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On the spatial distribution and nearest
neighbor distance between particles
in the water column determined from
in situ holographic measurements
E. MALKIEL1, J. N. ABRAS1, E. A. WIDDER2 AND J. KATZ1*
1
DEPARTMENT OF MECHANICAL ENGINEERING, JOHNS HOPKINS UNIVERSITY, 223 LATROBE HALL, 3400 N. CHARLES STREET, BALTIMORE, MD 21209, USA
2
AND BIOLUMINESCENCE DEPARTMENT, HARBOR BRANCH OCEANOGRAPHIC INSTITUTION, FORT PIERCE, FL
*CORRESPONDING AUTHOR:
34946, USA
[email protected]
Received July 11, 2005; accepted in principle September 21, 2005; accepted for publication November 21, 2005; published online November 30, 2005
Communicating editor: K. J. Flynn
A film-based holography system was used in conjunction with instrumentation that detected
bioluminescent thin layers to record the spatial distribution of zooplankton and their prey in the
Gulf of Maine, USA. The holocamera and instruments were mounted on the Johnson Sea Link
(JSL) in a setup that minimized the disturbance to the sample volume. More than 143 holograms
were automatically scanned to provide focused images of 5000–10 000 particles and their threedimensional coordinates in each 894 cm3 sample. The reconstructed volumes provided clear images
of intermingling copepods species, nauplii, Pseudonitzschia diatoms and particles in the 10 m–5 mm
size range. Spatial analysis of the nearest neighbor distance (NND) of the smallest particles showed
a random distribution, but detritus particles showed small-scale clustering in regions below
the pycnocline. A detritus maximum, found several meters below the pycnocline, at 20–30 m, was
determined to be caused by fecal pellets in various stages of degradation. This region also contained
elevated concentrations of calanoids, cyclopoids and harpacticoids. In one third of the cases, the
harpacticoids, Aegisthus sp, were attached to detritus.
INTRODUCTION
The current understanding of biological processes in the
open ocean is limited by our ability to observe and
measure processes as they occur. Recent studies have
demonstrated that plankton population dynamics
depend on the growth, reproduction and mortality that
occur in transient-localized patches, presumably because
the concentration of resources (nutrients, food, conspecifics) within patches is greater than in outlying
areas (Lasker, 1975; Mullin and Brooks, 1976; Munk
and Kiorboe, 1985; Cowles, 1998; Folt and Burns,
1999; Leising, 2001; Clay et al., 2004). Some of these
patches are <1 m thick, yet they may extend horizontally
for hundreds or even thousands of meters and can dissipate within hours or last as long as days (Dekshenieks
et al., 2001).
Traditional sampling methods such as net tows have
difficulty resolving spatial variations in concentration at
scales of <5 m and, therefore, underestimate the actual
concentrations in thinner patches. Consequently, a series
of increasingly sophisticated instruments have been
introduced in recent years to study the causes for the
formation and dissipation of these patches. Advances in
acoustic and bathyphotometric profilers (Holliday et al.,
1998; Widder et al., 1999) have greatly enhanced our
ability to locate concentrations of the larger zooplankton, while optical absorption profiling (Twardowski et al.,
1999) and two-dimensional fluorometry (Franks and
Jaffe, 2001) have aided in identifying chlorophyll maxima, indicating potential food concentrations for herbivores. Underwater video observations (Davis et al., 1996;
Tiselius, 1998) have been used to make zooplankton and
plankton counts, occasionally to species level, when
doi:10.1093/plankt/fbi107, available online at www.plankt.oxfordjournals.org
Ó The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
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supplemented with ocean truthing based on net tows
(Benfield et al., 1996). These methods can provide
detailed spatial correlations of in situ species concentration, which can be used to infer trophic dynamics.
Examination of the interaction of zooplankton with the
surrounding particle field requires measurements of the
three-dimensional spatial distribution of the plankton in
the immediate vicinity. Holography is the only technology
capable of achieving this goal, especially when the volume
of interest contains >100 000 particles (stereo and multicamera photography can track the three-dimensional
motion of up to 1000 particles). Unlike microscopy, holography maintains the same lateral resolution over a substantial depth of field (Vikram, 1992). This advantage has
led to the development of several submersible holography
systems, starting with a sample volume of a few milliliters,
to samples of 1 L and above (Carder et al., 1982; O’Hern
et al., 1988; Katz et al., 1999; Watson et al., 2001). In this
article, we use a submersible holography system to profile
the particle distributions and interparticle distances in
the Gulf of Maine, USA. The in situ measured threedimensional spatial distributions of various particle species
enable us to clarify predator–prey relationships in the natural
environment and elucidate particle aggregation processes.
The goal of the field study was to gain an understanding of the biological and physical factors that govern
zooplankton patchiness in the ocean. This joint effort
involved simultaneous deployment of instruments developed at the Harbor Branch Oceanographic Institution
(HBOI) and at The Johns Hopkins University (JHU).
The test site was selected based on results of prior field
studies reported in Widder et al. (Widder et al., 1999),
which detected the existence of thin layers with high
concentrations of the bioluminescent copepod Metridia
lucens in the Gulf of Maine. The bioluminescence imaging
system utilized in these studies was developed for locating
regions with high levels of bioluminescence and recording
the associated displays. In the study reported here, these
instruments were used in conjunction with the JHU holocamera to detect bioluminescent thin layers and guide site
selection for recording holograms. Both the bioluminescence imaging system and the holocamera were mounted
on the top forward section of an untethered manned
submersible, the Johnson Sea Link (JSL; Fig. 1A), described
in Liberatore et al. (Liberatore et al., 1997).
METHOD
Bathyphotometer and Spatial Plankton
Analysis Technique system
The HBOI equipment provided a means of locating high
concentrations of bioluminescent species in two steps, as
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described in detail by Widder and Johnsen (Widder and
Johnsen, 2000). First, a high-intake defined-excitation
bathyphotometer (HIDEX-BP) (Widder et al., 1993) was
deployed from the ship to obtain vertical profiles of stimulated bioluminescence along with temperature, conductivity and fluorescence. Then, target regions of high
bioluminescence intensity were probed with the JSL
submersible using the Spatial Plankton Analysis Technique (SPLAT) system (Widder and Johnsen, 2000). The
SPLAT screen was mounted on the upper work bar of
the JSL and can be seen on the left side of Fig. 1B. It
consisted of a 60 80 cm screen aligned perpendicularly
to the flow during forward motion in order to stimulate
bioluminescence. Its center was displaced 2 m laterally,
0.75 m forward and 0.5 m below the sample volume of the
holocamera. The occurrence and patterns of bioluminescence on this screen were recorded with an intensified
video camera. The plankton pump system (Critter Getter)
that is usually used on the JSL to pump plankton samples
simultaneous with video transects could not be accommodated on this mission because of the weight of the holocamera. In order to collect plankton samples from discrete
depths of interest, a 335-micron mesh plankton net was
attached to the exhaust of the HIDEX, and samples were
pumped for 5 min at 18 L s1. The plankton in these
samples were enumerated for comparison with abundance
estimates from holograms recorded at the same depths.
Holocamera
A sketch of the optical elements of the in-line submersible holocamera is presented in Fig. 1C, and details are
available in Katz et al. (Katz et al., 1999) and Malkiel
et al. (Malkiel et al., 1999, 2004). A spatially filtered and
collimated ruby laser beam illuminates the sample
volume. The interference pattern between the light scattered from the particles and the remaining (undisturbed)
part of the beam is transmitted through relay lenses and
recorded on 70-mm high-resolution holographic film.
These optical elements were mounted on top of the
JSL, at the same forward position as the SPLAT system,
but on the opposite side (Fig. 1B). This arrangement was
chosen in order to minimize the effect of the bulk of the
submersible on the fluid/flow in the sample volume
during forward or upward transects. The rest of the
components, including the control and power supply
electronics, were mounted in front of the submersible
in place of a robotic collection device, in order to minimize the pitch of the JSL during the recovery procedure,
when the optical system was located above the waterline.
The holocamera was designed to decrease the disturbance to the sample volume by positioning the sample
volume, a 287 mm long, 63 mm diameter cylindrical
region (894 cm3), between two port windows located at
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Fig. 1. Holocamera mounted on top of Johnson Sea Link (JSL) (A) oblique top view, (B) front view and (C) schematic view of optical components.
the tops of streamlined fairings (89 cm above the base).
In order to avoid disturbance of the sample volume, all
the holograms discussed in this article were recorded
during buoyancy driven, slow vertical ascents or during
powered (1 Kt) forward transects.
In the configuration shown in Fig. 1, the holocamera
was capable of recording 400 holograms per dive at a
maximum rate of 1 hologram (single or double exposure)
every 30 s. The system performance was monitored and
controlled, including the selection of timing for exposures,
by an operator sitting in the rear compartment of the JSL.
A laptop connected to the holocamera through an RS232 underwater link provided the means of communication. The timing of data acquisition was coordinated by
the operator/scientist sitting in the forward compartment
that could observe the SPLAT screen and direct the pilot.
In addition to the holograms, the holocamera contains
a Seabird, Seacat CTD, which was encased in the vertical fairings over the film drive. The CTD-analysed
water samples were pumped from a port located about
10 cm downstream from the camera window. The samples were also passed through an optical transmissometer
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(660 nm; 25 cm path length) and a dissolved oxygen
sensor. Data were also available for comparison from
another CTD, which was mounted on the side of the
JSL. The latitude and longitude locations of the submersible were recorded with a shipboard Trackpoint system.
Site and transect procedures
The dives detailed in this report took place in the Gulf of
Maine, at Wilkinson basin (42 380 N, 69 360 W), on 3–4
July 2000. Although the basin extends to a maximum
depth of 260 m, most of the holograms were recorded in
the euphotic zone. In this article, we report on the results
obtained in three dives. Two of them (dives 3249 and
3251) were performed at night (10:00 PM–1:00 AM,
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eastern daylight time [EDT]), and one (dive 3250) was
performed during the day (1:00 PM–3:00 PM). During
the night dives, bioluminescent layers were located using
real-time sensor feedback from the intensified video
(SPLAT) recordings. The submersible was trimmed to
neutral buoyancy just below a layer and then motored
forward to move out from under any water disturbed during the descent. A minimum amount of positive trim was
then applied, allowing the sub to drift up very slowly
through the layer, while a series of holograms were collected
in rapid succession. Alternatively, the submersible was held
at a constant depth, and holograms were recorded during
horizontal transects. The depth and order of all the holograms recorded during the three dives are shown in Fig. 2,
Fig. 2. Depth locations of recorded and analysed holograms for dives 3249–3251 (A–C).
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including indications on the number of exposures and
whether the data have been analysed. The vertical profiles
provided data on the plankton distribution throughout the
water column, whereas multiple holograms recorded at
several specific depths provided better sample statistics
and enabled us to examine the horizontal spatial variability.
Using the SPLAT system, the horizontal transects enabled
us to identify bioluminescent species present at a certain
depth.
Data analysis procedures
The exposed holographic film was removed from the
camera chamber after each dive and developed onboard
the ship. Each hologram could be reconstructed either
optically or numerically onboard to provide detailed, infocus images of the plankton throughout the sample
volume. The numerical reconstruction procedure,
detailed in Milgram and Li (Milgram and Li, 2002),
digitized the hologram using a scanner and then reconstructed specific regions of interest (ROI) at any desired
depth within minutes. The optical reconstruction method
consisted of placing the holograms in front of an
expanded and collimated He–Ne beam, which created
a three-dimensional image field of the original sample
volume. This reconstructed field could be then scanned
at high speed with a digital/video camera mounted on a
three-dimensional traversing system. Figure 3 demonstrates the resolution obtainable from optical reconstruction throughout the sample volume. Bristles with a
diameter of <3 mm on the setae of Calanus finmarchicus,
located 185 mm from the film plane (after being relayed
by the relay lenses, Fig. 1C), were clearly discernible.
Discrete spherical objects are more difficult to distinguish
from the background speckle noise and had to be at least
10 mm in diameter in order to be detected (Malkiel et al.,
1999).
Back in the laboratory, the central 20 5 5 cm3
part of each reconstructed hologram was scanned at a
resolution of 3.59 mm/pixel and at 1 mm intervals in
depth. Thus, each reconstructed volume was converted
to 200 planes, each with 14K 14K pixels. (This gives a
total size of 39.2 GB, but each scan was actually only 35
GB due to the exclusion of corner regions completely
outside the circular field of interest.) The images were
acquired using a 1K 1K 30 frames s1 (fps) digital
camera equipped with a microscope objective and
mounted on a precision three-dimensional motorized traversing system. Although the real sample volume extends
>243 mm that 20 cm of reconstruction represents, the
analysis avoided regions located in the immediate vicinity
of the windows (the 22 mm closest to the windows), which
included the boundary layer of the instrument. We also
avoided the outer perimeter of the reconstructed volumes
Fig. 3. Sample image of copepod and views of setal bristles. Scale in
panel A is 1 mm. Scale in panels B and C is 0.1 mm.
due to a decrease in resolution as a result of spherical
aberrations caused by the relay lenses.
Automated procedures were developed for acquiring,
processing and analysing the scanned images. Automation was essential for obtaining meaningful statistics from
the enormous digitized database, 5 TB for the 143
holograms analysed to obtain the particle distributions
discussed in this article. Details on the automated system, methods and associated procedures are presented
elsewhere (Malkiel et al., 2004). Here, we only briefly
summarize the type of data obtained and saved for
subsequent analysis. Each reconstructed volume typically contained 5000–10 000 particles. The automatic
scanning and analysis process found the particles and
recorded their in-focus images, their three-dimensional
coordinates and their sizes. The uncertainty in directions
perpendicular to the optical axis of the hologram was
about equal to the pixel resolution, i.e. 3.6 mm, whereas
the uncertainty in coordinates in the axial direction
was 100 mm. The entire scanning process took 4–6 h
per hologram and was performed without human
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intervention. After the analysis was completed, the images
of particles >50 mm, typically several hundred per
hologram, were examined and classified manually into
several groups (details follow). The copepods were
classified with the help of Trevor Myslinski at HBOI,
USA. For copepods >1 mm, we also recorded the threedimensional coordinates of some of their body parts
(e.g. antennae, head, tail), enabling us to determine
their orientation. The substantially more numerous
particles <50 mm were automatically relegated to the
classification of dinoflagellates, as discussed below.
ctenophore displays between 30 and 40 m. During the
course of the dive, the ctenophore displays spread into
the upper part of the water column, between 15 and 45 m,
while the copepod displays were concentrated between
45 and 55 m. A similar pattern was observed the next
night for dive 3251 where, during the initial part of the
dive, copepod displays were seen throughout the water
column between 10 and 60 m, and ctenophore displays
were concentrated between 35 and 45 m. By the end of
the dive, the ctenophore displays were seen between
10 and 50 m, and copepod displays were concentrated
below that.
RESULTS
Types and images of particles
Bioluminescence
A profile of stimulated bioluminescence made at 21:40,
50 min prior to the beginning of dive 3249 (Fig. 4A)
revealed bioluminescent thin layers associated with the
fluorescence peak, with the two brightest layers at 34
and 48 m located below the fluorescence maximum at
28 m. Another profile made at 2:50, 35 min after the
dive concluded, revealed a thin layer of intense bioluminescence associated with the fluorescence maximum at
46 m and a broad peak between 100 m and the deepest
depth of the profile at 150 m (Fig. 4B). The following
night a profile taken at 20:40, 50 min prior to dive 3251,
revealed thin layers of bioluminescence <100 m and
very little luminescence associated with the fluorescence
maximum (Fig. 4C). The profile taken 45 min after the
conclusion of dive 3251 at midnight revealed bioluminescent thin layers associated with the fluorescence peak
between 37 and 65 m, as well as at 118 m and between
240 and 250 m. There was also a broad peak of luminescence between 115 and 185 m (Fig. 4D). On both
nights, sunset was at 20:42, and the end of civil twilight
at 21:07. (‘End of civil twilight’ is defined as the point
when the center of the sun is 6 degrees below the
horizon, and skylight has dimmed to the point where
the brightest stars are visible under optimum atmospheric conditions.) Moonset on the night of dive 3249
was at 22:39, and the moon was a waxing crescent with
5% of the visible disk illuminated. Moonset on the night
of dive 3251 was at 23:29, and 11% of the visible disk
was illuminated.
SPLAT CAM recordings made during the dives
revealed that the primary sources of luminescence were
secretory displays characteristic of copepods (probably
the calanoid, M. lucens, and harpacticoid, Aegisthus sp.)
and the ctenophore, Euplokamis sp. (Widder et al., 1999).
Near the beginning of dive 3249, both ctenophore and
copepod displays were seen throughout the water column, between 8 and 50 m with the densest layer of
Three classes of copepods, belonging to the groups of
calanoid, cyclopoid and harpacticoid were present in the
reconstructed hologram volumes that were analysed.
Sample images of the predominant species along with
nauplii are shown in Fig. 5. Other than copepods, particles >50 mm were almost invariably detritus, examples
of which are shown in Fig. 6. Detritus particles were
classified either as fecal pellets from copepods and
euphasiids (Fig. 6A–D) or as microaggregates (Fig. 6E–G).
The latter classification was applied to amorphous
particles and agglomerations >50 mm. Many of the
detritus particles, e.g. Fig. 6D, were at least partially
outlined by a bright frilly edge. This appears to be the
signature of extracellular polymeric substance (EPS)
when it is recorded using in-line holography. Black
et al.’s (Black et al., 2001) in-line holographic recordings
of Xanthum gum (bacterial EPS) particles suspended in
water and attached to sand grains show such characteristics. Composed primarily of polysaccharides, EPS can
be produced by bacteria (Evans, 2000), attached to
detritus during the degradation process. Figure 6B is
one of numerous examples in our holograms that show
a fecal pellet with a partial frilly edge, presumably at the
onset of the degradation process consistent with the
observation that fecal pellets are typically host to large
bacterial colonies (Delille and Razouls, 1994; Hansen
and Bech, 1996; Thor et al., 2003). When found free
floating, particles of EPS have been referred to as microgels or transparent exopolymer particles (TEP) and may
also refer to diatom exudates having a similar nature
(Hoagland et al., 1993; Passow et al., 1994). Although it is
usually necessary to stain a filtrate in order to visualize
these particles (Passow and Alldredge, 1995), in-line
holography, being sensitive to slight variations in refractive index, is able to record images of these particles in
the laboratory (Black et al., 2001) or in situ, as shown in
Fig. 6E–G.
There were primarily two types of phytoplankton in
the recorded volume: dinoflagellates and colony-forming
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Fig. 4. Profiles of bioluminescence and florescence before and after night dives 3249 (A and B) and 3251 (C and D) obtained with the HIDEX
system.
diatoms The diatoms (Fig. 7) were identified as belonging to the Pseudonitzschia family, which species form
chains of partially overlapping slender (3–8 mm diameter; 33–160 mm long) cells (Tomas and Hasle, 1997).
The reconstructed images of the diatom colonies showed
that the cell diameters were <7 mm, but the background
was too noisy to determine the length of individual cells.
However, despite this, overlap of connecting cells could
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Fig. 5. Samples of the predominant copepods observed in sample volume. Calanoids: (A) Metridia lucens and (B) Calanus finmarchicus; cyclopoids: (C)
Oithona spinirostris and (D) Oithona similis; harpacticoids: (E) Aegisthus sp. and (F) various nauplii. Scale in A–D is 1 mm. Scale in E–F is 0.1 mm.
be inferred from the absence of a strong diffraction
pattern, which was found to form in out-of-focus reconstructions of colonies that had spaces between adjacent
cells (e.g. Skeletonema). Diatom chains also presented a
challenge for the automated scanning system. Owing to
their length (typically 25 mm), only small portions of a
chain would come into focus in certain scanned planes.
Thus, images of these diatom chains were difficult to
acquire automatically, and we made no effort to combine data obtained in different planes. Rather, small
subsections (32 mL) of 15 holograms spanning the
upper 60 m of dive 3249 were analysed manually. The
vertical distributions of Pseudonitzschia are shown in the
inset to Fig. 7. Each bar represents a different hologram,
including those >40 m that do not contain any diatoms.
The Pseudonitzschia appeared to be confined to the <40 m.
Subsequent day and night dives have similar distributions
(data not shown). In a number of cases (<10 %), as
demonstrated in Fig. 7B, the diatom chains were attached
to detritus. These chains were perhaps precursors of
larger flocs, but such large flocs were not present in the
holograms analysed to date.
Round isolated objects <50 mm, examples of which are
shown in Fig. 8, were classified as dinoflagellates,
although admittedly the resolution was not sufficient to
be certain that they were not microzooplankton or even
just small bits of detritus. We did not use net tows with a
fine enough screen mesh and did not collect water samples during the field tests that could have helped in
identifying the small particles. The focus of the cruise
was on the distribution of the larger copepods, and the
water sampling mechanism on the JSL was removed to
make room for the electronics of the holocamera. Ceratium
species (Fig. 8C), because of their prominent horns, were
the only dinoflagellate species that were clearly identified.
Statistics on vertical and size distributions
Figure 9 shows the distribution with depth of the particle
classes mentioned above for each of the three dives.
Each point represents the number of particles per unit
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Fig. 6. Detritus in various states of degradation. Fecal pellets from
copepods (A), euphausiids (B and C) and with transparent exopolymer
particles (TEP) (D). Panels E–G are microaggregates of more severely
degraded detritus. Scale is 0.1 mm.
volume detected in an individual reconstructed threedimensional field. The actual number of particles can
be calculated by multiplying the concentration by the
sample volume, 500 cm3. For reference, we also provide the vertical distributions of salinity, depth and temperature for each of the dives. The detritus, composed
mostly of microaggregates and fecal pellets, appeared
over the entire depth, but had the highest concentration
in the region just below the pycnocline, mostly due to the
increased number of fecal pellets. To highlight these
trends, we present both the entire detritus group and
then separate it into marine snow and fecal pellets.
The copepods, especially the cyclopoids, concentrated
near and above the pycnocline during the night and
reached levels as high as 22 copepods L1. During the
day, the cyclopoids were not located at the pycnocline
and were more or less spread uniformly in regions deeper than 20 m (note that we do not have day data from
above the pyncocline). The calanoids, mostly comprised
of C. finmarchicus, showed only a slight preference for the
region near or above the pycnocline during the night, at
the same elevation as the peak in the cyclopoids. However, they were still preferentially located in the region
just below the pycnocline during the day. The several
specimens of M. lucens identified in the sample volumes
were found at 6, 49 and 54 m during the first night. The
net data summarized in Table I confirms that the
concentration of the copepods (except for C. finmarchicus
on the first night at 44-m depth) was usually much
<0.1 L1. As will be discussed later, the occurrence of
the Metridia species was too sparse for meaningful statistics. The depth average abundance of other copepods in
our samples (suggested by the trendlines in Fig. 9)
appeared to be almost an order of magnitude greater
than what was collected in the net. The nauplii appeared
at all depths but were concentrated mostly near and
above the pycnocline during the first night dive. During
the day, they were concentrated below 40 m and almost
disappeared from the shallower water. The nauplii reappeared over the entire water column during the second
night dive, but their numbers still peaked below 40 m, at
the same elevations as the day peak.
Further insight into the behavior of the copepods can
be obtained by calculating for each of the copepod
species Lloyd’s index of patchiness (Lloyd, 1967; Pielou,
1973), defined as m*/m where m is the overall mean
number of individuals per sample volume (concentration) and m* is the mean concentration that an individual sees in a sample volume (excluding itself). This
index is widely used as a measure of the mean clustering
of individuals compared with random distributions. In a
purely random situation, where the statistics of finding
an individual in a sample follows a Poisson distribution,
the index of patchiness is one. In the more general case,
the probabilities of a certain concentration can follow a
negative binomial distribution, and the index can be
higher or lower than one. The results, computed using
the efficient fitting method of Bliss and Fisher (Bliss and
Fisher, 1953) for negative binomial distributions, are
presented in Fig. 10. For reference, each graph also
shows the number of holograms and copepods found
and used in the analysis. All the groups had aggregation
indices larger than one, indicating clustering. For one of
the night dives, the harpacticoids were aggregated 6.5
greater than random, but due to the low number of
individuals found, this result does not have a high significance level. In all of this article, we assume that a result
is significant if P < 0.05 in a two-tailed hypothesis test
(where P is the likelihood that this outcome is a result of
a standard variation of a population without aggregation
in this discussion). The cyclopoids, mainly consisting
of the species Oithona similis, were the only group with
a sufficient population to show statistically significant
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Fig. 7. (A) Pseudonitzschia diatoms brought into focus by combining images from multiple depths. (B and C) Detritus attached to the diatom chain.
Inset in panel C: depth distribution of Pseudonitzschia in dive 3249. Scale is 1 mm.
aggregation during the night. The aggregation did not
seem to persist as strongly, and the total number of these
copepods was much smaller during the day. The total
number of calanoids at night was 50% smaller (than
that of the cyclopoids), and the aggregation index
decreased to <2, but clustering still existed in all three
dives. The nauplii also tended to cluster but not at a
significant level.
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120- to 180-mm range, the concentration appeared to be
constant down to a depth of 30 m and then decreased
and remained at a lower level at greater depths. The
largest particles seemed to have a concentration maximum in the 20- to 30-m depth range, i.e. just below the
pycnocline. This peak is most evident when we restrict
our attention to the night data but persists in the day as
well (day data are not shown).
Nearest neighbor distances
Fig. 8. Example of unknown dinoflagellates (A and B) and of a
Ceratium tripos (C). Scale is 0.1 mm.
To compare the size distributions of particles to those
presented in the literature, we calculated the equivalent
diameter of the particles from the area of the threshholded image [de ¼ ð4A=Þ0:5 ]. The resulting instantaneous size distribution, calculated from the data
obtained in one of the reconstructed images, is presented
in Fig. 11A. Clearly, the small particles dominated the
total number. The existence of a peak at 30 mm with a
slight decrease in concentration at smaller sizes may be
affected in part by a reduction in the strength of signal as
the system reached its resolution limit (10 mm). However,
the peak was in the same range as many dinoflagellates,
including the red tide species Alexandrium tamaranse that
bloomed 60 miles up the coast (in the upstream direction of the Eastern Maine Coastal Current), 2 weeks
before our deployment (Thomas et al., 2003). For sizes
>30 mm, the decrease is incontrovertible, and the probability density up to 100 mm seems to be proportional
to de3 A least-squares power fit to this range,
n ¼ 2:7de2:97 , is also shown on the graph. For larger
sizes, the number of particles per individual hologram
was too small to be statistically accurate. Other reconstructed volumes (not shown) had similar trends. The
overall size distribution, composed from all the data of
the first night (Fig. 12), shows similar trends. When the
data in Fig. 10A are replotted as a volume distribution,
with Ve ¼ de3 =6, Fig. 11B clearly show that most of the
volume, a proxy for biomass, appears to be in classes
>100 mm. Jackson et al. ( Jackson et al., 1997), in a
simultaneous comparison of in situ particle sizing techniques, found similar composite spectra.
To examine the effect of depth on the characteristic
size of particles, one can divide the data into several size
ranges and plot their vertical distributions. The results
for the two night dives are presented in Fig. 12. As
is evident, particles <60 mm had a relatively uniform
vertical distribution. In the 60- to 120-mm range, there
was a slight concentration decrease with depth. In the
One of the goals of this project was to find possible
reasons that cause the copepods to aggregate in certain regions/depths. The large number of detected
dinoflagellates, which are a potential food source as
suggested by the literature (Mauchline, 1998), led us
initially to explore the possibility that the aggregation
was related to variations in the concentration of
dinoflagellates. The data however, especially Figs 9 and
12, indicate that unlike the copepods, the dinoflagellates
(or other small particles) were more or less distributed
uniformly in depth. For the more numerous particles, i.e.
dinoflagellates and detritus, one can use the data to
calculate the nearest neighbor distances (NNDs) between
particles over the sample volume. When the measured
NND is compared with that of randomly distributed
particles, the results provide a statistically robust method
for determining clustering trends. Conditional sampling
can then be used for examining the effect of environmental parameters, including presence of other particles,
on the spatial distributions of a certain object (Diggle,
1983).
Figures 13 and 14 compare the measured distributions
of NND with those obtained from ensemble averages of
randomly distributed particles. The random distributions were created by using a random number generator
to specify the coordinates of particles, while maintaining
the same mean concentration (total number of particles/
volume) as the measured data. To make the comparison
as accurate as possible, the simulated volume had exactly
the same shape and size as the scanned volume and
included only regions where the camera could possibly
detect particles, i.e. eliminating regions that were
blocked by the aperture of the hologram or by larger
particles. In the case of dinoflagellates, the process
included masking out portions of the reconstructed
volume, over the entire depth, that were obscured
by the diffraction pattern caused by the presence of
copepods and/or detritus. In the case of detritus, only
the regions directly behind or in front of copepods
were masked. The simulated dinoflagellate distributions
were further refined to account for spatial variations in
the measured concentration of small particles due to
slight reductions in image quality at the edge of the
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Fig. 9. Depth distribution of particles according to classification and associated CTD data for night dives 3249 (A) and 3251 (C) and a day dive 3250 (B). Filled diamonds represent pumped net
data.
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Table I: Copepod concentrations (N/m3) from 5400-L pumped net samples taken before
and after night dives
Date
4 July
4 July
Time
4:00
3:00
20:00
19:30
2:00
1:30
57
44
57
44
57
Depth (m)
Metridia lucens
44
4 July
4 July
5 July
5 July
9.1
6.5
55.2
34.4
8.7
176.3
22.6
16.7
25.0
22.6
31.1
Oithona sp.
0.0
14.6
28.3
14.6
14.6
19.8
Macrosetella sp.
0.0
0.2
0.0
0.0
0.0
0.0
Calanus finmarchicus
27.6
Fig. 10. Aggregation indices for the major groups of copepods, separated according to dive. Open bars indicate the day dive and hatched bars
the night dives. Error bars indicate 1 SD of this statistic.
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Fig. 11. Instantaneous size (A) and volume distribution (B) of particles in midst of detritus layer at a depth of 28.7 m, during a night dive.
Fig. 12. Variations of the number of particles with depth in several
size ranges for the two night dives 3249 and 3251. Each point represents a different hologram.
Fig. 13. Probability density function of the measured nearest neighbor distance (NND) between dinoflagellates (and other small particles)
as compared with simulation using randomly distributed particles with
the same mean concentration (dive 3249).
reconstructed volumes and at large distances from the
film plane (for details on resolution, see Malkiel et al., testing procedure (Freedman et al., 1980). This method is
2004). There was no need to apply the latter procedure based on calculating the ‘z score’, defined as
on the distribution of the (larger) detritus particles.
Figure 13 suggests that the distribution of NND of the
Xs Xr
z ¼ pffiffiffiffi
ð1Þ
dinoflagellates does not appear to differ significantly from
r N
that of the randomly distributed particles. For the detritus,
we provide sample distributions of NND (Fig. 14), one at a
depth of 36.5 m showing a marked deviation from the where X s is the mean NND of a sample, N is the
random simulation (Fig. 14A), while the other, at 5.6 m, number of particles in the sample, while X r is the
shows very little difference. To quantify the deviation from mean and r is the SD of the NND obtained from
the random distribution, we used a two-tailed hypothesis the random simulation. For a result to be statistically
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Fig. 14. Probability density function of nearest neighbor distance (NND) between detritus as compared with simulation using randomly
distributed particles with the same mean concentration at a depth of (A) 36.5 (dive 3251) and (B) 5.6 m (dive 3251).
significant, the z score should have a magnitude >2,
corresponding to probability of <5% (P < 0.05) that a
certain phenomenon happens by chance. A z > 2
indicates high likelihood of regular (spatially uniform)
mean distance between particles, whereas z < –2
indicates clustering. Vertical distributions of the z
scores for the two night dives are shown in Fig. 15.
Clearly, there was significant clustering of detritus in
many of the samples recorded at a depth of 20 m and
below. The trend is clearer in the cumulative z score
for each depth calculated by
M
P
M
P
Ni ðX s X r Þi
Ni
i¼1
zm ¼ i¼1
0:5
M
P
ð2r =N Þ1
i
ð2Þ
i¼1
where M is the number of samples at each depth. It is the
weighted average X s X r of all of the samples at a
depth divided by the standard error of this set. Clustering
Fig. 15. Cumulative nearest neighbor distance (NND) z scores for dives 3249 (left) and 3251 (right). Open symbols, based on instantaneous
distribution in a single hologram. Closed symbols, zm [equation (2)], the cumulative z score based on all samples at a particular depth.
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was evident at 20 m and below and at 13 m and below
for the first and second night dives, respectively. Note
that the center of the pycnocline (Fig. 9) also shifted
from 18 m in the first night to 13 m on the second
night. Thus, clustering of detritus only occurred at and
below the pycnocline. We return to these results in the
next section.
The NND between individuals within a class provides
statistically meaningful results only if the class has a sufficient number of particles. Because of the small number of
copepods within each sample, there was no value in calculating the NND between copepods. However, one can
calculate the statistics of the distance between copepods
and other densely populated particles in the sample
volume, i.e. dinoflagellates and detritus. With respect to
the dinoflagellates, their concentration in the vicinity of
calanoid copepods, defined as a 5 mm diameter, 20 mm
long cylindrical region surrounding the copepod, was
hardly different (by 1–2% at most) from the expected
concentration. Thus, the calanoid copepods did not show
a tendency to be located in regions with elevated concentrations of small particles.
To examine the statistics of the distance between
copepod and detritus, we divided the detritus into several size classes and calculated the NND between the
copepods and each size group separately. The results for
the three classes of copepods, presented in Fig. 16,
displayed several trends. First, as expected, except for a
few cases, there was an increase in NND with increasing
size of detritus. The likely primary cause for this trend
was reduction in the number/concentration of particles
with increasing particle size. Second, in the 145–285 mm
size range, there was an agreement between the NND
from the detritus to the cyclopoids and calanoids, but
both were 50% higher than the harpacticoid data. The
harpacticoids were thus preferentially in proximity to
detritus and in one third of the recordings actually
attached to some form of detritus. (The attached cases
were not processed as having a NND of zero, but rather
the distance to the next proximal detritus particle was
used in Fig. 16A.) A few examples are presented in
Fig. 17. Consistent with these observations, Fig. 9 also
shows that the harpacticoids tended to be located in
regions where detritus was present.
DISCUSSION
Plankton population dynamics depend on microscale
interactions. Documenting inhomogeneities in plankton
distributions at the microscale level is therefore a
valuable means of investigating processes that shape
pelagic marine ecosystems. It was the discovery of one
such inhomogeneity, the existence of thin layers of the
Fig. 16. Nearest neighbor distances (NND) between a copepod and
detritus particles, conditionally sampled according to particle size and
copepod species class. Each point represents a 70-mm bin. Error bars
are based on twice (P = 0.05) the SD of the data.
bioluminescent copepod, M. lucens (Widder et al., 1999)
that initially prompted this investigation. We postulated
that, using holographic imagery, we might be able to
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Fig. 17. Examples of harpacticoid Aegisthus sp. feeding off of detritus
(A–D) and engaging in mating behavior (E). Scale in panel A is 1 mm
and scale for panels B–E is 0.1 mm.
determine whether the aggregation of these copepods into
thin layers could be attributed to an avoidance response
to current shears or turbulent regions or might be due to
the copepods orienting to some food resource.
Based on the HIDEX-BP profiles that were collected
before and after each dive and the SPLAT recording
made during each dive, it was clear that the distribution
of bioluminescent plankton in the water column was
changing during the course of a dive. The difference
between profiles taken before and after each dive was
most pronounced for dive 3251 where the profile taken
before the dive was taken right at sunset, when the
evening assent of vertical migrators was still in progress.
For dive 3249, the profile taken before the dive was
taken an hour after sunset when the sunset assent should
have been largely complete. The analysed hologram
data of dive 3249 comes from the end of the dive
when the copepod displays were confined to 45–55 m
according to the SPLAT recording.
All the instruments involved identified/detected
M. lucens layers during the night. Net tows also showed
that the M. lucens in these layers were mostly (94–99%)
comprised of females. From the holographic images, we
have seen that at all depths, the population of calanoid
copepods consisted mostly of C. finmarchicus. Because of
the small number of detected M. lucens in the holograms
(a total of less than one dozen), we do not have sufficient
data to draw conclusions as to what attracts them to a
specific region layer1. However, the distributions of the
other particles indicated that the thin M. lucens layers
were not associated with microaggregates or with particles in the size class of dinoflagellates. Furthermore, the
concentration of these small particles (denoted as
dinoflagellates in this article) in the vicinity of all the
calanoid copepod species was also not significantly different from anywhere else in the reconstructed volumes.
Thus, the presence of these particles or dinoflagellates as
a potential prey did not seem to have a dominant effect
on the spatial distributions of the calanoids on the basis
of their overall concentration. However, the copepods
could have been aggregating to a subset of these particles
(e.g. a particular species of dinoflagellates) which
could not be distinguished from our data. Similarly, the
NNDs between calanoids and microaggregates do not
indicate preferred accumulation near or away from
microaggregates.
It is known, however, that M. lucens copepods
eat C. finmarchicus nauplii (Sell et al., 2001). We have
documented an instance of a nauplius near a
M. lucens (http://www.me.jhu.edu/lefd/shc/JSLtrip.htm),
and our recordings show M. lucens co-occurred with
C. finmarchicus. There were too few M. lucens individuals
in our holograms to derive any meaningful covariance
values, but in the region where M. lucens was mostly seen,
there were samples with elevated nauplii concentrations.
In one recording a M. lucens was identified at the surface
(5.7 m in dive 3249), and the nauplii concentration was
15.3 L1. These concentrations may themselves be due
to feeding on microalgae which are associated with the
fluorescent peak near this region (Fig. 4).
Other factors may affect M. lucens distribution. For
instance, the escape response of M. lucens is slower than
that of C. finmarchicus, forcing the former to undergo diel
migrations (Hays et al., 1997; Weatherby et al., 2000).
These observations are consistent with the observed lack
of M. lucens during the day and the formation of
bioluminescent layers of the species during the night. It
is also doubtful that the formation of the thin M. lucens
layer is related to mating, due to the predominance of
females and the dilute concentration (Widder et al.,
1999). Only once, in an early night dive, did we record
a pair of M. lucens in proximity at a depth of 93 m.
On the formation of copepod
and detritus layers
The data and visual observations on the C. finmarchicus
do show some interesting trends. During the two night
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dives, the highest concentrations of C. finmarchicus, 14
L1 the first night and 10 L1 the second night, were
found at the pycnocline and above. However, they were
present over the entire water column (Fig. 9). A night
dive from several days earlier (data not presented)
showed these copepods massing in the 7- to 10-m
depth range, while the pycnocline was at 8 m. The
only phytoplankton that we could distinguish clearly at
high concentration in this region, Ceratium tripos, has not
been listed in the literature as preferred food for
C. finmarchicus. Thus, it is doubtful that the Ceratium was
the cause for the aggregation. Other dinoflagellates and
small particles were distributed over the entire water
column.
The concentrations of the calanoids decreased significantly everywhere during the day, and the main bulk
was detected between 12 and 32 m, i.e. it extended well
below the pycnocline. Visual observations made from
the submersible during three separate day dives, consistently found high concentrations of calanoids, the only
species visible to the unaided eye, in the 20- to 30-m
depth range. The same conclusions can be reached by
visual inspection of holograms even without reconstruction (large particles can be seen directly), which are not
includedinthedatapresentedinthisarticle.The10-to30-m
range also contains a high concentration of dinoflagellates
(or other small particles), but they are widely distributed
over the entire water column and thus cannot explain
the preferred depth of the calanoids. Then, why do the
C. finmarchicus aggregate there? We try to examine several
possible contributing factors below.
The analysed holograms show that there was a clear
detritus maximum at the same depths as the calanoids,
due mostly to a peak in the fecal pellet concentrations.
The fecal pellets are found to degenerate into amorphous blobs of microaggregates, which had a less prominent maximum in the same region. A cursory visual
inspection of the exposures from all the dives indicates
that this detritus layer persisted for the entire cruise
period (7 days) and had a relatively sharp drop-off at
27–30 m, consistent with the distributions of the large
C. finmarchicus copepods, most notably during the day.
A significant portion of the fecal matter seems to be from
the calanoids themselves, although larger fecal pellets
were also observed. The cyclopoids, although present
in elevated concentrations in the same region, seem to
be an unlikely source of the fecal pellets, because of their
small size, 2–3 times smaller than the calanoids. The
main food source for these calanoids is not obvious, but
it does not appear to include detritus. Furthermore, the
NND from the calanoids to the detritus was not particularly low to suggest any special relationship. Thus, the
spatial distributions of particles do not point at an
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obvious reason for the copepod layer. One may speculate that the elevated concentration of typically large
detritus particles, which exceeded 200 L1, may have
decreased the ability of visual predators to detect the
copepods or may have even repelled these predators.
The causes for the formation of the detritus layer are
also not well understood. Furthermore, the detritus particles below the pycnocline showed a strong tendency to
cluster, as illustrated by the probability density function
of NND (Fig. 14A). Although diatoms have been previously linked to the formation of aggregates (Alldredge
et al., 2002), and sometimes seen in our images attached
to a cluster of detritus (Fig. 8B), the relatively rare
occurrence of such attachments suggests that the diatom
chain itself played a minor role in clustering process that
we observed. If it did contribute here, it would be
through the release of TEP material that would then
floc at a later time. The clustering of fecal pellets seems
to have happened on its own accord, perhaps due to
differential settling of large particles. A settling large
particle generates a wake with downward-induced
motion. A particle trapped within this wake is accelerated to a higher speed compared with quiescent settling
due to the combined effect of gravity and the downwash.
If the two particles have similar terminal velocity, they
will cluster. If the following particle has a lower terminal
speed, it may still separate from the larger one if the sum
of terminal speed and wake-induced motion is smaller
than the terminal speed of the leading particle. Close
particles moving side by side attract each other due to
potential flow (pressure) effects. Thus, a descending particle is expected to cluster with other particles falling at
the same speed (Wu and Manasseh, 1998). In the region
below the pycnocline (which shoals on the second night),
clustering is evident, while above it the distribution
seems to be random. We speculate that the random
distribution was caused by the higher turbulence in the
mixed region above the pycnocline.
The persistent localization of the harpacticoids,
Aegisthus sp., in the 20- to 30-m region does seem to
have trophic significance. The clearest evidence for a
trophic relationship between Aegisthus and detritus are
the images that show the harpacticoids attached to
fecal pellets and microaggregates (Fig. 17). Many benthic
harpacticoids are known detrivores (Hicks and Coull,
1983; Montagna, 1984; Perlmutter and Meyer, 1991).
The attachment to these particles may also stem from
the poor swimming capability of harpacticoids (J. R.
Strickler, Great Lakes WATER Institute, USA, personal
communication). As noted before, this behavior has
been seen in 33% of the images that contain this copepod. Furthermore, the NND between a harpacticoid
and detritus particles in the 145–280 mm size range is
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25–40% smaller than that for cyclopoids and calanoids.
Some of this decrease was undoubtedly due to the preferential location of the harpacticoids in the detritus
layer. It is unclear exactly on what the harpacticoids
were grazing. They may feed on the undigested content
of the fecal pellet, bacteria or bacteria products (TEP)
or even microzooplankton. Clusters of motile particles
<15 mm were observed around some images of detritus.
The distributions of the cyclopoids also show some
order. Their clustering is mainly related to their localization near the pycnocline at night. Their peak concentration (22 L1) shifted from 14 m on the first night to 6 m
on the second night, in accordance with the shoaling of
the pycnocline from 18 to 13 m. What attracts them to
this region is still unclear. Very often there is a chlorophyll maximum at a pycnocline (Condie, 1999), possibly
due to species with sizes below our resolution limit.
Because the cyclopoids are predatory, however, it is
more likely that they were orienting toward microzooplankton, which have sometimes been known to aggregate at such interfaces (Gifford, 2003). No high
concentration of particles of the right size was detected,
however. A possible explanation may be found in mesocosm experiments that show that copepods migrate to
haloclines, even in the absence of a target food source
(Lougee et al., 2002). As they suggest, the haloclineseeking behavior is probably a part of a feeding strategy,
which is likely to be successful due to the tendency of
particles to accumulate at interfaces. We propose that
the halocline may also serve as a marker in space, to
which a species might aggregate, and thereby increase
their chances for mating. Such aggregations occur preferentially at night, so as not to attract fish, which are
visual predators. Daylight brings a downward migration
to darker regions and an absence of such aggregations
(Fig. 9B).
On the effect and behavior of ctenophores
Cydippid ctenophores (Euplokamis sp.) were found to
occupy the region just above the M. lucens, namely at a
depth of 44 m on the first night and in the 26–38 m
range on the second night. Evidence on their concentration
peak comes mainly from the bioluminescence observations using the SPLAT system. In the two instances that
the holocamera captured images of these ctenophores,
at 47 (Fig. 18) and 32 m on the first night, harpacticoid
copepods (Clytemnestra sp. and Aegisthus sp.), which were
not very abundant in our holograms, were in proximity
to the ctenophores. It appears that the harpacticoids
were the ctenophore’s prey rather than parasites. For
instance, the second captured image shows (not shown
here) an Aegisthus sp. orienting toward and within a
body’s length of fecal pellet; its preferential food. It is
Fig. 18. Sample image of ctenophore Euplokamis (A) at 48 m near a
harpacticoid copepod Clytemnestra shown magnified in panel B. Scale
is 2 cm for panel A and 1 mm for panel B. Note the existence of
colloblasts on the tentacles.
also well established that the ctenophore Euplokamis sp.
feeds preferentially on copepods. The reason for the
proximity to the harpacticoids in particular is unclear.
The Euplokamis sp. might have a special affinity for
harpacticoids, or the sticky lobes of the tentacles may
resemble the marine microaggregates, to which harpacticoids have a tendency to attach.
One question is that ‘If the ctenophores prefer to prey
on copepods, why did they concentrate below the layer of
harpacticoids, in a region that had very few copepods?’
We can think of two reasons: Either the ctenophores
were responsible for the depletion of the copepods or
some mechanism/process prevented them from
approaching the harpacticoid layer. In searching for a
likely explanation for the latter, recall that the harpacticoids were mostly found in the 20- to 30-m depth range,
where the large detritus particles and fecal pellets had
the highest concentration. The 0–30 m range is also
where Pseudonitzschia diatoms were abundant. We speculate that the detritus and the long thin Pseudonitzschia
diatoms might have inhibited the migration of the ctenophores. Likely reasons for avoiding the detritus and
Pseudonitzschia layers include undesirable clinging of detritus or entanglement with the Pseudonitzschia as the tentacles sweep through the water. The detritus might
trigger unnecessary feeding responses since they are of
the same size as the harpacticoids. Clearer water at the
base of a detritus layer is more likely to keep the tentacles
clear of unwanted material. For harpacticoids migrating
below the detritus layer, the tentacle’s sticky lobes also
become attractive landing sites.
The particle-laden layer was not devoid of jellyfish
however. Several instances of other types of ctenophores
(probably Pluerobrachia sp.) and/or their tentacles were
recorded in the midst of the detritus layer (at a depth of
24 m) and at the pycnocline (at 10 and 12 m), but not in
the sets that we have analysed in detail. The SPLAT
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Fig. 19. A sample image from dive 3244 showing a ctenophore
(unidentified sp.) engulfing what may be a copepod at a depth of
18 m, i.e. within the detritus layer. One tentacle is straight, and the
other seems to be wrapped around the prey. A nauplius can be seen
near the straight tentacle, as further clarified in the magnified section.
Scale is 1 mm for panel A and 0.1 mm for panel B.
system identified the lobate ctenophore Bolinopsis infundibulum, which does not possess tentacles, at various depths
through the upper region (at 15, 30, 45 m) during the
afternoon transects. In Fig. 19, we show a ctenophore at
a depth of 18 m (dive 3244), which does not have
colloblasts (sticky cells) on its tentacles. Thus, the tentacles probably possess nematocysts (stinging cells), which
are below our resolution range. This ctenophore seems
to be in the act of engulfing an object (possibly detritus)
with one of its tentacles, while the other is extended.
Note the existence of a nauplius near the outstretched
tentacle. Extending the postulate from the previous
paragraph, species that do not have colloblasts may be
less sensitive to the presence of concentrated detritus,
which allows them to take advantage of the higher concentrations of copepods at shallower depths.
CONCLUSION
The detection of bioluminescence in the water column
using the HIDEX-BP and SPLAT system, along with
CTD profiling, provided context to holographic measurements of the spatial distributions and interactions between
particles. Although these instruments were brought
together to investigate bioluminescent thin layers, the
observations and subsequent analysis provided insight into
trophic processes and relationships between several types
of particles. We analysed in detail 200 reconstructed
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holograms, each providing the spatial distribution of
particles >10 mm within a sample volume of 1 L.
This article discusses several findings, the most striking
of which was the accumulation of calanoid, cyclopoid and
harpacticoid copepods in a detritus layer during the night.
The detritus concentration was two orders of magnitude
higher than that of the copepods. This layer was located
below the pycnocline, and the peak in detritus was a result
of a buildup of fecal pellets in various stages of aggregation and degradation. Most of the fecal pellets had sizes
and shapes consistent with those of calanoid copepods.
About one third of the harpacticoids (Aegisthus) were
attached to detritus, and the rest had NNDs from the
detritus that were lower than those of the other copepods.
High concentrations of Pseudo-nitzschia species were
found in shallow water (<35 m) during the day and
night dives. Some of these diatoms had lengths reaching
25 mm. In some cases, they were attached to detritus.
It is difficult to determine what attracted copepods to
the region of high detritus concentration, which also
contain Pseudonitzschia. In searching for a possible explanation, we note that the copepod and detritus layers
were bounded from below by a ctenophore layer (Eupokamis sp.). We speculate that these ctenophores avoid the
detritus to prevent fouling of their colloblasts and possibly also the long Pseudonitzschia diatoms, with which its
tentacles could become entangled. Thus, the copepods
may be safer within the detritus and Pseudonitzschia layer.
Metridia lucens, the bioluminescent calanoid species, was
found almost exclusively below the ctenophore layer,
and at low concentrations.
NND analyses indicated that the detritus had clustered distributions below the pycnocline, but appeared
to have a random distribution in the (presumably) mixed
region at shallower depths. The small particles, including
the dinoflagellates, had random spatial distributions at
all depths. Also, the present results provide no indication
of any relationship between the local concentration of
small particles and the presence of calanoid copepods.
There is, however, evidence that the copepods themselves were clustered during the night, with the cyclopoids being the most striking example.
Before concluding, we like to comment on the future
of in situ measurements using holography. The insight
provided by the reconstructed holograms is evident.
However, the process of recording holograms on film,
reconstructing them and scanning through the volume is
time consuming. Recent advances in digital imaging
now enable us to record digital holographic movies
(even at high speed) and reconstruct them numerically
(Malkiel et al., 2003) essentially in real time. However,
the digital holograms have a substantially lower resolution, or if the magnification is increased to maintain the
168
E. MALKIEL ETAL.
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SPATIAL DISTRIBUTION OF PARTICLES USING IN SITU HOLOGRAPHY
same resolution, the sample volume is smaller. As an
illustration of the difference, each cross section of the
optically reconstructed film hologram is scanned to a
14 000 14 000 pixel array, whereas a typical 15 fps
digital camera currently has a resolution of 2048 2048
pixels. Cameras operating at 2000 fps with resolutions of
1024 1024 pixels are also available. Thus, digital
holography offers many advantages (which we are now
implementing), albeit with a considerable compromise in
resolution.
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ACKNOWLEDGEMENTS
This project could not be completed without the enormous effort put forth by Stephen King, Yury Ronzhes
and Omar Alquaddoomi at JHU and David Smith and
the crew of the Johnson Sea Link at HBOI. We also
thank Steve Bernstein for his help with HIDEX-BP
deployments and data collection, Pascale-Emmanuelle
Lapernat for her help in identifying harpacticoid species
and Alice Alldredge for her assistance in identifying the
detritus. This research was supported by NSF grant
OCE-9107564(JHUpart),ONRgrantN00014-00-1-0176
(HBOI part) and HBOI contribution 1612.
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