Future Studies of Zooplankton Behavior: Questions ... - IngentaConnect

BULLETIN OF MARINE SCIENCE, 43(3): 853-872,1988

FUTURE STUDIES OF ZOOPLANKTON BEHAVIOR: QUESTIONS AND TECHNOLOGICAL DEVELOPMENTS H. J. Price, G.-A. Paffenhofer, C. M. Boyd, T. J. Cowles, P. L. Donaghay, W M. Hamner, W Lampert, L. B. Quetin, R. M. Ross, J. R. Strickler and M. J. Youngbluth ABSTRACT Important questions about zooplankton behavior include the evolutionary causes of vertical migration, Ihe influence of behavior in generating and maintaining patchiness, and the effect of various behaviors on energy budgets. Zooplankton feeding rates and the potential for selectivity among prey types also remain controversial. In many respects we have reached the limits of what can be inferred about these topics from traditional techniques such as before-and-after grazing experiments in the laboratory and sampling with nets at sea. A variety of recently developed or proposed techniques could be used to obtain more direct information on zooplankton behavior. Three-dimensional video systems which quantitatively track individual zooplankters over time have been developed for use in laboratories, mesocosms and in situ. Recently-developed mesocosms facilitate manipulation of environmental variables under conditions which allow large-scale behavioral responses such as vertical and horizontal migration. Increased availability of submersibles and ROYs, combined with advances in video technology such as image intensifiers, low-light level cameras and range-gated lasers, should make it possible to collect quantitative behavioral data on undisturbed zooplankters in situ. Yarious optical devices could provide data on the in situ availability of phytoplankton, dissolved chemical signals and turbulence at the microscales relevant to individual zooplankters. Application of neurophysiological techniques would allow determination of the chemical and mechanical signals which can be detected and the threshold levels required. Lastly, continuing improvements in oxygen and pH sensors might be combined with video observations to compile realistic time budgets of behavior in large volumes of water while simultaneously measuring metabolic rate over time.

There has been increasing recognition in recent years that zooplankton have elaborate behavioral repertoires, both inherited and learned, which allow them to respond to changing environmental conditions. The ultimate goals in studying these behaviors are to understand how natural selection has influenced behavior and how behavior influences community structure. Although our knowledge of zooplankton behavior has expanded rapidly over the last decade, many questions remain which will require technological advances to answer. In many respects we have reached the limits of what we can learn from traditional methods. We believe that not much more remains to be gained by attempts to infer zooplankton behavior from traditional before-and-after grazing experiments in the laboratory or by taking more net tows in lakes and at sea. The development of new technology has often overturned ideas generated from these traditional techniques. For example, the development of high-speed cinematography led to replacement of the idea that copepods are relatively simple filter-feeders with a much more complicated picture of how they detect, capture and reject cells. The flexibility of their feeding behavior was further demonstrated when the development of flow cytometry made it possible to show that selectivity can be achieved even among algal cells of the same species which differ in nitrogen content (Cowles et aI., in press). Similarly, previous inferences about the in situ 853

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BULLETIN OF MARINE SCIENCE, VOL. 43, NO.3, 1988

distribution of zooplankton and potential prey have been modified by recent visual observations and sampling procedures from submersibles and SCUBA divers. These observations indicate considerable fine-scale structure and patchiness, including very high abundances of single zooplankton species distributed within narrow depth intervals, the presence of particle-rich layers and dense patches of marine snow. Such structure had gone largely unrecognized despite decades of sampling with plankton nets and pumps. We have selected some of the major questions discussed at the Zooplankton Behavior Symposium held in April of 1987 in Savannah, Georgia (this volume), and describe both recently developed or proposed technology which could be used to answer them. Our goals are to increase the use of recent technological advances, and hopefully to inspire new developments by pointing out existing technological needs. We have organized the material into six main sections referring to approaches to answer various behavioral questions (3-dimensional visual observations; use of mesocosms; in situ observations) and to topics which require specific attention (micro scale environmental variability; sensory physiology; energetic costs of behavior). We do not intend to cover all future spheres of interest in zooplankton behavior, but wish to draw attention to several areas of major significance. 3-DIMENSIONAL

VISUAL

OBSERVATIONS

One technological need common to many of our questions about zooplankton behavior is the ability to track free-swimming individuals in three dimensions over time. Such an ability is needed to quantify the perceptive volume within which zooplankters can detect and respond to individual particles, an important prerequisite to predicting encounter rates with various particle types. Three-dimensional observations over time are also needed to determine how zooplankters alter their swimming behavior in response to food patches on a variety of scales. These data are required to determine the relative importance of physical factors versus behavioral aggregation in generating and maintaining zooplankton patchiness. Direct monitoring of the response of individual zooplankters to prey patches would also help indicate the degree of error in estimates offeeding rates and energy budgets caused by assumption of "average" homogeneous prey concentrations. Lastly, such direct visual observations would be useful in constructing time budgets, i.e., how do zooplankters partition their time between swimming at various speeds, "resting," feeding, mating and escaping predators? How does this partitioning vary with food availability and abundance of predators? This information is necessary to construct more realistic daily energy budgets. Two-dimensional optical systems are not adequate for obtaining quantitative data on movement in a 3-dimensional medium such as water. Three-dimensional optical systems have been used extensively to examine fish schooling and swimming behavior (Dill et aI., 1981; Potel and Wassersug, 1981; Klimley and Brown, 1983) but have only recently been applied to studies of zooplankton behavior. We describe below three recently developed systems for quantifying zooplankton behavior in different environments and on different spatial scales. In Laboratories. -A 3-dimensional system which resolves both free-swimming copepods and their prey items as sma]] as 10 /Lmhas been described by Strickler (1985). Two collimated beams intersect at 90°, and tracking of the animal in a 6 liter vessel is provided by an optical system which can move in the X and Y direction while the vessel moves in the Z direction. The system is a modification

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of a Schlieren optical pathway in which a collimated beam is focused by the condensing lens. At the focal point, a small black spot stops the light from reaching the image plane, resulting in a dark exposure. Light scattered by an object (e.g., a zooplankter) in the collimated beam will miss the black spot and reach the image plane, forming a light image on a black background. This system was primarily designed to determine the perceptive volume within which copepods detect and respond to individual particles. However, since it has a spatial resolution of 10 J.lm, it could also be used to examine the responses of ciliates to microscale aggregations of dinoflagellate prey or marine snow. In Mesocosrns. - The mechanisms which generate and maintain zooplankton swarms can also be addressed for larger zooplankters using 3-dimensional tracking of swimming behavior in mesocosms. A 3-D system has recently been used by Price (1987) in the tower tank at Dalhousie University to determine how the swimming behavior of euphausiids changes when they encounter a subsurface algal patch. This system uses two low-light level video cameras which focus on a volume of approximately 200 liters. An electronic screen-splitter is used to synchronize the output of the cameras and deliver images from both cameras onto opposite halves of a single video screen. Unlike many techniques of stereophotogrammetry, this system does not require knowledge of focal length, camera positions or orientations. Instead, a three-dimensional calibration grid is filmed suspended in the volume of water to be viewed and then removed prior to experiments. As long as the camera positions remain fixed, the known coordinates of this grid can later be used to obtain the 3-D coordinates of the zooplankters over time, using the direct linear translation method (Marzan and Karara, 1975; O'Brien et aI., 1986). In this system and the one developed by Strickler, 3-dimensional data are reconstructed via computer analysis after filming, i.e., only 2-dimensional images are viewed by the operator during filming. In situ. -A third system for collecting 3-D data has been developed for use on submersibles (Hamner et aI., in press). This is a commercially available system marketed by Stereographics Corporation and designed in part by Lipton (1982). Two video cameras are mounted parallel to each other in separate housings outside the submersible, and the images are ported inside to a single tape recorder onto which the video signals are multiplexed. The images are presented alternately on a single monitor via stereo image alteration using electro-optical shutters on the screen surface and on the glasses worn by the video operator. This separates the left and right images and creates stereopsis. The advantage of this system for use in submersibles is that the operator can view 3-D images as they are recorded, thereby insuring that the best possible information is collected during the dive. The disadvantage of the single monitor-recorder solution is that the equipment necessary to multiplex two video signals electronically onto one video frame is fairly expensive. Another 3-D optical system has been developed for shallow-water research by SCUBA divers (Hammer et aI., in press). Two video cameras are mounted in parallel at approximately human interocular distance between centers of lenses (70 mm). They produce left and right video images that are recorded on separate recorders. The advantages of this system for shallow water work are that small, relatively inexpensive cameras and recorders can be packaged in one portable underwater housing. Use of the calibration system described for mesocosms is not feasible at sea, so the system of Klimley and Brown (1983) was used for the submersible and SCUBA-based systems. Knowledge of the exact distances between the two cameras

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and from camera lens to film plate is required. Additionally, calibration is achieved prior to use by measuring the size and horizontal displacement in the paired images of a reference object of known size and distance from the cameras. If the cameras are stationary, accurate coordinates of positions in 3-D space can be obtained. If the cameras track a given individual, this animal becomes the reference point and movements of other individuals are calculated in relation to the moving reference point. Recalibration of the cameras is not necessary as long as the cameras are not moved in relation to one another. Improved Resolution of Images. -Currently the resolution of the 3-D systems for mesocosms and in situ work only allows tracking of larger zooplankters such as euphausiids and gelatinous forms. However, the resolution of video equipment is increasing rapidly, and filming of smaller zooplankters such as copepods and even ciliates may soon become possible. One problem with improved resolution is the need to obtain images at lightlevels which do not disturb the animals. Very low-light level cameras are now available which can produce useable images at 10-5 footcandles. Also, electronic image intensifiers can be used to amplify by several orders of magnitude the amount of light which reaches the video camera. The use of red or infrared lights is also possible, but its rapid attenuation in water requires the organisms to be fairly close to the light source. High-speed video systems are required to observe the movement of cilia and have been used successfully with bivalve larvae (Gallager, 1988) and tintinnids (Taniguchi and Takeda, 1988). These systems are currently available up to speeds of 2,000 frames per sec (reviewed in Cheetham and Scheirman, 1988). However, high speed systems have the disadvantage of requiring very high light levels, and of a loss of resolution of prey particles with increasing imaging rate. Another problem with improved resolution is obtaining sufficient magnification to observe smaller zooplankters such as ciliates and copepods at a distance. Longdistance microscopes have recently been developed which may be useful in overcoming this problem (Hyzer, 1982). Questar's QM 1 claims a resolution of 4 ~m at a distance of 1 m away. Application of such instruments for studying small zooplankton is currently limited by our inability to automatically track individuals. Since the field of view is quite small at such high magnifications, it is difficult to locate moving individuals and keep them within view for any period of time. Although motorized 3-D mounts are available, some means of automatically "locking in" an individual in the field of view would be desirable. An alternative is to have multiple high-resolution lenses, but that may be prohibitively expensive for most purposes. An automated tracking system would also be useful with lowermagnification lenses to follow larger, more rapidly swimming zooplankters. For in situ work, it would be desirable to focus on animals which are considerable distances from the submersible in order to minimize the effects of disturbance. Obviously, this creates problems due to the rapid attenuation of light in water and to backscattered light from particles in the intervening distance which severely reduces image contrast. Such problems might be overcome by the use of range-gated lasers (Dixon et al., 1984). These imaging systems use a 10-20 ns pulsed blue-green laser beam to illuminate an area up to 100 m away, and rangegated receivers to minimize interference from backscattered light. Image Analysis. -Another problem is the need for automated image analysis. The volume of data obtained by 3-dimensional systems is so large that manual digitization is severely limiting. If one were to manually digitize the movements of20 zooplankters in 3-D space, and record their positions via stop-frame analysis


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at 30 frames per sec, over a half-million file entries would be required to digitize 10 min of data. At one entry every 3 sec (far faster than most could manage) it would take about 6 months to digitize 10 min of video footage (Hamner et aI., in press). The prototype of the main automated tracking system used today is the "Bugwatcher" (Wilson and Greaves, 1979; Buskey, 1984). This system recognizes specific objects by preprocessing each video frame for contrast discontinuities, then outlines the object and summarizes its position in space as a single centroid. The centroid is then tracked automatically over time, relieving the operator of manually entering data. The system, therefore, sacrifices some visual information in order to accurately track movement. A commercial descendant of the Bugwatcher, The Expert Vision Integrated Motion Analysis System, is now available for 3-dimensional analysis. It is capable of tracking objects recorded by multiple cameras placed at any angle and through any transparent media or mixtures thereof (such as an air-water interface). Although this system greatly speeds data analysis in most instances, there are also several disadvantages. If dense concentrations of zooplankters are in a single field of view, the Expert Vision system gets confused when images are occluded and/or adjacent tracts become too precisely aligned. In this event the scientist can usually determine what actually happened by viewing the original video tape again and again at slower and slower speeds, then interactively editing the data files. Another disadvantage of the system is the loss of information that results from reducing successive images of each individual to a series of points moving in 3-D space. This prohibits automated measurements of body lengths, shapes and surfaces, and does not allow separation of various taxonomic groups. Such automated tracking systems do not currently have the ability to automatically analyze complex images. They work well for simple images with a fairly high level of contrast, e.g., outlines ofzooplankter's bodies versus the background. They are not applicable to images which have a complex gradation of shades which move over time, e.g., close-up views of feeding appendages and their response to incoming particles. This must still be accomplished by manual digitization, a process which can take several operator-hours to record a single second of a zooplankter's life. USE OF MESOCOSMS

Many patterns of zooplankton behavior, such as vertical or horizontal migrations, take place on scales which are impossible to replicate in the laboratory. Field studies of these behaviors are often difficult to interpret due to a variety of uncontrolled factors that affect the behavioral response and to difficulties in tracking the same group of animals over time. Mesocosm tanks which are intermediate in scale between traditional laboratory vessels and field experiments offer promising opportunities to examine zooplankton behavior. We describe below two recently developed mesocosms, one system for freshwater studies in West Germany and another for marine zooplankton in Virginia. We also discuss recent applications of several older marine mesocosm tanks in Nova Scotia. These latter tanks have been available for over a decade, but unfortunately have received limited use for zooplankton behavioral studies. Differences between the tanks at the various locations suggest that each is best suited to answering particular types of questions. Max Planck Institute of Limnology. - Two plankton towers designed by W. Lampert were recently erected inside the Max Planck Institute for Limnology in PIon,

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West Germany. These tanks were designed to provide the maximum possible control of environmental factors with the aim of distinguishing between the growing number of hypotheses on the ultimate (evolutionary) reasons for vertical migration (reviewed in Haney, 1988). The two stainless steel cylinders are 0.85 m in diameter and 11.5 m high and can be filled with unfiltered or filtered water from a nearby lake. The relatively small diameter was chosen to assure optimal environmental control from outside of the tanks. The heat control system attached to the wall of the tank is interrupted every 50 cm of height, so that a separate temperature control system can be plugged in. This allows construction of a thermocline at any point in the column. Since the temperature gradient is maintained by external controls, the shape of the thermocline can be designed by the experiments. Each tower has 22 sampling ports arranged vertically in 0.5 m distances. The ports are interchangeable adapters that contain a small diameter tube for sampling plankton, two tubes as inlets and outlets for local treatment of water inside the cylinder, and a temperature sensor. The inlets and outlets can be used to withdraw water from a specific depth, treat it (for example, enrich it with algae or remove oxygen) and feed it back to the original layer. This will allow creation of layers of specific conditions (e.g., deep chlorophyll layers). Natural and artificial light are provided from the top of the columns. Three pairs of windows (at I, 4, and 10m depth) arranged at a 90 angle allow three-dimensional observation of individual animals. A high-frequency echo sounder directed from the bottom of the tanks can also be used to monitor zooplankton distributions. The system allows simulation of specific conditions in the vertical structure of a lake and examination of the effect of single factors on zooplankton behavior. Since it also provides the potential to measure fitness parameters such as recruitment and mortality, it can be used to test ultimate factors. Typical questions with respect to vertical migration would be: Do the animals optimize their migration behavior in the tradeoff between increased food availability and increased mortality? Do they take the risk of being eaten when the food is scarce? Do they cease migrating to the surface when more food is available in the hypolimnion? If migration behavior can be controlled by external cues (light), it should be possible to estimate the costs of migration directly. Animals can probably be forced to migrate several times a day by short term light cycles, and their development can be compared to reversed migration behavior under the same thermal and nutritional conditions, if the light is introduced either from the top or from the bottom of the column. This would also provide insight into the role of light and gravity in regulating the direct movements. Direct observations of behavioral responses can be made, if a certain cue is restricted to a specific layer by external control. This cue may be inhibiting filamentous blue-green algae or the "smell" of a predator. The effect of selective feeding can be tested by caging predators near the surface or near the bottom of the tanks. Many more experiments of this kind can be imagined. It is important to all of them that the environment be completely controlled, so that a single factor can be varied and that two identical systems are available. This system may still not satisfy all requirements. The relatively small diameter, for example, is a compromise between avoiding "wall effects" and the need for easy handling and accurate control. Also, the relatively small diameter of the tank will not make it possible to replicate the angular light distribution found in the field (Steams, 1986). However, it is hoped that the tanks will serve as a center to attract investigators who feel that their ideas can only be tested using these fa0


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cilities. There will be no charge for the use of the system, and the quality of proposed research will be the only criterion for access. Dalhousie University. - Mesocosm tanks for marine organisms are available at Dalhousie University in Halifax, Nova Scotia, Scripps Institution of Oceanography in La Jolla, California and at The Marine Ecosystem Research Laboratory (M.E.R.L.) in Narragansett, Rhode Island. We will only discuss here the use of the tanks at Dalhousie. They are more suited to zooplankton behavioral studies than the others, offering a higher degree of environmental control of light and temperature, as well as numerous portholes for conducting visual observations. For a more detailed description of the tank at Scripps see Balch et al. (1978) and for the M.E.R.L. tanks see Santschi (1982). The technical aspects of the Dalhousie tanks have been described in detail by Conover and Paranjape (1977) and Balch et al. (1978). Briefly,there are two tanks, one 3.7 m in diameter and I0.5 m deep (tower tank) and another 15 m in diameter and 3.5 m deep (pool tank). Both can be filled with filtered or unfiltered seawater and/or dechlorinated freshwater which can be heated or cooled as required. Water inlet ports at six levels in the tower tank allow temperature and/or salinity stratification of the water column. Six metal halide lamps are located at the top of the tower tank, and 22 mercury lamps over the pool tank, each on separate timers. Some of the questions raised above about vertical migration in freshwater zooplankton could also be addressed with marine zooplankton in the tower tank. However, the large size of this tank means that the degree of environmental control is not as great as envisioned for the PIOn tanks. On the other hand, their large size makes both the tower and the pool tank suitable for examining the behavior of larger zooplankters such as euphausiids and gelatinous forms, as well as copepods. Although Conover and Paranjape (1977) suggested long ago that the Dalhousie tanks are perhaps best suited for collecting visual data on behavior in short-term experiments, this potential has been largely unutilized. A series of 26 windows line the tower tank, while in the pool tank, visual data can be obtained through a series of 22 windows or from a rotating bridge suspended over the tank. Mackie et al. (1981) used the portholes in the tower tank to examine vertical migration of Aurelia through visual counts of their distribution. Recent advances in video technology now make it possible to use these portholes to collect quantitative behavioral data on zooplankton. One question which could be addressed using these tanks is how swimming behavior affects the generation and maintenance of zooplankton patches on both horizontal and vertical scales. Price (1987) recently used the tower tank to visually monitor the response of euphausiids to the introduction of a subsurface algal patch. Three-dimensional data collection allowed determination of swimming speeds, path orientations, time spent swimming and sinking, frequency of turns and the net-to-gross displacement ratios of paths. Such data indicate whether zooplankton can rapidly locate an algal patch and how swimming behavior is altered to keep them within it. The minimum patch size or concentration of algae or exudates which elicits changes in swimming behavior could also be determined with this technique. Responses to meter-scale prey patchiness along horizontal dimensions could be examined by using the pool tank. Advances in video technology (see above) could be adapted for use in these tanks to improve resolution and directly monitor the behavioral response to food patches by smaller forms such as copepods, or by more transparent gelatinous groups.

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Another type of visual data which could be collected in these tanks is the time budget of various behaviors, as discussed under "Three-dimensional visual observations" above. These types of observations would be particularly valuable for gelatinous forms which show very abnormal behavior in small vessels. Both of the Dalhousie tanks are available for use by scientists from outside the University, with moderate user fees. Langley Research Center. -A tow tank at the NASA-Langley Research Center, Hampton, VA, has recently been adapted by P. Donaghay for studies of marine zooplankton. The tank has been modified to examine the mechanisms of generating, maintaining and dissipating plankton patchiness, particularly the interactions between zooplankton vertical migration and physical shear resulting from currents moving in different directions at various depths (Donaghay et aI., 1987; French and Donaghay, 1987). It is 875 m long, 7.3 m wide, 3.7 m deep and holds 21,000 m3 of water. Of course, it is not possible to maintain strict environmental control of all variables in such an enormous tank. However, in contrast to the mesocosms described above, this tank is large enough to maintain plankton patches in the presence of physical mixing processes similar to those that prevail in the ocean. The concrete tank can be filled with filtered or unfiltered seawater from an adjacent arm of lower Chesapeake Bay. It is enclosed in a building excluding sunlight, with artificial lights providing control oflight intensity and photoperiod. Vertical temperature and salinity structure can be controlled by the balance between source waters added to the exchange through the tank walls and artificial mixing. Mixing is provided by an automated stirrer that moves along the axis of the tank. Changes in mixer design and speed can be used to manipulate turbulence levels within oceanic ranges (as defined by dye studies). Surface and internal waves could also be used to induce mixing by refurbishing a wave machine. A motorized platform can be moved up and down the tank to provide a base for sampling physical, chemical and biological structure and making observations of zooplankton behavior. A plankton patch can be initiated in this system by first filling the tank with filtered sea water, then inoculating and developing a patch in a lOO-mlong plastic bag floated in one section of the tank. Light, nutrients and physical mixing conditions are then provided to support plankton growth in the bag relative to surrounding waters. Once the desired abundance differences are achieved, seawater densities between sections are balanced and the bag is removed, leaving the patch free to be experimentally manipulated. Since the composition of these patches is controlled by the inocula, this technique can be used to develop single or multiple patches along the tank axis composed of individual or species mixes of phytoplankton, microzooplankton, zooplankton and/or predators. Density or pumpdriven differential currents between surface and bottom layers can then be induced, and zooplankton behavioral responses observed over days to weeks. Since it is possible to both observe zooplankton behavior and measure changes in population dynamics and community composition, it should be possible to address the evolutionary consequences of behavior. Such an approach was discussed above for the PIon tanks as a means of estimating the costs and benefits of vertical migration in lakes. However, additional factors must be considered in marine systems since vertical migration interacts with currents moving at different depths. This should lead to vertical migrators at sea experiencing more temporally variable and unpredictable levels offood and predation pressure than do vertical migrators in lakes. Such unpredictability may have associated costs (Lampert and


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Muck, 1985; Donaghay 1988). Also, individuals that differ in the degree of vertical migration, average swimming velocity and/or oriented swimming may be differentially dispersed from a patch (Donaghay et aI., 1987). The Langley tank can be used to understand these complex interactions between physical and biological processes occurring in both vertical and horizontal dimensions. For example, do zooplankters use vertical migration through various shear layers to locate algal patches? Is vertical migratory behavior modified to keep zooplankton within food patches in areas where shear forces would otherwise disperse them? Patch structure and shear can be varied while simultaneously measuring zooplankton behavioral responses and the impact of such responses on recruitment. In addition to zooplankton behavioral studies, the scale of this tank would be appropriate for studies of higher trophic levels such as fish larvae. Efforts are currently underway to develop the system into a national facility that can be used by a number of investigators, with access determined by the quality of submitted proposals. IN SITU OBSERVATIONS

Ultimately, zooplankton ecologists would like to address many of the questions asked in the above sections with direct behavioral observation at sea. Such an ability is important to develop for several reasons. As mentioned in our introduction, recent in situ observations have indicated that the water column has considerably more fine-scale structure than is evident from pump and net samples. However, we know relatively little about the role of behavior in creating or responding to such structure. How do vertical and horizontal movement patterns contribute to observed concentrations of single species in narrow depth intervals (Alldredge et aI., 1984; Hamner et aI., 1987)? How do zooplankters respond to fine-scale structure of potential prey items such as layers of fecal pellets (Youngbluth et aI., in press), densely clustered colonial diatoms (Youngbluth et aI., 1987) or marine snow aggregates (Alldredge et aI., 1986)? In situ behavioral observations could suggest whether such heterogeneities may represent significant food sources for certain taxa. In situ behavioral observations would also be useful in addressing questions about vertical migration. As pointed out by Hamner (1988), in all the hundreds of published papers on vertical migration, no one has ever tracked an individual zooplankter in situ over a 24-h period. Such a study would not only provide data on how zooplankton budget their time between various behaviors, but would allow us to examine behavioral variability between individuals. In instances where only portions of populations appear to migrate, we cannot determine whether all individuals migrate up and down but do so out of phase, or whether there is truly a subpopulation which never migrates. Also, do individuals migrate up and down several times during the night depending on their level of gut-fullness (Simard et aI., 1986)? These types of questions cannot be answered by population level sampling techniques, but must be addressed by following individuals over time (Hamner, 1985). The only paper of which we are aware that followed the vertical migration of single invertebrate individuals over 24 h is that of Carlson et aI., (1984). They attached neutrally bouyant sonic transmitters to the shells of Nautilus. By setting each transmitter to different frequencies, individuals could be distinguished and tracked over time. With the increasing miniaturization of electronic components, it may be possible in the distant future to use such devices to follow large zooplankters such as euphausiids or gelatinous forms. In the meantime long-term

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visual tracking of single individuals seems possible, but obviously will require considerable effort and expense. In situ behavioral observations are also needed to verify existing behavioral data collected in laboratories. For example, considerable evidence has been presented at this symposium and elsewhere on the flexibility and complexity of zooplankton feeding behavior in the laboratory, where certain taxa have been observed to remotely detect particles, Additionally, many groups including the heteroflagellates, ciliates, copepods and euphausiids show chemosensory responses towards "patches" of prey or exudates in the lab. However, we still have virtually no direct information on the frequency of such responses at sea, where increased chemical and physical noise may interfere with detection and response. In situ quantitative behavioral data on feeding and swimming are particularly needed for gelatinous groups which tend to show abnormal behaviors in the laboratory due to handling effects and disturbance from the walls of the container. Submersibles and RO Vs. - Three basic categories of vehicles are available for in situ work. These include relatively large submersibles such as ALVIN,the JOHNSON SEALINKSand the PISCES,smaller one-person vehicles such as DEEPROVER,and unmanned remotely-operated vehicles (RaYs). The types and locations of presently active units are given in Hanson and Earle (1987). Of these, the large submersibles have the longest history of use for zooplankton research. Zooplankton scientists have primarily used these submersibles to obtain estimates of abundance at various depths (Mackie, 1985; Bowers, 1988; Mills and Goy, 1988) and for capturing and identifying fragile gelatinous forms. Useful behavioral observations have also been made, but have been primarily descriptive. Quantitative behavioral data can now be obtained from submersibles using 3-dimensional analysis systems described above. Further progress in quantifying behavior depends on the adaptation and creation of recording devices which can observe individuals without disturbance from the vessel's light or movement (Youngbluth, 1984). If currently available electronic image intensifiers can be improved and used in concert with low-light SIT and ISIT cameras (Wood and Potts, 1987), observations and measurements could be made at ambient light conditions. This practice would reduce unnatural phototactic responses associated with using incandescent light in the deep sea, and could document a wide variety of normal swimming, feeding and breeding behaviors. These devices should also enhance measurements of bioluminescent displays and allow experiments to examine the communication value of these signals between and among species (Widder et aI., 1986). Physical disturbance of the zooplankters can be minimized in vehicles with variable ballast control by trimming the vessel to be neutrally buoyant and allowing it to drift with a prevailing current. Also, the use of rangegated lasers (Dixon et aI., 1984) may eventually allow observations to be made at long distances from moving vehicles, where disturbance from the vessel's pressure wave is minimal. Zooplankton behavioral studies should be further aided by continuing development of sampling devices to capture and identify delicate, slow-moving forms as well as more robust, fast-swimming individuals (Youngbluth, 1984; Tietze and Clarke, 1986). Submersible-based sampling devices could be used to quantify finescale distributions of potential prey, such as microzooplankton concentrated at current shears (Townsend and Cammen, 1985) or fecal pellets stalled at pycnoclines (Youngbluth et aI., in press). Suction devices are also being developed which can selectively sample large quantities of marine snow (Davoll and Youngbluth, pers. comm.). Quantitative behavioral observations combined with quantitative

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sampling techniques should contribute greatly to our understanding of the role of such fine-scale patchiness. Fixed Arrays of Submerged Cameras. - In situ observations could also be obtained through the use of fixed cameras at various positions in the water column. This would also allow more long-term observations at multiple locations than is possible with submersibles or ROVs. Such a system is currently being developed by G.-A. Paffenhofer and J. R. Strickler for eventual deployment on the southeastern U.S. continental shelf. The system will be designed to determine the abundance, composition and behavior of zooplankton over periods of minutes to months in relation to environmental variables. Each recording unit in this system will consist of an infrared laser, collimating and condensing lenses, a video camera and recorder. These units will be placed at a minimum of three different depths on across-shelf and along shelf current meter arrays, providing synoptic data over three dimensions. Accompanying data will include current velocity and direction, temperature and in vivo chlorophyll concentration at each camera location. Due to the use of an infrared light source, recordings will only be made during dark hours. The resolution of the system will be around 30 to 40 /.Lm, sufficient to distinguish late copepodid and adult calanoids and adult cyclopoids. Pelagic tunicates should also be easily separated into their three major groups. Using a field of 10 cm diameter with a 5 cm depth of field, this system would sample a volume of about 1.5 m3·h-1 at a current speed of 10 cm·s-I• To prevent zooplankton aggregation near a moored platform, a more or less continuous flow of water past the array is needed. This requirement means that the system will not be suitable for some locations. However, flows from 5 to 20 cm· S-1 are regularly recorded at the 30 to 40 m isobath of the southeastern continental shelf. Thus this system should cause little disturbance to the behavior of small « 5 mm) zooplankters. Such a system will provide synoptic data on behavior and abundance of various zooplankters at a variety of depths and geographic locations. MEASUREMENT

OF MICROSCALE

ENVIRONMENTAL

VARIABILITY

One point which arose repeatedly at this symposium is the need to understand environmental variability on scales which are relevant to the behavioral process under observation. As indicated above, we currently have no direct information on the in situ frequency of chemosensory behaviors such as remote detection of single particles or oriented swimming relative to micro scale food patches. In situ behavioral observations would be greatly strengthened by data on chemical and physical variability at scales relevant to these processes, i.e., from a few hundred microns to several centimeters. Data on the variability of the biomass and physiological status of phytoplankton at these scales is also needed, particularly in light of recent evidence that the chemosensory response of both ciliates and copepods varies greatly with cell quality (Butler et al., 1988; Verity, 1988). Phytoplankton. -Our present ability to resolve distributional patterns of phytoplankton biomass in the field prevents us from examining spatial patterns smaller than approximately 1 m. To overcome this problem, a rapid high-resolution sampling system is currently under development by Cowles et al. (1987) which will identify variations in food characteristics at spatial and temporal scales which are more relevant to the behavior of individual zooplankters. The system uses laser/fiber optics technology to measure phytoplankton fluorescence properties

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from a free-fall-and-retrieval microstructure pro filer (Rapid Sampling Vertical Profiler [RSVP]) and from a towed thermistor chain. In the profiling mode, this system will provide centimeter-scale resolution of phytoplankton fluorescence coincident with and at the same sampling frequency as temperature, conductivity and velocity microstructure. In the towed configuration, it will obtain phytoplankton biomass estimates every two to four meters horizontally at multiple depths in the upper 100 m. The basic system consists of an argon laser which sends excitation light down a fiber optic cable attached to the microstructure profiler and/or the thermistor chain. The fluorescence spectrum emitted from the cells in the illuminated volume is collected and transmitted up the fiber to an optical multichannel intensified diode array detector. Up to 30 fluorescence spectra per second can be obtained as the microstructure profiler falls through the upper water column. The emission from the water Raman peak provides an external standard to check for signal attenuation in the fiber. Calibration studies with standard fluorometric techniques indicate that the system will detect chlorophyll concentrations of less than 0.05 micrograms per liter without optical enhancement of the fiber tip. The advantages oflaser/fiber optic technology are considerable; no underwater instrumentation, no severe power limitations, high rate of data accumulation, improved temporal and spatial resolution of fluorescence fields, shared use of laser and multichannel detector by multiple sensor systems, and enormous potential for fluorescence detection of different compounds within cells using specific optical sensors. Coupling these fluorescence techniques with the microstructure profiler and thermistor chain permits phytoplankton biomass estimates to be made on scales which are linked to micro scale physical processes and on scales which are more relevant to zooplankton food selection and detection processes. Dissolved Chemical Compounds. -In addition to the system described above for determining micro scale phytoplankton variability, considerable potential exists for the development of in situ optical techniques for measuring microscale variability of dissolved chemical compounds. Knowledge of the spatial and temporal variability of the chemical signals which zooplankters detect is essential to interpretations of their behavioral response (reviewed in Atema, 1987). Laser Raman spectroscopy is a common technique in analytical chemical laboratories (Graselli et aI., 1981), and has the potential for real-time in situ analysis of seawater (Smutz, 1985), This technique identifies the structure of complex compounds through characteristic shifts in the frequency of scattered light, providing chemical "fingerprints" of the illuminated sample volume. Quantitative estimates of the concentration of various compounds can also be obtained. Since the area of the laser beam can be extremely small, the technique may be used to characterize sample volumes as small as a few nannoliters. Using scanning Raman microprobes, these point estimates can be obtained in rapid succession in order to map out micro scale variations as a function of horizontal and vertical coordinates (Graselli et aI., 1981). Modification of these laboratory techniques for in situ use would allow determination of the spatial distribution and residence time of chemical cues under a variety of natural conditions. Turbulence. -Optical techniques also offer promising opportunities for measurements of micro scale turbulence on scales of a few cms down to a few hundred microns. Development of flow visualization techniques for use in the water column would be advantageous in that they are non-intrusive (relative to hot-wire anemometers, Gust, 1982), and could provide data concurrent with direct visual observations of behavior.


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Laser Doppler anemometry is a common optical technique for obtaining microscale velocity measurements in the laboratory (Buchave et aI., 1979) and has recently been used to study benthic boundary layer flows in situ (Agrawall and Belting, 1988). With this technique, fluid velocity is measured only at single point at any given instant. However, one question we would like to address is the distribution of micro scale (mm-size) eddies within a volume of water. Both modelling attempts and empirical data from wind tunnels suggest that microscale eddies are distributed very unevenly and occupy only a small percentage of a given volume (Kuo and Corrsin, 1971; Siggia, 1981). Such micro scale distributions have important implications for the residence time and predictability of chemical signals and for the zooplankter's behavioral response. To collect such data at sea we would like to obtain simultaneous determinations of fluid velocity throughout a specified volume of water. Other optical flow-visualization techniques might be adapted to provide this data, such as laser speckle photography, holography and interferometry (reviewed in Lauterbom and Vogel, 1984). Application of various non-intrusive optical techniques could eventually provide information on microscale variability in phytoplankton biomass, dissolved chemical constituents and fluid velocity concurrently with in situ behavioral observations. Such data would allow us to understand the interaction between zooplankters and their environment at scales which are relevant to the feeding and swimming behavior of individuals. SENSORY

PHYSIOLOGY

As mentioned in several of the above sections, there is now considerable behavioral evidence that zooplankton behavior is affected by dissolved chemical compounds or by chemical differences between food particles. Such behavioral data include observed differences in feeding rate, collection of animals in pipettes containing preferred odors, etc. These behavioral approaches are in contrast with studies that use a neurophysiological approach to answer questions such as "What chemicals can an animal detect, and at what concentrations?" Such neurophysiological studies have been carried out extensively on lobsters and other large crustaceans (Ache, 1982; Carr and Derby, 1986; Derby and Atema, 1987). The intent of these studies has generally been to elucidate the cellular physiology of taste and smell. The lobsters "nose" (the lateral branch of the first antenna) has been the basis of many of these investigations because it is a good neurophysiological preparation, characterized by receptor cells that extend as nerve tracts from the tip of the antenna, running several centimeters without synapses back to the brain of the lobster. The ability of a single antenna cell to detect specific odorants at known concentrations can now be studied with some ease. The obvious wish is to apply the techniques of the neurophysiologist to, for example, the relatively large first antenna of a copepod or a euphausiid to extend and interpret information derived from behavioral studies. Such applications are currently in the initial stages of development by C. M. Boyd. The underlying goal of such an effort is to gain an understanding of how zooplankton perceive and respond to predators, prey and mates. Atema and associates (Derby and Atema, 1982) have been successful in doing this for the American lobster, and their combined efforts of neurophysiology and behavioral studies are of the sort that are needed for smaller planktonic animals. The task of scaling down the techniques to the size of a typical zooplankter, however, is formidable. Most zooplankton will prove to be difficult subjects because of their small size

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and fragility. It is unlikely that any zooplankters will become a standard physiological preparation for these reasons, and it should continue to be the rule that basic problems of physiology are best studied on the best preparation available. However, we anticipate that combined behavioral-neurophysiological studies on a few selected zooplankters will evolve as experience demonstrates the feasibility and as results demonstrate the utility of the approach. Several questions seem well suited to the neurophysiological-behavioral approach. The ability of the copepod Euchaeta to detect and capture live prey in contrast to its failure to respond to the same prey when freshly killed suggests that this predator responds to mechanical disturbances (Yen, 1982). What are the threshold levels of this response, and does the predator distinguish between the different types of mechanical disturbances generated by various prey types? This copepod is fortunately large enough to permit application of a suction electrode to a dissected first antenna, and to record nerve signals in response to various intensities and frequencies of mechanical disturbance. One could therefore mimic the beats ofa swimming prey species with the appropriate vibrator and determine the ability of the predator to sense the proximity of the prey. One area of controversy which might be addressed with a neurophysiological approach is the hypothesis of Legier-Visser et al. (1986) that herbivorous copepods detect single algal cells via mechano- rather than chemoreception. They proposed that mechanoreceptors on the first antennae initially detect an incoming cell and elicit a capture response, although chemosensors on the mouth-parts still could function in post-capture rejection ofpartic1es. An herbivore as large as Neocalanus could serve as a neurophysiological preparation for which the levels of sensitivity could be established for various concentrations of dissolved chemicals or complex food extracts. Those responses could be contrasted with frequencies and intensities of mechanical vibrations. These thresholds of mechano- and chemoreception could be compared with the magnitude of appropriate cues emanating from a phytoplankton cell, as well as to the typical intensity of background "noise" of dissolved chemicals and physical disturbances. Rather strangely, all the literature in which sensory mechanisms of copepods, as well as other crustaceans, are considered seems to deal with the antennae and cephalothoracic appendages. However, comparisons of copepod morphology with the literature on insect sensory physiology suggest questions concerning the utility of plumose caudal appendages. Why does Euchaeta carry elaborate plumes on its caudal furcae? And, at the extreme, what is the function of the beautiful caudal plumes borne by females of Calocalanus pavo and Calocalanus plumulosus? Studies on analogous caudal structures of the cockroach (Plummer and Camhi, 1981) demonstrate that these structures are mechanosensors that detect the bow wave of an attacking frog. The correspondence is obvious and inviting. The emphasis in this section on Crustacea reflects the degree of work done on this group, and does not necessarily imply a dominant ecological importance. Studies of both behavior and neurophysiology of, for example, salps and medusae have lagged behind those of crustaceans, primarily due to their fragility. However, the neurophysiology ofmusc1e contraction has been examined in the hydromedusa Aglantha (Mackie, 1980; Mackie and Meech, 1985), and some ofthe technologies used might be adapted for use in studying sensory physiology of gelatinous forms. Ciliates may also be an excellent group for application of these techniques. Ciliates have been employed for decades in many physiological studies, but recently an extensive literature has developed based on genetic, biochemical and biophysical approaches (Hinrichsen and Schultz, 1988). Ecologists should be able to draw

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upon the background of information at the molecular level to understand the functioning of ciliates in situ. MEASURING

ENERGETIC

COSTS OF BEHAVIOR

For zooplankton, as for all animals, there are energetic costs for the various behaviors mentioned in the above sections, such as moving the feeding appendages, handling and rejecting particles, migrating vertically or horizontally, escaping a predator, and following a chemical trail to find a mate. Most, if not all of these behaviors are necessary if the animal is to survive and reproduce, but the use of energy for these behaviors alters the energy balance, leaving less energy for growth and reproduction. However, we know very little about how energy is partitioned on a behavioral level or about the role of energetic costs in determining when and if certain behaviors will occur. Until recently, estimating the energetic costs of most behaviors has been difficult due to problems both in the design of the apparatus and in the design of the experimental protocol. Technical advances such as the ability to make in situ observations of larger zooplankton from submersibles or in mesocosms and the ability to track movement in 3-dimensions now make it possible to construct realistic time-behavior budgets. If these observations can be coupled with detailed laboratory studies on the energetic costs of specific behaviors, then the time budgets can be expressed in terms of energy as well as behavior. Techniques for evaluating the energetic costs of particular behaviors in crustacean zooplankton are currently under development by L. B. Quetin and R. M. Ross. For example, the cost of swimming in pelagic crustaceans is thought to be second only to that of basic metabolism. However, swimming is often intimately tied to feeding, as is the case in euphausiids and sergestiid shrimps. The relationships between swimming speed, filtration activity and oxygen consumption must be quantified to evaluate the energetic cost or savings of behaviors such as migration and schooling. The technical difficulties associated with determining the swimming energetics of pelagic crustaceans are much greater than those for fish. Fish can be trained to swim at a constant known speed in a small area of a tunnel set off by electrified screens (Goldspink, 1977). Pelagic crustaceans do not swim as consistently as fish and frequently end up plastered to the screens. Departures from constant swimming may be for only short periods of time but destroy the animal. An annulus with no screens solves some problems but has its own attendant difficulties. First, to eliminate wall effects associated with swimming in an enclosed space the ratio of the cross section of the tunnel to the cross section of the animal should be no less than 50 and preferably 100 (A. Knowles, pers. comm.). The difficulty arises because the volume of the annulus needs to be small enough to allow us to measure decreases in oxygen over short time periods. In a necessary compromise in previous studies on euphausiids (Torres et al., 1982; Torres and Childress, 1983) this ratio was 10 to 12. A solution to these design problems is an annulus with a tunnel to animal cross section ratio of about 50, in conjunction with a small stable oxygen sensor (Lucisano et al., 1987). This oxygen sensor does not consume oxygen and responds quickly to changes in oxygen concentration. Water is driven around the annulus by a plexiglas ring that is magnetically coupled to an external drive ring attached to a variable speed motor. The dominant feature of the circulation is forward motion, but the circulation pattern can be thoroughly mapped (LaBarbera and Vogel, 1976).

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The second difficulty concerns generating the necessary behavior in the laboratory. In this instance we want the zooplankter to swim at a fixed and predictable rate both singly and in schools. Schooling and swarming behavior can be generated in many species with appropriate current, lighting and substrate manipulations (O'Brien et aI., 1986). For example Euphausia superba will swim steadily in response to water velocities above about 3 em· S-1 when able to orient to moving stripes (Quetin and Ross, pers. obs.) and will school in appropriate lighting conditions (Hamner, pers. obs.). Energetic costs for specific swimming velocities and feeding behaviors could be combined with time budgets of these behaviors obtained from field observations or observation in large enough volumes to avoid wall effects to construct more realistic daily energy budgets. This approach yields more precise estimates of the costs of certain behaviors than does measurement of respiration rate while the animal swims at its own chosen rate which may vary over time. If behavior varies during the period that respiration rate is measured, then the rate is an average due to all the behaviors and rates of swimming observed. The acute effects of specific behaviors on respiration rate are lost. However, such an approach cannot be used for organisms that cannot be stimulated to perform specific behaviors or that need large volumes to avoid wall effects, like the gelatinous zooplankton, or for behaviors that are not continuous. In these cases, simultaneously measuring the metabolic rates over time and recording the behaviors may be the only way to measure the energetic costs of behavior. Experimental manipulation of the environment may alter the proportions of time spent on different behaviors, such as swimming vs. "resting," swimming at different speeds, etc., thereby allowing estimation of energetic costs. There are technical problems that need to be overcome in order to measure the oxygen consumption of gelatinous zooplankton or even copepods in large enough vessels so that wall effects on behavior are minimal. Promising developments in both polarographic oxygen sensors and in the techniques of measuring pH suggest that eventually we will be able to measure the metabolic rates of organisms in volumes which are large compared to the animal. Oxygen sensors are needed that are stable over the long term (Hale, 1983) and sensitive, give reproducible results and can be operated under conditions that do not influence the behavior of the animal. The micro-cathode electrode (Mickel et aI., 1983) and the pulsed oxygen electrode (Langdon, 1984) require little ifany stirring and are ideal for measuring respiration rates where vigorous stirring could disturb the activity of the organism. The pulsed oxygen electrode has the additional advantages of being 4 to 8 times more sensitive than electrodes with continuous input and of remaining driftfree for several weeks. Changes in the pH are another measure of metabolic rate, as the carbon dioxide evolved during metabolism alters the pH. However, until recently the necessary precision in measuring pH was not available. Some investigators already claim that modem instruments give pH to within 0.003 pH units. In addition a spectrophotometric procedure outlined in Byrne (1987) that gives rapid, precise quantification of a buffer's (seawater) relation to a standard may enable us to measure pH to within 0.00 1 pH units. These improvements in the sensors must be coupled with the improved technology in video cameras for 3D systems (see above) to allow us to compile realistic time budgets of behaviors in large volumes of water while simultaneously measuring the metabolic rate over time. Combining detailed laboratory studies of the energetic costs of behavior with time budgets of observed behavior also promises to help evaluate the role of energetics in the feeding behavior of smaller zooplankton. The cost of various behaviors could be estimated by simultaneously quantifying one or two behaviors and oxygen consumption under conditions where the time budget ofthe behaviors

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varies. For example, since feeding behavior of different species can be stimulated with a vibrating probe (Feigenbaum and Reeve, 1977) or various chemicals (Hamner et aI., 1983), an apparatus could be designed for simultaneously measuring oxygen consumption and the behavior time-budget of a tethered (one-behavior) or free-swimming (two-behaviors) animal. Also, since some copepods vary the proportion of time spent moving their feeding appendages and the rate of movement depending on algal concentrations (Price and PaffenhOfer, 1986), it may be possible to quantify energetic costs of increased feeding rates in free-swimming animals using chemical extracts. Since the concept of a lower food concentration threshold below which feeding stops is based on the idea that energy used to feed is greater than the energy gained during feeding at low concentrations, quantifying the cost is of great interest. CONCLUSION

Exploration of the behavior of individual zooplankters and populations under undisturbed environmental conditions is necessary to understand how various species exist and co-exist in freshwater and at sea. Inferences about behavior from a species' morphology, before-and-after grazing experiments in the laboratory and net tows in the field cannot replace and will not be as indicative as continuous observation of an animal's activity. Information on environmental variability on micro- and fine scales will be needed to interpret observed behaviors, as well as information on internal factors such as sensory physiology and energetic costs. However, we need adequate tools and facilities to obtain such information. To keep expenses as low as possible and to make technological advances available to a large number of investigators, national research centers could be equipped to allow studies on certain aspects of zooplankton behavior. Each center would have a great depth of expertise in its specific field of application (e.g., submersible observations, mesocosms). This expertise would be readily available to zooplankton researchers who would like to conduct behavioral studies but would otherwise need vast resources and time to develop the needed technology. LITERATURE

CITED

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--and L. B. Quetin, 1982. A pressure vessel for the simultaneous determination of oxygen consumption and swimming speed in zooplankton. Deep-Sea Res. 29: 631--639. Townsend, D. W. and L. M. Cammen. 1985. A deep protozoan maximum in the Gulf of Maine. Mar. Ecol. Prog. Ser. 24: 177-182. Verity, P. G. 1988. Chemosensory behavior in marine planktonic ciliates. Bull. Mar, Sci. 43: 772782. Widder, E. A., S. A. Bernstein, J. F. Case and B. H. Robison. 1986. Bioluminescence patchiness, scaling and background based on image analysis of video recordings from a midwater submersible. EOS 67: 994. Abst. Wilson, R. S. and J. O. B. Greaves. 1979. The evolution of the bug system: recent progress in the analysis ofbio-behavioral data. Pages 251-272 in F. S. Jacoff, ed. Advances in marine environmental research. USA EPA (EPA-600/9-79-035). Wood, J. W. and G. W. Potts. 1987. Low-light level video system for use in underwater research. Int. Underwater Sys. Des. 9: 22-26. Yen, J. 1982. Sources of variability in attack rates of Euchaeta elongata Esterly, a carnivorous marine copepod. J. Exp. Mar. BioI. Ecol. 63: 105-117. ---, G.-A. Paffenhofer, T. G. Bailey and P. J. Davoli. 1987. Massive bloom of the colonial, gelatinous diatom Thalassiosira subtilis in upwelled waters: distribution, abundance, productivity and fate: a submersible study. EOS 68: 1724. Abst. --, T. G. Bailey, P. J. Davoli, C. A. Jacoby, P. I. Blades-Eckelbarger and G. A. Griswold. In Press. Epibenthic krill impact particle fluxes and food webs: detection by submersible. NOAA Symp. Series for Undersea Res. 6. Youngbluth, M. J. 1984. Manned submersibles and sophisticated instrumentation: tools for oceanographic research. Pages 335-344 in Proceedings of SUBTECH 1983 Symposium, Soc. Underwater Tech., London. DATEACCEPTED: May 9, 1988. ADDRESSES: (H.J.P.) Dalhousie University, Dept. of Oceanography, Halifax, NS B3H 4Jl, Canada; (G.-A.P.) Skidaway Institute of Oceanography, P.O. Box 13687, Savannah, Georgia 3/416; (C.M.B.) Dalhousie University, Dept. of Oceanography, Halifax, NS B3H 4Jl, Canada; (T.J.C.) College of Oceanography, Oregon State University, Corvallis, Oregon 97331; (P.L.D.) Graduate School of Oceanography. University of Rhode Island, Narragansett, Rhode Island 02882; (W.M.H.) Dept. of Biology, University of California, Los Angeles, California 90024; (W.L.) Max-Planck-Institutfiir Limnologie, Postfach 165, D-2320 PIon, Germany (FRG); (L.B.O. & R.M.R.) Marine Science Institute, University of California. Santa Barbara, Santa Barbara, California 93106; (J.R.S.) Boston University Marine Program, Marine Biological Laboratory, Woods Hole, Massachusetts 02543; (M.J. Y.) Harbor Branch Oceanographic Institution. 5600 Old Dixie Highway, Ft. Pierce, Florida 33450. PREsENTADDRESS: (H.J.P.) Monterey Bay Aquarium Research Institute, 160 Central Avenue, Pacific Grove, California 93950.