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RESEARCH ARTICLE J. H. Cohen R. B. Forward Jr. Diel vertical migration of the marine copepod Calanopia americana. II. Proximate role of exogenous light cues and endogenous rhythms Received: 26 August 2004 / Accepted: 7 January 2005 / Published online: 4 March 2005 ȑ Springer-Verlag 2005 Abstract The marine copepod Calanopia americana Dahl undergoes twilight diel vertical migration (DVM) in the Newport River estuary, North Carolina, USA, in syn- chrony with the light:dark cycle. Copepods ascend to the surface at sunset, descend to the bottom around mid- night, and make a second ascent and descent before sunrise. Behavioral assays with C. americana in the laboratory during fall 2002/2003 and summer 2004 investigated aspects of three hypotheses for the proxi- mate role of light in DVM: (1) preferendum hypothesis (absolute irradiance), (2) rate of change hypothesis (relative rates of irradiance change), and (3) endogenous rhythm hypothesis. Results suggest that C. americana responds to exogenous light cues consistent with its DVM pattern; changes in absolute irradiance evoked swimming responses that would result in an ascent at sunset and descent at sunrise, while relative rates of irradiance decrease at sunset (0.0046 s 1 ) evoked an ascent response, and relative rates of irradiance increase at sunrise (0.0042 s 1 ) evoked a descent response. Fur- thermore, C. americana expressed an endogenous rhythm in vertical migration that was positively corre- lated with field observations of twilight DVM. Collec- tively, these results indicate that both exogenous light cues and endogenous rhythms play a proximate role in twilight DVM of C. americana, providing redundancy in the causes of its vertical migration. Introduction Light is the primary proximate factor in diel vertical migration (DVM) of zooplankton (Ringelberg 1964, 1999; Forward 1976, 1988; Haney 1988). Since DVM has a diel period, its proximate cause should also have a diel period, as the light cycle does (Ringelberg 1995a). However, DVM is highly variable in space and time (Bollens and Frost 1989a, 1989b; Ohman 1990; Dagg et al. 1997), with non-photic environmental factors (e.g. temperature and food availability) influ- encing aspects of migratory behavior (Haney 1993). To clarify this, Ringelberg (1995b, 1999) suggested that environmental factors influencing proximate aspects of DVM should be classified in the following hierarchy: (1) primary causal factors associated with irradiance change over the diel cycle; (2) secondary causal factors including the presence of predators, food, and light adaptation conditions; and (3) tertiary causal factors including temperature, oxygen, and sub-surface chlo- rophyll, which are environmental gradients. This hierarchy recognizes light as the proximate cause of DVM, with the primary causal factor in vertical movement usually being light-mediated swimming behavior (photoresponses; Forward 1988; Ringelberg 1999). Secondary factors mediate photoresponses and influence the initiation of migration. These secondary factors include chemical cues from predators (Forward and Rittschof 1999, 2000), feeding state (Cronin and Forward 1980; Dagg 1985), and the level of light adaptation (Stearns and Forward 1984; Forward 1985). Tertiary factors influence swimming zooplank- ton, and may act as barriers to vertical movements (Sameoto 1984; Daro 1988; Harris 1988; Dagg et al. 1997). Communicated by J.P. Grassle, New Brunswick J. H. Cohen (&) R. B. Forward Jr. Duke University Marine Laboratory, Biology Department and Nicholas School of the Environment and Earth Sciences, Duke University, 135 Duke Marine Lab Rd., Beaufort, NC 28516, USA E-mail: [email protected] Tel.: +1-772-4652400308 Fax: +1-772-4680757 Present address: J. H. Cohen Marine Science Division, Harbor Branch Oceanographic Institution, 5600 U.S. 1N, Ft. Pierce, FL 34946, USA Marine Biology (2005) 147: 399–410 DOI 10.1007/s00227-005-1570-4

Diel vertical migration of the marine copepod Calanopia americana. II. Proximate role of exogenous light cues and endogenous rhythms

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RESEARCH ARTICLE

J. H. Cohen Æ R. B. Forward Jr.

Diel vertical migration of the marine copepod Calanopia americana. II.Proximate role of exogenous light cues and endogenous rhythms

Received: 26 August 2004 / Accepted: 7 January 2005 / Published online: 4 March 2005� Springer-Verlag 2005

Abstract The marine copepod Calanopia americanaDahlundergoes twilight diel vertical migration (DVM) in theNewport River estuary, North Carolina, USA, in syn-chrony with the light:dark cycle. Copepods ascend to thesurface at sunset, descend to the bottom around mid-night, and make a second ascent and descent beforesunrise. Behavioral assays with C. americana in thelaboratory during fall 2002/2003 and summer 2004investigated aspects of three hypotheses for the proxi-mate role of light in DVM: (1) preferendum hypothesis(absolute irradiance), (2) rate of change hypothesis(relative rates of irradiance change), and (3) endogenousrhythm hypothesis. Results suggest that C. americanaresponds to exogenous light cues consistent with itsDVM pattern; changes in absolute irradiance evokedswimming responses that would result in an ascent atsunset and descent at sunrise, while relative rates ofirradiance decrease at sunset (�0.0046 s�1) evoked anascent response, and relative rates of irradiance increaseat sunrise (0.0042 s�1) evoked a descent response. Fur-thermore, C. americana expressed an endogenousrhythm in vertical migration that was positively corre-lated with field observations of twilight DVM. Collec-tively, these results indicate that both exogenous lightcues and endogenous rhythms play a proximate role in

twilight DVM of C. americana, providing redundancy inthe causes of its vertical migration.

Introduction

Light is the primary proximate factor in diel verticalmigration (DVM) of zooplankton (Ringelberg 1964,1999; Forward 1976, 1988; Haney 1988). Since DVMhas a diel period, its proximate cause should also havea diel period, as the light cycle does (Ringelberg1995a). However, DVM is highly variable in space andtime (Bollens and Frost 1989a, 1989b; Ohman 1990;Dagg et al. 1997), with non-photic environmentalfactors (e.g. temperature and food availability) influ-encing aspects of migratory behavior (Haney 1993). Toclarify this, Ringelberg (1995b, 1999) suggested thatenvironmental factors influencing proximate aspects ofDVM should be classified in the following hierarchy:(1) primary causal factors associated with irradiancechange over the diel cycle; (2) secondary causal factorsincluding the presence of predators, food, and lightadaptation conditions; and (3) tertiary causal factorsincluding temperature, oxygen, and sub-surface chlo-rophyll, which are environmental gradients. Thishierarchy recognizes light as the proximate cause ofDVM, with the primary causal factor in verticalmovement usually being light-mediated swimmingbehavior (photoresponses; Forward 1988; Ringelberg1999). Secondary factors mediate photoresponses andinfluence the initiation of migration. These secondaryfactors include chemical cues from predators (Forwardand Rittschof 1999, 2000), feeding state (Cronin andForward 1980; Dagg 1985), and the level of lightadaptation (Stearns and Forward 1984; Forward1985). Tertiary factors influence swimming zooplank-ton, and may act as barriers to vertical movements(Sameoto 1984; Daro 1988; Harris 1988; Dagg et al.1997).

Communicated by J.P. Grassle, New Brunswick

J. H. Cohen (&) Æ R. B. Forward Jr.Duke University Marine Laboratory,Biology Department and Nicholas School of theEnvironment and Earth Sciences, Duke University,135 Duke Marine Lab Rd., Beaufort, NC 28516, USAE-mail: [email protected].: +1-772-4652400308Fax: +1-772-4680757

Present address: J. H. CohenMarine Science Division, Harbor Branch OceanographicInstitution, 5600 U.S. 1N, Ft. Pierce, FL 34946, USA

Marine Biology (2005) 147: 399–410DOI 10.1007/s00227-005-1570-4

Of the numerous hypotheses proposed to explain therole of light as the proximate cause of DVM, three seemthe most plausible: (1) the preferendum (isolume)hypothesis, (2) the rate of change hypothesis, and (3) theendogenous rhythm hypothesis. The historical back-ground and evidence for these hypotheses has beenextensively reviewed (Ringelberg 1964, Forward 1988).

The preferendum hypothesis states that zooplanktonfollow specific isolumes (i.e. levels of constant irradi-ance), which move vertically in the water column atsunrise and sunset, but otherwise remain at relativelyconstant depths. The tendency of zooplankton to remainclose to this moving level of light (e.g. by a suite ofnegative feedback phototactic/geotactic behaviors) ex-plains their evening ascent and morning descent duringnocturnal DVM. A variation of this hypothesis involveszooplankters that initiate a behavioral response, forexample phototaxis or geotaxis, leading to migrationwhen exposed to a specific absolute irradiance (Sweattand Forward 1985). Field and laboratory evidencesupports the preferendum hypothesis for DVM in somepelagic and mesopelagic species in deep environments(Boden and Kampa 1967; Frank and Widder 1997,2002), and in some shallower dwelling species in turbidwater where distinct isolumes can form (Forward 1985),but other proximate mechanisms are likely involved incueing migration of most shallow-water species (coastal,estuarine, freshwater, hypersaline) (Forward 1988).

The rate of change hypothesis suggests that verticalmovements in DVM are caused by behavioral responsesto the relative rate and direction of irradiance changefrom some non-fixed ambient adaptation intensity. Thishypothesis is supported by a variety of laboratory andfield studies suggesting that several zooplankton specieshave behavioral responses to relative rates of irradiancechange consistent with DVM, and rates of change thatinitiate photobehavior in the laboratory, as well as levelsof light adaptation providing maximal responsiveness ofthese behaviors, are similar to those occurring at sunriseand sunset in the field (Forward 1988; Ringelberg 1999).The most compelling evidence for the rate of changehypothesis comes from work on the freshwater cladoc-erans Daphnia spp. (Ringelberg 1999, and referencestherein), the zoea of the estuarine crab Rhithropanopeusharrisii (Forward 1985), and the estuarine/coastalcopepod Acartia tonsa (Stearns and Forward 1984).Both the ascent and descent of Daphnia spp. andA. tonsa involve behavioral responses in accordance withthe rate of change hypothesis. In R. harrisii only theascent phase of DVM involves a phototactic response torates of irradiance change; its descent follows anisolume.

Unlike the preferendum and rate of change hypoth-eses, the endogenous rhythm hypothesis involves lightacting indirectly as an entraining agent (zeitgeber) tosynchronize vertical migration with the diel light cycle(Rudjakov 1970; Dunlap et al. 2004). In a field studyon the marine copepod Calanus helgolandicus(pacificus), Enright and Honeggar (1977) suggested that

a pre-sunset rise of the migrating population was due toan endogenous rhythm, possibly in activity. Laboratorystudies examining endogenous vertical migrationrhythms in copepods and other migratory zooplanktonhave been equivocal. Harris (1963) reported a night-ac-tive endogenous vertical migration rhythm in Calanusfinmarchicus maintained under constant darkness in asmall tank. In contrast, several laboratory studies havefailed to find a similar rhythm in A. tonsa (reviewed byStearns 1983). Using a natural mixed zooplanktonassemblage in large troughs, Enright and Hamner (1967)observed DVM in several species under the ambientphotoperiod, few of which continued to migrate underconstant darkness. Thus, it appears that endogenousrhythms are not a universal proximate cause of DVM,but may be a factor for some species.

In laboratory experiments, we examined light as theprimary causal factor in twilight vertical migration ofCalanopia americana in the Newport River estuary(Cohen and Forward 2005) to answer the followingquestions: (1) Do absolute changes in irradiance affectvertical position of copepods in the water column? (2)Does C. americana exhibit photobehavior consistentwith twilight DVM upon stimulation with relative ratesof irradiance change that occur at sunset and sunrise? (3)Does C. americana possess an endogenous rhythm invertical migration behavior that could underlie DVM?

Materials and methods

Experiments testing responses to absolute irradiancechange

Calanopia americana Dahl were captured using a sta-tionary, 333-lm-mesh, 0.75-m plankton net, set prior tomaximum current on night-time flood tides near Beau-fort Inlet, N.C., USA (34�4¢N; 76�4¢W), in November2002. All net samples were brought to the laboratory,diluted with ambient seawater (salinity=36), and al-lowed to acclimate to the temperature of all experiments(23�C) under the ambient photoperiod of 10 h light:14 hdark with gentle aeration. Trials were conducted duringboth the day (0900–1200 hours) and night (2100–0100 hours). For trials conducted during the night,C. americana were selected for experiments the night ofcollection. Only healthy adult females were used to re-duce variation due to sex and growth stage (age); femaleC. americana are larger than males, and female cope-pods, in general, have greater energy requirements dueto egg production (Mauchline 1998). For trials con-ducted on the day after collection, the same selectionprocess occurred within 10 h of collection. Groups of 50C. americana were gently pipetted into 40 ml of aged100-kDa-filtered offshore seawater (salinity=36), inwhich they remained no longer than 2 h without fooduntil use in an experiment.

Aged 100-kDa-filtered seawater (hereafter FSW)was prepared by septic filtration (A/G Technology

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model UFP-100-C-4X2A) of offshore seawater to re-move biologically active molecules >100 kDa, andsubsequent aging for at least 1 week. This process pro-duces seawater with a consistent chemical compositionthat does not alter crustacean photoresponses (Rittschofet al. 1983; Forward and Rittschof 1999, 2000).

Groups of 50 copepods were transferred to an acryliccolumn (5·5·19 cm) filled with FSW, which was placedat the horizontal center of a water bath (50·50·25 cm)with inside walls painted flat black. The bath was filledwith deionized water at a level just below the upper edgeof the column. Light entered the bath from the top andpassed through a white acrylic diffuser plate, creating auniform overhead light field. The walls of the bath wereoutside the critical angle (zenith ±48.6�), as viewed fromthe bottom of the column. Testing in this type ofapparatus simulates the normal underwater angularlight distribution: a relatively bright light overhead withdiffuse surrounding irradiance and a darker bottom(Verheijen 1958, 1985; Forward et al. 1984). Zooplank-ton display abnormal photobehaviors in highly direc-tional light fields (e.g. Stearns and Forward 1984). Thelight source was a 750-W incandescent bulb, with irra-diance controlled using fixed neutral density filters andspectral composition limited to the blue-green spectrumby a Corning no. 4-96 filter (Kopp Glass). The spectralsensitivity of C. americana encompasses those wave-lengths maximally transmitted by the blue-green filter(475–550 nm; Cohen and Forward 2002). Irradiancecalibration of the laboratory apparatus was in photonsper square meter per second (at 500 nm; EG&Gmodel 550 radiometer), units similar in magnitude tothe ‘‘copelux’’ units described by Cohen and For-ward (2005). In the laboratory apparatus, an irradi-ance of 1.1·1017 photons m�2 s�1 corresponded to1.2·1017 copelux.

The stimulus series presented to a group of copepodsconsisted of either: (1) step increases in irradiance or (2)step decreases in irradiance. For step-increase trials,copepods were exposed to darkness for 15 min, afterwhich light was increased to 1·1010 photons m�2 s�1.Light was subsequently increased by 1 OD every 5 minby removing neutral-density filters from the light path.The final stimulus presented to the copepods was1·1016 photons m�2 s�1. For step-decrease trials, thestimulus order was reversed; the first stimulus was1·1016 photons m�2 s�1 (15 min), and the final stimuluswas darkness. Both day and night trials were replicatedfive times with different groups of copepods. This re-sulted in the following experimental treatments: (1) daystep increases, (2) day step decreases, (3) night step in-creases, and (4) night step decreases.

Copepods in the columns were recorded using aclosed-circuit video system, with illumination by far-redlight (maximum transmission=774 nm). Far-red lightdoes not alter or induce photoresponses in C. americana(Cohen and Forward 2002). The number of copepodsswimming in the upper 20% of the column was countedfrom video records at the end of the initial light

condition (15 min in either darkness or 1·1016 photonsm�2 s�1) and after 5 min at each subsequent irradiancelevel. Significant differences in the number of copepodsin the upper 20% of the column among irradiance levelswere tested for each treatment using one-factor re-peated-measures ANOVAs. Multiple comparisons weredone using a Dunnett’s test with the dark treatment as acontrol (q¢a(2) 32,8; Zar 1999).

Experiments testing responses to relative ratesof irradiance change

For experiments on relative rates of irradiance decrease(as occur at sunset), copepods were collected at night inOctober 2003 as described above. Healthy adult femaleC. americana were selected immediately upon returningto the laboratory; they were placed in FSW (salin-ity=36), fed an algal paste solution at 0.64 ml l�1 (3 mlconcentrated Brine Shrimp Direct brand ‘‘TahitianBlend’’ algal paste in 22 ml FSW), and allowed toacclimate overnight to the temperature of 23�C undergentle aeration. The ambient photoperiod of 11 hlight:13 h dark was maintained. Three hours prior tosunset on the day following collection, a group of 50copepods was added to an acrylic cuvette (4·4·5 cm)filled with FSW. Copepods remained in this conditionfor at least 1 h under cool-white fluorescent lighting(�5·1019 photons m�2 s�1). Copepods were then darkadapted for 2 h in a light-tight box before photore-sponses were tested.

For experiments on relative rates of irradiance in-crease (as occur at sunrise), copepods were collected atnight in July 2004 as described above, and gently aer-ated overnight. The following morning, healthy adultfemale C. americana were placed in the same feedingconditions described above. Copepods were maintainedin an incubator at 23�C under the ambient photoperiodof 14 h light:10 h dark with gentle aeration. Prior tosunrise the following morning, a group of 60 C.americana was added to an acrylic cuvette (4·4·5 cm)filled with FSW. Copepods were transferred to cuvettesunder dim red light, and therefore remained darkadapted.

For both relative increases and decreases in irradi-ance, a cuvette of dark-adapted C. americana was placedin the water bath described above, and using a modifiedversion of the light stimulus arrangement described forabsolute irradiance experiments, copepods were lightadapted at 8·1013 photons m�2 s�1. This adaptationlevel was used because it was greater than the visualthreshold of dark-adapted C. americana (1.3·1012 pho-tons m�2 s�1; Cohen 2004), but not so high as to causethe copepods to cease swimming and remain on thebottom of the cuvette (see absolute irradiance data be-low). Irradiances of �8·1013 photons m�2 s�1 occur atthe bottom of the Newport River estuary �0.5 h aftersunset, when the initial ascent of copepods in the watercolumn occurs, and >0.5 h prior to sunrise in surface

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waters, when copepods have descended to depth (Cohenand Forward 2005).

After 15 min of light adaptation, a series of eitherrelative irradiance decreases or increases was presented.Stimuli were continuous rates of change lasting 20 screated by a circular variable-neutral-density filter wheel(Oriel Instruments), coupled to a variable speed motor(1 rpm). The wheel was calibrated for relative rates ofirradiance change using the equation of Ringelberg(1964) in units of per second. Relative decreases werepresented in the following order: �0.0021, �0.0032,�0.0046, �0.0058, and �0.0068 s�1. Relative increaseswere presented in the following order: 0.0023, 0.0031,0.0042, 0.0055, and 0.0062 s�1. These rates were selectedbecause they encompass those occurring at twilight inestuarine/coastal water (Cohen and Forward 2005).After each stimulus, the wheel was returned to its pre-stimulus position, and copepods were given 5 min at theadaptation irradiance prior to the next stimulus.Preliminary experiments determined this interval be-tween stimuli did not alter the response upon repeatedstimulation at intermediate rates of decrease. Forrelative decreases, four replicate trials were conductedafter sunset (�1845 hours). For relative increases,five replicate trials were conducted prior to sunrise(�0600 hours).

Movement of copepods during the experiments wasrecorded as described above for absolute irradianceexperiments. Aspects of swimming behavior and orien-tation were later analyzed from video recordings using aPC-based motion analysis system (CellTrak software,Motion Analysis). Swimming behavior was analyzedduring a 3-s interval beginning 6 s after the start of astimulus (response), as well as 20 s prior to each stimulus(control) for the same duration under light-adaptationconditions (8·1013 photons m�2 s�1). The mean angulardirection of movement in the XY-plane for copepods inthe field of view (10–25 individuals) was determinedfrom digitized video recordings. Only copepods thatoriented in a significant direction while being observedwere used for analysis (Rayleigh’s z, a=0.01). The per-centage of copepods ascending (moving upward towardsthe stimulus light, ±30�) and descending (movingdownward away from the stimulus light, ±30�) wasdetermined. An increase in either the percentageascending or descending relative to the control valuesindicated a response to the stimulus.

For both experiments, one-factor repeated-measuresANOVAs were performed on control data to ensurethere were no significant differences (a=0.05) prior topooling controls for subsequent analyses (Zar 1999).This was the case for both experiments, so control valuesfor all stimuli in an experiment were pooled, yielding asingle control mean and standard error. Response datawere then analyzed using one-factor repeated-measuresANOVAs, including the controls as additional treat-ments. Where applicable, multiple comparisons weredone using a Dunnett’s test versus the control treatment(Zar 1999). A one-tailed statistical test was used, because

relative rates of irradiance change were expected to in-crease the percentage of copepods ascending relative tothe control for relative irradiance decreases and to in-crease the percentage descending for relative irradianceincreases.

Experiments testing endogenous rhythms

The presence of an endogenous vertical migrationrhythm in C. americana was evaluated by counting thenumber of copepods present in the upper half of col-umns maintained under constant darkness for �4 days.Copepods were collected at night as described above inSeptember and October 2002 and November 2003. Theywere either sorted immediately, or acclimated overnightto the temperature of all experiments (23�C) undergentle aeration, with the ambient photoperiod of 13–10 h light:11–14 h dark (depending upon experiment),and sorted the following day. Groups of either 55 or 100healthy female C. americana were transferred to acryliccolumns (5·5·19 cm) containing the following initialfeeding conditions: (1) 2.5·106 Rhodomonas sp. cells l�1,(2) 1.25·106 Rhodomonas sp. cells l�1, (3) 2.5·105 Rho-domonas sp. cells l�1, and (4) 50 freshly hatched Artemiafranciscana nauplii. Rhodomonas sp. algal cells and A.franciscana (or brine shrimp) were used as foodthroughout the experiments, as copepods survive well oneither diet when maintained under the ambient photo-period in the laboratory (J. Cohen, personal observa-tions). Rhodomonas sp. was tested at severalconcentrations to determine if rhythms were dependentupon food concentration. Rhodomonas sp. solutionswere prepared for each column by diluting an appro-priate volume of algal culture with FSW to a final vol-ume of 400 ml, whereas A. franciscana were addeddirectly to 400 ml of FSW. Columns were placed in frontof a far-red light (maximum transmission=774 nm) inan otherwise light-tight room, and the upper half of eachcolumn was imaged for 3–4 days using a closed-circuitvideo system with multiplexer (ATV DPX4) and time-lapse recorder (Panasonic AG-RT600A). The lightconditions were effectively constant darkness for C.americana as far-red light does not alter or inducephotoresponses in this copepod species (Cohen andForward 2002). The number of copepods present in theupper half of the columns was counted at 0.5-h intervalsfrom the time-lapse videos.

Three endogenous rhythm experiments were done.The first experiment began 3 days prior to the firstquarter of the lunar cycle (10 September 2002) andended on the first quarter (13 September 2002). Twocolumns were prepared, each with 100 C. americana.The initial feeding condition was 2.5·106 Rhodomon-as cells l�1, with no additional food provided duringthe experiment and no aeration. Columns were placedin darkness, and monitoring began at 1500 hours on 10September (sunset=1922 hours) and continued until0730 hours on 13 September. The second experiment

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began 1 day after the new moon (7 October 2002) andended 2 days prior to the first quarter (11 October2002). Six columns were prepared, each with 100 C.americana, with two columns at each of the followinginitial feeding conditions: 2.5·106, 1.25·106, and2.5·105 Rhodomonas cells l�1. No additional food wasprovided during the experiment, and there was noaeration. Columns were placed in darkness, and mon-itoring began at 2335 hours on 6 October (sun-set=1841 hours) and continued until 1300 hours on 11October. The third experiment began 1 day after thenew moon (24 November 2003) and ended 2 days priorto the first quarter (28 November 2003). Four columnswere prepared, each with 55 C. americana. The initialfeeding conditions were 50 freshly hatched A. francis-cana nauplii, with 50 nauplii added at haphazard timeseach day of the experiment. Each column was aeratedby a slowly bubbling pipette submerged halfway fromthe bottom of the column. Columns were placed indarkness, and monitoring began at 1700 hours on 24November (sunset=1657 hours) and continued until0900 hours on 28 November.

Endogenous rhythm time series were analyzed forsignificant periodicity using autocorrelation analysis,and period estimates (free-running period; Dunlap et al.2004) were obtained using maximum entropy spectral

analysis (Levine et al. 2002). Cross-correlation analysiswas used to determine the phase relationships amongendogenous rhythm time series, as well as between eachtime series and environmental cues that could poten-tially entrain the rhythm (tidal height, subjective irra-diance) (Wing et al. 1995). Endogenous rhythm timeseries were averages of replicate columns. Tidal heighttime series (meters above mean lower low water,MLLW) for the site where copepods were collected attimes corresponding to each experiment were obtainedfrom a computer program (Tides and Currents Pro,Nobeltech Nautical Software, 2001). Irradiance timeseries were step functions, with a maximum value (sub-jective day) from the time of sunrise through the time ofsunset and a minimum value (subjective night) from thetime of sunset through the time of sunrise.

Endogenous vertical migration rhythms recorded inthe laboratory were cross-correlated with the twilightDVM pattern described for C. americana in the NewportRiver estuary (Cohen and Forward 2005). Laboratorymeasurements of the percentage of copepods in the up-per half of columns for all replicates of the2.5·106 Rhodomonas sp. cells l�1 feeding condition(n=4) were averaged according to time relative to sub-jective sunset. This feeding condition was used becausemortality was the lowest (19–38%).

Results

Experiments testing responses to absolute irradiancechange

When initially placed in darkness for 15 min, >60% ofCalanopia americana were found in the upper 20% of thecolumn during both day and night experiments(Fig. 1a). Five-minute exposures to light with 1-OD in-creases, starting from 1·1010 photons m�2 s�1, resultedin significantly fewer copepods in the upper columnrelative to the dark percentage when absolute irradiancewas >1·1011 photons m�2 s�1 for daytime experiments,

Fig. 1a, b Calanopia americana. Vertical position in response toabsolute irradiance change. Percentage of copepods in the upper20% of the column plotted as a function of absolute irradiance.Absolute irradiance changes presented as: a step increases fromdarkness to 1·1016 photons m�2 s�1 and b step decreases from1·1016 photons m�2 s�1 to darkness. Arrows below x-axes showdirection of step changes in irradiance. Means and SE (n=5)plotted for experiments conducted during the day (open circles) andnight (solid circles). Gray asterisks (day experiments) and blackasterisks (night experiments) show responses at a given irradiancesignificantly different from the response in darkness (one-factorrepeated-measures ANOVAs with a Dunnett’s test for multiplecomparisons, q¢a(2) 32,8; Zar 1999). Control responses in darknessindicated by ‘‘C’’ on the plots. See Table 1 for individual teststatistics

403

and when absolute irradiance was >1·1014 photonsm�2 s�1 for experiments conducted during the night(Fig. 1a; Table 1). When these experiments were con-ducted in reverse (1-OD decreases; Fig. 1b), few C.americana were in the upper 20% of the column after theinitial 15-min exposure to 1·1016 photons m�2 s�1. Forexperiments conducted during the night, the percentageof copepods in the upper column increased as absoluteirradiance decreased until 1·1011 photons m�2 s�1,where the percentage of copepods in the upper columnwas the same as in darkness (Fig. 1b; Table 1). In con-trast, absolute irradiance decreases during daytimeexperiments did not result in an increase in copepods inthe upper column (Fig. 1b; Table 1). No mortality wasobserved during these experiments.

Experiments testing responses to relative ratesof irradiance change

Relative rates of irradiance decrease, simulating thoseoccurring during sunset in the Newport River estuary,evoked a stimulus-dependent ascent response in C.americana (Fig. 2a). The percentage of copepodsascending did not differ significantly among pre-stimuluscontrols (P=0.204), whereas significant differences ex-isted among rate of change stimuli (P=0.023). Relativeirradiance decreases evoked greater percentages of co-pepods ascending than non-stimulus controls for all ratestimuli except the slowest (�0.0021 s�1) (Fig. 2a). Onlyat �0.0046 s�1 was the ascent response significantlygreater than the pooled controls (q¢=2.738, P<0.05,Dunnett’s test). No mortality was observed during theseexperiments.

Relative rates of irradiance increase, simulatingthose occurring during sunrise in the Newport Riverestuary, appeared to evoke a stimulus-dependent des-cent response in C. americana (Fig. 2b). Significantdifferences were not found among pre-stimulus con-trols (P=0.562), and no significant differences existedamong descent responses to rate of irradiance increasestimuli in ANOVAs (P=0.138). However, relativeirradiance increases did evoke a greater mean per-centage of copepods descending than non-stimuluscontrols for 0.0031 and 0.0042 s�1 stimuli (Fig. 2b);the 0.0042 s�1 stimulus was significantly greater than

its paired pre-stimulus control (P=0.049, paired t-test)(Fig. 2b). No mortality was observed during theseexperiments.

Table 1 Calanopia americana. Statistical analysis of experimentstesting responses to absolute irradiance change. For each experi-ment, P-value for a one-factor repeated-measures ANOVA (RMA-NOVA) is provided. For each significant RMANOVA, multiple

comparisons were done using Dunnett’s test versus dark treatment(q¢a(2) 32,8; Zar 1999). For each irradiance level (photons m�2 s�1),Dunnett’s q¢-test statistic is provided. Significant test statistics de-noted by asterisks: **P<0.01, ***P<0.001

Experiment RMANOVA P-value Dunnett’s q¢

1·1010 1·1011 1·1012 1·1013 1·1014 1·1015 1·1016

Day Increases <0.001 0.733 3.787** 4.398*** 6.23*** 8.062*** 9.039*** 10.627***Night Increases <0.001 1.982 1.858 1.487 1.982 4.336*** 6.318*** 7.185***Day Decreases 0.614 — — — — — — —Night Decreases <0.001 1.739 1.642 4.444*** 3.575** 4.734*** 6.473*** 11.208***

Fig. 2a, b Calanopia americana. Response to relative rates ofirradiance change. a Percentage of copepods ascending (movingupward towards stimulus light, ±30�) plotted as a function ofrelative rate of irradiance decrease. Responses during light stimulidenoted by sold circles connected with a solid line; open circlesconnected by a dashed line denote control responses prior to eachstimulus. Means and SE (n=4) are plotted. Black asterisks showresponses significantly greater than pooled controls (one-factorrepeated-measures ANOVA with a Dunnett’s test for multiplecomparisons, q¢0.05(1) 34,6; Zar 1999). b Percentage of copepodsdescending (moving downward away from stimulus light, ±30�)plotted as a function of relative rate of irradiance increase. Meansand SE (n=5) are plotted as in panel a

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Experiments testing endogenous rhythms

An endogenous vertical migration rhythm with nightactivity was present regardless of feeding condition(Fig. 3). The general pattern of this rhythm was a peakin copepod abundance in the upper half of the columnsaround subjective sunset, an abundance decrease priorto midnight, and a less pronounced second upper col-umn abundance peak in the latter half of subjectivenight. Copepod activity in the columns decreased withtime under constant dark conditions during all experi-ments (Fig. 3). A potential cause of this decrease wasbehavioral incapacitation due to low or no feeding andsubsequent copepod mortality, which was assessed at theend of each experiment and was highly variable. Thelowest mortality (19–38%) was observed in 2.5·106 and1.25·106 Rhodomonas sp. cells l�1 feeding conditions,with higher mortality (60–100%) in 2.5·105 Rhodo-monas sp. cells l�1 and Artemia franciscana treatments.Accordingly, only data from the 2.5·106 and1.25·106 Rhodomonas sp. cells l�1 feeding conditionswere analyzed. All replicate time series for both of thesefeeding conditions were significantly periodic (autocor-relation coefficients >95% CI), and major MESAperiod estimates ranged from 21.9 to 24.6 h (aver-age=23.8 h, SD=1.11 h). Lesser MESA energies(�12 h) were present for all time series. Period estimates

did not differ significantly between feeding conditions(P=0.438, t-test).

Cross-correlation analysis was used to examine thephase relationship of endogenous rhythm time serieswith environmental variables that could potentially en-train rhythms (tidal height, subjective irradiance).Endogenous rhythm series with different feeding condi-tions differed in their phase relationship with time seriesof tidal height and subjective irradiance. For experi-ment 1 (10–13 September 2002, 2.5·106 Rhodomon-as sp. cells l�1), minimum values in the rhythm timeseries preceded maximum tidal heights by 1.5 h, asindicated by a negative cross-correlation (Table 2).Minimum values for this rhythm series also precededmaximum irradiance (middle of subjective day) by16.5 h (i.e. minimum rhythms at 0545 hours) (Table 2).For experiment 2 (7–11 October 2002), both the 2.5·106and 1.25·106 Rhodomonas sp. cells l�1 feeding condi-tions were significantly cross-correlated with tidal heightand irradiance. Minimum values in these rhythm seriesoccurred 4 and 4.5 h after maximum tidal heights and 2and 2.5 h after maximum irradiance (middle of sub-jective day), respectively (Table 2).

Cross-correlation analysis was also used to examinethe phase relationship among endogenous rhythm timeseries. Maximum tidal height during experiment 1 (10–13 September) occurred 2 h after maximum tidal height

Fig. 3a–c Calanopiaamericana. Copepodendogenous vertical migrationrhythms. Percentage ofcopepods in the upper half ofthe column shown as time seriesat 0.5-h intervals (solid circles,solid line); one of two replicatecolumns shown. a Experiment 1conducted 10–13 September2002, initial feeding condition2.5·106 Rhodomonas sp. cellsl�1; b experiment 2 conducted7–10 October 2002, initialfeeding condition2.5·106 Rhodomonas sp. cellsl�1; c experiment 2 conducted7–10 October 2002, initialfeeding condition1.25·106 Rhodomonas sp. cellsl�1. For all panels, tidal heightat the collection site duringexperiments, in meters aboveMLLW (right y-axis), plotted asa dashed line. Subjective night inthe field represented by grayshading

405

during experiment 2 (7–11 October) (lag=2 h, R-va-lue=0.969). Accordingly, cross-correlations of endoge-nous rhythm series conducted during these dates shouldhave a lag of 2 h if they were in phase with the tides.Significant cross-correlations (lag=�3 h) were foundbetween the endogenous rhythm series for experiment 1(10–13 September, 2.5·106 Rhodomonas sp. cells l�1)and both the 2.5·106 and 1.25·106 Rhodomonas sp. cellsl�1 feeding conditions tested in experiment 2 (7–11October), indicating that maximum values in theexperiment 1 series occurred 3 h prior to maximumvalues in the experiment 2 series (Table 2). The feedingconditions of experiment 2 were significantly cross-cor-related and in phase with one another (lag=0; Table 2).

Endogenous vertical migration rhythms recorded inthe laboratory (10–13 September and 7–11 October,2.5·106 Rhodomonas cells l�1 feeding condition, n=4)were cross-correlated with the twilight DVM patterndescribed previously (Cohen and Forward 2005) for C.americana in the Newport River estuary (Fig. 4). Whilethe scaling of these data are quite different (percentabundance in laboratory columns versus absoluteabundance in the field), both the laboratory measure-ments of endogenous vertical migration rhythms incolumns and field observations of copepod abundance at0.5 m depth show an abundance increase around sunset(maximum 0–1.5 h after sunset), followed by a sharpdecrease (minimum �4 h after sunset), then a secondabundance peak in the latter half of the night (5–10 hafter sunset). Cross-correlation analysis shows that thelaboratory and field data are slightly out of phase, withfield observations lagging behind laboratory measure-ments by 1 h (R-value=0.528).

Discussion and conclusions

The present study of Calanopia americana behavior inthe laboratory investigated aspects of three hypothesesfor the proximate role of light in DVM: (1) the prefer-endum hypothesis (absolute irradiance), (2) the rate ofchange hypothesis, and (3) the endogenous rhythmhypothesis. C. americana has behavioral responses to

exogenous light cues consistent with its twilight DVMpattern; changes in absolute irradiance evoke swimmingresponses that would result in an ascent at sunset anddescent at sunrise, while relative rates of irradiance de-crease occurring at sunset evoked an ascent response,and relative rates of irradiance increase occurring atsunrise likely evoked a descent response. Furthermore,C. americana expressed an endogenous rhythm in ver-tical migration that was positively correlated with fieldobservations of twilight DVM. Collectively, these resultssuggest that both exogenous light cues and endogenousrhythms could play a proximate role in the twilightDVM pattern of C. americana, providing redundancy infactors stimulating vertical migration.

These proximate cues can be combined into a theo-retical model for DVM in C. americana, shown graphi-cally in Fig. 5. The ascent of copepods at sunset resultsfrom a combination of: (a) the active phase of an

Table 2 Calanopia americana. Cross-correlation analysis matrix.Replicate endogenous vertical migration rhythm time series aver-aged for each feeding condition and cross-correlated with theirrespective tidal heights (meters above MLLW), irradiance (stepfunction, maximum during subjective day and minimum duringsubjective night), and with each other. Lags (h) correspond to thephase relationship of the series listed in the first column of the table

with respect to the series listed in the first row (i.e. negative lagindicates vertically listed series preceded horizontally listed series).Maximum R-values >95% white noise CI are provided in paren-theses after each lag;positive values indicate positive correlations(high values in both series) and negative values indicate negativecorrelations (high values in vertically listed series, low values inhorizontally listed series)

Tidal height Irradiance Expt 2, 7–11 October 2002,2.5·106 Rhodomonas sp. cells l�1

Expt 2, 7–11 October 2002,1.25·106 Rhodomonas sp. cells l�1

Expt 1, 10–13 September 2002,2.5·106Rhodomonas sp. cells l�1

�1.5 (�0.313) 16.5 (�0.440) �3.0 (0.710) �3 (0.654)

Expt 2, 7–11 October 2002,2.5·106Rhodomonas sp. cells l�1

4.0 (�0.334) 2.0 (�0.384) — 0.0 (0.879)

Expt 2, 7–11 October 2002,1.25·106Rhodomonas sp. cells l�1

4.5 (�0.285) 2.5 (�0.319) — —

Fig. 4 Calanopia americana. Endogenous vertical migrationrhythm measured in laboratory columns related to twilight dielvertical migration in the Newport River estuary. Percentage ofcopepods in the upper half of laboratory columns during subjectivenight for four replicates of the 2.5·106 Rhodomonas sp. cells l�1

feeding condition (solid line, left y-axis), averaged according to timerelative to sunset (0 h=sunset). Field observations (dotted line,right y-axis) of C. americana abundance (copepods 10 m�3) 0.5 mbelow the surface in the Newport River estuary (Cohen andForward 2005), similarly averaged according to time relative tosunset

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endogenous vertical migration rhythm, (b) upwardswimming at low absolute irradiance levels, and (c) up-ward swimming during fast relative rates of irradiancedecrease. The ‘‘midnight sink’’ is under endogenouscontrol, with descent resulting from either an activitydecrease (and passive sinking) or a satiation-drivendescent. The early morning ascent is likewise underendogenous control, resulting from either an activityincrease or a hunger-driven ascent. The sunrise descentresults from a combination of: (a) the inactive phase ofan endogenous vertical migration rhythm (and passivesinking), (b) descent at high absolute irradiance levels,and (c) descent during fast relative rates of irradianceincrease. Daytime residence in deep water or on thebottom is the result of the inactive phase of the endog-enous rhythm, high absolute irradiance suppressing anascent, and minimal relative rates of irradiance changethroughout the day.

C. americana behavioral responses to absolute irra-diance decreases are consistent with their ascent atsunset in accordance with the preferendum hypothesis,but are also affected by their endogenous rhythm. Theascent phase of DVM in both the chaetognath Sagittahispida and larvae of the freshwater culicid insect Cha-oborus punctipennis is initiated when ambient irradiancefalls below a threshold, resulting in a ‘‘release’’ of theseorganisms from the bottom into the water column atsunset by either negative geotaxis or an activity increase(Sweatt and Forward 1985; Swift and Forward 1988). Asimilar release scenario may occur in C. americana withirradiance decreases at sunset; copepod abundance0.5 m above the bottom of the Newport River estuarypeaked 15 min after sunset, when irradiance on thebottom was �1·1016 copelux and decreasing sharply(Cohen and Forward 2005). The behavioral responseexhibited by C. americana to absolute irradiance de-creases at night was not present when copepods weregiven the same stimuli during the day. At this time, C.americana are in the inactive phase of their endogenousrhythm. The lack of an ascent response upon suddenirradiance decreases would be advantageous for cope-pods on the bottom during the day, as it would prevent

them from ascending in the water column prior to sunsetif there were a sudden irradiance decrease (e.g. frompassing clouds).

The response of C. americana to absolute irradianceincreases is likewise consistent with their descent fromthe surface at sunrise in the field and the preferendumhypothesis. Irradiance 0.5 h prior to sunrise 0.5 m belowthe surface of the Newport River estuary was�5·1016 copelux (Cohen and Forward 2005). C. ameri-cana abundance at this depth had decreased to zeroprior to this time, which fits with laboratory observa-tions of few individuals at the surface of laboratorycolumns when irradiance was >1·1011 photons m�2 s�1during daytime experiments and 1·1014 photons m�2

s�1 during night-time experiments. Absolute irradiancewas similarly shown by Forward (1985) to control thedescent phase of DVM in zoea of the estuarine crabRhithropanopeus harrisii. The effect of step irradianceincreases during daytime laboratory experiments wasmore prominent than that during night-time experi-ments, which likely resulted from the night-activeendogenous rhythm of C. americana increasing thepercentage of copepods in the upper column at nightregardless of irradiance relative to the inactive (daytime)phase of the rhythm.

The ascent response of C. americana to relative ratesof irradiance decrease fits with the rate of changehypothesis for the ascent of copepods at sunset.According to this hypothesis, migrators should ascend inthe water column (e.g. by positive phototaxis) whenexposed to relative rates of irradiance decrease abovesome threshold (Forward 1988). Laboratory determi-nations of the threshold relative rate of irradiancechange (RRC) depend upon the adaptation irradiance,with zooplankters being the most sensitive when adaptedat irradiance levels to which they are normally exposedduring twilight (Ringelberg et al. 1967; Stearns andForward 1984; Forward 1985), as well as on the numberof stimuli tested (e.g. �0.0021, �0.0032, �0.0046,�0.0058, and �0.0068 s�1 in the present study). Thethreshold relative rate of irradiance decrease for C.americana adapted to 8·1013 photons m�2 s�1 in the

Fig. 5 Calanopia americana.Theoretical model for theproximate basis of twilight dielvertical migration in C.americana. Copepod abundancein the surface water plotted as afunction of time for a singlemigration cycle. Nightrepresented by gray shading

407

present study was �0.0046 s�1, which is intermediatewith respect to those of the two best-studied organismmodels for DVM when adapted to twilight irradiancelevels: Daphnia sp. (�0.0017 s�1, Ringelberg 1964) andRhithropanopeus harrisii (�0.0044 to �0.0086 s�1, For-ward 1985). The RRC threshold for C. americana issimilar, though slightly faster, than relative rates ofirradiance decrease on the bottom of the Newport Riverestuary when copepod abundance in the water columnwas increasing rapidly (�0.0032 to �0.0042 s�1; Cohenand Forward 2005). Part of this difference may be due tothe range of RRC stimuli given in the laboratory; RRCstimuli were at �0.0032 and �0.0046, yet a significantresponse may have occurred at an untested intermediatestimulus. The influence of other proximate factors (i.e.absolute irradiance and endogenous rhythms) may alsohave caused some copepods in the field to ascend in thewater column earlier than would be expected based so-lely on their response to relative irradiance decreases(e.g. Enright and Honeggar 1977). The ascent of C.americana will occur upon exposure to absolute irradi-ance <1·1016 photons m�2 s�1 during sunset, and theendogenous rhythm will result in copepods ascendingaround sunset.

C. americana appear to have a descent response torelative rates of irradiance increase occurring at sunrise,which likewise fits with the rate of change hypothesis,but further work is needed to determine a thresholdRRC. In the Newport River estuary, copepods haddescended from the surface prior to a relative rate ofirradiance increase of 0.0033 s�1 (Cohen and Forward2005), suggesting that if RRC played a role, the descentresponse likely occurred at rates >0.0033 s�1. C.americana had a heightened (though not significant)descent response at 0.0042 s�1. Further laboratory andfield measurements are necessary to confirm the role ofrelative rates of irradiance increase in the sunrise descentof C. americana.

The night-active circadian rhythm in vertical migra-tion of C. americana, when tested under constant dark-ness in laboratory columns, is consistent with its twilightDVM. A circatidal component to the rhythm (e.g.Forward and Cronin 1980) was not present, whichmatches DVM field observations lacking a tidal com-ponent (Cohen and Forward 2005). Copepod endoge-nous rhythms decayed in magnitude, but maintained arhythmic oscillation under constant dark conditions.This decay could have been due, in part, to copepodmortality (19–38% in 2.5·106 and 1.25·106 Rhodomon-as sp. cells l�1 feeding conditions), which did not appearto have resulted from insufficient food or dissolvedoxygen (J. Cohen, personal observations), and may haveresulted from an activity decrease as energy reservesdecreased due to low or no feeding. An alternativeexplanation for the decay under constant dark condi-tions is that removal of copepods from exposure to therhythmic environmental entraining stimulus, most likelythe light:dark cycle, resulted in a gradual loss of therhythm in the experimental organisms. This scenario is

common in studies of biological rhythms (Dunlap et al.2004), and was observed in a previous study of activityrhythms in several species of pontellid copepods(Champalbert 1978).

The involvement of an endogenous rhythm in C.americana DVM, particularly the ‘‘midnight sink’’, issuggested by the similarity between laboratory mea-surements of endogenous rhythms in columns(2.5·106 Rhodomonas sp. cells l�1) and field observa-tions of DVM. Laboratory and field data were slightlyout of phase, suggesting factors other than an endoge-nous rhythm alone were responsible for the ascent ofcopepods at sunset (e.g. absolute and relative changes inirradiance). Another difference among laboratory andfield data was that copepods in the laboratory remainednear the surface for longer into the early morning thandid copepods in the field when both were analyzed rel-ative to the time of sunset. This was likely a result of themonths in which these experiments were conducted andconsequently a difference in the photoperiod to whichcopepods were entrained; laboratory endogenousrhythm experiments were performed in September andOctober (length of night�11–12 h), whereas fieldobservations of DVM were made in July (length ofnight�10 h). Accordingly, copepods collected for labo-ratory experiments in fall would be entrained to a longerdark period, and therefore should have a longer activephase of the rhythm than copepods during summer fieldobservations.

In these experiments we did not assess whether theendogenous vertical migration rhythm of C. americanawas coupled with a nocturnal feeding rhythm or wassimply an activity rhythm. Endogenous rhythms infeeding are common in marine calanoid copepods(Mauchline 1998), but their role in DVM is unclear. Forsome copepod species, nocturnal feeding and DVM areindependent, each behavior with its own proximatecontrols (Stearns 1986; Pagano et al. 1993). For others,DVM appears related to a hunger/satiation cycle, withcopepods rising to feed in the upper water column, andsatiated individuals sinking (Pearre 1973, 2003; Mackasand Bohrer 1976; Harding et al. 1986; Pagano et al.1993). The ‘‘midnight sink’’ that occurred in laboratorycolumns could be due to a satiation-driven descent ofcopepods that initially ascended around subjective sun-set, with the second peak in abundance at the surfaceresulting from a second feeding bout. An alternativeexplanation for the ‘‘midnight sink’’ is that the rhythm isbased on a circadian oscillation in activity (Rudjakov1970). The presence of two peaks during the active phaseof circadian rhythms is often a property of a circadianoscillating system (Aschoff 1966). Activity rhythms forpontellid copepods (Anomalocera patersoni, Pontellamediterranea, and Labidocera wollastoni) were reportedby Champalbert (1978). He hypothesized that rhythmicoscillations in activity, synchronized by the light:darkcycle and hydrostatic pressure, underlie weak verticalmigrations in these purported neustonic species. It isunclear from the present study whether the endogenous

408

vertical migration rhythm of C. americana is related to anocturnal feeding rhythm or a nocturnal activityrhythm.

There is redundancy in the proximate role of light inC. americana DVM. While the primary causal factorcueing DVM has been investigated in relatively fewmigratory species, it has been recognized for some timethat no one photic stimulus is applicable for all species(e.g. Forward 1988). Furthermore, one type of photicstimulus may not be applicable for all aspects of theDVM in an individual species. The present study sup-ports this by showing that a unification of the prefer-endum, rate of change, and endogenous rhythmhypotheses best explain C. americana DVM. Future ef-forts seeking to identify and generalize the proximaterole of light in the DVM of zooplankton should assessall of these hypotheses, as the applicability of one neednot exclude the others.

Acknowledgements We thank P. Tester and S. Kibler for providingalgal cells, and R. Barber, S. Johnsen, D. Rittschof, andD. Steinberg for comments on an earlier draft of the manuscript.This material is based in part on research supported by theNational Oceanic and Atmospheric Administration (ECOHABgrant NA17OP2725 to R. Forward and P. Tester), with additionalfunding provided by the Oak Foundation.

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