RESEARCH ARTICLE

J. H. Cohen Æ R. B. Forward Jr.

Diel vertical migration of the marine copepod Calanopia americana.I. Twilight DVM and its relationship to the diel light cycle

Received: 26 August 2004 / Accepted: 7 January 2005 / Published online: 22 February 2005� Springer-Verlag 2005

Abstract Marine copepods commonly exhibit verticalmovements in the water column over the diel cycle,termed diel vertical migration (DVM), with the mostcommon pattern being an ascent in the water column tominimum depth around sunset and descent to maximumdepth around sunrise. The present study characterizedthe DVM pattern of the pontellid copepod Calanopiaamericana Dahl in the Newport River estuary (NorthCarolina, USA, in July 2003). The estuary is shallow andwell-mixed, and the study site (34�43¢N; 76�40¢W),1.5 km inside the estuary entrance, is unusual in lyingwithin a gyre where tidal currents are always in theseaward direction. Changes in C. americana verticalabundance were related to spectrally relevant changes inlight throughout the diel cycle. Simultaneous measure-ments of light and zooplankton abundance near thesurface (0.5 m depth) and near the bottom (0.5 m abovebottom) were made over one 4-h period and two 3-dayperiods during different phases of the tide. Theseobservations suggest that C. americana undertook twi-light DVM in the Newport River estuary; an ascent tothe surface occurred at sunset, followed by a descent tonear the bottom around midnight, with a second ascentto the surface and then descent to near bottom at sun-rise. DVM in C. americana was independent of the tidal

cycle, with the initial ascent in the water column atsunset possibly associated with relative rates of irradi-ance change. Copepod vertical movements were consis-tent with a night-active endogenous rhythm, andappeared independent of the abundance of predatorychaetognaths, Sagitta spp. In DVM studies withmigrators like C. americana that are broadly sensitiveto visible wavelengths of light, measuring photo-synthetically active radiation may be a reasonablealternative to measuring light in a spectrally relevantphotometric unit.

Introduction

Diel vertical migration (DVM) is extremely common inboth freshwater and marine zooplankton, particularlyamong copepods (Haney 1988; Longhurst and Harri-son 1989). Of the three recognized DVM patterns (seeHutchinson 1967), the simplest is an ascent in thewater column to minimum depth around sunset anddescent to maximum depth around sunrise, termednocturnal or normal DVM. Another pattern, reverseDVM, involves an ascent to shallow water at sunrisefollowed by a descent to deeper water at sunset. Thethird pattern, twilight DVM, involves an ascent to thesurface at sunset, a descent to deeper water aroundmidnight (the ‘‘midnight sink’’ sensu Cushing 1951),followed by a second ascent to the surface and thendescent to deeper water at sunrise. Variability in thepattern for any given species at a particular place andtime is likely related to plasticity in the behavioralresponses of copepods under variable ambient preda-tion pressures (Bollens and Frost 1989a, 1989b; Oh-man 1990), changing environmental conditions (Dagget al. 1997), and during different ontogenetic stages(Neill 1992).

Calanopia americana is a primarily coastal calanoidcopepod, with a subtropical to tropical range extending

Electronic Supplementary Material Supplementary material isavailable in the online version of this article at http://dx.doi.org/10.1007/s00227-005-1569-x.

Communicated by J.P. Grassle, New Brunswick

J. H. Cohen (&) Æ R. B. Forward Jr.Duke University Marine Laboratory, Biology Departmentand Nicholas School of the Environment and Earth Sciences,Duke University, 135 Duke Marine Lab Rd., Beaufort,NC 28516, USAE-mail: jcohen@hboi.eduTel.: +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: 387–398DOI 10.1007/s00227-005-1569-x

from Cape Hatteras, N.C., east to Bermuda, and southto Brazil (Bowman 1971). This copepod species is in thefamily Pontellidae, but is smaller than most pontellids(<2.0 mm in length; Fleminger 1956). Also unusual fora pontellid, C. americana is a very strong verticalmigrator with either a nocturnal or twilight patternlikely, as previous studies reporting this species in eithershallow sites inshore (<15 m water column depth) ordeeper sites offshore (>700 m water column depth) havefound it in greater abundance in surface waters at nightthan during the day (Clarke 1934; Bowman 1971; Turneret al. 1979). Clarke (1934) reported that both male andfemale C. americana in St. George’s Harbor, Bermuda(12.4–14 m depth), reside on the bottom during the day,and rise to the surface around sunset. This study had acoarse sampling design over two consecutive days(midday and early evening on day 1, early morning andearly evening on day 2), and therefore could not dis-tinguish between nocturnal and twilight DVM.

Light is generally regarded as the primary proximatecausal factor controlling the movement of migratorsduring DVM (Forward 1988; Ringelberg 1999). Strongevidence for the role of light comes from field obser-vations showing that migration usually occurs at twi-light, which is the time of day with the greatest relativechange in irradiance. Furthermore, laboratory obser-vations suggest that many zooplankton species respondto relative rates of irradiance change consistent withswimming during DVM (rate of change hypothesis).Physiological thresholds for these responses correlatewell with relative rates of irradiance change at twilightat depths inhabited by migrating zooplankton (For-ward 1988; Ringelberg 1995; Cohen and Forward2005). Other evidence for the role of light in DVMcomes from the observation that many pelagic zoo-plankton species maintain their depth at distinct levelsof irradiance throughout the diel cycle (isolumehypothesis; Boden and Kampa 1965, 1967; Frank andWidder 2002).

A common experimental approach in DVM studies isto correlate movements of migrators with surface or insitu light levels. A common problem, however, is thatthe light measured may be unrepresentative of the lightavailable to migrators. Light is typically measured asquantal flux at a specific wavelength (e.g. photons persquare meter per second per nanometer at 480 nm) or asquantal flux over the visible range [e.g. photosyntheti-cally active radiation (PAR), photons per square meterper second at 400–700 nm]. While quantal flux units areuseful, as they are relatively straightforward to obtainwith commonly used radiometers and can be compareddirectly with light measurements reported in otherstudies (e.g. Widder and Frank 2001), they do not ade-quately reflect the number of photons available to thevisual system of a given organism. The spectral sensi-tivity function (or photobehavioral responsivity) of anorganism is not equal at all wavelengths, as visual sys-tems are not sensitive to all wavelengths equally. Pho-toreception occurs via visual pigments with discrete

spectral absorption maxima, which are reflected in theoverall spectral responsivity of the visual system and inresulting photobehaviors (Archer 1999).

If light is to be related to vertically migratingorganisms, the most relevant light measurements shouldaccount in some way for the spectral sensitivity of theorganism under study. This can be done by either fil-tering the photodetector with a filter that simulates thespectral sensitivity of the study organism (Boden andKampa 1965, 1967; Frank and Widder 1997; Widderand Frank 2001), or by applying a wavelength-specificweighting factor to spectrally explicit light data thatcorrects these values based on how well the studyorganism detects light at that wavelength (Gal et al.1999). Cohen and Forward (2002) showed thatC. americana have photobehavioral responses at wave-lengths from 350 to 580 nm, but not at longerwavelengths. These data can be used to calculate wave-length-specific weighting factors that, when applied toenvironmental light data, allow light measurement in aphotometric ‘‘copelux’’ unit that is spectrally relevant toC. americana photobehavior.

The purpose of the present study was to characterizethe DVM pattern of C. americana in the Newport Riverestuary (N.C., USA). C. americana vertical abundancewas related to changes in light (copelux) throughout thediel cycle. Sampling included three sets of field obser-vations made during different phases of the tide. Cope-lux light data were compared with conventional lightunits.

Materials and methods

Study site

Measurements of Calanopia americana Dahl abun-dance and of light were made simultaneously from adock on the southeast corner of Piver’s Island at theDuke University Marine Laboratory, Beaufort, N.C.,USA, in July 2003. C. americana occurs near-shore atthis location during summer and fall, and is foundfurther offshore during the winter and spring (Bow-man 1971). The sampling site is �1.5 km inside theentrance to the Newport River estuary (34�43¢N;76�40¢W). This area experiences semi-diurnal tides,with neap and spring tidal amplitudes during the studyreaching �0.88 and �1.05 m above mean lower lowwater (MLLW), respectively. Maximum water columndepth during spring high tide at the study site was�4 m. Currents at this location are unusual; they donot oscillate between the seaward direction on ebb tideand the landward direction on flood tide, as expectedfor a typical estuary. Rather, the study site is in agyre, such that current direction is always seawardregardless of the tidal cycle, with current velocityexhibiting less change over the tidal cycle than pre-dicted for the site (W. Breedan and P. Howd,unpublished data).

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Three sets of observations (see Electronic Supple-mentary Material, S1) were conducted during July 2003:(1) 4-h preliminary observations from 1900 to2330 hours on 13 July (full moon, high tide=2052 -hours, sunset=2021 hours), (2) a 3-day observationperiod from 0430 hours on 18 July to 2230 hours on 20July (1–3 days prior to third-quarter moon, approachingneap tides, sunrise�0605 hours, sunset�2018 hours),and (3) a 3-day observation period from 0430 hours on25 July to 2230 hours on 27 July (2–4 days prior to newmoon, approaching spring tides, sunrise�0612 hours,sunset�2014 hours). The preliminary observations cap-tured DVM behavior during a single sunset ascent of themigrating population. The two 3-day observation peri-ods captured DVM behavior over three consecutivesunrise/sunset cycles. These multi-day observations wereconducted during lunar periods out of phase by 7 days(tides out of phase by �6 h), in order to test for a tidalcomponent to C. americana vertical migration behavior.

Environmental variables

Environmental variables including temperature, salinity,and downwelling irradiance were measured simulta-neously with copepod collections. Temperature andsalinity were measured 0.5 m below the surface (surface,0.5 mbs) and 0.5 m above the bottom (bottom, 0.5 mab)at regular intervals (mid-flood, high tide, mid-ebb, lowtide) during the day and at night by lowering a tem-perature/salinity probe (YSI 30) to the appropriatedepth.

Downwelling irradiance was measured using a cosine-corrected fiber optic spectroradiometer (USB2000,Ocean Optics; 350–1000 nm spectral response) coupledto a laptop computer. Irradiance measurements at 0.5 mdepth were made at 0.3 nm wavelength intervals from360 to 800 nm every 2 h throughout the day. Samplingfrequency was increased to every 5 min during twilight(sunrise or sunset ±2 h) as irradiance change was rapidduring these times. Irradiance values were too low be-fore �0550 hours and after �2100 hours to be detectedby the spectroradiometer. Light near the bottom wascalculated according to the Beer–Lambert law (Wein-berg 1976). Wavelength-specific attenuation coefficientsused in this calculation were determined for eachobservation period separately as either a single kk

determination (1830 hours, 13 July twilight observation)or by averaging several kk calculations (3 morning and 3evening for multi-day observations). Irradiance datafrom 360 to 740 nm were converted to a single value in‘‘copelux’’ units, which estimated the total quantal fluxover this wavelength range available for photobehaviorin C. americana, by integrating irradiance after weight-ing wavelengths by the photobehavioral response func-tion for this copepod species (Cohen and Forward2002). Relative rates of irradiance change (RRC) werecalculated using the equation of Ringelberg (1964) inunits per second.

Zooplankton sampling

Zooplankton were collected from two depths (surfaceand bottom) simultaneously using two gasoline-poweredpolypropylene centrifugal pumps (McMaster-Carrmodel 3068K3, 3.5 hp). For surface samples, one pumphad its intake hose 0.5 m below the surface (0.5 mbs),with its position maintained by a float at the surface andconcrete blocks on the bottom. For bottom samples, asecond pump had its intake hose 0.5 m above the bot-tom (0.5 mab), with its position maintained in a similarmanner. Intake hoses were fitted at their submerged endswith spring-loaded poppet check valves (McMaster-Carrmodel 46835K37) in order to maintain the prime of thepumps between samples. The check valves were fitted attheir outer ends with polyethylene strainers (McMaster-Carr model 44265K25, 6.35 mm·25.4 mm perforations)that excluded large debris from the intake flow. With thepumps running and the check valves and strainers inplace, suction was moderate and only detectable by ahuman observer when touching the surface of thestrainer. It was therefore assumed that the sample depthswere discrete, with the depths of the strainers repre-senting the depths at which zooplankton were collected.

Water from each depth was pumped through outflowhoses into 333-lm-mesh plankton nets (Sea-Gear). Thenets were submerged in large containers filled withambient estuarine water (�120 l), with the mouths of thenets maintained above the level of the water in thecontainers in order to reduce structural damage tozooplankters during sample collection. Nets were rinsedthoroughly in their surrounding water in order to washall material pumped into the net down to the cod ends.Sample material (>333 lm) was concentrated to 100 mltotal sample volume by decanting water from the samplethrough mesh windows (333 lm) in the cod ends. Thesamples were carefully transferred into 500-ml contain-ers with �20 ml of GF/F-filtered offshore seawater usedto rinse any material remaining in the cod ends into thesample bottles. Organisms in the samples were killedimmediately by adding 15 ml of borax-buffered 40%formalin stock solution to each sample bottle (Omoriand Fleminger 1976). After 2–4 days, samples weretransferred to 70% EtOH.

A sample consisted of all material >333 lm con-tained in water pumped continuously for 5 min. Pumpflow rates were calibrated by measuring the amount oftime necessary to pump 100 l of water. Average flowrates (n=5) for surface and bottom samples were 5.98and 5.20 l s�1, respectively. Therefore, during the 5-min sampling intervals, approximately 1.79 and1.56 m3 of water were pumped from the surface andbottom, respectively. Replicate samples from eachdepth were collected every 2 h throughout the day andnight. Sampling frequency was increased to every 0.5 hduring a 4-h period at twilight (sunrise or sunset±2 h), as copepods were expected to be changingtheir positions in the water column rapidly duringthese times.

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The entire content of each sample was sorted on aWard counting wheel (Wildco model 1810-E80).C. americana were identified according to Fleminger(1956). As C. americana abundance was lower thanexpected and no qualitative differences in male/femaleabundance patterns were observed, counts of adult malesand females were combined, and replicate samples weresummed for all statistical analyses. Chaetognaths, whichare important non-visual predators of copepods (Baierand Purcell 1997), were abundant in all samples, and thedominant chaetognaths, Sagitta spp., were enumerated(Michel 1984). Inadequate preservation of chaetognathsprecluded their identification to the species level.

Statistical analyses

Copepod abundance time series were analyzed usingautocorrelation and maximum entropy spectral analysisalgorithms (Levine et al. 2002) programmed in MAT-LAB (ver. 5, The Math Works). Total water-columnabundance of copepods (surface+bottom) waslog10(x+1) transformed prior to these analyses. Sum-med data were used because they best reflect the overallabundance of C. americana in the water column. Thesampling frequency varied throughout each time series(2-h intervals during the day and night, 0.5-h intervals attwilight), so data were linearly interpolated with a uni-form sampling frequency of 0.5 h. An interval of 0.5 hwas used because this was the highest frequency ofcopepod sampling. Autocorrelation analysis was used todetermine if each time series had significant periodicity;autocorrelation plots with peaks at lags >1 exceedingthe 95% CI of ±2/�N (N=sample size) were consideredsignificantly periodic (Chatfield 1989). The period esti-mates for time series with statistically significant peri-odicity were determined using maximum entropyspectral analysis (MESA), which fits an autoregressivemodel to the data and uses Fourier analysis of theautoregressive coefficients to construct a spectrum.Periodicity estimates were used to resolve potential tidaland diel signals in the time series. Autocorrelationanalysis and MESA period estimates were also calcu-lated for time series of Sagitta spp. abundance.

Cross-correlation analysis was used to determine thephase relationship between peaks in the 18–20 and 25–27July copepod abundance time series to test whether therewas a tidal component to C. americana DVM. Copepodabundance data were the same as described for auto-correlation and MESA analyses, except that they werenot log transformed. Similar cross-correlations werecalculated for Sagitta spp. abundance time series.Additional cross-correlation analyses were done betweeneach copepod abundance time series (18–20 and 25–27July) and its respective time series of tidal height andirradiance. Tidal height above MLLW for each 0.5-hinterval of the abundance series was obtained from acomputer program (Tides and Currents Pro, NobeltechNautical Software 2001). Light data were in copelux

units calculated as described above for a depth of 0.5 m.Cross-correlation algorithms following Wing et al.(1995) were used to determine R-values for lags of �10to 10 h. As all of the time series were autocorrelated (see‘‘Results’’, tidal height and light autocorrelation notshown), the 95% CIs used to evaluate the significance ofthe cross-correlations at each lag were constructed intwo ways, one with SEs assuming independent pointsand the other assuming autocorrelations of the series(Wing et al. 1995). The SE calculations resulting in themore conservative significance criterion were used forcalculating 95% CIs.

Results

Environmental variables

A range of atmospheric weather conditions occurredduring the field observations (see Electronic Supple-mentary Material, S1). The preliminary observationperiod (sunset, 13 July) was conducted during a fullmoon, but with little moonlight due to overcast skies.For the multi-day observation periods, a third-quartermoon rose in the latter half of the night during 18–20July, and a new moon rose early in the morning 25–27July. The amount of moonlight during these periods,while not measured, was likely variable given the vari-ability in cloud cover (see Electronic SupplementaryMaterial, S1).

Minimal differences in surface and bottom measure-ments of temperature and salinity suggest that overallthe water column was rather homogenous and stable(see Electronic Supplementary Material, S2). In general,surface water was slightly warmer and less saline thanbottom water. Water temperature during both multi-dayobservation periods ranged from 26�C to 28�C, with noobvious tidal signal when temperatures were plotted as afunction of tidal height (see Electronic SupplementaryMaterial, S2 a, b). A tidal signal was present in salinitymeasurements, as salinity generally increased by �2during both multi-day observation periods withincreasing tidal height (see Electronic SupplementaryMaterial, S2 c, d). Water temperature and salinity dur-ing the sunset observation on 13 July were intermediatewith respect to the data collected during the multi-dayobservations (surface: 26.8�C, salinity 34.4; bottom:26.8�C, salinity 34.5).

Irradiance data collected during a single twilight(1830–2240 hours, 27 July) were compared between thephotometric copelux unit, quantal flux as PAR, andquantal flux at 480 nm (the wavelength of maximalCalanopia americana photosensitivity; Cohen and For-ward 2002). This date was chosen because the night wasclear, and the irradiance data were representative oftwilight conditions. At both the surface (0.5 mbs;Fig. 1a) and on the bottom (0 mab; Fig. 1b), copeluxirradiance was similar to but less than PAR. Irradiancein units of quantal flux at 480 nm was much lower (two

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to three orders of magnitude) than either PAR or cop-elux (Fig. 1a, b). Relative rates of irradiance changecalculated for sunset on 27 July at the surface (0.5 mbs;Fig. 1c) and on the bottom (0 mab; Fig. 1d) showedlittle difference among the three units.

Copepod abundance time series

C. americana abundance during all observation periodsconsisted of diurnal periods with infrequent peaks andnocturnal periods with frequent distinct peaks (Fig. 2).In the preliminary observation (sunset on 13 July; datanot shown), copepods were present in surface (0.5 mbs)and bottom (0.5 mab) samples prior to sunset, butmaximum abundance was not reached until after sunset.An initial peak in bottom abundance (2030 hours) wasfollowed by a broader peak in surface abundance(maximum at 2130 hours). As surface abundance de-creased after 2130 hours, bottom abundance increased.In both multi-day observations (18–20 July and 25–27July) (Fig. 2a, b), C. americana were present in bottomsamples during every sunset and during most sunrises,but were absent during the middle of the night. Cope-pods occurred in bottom samples for only a short time attwilight (Fig. 2a, b). In surface samples, peaks in C.americana abundance also tended to occur around sun-set, with subsequent abundance peaks later in the night,commonly beginning around midnight and endingaround sunrise (Fig. 2a, b). The observed pattern of

abundance more closely matches the theoretical expec-tation for twilight DVM than for nocturnal DVM(Fig. 2c). Total water-column abundance (sur-face+bottom) time series for C. americana and a po-tential predator, the chaetognaths Sagitta spp., duringthe multi-day observations differed markedly; copepodabundance peaks occurred primarily at night, whereaschaetognath abundance peaks occurred during both dayand night (Fig. 3).

Autocorrelation analysis was used to test for sig-nificant periodicities in time series of total water col-umn C. americana abundance (surface+bottom).Abundance time series for both multi-day observationswere significantly autocorrelated (autocorrelationcoefficients >95% CI). MESA was used to determineperiod estimates and to quantify other potentiallymeaningful periodicities within the abundance timeseries. The MESA spectra for 18–20 July and 25–27July were similar, but not identical, with each having alarge approximately diel period (22.4 and 24.4 h,respectively) and several shorter periodicities (4–12 h)(Fig. 4a, b). These shorter periodicities were similar toseveral peaks (4.7–8 h) observed in the MESA spec-trum for theoretical twilight DVM plotted in Fig. 2c(Fig. 4c) and not in the spectrum for nocturnal DVM(Fig. 4d). Both multi-day time series for the chae-tognaths Sagitta spp. were also significantly autocor-related (autocorrelation coefficients >95% CI), withMESA period estimates of 13.1 h for both 18–20 Julyand 25–27 July.

Fig. 1a–d Comparison ofcopelux with commonirradiance units measured at 5-min intervals during sunset on27 July and relative rates ofirradiance change (RRC)plotted as a function of time.All irradiance measurementswere made at: a 0.5-m depthusing a cosine-correctedspectroradiometer. b Bottom(0 mab) values calculated from0.5-m measurements asdescribed in ‘‘Materials andmethods’’. RRC was calculatedfor irradiance values andplotted for: c 0.5-m depth and dbottom (0 mab). Data shown inthree units: (1) PAR (photonsm�2 s�1, integrated 400–700 nm, solid line), (2) copelux(normalized, integrated 360–740 nm, dashed line), and (3)quantal flux at 480 nm(photons m�2 s�1 nm�1 at480 nm, dotted line). Time ofsunset (SS) indicated in panel a

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The cross-correlation of total water column C.americana abundance time series for the 18–20 July and25–27 July observations was used to test for the effect oftidal time on vertical migration. These time series werein phase, with the only significant cross-correlationcoefficient (R> or <95% CI) being positive at a lag of0 h (Fig. 5; Table 1). This differed sharply from thecross-correlation of the tidal height time series duringthe 18–20 July and 25–27 July observations (Fig. 5),which contained a negative R-value maximum at a lag of0.5 h, and positive R-value maxima at lags of �6 and6.5 h. Cross-correlation analysis of the Sagitta spp.multi-day abundance time series (Fig. 5) revealed an outof phase relationship similar to that observed for the

cross-correlation of the corresponding tidal height timeseries; both of these cross-correlations contained a neg-ative R-value maximum near a lag of 0 h and positive R-value maxima at both positive and negative lags.

Other cross-correlations were calculated between C.americana abundance during both multi-day observa-tion periods and corresponding time series of irradiance(copelux) and tidal height, as well as Sagitta spp.abundance (Table 1). Copepod abundance was nega-tively cross-correlated with irradiance at lags of �0.5 hduring both multi-day observations. Copepodabundance during 18–20 July was not significantly cross-correlated with tidal height, whereas abundance andtidal height during 25–27 July had a weak negativecross-correlation at a lag of �7 h. When 18–20 JulyC. americana and Sagitta spp. abundance time serieswere cross-correlated with each other, lags of 0 and 4 hwere significant and positive, whereas a lag of 2 h wassignificant and negative (Table 1). When 25–27 Julyseries were similarly cross-correlated, lags of 0.5 and 8 hwere significant and positive (Table 1), with no signifi-cant negative lags.

Copepod abundance and irradiance change

C. americana abundance data for the same collectiontimes during the three observation periods were aver-aged and plotted as a function of collection time(Fig. 6a). Absolute irradiance (Fig. 6a) and relative rates

Fig. 2a–c Calanopia americana. Abundance during multi-dayobservations in surface (0.5 mbs) and bottom (0.5 mab) samples.a Copepod abundance from 0430 hours on 18 July to 2230 hourson 20 July in surface (solid line) and bottom (dotted line) samples.Sampling was 1–3 days prior to third quarter moon, approachingneap tides. Sunrise and sunset occurred at approximately 0605 and2018 hours, respectively, with night represented by gray shading.Data for males and females were pooled, and time series werelinearly interpolated at 0.5-h intervals. Abundance data plotted aslog10(x+1) copepods per 10 m3. Tidal height above MLLW alongupper portion of panel. b Copepod abundance from 0430 hours on25 July to 2230 hours on 27 July as in panel a. Sampling was 2–4 days prior to new moon, approaching spring tides. Sunrise andsunset occurred at �0612 and 2014 hours, respectively. c Expectedtime series for copepod abundance in surface samples if organismswere undergoing twilight (solid line) or nocturnal (dotted line) dielvertical migration

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of irradiance change (Fig. 6b) for the three periods werealso averaged by collection time and compared withaverages of copepod abundance. An initial narrow

abundance peak in bottom samples occurred at2030 hours, just after sunset (�2015 hours), followed bya broader abundance peak in surface samples centered at

Fig. 3a, b Calanopia americanaand Sagitta spp. Total water-column abundance[log10(x+1)-transformedsurface+bottom] of copepods(dashed line) and chaetognaths(solid line) during multi-dayobservations: a 18–20 July andb 25–27 July. Sunrise and sunsetoccurred at �0605 and2018 hours, respectively, withnight represented by grayshading. Tidal height aboveMLLW along upper portion ofeach panel

Fig. 4a–d Calanopiaamericana. Maximum entropyspectral analysis (MESA) ofcopepod abundance in multi-day time series. MESA spectrashowing power as a function ofperiod for: a 18–20 July totalwater-column abundance[log10(x+1)-transformedsurface+bottom], b 25–27 Julytotal water-column abundance,c theoretical twilight dielvertical migration (DVM),d theoretical nocturnal DVM.Period estimates (h) shown onplots for periodicities withrelative peaks in power. MESAvalues for twilight andnocturnal DVM calculated fortime series in Fig. 2c haveshorter period estimates thanexpected for a strictly dielpattern (24 h) due toasymmetry in beginning andending points of the series

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2100 hours. This increase in C. americana abundance inbottom and surface samples near sunset occurredas absolute irradiance was decreasing sharply andaccordingly the relative rate of irradiance decreasewas becoming maximally negative [bottom RRC(0 mab)=�0.0032 s�1 at 2030 hours, �0.0042 s�1 at2045 hours]. A second copepod abundance peak in thebottom samples coincided with a decrease in abundance

in the surface samples at 2200 hours. Relatively lowabundance occurred in both surface and bottom samplesuntil 0100 hours, with the onset of a second peak insurface abundance centered at 0230 hours. A finalabundance peak in the bottom samples occurred at0500 hours, as surface abundance was decreasing. By0530 hours, prior to there being enough light for mea-surement, surface abundance had decreased to zero. Theearliest measurements of relative rate of irradiance in-crease were at 0550 hours, when RRC at the surface(0.5 mbs) was 0.0033 s�1. By 0600 hours, just prior tosunrise (�0605 hours) bottom abundance had decreasedto zero [surface RRC (0.5 mbs)=0.0029 s�1].

Discussion and conclusions

Previous reports of vertical migration in the marinecopepod Calanopia americana suggested that this speciesundergoes DVM with night-time residence in surfacewaters (Clarke 1934; Bowman 1971; Turner et al. 1979).But, these studies were not designed to dissect the causesof DVM. The present study examined C. americanavertical migration in an estuarine system with semi-diurnal tides and related copepod vertical abundance tophysical variables including tidal height and light. Thesampling design allowed for: (1) the differentiation oftidally mediated versus light-mediated vertical migra-tions (nocturnal, twilight, or reverse DVM) and (2) thecorrelation of changes in copepod abundance at thesurface and near the bottom with changes in light. Pre-vious laboratory characterization of the spectral sensi-tivity of C. americana photobehavior (Cohen andForward 2002) allowed for light measurements to bemade in spectrally relevant ‘‘copelux’’ units, a photo-metric unit based on how well this species respondsbehaviorally to light of different wavelengths, whichwere compared to more conventional light units (PAR;quantal flux at 480 nm).

C. americana at the study site in the Newport Riverestuary undertook light-mediated twilight verticalmigration, with no apparent tidal component. Theoverall abundance of this species throughout theobservations was low (maximum abundance at a singlecollection time=35 copepods per 10 m3), with relativepeaks in both surface and bottom abundance occurringpredominantly in nocturnal and not diurnal samples. Ifthere was a tidal component to the vertical migration ofC. americana, then the cross-correlation of abundancetime series for the 18–20 July and 25–27 July observationperiods in consecutive weeks should have matched thetidal height cross-correlation and been out of phase by�6 h. However, this was not the case as the C. ameri-cana abundance time series were in phase with the onlysignificant R-value being positive at a lag of 0 h. Cross-correlations of each copepod abundance time series withits respective tidal height and irradiance time seriesconfirmed that vertical migrations were not consistentlyrelated to the tidal cycle. Only during the 25–27 July

Fig. 5 Calanopia americana and Sagitta spp. Copepod (solid line)and chaetognath (dashed line) abundance (surface+bottom) andtidal height (dashed-dotted line) in multi-day time series for 18–20July cross-correlated with their respective time series for 25–27July. Algorithms followed Wing et al. (1995), with cross-correlationcoefficient (R) computed for lags of �10 to 10 h. 95% CI (dottedlines) was used to evaluate significance of cross-correlations at eachlag

Table 1 Calanopia americana. Phase relationship of copepodabundance with respect to tidal height, irradiance, and Sagitta spp.abundance. Lags represent phase relationship of first time serieslisted with respect to second series; negative lag indicates first timeseries preceded second series; positive lag indicates first time serieslagged second series. Positive maximum R-values indicate positivecross-correlations (e.g. high abundance and high irradiance); neg-ative maximum R-values indicate negative cross-correlations (e.g.high abundance and low irradiance) (n.s. no significance)

Cross-correlated time series Lag, h (max.R)

18–20 July C. americana abundancevs. 25–27 July C. americana abundance

0.0 (0.554)

18–20 July C. americana abundancevs. 18–20 July irradiance (copelux)

�0.5 (�0.365)

18–20 July C. americana abundancevs. 18–20 July tidal height

n.s.

25–27 July C. americana abundancevs. 25–27 July irradiance (copelux)

�0.5 (�0.499)

25–27 July C. americana abundancevs. 25–27 July tidal height

�7.0 (�0.185)

18–20 July C. americana abundancevs. 18–20 July Sagitta spp. abundance

0.0 (0.194)4.0 (0.243)2.0 (�0.190)

25–27 July C. americana abundancevs. 25–27 July Sagitta spp. abundance

0.5 (0.303)8.0 (0.399)

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observation period was C. americana abundance signif-icantly cross-correlated with tidal height, with highcopepod abundance related to low tidal height at a lag of�7 h. This significant cross-correlation likely resultedfrom peaks in tidal height only occurring during daylightand not at night during these days, and not from cope-pods migrating with the tides. In contrast, copepodabundance time series were regularly and strongly cross-correlated with their respective diel light cycles; highcopepod abundance occurred during nocturnal periodsof low irradiance during both multi-day observationperiods.

Tidal migrations are common in some estuarinezooplankton, resulting in their retention in estuaries.The physical basis of this transport mechanism is the netseaward movement of the upper water column of apartially mixed estuary, and the net landward movementof the lower water column, as well as the bottom actingas a boundary layer reducing current velocity (Forwardand Tankersley 2001). A tidal swimming rhythm relatedto estuarine retention has been observed in the estuarinecopepod Eurytemora affinis (Hough and Naylor 1992).Field studies have also observed tidal migration patternsin E. affinis and several other estuarine copepod species(Woolridge and Erasmus 1980; Kimmerer et al. 1998,

2002; Rawlinson et al. 2004). However, tidal migrationwas absent in both laboratory and field studies on theestuarine/coastal copepod Acartia tonsa in the NewportRiver estuary, while diel vertical migration was clearlypresent (Stearns 1983). Similarly, the present studyfound no evidence for a tidal migration pattern in C.americana, but strong evidence for light-mediated DVM.This is not surprising as E. affinis is primarily limited toestuarine areas, and therefore exposed to high tidal flux,while A. tonsa and C. americana have distributions thatinclude estuarine and less tidally influenced oceanichabitats (Bowman 1971). The lack of a tidal migrationpattern in this field study predicts that laboratory studieswith C. americana should not detect circatidal rhythms(see Cohen and Forward 2005).

The pattern of light-mediated vertical migration ob-served for C. americana in the present study appears tobe twilight DVM. Cushing (1951) recognized the twilightpattern to be paradigmatic of DVM in the ocean. Thisview was shared by Pearre (1979, 2003), who suggestedthat the ‘‘midnight sink’’ occurs due to asynchronousmovements of individuals within the migrating popula-tion. In this model, some migrators ascend early in thenight, feed, and descend after they become satiated.These same migrators may ascend for a second feeding

Fig. 6a, b Calanopiaamericana. Nocturnal copepodabundance related toirradiance. a Abundance data[log10(x+1) copepods per10 m3) averaged for the samecollection times during threeobservation periods (13 July,18–20 July, and 25–27 July),and means with SE plotted as afunction of collection time forsurface (triangles; 0.5 mbs) andbottom (circles; 0.5 mab)samples; n=7 for samplescollected between 1830 and2230 hours and n=4 forsamples collected between 2300and 0630 hours. Also plotted isaverage absolute irradiance incopelux units (solid line, nosymbols) scaled to right y-axis.b Average relative rates ofirradiance decrease at sunset(left y-axis) and increase atsunrise (right y-axis) plotted forcopelux irradiance in units persecond. For both panels, lightdata at sunset are for thebottom (0 mab), whereas atsunrise they are for the surface(0.5 mbs). Night represented bygray shading

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bout later in the night, and/or a second group ofmigrators may ascend to feed at the surface, descendingat sunrise (Pearre 2003). However, as this model is basedon movements of individual migrators and not themigrating population, it cannot be directly tested usingthe sampling methods employed by most DVM fieldstudies (including the present study), which measure themean abundance of organisms at different depths overtime (Pearre 1979). The present study was designed tocapture some of the potential variability of individualmigrations by employing simultaneous sampling at twodepths (surface and bottom) with high frequency (0.5 or2 h), but we cannot claim to have captured the specificmovements of individual migrators.

Given the limitations of the study design, the ob-served distribution of C. americana is remarkably con-sistent with the model described above. In shallowsystems such as the Newport River estuary, C. ameri-cana most likely reside in the sediments during the day,resulting in low water-column abundance. Clarke (1934)reported that C. americana rapidly buries itself whenplaced into a laboratory container with mud. Lowdaytime copepod abundance in the water column in-creased at sunset, with an initial sharp increase in bot-tom samples, followed by an increase in surface samples,consistent with a synchronous ascent of migrators earlyin the night. A decrease in surface abundance was thenfollowed by a second, less dramatic, increase in bottomabundance, as would be expected for satiated copepodsreturning to the bottom asynchronously, resulting in a‘‘midnight sink’’. A second increase in surface abun-dance began at �0100 hours, consistent with earlymigrators ascending for a second feeding bout, or with asecond group of migrators ascending to feed at thesurface. Lastly, the predicted final descent of migratorsto the bottom at dawn can be inferred from the decreasein surface abundance prior to sunrise, followed by anabundance peak in bottom samples, then a return to lowdaytime abundance.

Moonlight has been shown to affect the vertical dis-tribution of some zooplankton species and not others(McFarland et al. 1999). In the present study, moonlightirradiance was too low to be measured, and thereforecould not be related directly to copepod abundance.However, there was variation among observation peri-ods in the moon phase, time of moonrise and moonset,and weather conditions. Despite this variation, thegeneral pattern of an increase in both surface and bot-tom C. americana abundance at sunset, followed byabundance peaks in the latter half of the night, wasconsistent throughout. This suggests that the night-timevertical distribution of C. americana, particularly the‘‘midnight sink’’, was in part controlled by other non-light factors.

Increases in C. americana abundance at sunset wereregular and dramatic. A proximate explanation for theseincreases could involve an ascent of C. americana inresponse to an exogenous light cue, or a shift from aninactive to an active phase of an endogenous behavioral

rhythm (Cohen and Forward 2005). Both absoluteirradiance (isolumes) and RRC values have been sug-gested as exogenous cues in the migration of crustaceanzooplankton (Boden and Kampa 1965, 1967; Forward1988; Ringelberg 1999; Frank and Widder 2002). Ourstudy was not designed to conclusively evaluate whetherC. americana were following an isolume; too few depthswere measured, and the difference in irradiance betweenthe surface and bottom (one order of magnitude) waslikely not large enough for a perceivable isolume. WhileRRC values could be an exogenous cue for the ascent ofC. americana at sunset, a potential role for RRC stimuliin the descent of copepods at sunrise is less clear.Copepod abundance increased near sunset as RRC onthe bottom (0 mab) was becoming maximally negative(�0.0042 s�1 at 2045 hours), whereas at sunrise cope-pods had descended prior to there being enough light forRRC measurements. Displacement velocity during boththe ascent and descent of the freshwater cladoceranDaphnia galeata x hyalina has been correlated withRRC, suggesting phototactic responses to these stimulicould cause its DVM (Ringelberg and Flik 1994).Likewise, phototactic responses to RRC appear tounderlie the ascent phase of DVM in zoea of the estu-arine crab Rhithropanopeus harrisii (Forward 1985) andin the estuarine copepod Acartia tonsa (Stearns andForward 1984). The potential role of RRC as a causalfactor in C. americana DVM will be discussed in asubsequent paper (Cohen and Forward 2005).

The light measurements discussed above were incopelux, a photometric unit that weights irradiance atwavelengths between 360 and 740 nm based on relativespectral sensitivity of C. americana determined in lab-oratory behavioral studies (Cohen and Forward 2002).A similar approach was used by Gal et al. (1999)studying the freshwater mysid Mysis relicta. These au-thors weighted irradiance measurements based onspectral sensitivity determined microspectrophotomet-rically and derived the photometric ‘‘mylux’’ unit,which they determined was better than either PAR orlux sensors in predicting mysid distributions, with luxsensors being more comparable to mylux than PARsensors. In the present study, copelux was comparedwith two other light units: (1) PAR, obtained by inte-grating irradiance data from 400 to 700 nm, and (2)quantal flux at 480 nm, which was the irradiance mea-sured at 480 nm, the wavelength at which C. americanadisplays maximum photoresponsiveness. As both cop-elux and PAR integrated irradiance over multiplewavelengths, these values were approximately two or-ders of magnitude greater than quantal flux at 480 nm.However, relative rates of irradiance change werecomparable for all units, and only differed at the end oftwilight, when maximum relative decreases were re-corded. For migrating organisms like C. americana withbroad spectral sensitivity, measuring irradiance as PARmay be a reasonable alternative to a photometric unit.The advantage of this is that light measurements wouldbe directly comparable to other studies, while the dis-

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advantage is that PAR may overestimate absoluteirradiance early in twilight and relative rates of irradi-ance change late in twilight.

The ultimate adaptive role of vertical migration iscommonly thought to be predator avoidance. Nocturnaland twilight migrators would have reduced predationpressure from visually orienting surface predators, suchas fish, by spending days in dark conditions at depth oron the bottom, entering surface waters only at night tofeed (Zaret and Suffern 1976; Ringelberg 1999). Reversemigration is generally thought of as a defense againstvertically migrating predators, often invertebrates,which themselves are undergoing nocturnal DVM as adefense against diurnal fish predators. This situation hasbeen most clearly documented in the marine environ-ment for the copepods Pseudocalanus spp., which un-dergo reverse DVM when predatory copepods andchaetognaths are present and migrating nocturnally(Ohman et al. 1983).

The estuarine/coastal chaetognaths Sagitta spp.were the only organisms collected during the presentstudy that would likely prey on C. americana. Incontrast to the twilight DVM pattern of C. americana,Sagitta spp. appeared to either undergo tidal verticalmigration, or, alternatively, were present primarilyoffshore and were brought into the sampling locationonly on rising tides. As Sagitta spp. abundance in thewater column was tidally mediated rather than light-mediated, there was substantial overlap of abundancepeaks in copepods and chaetognaths during the multi-day observations. Accordingly, it is not likely that theadaptive role of DVM in C. americana is for theavoidance of non-visual chaetognath predators. DVMmay play a role as a predator avoidance mechanism inC. americana with regard to predators not sampled inthe present study. Fish may have been able to avoidthe pumps or were blocked by the strainers, whilegelatinous zooplankters (e.g. ctenophores) may havebeen destroyed during pumping and not sampledadequately.

In the field, C. americana displayed twilight DVMwith no apparent tidal component. Copepods ascendedin the water column at sunset, a ‘‘midnight sink’’ oc-curred, and then there was a subsequent increase incopepods at the surface in the latter half of the nightwith descent by sunrise. This migration pattern couldresult at the proximate level from endogenous rhythms,behavioral responses to exogenous light stimuli, or acombination of the two. A companion paper (Cohenand Forward 2005) tests hypotheses concerning thesepossibilities.

Acknowledgements We thank E. Sinkhorn for her technical assis-tance, and R. Barber, S. Johnsen, D. Rittschof, and D. Steinbergfor comments on an earlier draft of the manuscript. This material isbased in part on research supported by the National Oceanic andAtmospheric Administration (ECOHAB grant NA17OP2725 to R.Forward and P. Tester), with additional funding provided by theOak Foundation.

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