ORP-81-3
ORP Contract
Report No. 81-3
Deep-Sea Food Web Analysis Using Immunological
Methods: Results of a Feasibility Study
Robert J. Feller
Belle W. Baruch Institute for
Marine Biology and Coastal Research,
Department of Biology, and the
Marine Science Program
University of South Carolina
Columbia, South Carolina 29208
July 1981
This report was prepared with support from the
Environmental Protection Agency, Office of Radiation
Programs, under Contract No. WA 80-8204
Project Officer :
Marilyn E. Varela
Office of Radiation Programs
U.S. Environmental Protection Agency
Washington, D. C. 20460
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
ORP Contract Report 81-3
2.
3. RECIPIENT'S ACCESSION NO.
128000
4. TITLE AND SUBTITLE
Deep-Sea Food Web Analysis Using Immunological Methods:
Results of a Feasibility Study
5. REPORT DATE
July 1981
6. PERFORMING ORGANIZATION CODE
(/306077
8. PERFORMING ORGANIZATION REPORT NO.
7. AUTHOR(S)
Robert J. Feller
'. PERFORMINQ.ORGANIZATION NAME AND; ADDRESS. •
U3.PRQGRAM ELEMENT NO. , ;.•-••
Belle W. Baruc.h Inst.
Research
for Marine Biology and Coastal
University of South Carolina
Columbia, SC 29208
11. CONTRACT/GRANT NO.
WA 80 - B 204
12. SPONSORING AGENCY NAME AND ADDRESS
Office of Radiation Programs
ANR-461
Washington, D.C. 20460
13. TYPE OF REPORT AND PERIOD COVERED
14. SPONSORING AGENCY CODE
200/03
15. SUPPLEMENTARY NOTES
16. ABSTRACT
Radioactive.waste disposal sites used in the past have been found to be leaking low
levels of radionuclides from containers placed on the sea bed. The potential exists
for food chain transport of radionuclides from deep ocean regions to man, but the
mechanisms by which such reverse transport upward can occur are largely unknown.
When biological samples are analyzed, it is frequently found that their stomachs
contain visually unidentifiable remains. Immunological gut analysis methods are
useful in identifying such remains. The ability of antibodies to discriminate among
proteins of different organisms depends on the degree to which a given antiserum
cross-reacts with antigens from each organism. The ability of antisera to shallow
water taxa to descriminate among deep-sea taxa was tested in hopes that these
antisera could discriminate among higher taxonomic levels of deep-sea organisms.
Preliminary tests using protein extracts of mid-water planktonic animals were
successful and revealed high affinities among shallow-water and mid-water species
of the same taxon. It is concluded that the immunological method may provide higher-
order taxon information for predator-prey interactions among deep-sea organisms.
This level of discrimination may provide data which could not be gathered using
traditional methodologies.
17.
KEY WORDS AND DOCUMENT ANALYSIS
a.
DESCRIPTORS
b.lDENTIRERS/OPEN ENDED TERMS
c. cos ATI Field/Group •
Ocean Dump Sites
Radionuclides
18. DISTRIBUTION STATEMENT
Released Unlimited
19. SECURITY CLASS (ThisReport)
unrestricted
21. NO. OF PAGES
20. SECURITY CLASS (Thispage)
unrestricted
22. PRICE
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NOTICE
mis report was prepared by Belle W. Beruch Institute for Marine
biology and Coastal Research, Department of Biology, and the Marine
Science Program, University of South Carolina for the United States
environmental Protection Agency's Office of Radiation Programs (ORP)
under Contract No. WA 80-B204. ORP has reviewed it, and the contractor
has responded to our comments. We are publishing this report because of
its useful information. We have not verified all of the results
ourselves, however; nor have we applied our own editorial standards to
the text. OrtP does not necessarily publish all of the contractor reports
it receives.
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TABLE OF CONTENTS
PAGE
List of Figures and Tables 3
Abstract ; 4
Introduction , 5
Methods 12
Results 14
RV Endeavor cruise 14
Formalin preserved materials '. 14
Iminunodiffusion specificity tests 16
Summary 20
Recommendations 21
Acknowledgements '. 21
References 22
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LIST OF TABLES AND FIGURES
FIGURES PAGE
1. Schematic diagram of the immunological method of
stomach content analysis 9
TABLES
1. Summary of methods for studying predator - prey inter-
actions '
3. List of organisms used in specificity tests 13
3. Bottom fauna collections from cruise EN-053, RV Endeavor.. 15
4. Precipitin reactions observed in specificity tests between
antisera to shallow-water taxa and -mid-water organisms
extracts ^
5. Additional specificity test results 19
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Deep-Sea Food Web Analysis Using Iramunological
Methods: Results of a Feasibility Study
Robert J. Feller
ABSTRACT
Radioactive waste disposal sites used in the past have been
found to be leaking low levels of radionuclides from containers
placed on the sea bed. The potential exists for food chain
transport of radionuclides from deep ocean regions to man, but the
mechanisms by which such reverse transport upward can occur are
largely unknown. Biologically mediated pathways could enhance
dispersal rates of radionuclides from deep-sea sediments, but
sampling difficulties in this remote environment render many
potentially useful food web analysis methods useless. When
biological samples are analyzed, it is frequently found that their
stomachs contain visually unidentifiable remains. Immunological
gut analysis methods are useful in identifying such remains.
The ability of antibodies to discriminate among proteins of
different organisms depends on the degree to which a given
antiserum cross-rstcts with antigens from each organism. In low
diversity shallow-water benthic communities, it is possible to
make antisera specific to each target organism, but there are far
too many species in the deep, sea to ever produce highly specific
antisera. Thus the ability of antisera to shallow-water taxa to
discriminate among deep-sea taxa was tested in hopes that these
aatisera could discriminate among higher taxonomic levels of
deep-sea organisms. Preliminary tests using protein extracts of
mid-water planktonic animals were successful and revealed high
affinities among shallow-water and mid-water species of the same
taxon. It is concluded that the immunological method may provide
higher-order taxon infprmation for predator-prey interactions
among deep-sea organisms. This level of discrimination may
provide data which could not be gathered using traditional
methodologies.
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INTRODUCTION
The EPA Office of Radiation Programs has for the past several
years been conducting comprehensive site specific oceanographic
surveys at radioactive waste disposal sites used by the U.S. in
the past. These survey activities have been conducted pursuant to
the Marine Protection, Research and Sanctuaries Act of 1972, as
Amended, which authorizes EPA to regulate all ocean disposal
activities, including the disposal of radioactive waste not prohi-
bited by law. Under the provision of the Act, EPA is also
required to establish and apply criteria for reviewing and evalu-
ating permit applications. To date, EPA has issued no permits for
'ocean disposal of low-level radioactive waste.
The Office of Radiation Programs has investigated the four
major sites used for radioactive waste disposal in the past.
These include two sites, at 900m and 1700m in the Pacific, and two
at depths of 2800m and 3800m, in the Atlantic. In each of these
sites, waste packages were located with the use of a submersible,
and low levels of radionuclides were found to either be leaking or
leaching from the containers.
One of the essential parameters yet to be addressed on a
comprehensive basis is that of the potential for food chain
transport of radionuclides from deep ocean regions upward to man.
The primary focus of scientists in the past has been upon energy
transfer downward through the water column. It is essential that
an integrated approach be developed for identifying reverse
transport mechanisms because of' the complex interactions which
take place between the organisms of the sea, their environment,
and people. Research is needed to identify and evaluate:
a. The possible interrelationships among deep-sea, mid-
water, and surface communities;
b. Factors which will assist in translating concentrations
of radionuclides in seawater and bottom sediments into
concentrations that will result in marine organisms; and,
c. Approaches to predict and analyze critical pathways to
man.
This research may assist EPA in devel'^ping the technical
basis and requirements for establishing regulations and evaluating
permit applications for ocean dumping of other than high-level
radioactive waste.
Given a point source of radionuclide leakage on the.deep-sea
floor, questions arise concerning the possible pathways $y which
these contaminants could reach man (Angino, 1977). Aside from
diffusional and advective transport of radionuclides in soluble or
fine particulate phase, it seems reasonable to suppose that bio-
logically-mediated pathways also exist which could enhance
,dispersai rates.
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The predominance of predatory, scavenging and deposit-feeding
modes in the deep-sea increases the likelihood that radionuclides,
if sorbed onto sediment particles, could enter the food web and be
rapidly moved away from a leakage area (Sanders and Hessler, 1969;
Hessler, 1974; Grassle et al., 1975). For example, numerically
dominant deposit-feeding organisms such as polychaetes might
ingest, assimilate, and thus bioconcentrate sediment-sorbed radio-
nuclides. A highly motile or vertically migrating predator on
polychaetes (fishes or amphipods, for example) could in turn
translocate these materials a considerable distance from the site
of ingestion (Bureau et al, 1979). In addition to such predator-
prey relationships, biologically mediated mobilization of buried
radioactive waste is possible as a consequence of simple
.sediment-moving activities of bottom-dwelling organisms.
Burrowing behavior, subsurface deposit-feeding, and aqueous
ventilation of burrow structures (for respiration and metabolite
excretion) can increase the exposure of buried materials and
increase their solubilization rates (Hessler and Jumars, 1977).
Assuming, then, that deposit-feeders and motile predators can
mobilize buried or leaked radionuclides, transfer pathways
involving these types of organisms must be identified in order to
predict transfer rates. Numerous methods exist for identifying
predator-prey relationships (Kiritani and Dempster, 1973), but the
deep-sea environment constrains the application of many of them,
especially observational and "tracer" or labelling methods.
Stomach contents analysis of deep-sea organisms might then seem to
be a prime candidate for application to the problem, but, unfortu-
nately, many of the same difficulties encountered in the visual
analysis of deposit-feeders and motile predators in shallow waters
apply to deep-sea taxa as well. That is, large portions of the
gut contents are recognizable only as fluidized amorphous masses.
Fish stomach contents are more easily identified than those of
deposit-feeders (prey are typically ground up), but deep-sea
fishes often regurgitate or otherwise lose their ingesta upon
capture and retrieval to the surface. Examination of such
specimens rarely reveals the presence of intact, identifiable
prey. Deep-sea scavengers' stomachs are frequently found full or
even distended with unidentifiable "meat" (Dahl, 1979).
Alternatives to visual analysis of stomach contents are few
(Table 1). Chemical analysis for specific elements or measurement
of bioaccumulations of specific elements is possible, of course,
and can provide valuable information on the distribution routes of
target elements. The collection of fresh specimens for analysis
is very expensive, however, and the effects of biological
fixatives may render chemical tracer methods useless.V' Recent
application of serological methods for analysis of food web
connections in benthic organisms by Feller et al. (1979) may hold
promise for tracing biomass fluxes among deep-sea taxa. The
methods are particularly useful in cases where stomach contents
are morphologically unrecognizable. The basic concepts of the
immunological method are shown in Figure 1.
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TABLE 1. An abbreviated summary of methods for the analysis of predator-prey
food web interactions and their potential use in the deep-sea.
METHOD
Direct observation
Predator exclusion
Laboratory prey
offerings
Tracer or label
experiments
Chemical analyses of
stomach contents
Fluorescence analyses
Bioaccumulation studies
Carbon isotope ratios
Hydrogen isotope ratios
COMMENTS POTENTIAL
Possible, but very expensive, as Low
remote sensing devices or deep
submersibles required; not likely
to yield even qualitative data on
predation processes; visual obser-
vation will be size-biased; data for
epifauna difficult to extrapolate to
infauna.
Usually do not provide unequivocal Very low
results on soft-bottoms; out of the
question for deep-sea where predators
are generally not well-identified.
We cannot reliably collect and/or Low
maintain deep-sea fauna in the lab;
extrapolation to field difficult.
Recovery of labelled prey would be Very Low
essentially zero in the deep-sea.
Could provide qualitative data on Moderate
food sources, but such data are
generally unspecific; requires
elaborate equipment and technical
skills; biased by whatever types
of animals are examined (true for
any method).
May work for pelagic species at Low
mid-depths, but not likely to work
for benthic species as method
requires presence of chlorophyll.
Variable in quality and difficult High
to interpret, but possible" to
follow gross patterns of biomass
flux; correct choice of target V
elements or compounds not easy.
Useful where plant or detrital •• '/" Lev;
material serves as food; poor
choice in deep-sea because ratios
are unknown for most taxa.
Untested in marine environment and Low
susceptible to gut content
contamination; less sensitive than
carbon isotope ratio method.
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TABLE 1. (cont'd)
METHOD COMMENTS POTENTIAL
Mouth part morphology Allows only broad classification Low
of animals into feeding types;
useless for tracing fluxes.
Visual stomach content Probably the most reliable technique Very high
analyses in spite of its potential biases;
stomachs of ingested prey should
also be examined.
Imraunological methods Worth testing on fresh-frozen Unknown
specimens to see if cross-reactions
are phylogenetically faithful across
taxa from shallow to deep habitats.
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I
10
I
Figure 1. Schematic diagram of the immunological method for
stomach content analysis (modified from Greenstone, 1979).
Harpacticoid copepods (H) are cleared of gut contents and ground
in saline. Their soluble proteins are injected into a rabbit
whose immune system creates antibodies to the copepod proteins.
The Y-shaped antibodies are harvested from the rabbit's blood by
centrifugation to remove red blood cells (rbc). This antiseruni is
shape-specific and will combine with harpacticoid antigens to form
precipitin lines within an agar matrix. The stomach contents of
suspected predators are assayed for the presence of harpacticoid
proteins using an immunodiffusion test in agar.
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Argyropelecus sp., a common mesopelagic hatchet fish, is shown
with stomach everted. The reduction in pressiyre as the animal was
brought to the surface has caused the stomach to balloon out of
its mouth. Incomplete eversion often leaves a fluid residue in
the stomach which would be amenable to immunological analysis.
(from Fig. 227, Deep Ocean, P. J. Herring and M. R. Clarke, eds. ,
Praeger Publishers, New York, 1971)
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Serological assays require a modest stock of taxon-specific
antibodies with which to test for the presence of prey antigens
(proteins, carbohydrates, fatty acids, etc.) in predators
stomachs. Antibody specificity is highest whenever an antibody
recognizes and reacts only with its target antigen - this is
seldom the case when antibodies are made by injecting whole-
organism extracts into a mammalian host such as a rabbit. That
is, an antiserum prepared to recognize antigenic proteins from a
species of bivalve may also (and usually does) recognize and react
to some extent with antigens from another species of bivalve and
to a lesser degree with other more distantly related molluscs.
Such cross-reactions may be used to advantage in the stomach
analysis of deep-sea organisms under the assumption that similar
taxa (phylum, order, family, etc.) from shallow water share
antigenic components with their deep-sea relatives. It is
obviously too expensive to collect a sufficient diversity or
quantity of live specimens from abyssal depths with which to
prepare antibodies to test the assumption on a grand scale. But
the existence of a variety of antibodies to shallow water benthic
invertebrate taxa (e.g., Annelida, Mollusca, Arthropoda, and many
other lower-order taxa) allows alternative approaches to this
otherwise expensive problem.
In conjunction with investigations of predator-prey inter-
actions among shallow-water marine organisms, an antiserum was
successfully prepared in rabbits by injecting them with
whole-organism extracts of adult grass shrimp, Palaemonetes pugio,
which had been preserved in a 5% formalin-seawater solution for
nearly five months. This was a somewhat surprising discovery,
especially since formaldehyde polymerizes antigenic proteins so
readily (Jones, 1976). The anti-P. pugio antiserum was of course
not as sensitive and specific as antiserum prepared using fresh or
fresh-frozen shrimp extract, but it retained sufficient
specificity for recognition of higher order taxa. That is, the
antiserum cross-reacted with several other crustaceans but not
with the annelids or bivalve molluscs tested. Further, antiserum
prepared with fresh shrimp also appeared to retain the ability to
discriminate among higher order taxa when tested against
formalin-preserved material. This suggested that it might be
possible to examine the stomachs of formalin-preserved deep-sea
specimens using an extant battery of anti^era prepared with
extracts from fresh, shallow-water organisms" Results obtained
might provide predator-prey data at only a high order taxonpmic
level, but even this type of information is sorely lacking.- for
abyssal animals. Such coarse data still might identify key
predator-prey links worthy of more detailed study in the. future.
Some fraction of the large number of deep-sea specimen*'reposited
in various oceanographic institutions and museums would have to be
made available for serological examination towards this end.
It was thus proposed to examine the nature of serological
cross-reactions among organisms collected from the deep-sea with
antibodies prepared against whole-organism saline extracts of
shallow-water benthic invertebrates. These studies were designed
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to specifically test the feasibility of using imraunological
methods to examine the stomach contents of deep-sea predators,
scavengers, and deposit-feeding organisms.
A cruise aboard RV Endeavor, University of Rhode Island, to
collect deep-sea fauna for immunological testing was unsuccessful
in this regard, as little time was available for biological
sampling under the prevailing weather and scheduling of other
worker's tasks (Laine et al., 1980). However, through the
cooperation of Dr. Bruce Robison, University of California at
Santa Barbara, a variety of mid-water organisms were donated for
testing. This report concerns results of specificity tests using
antisera to shallow-water benthic invertebrates from both Puget
Sound, Washington, and North Inlet, South Carolina, and whole-
organism saline extracts of the mid-water specimens donated by Dr.
Robison. Comments are also directed towards the feasibility of
analysing the stomach contents of formalin preserved specimens,
the applicability of other methods of food web analysis ia the
deep-sea, and recommendations for further research.
METHODS
Antisera to shallow-water organisms were prepared by
injecting whole-organism extracts of a given invertebrate species
into white, female, New Zealand rabbits. The extracts were
prepared by grinding freshly-collected animals (whose guts had
been cleared for 24 hr) in 5 mM TES [N - tris (hydroxymethyl)
methyl-2-aminoethane sulfonic acid], 30 mM NaOH, and 150 mM NaCl
at pH 7.3. The TES-saline protein mixture was centrifuged at 1000
x g and the supernate stored at -20°C. This soluble protein
extract served as antigen for the injection series following the
protocol of Feller et al. (1979). Serum collected from the
rabbits was stored at -20 C until use.
Mid-water animals donated by Dr. Robison were sorted from
trawl catches in the Santa Cruz basin at depths of about 1200 m
and frozen intact on board ship. They were mailed air freight to
Columbia, S.C., and arrived still frozen on dry ice. No thawing
was known to have occurred during handling or shipment. The
numbers of each organism solubilized in TES-saline, the volumes of
TES-saline used, and other comments regarding"preparation of the
mid-water organism extracts for testing are very similar, to
procedures used in preparing extracts of the shallow-water
organisms (Table 2). Total protein concentrations of the various
extracts were not measured but probably ranged from 1.- to 10 mg
total protein per ml. Each species was ground with glass 'beads in
a cold mortar and pestle for approximately 1 min. The soluble
protein slurry was then centrifuged at 1000 x g for 10 min and the
supernate stored at -20°C until tested.
To test for the presence of cross-reactions between soluble
protein extracts of mid-water species aad antisera to shallow-
water organisms, an immunodiffusion technique was used.
Microscope slides (25 x 75 mm) were coated with 1.2 ml of 0.5%
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TABLE 2. Mid-water organisms utilized in specificity test.
ORGANISM NO.
Gausia princeps 35
(copepod)
Euphausia pacifica 14
(euphausiid)
Sergestes sitnilis 8
decapod)
Cystisotna sp.
(amphipod)
Phronima sp.
(amphipod)
Cranchiid squid
Cyclothone acclindens 4
(pisces)
Stenobrachius leucopsaurus 1
(pisces)
Triphoturus mexicanus 1
(pisces)
Eucopia sp. 10
(decapod)
Pasiphaea emarginata 4
(amphipod)
Hymenodora debilis 4
(decapod)
ml TES
3.0
2.0
2.0
1.0
1.0
2.0
3.0
3.0
4.0
5.0
8.0
6.0
COMMENTS
intact adult females
intact adults; 15mra
total length each
Tail meat only, no
exoskeleton; 1.2-
2.5 cm total length
intact animals with
visually empty guts
seawater frozen inside
exoskeleton
intact animals plus
barrel; visually
empty guts
4.5 cm total length;
intact, empty gut
tail meat only
6.5 cm total length;
tail meat only
6.8 cm total length;
tail meat only
intact animals; 2.0-
2.5 cm total length
tail meat only; 7.0-
7.8 cm total length
tail meat only; 4.0-
7.0 cm total length
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agarose \(in 8 mM veronal, 40 raM ' sodium veronal, 0.25% Triton
X-100, 0.01% sodium azide). A plastic template with 4 wells
surrounding a central well served as point sources for the
diffusion of extracts and antisera through the agarose.
Typically, 10-15 pi of extract was added to the center well with
four different antisera (15 |Jl per well) in the surrounding wells.
Each test was duplicated. Diffusion proceeded ac room temperature
for 48 hr. Slides were washed in saline to remove unprecipitated
proteins and in distilled water to remove salts:. After washing,
each slide was dried and stained with Coomassie Brilliant Blue R.
Precipitin lines were examined and counted using back-lighting
through opaque glass.
RESULTS
RV Endeavor cruise
Despite time and equipment limitations, two bottom samples
were collected with a geologist's sphincter core during cruise
EN-053, 11 August 1980. Geologists operated both cores which,
upon retrieval, were routinely siphoned to remove overlying water
(25-30 cm deep). Most of this water was collected and examined
for fauna. It is unknown to what extent this material was
contaminated by surface waters during the 1.5 hr retrieval period.
The surface 1 cm of sediment was collected from each core, with
one-third frozen on dry ice, one-third preserved in 0.5%
formalin-seawater (v/v), and one-third preserved in 0.5%
gluteraldehyde-seawater (v/v). All sediments were washed through
a 44 micrometer mesh. The cores were severely winnowed when they
reached deck, and therefore, any quantification on an areal basis
will be underestimated by an unknown amount.
Abundances recorded fell within the range reported for
meiofauna in the area (Coull et al., 1977), and harpacticoid
copepod diversity was also high as expected (e.g., Thistle, 1978).
An insufficient biomass of any taxon was collected in the frozen
fraction to test with shallow-water antisera.
Notable aspects of the two core samples examined were the
dominance of agglutinated foraminifera (though very few if any may
have been alive), absence of macrofanna* (not particularly
surprising for such small samples at abyssal depths), the presence
of a molt of Microsetella, a surface-dwelling planktonic harpacti-
coid copepod (if it was not due to surface water contamination, it
would have taken weeks to reach the bottom), and the absence of
any obvious macrofaunal-scale features on the sediment surface (no
tubes, tracks, or biogenic structures).
Formalin preserved materials
Attempts to utilize antisera prepared against fresh or
fresh-frozen organism protein extracts to detect specific proteins
in formaldehyde-preserved specimens were initially encouraging.
However, sample size was too small, and when additional and more
extensive tests were performed, the immuno-assay became an
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TABLE 3. Summary of bottom fauna collections using 1 m long, 21.6 cm inside
diameter, sphincter core (366 cm ), RV Endeavor cruise EN-053, 11
August 1980, location 1.
SAMPLE NO.
SC #4
SC #6
OVERLYING WATER:
LATITUDE
32 44.6N
32 46.5N
LONGITUDE
70 43.2W
70 44.1W
SEDIMENT (upper 1 cm):
Nematoda
Harpacticoida
Calanoida
Nematoda
Harpacticoida
Radiolaria
Foraminifera tests
TIME (Z)
0134
0730
SC #4
4
2
SC #4
3**
128*
3322
DEPTH (m)
5348
5346
SC #6
***
Nearly all were bits and pieces of tests, with fewer than
0.1% intact; unable to determine if any were alive when
collected since no vital stains were used; sieving was too
gentle to have broken intact specimens, thus likely that
much fewer than-0.-l% were alive.
Most were nearly intact or easily recognized pieces.
No two organisms were the same species; mostly from family
Cletodidae (Eurycletodes spp.) and Ameridae; a single molt of
Microsetella sp. occurred in SC #4 sediment; SC #6 contained
a gravid female from family Cletodidae.
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unreliable methodology. Although formalin preservation
effectively cross-links proteins and prevents their denaturation,
the immunoreactive sites on the proteins may retain a conformation
which is still recognizable to antisera. However, the extent to
which this conformation remains constant is essentially unknown,
and even slight shifts in the pH of the preservative medium may
alter the shape of the protein molecules. It is thus difficult to
ascertain whether reactions observed are due to true antigen-
antibody interactions or to reactions caused by alterations in
molecular shape. The lack of standardized preservation methods
among different reseachers complicates ' the picture considerably,
for proteins may obtain varying .degrees of immunoreactivity
depending upon formalin strength, buffering capacity, and
preservation temperature. It is tempting to think that preserved
materials from :.he deep-sea may retain enough reactivity for use
with antibody recognition, but reliability is too low for any
practical application in food web or taxonomic studies. I have
abandoned any further testing of preserved material.
Immunodiffusion specificity tests
The cross-reaction tests between soluble protein extracts of
the mid-water organisms and antisera to shallow-water benthic
invertebrates were very successful. They revealed the following
relevant features (Table 4):
1) antisera to shallow-water organisms recognize similar
antigenic proteins in mid-water animals;
2) this recognition, as measured by the numbers of
precipitin lines formed, was always less intense than
the respective self-reactions;
3) many of the mid-water animal extracts did not react with
some of the antisera, i.e., no precipitin lines formed
in the agarose;
4) those mid-water animals whose extracts did cross-react
did so along classical phylogenetic lines.
Thus, the antisera were predominately taxon-specific, the only
strong exception being the Hobsonia antiserum which recognized
antigenic proteins from mid-water crustaceans. This specificity
at higher taxonomic levels coupled with broad cross-reactivity
within a given taxonomic level is an ideal property for antisera
which might be used as a gross assay tool in deep-sea food web
studies. Furthermore, many of the antisera tested were prepared
using whole-organism extracts of species from the west coast of
the United States (Puget Sound, Washington), so that cross-
reactivity and taxon specificity was apparently independent of
whether the antisera were from widely separated geographic areas.
More detailed evidence along these lines is presented by Feller
and Gallagher (in preparation).
Additional but less extensive tests were performed using only
nine of the nineteen antisera in Table 4 with extracts of T.
mexicanus, Eucopia sp., P. emarginata, and H. debilis (Table 5).
These tests also revealed the same features as outlined in points
1 through 4 above. This further enhances the generality of the
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TABLE 4 Maximum number of precipitin lines observed in antigen-antibody reactions between whole-organism
extracts of mid-water organisms and antibodies prepared against extracts of shallow-water taxa. Few if
any cross-reactions are extensive when compared to the number of lines observed in self-reactions.
— • WHOLE-ORGANISM EXTRACTS OF:
ANT I SERA
Crustacea
Amphipoda
Decapoda
Copepods
Ostracoda
Mollusca
Bivalvia
Gastropoda
Annelida
Polychaeta
Oligochaeta
Nematoda
A - Phronima sp
TO:
: Corophium salmonis
Eogammarus confervicolous
: Callinectes sapidus
Penaeus setiferus
Palaemonetes pugio
Crangon franciscorum
Uca pugnax
Uca pugilator
: Huntemannia jadensis
: Ostracoda spp.
: Mercenaria mercenaria
Crassostrea virginica
Tagelus plebius
Genkensia demissa
: Littorina ir^rata
: Diopatra cuprea
Hobsonia florida
: Oligochaeta spp.
: Diplolaimella chitwoodi
. (amphipod) D - Gausia
A
2
3
4
6
2
1
1
4
5
2
1
1
3
2
B
1
2
2
5
3
2
2
2
2
2
princeps (cc
B - Cystosoma sp. (amphipod) E - Euphausia
pacifica
C
1
4
9
5
1
1
5
5
>pepod)
(euphausiid)
IT J\
D
2
2
1
1
2
1
3
1
1
2
G -
H —
E
1
2
6
8
3
3
1
2
1
F
2
3
1
1
2'
1
3
2
G H Self
7
8
12
15
1 13
12
11
14
12
7
13
12
12
15
12
11
8
12
6
Stenobrachius Leucopsaurus (pisces)
Cyclothone acclindens (pisces)
C - sergestes similis (decapod)
- 17 -
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Representative taxa used for cross-
reaction tests:
A)
B)
C)
Cystisoma sp., a large (10 cm)
hyperiid amphipod;
Phronima sp., a pelagic amphipod
that lives within the empty barrel
of a^siphonophore;
Cyclothone sp., a numerically
dominant meso - and bathypelagic
fish genus.
(from Figs. 171, 1-72,^ 185, Deep Oceans,
P. J. Herring and M. R. Clarke, eds,
Prager Publishers, New York, 1971)
- 18 -
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TABLE 5. Maximum number of precipitin lines observed in cross-reaction tests
between antibodies to shallow-water benthic invertebrates and whole-
organism extracts of mid-water organisms.
WHOLE-ORGANISM EXTRACTS OF:
ANTISERA TO:
Mollusca
Bivalvia :
Gastropods :
Annelida
Polychaeta :
Mercenaria mercenaria
Crassostrea virginica
Littorina irrorata
Diopatra cuprea
4
2
4
2
2
1
K
1
3
Self
Crustacea
Decapods :
Ostracoda :
Callinectes sapidus
Penaeus setiferus
Palaemonetes pugio
Uca pugilator
Ostracoda spp.
6
7
5
4
6
8
5
6
6
9 1
6
8
12
15
13
14
7
13
12
12
11
I - Eucopia sp. (decapod)
J - Hymenodora debilis (decapod)
K - Pasipfaaea emarginata (amphipod)
L - Triphoturus mexicanus (pisces)
- 19 -
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observed antiserum specificity at higher taxonoroic levels with
broad cross-reactivity within taxonoraic levels.
SUMMARY
The immunological approach to food web analysis in the
deep-sea merits further testing for the following reasons:
a) the method works for terrestrial and/or aquatic
communities (Boreham and Ohiagu, 1978);
b) the method is extremely sensitive and can detect very
low concentrations of protein (|Jg to mg per ml);
c) cross-reactions among shallow-water antigens and their
homologous antibodies reflect traditional taxonomic
similarities;
d) preliminary tests utilizing antisera to shallow-water
species were successful in detecting antigenic proteins
from taxonomically related mid-water planktoiiic
organisms; *••
e) antibody affinities are highest between shallow-water
and mid-water species of the same taxa;
f) cross-reactions among similar taxa from the west coast
and east coast also reflect traditional taxonomic
similarities (manuscript in preparation).
These preliminary findings could. not be more encouraging.
They indicate that it may be possible to analyse the stomachs of
deep-sea predators and easily determine which taxonomic groups
they had been eating. With some refinement it may be possible to
determine that, for instance, a crustacean meal was amphipod and
not decapod. Many marine fish predators contain visible masses of
organic material or "meat" which- cannot be identified. The
development of the immunological method now has a high probability
of offering a means by which such stomach material may be
identified. Although the deep-sea biological community is too
diverse to ever hope that specific identifications could be made,
this higher-order taxon information will be invaluable in
providing direct evidence for predator-prey interactions that
could perhaps never be determined using traditional methodology.
'*.
Visual analysis of stomach contents should, of course, always
be performed in conjunction with any immunological analysis. .. But
because this technique is so sensitive, we may also be able to
examine the stomach contents of the ingested prey themselves and
determine secondary or second-order predator-prey interactipns.
Relative to other's efforts at deep-sea food web analysis,
the immunological method thus offers not simply an alternative
approach but a complimentary technique which can give information
when other methods fail. The major disadvantage, however, is that
specimens must be examined in either the fresh or fresh-frozen
state. Other methods, especially visual ones, can utilize
formalin preserved material. However, since most deep-sea
- 2.0 -
-------
collections are made from ships of substantial size, most are
likely to have sufficient cold-storage capacity to preserve
catches in the frozen state.
RECOMMENDATIONS
Deep-sea food chain transfer studies should begin, of course,
with a competent review of published and "gray" literature
pertaining to stomach content analysis in deep-sea organisms.
There are very few of these. A review of literature pertaining to
estimates of biomass and abundance of deep-sea fauna (there are
considerably more of these and they vary in quality to such an
extent that many are uninterpretable and useless) will provide a
baseline against which future estimates from proposed dump sites
may be compared. No estimate of biomass, however, can provide
anything more than a broad, subjective indication that certain
taxa may or may not enter food webs. Direct evidence is
necessary.
The interests of EPA would be best served at this time by
interacting with active deep-sea researchers and providing them
nominal support to report their food-web findings from various
deep-sea oceanic provinces. I believe EPA is now doing this very
well, with the exception that, to my knowledge, no strictly
biologically oriented cruises to the dump sites have been
organized. Samples should be taken from the areas of interest
utilizing box cores for sediment biota, bottom trawls, bait-
trapping, microbial, and bioenergetic studies (e.g., oxygen
consumption). These are all expensive propositions. Stomach
content analysis of specimens continues to provide the most
informative and reliable data for food-web studies. Hyslop (1980)
reviews these visual methods with emphasis on their quantisation.
Such traditional methodology suffers the typical limitations
imposed when organisms sampled contain no visually recognizable
remains in their stomachs.
ACKNOWLEDGEMENTS
This feasibility study could not have been done without the
competent technical assistance of C. Mcllvaiae, J. Dorsch, and A.
Evjen. Special gratitude is extented to Dr. Bruce Robison for-his
generous donation of mid-water specimens. I would also like to
thank the crew of RV Endeavor, University of Rhode Island, for
their professionalism and willingness to help under sometimes
adverse conditions. Dr. E. Laine, Chief Scientist •''on' cruise
EN-053, was especially helpful. This work was sponsored by the
Environmental Protection Agency, Office of Radiation Programs,
with Dr. Marilyn Varela project officer. Antisera utilized in
specificity tests were made available through grants OCE 76-81221
and OCE 79-19473 from the Biological Oceanography Section,
National Science Foundation.
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