow in a chemical choice apparatus. The apparatus (Fig. 1) consisted of a central cylindrical (650 ml volume) chamber having a center standpipe and four peripheral cylindrical (550 ml volume) chambers situated at 908 to each other. The peripheral chambers were connected to the central tank by polyacrylate tubing (ID 1.5 cm). Stimulus water flowed by gravity from constant pressure reservoirs through flow meters and flow control valves into the peripheral chambers, then into the central chamber via the tubing and finally exited through the center standpipe. For the experiments, only two peripheral chambers situated 1808 apart were used. The other two chambers were blocked. During the control

Fig. 1. Diagrammatic representation (A. top view; B. side view) of the chemical choice apparatus. Dashed arrows indicate the direction of water flow from the peripheral / radial chambers into the central chamber and out the central standpipe.

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experiment, the two peripheral chambers received Offshore Water. For all other trials, two different waters were tested simultaneously and each water entered opposite peripheral chambers. The chamber receiving a specific water was changed between trials. For the experiments, intermolt or premolt megalopae were initially confined in the center chamber with a removable cylindrical plastic sleeve. Once the water flow into the peripheral chambers was adjusted to be 15 ml / min, the light was extinguished, and post-larvae were released by slowly removing the sleeve. After 30 min in darkness, flow was stopped and post-larvae in the center chamber, connecting tubes and peripheral chambers were counted. Those megalopae that remained in the center chamber were considered unresponsive. Responsiveness to water from a specific peripheral chamber was quantified as the proportion of megalopae in the peripheral chamber and associated connecting tube. A total of 36 tests (18 with intermolt and 18 with premolt megalopae) were conducted. For each molt stage, there were three control tests with Offshore Water entering both peripheral chambers, and five paired tests each with (1) Offshore Water and Seagrass Odor Water, (2) Offshore Water and Predator Odor Water and (3) Seagrass Odor water and Predator Odor water. For each control tests 32 megalopae were tested simultaneously, whereas 50 were tested simultaneously for each paired test. Choice chamber data were analyzed for attraction to the different test waters using a Dunnett test for proportions (Zar, 1984).

5. Results

5.1. Visual experiments: orientation to solid targets A megalopa was considered as responding to a visual cue, if it swam from the center of the arena to the surrounding wall within 60 sec. For analysis, data were separated into direction of movement and the proportion of megalopae responding. Directional responses were analyzed by circular statistics to determine if intermolt and premolt megalopae showed direction movement relative to the position of the different size solid targets. In each control condition with a completely white surrounding collar (Tables 1 and 2) intermolt and premolt megalopae moved uniformly, which indicates there were no directional cues in the arena. Intermolt megalopae responded significantly by moving away from the center of all black targets except 58 and 3508 in Offshore Water, and all targets except 3508 in Seagrass Odor Water (Table 1). They were less directed when exposed to narrow targets in Predator Odor Water as significant orientation away did not occur to 5–108 targets. The exception to orientation away from dark targets occurred in the presence of the 3508 target. Megalopae reversed direction and oriented away from the 108 white sector toward the middle of the black sector in Seagrass Odor Water and Predator Odor Water and did not orient in Offshore Water (Table 1). The general orientation pattern of premolt megalopae to solid targets was similar to that for intermolt megalopae. Premolt megalopae oriented away from all targets except

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Table 1 Orientation of Intermolt megalopae to solid targets in Offshore Odor, Seagrass Odor and Predator Odor Waters Target (8) Offshore Water Control 5 7.5 10 15 20 90 180 350 Seagrass Water Control 5 7.5 10 15 20 90 180 350 Predator Water Control 5 7.5 10 15 20 90 180 350

n

E (8)

a (8)

Deviation from expected

r

V,u

Sig.

49 30 50 46 50 47 47 50 44

21.00 182.50 183.75 185.00 187.50 190.00 225.00 270.00 355.00

– – 152.05 185.67 200.52 211.03 224.97 258.08 –

– – 31.70 0.67 13.02 21.03 0.03 11.92 179.39

0.02 0.31 0.54 0.58 0.35 0.54 0.63 0.80 0.09

0.09 0.50 4.56 5.60 3.41 4.88 6.12 7.82 2 0.87

ns ns *** *** *** *** *** *** ns

43 40 43 35 40 36 36 45 34

80.00 182.50 183.75 185.00 187.50 190.00 225.00 270.00 355.00

– 190.63 189.94 187.52 204.92 160.89 209.03 252.88 131.15

– 8.13 6.19 2.52 17.42 29.11 15.97 17.12 223.85

0.17 0.32 0.37 0.38 0.43 0.32 0.24 0.82 0.45

1.35 2.82 3.40 3.18 3.67 2.34 1.96 7.42 2.69

ns ** *** ** *** * * *** **

41 29 42 29 44 39 42 33 38

94.00 182.50 183.75 185.00 187.50 190.00 225.00 270.00 355.00

– – – – 195.62 217.88 208.77 258.45 173.88

– – – – 8.12 27.88 16.24 11.55 6.12

0.15 0.17 0.19 0.12 0.22 0.47 0.60 0.67 0.55

1.20 1.24 1.23 0.89 2.02 3.69 5.26 5.37 4.83

ns ns ns ns * *** *** *** ***

Symbols: Target (8) – angle subtended by the target; Control – all white surround; n – sample size; E (8) – expected direction of orientation, which is directly away form the center of the target; a (8) – mean direction of orientation; Deviation from expected – angular difference between the expected angle (E) and the orientation direction (a); r – mean vector length; V,u – calculated statistic for the V test; Sig. – indicates whether megalopae were either not significantly oriented (ns) or had significant orientation at the P , 0.05 (*), P , 0.01 (**) or P , 0.001 (***) levels.

58 and 3508 in Offshore Water, all targets but 108 and 3508 in Seagrass Odor Water, and all targets but 58, 908, and 3508 in Predator Odor Water (Table 2). In all three types of water, they oriented away from the white 108 sector when the 3508 target was present (Table 2).

5.2. Visual experiment: orientation to vertically striped targets Targets with vertical strips were tested as mimics for the pattern of seagrass stalks and perhaps a school of small fish. Patterns with overall widths of 208 and 908 were used because in most cases intermolt and premolt megalopae oriented away from solid dark

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Table 2 Orientation of Premolt megalopae to solid targets in Offshore, Seagrass Odor, Predator Odor Waters Target (8) Offshore Water Control 5 7.5 10 15 20 90 180 350 Seagrass Water Control 5 7.5 10 15 20 90 180 350 Predator Water Control 5 7.5 10 15 20 90 180 350

n

E (8)

a (8)

Deviation from expected

r

V,u

Sig.

43 28 31 34 32 41 45 35 31

75.00 182.50 183.75 185.00 187.50 190.00 225.00 270.00 357.00

– – 183.85 176.15 232.37 180.00 201.58 266.64 167.34

– – 0.10 8.85 44.87 10.00 23.42 3.36 187.66

0.148 0.243 0.506 0.560 0.299 0.461 0.444 0.745 0.346

0.365 1.500 4.175 4.175 1.845 4.108 3.863 6.109 2 2.703

ns ns *** *** * *** *** *** **

36 31 36 39 46 36 39 38 39

255.00 182.50 183.75 185.00 187.50 190.00 225.00 270.00 355.00

– 194.50 198.25 – 165.59 179.55 238.24 248.83 174.36

– 12.00 14.50 – 21.91 10.45 13.24 21.17 180.64

0.137 0.364 0.510 0.213 0.248 0.344 0.488 0.611 0.299

1.099 2.805 4.193 0.693 2.207 2.874 4.198 4.970 2 2.640

ns ** *** ns * ** *** *** **

42 47 41 39 42 39 20 18 20

140.00 182.50 183.75 185.00 187.50 190.00 225.00 270.00 355.00

– – 204.48 157.65 187.26 171.25 – 252.95 168.60

– – 20.73 27.35 0.24 18.75 – 17.05 186.40

0.250 0.067 0.240 0.319 0.376 0.331 0.347 0.810 0.331

0.731 0.586 2.035 2.506 3.443 2.768 1.085 4.648 2 2.083

ns ns * * *** ** ns *** *

Symbols are as described for Table 1.

targets of these sizes (Tables 1 and 2). For comparison, responses to 208 and 908 solid targets (from Tables 1 and 2) are included in Tables 3 and 4. The striped targets had patterns of either alternating 28 black and white stripes (N 5 narrow pattern) or alternating 48 white stripes combined with 28 black stripes (W 5 wide pattern). Orientation by intermolt megalopae to all three types of vertical stripes (20N, 90N, 90W) was significant and away from the targets (Table 3). In contrast, five of the nine tests with premolt megalopae and stripes had no significant orientation (Table 4). Premolt megalopae failed to orient to the 908 narrow pattern in Offshore Water and the 208 narrow pattern in Seagrass Odor Water. The most dramatic behavioral change was the general absence of orientation in Predator Odor Water, which indicates that in the presence of a vertical stripe pattern in Predator Odor Water premolt megalopae swim in all directions. Direct observation indicated that these megalopae either remained motionless or swam rapidly in a random direction but upon encountering the arena wall, they stopped swimming, pulled in their legs and remained motionless. In the presence of

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Table 3 Orientation of Intermolt megalopae to solid and striped targets in Offshore, Seagrass Odor, and Predator Odor Waters Target (8)

n

Offshore Water 20SOL 47 20NST 44 90SOL 47 90NST 34 90WST 45 Seagrass Water 20SOL 36 20NST 36 90SOL 36 90NST 27 90WST 41 Predator Water 20SOL 39 20NST 44 90SOL 42 90NST 44 90WST 36

E (8)

a (8)

Deviation from expected

r

V,u

Sig.

190 190 225 225 225

211.03 191.47 224.97 200.71 189.55

21.03 1.47 0.03 24.29 34.45

0.54 0.56 0.63 0.47 0.40

4.88 5.20 6.12 3.52 3.02

*** *** *** *** **

190 190 225 225 225

160.89 173.86 209.03 207.50 195.04

29.11 16.14 15.97 17.50 29.96

0.32 0.33 0.24 0.29 0.44

2.34 2.68 1.96 2.04 3.46

* ** * * ***

190 190 225 225 225

217.88 185.25 208.77 217.31 251.02

27.88 4.76 16.24 7.69 26.02

0.47 0.37 0.60 0.54 0.22

1.20 1.24 1.23 0.89 2.02

*** *** *** *** *

Symbols: Target (8)-target description; 20SOL-208 solid target; 20NST-208 narrow striped target with vertical black and white stripes 28 wide; 90SOL-908 solid target; 90NST-908 narrow striped target with narrow vertical black and white stripes 28 wide; 90WST-908 wide striped target with alternating black lines 28 wide and white lines 48 wide. The other symbols are as described for Table 1.

Table 4 Orientation of Premolt megalopae to solid and striped targets in Offshore, Seagrass Odor and Premolt Odor Waters Target (8)

n

Offshore Water 20SOL 41 20NST 39 90SOL 45 90NST 46 90WST 41 Seagrass Water 20SOL 36 20NST 31 90SOL 39 90NST 35 90WST 45 Predator Water 20SOL 39 20NST 39 90SOL 20 90NST 22 90WST 48

E (8)

a (8)

Dev. from expected

r

V,u

Sig.

190 190 225 225 225

180.00 177.61 201.58 – 226.78

10.00 12.39 23.42 – 1.78

0.461 0.234 0.444 0.225 0.236

4.108 2.020 3.863 1.352 2.133

*** ** *** ns *

190 190 225 225 225

179.55 – 238.24 190.72 189.20

10.45 – 13.24 34.28 35.80

0.344 0.141 0.488 0.328 0.316

2.874 1.092 4.198 2.268 2.431

** ns *** * *

190 190 225 225 225

171.25 – – – –

18.75 – – – –

0.331 0.247 0.347 0.252 0.029

2.768 1.113 1.085 1.046 0.007

** ns ns ns ns

Symbols are as described for Table 3.

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Table 5 Dunnett test for proportions of Intermolt megalopae responding to similar size striped and solid targets in different test waters Water type

Test comparison

q

Significance

Offshore Water

20NST (69.73) vs. 20SOL (75.82) 90NST (55.55) vs. 90SOL (75.82) 90NST (55.55) vs. 90WST (71.57) 20NST (58.05) vs. 20SOL (58.05) 90NST (69.73) vs. 90SOL (66.42) 90NST (69.73) vs. 90WST (58.05) 20NST (69.73) vs. 20SOL (62.03) 90NST (47.29) vs. 90SOL (58.05) 90NST (47.29) vs. 90WST (64.90)

2.04 6.33 4.95 0.67 1.08 3.65 2.46 2.97 5.00

ns P , 0.01 P , 0.01 ns ns P , 0.01 P , 0.05 P , 0.05 P , 0.01

Seagrass Odor Water

Predator Odor Water

Target symbols are as described in Table 3. The target size and type, arcsin transformed mean percent response (in parentheses), absolute Dunnett statistic (q) and statistical significance are shown. ns indicates not significant.

Offshore and Seagrass Odor Waters megalopae continued to swim after encountering the arena wall.

5.3. Visual experiments: comparison of responses to solid and vertical striped targets To determine if responses to solid and striped targets differed, the proportion of megalopae showing directional responses were compared for the same size targets. In Offshore Water, the percent response of intermolt megalopae to 208 narrow striped (20NST) and 208 solid (20SOL) targets were not significantly different (Table 5). However, responses to the different 908 targets were statistically different (Table 5). In Seagrass Odor Water, the only statistical difference was between 908 narrow stripes (90NST) and 908 wide stripes (90WST). In Predator Odor Water, all three comparisons were statistically different (Table 5). Thus, intermolt megalopae showed different responses to different targets of the same size and the pattern of responses varied with water type. In contrast, the response pattern of premolt megalopae was similar in all water types (Table 6). There were no significant differences in responses to (1) 208 solid (20NST) and 208 narrow striped (20NST) targets and (2) 908 narrow striped (90NST) and 908 wide stripes (90WST) targets in all water types. However, there was a consistent difference between responses to the 908 narrow striped target (90NST) and the 908 solid target (90SOL). Thus, premolt megalopae responded differently to 908 targets but the response patterns did not change with water type.

6. Chemical choice experiments Intermolt and premolt megalopae were tested for directed movement in the dark toward the source of flowing water containing different odors. The null hypothesis was that megalopae were equally likely to enter either peripheral chamber in the chemical

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Table 6 Dunnett test for proportions of Premolt megalopae responding to similar size striped and solid targets in different test waters Water type

Test comparison

q

Significance

Offshore Water

20NST (73.57) vs. 20SOL (71.57) 90NST (73.57) vs. 90SOL (64.09) 90NST (62.03) vs. 90WST (64.90) 20NST (41.55) vs. 20SOL (39.23) 90NST (41.55) vs. 90SOL (78.46) 90NST (62.05) vs. 90WST (62.3) 20NST (55.79) vs. 20SOL (62.03) 90NST (56.79) vs. 90SOL (71.57) 90NST (51.94) vs. 90WST (58.05)

0.67 2.84 0.90 0.53 10.10 0.00 1.58 4.60 1.75

ns P , 0.05 ns ns P , 0.01 ns ns P , 0.01 ns

Seagrass Odor Water

Predator Odor Water

Target symbols are as described for Table 3. The target size and type, arcsin transformed mean percent response (in parentheses), absolute Dunnett statistic (q) and statistical significance are shown. ns indicates not significant.

choice apparatus. The control situation had Offshore Water entering both peripheral chambers. There was no significant difference between the proportion of megalopae attracted to either source of Offshore Water (control) for premolt and intermolt megalopae, which indicates there was no orientation bias in the test apparatus (Table 7). When Offshore Water was paired with Seagrass Odor Water, there was no significant difference between the mean proportions of megalopae attracted to Offshore Water and Seagrass Odor Water for both premolt and intermolt megalopae (Table 7). In contrast, when Predator Odor Water was paired with either Offshore or Seagrass Odor Waters, Table 7 Comparison of the mean percentages of intermolt (A) and premolt (B) megalopae attracted to Offshore, Seagrass Odor and Predator Odor Waters in the chemical choice chamber Chambers 1 A. Intermolt Offshore Water Offshore Water Offshore Water Seagrass Water B. Premolt Offshore Water Offshore Water Offshore Water Seagrass Water

Mean

Chambers 2

Mean

q

Null hypothesis

35.67 39.82 44.43 41.55

Offshore Water Seagrass Water Predator Water Predator Water

33.83 33.21 15.34 15.34

0.63 0.88 2.95 3.45

accept accept reject reject

33.21 36.87 46.15 33.21

Offshore Water Seagrass Water Predator Water Predator Water

39.82 35.06 21.13 19.40

1.39 0.71 3.61 2.24

accept accept reject reject

Chambers 1 indicate that water entered one peripheral chamber and mean is the arcsin transformed mean percentages attracted to that chamber. Chambers 2 indicates the water entered the opposite peripheral chamber and mean is the corresponding mean percentage of attracted megalopae. The null hypothesis was that equal proportions of megalopae entered the two peripheral chambers. Percentages were arcsin transformed and compared by a Dunnett test for proportions with q (absolute value) as the test statistic. The hypothesis was accepted (last column), if there was no difference in the proportions and rejected if there was a difference (P , 0.05). The sample size for the control (Offshore Water vs. Offshore Water) was 3 and for the other experiments was 5.

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significantly fewer intermolt (Table 7A) and premolt (Table 7B) megalopae were attracted to the Predator Odor Water. Thus, megalopae avoided water containing predator odor.

7. Discussion The post-larva is a critical stage in the life cycle of the blue crab Callinectes sapidus because it must be transported from offshore areas into estuaries where it settles and metamorphoses. The present study tested the hypothesis that intermolt and premolt megalopae have different behavioral responses to visual and chemical cues. Intermolt megalopae are primarily involved in horizontal transport toward and within estuaries (e.g. Epifanio, 1995; De Vries et al., 1994; Olmi, 1994), while premolt megalopae should be at or near settlement sites in estuaries. Changes in behavior with molt stage should reflect changes in the selective pressures within different environments, whereas uniform behavior by both molt stages implies a common function(s) during transport and at settlement sites. Chemical cues present during onshore transport from offshore areas were tested by exposing megalopae to Offshore Water. Alternatively, chemical cues present at settlement sites in estuaries were odors from aquatic vegetation and predators. Since megalopae preferentially settle in seagrass beds (Orth and van Montfrans, 1987; Olmi et al., 1990), odor from the seagrass Zostera marina was tested to represent chemical cues from a settlement habitat. Odor from the planktivorous fish Fundulus heteroclitus was tested as a predator odor because planktivorous fishes frequently occur in settlement habitats and this species commonly inhabits shallow estuarine areas. Since responsiveness usually varies with odor concentration, the test concentrations were based upon results from past studies of the concentrations of dissolved substances from Z. marina (Forward et al., 1996) and F. heteroclitus (R. Forward unpublished data) that affect metamorphosis of blue crab megalopae and the effects of F. heteroclitus odor on crustacean photoresponses involved in diel vertical migration (McKelvey and Forward, 1995). Directional orientation of intermolt and premolt megalopae to solid dark targets was similar in all test waters (Tables 1 and 2). Targets of 108 and less evoked variable orientation responses, while those between 208 and 1808 consistently induced an avoidance response in which megalopae swam directly away from the middle of the solid target. This behavior is probably a predatory avoidance response in which megalopae respond to large dark objects as predators. Similar responses are observed in ´ et al., 1995a), adult other crustaceans such as megalopae of mangrove crabs (Dıaz hermit crabs (Orihuela et al., 1992) and adult blue crabs (Woodbury, 1986). Upon exposure to the 3508 target, orientation of both intermolt and premolt megalopae reversed, as they swam away from the 108 white sector toward the middle of the black sector. This response may represent shelter seeking behavior. The 108 white sector could be the entrance to a shelter, and megalopae are moving further into the shelter by swimming away from the entrance. In contrast, the behavior of intermolt and premolt megalopae was different when

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37

presented with patterns that simulated stalks of seagrass. Intermolt megalopae oriented away from all patterns in all water conditions (Table 3). Premolt megalopae generally oriented away in Offshore and Seagrass waters but failed to move in a consistent direction in the presence of Predator Odor Water (Table 4). The absence of orientation by either intermolt or premolt megalopae toward a striped pattern when Seagrass Odor was present suggests that vision is not used for orientation toward seagrass beds. The absence of this visual response is consistent with the observation that up estuary transport and probably settlement in specific sites primarily occurs at night (Dittel and Epifanio, 1982; Brookins and Epifanio, 1985; Mense and Wenner, 1989; Little and Epifanio, 1991; De Vries et al., 1994; Olmi, 1994) when visual cues are absent. In fact, orientation away from the seagrass pattern in the presence of seagrass odor suggests that settlement in seagrass beds during the day would be inhibited by visual cues. Several results indicate that visually directed predator avoidance responses vary with molt stage. First, in the presence of predator odor water intermolt megalopae swam away from striped targets (Table 3), whereas premolt megalopae show non-directional responses (Table 4). Second, when considering the proportions of megalopae that responded in different test situations, the significant difference for intermolt megalopae varied with water type (Table 5). In contrast, the pattern of statistical significance for responses of premolt megalopae was consistent in all water types (Table 6). When considering specific responses, intermolt megalopae orient away from all large dark objects and patterns (except the 3508 target) in all types of water (Tables 1 and 3). Since megalopae are probably transported from offshore waters into estuaries in the intermolt stage, they are consistently in the water column and a uniform directional predator avoidance response may be expected in different water types. In contrast, premolt megalopae do not occur in offshore areas (Wolcott and De Vries, 1994) and are more likely to occur at benthic settlement sites in estuaries, such as seagrass beds (Orth and van Montfrans, 1987; Olmi et al., 1990). In the absence of predator odor, premolt megalopae responded to targets by swimming away. In the presence of predator odor, the range of behavioral responses expands. When presented with a solid target, premolt megalopae either remained motionless or swam rapidly away. Either response would serve to avoid predators. The results with targets resembling the underwater pattern of seagrass stalks in the presence of Predator Odor (Table 4) suggests that the pattern did not mimic seagrass but rather appeared as a school of small fish to the megalopae. Premolt megalopae responded to this stimulus with a startle response in which they swam randomly in all directions. The response of megalopae in the chemical choice chamber indicates (1) they can move relative to a chemical cue in a current stream and (2) both molt stages of megalopae have the same responses to the test chemical cues. Both molt stages failed to discriminate between Offshore Water and Seagrass Odor water, which suggests that arrival at seagrass beds does not involve chemically cued up-current swimming. In addition, both molt stages avoided water containing predator odor, which suggests settlement would not occur in areas having levels of fish odor as tested in the present study. These studies support the hypothesis that intermolt and premolt blue crab megalopae have different behavioral responses to visual and chemical cues. The results suggest that

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neither molt stage uses visual nor chemical cues for oriented movement toward settlement sites, such as seagrass beds. In contrast, megalopae have a variety of predator avoidance responses. Both molt stages avoided water containing predator odor and swam away directly away from large dark objects, which might represent predators. Premolt megalopae have additional responses in that they can also remain motionless in the presence of predator odor, or may move in random directions in a seagrass bed. Thus, most of the behavioral responses to chemical and visual cues observed in the present study can be interpreted as being involved in predator avoidance.

Acknowledgements This research was supported by funds from the National Science Foundation under grant [ OCE-9216629 (to RBF and DR) and from the Instituto Venezolano de Investigationes Cientifiicas (IVIC) Caracas Venezuela (to HD and BO). We thank D. Blondel for his technical assistance.

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