fbq164.pdf - Oxford Academic

Pheromone trail following in threedimensions by the freshwater copepodHesperodiaptomus shoshone

JEANNETTE YEN 1*, JENNIFER K. SEHN 1, KIMBERLYCATTON 1†, ANDREW KRAMER 2‡ AND ORLANDO SARNELLE 2

1SCHOOL OF BIOLOGY, SCHOOL OF CIVIL AND ENVIRONMENTAL ENGINEERING, GEORGIA INSTITUTE OF TECHNOLOGY, ATLANTA, GA 30332-0230, USA AND2

DEPARTMENT OF FISHERIES AND WILDLIFE, MICHIGAN STATE UNIVERSITY, EAST LANSING, MI, USA

†PRESENT ADDRESS: DEPARTMENT OF CIVIL ENGINEERING, COLORADO STATE UNIVERSITY, FORT COLLINS, CO, USA.

‡PRESENT ADDRESS: ODUM SCHOOL OF ECOLOGY, UNIVERSITY OF GEORGIA, ATHENS, GA 30602, USA.

*CORRESPONDING AUTHOR: [email protected]

Received September 11, 2010; accepted in principle December 6, 2010; accepted for publication December 7, 2010

Corresponding editor: Roger Harris

Finding mates can pose a particular problem for obligately sexual planktonicorganisms, resulting in a variety of adaptations to ensure sufficient mating.Several types of mate-finding behavior have been observed in marine cope-pods, but the one most effective at low population density, following a phero-mone trail, has not been observed in freshwater copepods. Using three-dimensional (3D) videography, we show that males of the large-bodied alpinespecies Hesperodiaptomus shoshone follow pheromones in the female’s trail. Usinga trail mimic comprised of female-conditioned water, we found that males fol-lowed female scent without the presence of the female. This behavior wasreduced when the female scent was diluted, suggesting that the male’s behav-ior can be modified by the intensity of the chemical signal. Analyses of the3D trajectories of copepods that formed mating pairs indicate that the maledoes not make a direct approach to the female, as might be expected if herelied purely on hydrodynamic or visual cues. Instead, males that are.0.5 cm from females react to crossing female trails by making an abruptturn and spending more than 2 s following the female’s trail. Furthermore,flow field analysis showed that at this distance it was unlikely that copepodscould distinguish the hydrodynamic signal from the background flow. This isthe first demonstration of chemical trail following in a freshwater copepod andhas important implications for encounter rates and viable population densitiesin similar species.

KEYWORDS: calanoid copepods; mate tracking; Hesperodiaptomus shoshone;pheromone

I N T RO D U C T I O N

A persistent population of sexually reproducing organ-isms depends on sufficiently high encounter ratesbetween females and males. Planktonic copepods at low

density in the open waters of lakes or oceans are oftenseparated by 100’s of body lengths, making directrandom encounters infrequent (Gerritsen, 1980;

doi:10.1093/plankt/fbq164, available online at www.plankt.oxfordjournals.org. Advance Access publication January 11, 2011

# The Author 2011. Published by Oxford University Press. All rights reserved. For permissions, please email: [email protected]

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Buskey, 1998). As a result, active searching combinedwith the ability to detect potential mates from a distancemay be required to produce a reasonable chance ofencounters between sexes (Gerritsen and Strickler,1977; Haury and Yamazaki, 1995; Kiørboe, 2006).Some marine copepods increase perceptual distancebeyond physical contact via hydrodynamic cues(Strickler, 1998), whereas males of other species followchemical trails produced by females (Doall et al., 1998;Tsuda and Miller, 1998; Kiørboe and Bagøien, 2005).Because hydrodynamic signals rapidly decay (Yen et al.,1998), pheromone trails that can be followed over manybody lengths should lead to larger improvements inencounter rates between potential mates (Gerritsen andStrickler, 1977; Kiørboe and Bagøien, 2005).

Although freshwater copepods experience the samechallenges in locating mates as marine species, theirability to increase the encounter rate with trails andchemical signals is not well understood. Watras (Watras,1983) suggested that in the calanoid copepod Diaptomus

leptopus, male preference for gravid females was basedon the detection of a chemical signal, but only observedmales pursuing females over a distance of one or twobody lengths. Males in this species increased swimmingspeed in the presence of gravid females (van Leeuwenand Maly, 1991) which could lead to higher encounterrates of males with females (Gerritsen, 1980; Kiørboe,2006) but also increases the risk of encountering preda-tors (Kiørboe, 2008). More recent research on anotherfreshwater copepod, Leptodiaptomus ashlandi, also foundevidence for increased swimming speed in response to achemical signal, but not for directional tracking offemales (Nihongi et al., 2004). Because marine specieswith the greatest ability to locate mates from a distanceuse trail following (Kiørboe and Bagøien, 2005), it isimportant to determine if this mechanism is present infreshwater species. If not, freshwater species must main-tain higher population densities or employ mate findingbehavior distinct from their marine relatives.

The bioenergetics of pheromone production and itsdiffusion rate in water implies that larger animals aremore likely to employ pheromones in mate location(Dusenberry and Snell, 1995). Large, freshwater diapto-mid copepods are common in alpine lakes in the SierraNevada and Rocky Mountains where fish predators arenot naturally present (Anderson, 1980). More recently,fish stocking has led to the local extinction of somelarge calanoid copepod species, includingHesperodiaptomus shoshone and H. arcticus (Knapp et al.,2001; Parker et al., 2001; Sarnelle and Knapp, 2004).These copepod populations sometimes fail to recoverfollowing fish removal despite the presence of diapaus-ing egg banks, presumably because of an Allee effect

stemming from mate limitation (Sarnelle and Knapp,2004; Kramer et al., 2008). The ability of these speciesto recolonize will depend on the critical density forrecovery which will depend on the encounter rate ofmales and females (Kramer et al., in press). Here, wedescribe the mate tracking behavior of H. shoshone in thefreshwater environment and provide evidence that thiscopepod uses chemical trail tracking in mate location,increasing the probability of mate encounter for theseobligately sexual organisms living a relatively vast three-dimensional (3D) habitat.

M E T H O D

Animal collection and maintenance

Hesperodiaptomus shoshone individuals were collected witha plankton net from Dissertation Lake (lake 52121,3615 m altitude, 378160200N, 11884103300W). Adults ofthis species are available for �1 month/year, from thetime the lake thaws in July until the lake freezes inSeptember. Immediately after collection, males andfemales were sorted into separate containers prefilledwith lake water. The male can be identified with a handlens based on its geniculated antennule, which is usedto grasp the female prior to mating. Copepods wereshipped in insulated containers on ice overnight toGeorgia Institute of Technology in Atlanta, GA, USA.Cultures were maintained at 128C in an incubationroom and were fed concentrated Tetraselmis spp. andRhodomonas lens phytoplankton along with rotifers andlow-salinity (5 ppt) Artemia nauplii. Males and femaleswere maintained in separate containers. Averageprosome lengths of adults in the collection were 2.46+0.13 mm (males) and 2.63+ 0.13 mm (females).Experiments were initiated within 4 days of collection.

Experimental observation

Mating behaviors of adult male and female H. shoshone

were observed using 3D Schlieren laser videography asdeveloped by Strickler (Strickler and Hwang, 1998) andfurther described by Doall et al. (Doall et al., 1998).Briefly, two video cameras were mounted to giveorthogonal views (x–z and y–z) of the experimentaltank. Using the common z-axis, the two orthogonalviews were superimposed and slightly offset to facilitatethe measurement of the 3D position of each animal.The vertical axis, common to both superimposedorthogonal views, was designated the z-axis.Visualization using the Schlieren optics relies on differ-ences in the refractive indices between the water and

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the copepods. Copepods were visible as white silhou-ettes illuminated by an infrared laser against a blackbackground. Copepods did not respond to this wave-length of light.

The size of the experimental tank ranged from 0.7 to3.0 L. Filtered, fresh water from Lake Lanier, locatedjust outside of Atlanta, was maintained at 128C in thetank by circulating distilled water in a large water jacketthrough a refrigeration unit. The pH was adjusted tomatch that of the source lake. Animal numbers rangedfrom 5 to 15 males and 5 to15 females per observationperiod.

At the beginning of each observation period,H. shoshone males were spooned into an experimentalvessel. Male behavior in the absence of females wasobserved for 20 min at the beginning of each exper-iment. After 20 min, one female for each male wasadded to the observation container. Mating interactionswere recorded for 2–4 h.

Trail-mimic bioassay

To determine if H. shoshone would follow a chemicaltrail in the absence of the females, males were exposedto chemical trails created from female-conditionedwater. To create chemical trails of H. shoshone, femaleswere placed in beakers containing filtered spring waterat a density of 1 animal per 10 mL. Females wereallowed to condition the water overnight (between 8and 10 h). After incubation was completed, female-conditioned water was transferred to sterile plastic vialsand frozen. Prior to chemical trail bioassays, female-conditioned water was thawed and was doubly-labeledwith high-molecular weight dextran to increase thedifference in the refractive index, enabling imaging ofthe trail using the Schlieren optics. To test the effect ofcue intensity, the conditioned water was first dilutedwith non-conditioned spring water (Crystal Spring) to50 and 10% the original strength. Control trails con-tained filtered spring water and dextran. Trail-mimicbioassays were conducted with 20 reproductivelymature H. shoshone (California) males in a 3-L tankaccording to the methods described by Yen et al. (Yenet al., 2004). Trails were fed into the tank by an elec-tronic syringe pump via capillary tubes at0.01 mL min21, resulting in a trail velocity of0.2 mm s21. The speed of flow of mimic is much lessthan the speed of the female (1.4 cm s21) and unlikelyto be a source of a hydrodynamic signal. Once thetrails were fully developed (after a few minutes), maleswere added into the tank. Trail following behavior wasvisualized using the Schlieren optical system (see above).Trials were conducted for 2 h. Upon trial termination,

animals were returned to their stock cultures anduntested copepods were used in replicates. The exper-imental tank was rinsed with distilled water and all syr-inges and capillary tubes were rinsed with acetone anddistilled water and allowed to dry between trials toremove any chemical scent.

Digital tracking of swimming trajectories

Mating interactions of H. shoshone were demonstrated bythe pursuit and/or capture of females by males. Thesex of animals was not obvious from the recording, sowhen smoothly swimming pairs remained together formore than one second in the video sequences, thepursuer was assumed to be a male and the pursued afemale and analyzed for this study of male-femalemating interactions. This assumption was based on thefollowing observations. In female-only observations, notrackings were observed. In male-only observations,couplings were infrequent: with 10 males, one couplingwas noted in an hour, whereas when 10 females wereadded to the experimental vessel with the same 10males, 5 couplings were noted in half an hour. Anothertrial with 20 males showed no couplings during 45 min.Pairings also were distinct: male–male pairings wereshort with rapid movements and antennal flickingwhereas male–female pairings remained attached forlonger periods with calm swimming.

A total of 16 h of observation were recorded with acharged-coupled device camera in VHS tape format.Mating events that occurred centrally in the tank andproviding more than 1 s of tracking were chosen for tra-jectory analysis (n ¼ 11). Analog video clips containingmating interactions were converted to a digital formatusing a dpsVelocity 8 (Leitch, Ackworth, GA, USA)interface between the video recording VCR and anIBM-compatible computer as a sequence of digitalimage frames at 33.3 ms intervals. Scion Image (ScionCorporation, Frederick, MD, USA) video analysis soft-ware was used to track the position of the rostrum ofindividuals on a Cartesian coordinate system. Positionmeasurements were taken for each frame, resulting in atemporal resolution of 33.3 ms. A 10.0-mm calibrationmeasurement from the video was used to convertpixel measurements into millimeters. Calibrated pos-ition measurements were used in subsequent quantitat-ive analyses of swimming trajectories and matinginteractions.

Swimming speed

The temporal sequence of swimming behaviors wasclassified into three stages with respect to pursuit and

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capture: “before” initiation of mate pursuit, “during”pursuit and “after” mate capture. Swimming speeds ofindividuals performing these behaviors were calculatedas the distance between consecutive 3D positionsdivided by the time elapsed between those two positions(i.e. 33.3 ms). The distance d between points was calcu-lated using the x, y, z coordinates in the followingequation:

d ¼ ððx2 � x1Þ2 þ ðy2 � y1Þ2 þ ðz2 � z1Þ2Þ1=2:

The average male and female swimming speeds before,during and after pursuit were calculated as the meansof consecutive 33.3 ms interval speeds.

Perceptive distance

Two perceptive distances were calculated for eachmating event: chemical and hydrodynamic. The chemi-cally mediated perceptive distance was calculated as thedirect linear distance between the male and the femaleat the initiation of mate pursuit behavior. The hydro-dynamic perceptive distance was calculated as the lineardistance between the male and the female when themale lunges to catch the female. At this distance, thehydrodynamic wake created by the female providesthe directional stimulus to evoke the high-speed lungethat results in mate capture (Yen et al., 1992, 1998). Theincrease in perceptive ability conferred by using chemi-cal perception versus hydrodynamic perception wascharacterized as the ratio of chemical perceptive dis-tance to hydrodynamic perceptive distance for eachmating event.

Event durations

The age of the trail at the time of detection was calcu-lated as the difference between the time when thefemale was positioned at the detection point to the timewhen the male initiated pursuit from this point. Thetime tracking was taken to be the time elapsed betweenmale initiation of pursuit and attempted capture of thefemale. The time coupled was recorded as the timeelapsed between successful capture of the female andseparation of the mating pair or disappearance of themating pair from the field of view.

Biologically generated flow

The hydrodynamic trail generated by a free-swimming,female H. shoshone individual was quantified using anParticle Image Velocimetry (PIV) system. The copepodswere housed in a 6 � 6 � 15 cm glass container filled

with Crystal Geyser spring water and seeded with tita-nium dioxide. The tank containing the copepods wasplaced inside a 15-cm cubic glass recirculation tankwhere deionized water was recirculated from the tankinto a Fisher Scientific chiller that maintained a temp-erature of 138C. To visualize the flow fields, a planarlaser sheet (width of 0.7 mm) was produced from apulsed, infrared Oxford laser (model HSI -500) to illu-minate the titanium dioxide particles. A high-resolution(1280 � 1024 pixel) VDS Vosskuhler CMC-1300 digitalcamera was placed parallel to the laser sheet.High-resolution pictures were taken at a frequency of50 Hz. Since the laser beam was pulsed with a displace-ment of 7 ms, the camera captured sequential imageswith a 7-ms time displacement. A smaller camera thatwas used to monitor the location of copepods in thelaser sheet was located perpendicular to the VDSVosskuhler digital camera. Data were only collectedwhile copepods were swimming within the laser sheet.A more detailed description of this set-up is available inCatton et al. (Catton et al., 2007).

PIV is a non-intrusive method used to quantify vel-ocity fields by measuring the displacement of seedingparticles over a time interval (Raffel et al., 1998). Thedisplacement of the particles is found by comparing thelocation of the particles between image pairs in 32 � 32pixel interrogation windows using a cross-correlationanalysis. To calculate the velocity, the displacement ofthe particles is divided by the time displacementbetween the two images. The velocity field was vali-dated by comparing a local grid value to the medianvalue of a neighboring 3 � 3 grid. Typically, less than1% of the velocity vectors within the velocity field wereidentified as bad vectors and replaced with a spatiallyinterpolated value (Westerweel, 1994; Nogueira et al.,1997). A Cartesian coordinate system was used wherex denotes the horizontal direction and y denotes the ver-tical direction, and where y values increase from thebottom of the tank to the top of the tank. The u and v

velocity components were calculated as:

u ¼ @x

@tand v ¼ @y

@t

The shear strain rate has been identified as a fluid com-ponent that elicits a behavioral response in copepods.Shear strain rate values .0.4 s21 cause an escape reac-tion in certain copepod species (Fields and Yen, 1997;Kiørboe et al., 1999). Thus, a shear strain rate value of0.4 s21 was used to determine the spatial extent of thehydrodynamic trail:

exy ¼1

2

@v

@xþ @u

@y

� �


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The shear strain rate is only one component of thestrain rate tensor, although it has been shown to be thelargest component in copepod flow fields (Catton et al.,2007). However, the orientation of the copepod in theflow field will determine what components of the strainrate tensor are potentially sensed by the copepod sensorarray. The maximum deformation rate is a measure ofthe maximum strain rate in the fluid that could besensed by the copepod (Kiørboe and Visser, 1999). Themaximum deformation rate is determined by solvingfor the eigenvalues (l1, l2) of the strain rate tensorusing the equation below and taking the maximum ofthe two eigenvalues. Kiørboe and Visser (Kiørboe andVisser, 1999) found that copepods in both Couette flowand siphon flow respond with escape behaviors to amaximum deformation rate in the fluid of �0.4 s21.Therefore, 0.4 s21 has been set as the thresholdmaximum deformation rate to determine the spatialextent of the hydrodynamic disturbance:

detexx � l1 exy

exy eyy � lx

�� ¼ 0

R E S U LT S

Qualitative description

A mating event is identified by the observation of twocopepods attached to each other via their antennule orthoracic appendages. Copepods attached by theirmouthpart appendages were considered to be engagedin a predation event and therefore were not included asa mating event. Analyses of the events preceding the

observed pair formation provided the followingsequence of events. (i) The male crosses the path of thefemale and then adjusts his bearing to follow thefemale’s path (the female copepod can be over 10 bodylengths ahead of the male copepod when pursuitbegins). (ii) The male accelerates and catches up to thefemale in a matter of seconds. (iii) Once within a fewmillimeters or 1–2 body lengths from the female, themale copepod lunges in her direction and captures herwith his geniculated antennule. [Supplementarymaterials online provide 3D animations of visualizedtrajectories of two mate tracking events. The analysis of105 additional 3D trajectories of mate-tracking pairs byKramer et al. (in revision) support the results presentedhere.]

Analyses of 3D trajectories of the male and femalecopepods involved in pair formation showed that malesfollow the path of the female. Instead of swimmingdirectly toward potential mates, H. shoshone malesclosely follow the previous swimming trajectory offemales (Fig. 1). Note the overlap in the male trajectoryon the female trajectory. This is considered the trackingevent. In Fig. 1A, the male is able to stay on track,whereas in Fig. 1B, the male loses track of the femalebut is able to re-intersect her path to continue followingit for his subsequent successful capture of the femalecopepod.

Trail mimics

Hesperodiaptomus shoshone males are able to follow a trailmimic that contains the scent of female conspecifics(Fig. 2). In these experiments, males encountering trailscontaining female-conditioned water followed themnearly half the time, and never followed control trails

Fig. 1. Graphical representations of 3D mating trajectories of H. shoshone. (A) Typical mating trajectory. (B) Trajectory where male leaves thetrail but is able to relocate the trail and continue tracking. Male and female trajectories are shown in grey and black, respectively. Start positionsare labeled as “Sm” (male start position) and “Sf ” (female start position). A male exhibits normal cruising behavior until he detects a female’schemical signal (Dm). At the point of signal detection, the female is �5 mm ahead of the male (Df). After the male detects the female, he turns,finds the female’s chemical trail and begins tracking (Tm). Female position at initial time of male tracking is denoted “Tf ”. As the male tracksthe female’s trail, he speeds up until he catches her and copulation begins (C). [Supplemental materials provide 3D animations of visualizedtrajectories of the mate tracking events presented in Fig. 1A and B. Male and female trajectories are shown in red and blue, respectively.]


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containing only filtered water and dextran (Table I). Asthe female-conditioned water was diluted, the tendencyto follow trails decreased (Table I). Once males intersectthe trail they tend to swim up, following it to the source(95% of 20 documented trail follows).

The average distance the trail mimic was followed(3.16+ 1.8 cm) was similar to that in the live trials(3.84+ 3.08 cm, Kolmogorov–Smirnoff (K–S) test for

non-normal data, D ¼ 0.2222, P ¼ 0.803; Zar, 2009).Males spent the same amount of time following a trailmimic (2.8+ 1.3 s) as they did the trails in live trials(2.4+ 2.3 s, K–S test, D ¼ 0.4495, P ¼ 0.1267). Theseobservations indicate that males follow trail mimics in amanner similar to how they follow female copepods.

Kinematic analyses

Swimming speedBefore locating the female trail, male H. shoshone swim-ming speed (1.25+ 0.14 cm s21) was similar to femaleswimming speed (1.36+ 0.25 cm s21, K–S test, D ¼

0.3636, P ¼ 0.4792, n ¼ 11). After detecting thefemale’s trail, male swim speed increased significantly(1.67+ 0.21 cm s21, K–S test, D ¼ 0.8182, P ¼

0.0006549, n ¼ 11). Males accelerated further to 6.8+2.8 cm s21 (n ¼ 11) in the final lunge that resulted incoupling with the female (at point C in Fig. 1). Oncecoupled, mating pairs of H. shoshone swam an average of1.58+ 0.39 cm s21 (n ¼ 10). The female appears tolead the pair because the male must use his appendagesto capture the female and transfer the spermatophore.

Perceptive distanceMales can respond to females by following trails or byreacting to hydrodynamic wakes (Table II). The averagedistance that a male is from the female when he beginstracking is 0.5 cm (range ¼ 0.31–0.96 cm), whereas theaverage distance he is when executing the mate capture

Table I: The frequency that an encountered trail mimic (Yen et al., 2004) is followed by the maleplanktonic copepod H. shoshone

Medium # encounters # trail follows % follows Duration of trial (h)

Female-scented lake water 13 6 46.2 250% dilution 78 6 7.7 690% dilution 63 1 1.6 6Controla 11 0 0 2

aControl contained spring water (Crystal Spring) and dextran.

Table II: Detection distances and trail following behavior in H. shoshone

Perceptive distance (cm)a

Trail age (s) Pursuit distance (cm) Time tracking (s)Chemical Hydro-dynamic Ratio

Mean 0.529 0.250 2.337 0.57 3.838 2.40SD 0.224 0.070 1.390 0.39 3.082 2.28n 11 11 11 11 11 11Min–max 0.31–0.96 0.14–0.37 0.97–5.7 0.13–1.3 1.59–11.5 0.87–8.63

aPerceptive distance is the straight line distance between male and female at the initiation of trail following (chemical) or coupling behavior(hydrodynamic).

Fig. 2. Following a trail mimic: orthogonal pairs of 2D images of amale H. shoshone following a trail mimic containing the scent of thefemale. There is no female copepod present and the speed of the trailmimic is 15% the natural swimming speed of female copepods. Nomales followed the first trail (lines a and c) and trail was not disturbed.The second trail (lines b and d) deformed as the male swims up thetrail after 2, 4 and 5 s. Within 5 s, the male copepod has stayed ontrack of the scented trail mimic for 8 cm.


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is 0.25 cm (range ¼ 0.14–0.37 cm) from the female.Hence, males of H. shoshone more than doubled the dis-tance through which they were able to perceive poten-tial mates by using chemically mediated detection.

Event durationsTrail age at the time of detection varied in a range from0.13 to 1.3 s (Table II). Pursuit distance (1.59–11.5 cm)and time spent tracking (0.87–8.63 s) was variable(Table II) since it depended on trail age and on maleand female swimming speeds. Once mating wasinitiated, pairs could remain coupled for long periods(105+ 235 s, n ¼ 10) with one pair remaining togetherlonger than 12 min.

Biologically generated flow

The velocity field created by a female H. shoshone isshown in Fig. 3. To generate motion, fluid was

entrained from above the copepod by the swimmingappendages on the ventral side of the organism andexpelled downward along the body of the copepod.Females produced a velocity disturbance of �1 cm inlength and 0.9 cm in diameter. The trail decay time,or time period between maximum velocity to back-ground velocity (0.025 cm s21), was estimated to be0.7 s. Re-plotting the velocity vectors (in cm s21)coupled with a contour plot of shear strain rates(equal to or greater than +0.05 s21 in colored con-tours; Fig. 4A) defines the hydrodynamic structurethat can be perceived. Contours were included atshear strain rates of +0.05 s21 and +0.4 s21 asthese are two strain rate thresholds that are related tochanges in behavior in turning and escapingresponses (Fields and Yen, 1997; Kiørboe et al., 1999;Woodson et al., 2005). Coincidentally, the averagebackground shear strain rate prior to the entry of thecopepod was +0.05 s21 (n ¼ 4) and, as shown inFig. 4A, this threshold level did not adequately definethe spatial extent of the hydrodynamic disturbance. Atdistances .0.4 cm (Table III, Fig. 4A), the strain rate(x– y) is below the threshold response level and close tothe background levels. The plot of the maximum defor-mation rate was similar to the plot of the shear strainrate, but the boundaries of the fluid disturbance weremore clearly demarcated (Fig. 4B). Due to the additionof the linear strain rate, the maximum trail length waslarger when considering the maximum deformationrate but still under a value of 0.6 cm (Table III). Theprofiles of the maximum deformation rate along thepath projections denoted by the black lines in Fig. 3 andFig. 4 show that in two of the three positions, the hydro-dynamically perceived trail length was ,0.2 cm, so inmost cases hydrodynamic perception would have tooccur at distances ,0.6 cm. Thus, the male copepodwould need to be closer than 0.6 cm from the female tobe able to discern her wake by hydrodynamic sensing.

Fig. 3. A contour plot of the instantaneous velocity field produced bya free-swimming H. shoshone. The colored contours represent the areawhere the velocities are .0.025 cm s21, the background value ofvelocity prior to the passage of the copepod in the field of view. Theblack lines represent the path of the trail at 08, 458 and 908.

Fig. 4. Contour plots of velocity vectors in cm s21 and shear strain rate field (A) and maximum deformation rate (B) in colored contours.Strain rates �0.05 s21 are plotted in colored contours. The black lines represent the path of the trail at 08, 458 and 908.


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D I S C U S S I O N

Remote mate detection: chemical orhydrodynamic cues?

We found that males of the freshwater copepod speciesH. shoshone followed trail mimics containing femalescent, demonstrating that a chemical signal can guidethe male copepod to the female. Trail following behav-ior was reduced when the female scent was diluted,indicating that the male’s behavior was sensitive to theintensity of the chemical signal. Using 3D videography,we confirmed that the freshwater copepod H. shoshone

followed the female along her path. In darkness, malesof H. shoshone would track conspecific female copepodsoften and form persistent mating pairs. Analyses of the3D trajectories of male and female copepods thatformed mating pairs showed that the male copepod didnot make a direct approach to the female as might beexpected if the copepod relied on hydrodynamic orvisual cues. Instead, when the male crossed the femaletrail, he made an abrupt turn and spent several secondsfollowing the female’s trail prior to capture. On average,the male crossed the trail at distances greater than thatat which the hydrodynamic signal matches that of thebackground flow (.0.5 cm).

These multiple lines of evidence confirm maleH. shoshone can use a chemical trail to detect females,but the male’s behavior just prior to capture suggeststhat hydromechanical cues also are used during matetracking. Previous studies have shown that copepods be-haviorally respond to strain rates or the spatial gradientof velocity, rather than the velocity magnitude.Typically, a strain rate value of 0.5 s21 is used as thethreshold for copepod escape behavior (Fields andWeissburg, 2005). However, recent research (Woodsonet al., 2005) shows that the threshold for copepodturning behavior may be an order of magnitude lowerin intensity (0.05 s21). Here, we have often found thatH. shoshone can begin trail following when .1.5 cmfrom the female on trails older than 1 s. The flow fieldanalysis shows that, at this distance, the strain rate is notdifferent from the background and this supports the

conclusion that males are responding to a chemical cue.However, once engaged in pursuit, the male can experi-ence hydrodynamic strain rates .0.5 s21 in close proxi-mity to the female, which may guide his final lungebefore capture. We conclude that H. shoshone probablyrelies on both chemical and hydromechanical cues toguide the male to a female.

The mating behavior of H. shoshone contrasts withthat of Leptodiaptomus ashlandii (Nihongi et al., 2004),another member of the family Diaptomidae. TheDiaptomidae is a wholly fresh water family derivedfrom calanoids originating in the marine environment(Boxshall and Jaume, 2000). Female diaptomids do notstore sperm (like other members of this superfamilyDiaptomoidea) and must mate again for each batch ofeggs, which intensifies the need to find suitable mates tomaximize reproductive success. Leptodiaptomus ashlandii

males do not swim directly to mates nor along thefemale’s path. Instead, they may advect scent into theirfeeding current flow field from a female swimmingnearby, thus detecting a diffuse chemical signal thatevokes an increase in swimming speed. Leptodiaptomus

ashlandii also does not respond to fluid mechanical cuesby tandem hopping, as noted for another freshwatercopepod, Cyclops scutifer (Strickler, 1998). Hesperodiaptomus

shoshone inhabits environments having exceedingly lowproductivity (alpine lakes) and so occurs at densities thatare low relative to most other copepods. These factorsseem likely to select for effective strategies to ensuremating.

Remote detection of females is an efficient means ofimproving reproductive success by increasing the prob-ability of mating encounters (Kiørboe, 2006). Encounterrates can be increased by other tactics as well, includingaggregative behavior (Ambler et al., 1996) and fasterswimming speeds (Kiørboe, 2008; Kramer et al., inpress). However, whereas aggregative behavior promotesconspecific encounters, it also may increase intraspecificcompetition. Likewise, faster swimming may increaseencounters with predators as well as mates, where fastmovements can make copepods more conspicuous tovisual predators. Faster swimming also is correlated

Table III: The spatial extent of the hydrodynamic disturbances generated in the side view byfree-swimming, female H. shoshone

nn

Trail length (cm)

Velocity extent Shear strain rate extent Maximum deformation rate extent

Female H. shoshone 4 0.95+0.35 0.43+0.24 0.59+0.22

The threshold values that were used to define the length of the hydrodynamic trail for velocity, shear strain rate and maximum deformation rate were0.025 cm s21, 0.4 s21, 0.4 s21, respectively. The spatial extents are presented as the mean maximum length of the disturbance greater than thethreshold value+SD.


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with larger body size (Kiørboe, 2008), a major factordriving a zooplankter’s vulnerability to visually feedingfish (Brooks and Dodson, 1965), the dominant zoo-planktivores in most lakes. Neither of these two alterna-tive tactics is specific to improving interactions withmates. As shown here, the use of chemical and hydro-mechanical cues improves encounter rates with mateswithout these drawbacks, although there are likelymetabolic costs for the chemical production-detectionsystem. Additionally, there is a chance, though notdocumented, that predators might eavesdrop on thesecues.

Animals that can increase the probability of matingencounter via remotely detected cues should be moreable to establish populations from low-density coloniza-tion events. Thus, chemical detection of mates can beseen, at the population level, as a strategy that mitigatesthe negative effects of low density on population growth(i.e. Allee effects, Courchamp et al., 2008), by reducingthe “critical density” for positive population growth(Gerritsen, 1980; Kiørboe, 2006). Using behavioral dataon perceptive distances and swimming speed, alongwith a detailed encounter model, we are able to esti-mate the critical density for colonization events in thiscopepod species (Kramer et al., in press).

Conclusions

With this study, we increase the set of calanoid familieshaving members that rely on diffusible chemicals totrack mates to three: Temoridae, Centropagidae andnow Diaptomidae, all of which are grouped into thesuperfamily Diaptomoidea. Within this superfamily, thepheromone trail following behavior differs for the fresh-water and marine species of copepod. The chemicaltrails of the freshwater diaptomid H. shoshone are shorter(3.8 cm max) and their trail follows of 2.4 s are brieferthan those of the marine species. Temora longicornis

tracked trails for distances exceeding 13 cm, with resi-dence times in the trail of up to 5.5 s (Doall et al.,1998). Centropages typicus performed similarly, tracking upto 17 cm for 5.7 s (Bagøien and Kiørboe, 2005). MaleH. shoshone follow females closely, usually remaining,1 cm (�3 BL) from the female, whereas males of themarine species T. longicornis are often separated fromtheir females by 3 cm (�25 BL; Doall et al., 1998),suggesting either weaker sensitivity to chemicals by H.

shoshone or less stability of the freshwater pheromone.Our results demonstrate, for the first time, that chemi-cally mediated mate tracking behavior occurs in a largefreshwater copepod. This behavior may help to resolvethe paradox of this species’ widespread distribution inisolated alpine lakes despite the operation of Allee

effects limiting population establishment (Kramer et al.,2008).

S U P P L E M E N TA RY DATA

Supplementary data can be found online at http://plankt.oxfordjournals.org.

AC K N OW L E D G E M E N T S

The collection in the Sierra Nevada would not havebeen possible without the help of Roland Knapp.Collecting permits were provided by the Inyo andSierra National Forests, and the California Departmentof Fish and Game. We thank Rachel Lasley and HiteshPatel for their contribution to this research. We thankthe help of two reviewers.

F U N D I N G

This research was financially supported by a NationalScience Foundation Graduate Research Fellowship,National Science Foundation grants DEB-9629473,DEB-0075509, EHR-0114400, OCE-0728238, anREU supplement, VESR Graduate Student grant, anda Sigma-Xi Grant-in-aid of Research.

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