Jointed Setae – Their Role in Locomotion and Gait Transitions In

Journal of Experimental Marine Biology and Ecology, L 228 (1998) 273±290

Jointed setae ± their role in locomotion and gait transitions in polychaete worms

Rachel Ann Merz* , Deirdre Renee Edwards Department of Biology, Swarthmore College, Swarthmore, PA 19081, USA Received 15 August 1997; received in revised form 3 February 1998; accepted 7 February 1998

Abstract

Many families of polychaete worms have jointed setae in which the joint is external to the body and is not directly controlled by muscles or nerves. We assessed the role of these specialized structures in the hesionid polychaete, Ophiodromus pugettensis, by examining speed, step length, stride distance, stride frequency and gait transitions in worms with and without setal joints. Individual worms were videotaped while they moved over sandy surfaces at a range of speeds. The worms were then anaesthetized and all their compound setae were trimmed either distally or proximally to the setal joints. After two days of recovery the worms were videotaped a second time while they again moved over sandy surfaces at a range of speeds. From the video tapes we analyzed their locomotory performance before and after setal ablation. Animals in which the setae were shortened but in which the joint was left intact showed no consistent change in speed, step length, stride distance, stride frequency or gait transitions. Animals in which the joint had been removed both changed gaits at slower speeds (walking to undulatory walking and undulatory walking to swimming) and showed a signi®cant decrease in maximum swimming speeds and stride distance. A subset of data containing only cases where the worms were moving at the same speed in the same gait before and after setal ablation was analyzed. In these instances, after the removal of the joint, the worms had signi®cantly smaller stride distances and compensated for this by increasing stride frequency. In O. pugettensis, the undulatory walking gait is analogous to the trot±gallop transition in quadrupedal mammals because the animal switches from moving the appendages on a relatively rigid body to using a combination of body ¯exion and appendage movement to achieve propulsion, however, unlike quadrupedal mammals this transition takes place over a wide range of speeds and at different sites on the body as speed increases. These experiments indicate that jointed setae may be important both in allowing a worm to better control setal contact and traction with the substrate as well as in altering the effectiveness of its swimming stroke.  1998 Elsevier Science B.V. All rights reserved.

Keywords: Annelids; Polychaete worms; Ophiodromus pugettensis; Setae; Locomotion; Gaits

*Corresponding author. Tel.: 1 1 610 3288051; fax: 1 1 610 3288663; e-mail: [email protected]

1. Introduction

Polychaete annelids typically have dorsal and ventral arrays of chitinous setae associated with the parapodia of most segments (Fig. 1). Although these familiar structures are used extensively for identi®cation by taxonomists, there is relatively little known about the way they function on living worms (although, see Mettam, 1971, 1984; Roy, 1974; Knight-Jones and Fordy, 1979; Knight-Jones, 1981; Woodin and Merz, 1987). One major variety of these structures, known as jointed or compound setae, are found in 28 polychaete families (Fauchald, 1977; Fauchald and Rouse, 1997). Com- pound setae are associated with mobile or discreetly mobile worms but never with sedentary polychaetes (as de®ned by Fauchald and Jumars, 1979). Each compound seta is the product of a single cheatoblast and associated follicular cells and extends out of the body from the setal sac within a parapodium (Bobin, 1947; Bauchot-Boutin and Bobin, 1954; Schroeder, 1967, 1984; O'Clair and Cloney, 1974; (Fig. 1)). In compound setae, the joint has a socket in which the distal blade of the seta is typically anchored by both a ligament and a hinge (Gustus and Cloney, 1973) (Fig. 2). A functioning joint, therefore, is external to the body and is neither directly controlled

Fig. 1. SEM of a midbody parapodium of O. pugettensis, viewed from a ventral anterior position. The notopodium is relatively reduced. Compound setae can be identi®ed throughout the neuropodium, scale bar is 200 mm. R.A. Merz, D.R. Edwards / J. Exp. Mar. Biol. Ecol. 228 (1998) 273 ±290 275

Fig. 2. SEM of morphological details of the compound setae of O. pugettensis. (a): Tip of the distal blade, scale bar 5 1 mm. (b): Ventral view of two unbent setal joints, scale bar 5 10 mm. (c): Lateral view of two setal joints with their distal blades displaced slightly to the side, scale bar 5 2 mm. (d): View of the socket of the setal shaft, the base of the distal blade and the ligamentous attachment, scale bar 5 2 mm. by muscles nor innervated by nerves (Gustus and Cloney, 1973). The way in which compound setae bend at the joint is controlled by the shape of the cup and the attachment of the ligament (Gustus and Cloney, 1973; Schroeder, 1984; Merz and Woodin, 1987) (Fig. 2). We are aware of no report in the literature of the direct observation or test of the function of these setae (Schroeder, 1984). Gustus and Cloney (1973) suggest that based on morphology they expect the blade to move relative to the shaft, although it has no intrinsic power. When the seta is thrust against a substrate the 276 R.A. Merz, D.R. Edwards / J. Exp. Mar. Biol. Ecol. 228 (1998) 273 ±290 joint would allow ¯exibility, albeit con®ned to some degree by the hinge and ligament. This ¯exibility would presumably confer increased friction and reduce slippage. To understand if and how these structures function in polychaete locomotion, we compared the locomotory performance of worms with unaltered setae to the same animals' performances after the compound setae had been trimmed either distally or proximally to the joint (the distal treatment is essentially a control for the process of trimming and the effect of shortening setae; the proximal treatment examines the role of the joint per se). If the joint is crucial in locomotion, then we expected a diminution in performance when it is removed. Merely shortening the setae could also have a negative effect on locomotion; if that is true, then animals with setae that are ablated distal to the joint should have a diminished performance. For our test animal we chose the hesionid polychaete, Ophiodromus pugettensis (5 Podarke pugettensis) which lives from British Columbia to the Gulf of California. It is an active, mobile worm that is found in a variety of habitats including muddy bays, rocky shores, among fouling organisms on ¯oats and pilings and in the subtidal to the continental shelf (Morris et al., 1980; Kozloff, 1983). It can be free living with a diet of small invertebrates (Shaffer, 1979; also see Oug, 1980) or can live as a facultative commensal with a number of different partners (e.g., within the ambulacral grooves of star®sh (mainly Pateria) (Hickok and Davenport, 1957; Lande and Reish, 1968), on the holothurian Protankyra bidentata (Okuda, 1936), with the terebellid polychaete Eupolymnia heterobranchia (Shaffer, 1979), or on hermit crabs together with a nereid polychaete (Berkeley and Berkeley, 1948)). O. pugettensis was selected for this project because (1) it readily displays a variety of locomotory gaits, (2) it naturally lives and moves on a variety of substrates, (3) it has a morphology and size that make it amenable to experimental alteration and (4) it readily adapts to life in the laboratory (often occurring as an accidental resident in sea-water tables).

2. Materials and methods

2.1. Collection and housing of animals

Worms were collected by hand during low tide from the mud ¯ats of Garrison Bay, San Juan Island, Washington. They were found crawling on a variety of substrates including the surface of the mud, under and on cockle and clam shells, in mats of algae, and on an old sock. After collection the worms were transferred to sea-water tables at the Friday Harbor Laboratories where they were housed with Enteromorpha and Ulva collected at the same site and presumably inhabited by the copepods and other small invertebrates that are reported to make up the diet of O. pugettensis (Shaffer, 1979). Even though O. pugettensis is often found in the sea-water tables at the laboratories and appears to live well under those circumstances, for these experiments we used only animals that we had collected from the ®eld within the previous ten day period. R.A. Merz, D.R. Edwards / J. Exp. Mar. Biol. Ecol. 228 (1998) 273 ±290 277

2.2. Videotaping techniques

We used only active worms whose setae, parapodia and body wall appeared to be undamaged. The worms were relatively similar in size (mean length 21.0 mm, S.D.61.6 mm, n 5 15). Each worm was ®lmed both before and after setal trimming (see below) in a 7.2 3 7.2 cm plastic container in which there was a layer of ®ne grain white sand covered with about 1 cm of sea-water. We used a Sony DXC-107 CCD color video camera attached to a Pentax macrolens mounted above the ®eld of view which was illuminated with a GenRad GR 1546 stroboscope held 7.5 cm above the substrate, ¯ashing at 60 ¯ashes/second. The images were taped at 30 frames/second (this rate was con®rmed during data collection). Each taping sequence began by video recording a millimeter scale placed on the sand surface. At a minimum, each worm was recorded until it completed at least four episodes of walking, undulatory walking and swimming. If necessary, worms were gently nudged with a blunt probe to encourage them to move through the ®eld of view.

2.3. Video analysis

We used only episodes in which a worm moved at a continuous speed and was in focus throughout the measurement period. For each test episode, the frame by frame video image of the progress of a worm was traced onto acetate sheets. Worm speed was measured by counting the number of frames it took a worm to move along a measured distance. We attempted to analyze each worm throughout its entire speed range. Along with the speed, the gait used in each episode was identi®ed. Preliminary observations revealed that O. pugettensis has two distinct gaits (walking and swimming), with a transitional gait between them (undulatory walking) (Fig. 3). We considered an animal to be walking if its body was held in a relatively rigid linear position and its parapodia moved independently of the motion of the body, regularly contacting the sediment during the step cycle. A swimming worm was propelled by whole-body undulating waves that originated in the tail and were coordinated with parapodial movement. During swimming there was no contact between the solid substrate and the body. Any gait that contained elements of both walking and swimming was considered an undulatory walk In this gait the posterior portion of body undulated while anterior section remained relatively rigid. As speed increased, the amount of body undergoing ¯exion increased (Fig. 3). To examine the position and timing of parapodial movements at different speeds, selected parapodia and their associated scale bar were traced onto acetate sheets from the video images. Typical parapodia on the right and left side of the body were selected for analysis, based on their midbody position and their clear visibility as the animal moved through the ®eld of view. We measured the maximum distance that occurred between these selected parapodia and their adjacent neighbors (5 step length) for as many locomotory cycles as were visible in a given run (where a locomotory cycle is measured as one complete sequence of limb movements from initiation of the power stroke extension through the recovery stroke back to where the selected limb resumes its initial position relative to the body). These values were then averaged for each run to give an 278 R.A. Merz, D.R. Edwards / J. Exp. Mar. Biol. Ecol. 228 (1998) 273 ±290

Fig. 3. Tracings of sets of six consecutive video frames of the same unmodi®ed worm, displaying three different gaits. In each case the worm is moving from left to right, the vertical line was added to the image to provide a reference point to evaluate the progress of the animal, the shaded boxes highlight the movement away from that line, the time elapsed is 0.2 s. The walking speed was 3.75 mm s21 , the undulatory walking speed was 10.71 mm s21 and the swimming speed was 19.57 mm s21 .

estimate of step length at a given speed. We measured stride distance (distance traveled in one complete locomotory cycle) by monitoring how far these target parapodia traveled parallel to the axis of movement during a single locomotory cycle for as many cycles as were visible in a given run; these values were averaged to estimate stride distance at a given speed for an individual worm. The number of frames between repeated stages of the locomotory cycle were counted, averaged for a single worm moving at a given speed and used to calculate stride frequency.

2.4. Ablation techniques

To trim setae, individual worms were anaesthetized by isolating them in a petri dish with a small amount of sea-water. Isotonic MgCl was slowly dropped into the water until the worms were quiescent (usually a matter of a few minutes). While viewing the animals under a dissection microscope, the setae were trimmed with iridectomy scissors. All the compound setae on an individual worm were trimmed either immediately distal or proximal to the joints (depending on the assigned treatment regime) (Fig. 4). The distal and proximal cuts removed approximately 9 and 12%, respectively, of the total setal length. These distances were measured on 20 setae selected at random from ®ve mid-body parapodia of a preserved animal (21 mm in length). The mean total setal length for these setae was 1.066.13 mm; mean distance to the point of distal cut was 0.106.03 mm, and the mean distance to the point of a proximal cut was 0.136.03 mm). The ablation procedure took about 10 to 20 min. The ¯uid around the worm was then exchanged with fresh sea-water. When the worm began to move it was returned to the R.A. Merz, D.R. Edwards / J. Exp. Mar. Biol. Ecol. 228 (1998) 273 ±290 279

Fig. 4. Schematic illustration of the experimental modi®cations of the jointed setae of O. pugettensis (worm illustrations modi®ed from Uschakov, 1955; Banse and Hobson, 1974; Shaffer, 1979). The worm is illustrated from the dorsal view. The parapodia are drawn from the anterior view.

sea table and transferred to a small ¯oating plastic container made of ®ne mesh screening that allowed fresh sea-water to move constantly through the container. Within a few minutes worms typically regained their normal posture and behavior. Only those worms that appeared to have no behavioral changes or physical damage from this treatment were subsequently used for comparison. The worms were allowed to recover for two days before being videotaped for a second time, after which they were held for a period of 1 to 2 weeks to con®rm that they continued to show no side effects of the ablation procedure. The worms were then preserved in a 10% buffered formalin solution.

2.5. Scanning electron microscopy

To examine setal morphology, unaltered formalin ®xed specimens were dehydrated in an ethanol series and held in 100% ethanol for a minimum of 24 h. Dehydrated specimens were then placed in a small amount of hexamethyldisilazane which was allowed to evaporate under a fume hood at room temperature overnight (Dykstra, 1993). After the samples were completely dry, they were mounted on aluminum stubs, and coated with gold±palladium in a Technics Hummer V Sputter Coater. Specimens were examined in a JEOL JSM 35 C scanning electron microscope. 280 R.A. Merz, D.R. Edwards / J. Exp. Mar. Biol. Ecol. 228 (1998) 273 ±290

2.6. Statistical analysis

To insure that there were no initial performance differences between the treatment groups, the initial top speeds reached at each gait were compared using unpaired, two-tailed t-tests. The top speeds reached at a given gait before and after setal manipulation were compared within each treatment group using paired, two-tailed t-tests. To examine what elements of the locomotory cycle might be affected by setal manipulation, we compared step length, stride distance and stride frequency at the top speed reached at a given gait before and after setal manipulation. Within each treatment group we used paired, two-tailed t-tests. The only consistently signi®cant effects were associated with worms with setae trimmed proximally to the joint. Within that group we extracted all the records (n 5 29) for instances in which an individual worm had been recorded moving at the same speed (de®ned as two locomotory bouts with a measured speed within 1 mm s21 of each other) in the same gait (walking, undulatory walking or swimming) before and after the joints were removed. For these samples we compared whether step length, stride duration or stride frequency had increased or decreased compared to the original performance of the unmodi®ed worm. The frequencies of the response of each of these variables were compared with a G-test using William's correction for the two-cell case and assuming a null hypothesis of 0.50 frequency of occurrence in each instance (Sokal and Rolf, 1995).

3. Results

3.1. Characterization of locomotory cycle

O. pugettensis increase their speed of locomotion by altering different elements of the locomotory cycle. One element of this increase in speed comes from changes in the maximum distance between any two adjacent parapodia (5 step length) during the locomotory cycle. (This change in distance between parapodia is due to changes in the relative positions of the parapodia but not changes in whole segment length, which when measured at the middorsum of an individual remains constant over the range of speeds exhibited in any particular gait.) At very slow speeds the maximum distance between adjacent parapodia is small, and as speed increases so does this distance. For all ®fteen pre-operative worms this inter-parapodial step length reaches a maximum after which it no longer increases with speed (an example for a single individual is given in Fig. 5(a)). Another mode by which speed is increased is for the animals to increase stride distance ± the distance traveled by the body from one foot fall to the next. This increase in distance is accomplished both by the size of the ``step'' (as described above), and, also by the amount the body is pushed forward by the action of other segments when a given parapodium is in its recovery phase. In about half of the unmodi®ed worms we observed, this trait increased throughout the observed range of speeds (Fig. 5(b)). In the other half, this quantity leveled off to a fairly constant maximum value at the highest speeds. Worms can also increase their rate of locomotion by increasing stride frequency (the number of strides per second). Again, about half of the unmodi®ed worms we R.A. Merz, D.R. Edwards / J. Exp. Mar. Biol. Ecol. 228 (1998) 273 ±290 281

Fig. 5. The relationship between step length, stride distance and stride frequency with speed for a representative unmodi®ed worm (number 13, Fig. 7). Circles indicate walking, squares indicate undulatory walking, and triangles indicate swimming.

observed increased stride frequency throughout the range of speed, whereas the others reached a plateau above which stride frequency did not increase (Fig. 5). Worm gaits, as de®ned by our other criteria (see Methods and Materials), were not de®nitively associated with speci®c changes in these elements of the locomotory cycle (Fig. 5). In most cases, however, the maximum inter-parapodial step length was achieved in either faster walking speeds or in the slower speeds of the undulatory- 282 R.A. Merz, D.R. Edwards / J. Exp. Mar. Biol. Ecol. 228 (1998) 273 ±290 walking gait. Likewise, if stride distance and stride frequency leveled off, it was always within the swimming gait.

3.2. Comparison of locomotion in worms with and without joints

There was no signi®cant difference in the locomotory performance (as measured by the maximum speed at each gait) between the treatment groups before the setae were trimmed (walking, df 5 13, t 5 0.574, P 5 0.5756; undulatory walking, df 5 13, t 5 0.422, P 5 0.6800; swimming, df 5 13, t 5 1.322, P 5 0.2089). When setae were

Table 1 The results of paired two-tail t-tests comparing the locomotory performance for O. pugettensis, before and after their compound setae have been trimmed either distal or proximal to the joints Mean (S.D.) of Mean df t-value P-value maximum values difference for untrimmed after worms trimming Distal treatment (n 5 7) Walking speed (mm s21 ) 8.78 (6.39) 2 2.93 6 2 1.463 0.1938 step length (mm) 0.92 (0.16) 0.01 6 0.104 0.9204 stride distance (mm) 3.15 (0.91) 2 0.88 6 2 2.740 0.0338 stride frequency (strides s21 ) 2.38 (0.82) 0.50 6 3.066 0.0220 Undulatory walk speed (mm s21 ) 21.47 (4.77) 2 2.54 6 2 1.054 0.3324 step length (mm) 1.04 (0.15) 0.07 6 1.211 0.2715 stride distance (mm) 5.21 (0.82) 2 0.57 6 2 1.023 0.3456 stride frequency (strides s21 ) 4.09 (0.63) 2 0.19 6 2 0.191 0.5126 Swimming speed (mm s21 ) 43.75 (7.37) 0.67 6 0.240 0.8181 step length (mm) 1.17 (0.22) 2 0.07 6 2 1.406 0.2093 stride distance (mm) 8.33 (1.80) 0.12 6 0.166 0.8737 stride frequency (strides s21 ) 5.56 (0.88) 0.02 6 0.031 0.9765 Proximal treatment (n 5 8) Walking speed (mm s21 ) 10.47 (5.04) 2 4.20 7 2 2.518 0.0399 step length (mm) 1.04 (0.17) 2 0.01 7 2 0.307 0.7676 stride distance (mm) 3.15 (1.15) 2 0.86 7 2 2.466 0.0431 stride frequency (strides s21 ) 2.89 (0.61) 2 0.28 7 2 0.833 0.4324 Undulatory walk speed (mm s21 ) 22.79 (6.95) 2 7.99 7 2 3.844 0.0063 step length (mm) 1.09 (0.17) 0.00 7 0.023 0.9822 stride distance (mm) 5.75 (2.13) 2 1.84 7 2 3.109 .0171 stride frequency (strides s21 ) 3.98 (0.56) 2 0.05 7 2 0.169 0.8706 Swimming speed (mm s21 ) 50.16 (10.79) 2 11.66 7 2 5.892 0.0006 step length (mm) 1.12 (0.14) 2 0.05 7 2 1.048 0.3294 stride distance (mm) 9.24 (2.25) 2 2.26 7 2 2.825 0.0256 stride frequency (strides s21 ) 5.70 (0.94) 2 0.31 7 2 0.798 0.4510 In each case the values described and compared are those at the top speed of a given gait. R.A. Merz, D.R. Edwards / J. Exp. Mar. Biol. Ecol. 228 (1998) 273 ±290 283 shortened, but the joints were left intact, locomotory performance as measured by top speed at each gait was unchanged (Table 1). In contrast, when the joints were removed from the setae, worms did not move as fast at a particular gait and thus switched to the next gait at lower speeds (Table 1, Fig. 6). Examples of the performance of two typical individuals are illustrated in Fig. 6. In the case of the animal with the distal ablation, there is a similar range of speeds exhibited within each gait, and the maximum speed at a particular gait is quite similar before and after the removal of the setal tip (Fig. 6a). In contrast, when a worm has had its joint removed, it has a slower maximum speed at each gait and subsequently switches to the next faster gait at a lower speed (Fig. 6b). A comparison of the change in the top locomotory speed for each animal at each gait before and after setal ablation reveals that the animals that lost only the setal blade distal to the joint had no particular pattern of change (striped bars, Fig. 7). About half of these

Fig. 6. Examples of changes in locomotory performance after ablation of setae either distal or proximal to the joint for two worms. (a): The setae of this worm (number 10, Fig. 7) were trimmed distally, leaving the joint intact. Its initial performance is indicated by the gray triangles and its posttrimming performance by the black triangles. There is little difference in performance in top speed at a given gait or in the speeds at which the worm changes gaits. (b): The setae of this worm (number 9, Fig. 7) were trimmed just proximal to the joint. Its initial performance is indicated by gray circles and its posttrimming performance by black circles. Without the joint, the worm's top speed at a given gait is diminished and it switches to the next gait at a slower speed. 284 R.A. Merz, D.R. Edwards / J. Exp. Mar. Biol. Ecol. 228 (1998) 273 ±290

Table 2 The results of a G-test comparison of the difference in step length, stride distance and stride frequency for 29 instances when the same speed and gait (walking, undulatory walking or swimming) were recorded in a worm before and after the setal joints had been removed Number of Number of Number of G value P value positive negative positive or responses responses negative responses predicted by the null hypothesis Step length 15 14 14.5 0.0344 . 0.750 Stride distance 8 21 14.5 2 8.0338 , 0.005 Stride frequency 21 8 14.5 8.0338 , 0.005 worms showed a faster performance at a given gait before the ablation and about half the worms actually had faster performances after the ablation. The pattern for those animals that had the joint removed is distinctly different ± in almost all cases top speed for a particular gait was lower after ablation than before (black bars, Fig. 7). When we compared differences in step length, stride distance and stride frequency at the top speed for each animal at each gait we found that the two treatment groups had different patterns. The animals that retained their setal joints did not show any consistent signi®cant change in these variables after distal trimming (Table 1). In the walking gait, although neither speed nor step length was signi®cantly different after trimming the setae, there was a signi®cant decrease in stride distance and an increase in stride frequency (Table 1). For the undulatory-walking and swimming gaits there were no signi®cant differences in any of the locomotory characteristics that were measured. In contrast, the animals in which the joints had been removed had a stride distance that was consistently signi®cantly smaller after joint ablation in each gait (Table 1). There were no signi®cant differences in either step length or stride frequency at the gait transitions for these jointless-worms (Table 1). Because we know that the top speed at a particular gait is diminished for worms without their joints, we also compared step length, stride distance and stride frequency for 29 cases where a proximally trimmed worm had been recorded moving at the same speed (within 1 mm s21 ) within a gait (any gait) before and after treatment. We found that these animals have no difference in step length before and after ablation, but do have a signi®cantly diminished stride distance and a signi®cantly faster stride frequency (Table 2). Thus, these joint-less worms have diminished forward progress with each stride cycle after their setae are trimmed. Therefore, to attain the same speed at the same gait they increase the rate of the stride cycle

4. Discussion

The value of the conclusions that can be drawn from this experiment hinge on whether or not the effects seen by ablation of the setae proximal to the joint are a product of the lack of the joint or a result of the setae being shorter. Two things argue that the effect is due primarily to the loss of the joint. The ®rst is that these results are R.A. Merz, D.R. Edwards / J. Exp. Mar. Biol. Ecol. 228 (1998) 273 ±290 285

Fig. 7. Comparison of the change in the top locomotory speed for each gait before and after setal ablation for all worms. In the animals with distal ablations but intact joints there was no consistent pattern of change (diagonal striped lines). In the animals with proximal ablations there was a general diminution of performance after the joint was removed as measured by the maximum speed at a given gait (solid black bars). compared with those from animals whose setae were shortened without loosing the joint. These animals lost about 9% of their total setal length and showed no consistent signi®cant change in any of the locomotory parameters that were measured and had no signi®cant diminution in speed. The second argument addresses the concern that the worms with jointless setae did still have slightly shorter setae than did the worms with proximally trimmed setae. The jointless worms' setae were shortened by approximately 12% of their total length. We suggest that it is unlikely that this estimated 3% difference in setal length (between the treatment groups) is responsible for the difference in performance between worms with and without joints because the setal bundle is a dynamic structure that can be extended or withdrawn depending on the muscular action within the parapodium. In particular, the setae within polychaete parapodia are bundled together and held within setal sacs. There can be several setal sacs within a single parapodium. Each setal 286 R.A. Merz, D.R. Edwards / J. Exp. Mar. Biol. Ecol. 228 (1998) 273 ±290 sac has an attachment to intrinsic parapodial protractor muscles that run between the setal sac and the parapodial wall (Mettam, 1967, 1971; Lawry, 1971). Setal sacs also have a connective tissue attachment to an aciculum, a robust internal seta that is attached to intrinsic parapodial retractor and protractor muscles. Contraction of the chaetal retractor muscles pulls the aciculum deeper into the parapodium and subsequently causes the setal sac and its associated setae to withdraw. Contraction of the protractor musculature pulls the setal sac towards the parapodial surface, causing the setae to be extended and the aciculum to move into its more distal position (Mettam, 1967, 1971; Lawry, 1971). Thus, the movement of the setae is accomplished indirectly by muscular attachment to the setal sac and aciculum rather than attachment of muscles to individual setae and, to a large degree, the extent of the external extension of setae is under the control of the worm. When we observed O. pugettensis under a dissection microscope moving at a range of speeds over natural substrates we could see the movement of the setal bundles. It was rare that the setae ever appeared to be fully extended; these details, however, were more dif®cult or impossible to see at the fastest speeds. Thus, we expect that for most of its speed range, O. pugettensis can easily compensate for a 3% difference in setal length and therefore the difference in performance of the two treatment groups is associated with the presence or absence of the joint rather than the difference in setal length. So, in what way do passive joints at the ends of setae function? Our suggestion is that when the parapodia are in cyclic contact with the surface, these joints allow the setal tips to bend in such a way as to increase the setal contact with the substrate during the power stroke. This allows a parapodium to have more effective ground contact with an unpredictably irregular surface. During the recovery stroke when the setae are lifted off the substrate, the joints passively resume their normal position to be recon®gured during the next contact with the surface. We have observed this phenomenon many times. That it is likely to contribute to the effective locomotion of the worm is demonstrated by the fact that worms that lack these joints have diminished stride distances (distance the body is moved forward with each locomotory cycle) even though step lengths (the maximum distance between adjacent parapodia during the locomotory cycle) are not signi®cantly different (Tables 1 and 2). Because of reduced traction, each cycle of the limbs with jointless setae produces less forward movement of the worm than when the joints are intact (Tables 1 and 2). For an aquatic pedestrian that must contend with drag and buoyancy in addition to gravity, the ability to maintain contact with the substratum is particularly important (Martinez, 1996), and jointed setae may greatly increase the effectiveness of contact of each parapodial step. An additional issue is the role of sculpture or teeth on the distal blades of setae. Their shape suggests that they might have a role in increasing the friction between setae and the substratum. The fact, however, that the distally trimmed worms which lacked the portion of the blades with serrations did not suffer a signi®cant diminution in locomotory performance suggests that these features did not contribute signi®cantly to performance in this instance. But, since these experiments only measured performance on sand, it may be that the serrations are more important in providing traction on different surfaces. Because we have not been able to observe closely the action of these jointed setae during swimming, we have less understanding of how the joints function in this gait. R.A. Merz, D.R. Edwards / J. Exp. Mar. Biol. Ecol. 228 (1998) 273 ±290 287

Rough-bodied polychaetes, like O. pugettensis swim by means of drag-based thrust production (Clark and Tritton, 1970; Clark, 1976; Vogel, 1994). Given this mode, one could hypothesize that the setal joints make the power and recovery strokes more effective. In the power stroke the setae could be fully extended increasing the amount of water pushed by the moving parapodium. In the recovery stroke the joints would allow the setae be folded back against the body, diminishing the drag on the parapodium as it moves forward. A similar mechanism has been demonstrated for the passively controlled setal blades of the aquatic mite Limnochares americana (Barr and Smith, 1979). There are a couple of problems, however, with this interpretation for swimming O. pugettensis. The ®rst is that the worms with trimmed setae, but intact joints, do not have a signi®cant decrease in swimming performance (Fig. 7, Table 1). In that case, the setal blades aren't available to assist the rest of the parapodium to push the water during the power stroke, and so we would expect those animals with shortened setae to be slower at this gait if this were the mechanism by which jointed setae function in swimming. Second, an examination of pelagic worms reveals that although many are reported to have jointed setae (e.g., Alciopidea, Lopadorhynchidae, Pontodoridae; Fauchald, 1977) the joints of the compound setae of at least one alciopid, Rhynchonerella, are reinforced in such a way that they are not particularly ¯exible in any way at the joint (pers. obs). In species that go through a metamorphosis from a benthic form to a reproductive swimming epitoke, the new swimming setae are dramatically modi®ed. The blades are ¯attened and enlarged into an oar-shaped structure, the joint socket is much deeper and narrower (Schroeder, 1967). This change in morphology substantially stiffens the joint and restricts ¯exure of the setae at the joint (pers. obs.). Thus, at least some polychaetes that do a substantial amount of swimming (as opposed to the presumably rare escape-type swimming exhibited by O. pugettensis) have setae that lack or have lost the ability to bend at a predetermined joint. This still leaves us with the problem of why a worm with setae with intact joints but lacking blades doesn't show a diminution in swimming performance, whereas, the loss of joint and setal blade results in a signi®cant loss in ability (Fig. 7, Table 1). It would be interesting to experimentally ``lock'' the joint so that ¯exure at it is not possible, however, the small size of the structures and the necessity of animals remaining in sea-water has thwarted our attempts to glue or stiffen the setae at the joints. It may be that high-speed close-up video will reveal what the normal motion of the intact parapodia and setae are during swimming strokes and will thus give a clue to function of the joint under these circumstances. As polychaetes move from a stationary position through walking to swimming, they display different gaits. During walking, the worm's body is resting on the substratum and forward progress is a result of the sequential action of individual parapodia moving in a step cycle along the body. The intrinsic muscles of a parapodium lift it from the substrate, move it obliquely forward where the tip of the neuropodium contacts the substrate ( point d' appui, Foxon, 1936) and then pushes against the surface in a power stroke causing the associated section of the worm's body to pivot over the point of contact. (This process has been described by a number of authors including Gray, 1939; Mettam, 1967, 1971, 1984; Lawry, 1971). In swimming, the worm's body is moving through the water (only rarely or incidentally contacting the substrate) and whole body waves caused by the alternate contractions of the longitudinal muscles provide 288 R.A. Merz, D.R. Edwards / J. Exp. Mar. Biol. Ecol. 228 (1998) 273 ±290 propulsion. In some cases these body waves are coordinated with power and recovery strokes of the parapodia (Gray, 1939, 1968; Clark and Clark, 1960; Clark and Tritton, 1970; Clark, 1976). The neuropodia are no longer in contact with the ground and there is no sense of ``poling''. At least in some worms the action of the parapodia augments the ¯ow of water along the length of the worm and adds to the propulsive stroke (Gray, 1968; Clark and Clark, 1960; Clark and Tritton, 1970; Clark, 1976). Between these two extreme gaits, different species of polychaetes may exhibit an intermediate gait in which the weight of the body is still largely on the substrate, the parapodia continue to move through their step patterns and where the extra-parapodial contractions of the longitudinal musculature cause large whole body sinusoidal movement. In some cases authors have named and described this intermediate gait (e.g., ``rapid crawling'', Gray, 1939) in others its existence is only implied by the description of the worm's locomotion (Manton, 1973). This intermediate gait in O. pugettensis (``undulatory walking'') is characterized by the anterior segments continue to perform walking movements with individual parapodia while the posterior segments exhibit body waves from the contraction of longitudinal muscles. This gait is transitional along the worm's body ± as the worm's speed increases the fraction of the body participating in the undulatory movement increases, moving anteriorly until the whole body is consumed in the contractions and the animal is swimming. Although each of these three gaits operates over a range of speeds and is uniquely and clearly identi®able, the undulatory walking gait is transitional between the other two. The undulatory walking gait in Ophiodromus is analogous to the trot-gallop transition in small quadrupedal mammals in the sense that the animal switches from locomotion where the appendages move on a relatively rigid body to using a combination of body ¯exion and appendage movement to achieve propulsion. In mammals (Heglund et al., 1974; Heglund and Taylor, 1988) (with many fewer legs than polychaetes), or an arthropod with a stiff body (e.g., crabs) (Blickhan et al., 1993; Full and Weinstein, 1992) the trot-gallop transition takes place at a relatively speci®c speed. In polychaetes such as Ophiodromus that have many pairs of appendages and a ¯exible body, this transition may take place over a wider range of speeds and at different places on the body of the individual as its speed increases. Other species of polychaetes, depending on their morphology and habitat, have variations on walking and swimming gaits. In the relatively stiff-bodied amphinomid, Chloeia, swimming is achieved by an increase in the rate of the power-stroke of the parapodia without any undulation of the body (Mettam, 1984). Locomotion in these aquatic pedestrians is challenged by a wider variety of physical forces and a different scale of landscape topography than is typical of the more widely studied terrestrial vertebrates. In addition, the polychaete body is replete with structures whose functions are largely unknown even though they may be associated with everyday activities. The data in this paper demonstrate that joints of compound setae have a crucial role in effective locomotion for at least some polychaetes and that considerations of gaits and gait transition need to include the possibilities offered by the complex modular morphology of their ¯exible bodies.

Acknowledgements

We would like to thank Friday Harbor Laboratories and Dennis Willows for providing R.A. Merz, D.R. Edwards / J. Exp. Mar. Biol. Ecol. 228 (1998) 273 ±290 289 facilities as well as the Schwartz family for allowing us to collect animals from their dock. Brian Clark, Barbara Best and Sally Woodin provided interesting, helpful discussions about locomotion and worms. Two anonymous reviewers provided detailed and stimulating comments; Anne Rawson and Brian Clark gave editorial suggestions. Jacob Weiner and Peter Stoll provided advice about statistics. Emi Horikawa and Meg Spencer of the Swarthmore Science Library helped with obtaining a variety of references and Tom Bradley graciously helped with Russian transliteration. This work was funded by a Swarthmore Faculty Research Grant to R.A.M. and a Howard Hughes Medical Institute Undergraduate Biological Science Education Program Grant (No 71194- 505802) to D.R.E.

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