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Force production during pereiopod power strokes in Calanus finmarchicus
Journal of Marine Systems 49 (2004) 133 – 144 www.elsevier.com/locate/jmarsys
Force production during pereiopod power strokes in Calanus finmarchicus P.H. Lenz *, A.E. Hower, D.K. Hartline Be´ke´sy Laboratory of Neurobiology, Pacific Biomedical Research Center, University of Hawaii Manoa, 1993 East-West Road, Honolulu, HI 96822, USA Received 18 October 2002; accepted 7 May 2003 Available online 14 March 2004
Abstract Copepods achieve the dramatic speeds of 300 – 1000 body lengths/s during escape reactions. We investigated the details of this behavior in Calanus finmarchicus. Pereiopod movements during the power strokes were monitored in tethered individuals using high-speed video, while simultaneously measuring force production. At 8 – 10 jC, the power strokes for each pair of pereiopods registered as separate peaks in the force record, with the largest force being produced by the fourth and third pairs. Peak forces of 500 – 600 AN were attained within 3.5 ms of initiation of the power stroke sequence. During the power strokes, the pereiopods maximized their surface area by splaying distal segments and setae. During the return stroke, the pereiopods and the setae collapsed, minimizing surface area and thus generating only a weak reverse force. During power stroke sequences in free swimming C. finmarchicus, multiple peaks in acceleration (200 m s 2) corresponded to the power strokes of different pereiopod pairs. During the escape, C. finmarchicus produced maximum swimming speeds of 800 mm s 1 and an estimated muscle-mass specific power output of 300 W kg 1. Comparisons to other organisms indicate that this behavioral performance is particularly powerful and fast. How the copepods achieve this performance remains to be determined. D 2004 Elsevier B.V. All rights reserved. Keywords: Kinematics; Energetics; Escape behavior; Startle reflex; High speed video
1. Introduction The pursuit of understanding the dynamics of biological communities through detailed modeling efforts depends on an accurate representation of an animal’s behavior. However, given the relative paucity of behavioral data on plankton, the contribution of behavior is often underestimated in plankton population dynamics models (review: Yamazaki et al., 2002). * Corresponding author. Tel.: +1-808-956-8003; fax: +1-808956-6984. E-mail address: [email protected] (P.H. Lenz). 0924-7963/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jmarsys.2003.05.006
Behavior modifies the way in which animals interact with each other, with the community and with the environment (e.g., Forward and Ritschoff, 2000). The summation of individual behavioral responses may affect growth, reproductive and survival rates on the population level (e.g., Paffenho¨fer et al., 1995; Saiz et al., 1992). Copepods, the most abundant metazoan plankton in the oceans, are an important link in the food chain, being preyed upon by a wide variety of invertebrate and vertebrate predators. Copepods respond to threats with escape responses that propel them out of harms way at 500 body lengths/s (e.g., Strickler, 1975; Buskey et al., 2002). Species-specific
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differences in the escape behavior include stimulus thresholds, response latencies, swim velocities and accelerations (Fields and Yen, 1997; Lenz and Hartline, 1999; Lenz et al., 2000; Viitasalo et al., 2001; Buskey et al., 2002). The specific characteristics of the startle response in a species of copepod may ultimately determine the effectiveness of the escape from any particular predator (see Eiane et al., 2002). The escape is generated by the metachronal sequence of power strokes of the swimming legs (Storch, 1929; Strickler, 1975). During the return stroke, the swimming legs minimize their surface area and move synchronously to their original position (Strickler, 1975; Alcaraz and Strickler, 1988). The metachronal pattern moves water efficiently with minimal interference between pereiopods (Van Duren and Videler, 2003). This sequence of power and return strokes is produced multiple times in quick succession during an escape (Lenz and Hartline, 1999). Calanoid copepods with prosome lengths of 2 –3 mm (Calanus helgolandicus: Svetlichnyy, 1987; Undinula vulgaris: Lenz and Hartline, 1999) produce forces of 500 –1000 AN and energy outputs estimated at 8 10 7 J during the pereiopod power stroke sequence. Per gram of muscle, this is one of the higher rates of energy output yet reported in cold-blooded animals. The organization of the pereiopods (musculature, segmentation and setation patterns) is conserved in the calanoids (Boxshall, 1985; Huys and Boxshall, 1991). Maximizing and minimizing of the surface area during the power and return strokes has been described in other copepod species, as well as other aquatic invertebrates (Strickler, 1975; Morris et al., 1985; Alcaraz and Strickler, 1988; Wootton, 1999). Detailed temporal sequences of force development during the power strokes can be obtained with submillisecond resolution using force transducer records (Svetlichnyy, 1987; Alcaraz and Strickler, 1988; Lenz and Hartline, 1999). These show that copepods can generate a range of behavioral responses, as reflected in variations in the force-development time course. Quantitative differences in the force transients are a result of differences in the appendage movements during the power strokes. Technical difficulties involved in visualizing copepod swimming behavior as well as in measuring force production at a high temporal resolution have limited the ability to make the observations simultaneously in a single study (but
see Svetlichnyy, 1987; Alcaraz and Strickler, 1988). In the current paper, we recorded force production during the escape while recording the appendage movement in tethered Calanus finmarchicus using high-speed video. We examine how differences in the power and return strokes are translated into specific patterns of force production. Furthermore, we examine how these patterns translate into propulsive movement in free-swimming C. finmarchicus escaping from a mechanical stimulus.
2. Materials and methods 2.1. Collection and maintenance of copepods C. finmarchicus were collected in Frenchman Bay off Mount Desert Island (44j25.7VN, 66j11.8VW) by towing a 0.5-m diameter net (353-Am mesh) at subsurface from a slow moving boat (2001). In 2002, C. finmarchicus were collected with a net (500 Am mesh, 1 m diameter) towed vertically from 150 m depth in the Gulf of Maine off Mt. Desert Rock. Plankton collections were immediately diluted and, within 1 h, C. finmarchicus individuals were sorted into 3.5- and 1.75-l jars. Animals were kept at 7 – 9 jC in a 12h light – dark cycle. They were fed a mixture of Tetraselmis sp., Gymnodinium sanguinium (strain B4), Heterocapsa triquetra (strain HT984) and Oxyrrhis marina (strain CCMP1795) three times per week (inocula obtained from Dr. R. Campbell, U. Rhode Island). Individuals were kept up to 6 weeks in the jars prior to the behavioral experiments. During this time, a number of individuals molted from a copepodite stage 5 (CV) to adult. Only responsive individuals with intact antennae were used in the experiments. 2.2. Experimental set-up The set-up for the behavior experiments has been described in detail elsewhere (Lenz and Hartline, 1999). Briefly, individual copepods (prosome length: 2.5 –2.8 mm) are tethered to a force transducer with cyanoacrylate adhesive (KrazyGlue, Borden, Columbus, OH) and transferred to the experimental dish. In the dish, the tethered copepod was positioned ca. 2 mm from the edge of a 3-mm diameter sphere (2001) or cylinder (2002). The sphere was attached to a
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Fig. 1. Example of force transient produced by an adult male C. finmarchicus in response to a hydrodynamic stimulus. The hydrodynamic stimulus, 1.5 cycle at 700 Hz of vertical movement of a 3-mm sphere, was initiated at T0. Pr or preparatory movement was the initial disturbance in the force record prior to the force transient. Lat. or latency was measured as the time delay between the beginning of the stimulus and the propulsive force as shown. Force transients had multiple peaks (arrows) and Fmax was measured as the maximum force attained during one or more peak (typically second or third peak). Duration of transient was measured as the time interval from the beginning of the propulsive force to the abrupt termination of the force transient. The rise time (R) indicates the initial period of rapid force development. Cycle was measured as the time between the first peaks in force between two successive kicks.
piezoelectric transducer (Burleigh PZL-060, Exfo, Victor, NY), which was computer driven. The rapid vertical movement of this sphere produced a hydrodynamic disturbance, which decayed with distance from the center of the sphere following the dipole equations (Gassie et al., 1993). The typical stimulus used in these experiments was a trapezoidally modulated 1.5 cycle sine wave of 700-Hz frequency with a maximum vertical excursion of the sphere of 34.5 Am. The force produced during the power strokes was viewed on an oscilloscope and recorded in a computer data file (CSCOPE data acquisition program by Dr. Brad Jones, computer specialist UH; Gassie et al., 1993; Lenz and Hartline, 1999). For each experiment, we determined latency, rise time, peak forces, kick duration, timing of return stroke, number of kicks and kick frequency (Fig. 1). Temporal resolution for the force transducer was set to 0.02 ms. The latency or response delay was measured as the time between stimulus onset (0 ms) and the beginning of the forward propulsive force. A preparatory movement was typical, and occurred as disturbances (as both
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negative and positive forces) just prior to the rapid rise in forward propulsive force. Simultaneously, behavior of the animal was monitored with a high-speed video camera (Kodak Motioncorder Analyzer Model 3000, Rochester, NY) mounted on a dissecting microscope (6 ). Viewing of the copepod was done through the side of the experimental dish at right angles to the hydrodynamic stimulus and the animal attached to the force transducer. Video images were recorded at 1000 frames/s with shutter speed typically 0.001 – 0.0003 s. The stimulus, data acquisition from the force transducer and video recording were synchronized to the nearest 1 ms with data being recorded for 0.7 – 1 s after the stimulus was activated. In addition, we recorded at least 0.3 s of video data prior to the stimulus. The trigger signal from the stimulus ‘‘marked’’ the frame following the signal as the time-origin (t = 0) for the video sequence. Times assigned to the video frames by the camera were thus randomly up to 1 ms delayed compared to force-record times referenced to the stimulus. For some purposes, it was desirable to estimate the timing of video frames with respect to force records more closely than 1 ms. This was done by finding a set of nearly identical force records (e.g., Fig. 2), for which it was presumable that appendage movements were also identical. Corresponding sets of angle measurements for one pair of pereiopods (usually P3) straddling the 90j point were used to interpolate
Fig. 2. Five superimposed force records from a single individual adult male C. finmarchicus in response to a brief hydrodynamic stimulus. Experimental temperature: 8 – 9 jC.
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the time of occurrence of the 90j point in the video sequence. These times varied by 1 ms in the video record, as expected from the random synchronization of the video frames to the stimulus. The relative timing of any particular video sequence from the set within the 1-ms span so determined, allowed the correction of its timing with respect to the stimulus. Raw angles of pereiopods and other appendages with respect to the body axis were measured frame by frame. Except for the 0j, 90j and 180j, actual angles deviated somewhat from measured angles owing to the line of view of the camera not being exactly perpendicular to the plane of pereiopod movement. Scales showing geometric corrections for this are given on the right side of the figures. Temperature in the experimental dish was monitored and kept at 8 – 10 jC, which corresponded to the maintenance temperature for these animals in the laboratory. We worked primarily with adult males, and their typical response to a short hydrodynamic stimulus was one ‘‘kick’’, corresponding to a single metachronal sequence of pereiopod power strokes. The force records were analyzed as indicated in Fig. 1. Video records were analyzed frame-by-frame noting appendage movements. Movement of the urosome, first antennae, cephalic appendages, thoracic segments and pereiopods were noted and correlated with the force record. Escape responses in free-swimming C. finmarchicus were elicited using a 3-mm cylindrical bar attached to the piezoelectric transducer (for details, see Buskey et al., 2002). Video images were analyzed frame-by-frame for appendage movements by the copepod, swim trajectory, acceleration and swim velocities. 2.3. Pereiopod morphology Pereiopod pairs were dissected from preserved animals (5% formalin) and mounted on slides in glycerin. Pereiopods were examined and photographed through a compound inverted microscope (Nikon Diaphot, Tokyo, Japan) with a digital camera (Nikon CoolPix, Tokyo, Japan). Examination of pereiopods included measurements of the lengths of segments (coxa, basis, endopod and exopod), enumeration and measurements of setae and characterizing setae by their setulation patterns. Drawings of the
pereiopods were made from digital photographs and video images. Digital photographs were used to draw the structural details of the pereiopods, including size and shape of segments and setae. The video images were used to compute the position of the segments and setae during the power and return strokes.
3. Results In response to a sufficiently large hydrodynamic stimulus, C. finmarchicus responded after a brief latency with an escape composed of one or more force transients. Each force transient (or ‘‘kick’’; Lenz and Hartline, 1999) corresponded to a set of power strokes by the pereiopods. In C. finmarchicus, the type of response (single vs. multiple kick) depended on the magnitude of the stimulus, experimental temperature, developmental stage and sex. At 8 – 10 jC, adult males typically responded with a single force transient to a brief and small hydrodynamic stimulus (up to three times threshold). Individual experimental animals often responded in a consistent pattern from one stimulus presentation to the next (Fig. 2). The mean duration of the force transients in this individual lasted 12.2 ms (S.D. = 0.5, n = 18). The mean duration of force transients in tethered adult male C. finmarchicus ranged from 8.5 to 12.3 ms at 8 – 10 jC (11 experiments). The coefficient of variation for the duration of the force transient was less than 5% in most experiments (max. coefficient of variation: 8.3%). The first evidence of movement as signaled by a small change in the force record occurred typically between 2 and 3 ms after the stimulus onset (Fig. 3). A comparison among force records from different responses showed that this change in the force was a consistent feature (Figs. 2 and 3), and it corresponded to the ‘‘preparatory movements’’ observed in U. vulgaris (Lenz and Hartline, 1999). To determine what physical movements might contribute to this force, we examined the corresponding video frames (t = 2– 4 ms). The earliest major movement detected was the maxilliped being folded against the body (t = 3 ms in Fig. 3), accompanied by what appeared to be a general tensing of thoracic segments and followed closely by folding of the other cephalic appendages. The folding of the cephalic appendages was completed during the next 2 ms (Fig. 3). The first
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Fig. 3. Early force transients and appendage movements in a C. finmarchicus adult male. Top: video frames corresponding to times 2, 3 and 4 ms following triggering pulse activating a brief hydrodynamic stimulus. First detectable movement by maxilliped (M). Middle graph: raw angular displacement of four appendages with respect to body axis. Time has been adjusted by adding 150 As to video frame times to bring them into register with the force record below. Separate scale on right gives approximate adjustment for parallax for urosome and pereiopod (P5) due to pivot axis of appendage not being along the line of view (ventral side rolled ca. 65j toward camera). Corrections for antennae and maxilliped are less. Lower graph: force record following onset of stimulus (inset). A small artifact partly obscuring the initial response has been digitally subtracted from the record. Inset shows time-course of stimulus displacement.
antennae, fifth pereiopod pair (P5) and urosome moved significantly during the next ms (Figs. 3,4). The initial rapid rise in propulsive force from 0 to 200 AN occurred between 3 and 4 ms, which coincided with the beginning of the first power stroke (P5). The first antennae continued to retract. Since the animal was tethered, this retraction was completely dependent on muscle contractions. In a free-swimming animal, in contrast, the forward
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movement through the water normally would contribute to the folding of the first antennae (see below). Between 4 and 5 ms, the force increased from 200 to over 400 AN, the power stroke of P5 was completed and the first movement in P4 was observed. Thus, the maximum rate of force development, 0.2 N s 1 between 3 and 6 ms, corresponded to the combination of the power strokes of P5 and P4 (Fig. 4). Force transients produced by C. finmarchicus adult males had maximum forces of 640 AN and mean durations of 10.3 ms (five different individuals). The initial rise in propulsive force was followed by four individual peaks. These peaks as shown in the video frames corresponded to the power strokes of the individual pereiopod pairs in metachronal posterior to anterior sequence: P4, P3, P2 and P1. No additional movements were detected in the cephalic appendages. The urosome was elevated by up to 90j or more early in the power stroke sequence, then relaxed to its extended position. As the sequence continued the thoracic segments were progressively hypoextended reaching a maximum at the end of the P1 stroke (Fig. 4, bottom panel). The force correlated with and clearly is produced by the power strokes of the pereiopods. During the power strokes, the pereiopods splayed to maximize their surface area, achieving a fin-shaped form. The coxa and the intercoxal sclerite maintained a fixed Ushape. The basis, however, bent outward to separate the paired endopods and their setae. The exopod segments also bent outward and the setae spread in a fan shape. Estimated surface areas for the pereiopods during the power strokes are given in Table 1. The excursion of each pereiopod pair lasted between 3 and 5.5 ms, with the subsequent pair initiating its power stroke while the previous pair was nearing completion of its stroke (Fig. 4). In one typical experiment (Fig. 4), the intervals between the power strokes of the pereiopod pairs ranged from 2 to 3 ms with a longer delay usually occurring between P3 and P2. For most of the duration of each stroke, an approximately constant angular velocity was maintained (Table 2). Reynolds numbers of over 1000 were calculated for the individual pereiopods during the power stroke (Table 2). The completion of the last power stroke was characterized by a rapid decline to zero force after the last peak and occurred
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Fig. 4. Force record, appendage positions and corresponding video frames from 0 to 35 ms after a brief hydrodynamic stimulus during the power and return strokes. Same run as in Fig. 3. Note that the peaks in the force record are separated by 2 – 3 ms, and they correspond to pereiopod position near the perpendicular. Symbols for pereiopods: P5—open triangle, P4—filled triangle, P3—open diamond, P2—open squares, P1— open circles. During the return stroke, pereiopods return synchronously (filled circles). Bottom plot indicates angular positions of other appendages and the thoracic segments. Modified angular scale on right indicates approximate compensation for parallax owing to the 65j roll of the ventral side toward the camera. Compensation needed for appendages moving in the animal’s horizontal plane is smaller than for pereiopods. Insets show video frames of a pereiopod pair during the power (left) and return (right) strokes.
at 15 –16 ms after the initiation of the hydrodynamic stimulus (Fig. 4). When the animal’s response was limited to a single kick, the recovery period was slow and characterized by very small negative forces (Fig. 4). Prior to and overlapping with the return strokes of the pereiopods, we observed the slow return of the thorax. The initial movement in the pereiopods was noted at ca. 19 ms, and the synchronous return stroke of the pereiopods was complete at 25 ms. A slight negative force of less than 50 AN was measured at ca. 20 ms corresponding to the return stroke (Fig. 4). This minimization of this negative force was achieved by minimizing the surface area of the pereiopods: the coxa, basis, exopod and endopod were linearly arranged, with the setae
overlapping in a narrow central region. The exopod setae came together at the tip in a triangular pattern. Estimates of the surface areas of the pereiopod pairs during the power and return strokes are given in Table 1. Negative forces were further decreased by a bend between the basis and the endopod and exopod as the pereiopods moved forward reducing the drag. Finally, the antennae (first and second) and the maxillipeds returned to their original positions after the pereiopod return strokes (Fig. 4). We observed some modifications in the sequence of appendage movements when C. finmarchicus responded to a stimulus with multiple kicks (Fig. 5). At 8 –10 jC, the frequency of these multiple kick responses ranged from 40 to 60 Hz. In these cases, the
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Table 1 Relative surface areas of pereiopod pairs during the power and return strokes Pereiopod pair
Surface area (mm2) Power stroke
Return stroke
P1 P2 P3 P4 P5
0.46 0.69 0.78 0.98 0.46
0.21 – 0.31 0.30 – 0.46 0.33 – 0.49 0.45 – 0.67 0.21 – 0.31
Lengths and widths of segments and setae were measured digital micrographs of dissected pereiopods. The degree of spreading and collapsing of segments and setae was estimated from video images of tethered copepods during the power and return strokes (Fig. 4). The smaller area for the return stroke assumes a complete overlap of the setae into a triangular shape, whereas the larger area assumes a more rectangular shape.
initial power stroke sequence remained unchanged. However, the return stroke started immediately after the completion of the last power stroke of P1 (12 ms, Fig. 5). In the example shown in Fig. 5, the synchronous return stroke was completed in 6 ms, with a maximum negative force of 100 AN recorded at 17.5 ms. 2.5 ms later, the second power stroke of P5 had already started. The first antennae and cephalic appendages remained in their retracted positions between the first and second series of power strokes, but we observed some flexing of the cephalosome during the return stroke. In Fig. 5, the copepod responded with two force transients. After the second force transient,
Table 2 Peak angular velocities calculated from the linear portion of the pereiopod angle measurements during the power strokes (see Fig. 5) Pereiopod
Angular velocity (rad s 1)
S.D.
Max. width (mm)
Re
P1 P2 P3 P4 P5
700 580 640 800 1060
27 19 38 20 40
1.058 1.316 1.414 1.550 1.122
1100 1400 1700 1600 1400
Angular velocities were calculated for four different responses in an adult male C. finmarchicus with a force transient duration of 12.2 ms at 8 – 10 jC. Maximum width of pereiopod was calculated from measured dimensions of the pereiopods and the spread seen on video images. Angular velocity was assumed to be constant throughout the power stroke for the calculation of Reynolds numbers.
Fig. 5. Force production (bottom) and pereiopod position (top) for a double kick. Temperature: 10.65 jC. Scale at right gives angular parallax correction for the animal positioned with ventral side rotated 45j toward the camera. Symbols for pereiopods same as in Fig. 4 (P5: open triangle, P4: filled triangle, P3: open diamond, P2: open squares, P1: open circles). Pereiopod position during return stroke is a group average since individual pereiopods could not be resolved (filled circles). Video frame time adjusted to make the P1 force peak to correspond to a 90j angle. Animal’s ventral direction rotated 45j toward camera.
the return sequence was similar to the one described for the single kick response: a delayed and significantly slower return stroke by the pereiopods, a smaller negative force, and the return of the other appendages to their original position (Fig. 4). Free-swimming C. finmarchicus responded to the brief hydrodynamic stimulus with a fast start escape response. Fig. 6 shows the escape trajectory produced by the first kick of a multiple-kick response by a CV C. finmarchicus. Visualization of the animal movement was not as detailed as in the tethered animal, in particular the cephalic appendages could not be discerned. The power stroke sequence was faster in the free-swimming animals (6– 7 ms compared to 8.5 – 12 ms), as had been noted for C. helgolandicus by Svetlichnyy (1987). The first movement was detected in the first antennae as the animal turned away from the stimulus (Fig. 6A). Almost simultaneously, we detected the beginning of
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Fig. 6. Reaction pattern of a free-swimming C. finmarchicus CV to a similar hydrodynamic stimulus as used for tethered animals (vertical movement of 3-mm cylinder 1.5 cycle, 700 Hz). (A) Superposition of video images at 5-ms intervals following stimulus (‘‘cyl’’ seen lower left corner). (B) Plot of position of the rostrum of the animal shown in A at 1-ms intervals. (C) Plot of velocity of the rostrum at 1 ms intervals calculated as the distance between two consecutive positions divided by 1 ms. (D) Plot of acceleration of the rostrum at 1-ms intervals calculated similarly. Grey bars indicate approximate time intervals for power strokes of pereiopod pairs.
the first power stroke. The initial rapid rise in acceleration from 0 to 200 m s 2 coincided with the power strokes of P5 closely followed by P4. The first antennae were streamlined with the cephalothorax by the 6-ms frame. A consistent deceleration was observed between the power strokes of P3 and P2, which correlated with a relatively long interval between power strokes of these two pairs of pereiopods also observed in the tethered copepods. The power stroke of P1 occurred during the deceleration following the last peak. The velocity of the copepod increased throughout the power stroke sequence reaching a maximum of 800 mm s 1 at 8 ms. Subsequently, the velocity slowly declined as the animal continued to coast through the water. During a period of 14 ms, the copepod traveled ca. 7 mm, which translates into an average velocity of 500 mm s 1. A second power stroke sequence followed and the animal left the field of view.
4. Discussion Similar to other aquatic invertebrates, copepods employ two distinct forms of locomotion. The first, used for slow swimming, involves the rhythmic beating of the cephalic appendages (Strickler, 1982). Many of the filter-feeding copepods gather food particles during this slow locomotory activity (Koehl and Strickler, 1981; Poulet and Gill, 1988). Power strokes of the thoracic swimming legs, or pereiopods, are used in ‘‘hops’’ (Tiselius and Jonsson, 1990; Buskey et al., 2002), as well as in escape responses (Strickler, 1975). This metachronal sequence of power strokes is modulated. Relatively small velocities and accelerations are generated during the hop and sink behavior (Buskey et al., 2002). In contrast, during an escape, the copepod responds to a threat with a ‘‘fast start’’ reaction with a short latency and rapid rise in swim velocity as shown in tethered and free-swim-
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ming individuals (Lenz and Hartline, 1999; Lenz et al., 2000; Buskey et al., 2002; Buskey and Hartline, 2003). The responses we recorded for C. finmarchicus in response to the hydrodynamic stimulus were the strong, ‘‘fast start’’ type. As expected in drag-based swimming, the peaks of force corresponded to the near-perpendicular position of the pereiopod with respect to the body axis. Based on this evidence, there appears to be no contribution from jet propulsion. This is not unexpected given that the splayed pereiopods extend well beyond the lateral margins of the urosome in their remoted (end of power stroke) position. At 8– 10 jC, the measured angular velocities in C. finmarchicus ranged from 580 to over 1000 rad s 1. The pereiopod pair that initiates the power stroke sequence (P5) had the highest angular velocities. The lowest angular velocities were measured for P2 and P3, which also have the largest surface area. The power strokes in C. finmarchicus (Table 2) were comparable to those in C. helgolandicus (2.5 –2.8 mm prosome length; angular velocities < 900 rad s 1; Svetlichnyy, 1987) and faster than in the smaller Cyclops scutifer (0.7 mm prosome length), where angular velocities ranged from 480 to 610 rad s 1 at 20 jC (Alcaraz and Strickler, 1988). Applying scaling effects measured in dytiscid water beetles using power strokes (Nachtigall, 1977), a significantly slower angular velocity would be predicted for the larger C. finmarchicus (2 –3 mm prosome length). This suggests that C. finmarchicus is performing at a higher muscular output rate than C. scutifer. Mechanical power output of muscle has been estimated for many different organisms using one of three methods: (1) measurements of metabolic rates (respiration rates) during locomotion, (2) physiological measurements of work loops in semi-isolated muscles and (3) determination of costs calculated from kinematics of limb movements (Josephson, 1997). Using the latter approach, we can apply the methods of Blake (1986) to the splayed paddle-shaped pereiopod pairs (see inset Fig. 4). The total resistive work predicted (assuming no added-mass effects) can be calculated from Eq. (11) of Blake (1986) (multiplying P˜ by tp):
W ¼ P˜ tp ¼ K
R Z tp
Z 0
0
m2n Xcrdrdt
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where K = 1/2qCd (q is the density of water and Cd is the coefficient of drag), R is the length of the appendage, X is the angular velocity of the appendage, mn is the velocity of an element of appendage area normal to its surface, c is the chord of the appendage perpendicular to its long axis and tp is the time the power stroke takes. For angular velocities given in Table 2 with a sweep angle of 150j and a coefficient of drag of unity, this comes to 1.2 AJ. This is comparable to the 0.8 AJ reported for C. helgolandicus (Svetlichnyy, 1987) and U. vulgaris (Lenz and Hartline, 1999). 1.2 AJ at a repeat frequency of 50 Hz gives power outputs of 60 AW. Although the exact ratio of power stroke muscle to body mass is unknown for C. finmarchicus, 20% of wet weight (0.2 mg) is a conservative estimate (Bennet-Clark and Lucey, 1967). This would give a muscle-mass specific power output of 300 W kg 1. This is similar to that calculated for another copepod (Undinula vulgaris), but significantly higher than has been found in other organisms (Table 3). Fast-start responses in aquatic organisms are generated via C-and S-starts in fishes and some invertebrates, jet propulsion in squids, tail-flips in lobsters and other malacostracans and metachronal sequences of power strokes in copepods. As shown in Fig. 7, maximum velocities produced during fast-start escape responses increase as a power function of animal length in fishes ( y = 45.3x0.68, r2 = 0.830). The escape performances of lobsters (Nephrops norvegicus: Stentiford et al., 2000; Panulirus interruptus: Nauen and Shadwick, 1999), crayfish (Orconectis virilis: Webb, 1979), Daphnia pulicaria (Brewer et al., 1999), squid (Loligo vulgaria: Packard, 1969), pteropod (Clione limacine: Satterlie et al., 1977), mosquito larvae (Culex pipiens: Brackenbury, 1999), damselfly larvae (Enallagma spp.: McPeek et al., 1996) and tadpoles (Limnodynastes peronii: Wilson and Franklin, 2000) fall close to the fish maximum velocity vs. animal length relationship. In contrast, maximum velocities for the shrimp (Crangon crangon: Arnott et al., 1998; Pandalus danae: Daniel and Meyho¨fer, 1989) and the copepods (Acartia tonsa, A. lilljeborgii: Buskey et al., 2002; C. finmarchicus: present study; Euchaeta rimana: Yen and Strickler, 1996) are higher than what would be expected from length-velocity relationship of the fishes (Fig. 7). The copepods outperform the fishes by approximately an order of magnitude. This
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Table 3 Mechanical muscle power output in organisms with high energy output Organism
Species
Muscle
Temp. (jC)
Power (W kg 1)
Source
Copepod
Undinula vulgaris Calanus finmarchicus Athous haemorrhoidalis Manduca sexta
Pereiopod remotor Skeletal M4 Indirect flight
20 – 22, 8 – 10 25 40
400 300 145 90
Neoconocephalus triops Bombus terrestris
Metathoracic wing Asynchronous flight muscle Flight
30
266
Lenz and Hartline (1999), present study Evans (1973) Stevenson and Josephson (1990) Josephson (1984)
40
100
Josephson (1997)
24
80
Iliofibularis Myotomal
42 20
154 143
Soleus Extensor digitorum longus Flight
37 35 40
94 107 111 – 171
Click beetle Hawkmoth Katydid Bumblebee Fruit fly Lizard Fish
Drosophila melanogaster Dipsosaurus dorsalis Serranus cabrilla
Mouse
Mus musculus
Birds
Various
high performance in the free-swimming copepods is in agreement with the high muscular output estimated for the power stroke (Table 3). Thus, these two independent methods of quantifying behavioral per-
b
Fig. 7. Maximum swimming velocities plotted against animal size. Different taxa are represented by different symbols: — calanoid copepods (present study; Yen and Strickler, 1996; Buskey et al., 2002), — cladocerans (Brewer et al., 1999), w — malacostracans (Webb, 1979; Nauen and Shadwick, 1999; Stentiford et al., 2000), x — shrimp (Daniel and Meyho¨fer, 1989; Arnott et al., 1998), +— aquatic insect larvae (McPeek et al., 1996; Brackenbury, 1999), 5 — mollusks (Packard, 1969; Satterlie et al., 1977), q—tadpole (Wilson and Franklin, 2000), o — fishes (Williams et al., 1996; Domenici and Blake, 1997; Shepherd et al., 2000). Regression line ( y = 45.3x0.68, r2 = 0.830) computed for fish data only.
Lehmann and Dickinson (1997) Swoap et al. (1993) Wakeling and Johnston (1998) Askew and Marsh (1998), James et al. (1995) Marden (1987), Ellington (1991)
formance support the conclusion that the copepods have an exceptional escape response. How they achieve this performance is still uncertain, and it may be indicative of specializations in muscle functions at the cellular and sub-cellular level that may be unique to this group of animal. Successful predator evasion depends on the timely detection of an approaching threat and a timely and effective locomotory response. Using a siphon stimulus, Viitasalo et al. (2001) quantified the response distance as well as the effectiveness of the escape. In their experimental set-up, the distance from the siphon before an escape was initiated combines detection and latency, whereas the behavioral response quantifies the locomotory performance of the escape. Not surprisingly, species differed in both escape distance and effectiveness, which in turn affected individual species’ ability to escape from predatory attacks (Viitasalo et al., 1998). Hays et al., (1994, 1997) suggest that C. finmarchicus’ dominance and ability to persist in the surface layers is due to a particularly effective escape behavior. Indeed, C. finmarchicus has a myelinated nervous system with a shorter reaction time than the non-myelinated vertically migrating Augaptiloids (Lenz et al., 2000). However, the locomotory contribution to the escape may be just as important. Compared to other species studied, C. finmarchicus
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appears to have a particularly powerful and fast motor response.
Acknowledgements We would like to thank Bob Campbell, Ted Durbin, Pat Hassett and Melissa Wagner for assistance with algal cultures and information on maintenance of C. finmarchicus; Hinano Akaka, Brad Jones, Brian Kodama, Wilson McCord, Ted Murphy and Gabriel Rodrigues for critical technical support; Ian Cooke and Pat Hassett for loan of equipment; and the staff at Mount Desert Island Biological Laboratory (MDIBL) for assistance with logistics. Thanks also to Josef Ackerman, who provided many useful suggestions that greatly improved the manuscript. This work was supported by the Salisbury Cove Research Fund of MDIBL and National Science Foundation OCE99-06223.
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Storch, O., 1929. Die Schwimmbewegung der Copepoden, auf Grund von Mikro-Zeitlupenaufnahmen analysiert. Verhaltungen der Deutschen Zoologischen Gesellschaft. Zoologischer Anzeiger Supplement 4, 118 – 129. Strickler, J.R., 1975. Swimming of planktonic Cyclops species (Copepoda, Crustacea): pattern, movements and their control. In: Wu, T.Y.-T., Brokaw, C.J., Brennan, C. (Eds.), Swimming and Flying in Nature, vol. 2. Plenum Press, New York, pp. 599 – 613. Strickler, J.R., 1982. Calanoid copepods, feeding currents, and the role of gravity. Science 218, 158 – 160. Svetlichnyy, L.S., 1987. Speed, force and energy expenditure in the movement of copepods. Oceanology 27, 497 – 502. Swoap, S.J., Johnson, T.P., Josephson, R.K., Bennett, A.F., 1993. Temperature, muscle power output and limitations on burst locomotor performance of the lizard Dipsosaurus dorsalis. The Journal of Experimental Biology 174, 185 – 197. Tiselius, P., Jonsson, P.R., 1990. Foraging behaviour of six calanoid copepods: observations and hydrodynamic analysis. Marine Ecology Progress Series 66, 23 – 33. Van Duren, L.A., Videler, J.J., 2003. Escape from viscosity: the kinematics and hydrodynamics of copepods foraging and escape swimming. The Journal of Experimental Biology 206, 269 – 279. Viitasalo, M., Kiørboe, T., Flinkman, J., Pedersen, L.W., Visser, A.W., 1998. Predation vulnerability of planktonic copepods: consequences of predator foraging strategies and prey sensory abilities. Marine Ecology Progress Series 175, 129 – 142. Viitasalo, M., Flinkman, J., Viherluoto, M., 2001. Zooplanktivory in the Baltic Sea: a comparison of prey selectivity by Clupea harengus and Mysis mixta, with reference to prey escape reactions. Marine Ecology Progress Series 216, 191 – 200. Wakeling, J.M., Johnston, I.A., 1998. Muscle power output limits fast-start performance in fish. The Journal of Experimental Biology 201, 1505 – 1526. Webb, P.W., 1979. Mechanics of escape responses in crayfish (Orconectes virilis). The Journal of Experimental Biology 79, 245 – 263. Williams, P.J., Brown, J.A., Gotceitas, V., Pepin, P., 1996. Developmental changes in escape performance of five species of marine larval fish. Canadian Journal of Fisheries and Aquatic Sciences 53, 1246 – 1253. Wilson, R.S., Franklin, C.E., 2000. Effect of ontogenetic increases in body size on burst swimming performance in tadpoles of the striped marsh frog Limnodynastes peronii. Physiological and Biochemical Zoology 73, 142 – 152. Wootton, R.J., 1999. Invertebrate paraxial locomotory appendages: design, deformation and control. The Journal of Experimental Biology 202, 3333 – 3345. Yamazaki, H., Mackas, D.L., Denman, D.L., 2002. Coupling smallscale physical processes with biology. In: Robinson, A.R., McCarthy, J.J., Rothschild, B.J. (Eds.), The Sea, vol. 12. Wiley, New York, pp. 51 – 112. Chap. 3. Yen, J., Strickler, J.R., 1996. Advertisement and concealment in plankton: what makes a copepod hydrodynamically conspicuous? Invertebrate Biology 115, 191 – 205.
Force production during pereiopod power strokes in Calanus finmarchicus P.H. Lenz *, A.E. Hower, D.K. Hartline Be´ke´sy Laboratory of Neurobiology, Pacific Biomedical Research Center, University of Hawaii Manoa, 1993 East-West Road, Honolulu, HI 96822, USA Received 18 October 2002; accepted 7 May 2003 Available online 14 March 2004
Abstract Copepods achieve the dramatic speeds of 300 – 1000 body lengths/s during escape reactions. We investigated the details of this behavior in Calanus finmarchicus. Pereiopod movements during the power strokes were monitored in tethered individuals using high-speed video, while simultaneously measuring force production. At 8 – 10 jC, the power strokes for each pair of pereiopods registered as separate peaks in the force record, with the largest force being produced by the fourth and third pairs. Peak forces of 500 – 600 AN were attained within 3.5 ms of initiation of the power stroke sequence. During the power strokes, the pereiopods maximized their surface area by splaying distal segments and setae. During the return stroke, the pereiopods and the setae collapsed, minimizing surface area and thus generating only a weak reverse force. During power stroke sequences in free swimming C. finmarchicus, multiple peaks in acceleration (200 m s 2) corresponded to the power strokes of different pereiopod pairs. During the escape, C. finmarchicus produced maximum swimming speeds of 800 mm s 1 and an estimated muscle-mass specific power output of 300 W kg 1. Comparisons to other organisms indicate that this behavioral performance is particularly powerful and fast. How the copepods achieve this performance remains to be determined. D 2004 Elsevier B.V. All rights reserved. Keywords: Kinematics; Energetics; Escape behavior; Startle reflex; High speed video
1. Introduction The pursuit of understanding the dynamics of biological communities through detailed modeling efforts depends on an accurate representation of an animal’s behavior. However, given the relative paucity of behavioral data on plankton, the contribution of behavior is often underestimated in plankton population dynamics models (review: Yamazaki et al., 2002). * Corresponding author. Tel.: +1-808-956-8003; fax: +1-808956-6984. E-mail address: [email protected] (P.H. Lenz). 0924-7963/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jmarsys.2003.05.006
Behavior modifies the way in which animals interact with each other, with the community and with the environment (e.g., Forward and Ritschoff, 2000). The summation of individual behavioral responses may affect growth, reproductive and survival rates on the population level (e.g., Paffenho¨fer et al., 1995; Saiz et al., 1992). Copepods, the most abundant metazoan plankton in the oceans, are an important link in the food chain, being preyed upon by a wide variety of invertebrate and vertebrate predators. Copepods respond to threats with escape responses that propel them out of harms way at 500 body lengths/s (e.g., Strickler, 1975; Buskey et al., 2002). Species-specific
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differences in the escape behavior include stimulus thresholds, response latencies, swim velocities and accelerations (Fields and Yen, 1997; Lenz and Hartline, 1999; Lenz et al., 2000; Viitasalo et al., 2001; Buskey et al., 2002). The specific characteristics of the startle response in a species of copepod may ultimately determine the effectiveness of the escape from any particular predator (see Eiane et al., 2002). The escape is generated by the metachronal sequence of power strokes of the swimming legs (Storch, 1929; Strickler, 1975). During the return stroke, the swimming legs minimize their surface area and move synchronously to their original position (Strickler, 1975; Alcaraz and Strickler, 1988). The metachronal pattern moves water efficiently with minimal interference between pereiopods (Van Duren and Videler, 2003). This sequence of power and return strokes is produced multiple times in quick succession during an escape (Lenz and Hartline, 1999). Calanoid copepods with prosome lengths of 2 –3 mm (Calanus helgolandicus: Svetlichnyy, 1987; Undinula vulgaris: Lenz and Hartline, 1999) produce forces of 500 –1000 AN and energy outputs estimated at 8 10 7 J during the pereiopod power stroke sequence. Per gram of muscle, this is one of the higher rates of energy output yet reported in cold-blooded animals. The organization of the pereiopods (musculature, segmentation and setation patterns) is conserved in the calanoids (Boxshall, 1985; Huys and Boxshall, 1991). Maximizing and minimizing of the surface area during the power and return strokes has been described in other copepod species, as well as other aquatic invertebrates (Strickler, 1975; Morris et al., 1985; Alcaraz and Strickler, 1988; Wootton, 1999). Detailed temporal sequences of force development during the power strokes can be obtained with submillisecond resolution using force transducer records (Svetlichnyy, 1987; Alcaraz and Strickler, 1988; Lenz and Hartline, 1999). These show that copepods can generate a range of behavioral responses, as reflected in variations in the force-development time course. Quantitative differences in the force transients are a result of differences in the appendage movements during the power strokes. Technical difficulties involved in visualizing copepod swimming behavior as well as in measuring force production at a high temporal resolution have limited the ability to make the observations simultaneously in a single study (but
see Svetlichnyy, 1987; Alcaraz and Strickler, 1988). In the current paper, we recorded force production during the escape while recording the appendage movement in tethered Calanus finmarchicus using high-speed video. We examine how differences in the power and return strokes are translated into specific patterns of force production. Furthermore, we examine how these patterns translate into propulsive movement in free-swimming C. finmarchicus escaping from a mechanical stimulus.
2. Materials and methods 2.1. Collection and maintenance of copepods C. finmarchicus were collected in Frenchman Bay off Mount Desert Island (44j25.7VN, 66j11.8VW) by towing a 0.5-m diameter net (353-Am mesh) at subsurface from a slow moving boat (2001). In 2002, C. finmarchicus were collected with a net (500 Am mesh, 1 m diameter) towed vertically from 150 m depth in the Gulf of Maine off Mt. Desert Rock. Plankton collections were immediately diluted and, within 1 h, C. finmarchicus individuals were sorted into 3.5- and 1.75-l jars. Animals were kept at 7 – 9 jC in a 12h light – dark cycle. They were fed a mixture of Tetraselmis sp., Gymnodinium sanguinium (strain B4), Heterocapsa triquetra (strain HT984) and Oxyrrhis marina (strain CCMP1795) three times per week (inocula obtained from Dr. R. Campbell, U. Rhode Island). Individuals were kept up to 6 weeks in the jars prior to the behavioral experiments. During this time, a number of individuals molted from a copepodite stage 5 (CV) to adult. Only responsive individuals with intact antennae were used in the experiments. 2.2. Experimental set-up The set-up for the behavior experiments has been described in detail elsewhere (Lenz and Hartline, 1999). Briefly, individual copepods (prosome length: 2.5 –2.8 mm) are tethered to a force transducer with cyanoacrylate adhesive (KrazyGlue, Borden, Columbus, OH) and transferred to the experimental dish. In the dish, the tethered copepod was positioned ca. 2 mm from the edge of a 3-mm diameter sphere (2001) or cylinder (2002). The sphere was attached to a
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Fig. 1. Example of force transient produced by an adult male C. finmarchicus in response to a hydrodynamic stimulus. The hydrodynamic stimulus, 1.5 cycle at 700 Hz of vertical movement of a 3-mm sphere, was initiated at T0. Pr or preparatory movement was the initial disturbance in the force record prior to the force transient. Lat. or latency was measured as the time delay between the beginning of the stimulus and the propulsive force as shown. Force transients had multiple peaks (arrows) and Fmax was measured as the maximum force attained during one or more peak (typically second or third peak). Duration of transient was measured as the time interval from the beginning of the propulsive force to the abrupt termination of the force transient. The rise time (R) indicates the initial period of rapid force development. Cycle was measured as the time between the first peaks in force between two successive kicks.
piezoelectric transducer (Burleigh PZL-060, Exfo, Victor, NY), which was computer driven. The rapid vertical movement of this sphere produced a hydrodynamic disturbance, which decayed with distance from the center of the sphere following the dipole equations (Gassie et al., 1993). The typical stimulus used in these experiments was a trapezoidally modulated 1.5 cycle sine wave of 700-Hz frequency with a maximum vertical excursion of the sphere of 34.5 Am. The force produced during the power strokes was viewed on an oscilloscope and recorded in a computer data file (CSCOPE data acquisition program by Dr. Brad Jones, computer specialist UH; Gassie et al., 1993; Lenz and Hartline, 1999). For each experiment, we determined latency, rise time, peak forces, kick duration, timing of return stroke, number of kicks and kick frequency (Fig. 1). Temporal resolution for the force transducer was set to 0.02 ms. The latency or response delay was measured as the time between stimulus onset (0 ms) and the beginning of the forward propulsive force. A preparatory movement was typical, and occurred as disturbances (as both
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negative and positive forces) just prior to the rapid rise in forward propulsive force. Simultaneously, behavior of the animal was monitored with a high-speed video camera (Kodak Motioncorder Analyzer Model 3000, Rochester, NY) mounted on a dissecting microscope (6 ). Viewing of the copepod was done through the side of the experimental dish at right angles to the hydrodynamic stimulus and the animal attached to the force transducer. Video images were recorded at 1000 frames/s with shutter speed typically 0.001 – 0.0003 s. The stimulus, data acquisition from the force transducer and video recording were synchronized to the nearest 1 ms with data being recorded for 0.7 – 1 s after the stimulus was activated. In addition, we recorded at least 0.3 s of video data prior to the stimulus. The trigger signal from the stimulus ‘‘marked’’ the frame following the signal as the time-origin (t = 0) for the video sequence. Times assigned to the video frames by the camera were thus randomly up to 1 ms delayed compared to force-record times referenced to the stimulus. For some purposes, it was desirable to estimate the timing of video frames with respect to force records more closely than 1 ms. This was done by finding a set of nearly identical force records (e.g., Fig. 2), for which it was presumable that appendage movements were also identical. Corresponding sets of angle measurements for one pair of pereiopods (usually P3) straddling the 90j point were used to interpolate
Fig. 2. Five superimposed force records from a single individual adult male C. finmarchicus in response to a brief hydrodynamic stimulus. Experimental temperature: 8 – 9 jC.
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the time of occurrence of the 90j point in the video sequence. These times varied by 1 ms in the video record, as expected from the random synchronization of the video frames to the stimulus. The relative timing of any particular video sequence from the set within the 1-ms span so determined, allowed the correction of its timing with respect to the stimulus. Raw angles of pereiopods and other appendages with respect to the body axis were measured frame by frame. Except for the 0j, 90j and 180j, actual angles deviated somewhat from measured angles owing to the line of view of the camera not being exactly perpendicular to the plane of pereiopod movement. Scales showing geometric corrections for this are given on the right side of the figures. Temperature in the experimental dish was monitored and kept at 8 – 10 jC, which corresponded to the maintenance temperature for these animals in the laboratory. We worked primarily with adult males, and their typical response to a short hydrodynamic stimulus was one ‘‘kick’’, corresponding to a single metachronal sequence of pereiopod power strokes. The force records were analyzed as indicated in Fig. 1. Video records were analyzed frame-by-frame noting appendage movements. Movement of the urosome, first antennae, cephalic appendages, thoracic segments and pereiopods were noted and correlated with the force record. Escape responses in free-swimming C. finmarchicus were elicited using a 3-mm cylindrical bar attached to the piezoelectric transducer (for details, see Buskey et al., 2002). Video images were analyzed frame-by-frame for appendage movements by the copepod, swim trajectory, acceleration and swim velocities. 2.3. Pereiopod morphology Pereiopod pairs were dissected from preserved animals (5% formalin) and mounted on slides in glycerin. Pereiopods were examined and photographed through a compound inverted microscope (Nikon Diaphot, Tokyo, Japan) with a digital camera (Nikon CoolPix, Tokyo, Japan). Examination of pereiopods included measurements of the lengths of segments (coxa, basis, endopod and exopod), enumeration and measurements of setae and characterizing setae by their setulation patterns. Drawings of the
pereiopods were made from digital photographs and video images. Digital photographs were used to draw the structural details of the pereiopods, including size and shape of segments and setae. The video images were used to compute the position of the segments and setae during the power and return strokes.
3. Results In response to a sufficiently large hydrodynamic stimulus, C. finmarchicus responded after a brief latency with an escape composed of one or more force transients. Each force transient (or ‘‘kick’’; Lenz and Hartline, 1999) corresponded to a set of power strokes by the pereiopods. In C. finmarchicus, the type of response (single vs. multiple kick) depended on the magnitude of the stimulus, experimental temperature, developmental stage and sex. At 8 – 10 jC, adult males typically responded with a single force transient to a brief and small hydrodynamic stimulus (up to three times threshold). Individual experimental animals often responded in a consistent pattern from one stimulus presentation to the next (Fig. 2). The mean duration of the force transients in this individual lasted 12.2 ms (S.D. = 0.5, n = 18). The mean duration of force transients in tethered adult male C. finmarchicus ranged from 8.5 to 12.3 ms at 8 – 10 jC (11 experiments). The coefficient of variation for the duration of the force transient was less than 5% in most experiments (max. coefficient of variation: 8.3%). The first evidence of movement as signaled by a small change in the force record occurred typically between 2 and 3 ms after the stimulus onset (Fig. 3). A comparison among force records from different responses showed that this change in the force was a consistent feature (Figs. 2 and 3), and it corresponded to the ‘‘preparatory movements’’ observed in U. vulgaris (Lenz and Hartline, 1999). To determine what physical movements might contribute to this force, we examined the corresponding video frames (t = 2– 4 ms). The earliest major movement detected was the maxilliped being folded against the body (t = 3 ms in Fig. 3), accompanied by what appeared to be a general tensing of thoracic segments and followed closely by folding of the other cephalic appendages. The folding of the cephalic appendages was completed during the next 2 ms (Fig. 3). The first
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Fig. 3. Early force transients and appendage movements in a C. finmarchicus adult male. Top: video frames corresponding to times 2, 3 and 4 ms following triggering pulse activating a brief hydrodynamic stimulus. First detectable movement by maxilliped (M). Middle graph: raw angular displacement of four appendages with respect to body axis. Time has been adjusted by adding 150 As to video frame times to bring them into register with the force record below. Separate scale on right gives approximate adjustment for parallax for urosome and pereiopod (P5) due to pivot axis of appendage not being along the line of view (ventral side rolled ca. 65j toward camera). Corrections for antennae and maxilliped are less. Lower graph: force record following onset of stimulus (inset). A small artifact partly obscuring the initial response has been digitally subtracted from the record. Inset shows time-course of stimulus displacement.
antennae, fifth pereiopod pair (P5) and urosome moved significantly during the next ms (Figs. 3,4). The initial rapid rise in propulsive force from 0 to 200 AN occurred between 3 and 4 ms, which coincided with the beginning of the first power stroke (P5). The first antennae continued to retract. Since the animal was tethered, this retraction was completely dependent on muscle contractions. In a free-swimming animal, in contrast, the forward
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movement through the water normally would contribute to the folding of the first antennae (see below). Between 4 and 5 ms, the force increased from 200 to over 400 AN, the power stroke of P5 was completed and the first movement in P4 was observed. Thus, the maximum rate of force development, 0.2 N s 1 between 3 and 6 ms, corresponded to the combination of the power strokes of P5 and P4 (Fig. 4). Force transients produced by C. finmarchicus adult males had maximum forces of 640 AN and mean durations of 10.3 ms (five different individuals). The initial rise in propulsive force was followed by four individual peaks. These peaks as shown in the video frames corresponded to the power strokes of the individual pereiopod pairs in metachronal posterior to anterior sequence: P4, P3, P2 and P1. No additional movements were detected in the cephalic appendages. The urosome was elevated by up to 90j or more early in the power stroke sequence, then relaxed to its extended position. As the sequence continued the thoracic segments were progressively hypoextended reaching a maximum at the end of the P1 stroke (Fig. 4, bottom panel). The force correlated with and clearly is produced by the power strokes of the pereiopods. During the power strokes, the pereiopods splayed to maximize their surface area, achieving a fin-shaped form. The coxa and the intercoxal sclerite maintained a fixed Ushape. The basis, however, bent outward to separate the paired endopods and their setae. The exopod segments also bent outward and the setae spread in a fan shape. Estimated surface areas for the pereiopods during the power strokes are given in Table 1. The excursion of each pereiopod pair lasted between 3 and 5.5 ms, with the subsequent pair initiating its power stroke while the previous pair was nearing completion of its stroke (Fig. 4). In one typical experiment (Fig. 4), the intervals between the power strokes of the pereiopod pairs ranged from 2 to 3 ms with a longer delay usually occurring between P3 and P2. For most of the duration of each stroke, an approximately constant angular velocity was maintained (Table 2). Reynolds numbers of over 1000 were calculated for the individual pereiopods during the power stroke (Table 2). The completion of the last power stroke was characterized by a rapid decline to zero force after the last peak and occurred
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Fig. 4. Force record, appendage positions and corresponding video frames from 0 to 35 ms after a brief hydrodynamic stimulus during the power and return strokes. Same run as in Fig. 3. Note that the peaks in the force record are separated by 2 – 3 ms, and they correspond to pereiopod position near the perpendicular. Symbols for pereiopods: P5—open triangle, P4—filled triangle, P3—open diamond, P2—open squares, P1— open circles. During the return stroke, pereiopods return synchronously (filled circles). Bottom plot indicates angular positions of other appendages and the thoracic segments. Modified angular scale on right indicates approximate compensation for parallax owing to the 65j roll of the ventral side toward the camera. Compensation needed for appendages moving in the animal’s horizontal plane is smaller than for pereiopods. Insets show video frames of a pereiopod pair during the power (left) and return (right) strokes.
at 15 –16 ms after the initiation of the hydrodynamic stimulus (Fig. 4). When the animal’s response was limited to a single kick, the recovery period was slow and characterized by very small negative forces (Fig. 4). Prior to and overlapping with the return strokes of the pereiopods, we observed the slow return of the thorax. The initial movement in the pereiopods was noted at ca. 19 ms, and the synchronous return stroke of the pereiopods was complete at 25 ms. A slight negative force of less than 50 AN was measured at ca. 20 ms corresponding to the return stroke (Fig. 4). This minimization of this negative force was achieved by minimizing the surface area of the pereiopods: the coxa, basis, exopod and endopod were linearly arranged, with the setae
overlapping in a narrow central region. The exopod setae came together at the tip in a triangular pattern. Estimates of the surface areas of the pereiopod pairs during the power and return strokes are given in Table 1. Negative forces were further decreased by a bend between the basis and the endopod and exopod as the pereiopods moved forward reducing the drag. Finally, the antennae (first and second) and the maxillipeds returned to their original positions after the pereiopod return strokes (Fig. 4). We observed some modifications in the sequence of appendage movements when C. finmarchicus responded to a stimulus with multiple kicks (Fig. 5). At 8 –10 jC, the frequency of these multiple kick responses ranged from 40 to 60 Hz. In these cases, the
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Table 1 Relative surface areas of pereiopod pairs during the power and return strokes Pereiopod pair
Surface area (mm2) Power stroke
Return stroke
P1 P2 P3 P4 P5
0.46 0.69 0.78 0.98 0.46
0.21 – 0.31 0.30 – 0.46 0.33 – 0.49 0.45 – 0.67 0.21 – 0.31
Lengths and widths of segments and setae were measured digital micrographs of dissected pereiopods. The degree of spreading and collapsing of segments and setae was estimated from video images of tethered copepods during the power and return strokes (Fig. 4). The smaller area for the return stroke assumes a complete overlap of the setae into a triangular shape, whereas the larger area assumes a more rectangular shape.
initial power stroke sequence remained unchanged. However, the return stroke started immediately after the completion of the last power stroke of P1 (12 ms, Fig. 5). In the example shown in Fig. 5, the synchronous return stroke was completed in 6 ms, with a maximum negative force of 100 AN recorded at 17.5 ms. 2.5 ms later, the second power stroke of P5 had already started. The first antennae and cephalic appendages remained in their retracted positions between the first and second series of power strokes, but we observed some flexing of the cephalosome during the return stroke. In Fig. 5, the copepod responded with two force transients. After the second force transient,
Table 2 Peak angular velocities calculated from the linear portion of the pereiopod angle measurements during the power strokes (see Fig. 5) Pereiopod
Angular velocity (rad s 1)
S.D.
Max. width (mm)
Re
P1 P2 P3 P4 P5
700 580 640 800 1060
27 19 38 20 40
1.058 1.316 1.414 1.550 1.122
1100 1400 1700 1600 1400
Angular velocities were calculated for four different responses in an adult male C. finmarchicus with a force transient duration of 12.2 ms at 8 – 10 jC. Maximum width of pereiopod was calculated from measured dimensions of the pereiopods and the spread seen on video images. Angular velocity was assumed to be constant throughout the power stroke for the calculation of Reynolds numbers.
Fig. 5. Force production (bottom) and pereiopod position (top) for a double kick. Temperature: 10.65 jC. Scale at right gives angular parallax correction for the animal positioned with ventral side rotated 45j toward the camera. Symbols for pereiopods same as in Fig. 4 (P5: open triangle, P4: filled triangle, P3: open diamond, P2: open squares, P1: open circles). Pereiopod position during return stroke is a group average since individual pereiopods could not be resolved (filled circles). Video frame time adjusted to make the P1 force peak to correspond to a 90j angle. Animal’s ventral direction rotated 45j toward camera.
the return sequence was similar to the one described for the single kick response: a delayed and significantly slower return stroke by the pereiopods, a smaller negative force, and the return of the other appendages to their original position (Fig. 4). Free-swimming C. finmarchicus responded to the brief hydrodynamic stimulus with a fast start escape response. Fig. 6 shows the escape trajectory produced by the first kick of a multiple-kick response by a CV C. finmarchicus. Visualization of the animal movement was not as detailed as in the tethered animal, in particular the cephalic appendages could not be discerned. The power stroke sequence was faster in the free-swimming animals (6– 7 ms compared to 8.5 – 12 ms), as had been noted for C. helgolandicus by Svetlichnyy (1987). The first movement was detected in the first antennae as the animal turned away from the stimulus (Fig. 6A). Almost simultaneously, we detected the beginning of
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Fig. 6. Reaction pattern of a free-swimming C. finmarchicus CV to a similar hydrodynamic stimulus as used for tethered animals (vertical movement of 3-mm cylinder 1.5 cycle, 700 Hz). (A) Superposition of video images at 5-ms intervals following stimulus (‘‘cyl’’ seen lower left corner). (B) Plot of position of the rostrum of the animal shown in A at 1-ms intervals. (C) Plot of velocity of the rostrum at 1 ms intervals calculated as the distance between two consecutive positions divided by 1 ms. (D) Plot of acceleration of the rostrum at 1-ms intervals calculated similarly. Grey bars indicate approximate time intervals for power strokes of pereiopod pairs.
the first power stroke. The initial rapid rise in acceleration from 0 to 200 m s 2 coincided with the power strokes of P5 closely followed by P4. The first antennae were streamlined with the cephalothorax by the 6-ms frame. A consistent deceleration was observed between the power strokes of P3 and P2, which correlated with a relatively long interval between power strokes of these two pairs of pereiopods also observed in the tethered copepods. The power stroke of P1 occurred during the deceleration following the last peak. The velocity of the copepod increased throughout the power stroke sequence reaching a maximum of 800 mm s 1 at 8 ms. Subsequently, the velocity slowly declined as the animal continued to coast through the water. During a period of 14 ms, the copepod traveled ca. 7 mm, which translates into an average velocity of 500 mm s 1. A second power stroke sequence followed and the animal left the field of view.
4. Discussion Similar to other aquatic invertebrates, copepods employ two distinct forms of locomotion. The first, used for slow swimming, involves the rhythmic beating of the cephalic appendages (Strickler, 1982). Many of the filter-feeding copepods gather food particles during this slow locomotory activity (Koehl and Strickler, 1981; Poulet and Gill, 1988). Power strokes of the thoracic swimming legs, or pereiopods, are used in ‘‘hops’’ (Tiselius and Jonsson, 1990; Buskey et al., 2002), as well as in escape responses (Strickler, 1975). This metachronal sequence of power strokes is modulated. Relatively small velocities and accelerations are generated during the hop and sink behavior (Buskey et al., 2002). In contrast, during an escape, the copepod responds to a threat with a ‘‘fast start’’ reaction with a short latency and rapid rise in swim velocity as shown in tethered and free-swim-
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ming individuals (Lenz and Hartline, 1999; Lenz et al., 2000; Buskey et al., 2002; Buskey and Hartline, 2003). The responses we recorded for C. finmarchicus in response to the hydrodynamic stimulus were the strong, ‘‘fast start’’ type. As expected in drag-based swimming, the peaks of force corresponded to the near-perpendicular position of the pereiopod with respect to the body axis. Based on this evidence, there appears to be no contribution from jet propulsion. This is not unexpected given that the splayed pereiopods extend well beyond the lateral margins of the urosome in their remoted (end of power stroke) position. At 8– 10 jC, the measured angular velocities in C. finmarchicus ranged from 580 to over 1000 rad s 1. The pereiopod pair that initiates the power stroke sequence (P5) had the highest angular velocities. The lowest angular velocities were measured for P2 and P3, which also have the largest surface area. The power strokes in C. finmarchicus (Table 2) were comparable to those in C. helgolandicus (2.5 –2.8 mm prosome length; angular velocities < 900 rad s 1; Svetlichnyy, 1987) and faster than in the smaller Cyclops scutifer (0.7 mm prosome length), where angular velocities ranged from 480 to 610 rad s 1 at 20 jC (Alcaraz and Strickler, 1988). Applying scaling effects measured in dytiscid water beetles using power strokes (Nachtigall, 1977), a significantly slower angular velocity would be predicted for the larger C. finmarchicus (2 –3 mm prosome length). This suggests that C. finmarchicus is performing at a higher muscular output rate than C. scutifer. Mechanical power output of muscle has been estimated for many different organisms using one of three methods: (1) measurements of metabolic rates (respiration rates) during locomotion, (2) physiological measurements of work loops in semi-isolated muscles and (3) determination of costs calculated from kinematics of limb movements (Josephson, 1997). Using the latter approach, we can apply the methods of Blake (1986) to the splayed paddle-shaped pereiopod pairs (see inset Fig. 4). The total resistive work predicted (assuming no added-mass effects) can be calculated from Eq. (11) of Blake (1986) (multiplying P˜ by tp):
W ¼ P˜ tp ¼ K
R Z tp
Z 0
0
m2n Xcrdrdt
141
where K = 1/2qCd (q is the density of water and Cd is the coefficient of drag), R is the length of the appendage, X is the angular velocity of the appendage, mn is the velocity of an element of appendage area normal to its surface, c is the chord of the appendage perpendicular to its long axis and tp is the time the power stroke takes. For angular velocities given in Table 2 with a sweep angle of 150j and a coefficient of drag of unity, this comes to 1.2 AJ. This is comparable to the 0.8 AJ reported for C. helgolandicus (Svetlichnyy, 1987) and U. vulgaris (Lenz and Hartline, 1999). 1.2 AJ at a repeat frequency of 50 Hz gives power outputs of 60 AW. Although the exact ratio of power stroke muscle to body mass is unknown for C. finmarchicus, 20% of wet weight (0.2 mg) is a conservative estimate (Bennet-Clark and Lucey, 1967). This would give a muscle-mass specific power output of 300 W kg 1. This is similar to that calculated for another copepod (Undinula vulgaris), but significantly higher than has been found in other organisms (Table 3). Fast-start responses in aquatic organisms are generated via C-and S-starts in fishes and some invertebrates, jet propulsion in squids, tail-flips in lobsters and other malacostracans and metachronal sequences of power strokes in copepods. As shown in Fig. 7, maximum velocities produced during fast-start escape responses increase as a power function of animal length in fishes ( y = 45.3x0.68, r2 = 0.830). The escape performances of lobsters (Nephrops norvegicus: Stentiford et al., 2000; Panulirus interruptus: Nauen and Shadwick, 1999), crayfish (Orconectis virilis: Webb, 1979), Daphnia pulicaria (Brewer et al., 1999), squid (Loligo vulgaria: Packard, 1969), pteropod (Clione limacine: Satterlie et al., 1977), mosquito larvae (Culex pipiens: Brackenbury, 1999), damselfly larvae (Enallagma spp.: McPeek et al., 1996) and tadpoles (Limnodynastes peronii: Wilson and Franklin, 2000) fall close to the fish maximum velocity vs. animal length relationship. In contrast, maximum velocities for the shrimp (Crangon crangon: Arnott et al., 1998; Pandalus danae: Daniel and Meyho¨fer, 1989) and the copepods (Acartia tonsa, A. lilljeborgii: Buskey et al., 2002; C. finmarchicus: present study; Euchaeta rimana: Yen and Strickler, 1996) are higher than what would be expected from length-velocity relationship of the fishes (Fig. 7). The copepods outperform the fishes by approximately an order of magnitude. This
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Table 3 Mechanical muscle power output in organisms with high energy output Organism
Species
Muscle
Temp. (jC)
Power (W kg 1)
Source
Copepod
Undinula vulgaris Calanus finmarchicus Athous haemorrhoidalis Manduca sexta
Pereiopod remotor Skeletal M4 Indirect flight
20 – 22, 8 – 10 25 40
400 300 145 90
Neoconocephalus triops Bombus terrestris
Metathoracic wing Asynchronous flight muscle Flight
30
266
Lenz and Hartline (1999), present study Evans (1973) Stevenson and Josephson (1990) Josephson (1984)
40
100
Josephson (1997)
24
80
Iliofibularis Myotomal
42 20
154 143
Soleus Extensor digitorum longus Flight
37 35 40
94 107 111 – 171
Click beetle Hawkmoth Katydid Bumblebee Fruit fly Lizard Fish
Drosophila melanogaster Dipsosaurus dorsalis Serranus cabrilla
Mouse
Mus musculus
Birds
Various
high performance in the free-swimming copepods is in agreement with the high muscular output estimated for the power stroke (Table 3). Thus, these two independent methods of quantifying behavioral per-
b
Fig. 7. Maximum swimming velocities plotted against animal size. Different taxa are represented by different symbols: — calanoid copepods (present study; Yen and Strickler, 1996; Buskey et al., 2002), — cladocerans (Brewer et al., 1999), w — malacostracans (Webb, 1979; Nauen and Shadwick, 1999; Stentiford et al., 2000), x — shrimp (Daniel and Meyho¨fer, 1989; Arnott et al., 1998), +— aquatic insect larvae (McPeek et al., 1996; Brackenbury, 1999), 5 — mollusks (Packard, 1969; Satterlie et al., 1977), q—tadpole (Wilson and Franklin, 2000), o — fishes (Williams et al., 1996; Domenici and Blake, 1997; Shepherd et al., 2000). Regression line ( y = 45.3x0.68, r2 = 0.830) computed for fish data only.
Lehmann and Dickinson (1997) Swoap et al. (1993) Wakeling and Johnston (1998) Askew and Marsh (1998), James et al. (1995) Marden (1987), Ellington (1991)
formance support the conclusion that the copepods have an exceptional escape response. How they achieve this performance is still uncertain, and it may be indicative of specializations in muscle functions at the cellular and sub-cellular level that may be unique to this group of animal. Successful predator evasion depends on the timely detection of an approaching threat and a timely and effective locomotory response. Using a siphon stimulus, Viitasalo et al. (2001) quantified the response distance as well as the effectiveness of the escape. In their experimental set-up, the distance from the siphon before an escape was initiated combines detection and latency, whereas the behavioral response quantifies the locomotory performance of the escape. Not surprisingly, species differed in both escape distance and effectiveness, which in turn affected individual species’ ability to escape from predatory attacks (Viitasalo et al., 1998). Hays et al., (1994, 1997) suggest that C. finmarchicus’ dominance and ability to persist in the surface layers is due to a particularly effective escape behavior. Indeed, C. finmarchicus has a myelinated nervous system with a shorter reaction time than the non-myelinated vertically migrating Augaptiloids (Lenz et al., 2000). However, the locomotory contribution to the escape may be just as important. Compared to other species studied, C. finmarchicus
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appears to have a particularly powerful and fast motor response.
Acknowledgements We would like to thank Bob Campbell, Ted Durbin, Pat Hassett and Melissa Wagner for assistance with algal cultures and information on maintenance of C. finmarchicus; Hinano Akaka, Brad Jones, Brian Kodama, Wilson McCord, Ted Murphy and Gabriel Rodrigues for critical technical support; Ian Cooke and Pat Hassett for loan of equipment; and the staff at Mount Desert Island Biological Laboratory (MDIBL) for assistance with logistics. Thanks also to Josef Ackerman, who provided many useful suggestions that greatly improved the manuscript. This work was supported by the Salisbury Cove Research Fund of MDIBL and National Science Foundation OCE99-06223.
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