HANDBOOK OF SCALING METHODS IN AQUATIC ECOLOGY MEASUREMENT, ANALYSIS, SIMULATION - PART 3 doc

149 10 Signaling during Mating in the Pelagic Copepod, Temora longicornis Jeannette Yen, Anne C. Prusak, Michael Caun, Michael Doall, Jason Brown, and J. Rudi Strickler CONTENTS 10.1 Introduction 149 10.2 Methods 150 10.2.1 Trail Visualization 150 10.2.2 Copepod Pheromones 151 10.3 Results and Discussion 151 10.3.1 Scent Preferences 151 10.3.2 Tracking Behavior 154 10.3.3 Quantitative Analyses of Trail Structure and Odorant Levels 155 10.4 Conclusion 157 Acknowledgments 158 References 158 10.1 Introduction In 1973, Katona depicted the mate-Þnding response of copepods as occurring when the male copepod detects the edge of the diffusing cloud of pheromone emanating from the female at a distance of 4 mm. Here, the process of diffusion is needed to transport the signal molecules to the sensors of the male copepod to alert him of the presence and location of a female copepod. However, using the equation for the characteristic diffusion time (Dusenbery, 1992; t = r 2 /4D, where the diffusivity coefÞcient D = 10 –5 cm 2 /s for small chemical molecules), it would take approximately 45 min for the pheromone to diffuse this distance. It is unlikely the female copepod would remain in the same three-dimensional (3D) position in the ocean for that length of time. Instead, in 1998, we (Doall et al., 1998; Weissburg et al., 1998; Yen et al., 1998) reported that male copepods detect discrete odor trails left in the wake of the swimming diffusion of the odor trail to molecular processes. Diffusion does not transport the pheromone to the male and, instead, acts to restrict odor dispersion. The scent persists as a coherent trail, with little dilution of signal strength, and hence remains detectable for a period that gives enough time for the male to encounter it. In the case of the copepod Temora longicornis, this aquatic microcrustacean could Þnd trails that were less than 10.3 s old. Trails were followed for distances as long as 13.8 cm (~100 body lengths), greatly extending the encounter volume of the copepod (Gerritsen and Strickler, 1977). Past studies showed that copepods could detect signals only within a few body lengths (see Haury and Yamazaki, 1995, for a review). Our Þndings show the perceptive distance can be 10 to 100 times greater. © 2004 by CRC Press LLC female copepod (Figure 10.1). Within this low Reynolds number regime, viscosity limits the rate of 150 Handbook of Scaling Methods in Aquatic Ecology: Measurement, Analysis, Simulation Documenting copepods following 3D aquatic trails provoked the question of the species speciÞcity of chemical trails. A capability to discriminate the scent of conspeciÞcs enables mate preference, important in maintaining species integrity (Palumbi, 1994; Orr and Smith, 1998; Higgie et al., 2000). For such widely dispersing species as aquatic marine organisms, an understanding of mate recognition, and the mechanisms for mate Þnding and mate choice, may be key steps in the cascade that enforces reproductive isolation in pelagic environments. To evaluate odor preference in copepods, we developed a bioassay, based on the behavioral response of trail following. The bioassay relies on trail visualization (a Schlieren optical path: Strickler and Hwang, 1998; Strickler, 1998) so that we can see the trail and see which trail the copepod follows. With this technique, we saw male copepods follow scent trails containing the odor of their conspeciÞc female copepods. We also saw how the male disturbed the odor trail, making it less likely that another competing male would Þnd and follow the trail. 10.2 Methods 10.2.1 Trail Visualization To visualize the trail, we mixed high-molecular-weight dextran with Þltered seawater to change the refractive index of the trail and used Schlieren optics to see the trail (Strickler and Hwang, 1998; Strickler, 1998). For this optical path, an infrared laser light, focused through a pinhole spatial Þlter, was expanded and diverted with a large (8-in.) spherical mirror to pass through and illuminate an experimental vessel Þlled with Þltered seawater. The collimated light passed through the vessel, was collected on another large (8-in.) spherical mirror, and focused onto a small pointlike matched Þlter. The matched Þlter prevented the light from reaching the image plane and the CCD image looked black. When a phase object was introduced into the experimental vessel, it diffracted the light rays so the rays passed by the matched Þlter and reached the image plane. Phase objects, like the translucent copepod or the trail, were visualized as bright silhouettes against a black background of the image captured by the infrared-sensitive CCD video camera. A 1-mm-wide trail was created by dispensing ßuid from a Þne pipette tip (Eppendorf) to ßow down into the 4-l observation vessel Þlled with Þltered seawater (28 ppt). The pipette was gravity fed, via thin FIGURE 10.1 (Color Þgure follows p. 332.) (A) Mate-tracking by the copepod, T. longicornis (1.2 mm prosome length). The male copepod (thin trail) Þnds the trail of the female copepod (thick trail) when the trail is 5.47 s old and the female is 3.42 cm distant from him (nearly 30 body lengths). Upon encounter, the male spins to relocate the trail, then accelerates to catch up with the female. The male copepod follows the path of the female precisely in 3D space. When within 1 to 2 mm of the female, the male pauses quietly, then pounces swiftly to capture his mate for the transfer of gametes packaged in a ßasklike spermatophore. During copulation, the mating pairs spin and remain together for a few seconds or more. (B) Mate-tracking by the copepod, T. longicornis: backtracking. The male copepod (thin trail) Þnds the trail of the female copepod (thick trail) because of a strong cross-plume odor gradient. The female is 1.30 cm distant from him. Upon encounter, the trail is 2.3 s old and the male follows the trail in the wrong direction, away from the female, because of a weak along- plume gradient. When initially following the trail, the male smoothly and closely follows the trajectory of the female. After 1.27 s when he is 24.4 mm from his mate and the trail is 6.7 s old, the male turns around and backtracks. When backtracking, he follows the female path erratically, casting back and forth over the trail. After reaching his intersection point, the male copepod resumes smooth close-following of the undisturbed female path. (From Doall, M. et al., Philos. Trans. R. Soc. Lond., 353, 681, 1998. With permission.) Speed (mm/s) 10-20 20-30 30-40 40+ 5-10 a' t=0 s b t=6.00 s c t=9.67 s b' a t=2.067 s a' b t=5.47 s c t=7.00 s b' a t=0 s Z (cm) X (cm) X (cm) Y (cm) Y (cm) 0-5 B A © 2004 by CRC Press LLC Signaling during Mating in the Pelagic Copepod, Temora longicornis 151 tubing, from a beaker Þlled with a mixture of seawater and dextran (MW of 500,000 at 0.5 g/100 ml). The difference in the refractive index of the dextran–seawater mixture sinking down through the seawater could be detected, using the Schlieren optical path. When this mixture passed through the plain seawater, a Þne trail could be observed as a bright vertical line on the video image (Figure 10.2). 10.2.2 Copepod Pheromones To test the attractiveness of waterborne odorants, different scents were obtained by putting different copepod types (stage, sex, species) in the dextran-labeled water and using this conditioned water to create the doubly labeled trails. For these scents, the same number of copepods (20) would be added to the same volume of dextran-labeled seawater (20 ml) in the beaker, to introduce comparable odorant levels to the conditioned water. Copepods that had spent the day in this dextran–seawater mixture had no detrimental effects as they lived for days after being transferred back to plain seawater with phyto- planktonic food ( Rhodomonas sp.). The rate of inßow of the doubly labeled water, less than 3 mm/s and more often 1 mm/s, was adjusted by varying the height of the beaker of conditioned water that was gravity-fed into the tank. This small beaker was Þlled from a stock of unscented dextran-labeled seawater. The observation vessel held 20 to 50 male copepods to test their interest in the scented trails. Female copepods do not mate-track. 10.3 Results and Discussion 10.3.1 Scent Preferences When offered the choice of trails with dextran-only vs. trails with dextran and female copepod scent, the male copepods followed the trail with the scent of their conspeciÞc 80% of the time and the unscented FIGURE 10.2 (Color Þgure follows p. 332.) (A) Visualized trail and upwardly directed trail following. A 1-mm-wide trail was created by dripping ßuid from a Þne pipette tip (Eppendorf) to ßow into the 4-l observation vessel Þlled with Þltered seawater (28 ppt). The pipette was gravity-fed, via thin tubing, from a beaker Þlled with a mixture of seawater and dextran (MW of 500,000 at 0.5 g/100 ml). The difference in the refractive index of the dextran–seawater mixture sinking down through the seawater could be detected, using a Schlieren optical path. The trail on the left is the undisturbed trail. The disturbed chevron-dotted trails on the right show how the trail structure changes at various time intervals after the male copepod T. longicornis follows the trail. Copepod indicated at the upper left on last trail, colored orange (B) Following a curvy trail: When following the scent in the trail, T. longicornis also can stay on the track of curved trails, making the same turns as taken by the trail. The undisturbed curved trail on left was followed for 1.13 s by the male copepod, colored orange in panel on right. Note how trail structure changes in wake of swimming male. © 2004 by CRC Press LLC 152 Handbook of Scaling Methods in Aquatic Ecology: Measurement, Analysis, Simulation trail less than 10% of the time (Table 10.1A). The dextran in the trail did not preclude the male’s interest in the trail with the female odors. To determine when, during the reproductive maturation period of the female, the scent was produced at sufÞcient concentrations, we offered the choice of dextran trails with the scent of conspeciÞc females that had oocytes within their oviducts vs. females without oocytes. Chow-Fraser and Maly (1988) found for the freshwater copepod, Diaptomus, more males attempted to mate with gravid than with nongravid females. Here, we found no preference for T. longicornis based on scent-tracking (Table 10.1A). Barthélémy et al. (1998) note that a congener, T. stylifera, requires a separate fertilization event prior to each clutch of eggs, which may explain why both gravid and nongravid females were attractive to their male mates. Other copepods are able to store sperm for days to weeks; these copepods, which do not need to Þnd mates for every clutch, may be more attractive as virgins or nongravid females (Kelly et al., 1998). When given the choice to follow trails scented with the odor of conspeciÞc females (combined gravid and nongravid), conspeciÞc males, copepods of another species ( Acartia hudsonica), or dextran-only trails, varying degrees of preference were discovered. Preference, evaluated as the likelihood to follow an encountered trail, could be ranked from most preferred to least interesting: conspeciÞc females, conspeciÞc males, the other species, the unscented trail (Figure 10.3, Table 10.1B). As an additional test of preference, we also measured the tracking speeds of the male along the trail (Table 10.1B). Males would track conspeciÞc female-scented trails at 15 mm/s, which was faster than their tracking speed along trails scented with conspeciÞc male odor. For the rare event of tracking the dextran-only unscented trail, the tracking speed (5 mm/s) was just above the ßow speeds of the trail (1 to 3 mm/s). For the trails with higher ßow speeds, there was a slightly greater probability that the male would show disinterest (nick, cross, or escape from the trail; Table 10.1). Flow speeds now are kept below 1 mm/s to reduce the likelihood that the hydromechanical ßow disturbance would elicit an escape (Fields and Yen, 1997). The relative tracking speed on preferred trails (copepod swimming speed plus trail ßow speed) was slightly slower than natural trail-following speeds (Doall et al., 1998). FIGURE 10.3 (Color Þgure follows p. 332.) (A) Scent preferences of T. longicornis. Male copepods of T. longicornis were offered seven choices of scent trails (from left to right: Female, Male, Acartia, Dextran, Male, Female, Acartia). The male copepod showed an 80% preference to follow trails scented with the odor of conspeciÞc females (choice 6). Trails were followed to the source. The source of the odor was a stock of dextran-mixed Þltered seawater within which 20 females were swimming. The conditioned water ßowed through Þne pipettes down through the observation vessel, Þlled with Þltered seawater and 20 to 50 male copepods. Schlieren optics visualized the ßow patterns. (B) Downwardly directed trail-following. The trail on the left is the undisturbed trail. The disturbed trail on the right was the same trail that was followed down to the source by a male copepod, appearing in the lower right, colored orange. Trails were created by allowing scented seawater to ßow out of Þne pipettes at the bottom of the observation vessel. The vessel was Þlled with a mixture of dextran (20 to 40 g/4 l of 500,000 MW dextran) and seawater to create the difference in refractive index necessary to visualize the trail using Schlieren optics. The plain seawater would ßoat up to create the trail. Flow speed of 4.43 mm/s can be calculated by the upward movement of the bright bubble. © 2004 by CRC Press LLC Signaling during Mating in the Pelagic Copepod, Temora longicornis 153 Even though dextran-only trails were rarely followed, the copepod does not avoid dextran because scented dextran trails were followed. In most cases, the male copepod would encounter the scented trail and follow the trail up to the source. On occasion, the male would follow the trail down and away from the source and frequently would turn around. To determine if gravity inßuenced tracking direction of the male copepod, we designed the reverse situation. Here, seawater with and without the scent of other copepods was used to form trails in a tank Þlled with dextran-labeled water (20 to 40 g dextran/4 l). The trails began now at the bottom of the tank, as the dextranless water would ßoat up to the surface of the tank Þlled with dextran-labeled water. When the male encountered the trail, he would follow it 91.7% (11 trails followed/12 female trails encountered) of the time to the source, at the bottom of the From these analyses of trail following up or down to the source and the speed at which the trails were followed, we conclude that the behavior of mate-tracking by T. longicornis is chemically mediated and tracking direction is not determined by gravity. We found that most trails were followed to the source, indicating that the copepods are either able to detect the odor gradient or can detect the directional ßow in the sheared slow ßow of the manufactured trails. We also conclude that these male copepods can discern differences in scents between sexes and between species. However, these male T. longicornis copepods do not follow female trails exclusively, questioning the speciÞcity of the pheromone. Male copepods would not only follow female trails, but also male trails. Here, we would like to describe an event showing the adaptive value of following one’s own track. When studying mate-tracking, we would start with male-only swarms of copepods (Doall et al., 1998). In these swarms, there was the infrequent event when a male would follow the trail of another male but upon contacting the male, he would release him immediately. More interestingly, another event in swarms of mixed sex showed a male copepod that followed the trail of the female, but somehow got derailed and wandered off the trail. After a couple seconds, he turned around, retraced his “steps,” found the female trail, and followed it to her to form the spinning mating pair. Hence, an ability to self-track allowed this copepod to Þnd his mate successfully. The speciÞcity of the diffusible pheromone is further questioned by our observations of male T. longicornis following the trails scented with the odor of another species, Acartia tonsa. This behavior TABLE 10.1 Scent Choice by Male Copepods of the Species Temora longicornis Source of Scent Preference = # follows/# encounter [%] % Escapes Tracking Speed Flow Speed A. Response to Trails with the Scent of Females of Different Reproductive State and of Males Gravid female Tl 43/54 [80%] 7.41% 13.9 2.77 Nongravid female Tl 21/30 [70%] 6.67% 19.2 3.07 Male Tl 4/20 [20%] 10.0% 17.3 2.54 Dextran-only 0/4 [0%] 23.8% — 2.95 B. Response to Trails with Scent from Different Sexes and Species of Copepod Female Tl 31/39 [80%] 0 15.8 1.29 Male Tl 10/19 [52%] 0 10.1 1.28 Acartia tonsa 1/8 [12%] 0 13.6 1.0 Dextran-only 1/11 [9%] 0 5.7 1.32 Note: Random encounters by freely swimming male copepods of the species T. longicornis [Tl] with scent trails would evoke a response to the trail. Vertical scent trails were created from dextran-labeled seawater ßowing down through Þne pipette tips into the observation vessel Þlled with Þltered seawater (28 ppt). Male copepods, given a choice of trails scented with the odors of different types of copepods, would follow the trail traveling at an accelerated swimming speed or show disinterest (escape from, nick, or just cross the trail). Preference for a trail type is presented here as (# trails followed/# trails of that type encountered). Disinterest is presented as (# escapes/# encounters). Swimming speeds while trail following were compared to ßow speeds of the trail (mm/s). Experiments in A represent responses to the scent of conspeciÞcs. Experiments in B include responses to scents from a copepod of another genus. © 2004 by CRC Press LLC tank (Figure 10.3). 154 Handbook of Scaling Methods in Aquatic Ecology: Measurement, Analysis, Simulation suggests another possible scenario: as Temora is an omnivorous copepod, could it be responding to this scent as a possible means to track its prey? Prey tracking recently has been observed for catÞsh detecting the hydrodynamic wakes of goldÞsh prey (Pohlmann et al., 2001). Remote detection via the diffusible pheromone is one means of mate recognition. Other possible moments in mate recognition involve contact pheromones (Snell and Carmona, 1994) or a key-and-lock Þt of the complex coupling plate (Fleminger, 1975; Blades, 1977). However, there are copepod species that do not have coupling devices on their spermatophore that mirror the genital structure of the conspeciÞc female and only have simple means to cement the base to the female genital area. It also is not known how many copepods secrete contact pheromones, as this has been conÞrmed for only one species of benthic harpacticoid copepod (Ting et al., 2000), which exhibits precopulatory mate-guarding over extended periods of time. It is likely that decisions at every step in the mating process contribute to Þnal species recognition. 10.3.2 Tracking Behavior When the male copepod of T. longicornis detects a trail, he follows it in either direction, going to the scent source æ the female copepod æ or away from her. In one example where the male correctly tracked to the female, the position of the male copepod was 1.02 ± 0.34 SD (n = 45) mm from the central axis of the 3D trajectory taken by the female seconds earlier, indicating spatially accurate tracking way, away from the source of the scent: the female copepod. After 1.27 s, he reoriented and returned backtracking showed that when the male Þrst follows the trail, his positions precisely overlap the positions in the 3D trajectory taken by the female seconds earlier. The distance from the central axis of the female trail is close to 1 mm. When he turns around, we Þnd that the 3D trajectory of the male copepod no longer matches the path of the female. Instead, the male backtracks the disturbed trail very erratically, FIGURE 10.4 (Color Þgure follows p. 332.) Changes in scent trail shape caused by tracking behavior of male T. longicornis. (A) Trail structure changes from a smooth undisturbed vertical trail (left) to a disturbed trail (same portion of the trail shown on right) after the passage of the male copepod of T. longicornis. (B) Close-up of the disturbed trail. The red dotted line deÞnes the hypothesized helical trail left by spiraling movements of the male while tracking the trail. The spiral is longer, thinner, and has a greater surface area than the smooth trail. The action of molecular diffusion can dilute smooth and helical trail; radius of smooth and helical trail, slope of spiral structure of helix. For each 180∞ segment of the helix, the radius and slope were repetitively calculated to reconstruct the helix so it cuts through the brightest spots of the image. Scale: 6.8 pixels/mm. 10 20 30 40 50 60 70 80 90 0 20 40 60 Time = 0 sec Time = 1.33 sec AB © 2004 by CRC Press LLC the pheromone more quickly. Quantitative measurements of trail geometry (for Table 10.3 calculations) include: length of along the trail back to the female to capture her. Further analyses (Table 10.2) of this behavior of (Figure 10.1A). In another example (Figure 10.1B), the male found and followed the trail in the wrong Signaling during Mating in the Pelagic Copepod, Temora longicornis 155 casting back and forth over the original location of the female trail at distances twice as far from the central axis of the path. When backtracking, he casts at an average course angle of more than 70° in either the x–z or y–z direction, which is greater than the average course angle of 46° taken when following the trail initially. A tightening of his course angles to 15° begins after passing his initial intersection with the trail. Here, he again precisely follows the trail, remaining at distances of close to 1 mm from the axis of the path. Weissburg et al. (1998) found that this pattern of counterturns enabled the copepod to stay near or within the central axis of the odor Þeld. The male copepod’s casting behavior suggested that the trail was disturbed by his own swimming activity, making the trail more difÞcult to follow. Casting behavior also has been noted when the female copepod swims slowly or hovers (Figure 3b in Doall et al., 1998), producing a trail so thick, the male wanders back and forth between the edges. When the female copepod hops and creates a diffuse cloud, male casting behavior also is observed (Figure 5a in Doall et al., 1998) and the male can lose the trail, suggesting that the female hop diluted her scent to levels below the threshold sensitivity of his chemical receptors. These observations led us to hypothesize that the trail structure has changed in the wake of the tracking by the male copepod. To test this hypothesis, we relied on our method of small-scale ßow visualization to document changes in the structure of the trail, evoking the behavioral response of trail following by placing the scent of the female in the trail we created. By visualizing the trail, we saw how the tracking structure in the wake of the tracking male copepod. The undisturbed trail is a smooth vertical line and we presume from the two-dimensional (2D) image that the trail is a 3D cylinder. When the male copepod intersects the trail, he spins and turns to relocate the trail. He then accelerates to three times higher than his normal swimming speed as he follows the trail. Detailed analysis of the structure of the disturbed trail reveals a herringbone 2D pattern. We can imagine that the male copepod swims around the trail, deforming it into a 3D helix. Tsuda and Miller (1998) saw a larger male copepod of the species Calanus marshallae that would tilt back and forth as he followed the scent trail of his female. The tilting appears to allow the copepod to insert one antennule into the odor trail and one antennule out of it, thus assessing location by bilateral comparison. As these were 2D observations of the large copepod, we make the assumption that this tilting occurs in 3D, suggesting that the copepod spiraled around the trail. Spiraling while swimming fast has been observed during the escape response of the large Euchaeta norvegica (Yen et al., 2002). Spiraling by the mate-searching copepod Oithona davisae has been suggested to help the male locate a pheromone source more accurately and to promote diffusion of the pheromone to prevent other males from pursuing the source (Uchima and Murano, 1988). 10.3.3 Quantitative Analyses of Trail Structure and Odorant Levels To deÞne the characteristics of the disturbed trail, we assume that the brightness of the Schlieren image represents the location of the chemical trail, where areas with similar brightness represent similar concentrations (Gries et al., 1999). Here, we assume the structure of the deformed trail to resemble a TABLE 10.2 Kinematics of Backtracking in a Mate-Seeking Copepod, Temora longicornis Mate-Tracking of T. longicornis AWAY from Female (n = 39) Back TO Female on Disturbed Trail (n = 34) TO Female on Undisturbed Trail (n = 23) Normal Swimming (n = 40) Velocity (mm/s) 33.9 ± 9.7 50.4 ± 10.6 33.5 ± 12.4 14.4 ± 2.78 Course angle (x–z) 46.4 ± 41.5 73.3 ± 50.7 15.3 ± 15.6 Course angle (y–z) 42.3 ± 37.0 71.3 ± 43.1 26.2 ± 18.5 Track distance (mm) 1.16 ± 0.49 2.09 ± 1.14 1.15 ± 0.66 Note: The velocity, course angle (in both x–y and x–z directions), and track distance (closest distance between 3D trajectory of the male copepod and central axis of female trajectory) are given for the event when the male copepod crossed the trail of the female copepod, but went the wrong way: away from the source of the scent, the female. While tracking and retracing his steps, course angles and track distance nearly double in value, indicating the erratic casting behavior exhibited by the male as he returns along the disturbed trail. © 2004 by CRC Press LLC male disturbed the scented trail. Close analyses of the trail (Figure 10.4) show dramatic changes in trail 156 Handbook of Scaling Methods in Aquatic Ecology: Measurement, Analysis, Simulation 3D helix, where the relocation of odorant due to copepod movements producing an outward curve of the helix equals the amount relocated on an inward curve of the 3D structure. Using geometric and image processing techniques, we estimated the length, volume, and surface area of the scent trail before and after the copepod swam through it (Table 10.3). The undisturbed trail was assumed to be a cylinder. A section of the image of this cylinder was isolated; length and diameter measurements were taken and used to calculate volume and surface area. The disturbed trail was assumed to have a helical character. A section of the image of the disturbed trail corresponding to the section of the image of the undisturbed trail was isolated. This image section was thought of as a 2D projection of the helical trail onto the CCD imager: the bright spots of the image were assumed to indicate points where the helix crosses the plane of the image projection. Taking these bright spots as data points, an “adaptive helix” was Þt to the image in 180° segments as follows. Two successive bright spots were located and their positions used to determine parameters for a helical line segment passing from the Þrst to the second bright spot (Figure 10.4). Next the third bright spot was located and its position was used in conjunction with the second bright spot’s position to generate the parameters for another helical line segment to link the second and third bright spots. Proceeding in a similar fashion produced a 3D helical line joining the bright spots of the scent trail. A consequence of the construction of this helical line was that each 180° segment was itself naturally divided into many smaller subsegments and this characteristic was exploited to calculate length, volume, and surface area. The length was found by summing the lengths of each individual subsegment. For the remaining measurements, each subsegment was taken to be the axis of a short cylinder, the diameter of which was found by measuring several such “diameters” in the image and averaging. The volume and surface area of the whole helical scent trail then were found by summing the volumes and surface areas of the individual cylinders thus deÞned. TABLE 10.3 Geometric Changes in Trail Structure Dilutes Chemical Signal Scent Trails Smooth Trail (A) Disturbed Trail (A) Natural Trail (B) Disturbed Trail (B) Initial diameter a 0.14 cm ± 5.6% SE (n = 82) 0.083 cm ± 17.3% SE (n = 15) 0.05 cm 59% Initial length 1.2 cm 2.9 cm 0.10 cm 240% b Initial V 1.09 ¥ 10 –2 cm 3 1.6 ¥ 10 –2 cm 3 2.0 ¥ 10 –4 cm 3 1.6 ¥ 10 –4 cm 3 Initial SA 5.4 ¥ 10 –1 cm 2 7.5 ¥ 10 –1 cm 2 1.6 ¥ 10 –2 cm 2 2.2 ¥ 10 –2 cm 2 Initial C 100% 100% 100% 100% Final radius 0.09 cm 0.06 cm 0.05 cm 0.04 cm Final length 1.25 cm 2.91 cm 0.14 cm 0.277 cm Final V 0.0318 cm 3 0.0363 cm 3 8.9 ¥ 10 –4 cm 3 10.4 ¥ 10 –4 cm 3 Final SA 0.707 cm 2 1.15 cm 2 4.0 ¥ 10 –2 cm 2 6.1 ¥ 10 –2 cm 2 Final C 59% 43% 22% 15.2% Volume (V) = pr 2 h; surface area (SA) = 2prh. Possible initial odorant concentration (C) = 10 –5 M (Poulet and Ouellet, 1982). Length increment = characteristic diffusion length = SQRT[4Dt]; t = time. D = diffusion coefÞcient = 10 –5 cm 2 /s for small chemical molecules (Jackson, 1980). Note: The initial volume (V), surface area (SA), and pheromone concentration (C) of the smooth and disturbed trail mimic (A) and natural trail (B) were compared to the Þnal volume (V), surface area, and concentration after 10 s of molecular diffusion (radius increases by 0.2 cm). The geometric measurements of the undisturbed natural trail are from Table 2 in Yen et al. (1998). a Using MATLAB image analytical techniques, the edges were determined as that corresponding to a threshold luminance value of 40%. As the entire trail was only 10 pixels wide, different thresholds will yield different values. b the central axis of the trajectory of the female copepod. Similarly, after tracking, we found here that the trail expanded 2.37 times wider. © 2004 by CRC Press LLC As noted in Table 10.1, when backtracking natural trails, the male casts at nearly twice the distance from r = radius of cylinder; h = length of cylinder (see Figure 10.4 for illustration). Signaling during Mating in the Pelagic Copepod, Temora longicornis 157 disturbed trail was 60% thinner, 2.4 times longer, with a surface area 40% greater than the original trail. Although the helix extends over the same linear length occupied by the trail, it expands to a larger helix width and therefore may change the probability of encounter. Once encountered, the next male would have to manage to stay on the track of a thinner trail and also follow a much longer trail. Copepods are female copepod so if they found the helix, they could spend extra time following every helical loop or take longer casts suggesting exploration of a more diffuse odorant cloud. Comparing the volume of the smooth trail to that of the helical trail, we found the volume of the trail decreased by only 17%. The similarity in trail volume suggests that, at these Reynolds numbers, turbulent eddy diffusion is limited by viscous forces. The Reynolds number for the male copepod, while swimming along natural trails, can reach up to values of 60 (maximum tracking velocity = 50 mm/s in contrast to the males’ normal swimming velocity of 10 mm/s), quite different from the initial Reynolds number for the trail of 7 (swimming speed of female copepod = 6 mm/s). Here, the Reynolds number of the manufactured trail is close to 1 and that in the wake of the male is between 15 and 20. At these Reynolds numbers, diffusion acts by molecular processes only to slowly disperse the odor. The spiraling copepod reshapes the odor trail but there appears to be little dilution of trail contents. To determine if the odorant levels would differ after diffusion from a smooth vs. helical trail, we calculated how much larger the trail would become after 10 s of molecular diffusion (radius increases by 0.2 cm after 10 s). It is possible that adjacent loops of the helix would diffuse and fuse to reform the smooth trail. To estimate the time needed for this change to occur, we measured the average separation distance between loops of the helix as 1.92 mm. The loops would need to diffuse 0.96 mm to meet the next loop. This would take more than 3 min. Because trails older than 10 s are rarely followed, it is not necessary to consider the coalescing of odors between loops of the helix. Instead, we considered the change in concentration if the helix were a straight cylinder to compare to the change in concentration of the undisturbed smooth trail. We found that, after 10 s, the Þnal odorant concentration was 59% of the original level (= 100%) in the smooth trail and 43% of the initial concentration in the disturbed trail, with a 60% increase in surface area over the original trail. For natural mating trails, the oldest trail followed by the male T. longicornis was 10.3 s old (Doall et al., 1998). This suggests that trails of this age stimulate the copepod sensors just at their threshold. Trails any older are undetectable. Similarly, the backtracking copepod turned around when the trail age was 6.7 s old, close to the time limit beyond which diffusion reduced the concentration levels below the detection threshold. Considering the geometry of a natural trail (from Table 2 in Yen et al., 1998), we calculated that the odorant would decline to 22% of the original odorant levels excreted into the natural trail after 10 s. We conclude that 22% is the threshold for detection by copepod chemoreceptors. If we now disturb the natural trail in a geometrically similar fashion as was determined for the post-tracking manufactured scent trail, the odorant level in post-tracking helical natural trail would decline to 15.2% of the original levels after 10 s (Table 10.3B). This is less than the 22% threshold level. Therefore, in addition to becoming a longer and more convoluted trail, the odor became less distinct where odorant concentrations may drop below the threshold sensitivity of the male’s receptors. These structural changes, along with the changes in odorant levels in the disturbed trail plus possible chemical degradation of pheromones over time, have the potential to confound the tracking ability of another chemoreceptive plankter. Hence, by disturbing the trail due to his scent-tracking behavior, the male may lower the possibility that another competing mate or threatening predator will Þnd his female. 10.4 Conclusion To summarize, the study of mate-tracking, using this new behavioral bioassay along with the observation of 3D copepod trajectories in a 4-l container, indicates that T. longicornis is able to detect and follow scent trails. As he follows these pheromonal trails to his mate, the male copepod disturbs the signal and effectively lowers the possibility that another mate or chemoreceptive predator will Þnd his female. However, for T. longicornis, mate recognition by remote detection of a diffusible pheromone is not certain. Temora longicornis appears capable of following different trail types. To understand this seeming © 2004 by CRC Press LLC We compared the structure of the trail before and after tracking (Table 10.3A) and found that the able to follow curvy natural (Doall et al., 1998) and manufactured (Figure 10.2B) scent trails of the 158 Handbook of Scaling Methods in Aquatic Ecology: Measurement, Analysis, Simulation lack of speciÞcity, we consider the biology of sparse populations. Plankton, although common, form sparse populations because they are widespread at low densities; they seldom encounter each other and so are effectively rare (Gerritsen, 1980). In sparse populations, the probability that mates encounter each other is reduced, resulting in reduced birth rate and reduced population growth rates. If the population density is low enough, mating encounters are so rare that few individuals reproduce to sustain the population, leading to possible extinction. Because extinction is such a strong selective force, adaptations will be favored that increase the probability of mating encounters. Here it is adaptive to follow any trail because the probability of encountering anything is low for these small (1 to 10 mm), slowly swimming (1 mm/s), widely dispersed (1/m 3 ), aquatic microcrustaceans with limited ranges for their sensory Þeld (1 to 100 body lengths). As copepods are considered some of the most numerous multicellular organisms on earth (Humes, 1994), this study of their mating strategies has elucidated their reliance on unusually precise mechanisms for survival and dominance. Acknowledgments This work was supported by National Science Foundation Grants OCE-9314934 and OCE-9402910 to J.Y. and J.R.S., with continued support to J.Y. from IBN-9723960. All thank the editors: Laurent Seuront and Peter Strutton. References Barthélémy, R M., C. Cuoc, D. DeFaye, M. Brunet, and J. Mazza. 1998. Female genital structures in several families of Centropagoidae (Copepoda: Calanoida). Philos. Trans. R. Soc. Lond. B, 353: 721–736. Blades, P.I. 1977. Mating behavior of Centropages typicus (Copepoda: Calanoida). Mar. Biol. (Berlin), 40: 47–64. Chow-Fraser, P, and E.J. Maly. 1988. Aspects of mating, reproduction, and co-occurrence in three freshwater calanoid copepods. Freshw. Biol., 19: 95–108. Doall, M.H., S.P. Colin, J.R. Strickler, and J. Yen. 1998. Locating a mate in 3D: the case of Temora longicornis. Philos. Trans. R. Soc. Lond., 353: 681–689. Dusenbery, D.B. 1992. Sensory Ecology. New York: W.H. Freeman. Fields, D.M. and J. Yen. 1997. The escape behavior of marine copepods in response to a quantiÞable ßuid mechanical disturbance. J. Plankton Res., 19: 1289–1304. Fleminger, A. 1975. Taxonomy, distribution, and polymorphism in the Labidocera jollae group with remarks on evolution within the group (Copepoda: Calanoida). Proc. U.S. Natl. Mus., 120: 1–61. Gerritsen, J. 1980. Sex and parthenogenesis in sparse populations. Am. Nat., 115: 718–742. Gerritsen, J. and J.R. Strickler. 1977. Encounter probabilities and community structure in zooplankton: a mathematical model. J. Fish. Res. Board Can., 34: 73–82. Gries, T., K. Johnk, D. Fields, and J.R. Strickler. 1999. Size and structure of “footprints” produced by Daphnia: impact of animal size and density gradients. J. Plankton Res., 21: 509–523. Haury, L.R. and H. Yamazaki. 1995. The dichotomy of scales in the perception and aggregation behavior of zooplankton. J. Plankton Res., 17: 191–197. Higgie, M., S. Chenoweth, and M.W. Blows. 2000. Natural selection and the reinforcement of mate recognition. Science, 290: 519–521. Humes, A.G. 1994. How many copepods? Hydrobiologia, 292/293: 1–7. Jackson, G.A. 1980. Phytoplankton growth and zooplankton grazing in oligotrophic oceans. Nature, 284: 439–441. Katona, S.K. 1973. Evidence for sex pheromones in planktonic copepods. Limnol. Oceanogr., 18: 574–583. Kelly, L.S., T.W. Snell, and D.J. Lonsdale. 1998. Chemical communication during mating of the harpacticoid Tigriopus japonicus. Philos. Trans. R. Soc. Lond., 353: 737–744. Orr, M.R. and T.B. Smith. 1998. Ecology and speciation. TREE, 13: 502–506. Palumbi, S.R. 1994. Genetic divergence, reproductive isolation and marine speciation. Annu. Rev. Ecol. Syst., 24: 547–572. © 2004 by CRC Press LLC [...]... formation and maintenance Eos, 65: 731 732 Poulet, S.A and Ouellet, G 1982 The role of amino acids in the chemosensory swarming and feeding of marine copepods J Plankton Res., 4: 34 136 1 â 2004 by CRC Press LLC 180 Handbook of Scaling Methods in Aquatic Ecology: Measurement, Analysis, Simulation Price, H.J 1989 Swimming behavior of krill in response to algal patches: a mesocosm study Limnol Oceanogr., 34 : 649659... carrying capacity.) This interpretation allows the possibility of a conceptually simple link between the gradient in stimulus (say, light intensity or chemical concentration) and a gradient in the behavioral response â 2004 by CRC Press LLC 178 Handbook of Scaling Methods in Aquatic Ecology: Measurement, Analysis, Simulation 11.7 Conclusion The members of phototactic swarms of Daphnia and Temora in the... (stock -in- area against recruits-to-the-area) were obtained by plotting data values from a certain year (Nt) against values the year after (Nt+1) Crosscorrelations were used to determine the degree of correspondence between the series Furthermore, data values were ịtted both by linear regressions through the origin (to determine the replacement line or recruitment needed to replace the stock-at-spatial-location)... CRC Press LLC 166 Handbook of Scaling Methods in Aquatic Ecology: Measurement, Analysis, Simulation The strength of the attractive force does, however, have a very direct bearing on the steady-state swarm size x 2 For large t, x 2 approaches the value x2 = u2 w2 (11.12) (Uhlenbeck and Ornstein, 1 930 ) Thus swarm size is inversely proportional to the strength of the attractive forcing When w is large... spatial variance x 2 of an individuals path also measures the size of the swarm A standard result in the theory of diffusion (Okubo, 1980) is that under these assumptions, for large t, x 2 increases as x 2 ặ 2 Dt â 2004 by CRC Press LLC (11.1) Autocorrelation Handbook of Scaling Methods in Aquatic Ecology: Measurement, Analysis, Simulation Autocorrelation 164 Diffusion Time lag Swarming Time lag FIGURE... in both its mean slope (which is not signiịcantly different from the predicted value of zero) and its mean standard â 2004 by CRC Press LLC 174 Handbook of Scaling Methods in Aquatic Ecology: Measurement, Analysis, Simulation TABLE 11.1 Dynamic and Kinematic Parameters along the Axes of Swarming Motion for the Daphnia and Temora Experiments Source Damping coefịcient Concentration coefịcient Power of. .. scope of the present chapter Skipjack tuna appears to be able to adapt the feeding strategy to environmental conditions preying upon what it encounters (Roger, 1994a, b) and the 18C isotherm and 3 ml oxygen per liter isoline are considered lower limiting factors (Piton and Roy, 19 83) The exploitation rate on 1 83 â 2004 by CRC Press LLC 184 Handbook of Scaling Methods in Aquatic Ecology: Measurement, Analysis,. .. (11. 23) (Haury and Weihs, 1976) and k~ r A 1 C w u 2 d rz V (11.24) where u is the velocity, V the volume, A the frontal cross section, rz the density, and Cd the drag coefịcient of a zooplankter moving through water of density rw We can assume that the ratio of water to animal â 2004 by CRC Press LLC 176 Handbook of Scaling Methods in Aquatic Ecology: Measurement, Analysis, Simulation 1 0 -0 .5 1 0 -0 .5... in Strickler and Hwang (1998) and Strickler (1998); digitization methods are Light shaft z y x FIGURE 11.2 Geometry of the tank and light system â 2004 by CRC Press LLC 168 Handbook of Scaling Methods in Aquatic Ecology: Measurement, Analysis, Simulation described in Doall et al (1998) The light source in the Daphnia experiment was a 3- mW argon laser (wavelength 488 and 514.5 nm), which produced a collimated... mechanisms of chemosensory mate tracking by the copepod Temora longicornus Philos Trans R Soc London B, 35 3: 701712 Williamson, C 1981 Foraging behavior of a freshwater copepod: frequency changes in looping behavior at high and low prey densities Oecologia, 50: 33 233 6 Wishner, K., Durbin, E., Durbin, A., Macaulay, M., Winn, H., and Kenney, R 1988 Copepod patches and right whales in the Great South Channel off . Within this low Reynolds number regime, viscosity limits the rate of 150 Handbook of Scaling Methods in Aquatic Ecology: Measurement, Analysis, Simulation Documenting copepods following 3D aquatic. analyses of the trail (Figure 10.4) show dramatic changes in trail 156 Handbook of Scaling Methods in Aquatic Ecology: Measurement, Analysis, Simulation 3D helix, where the relocation of odorant. LLC required by kinematic considerations, as discussed above and illustrated in Figure 11.1: 166 Handbook of Scaling Methods in Aquatic Ecology: Measurement, Analysis, Simulation The strength of the attractive