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CHEMICAL ATTRACTANTS OF THE'FLORIDA SPINY' LOBSTER,
PANULIRUS ARGUS
by
Barry W. Ache, Bruce R. Johnson and Edward Clark
Department of Biological Sciences
Florida Atlantic University
Boca Raton, Florida 33431
TECHNICAL PAPER NO. 10
October 1978
Florida Sea Grant
CHEMICAL ATTRACTANTS OF THETFLORIDA SPINY,LOBSTER,
PANULIRUS ARGUS
by
Barry W. Ache, Bruce R. Johnson and Edward Clark
Department of Biological Sciences
Florida Atlantic University
Boca Raton, Florida 33431
TECHNICAL PAPER NO. 10
October 1978
The information contained in this paper was
developed under the auspices of the Florida Sea Grant
College Program, with support from the NOAA Office of
Sea Grant, U.S. Department of Commerce, grant numbers
04-5-158-44 and 04-6-158-44055. This document is a
Technical Paper of the State University System of
Florida Sea Grant College Program, 2001 McCarty Hall,
University of Florida, Gainesville, FL 32611.
Technical Papers are duplicated in limited quantities
for specialized audiences requiring rapid access to
information which may be unedited.
INTRODUCTION
It is now well established that chemical stimuli control diverse types of
behavior among marine organisms (Bardach, 1975). The chemical stimuli controlling
feeding behavior are at least in part simple organic compounds such as amino
acids and their derivatives. It follows that such compounds, once evaluated for
their activity and appropriately packaged, could provide an effective, economical,
and convenient artificial bait for economically important marine organisms.
The present study evaluates the potential of simple organic compounds as attractants
for the Florida spiny lobster, PanuZirus argus.
Two issues introduce some question as to the potential role of simple organic
compounds as feeding attractants. As noted by Carr et al. (1974), since most
studies implicating simple organic as feeding stimulants have not systematically
eliminated macromolecules as active feeding stimulants by "working down" from
complete extracts of potential food organisms, small organic molecules may not
entirely account for feeding competence in marine organisms. Secondly, "feeding"
is a multicomponent behavior involving the more or less discrete stages of food-
finding, food handling, biting, etc., each of which is potentially controlled by
novel chemical (or non-chemical) cues. It remains, however, that relatively
simple organic substances at least in part regulate feeding-associated activities
in decapod crustaceans as the spiny lobster (McLeese, 1970; Kay, 1971; Shelton
and Mackie, 1971; Mackie, 1973; Carr and Gurin, 1975; Hindley, 1975; Hamner and
Hamnert, 1977; Hartman and Hartman, 1977). In the one species of decapod subjected
to systematic analysis of feeding behavior, the shrimp, Palaemonetes pugio, low
molecular weight components account entirely for the stimulatory capacity of
three food extracts out of six tested-(Carr and Gurin, 1975).
Present research suggests chemosensitivity in decapod crustaceans is complex,
minimally being partitioned between three sets of receptors, the antennules, the
mouthparts, and the periopod dactyls. The antennules are characteristically
ascribed to low threshold chemoreception, the initial alerting and possibly
orienting stages of chemically-elicited feeding (Maynard and Dingle, 1963;
Hazlett, 1971). Certainly ablation of the lateral antennular filaments disrupts
orientation towards sources of dissolved substances (McLeese, 1973; Ache, 1975),
a phenomenon also demonstrated in P. argus (Reeder and Ache, In preparation).
Physiological evidence verifies the presence of low threshold chemoreceptors in
the antennular filaments of P. argus, receptors responsive to not only extracts
of potential food organisms (Ache et al., 1976) but to solutions of single amino
acids, as well (Laverack, 1964; Levandowsky and Hodgson, 1965; Johnson and Ache,
1978). While the evidence supporting distance chemosensitivity in the lobster
antennule isn't conclusive, it suggests this appendage as a logical site for
further analysis of such sensitivity.
Interest in the concept of defining artificial attractants or "baits" for
commercially important marine organisms was facilitated by a Sea-Grant-sponsored
symposium on potfishing and artificial baits (Jaeger, 1972). The basic concept
was extended by Hancock (1974) who suggested repellants of predatory species,
substances documented behaviorally, but yet to be characterized chemically,
might enhance a particular fishery's catch-effort as much as attractant compounds.
Previous workers have attempted to define artificial baits for commercially
important decapod crustaceans with varying success. Trimethylamine solutions
caught four times more American lobsters than unbaited traps (Moody, unpublished
technical report, Bio-dynamics, Inc.). The amino acid glycine enhanced trapping
the West coast dungeness crab, but not as well as natural baits (Allen et al.,
1975). Studies in progress to define a chemical substance suitable for potfishing
the western Australian rock lobster (P. longipes) are still inconclusive (R. Kage,
personal communication). A related finding is that specific amino acids attract
3 species of marine fish when dispensed in the natural habitat (Sutterlin, 1975).
Field tests are confounded, however, by the additional requirement of an'effective
release vehicle, a vehicle that releases at suprathreshold concentration through-
out the fishing interval. Suitable technology has yet to be applied on any large
scale to releasing artificial attractants in aquatic environments, but exists in
a number of forms of microencapsulation and slow release gels and polymers (e.g.,
Baker and Lonsdale, 1975). Allen et al. (1975) suggested and tested polyacrylamide
gels as releasants for amino acid solutions and extracts of natural baits.
The rationale of the present project is to survey a relatively large number
of chemical compounds for their ability to stimulate the antennular chemoreceptors
of the spiny lobster. Included among the most stimulatory compounds should be
those compounds, if any, that function as behavioral attractants. Their potential
as attractants is then verified in an olfactometer designed to assay the chemo-
toxic component of feeding behavior. Those compounds most attractive in the
olfactometer study are then field tested together with presently-used natural
baits for subsequent comparison of catch (and cost) effectiveness. This latter
step requires the definition of an effective releasant, which constitutes the
last aspect of the project.
METHODS
Animals Specimens of the lobster, Panulirus argus, were obtained locally from
a commercial lobsterman and maintained (1) in 150 gal. recirculating seawater
tanks in the laboratory for physiological analysis or (2) in running seawater
holding facilities for behavioral analysis. Lobsters were fed frozen pink shrimp
every third day unless noted otherwise. At no time during the capture, handling
or holding were the animals exposed to air for periods exceeding 30 sec.
personal communication). A related finding is that specific amino acids attract
3 species of marine fish when dispensed in the natural habitat (Sutterlin, 1975).
Field tests are confounded, however, by the additional requirement of an'effective
release vehicle, a vehicle that releases at suprathreshold concentration through-
out the fishing interval. Suitable technology has yet to be applied on any large
scale to releasing artificial attractants in aquatic environments, but exists in
a number of forms of microencapsulation and slow release gels and polymers (e.g.,
Baker and Lonsdale, 1975). Allen et al. (1975) suggested and tested polyacrylamide
gels as releasants for amino acid solutions and extracts of natural baits.
The rationale of the present project is to survey a relatively large number
of chemical compounds for their ability to stimulate the antennular chemoreceptors
of the spiny lobster. Included among the most stimulatory compounds should be
those compounds, if any, that function as behavioral attractants. Their potential
as attractants is then verified in an olfactometer designed to assay the chemo-
toxic component of feeding behavior. Those compounds most attractive in the
olfactometer study are then field tested together with presently-used natural
baits for subsequent comparison of catch (and cost) effectiveness. This latter
step requires the definition of an effective releasant, which constitutes the
last aspect of the project.
METHODS
Animals Specimens of the lobster, Panulirus argus, were obtained locally from
a commercial lobsterman and maintained (1) in 150 gal. recirculating seawater
tanks in the laboratory for physiological analysis or (2) in running seawater
holding facilities for behavioral analysis. Lobsters were fed frozen pink shrimp
every third day unless noted otherwise. At no time during the capture, handling
or holding were the animals exposed to air for periods exceeding 30 sec.
Physiological Assay Antennular chemosensitivity was assayed using the distal
5-6 cm section of the lateral filament excised from adult lobsters and arranged
for electrophysiological recording in a lucite recording chamber (Fig. 1) as
described in earlier reports from our laboratory (e.g., Ache et al.,1976).
PC
Fig. 1 Preparation chamber for recording
from excised antennules Of P. argus. A,
antennule; RC, recording compartment; SC,
SC stimulating compartment; S, septum; PC,
perfusion cannula; SW IN, seawater inlet;
SP, stimulus port; R, recording electrode.
S-SW IN
-S-CR RC
In this device, 50 pl volumes of potential stimulants injected into a 10 ml/min
carrier flow of artificial seawater contact the preparation 0.4 sec post injection,
rapidly peak to a maximum concentration approximately 10-1 times neat and sub-
sequently tail off over the following 7 sec. All substances tested as potential
stimulants were reagent grade purity. Solutions of test substances were prepared
within 3 hr of use in reagent-grade artificial seawater (M.B.L. formula) and their
pH adjusted to 7.6, the value of the seawater carrier flow that continuously washed
the preparation. Pure test chemicals were prepared for delivery at three con-
centrations, 10-3, 10-4, and 10-5 molar, unless noted otherwise. A few compounds
of unknown molecular weight were prepared on a wt/volume basis as noted in the
results section.
5
Nerve bundles containing 1-20 active chemosensory neurons were teased free
of the main antennular nerve for recording. While recording neural activity
(action potentials) from such a bundle, the preparation was stimulated sequentially
with 50 pl volumes of standard shrimp extract (SSE)*, three test chemicals at
a single concentration and a terminal replication of SSE. The sequence was then
repeated with each of the remaining two concentrations of test chemicals. A
1.5 min wash period separated each test chemical application. A new nerve bundle
was then teased free for recording and the stimulant series repeated. Each series
of three test chemicals (at 3 concentrations) was evaluated on a minimum of 5
nerve bundles obtained from no less than two different antennular preparations.
The recorded action potential trains were passed through a window dis-
criminator, adjusted to include all chemosensory activity and to discard any
concomitant non-chemosensory activity, and inputed via a Schmitt trigger to an
electric counter (Haer 7400 series) with the clock set at a bin width (0.5-5 HZ.)
appropriate for the total number of spikes to be counted. The counter output
was displayed as an instantaneous histogram on a storage oscilloscope and the
activity recorded from a given bundle was calculated from the histogram's area
within the time period the standard stimulant's activity remained above baseline
in that bundle. Post stimulus activity of a given bundle was so quantified for
each application of each of the three or, where noted, two, applied concentrations.
To allow for across-bundle comparison, these values had to be normalized to some
standard value, selected as a bundle's mean response to the initial and terminal
*SSE consisted of 1 gm peeled pink shrimp (Peneaus duorarum) abdominal muscle
homogenized in 2 ml artificial seawater, diluted 10 fold and subsequently
centrifuged to obtain a clear solution.
applications of SSE. The resulting ratio is referred to herein as the "activity
ratio". Activity ratios for all applications of a particular test chemical at
all concentrations were then averaged unweightedd mean) to generate a "mean
activity ratio" for that test chemical. Such "mean activity ratios" are
reported herein as representative of a compound's ability to stimulate antennular
chemoreceptors.
Behavioral Assay Those test chemicals eliciting larger neural responses (higher
mean activity ratios) were selected as candidate attractants and assayed
behaviorally for their ability to elicit chemotaxis in intact organisms. Several
assay devices, including two of "Y maze" design, were rigorously evaluated before
selecting the device used as being most compatible with the "skittish" nature of
P. argus and our interest to assay attractants per se, not necessarily generalized
feeding stimulants. The system consisted of six, 1.5 meter diameter plastic pools
arranged in a circle around a plastic head tank, all draining into a 4.6 meter
diameter tank. (Fig. 2) A continuous flow of unfiltered natural seawater
drained into the head tank, containing a standpipe to maintain uniform head pressure.
SFig. 2 Circular olfacto-
.. --.:., meters used to assay the
-. -.--. ,_,_ :-" 'behavioral attractiveness
of selected test chemicals
to P. argus. See text for
details.
.. .. i*
S.. .
Water gravity-fed from the head tank to each test pool via tygon tubing in
turn attached to a bent glass tube fixed to the side of the pool so as to direct
flow of water (14.7 liter/min) parallel to the bottom. In each pool, one half
of a cement block at the down stream end of the pool served as a habitat for
the lobster. A potential attractant was delivered to each pool via a small
diameter tygon tube inserting into each inlet tubing about 50 cm from the tube's
end. This arrangement allowed for metered pumping (60 ml/min) of potential
attractant solutions into the inlet flow of water where it was mixed by turbulence
to a concentration 6 x 10-3 times the starting concentration before discharging
into the tank. The flow established a chemical gradient across the pool such
that the concentration reaching the cement block was 2 x 10-4 times the starting
concentration, i.e., a 1.4 x 10 1 dilution across the diameter of the pool.
A test was preceded by introducing one test animal per pool, five days
prior to testing and two days after being caught. During this "settling in"
period, the animal was starved to heighten any response to potential attractants.
All work was done between 1800-0400 hr under diffuse flourescent light. A
potential attractant pumped into one pool reached the animal with a 10-12 sec
latency as evidenced by dye observation. Lobsters which walked upstream to
the immediate vicinity of the inlet tube within 4.0 min were scored as "responding".
Those not meeting this criterion were scored as "not responding". Time to
criterion was noted, as were the time to arousal and the time to initiate loco-
motion. Any unusual behavior was noted if it occurred. Repetition of this
sequence in the five other olfactometers constituted one "run". A single
potential attractant was tested on a single group of six "naive" lobsters over
two successive evenings. An evening's work started with two runs of shrimp
extract* to provide a measure of that group's general level of responsiveness
to chemical stimulation. A series of six runs were then made with the test
substance at three concentrations, 10-2, 10-3, and 10-4 g/liter (where solubility
allowed, otherwise as noted in results), in counterbalanced sequence so that
the three concentrations occurred in every possible order. This protocol was
repeated the second evening to provide a total of 72 presentations (to six
organisms) of each potential attractant. A new group of six lobsters was used
for each substance tested to eliminate any possible effects of conditioning
introduced by repetitive testing and/or feeding, required for long-term main-
tenance in the laboratory.
Data analysis is based on the responsiveness of each animal to the test
substance. The number of responses (criteria met) of each animal to the
test substance at one concentration (max possible = 4) is compared to that
animal's number of responses to standard shrimp extract and expressed as a
percentage. Animals not responding at all to standard shrimp extract are
excluded from statistical analysis. Percent scores thus obtained are tested
for significance in an analysis of variance (unequal group size, unweighted
mean). The analysis indicated significant concentration-dependent effects, as
expected; these were localized to the highest concentration used (10-2 gm/liter).
*Shrimp extract was prepared from 50 gm wet weight headed, shelled P. duorarum
homogenized in 200 ml artificial seawater. The resulting homogenate was centri-
fuged for 30 min at 12,000 G. The clear supernatant was decanted and frozen until
use. For use, the supernatant was thawed and brought up to 1,000 ml volume with
filtered natural seawater to provide a 50 gm/liter stock solution. One hundred
ml aliquots of the stock solution were diluted to 1,000 ml to produce a 5 gm/liter
standard shrimp extract concentration used in all behavioral assays. (This
solution was diluted in the apparatus to a final concentration of 10-3
gm/liter at the animal.)
9
Rankings were therefore computed for responses to the 10-2 gm/liter runs only.
Substances were ranked for relative attractiveness at this concentration by
comparing the mean percentage of responses, obtained by summing the scores of
all animals for a given substance and dividing the sum by the number of animals
tested with that substance.
Releasant Assay To evaluate the appropriateness of gels as dispensers for
attractant compounds, the materials' leach rates were measured in a continuous-
flow apparatus designed for this purpose. (Fig. 3) The retentiveness of samples
S -
f .- .-_ _. .. --.-
Fig. 3 Apparatus used to measure leaching rates of various potential
releasants. Flow rates into each test container can be regulated
and balanced via flow-control valves.
for the water-soluble dye uranine (sodium fluorescein) was determined coloro-
metrically by measuring dye concentration as a function of time in the effluent
surrounding each sample. A continuous, regulated flow of tap water into each
test container maintained maximum diffusion gradients around test samples.
The gels were placed in perforated 9 x 9 cm diam, screw cap, polyethylene
wide-mouth jars (Seapro, Inc., Rockland, Maine 04841), in trn suspended in the
test containers.
Laminated, controlled-release dispensers (Hercon brand, Herculite Protective
Fabrics Corp., New York, NY 10010) were also tested as potential release
vehicles. These represent a modified formulation of a plastic dispenser originally
developed for insect attractants (Beroza et al, 1974, Hardee et al.,1975), in
which a polymer matrix containing the dry chemical of interest is contained
between two protective barrier layers to form a 20 x 28 cm sheet-type dispenser
about 0.2 cm thick. (Fig. 4) Two forms of this dispenser were tested. One form
Controlled amounts of pesticide move from reservoir
layer to sustain active surface
Protective plastic barrier
Pesticide reservoir layer
Protective plastic barrier
Pressure-ensitive adhesive
Fig. 4 Diagramatic representation of the Hercon brand controlled-
release dispensers used to release attractant compounds in field
trials. Actual size, approximately 20 x 28 cm x 0.2 cm thick. The
pressure-sensitive adhesive layer was eliminated on the dispensers
used in the present study.
(Type A) used 5-mil polyvinyl chloride film as the barrier layers. The second
form (Type B) used unbleached muslin as the barrier layer, essentially exposing
the reservoir matrix directly to the environment. The amount of dry chemical
contained in these dispensers varied between 1 and 10 gm and is noted appropriately
in the Results section. Leach rates of these dispensers were laboratory assayed
by weight loss rather than colorometrically. Thirty, one inch squares were
suspended in the test containers on a wire loop with space between samples
to allow flushing. Weight loss was recorded daily and expressed as a percentage
of the average weight of dry attractant in a one inch square of the laminate.
One attractant, citric acid, was also released in field studies from a
natural source, dried citrus pulp (Life Guard Brand, Silver springs Citrus
Cooperative, Winter Garden, FL 32787). This material, available as a by-
product of Florida's citrus industry is a mixture of dried seeds, small pieces
of dried rind, and pelletized, dried pulp, commonly used as an agricultural
feed supplement. It was not assayed prior to field testing; the actual rate
of release of citrate from this material is unknown.
Field Tests The most attractive test chemicals were field tested using
the standard latch-trapping techniques common to the S. Florida fishery.
Traps were set on a sand/patch reef bottom, 18-21 meters deep, just offshore
from the Hillsborough Inlet, Broward County, Florida, in trawls of six traps
(1976) or 12 traps (1978). Traps were placed at the reef edge, and returned
as close to the same location as possible each time. The number of lobsters
caught with a given test attractant was compared to that caught with a control
bait, cowhide strips*. Baits were placed either in 25 x 12 x 2 cm wire mesh
bait baskets (cowhide, laminate sheets) or in perforated polyethylene containers
(agar, gels, dried citrus pulp). These in turn were secured to the top center
support of the traps. Traps with test baits were alternated with control-baited
*Accurate quantification of cowhide "odor", required for rigorous control, was
impossible since its fishing effectiveness appears correlated with its state
of decay. Bait baskets were initially filled to capacity and kept filled by
adding new material as needed each time the trap was pulled. Thus the
cowhide complement of a trap included material in all states of decay for
all but the first test.
traps within a trawl. The even number of traps/trawl assured both test and
control traps were equidistant from the trawl ends, thus compensating possible
end-effect bias. Forty-eight (24 test, 24 control) were fished in field experi-
ments 1-10; 94 in experiments 11-14. Following an initial four day "soak",
trawls were pulled every four days, weather allowing, the catch recorded
(size, sex, molt condition, female reproductive state), and the lobsters re-
turned to the habitat. Data were field-logged on magnetic tape and subsequently
transcribed in the laboratory.
RESULTS
Physiological Assay In all, 102 compounds were assayed physiologically.
Eighty-four of these met criterion on final analysis for numbers of bundles
and preparations tested. Mean activity ratios obtained for these chemicals
are summarized in Tables 1 and 2. The compounds tested include those reported
in the literature as (1) adequate stimulants for chemoreceptors of aquatic
organisms or (2) components of blood, muscle and/or urine of common marine
invertebrates and fishes. Some related analogs of these substances were also
included. Taurine (Table 1) was the most stimulatory single chemical tested,
with a mean activity ratio of 0.75 relative to that of standard shrimp extract.
Of interest was that the 10 highest ranking chemicals represent different classes
of compounds (e.g., fatty acids, amino acids, quatinary amines). Conversely,
single classes of compounds included strongly, weakly and moderately stimulatory
members (e.g., taurine 0.75; L-asparagine 0.16). No general correlation
existed between molecular type and stimulatory capacity. Hydrolysates of
proteins or protein mixtures proved moderately stimulatory at the concentrations
tested (Table 2::),although they are difficult to relate to the activities of
single chemicals on a molar basis. On a wt/vol basis, the complex compounds
traps within a trawl. The even number of traps/trawl assured both test and
control traps were equidistant from the trawl ends, thus compensating possible
end-effect bias. Forty-eight (24 test, 24 control) were fished in field experi-
ments 1-10; 94 in experiments 11-14. Following an initial four day "soak",
trawls were pulled every four days, weather allowing, the catch recorded
(size, sex, molt condition, female reproductive state), and the lobsters re-
turned to the habitat. Data were field-logged on magnetic tape and subsequently
transcribed in the laboratory.
RESULTS
Physiological Assay In all, 102 compounds were assayed physiologically.
Eighty-four of these met criterion on final analysis for numbers of bundles
and preparations tested. Mean activity ratios obtained for these chemicals
are summarized in Tables 1 and 2. The compounds tested include those reported
in the literature as (1) adequate stimulants for chemoreceptors of aquatic
organisms or (2) components of blood, muscle and/or urine of common marine
invertebrates and fishes. Some related analogs of these substances were also
included. Taurine (Table 1) was the most stimulatory single chemical tested,
with a mean activity ratio of 0.75 relative to that of standard shrimp extract.
Of interest was that the 10 highest ranking chemicals represent different classes
of compounds (e.g., fatty acids, amino acids, quatinary amines). Conversely,
single classes of compounds included strongly, weakly and moderately stimulatory
members (e.g., taurine 0.75; L-asparagine 0.16). No general correlation
existed between molecular type and stimulatory capacity. Hydrolysates of
proteins or protein mixtures proved moderately stimulatory at the concentrations
tested (Table 2::),although they are difficult to relate to the activities of
single chemicals on a molar basis. On a wt/vol basis, the complex compounds
Table 1 Mean activity ratios of
lateral chemoreceptors.
various pure chemicals to P. argus
Chemical X activity Chemical X activity
ratio ratio
Taurine
L-ascorbic acid
Proprionic acid
n-Valeric acid
L-glutamine
n-Butyric acid
B-alanine
Homarine
Creatine H20
Nicotinic acid
AMP
NH4C1
Histamine
Trimethylamine HC1
Betaine
L-proline
Glycine
L-histidine
L-isoleucine methyl
Fumaric acid
Hydroxy-L-proline
D-Galactose
L-lysine
a-picolinic acid
L-methionine
D ribose
L-valine
L-tyrosine
L-carnosine
Putrescine
0-phospho-L-serine
DL-lactic acid
a-lactose
L-citrulline
Glutathione
L-aspartic acid
i-inositol
Quinine
L-tryptophan
Guanidine HC1
L-leucine
L-malic acid
B-lactose
ester HC1
.75
.68
.68
.65
.61
.60
.57
.57
.56
.54
.54
.54
.52
.51
.51
.51
.50
.50
.50
.49
.47
.46
.44
.44
.43
.43
.42
.41
.41
.40
.39
.39
.39
.39
.38
.38
.38
.37
.36
.35
.34
.34
.34
L-glutamic acid
DL-ornithine
L-arginine
Maltose
D-fructose
Acetylcholine chloride
Inosine
Sucrose
Citric acid
D-glucose
DL-carnitine HC1
L-serine
UMP
AT P
L-alanine
N-acetyl-D-glucosamine
Urea
Hypoxanthine
r-amino-n-butyric acid
L-cysteine
2-aminoethanol
Levulinic acid
Sodium saccharin
Succinic acid
Sarcosine
Pyruvic acid
Phosphoethanolamine
D-mannitol
Adenosine
L-asparagine
Coumarin
IMP
D-sorbitol
Adenine
5-hydroxytryptamine
13
.33
.33
.32
.32
.31
.31
.31
.31
.31
.30
.29
.27
.27
.26
.26
.24
.24
.23
.23
.22
.22
.22
.21
.20
.20
.19
.19
.18
.17
.16
.16
.13
.13
.13
.11
Table 2 Mean activity ratios of various complex compounds to P. argus
lateral antennular chemoreceptors.
Compound Concentrations X activity
(gm/liter) tested ratio
Lactalbumin hydrolysate 0.046, 0.46, 4.6 .67
Bovine albumine 0.046, 0.46, 4.6 .57
Casein hydrolysate 0.038, 0.38, 3.8 .45
Ficoll 0.040, 0.40, 4.0 .39
Trypticase soy casein hydrolysate 0.046, 0.46, 4.6 .39
Hemoglobin (bovine) 0.068, 0.68, 6.8 .19
Taurine (from Table I) 0.013, 0.13, 1.3 .75
are 3-5 times more concentrated than, say, taurine, but have lower mean activity
ratios, even the most active of the group, lactalbumin hydrolysate (0.67 vs
0.75 for taurine). Rigorous comparison between the two groups, however, is
restricted by the possibility of anomolous dose/response relationships.
Behavioral Assay Nineteen single stimulatory substances and three mixtures
were assayed for their ability to elicit oriented locomotion in intact organisms.
Single compounds assayed behaviorally included the most stimulatory compounds
overall as well as the most stimulatory compound of each molecular type, unless
excessive cost or limited solubility necessitated using another representative
of a molecular type. Of the 19 single substances tested, 17 (proprionic acid
and 6-alanine runs were incomplete) were analyzed statistically. Mixtures
assayed behaviorally included (1) natural shrimp extract* prepared on the
same wt:vol basis as the dry single compounds, (2) a mixture of amino acids at
*Not to be confused with the single concentration of shrimp extract used as a
reference stimulant for each of the behavioral assays as described in Methods.
their component concentrations in the shrimp extract (Mixture A see Table 3)
and (3) an equal part mixture of the four most attractive single compounds
(Mixture B). The response to standard shrimp extract, 67.9% of all animals
tested (72.6% of those moving in at least 1 trial), verified its non-saturating
concentration, as estimated from preliminary experiments. Table 4 summarizes
the behavioral response data. Analysis of variance indicated overall signifi-
cance among chemicals tested, an overall concentration effect, and only a
weak interaction effect (Table 5).
Of the single compounds, citric acid attracted the greatest percentage of
lobsters relative to standard shrimp extract at the high and medium concentrations.
Overall significant differences occurred only at the highest concentration, however.
It was selected to rank order at this concentration since the outcome would be:
little affected by addition of medium and low values in view of the significant
interaction effect. Visual inspection of Table 4 verifies this. Other attractive
single substances are L-ascorbic acid (112.5%), succinic acid (108.3%), glycine
(98.5%), and trimethylamine HC1 (90.3%). Limited solubility of three compounds
valericc acid, egg albumin, nicotinic acid) prevented testing them at the same
concentrations as other substances, thus negating direct comparison of their
attractiveness. Overall, however, they appear less attractive than the highest
ranking compounds. No stimulatory single substance elicited repulsion in the
behavioral assay, although since the apparatus was not designed to test the
repulsion per se, a subtle repulsive effect could have gone unnoticed. It is
concluded from these studies that citric acid is the most attractive single
chemical of the group assayed, and therefore is the most likely substance for
field testing.
Table 3 Amino acids comprising abdominal tissue of Penaeus duoraruma
A.A. uM/ml A.A. yM/ml
Glycine 15.62 Isoleucine 0.176
Taurine 5.5 Methionine 0.173
Alanine 3.54 Asparagine 0.167
Proline 1.904 Tyrosine 0.153
Arginine 1.473 Aspartic Acid 0.138
Glutamine 1.053 Threonine 0.106
Valine 0.384 Phenylalanine 0.090
Leucine 0.317 Lysine 0.081
Glutamic Acid 0.275 Histidine 0.054
Serine 0.334
aFormulation supplied by Dr. Kenneth Blumenthal, University
Table 4 -
of Florida.
Response (oriented locomotion towards a source of ...) of intact
P. argus to selected stimulatory substances at three concentrations,
expressed as a percentage of response to standard shrimp extract.
Compound % response at concentrations of:
10-2 gm/liter 10-3 gm/liter 10-4 gm/liter
Shrimp extract 200.0 100.0 66.8
Citric acid 161.0 108.3 44.5
Mixture B 115.2 86.2 84.7
L-ascorbic acid 112.5 54.2 27.8
Succinic acid 108.3 45.8 33.2
Glycine 98.5 52.8 32.0
Trimethylamine HC1 90.3 41.7 41.7
Betaine 87.5 70.8 16.7
Mixture A 87.5 62.5 112.5
Taurine 83.3 63.8 59.7
L-glutamic acid 77.8 63.8 30.5
2-Aminoethanol 65.0 31.6 10.0
L-lysine 54.2 20.8 33.3
L-aspartic acid 54.2 33.3 25.0
Casein hydrolysate 45.8 20.8 25.0
Sucrose 45.0 30.0 50.0
a-lactose 40.3 33.3 32.0
Valeric acid* 40.2 5.5 5.5
Egg albumin* 33.2 40.0 51.6
Nicotinic acid* 20.0 5.0 5.0
*See p. 17, top
*Solubility limited the concentrations tested of these chemicals to:
Nicotinic acid 3 x 10-3 3 x 10-4 3 x 10-5
Egg albumin 6 x 10-4 6 x 10-5 6 x 10-6
Valeric acid 6 x 10-3 6 x 10-4 6 x 10-5
Table 5 Analysis of variance of the results presented in Table 4*.
Source of variance.... Calculated F score required
F score for significance
(p 2 .01)
1. among substances, overall 3.01 2.35F. (15,60)
F.01
2. among concentrations of
single substances 33.23 4.61F.0 (2,,'.)
3. interaction of 1. & 2. 1.62 1.46 (30, o)
F.05
4. among substances, high
concentration 2.51 2.35
5. among substances, medium
concentration 0.97 2.35 (N.S.)
6. among substances, low
concentration 0.17 2.35 (N.S.)
*Excluding nicotinic acid, egg albumin,
limited solubility.
and valeric acid due to their
Of the mixtures assayed behaviorally, the complete shrimp extract attracted
the greatest percentage of lobsters (at the high test concentrations) relative
to the standard shrimp extract. It also attracted more lobsters than did
the most attractive single compound, citric acid (200% vs 161%). The amino
acid component of the shrimp extract (Mixture A) was less attractive than the
complete extract (87.5% vs 200%). Combining the four most attractive single
compounds citric acid, ascorbic acid, succinic acid and glycine into
Mixture B attracted fewer lobsters than did the most attractive single substance
by itself (115.2% vs 161% for citirc acid). Mixture B elicited a response
(115.2%) about the same as the mean of its four component substances tested
individually (120%).
Releasant Assay Gelatin redissolved in seawater within a few hours of
solidifying, thereby proving it ineffective as a releasant. Polyacrylamide
gels, prepared as described by Allen et al. (1975), would not set when combined
with our attractant solutions. In light of the neurotoxic effects of the
acrylamide monomer, this alternative was not pursued further in the present
study. Agar gels (Difco bacteriological agar, technical grade) remained stable
in seawater and, contained in polyethylene bait containers,.resisted damage
from handling. Dye concentration from 10 cm diam X 3 cm thick discs of 1.5%
agar decremented 2 orders of magnitude over 3 days. Increasing agar concen-
tration to 5%, increased dye retention slightly, with dye concentrations decre-
menting 1 1/2 orders of magnitude over 3 days. Containing 1.5% agar discs in
plastic mesh bags instead of the polyethylene bait containers did not alter
release rates, indicating the container itself was not rate limiting. Stronger
acidic attractants (e.g., citric acid) prevented hardening of the agar discs
when prepared by dissolving powdered agar in the attractant solution. This
problem was overcome by buffering attractant solutions (NaHC03) to pH 7.5.
Two formulations of the-plastic laminate were tested. One (Type A), prepared
with 18-20% citric acid by weight, lost 30% of its contained dry attractant over
5 days. The second (Type B), prepared with 17% citric acid by weight, lost
30% of its contained dry attractant over 5 days.
Field Assay An initial series of 8 trawls placed varying distances offshore
localized the greatest catches (1976) to the 18-21 meter depth zone subse-
quently used in the present study. Table 6 summarizes the results of the
field trials. Experiments 1-3 test the effectiveness of two concentrations
of citric acid as an attractant when released from agar discs.. The low
individually (120%).
Releasant Assay Gelatin redissolved in seawater within a few hours of
solidifying, thereby proving it ineffective as a releasant. Polyacrylamide
gels, prepared as described by Allen et al. (1975), would not set when combined
with our attractant solutions. In light of the neurotoxic effects of the
acrylamide monomer, this alternative was not pursued further in the present
study. Agar gels (Difco bacteriological agar, technical grade) remained stable
in seawater and, contained in polyethylene bait containers,.resisted damage
from handling. Dye concentration from 10 cm diam X 3 cm thick discs of 1.5%
agar decremented 2 orders of magnitude over 3 days. Increasing agar concen-
tration to 5%, increased dye retention slightly, with dye concentrations decre-
menting 1 1/2 orders of magnitude over 3 days. Containing 1.5% agar discs in
plastic mesh bags instead of the polyethylene bait containers did not alter
release rates, indicating the container itself was not rate limiting. Stronger
acidic attractants (e.g., citric acid) prevented hardening of the agar discs
when prepared by dissolving powdered agar in the attractant solution. This
problem was overcome by buffering attractant solutions (NaHC03) to pH 7.5.
Two formulations of the-plastic laminate were tested. One (Type A), prepared
with 18-20% citric acid by weight, lost 30% of its contained dry attractant over
5 days. The second (Type B), prepared with 17% citric acid by weight, lost
30% of its contained dry attractant over 5 days.
Field Assay An initial series of 8 trawls placed varying distances offshore
localized the greatest catches (1976) to the 18-21 meter depth zone subse-
quently used in the present study. Table 6 summarizes the results of the
field trials. Experiments 1-3 test the effectiveness of two concentrations
of citric acid as an attractant when released from agar discs.. The low
catches with citric acid indicate this system is ineffective. Experiment 4,
identical to experiments 2 and 3 except that betaine (as the hydrochloride)
is substituted for citric acid as the potential attractant, tests the possibility
that the agar discs may provide an effective release vehicle if used with another
type of compound. The relatively higher catch (33% vs 2-8% of expts. 2,3) with
betaine suggests that citrate may be limiting in the first experiments, but
this difference must be considered re inter-trial variability. Experiments 5-7
show that within the test design, unbaited traps can fish as well (expts. 5,7)
or much better (expt. 6) than the artificially baited traps, thereby invalidating
the significance of the differences obtained in experiments 1-4.
Technology exists to produce more controlled release than that possible using
agar blocks by dispersing the attractant in various types of polymeric matrices.
Experiments 8-12 test the effectiveness of citric acid as an attractant in one
form of polymeric release system, a laminated sheet. Two formulations of the
polymer were tested (see Methods). While laminated dispensers of the first
formulation (Type A) containing 1 gm (expt.8) and 10 gm (expts. 9, 10) citric
acid caught more lobsters than did the citric acid/agar disc system, the number
of lobsters caught was less than that caught with cowhide. Again, the percentages
caught overall in experiments 8-10 did not exceed the percentages caught overall
in unbaited traps (expts. 5-7), indicating that variations in the catch effect
were likely due to parameters other than those controlled in the present experi-
ments. This idea is supported by the lower catch with 10 gm of citric acid
(expt. 9) than with 1 gm of the attractant (expt. 8).
Experiments 11 and 12 test the effectiveness of the second formulation (Type B)
of the citric acid/polymeric sheet release system. These experiments (see Methods)
fished about twice as many traps as previous experiments in an attempt to offset
20
the large variations in catch effort characteristic of experiments 1-10. In
both experiments the number of losters caught was less than that caught with
cowhide. In these trials, however, the total catch was too low for meaningful
results. The limited supply of laminated dispensers available precluded further
testing of the citric acid/polymer system.
Experiments 13 and 14 test the effectiveness of a natural source of citric
acid available as a by-product of Florida's citrus industry, dried citrus pulp.
While the number of lobsters caught with citrus pulp is not significantly lower
than that caught with cowhide, the small size of the total catch again precludes
drawing any meaningful conclusion from these results. Further testing of the
dried citrus pulp was not attempted.
Table 6 Results of field trials with selected chemical attractants for
P. argus.
Attractant
Tested
Citric acid
Citric acid
Citric acid
Betaine HC1
none(empty)
none(empty)
none(empty)
Citric acid
Citric acid
Citric acid
Citric acid
Citric acid
Dried citrus
pulp
Dried citrus
pulp
Attractant No. Caught
Concentration* Releasant Test Control
2 gm Agar 9 77
20 gm 1 60
20 gm 3 38
20 gm 14 42
18 109
36 41
23 69
1 gm laminate A 25 63
10 gm 10 36
10 gm ** 16 66
7.5 gm laminate B 1 7
7.5 gm 3 26
5 9
4 7
*per trap
**Laminate A
also included a surfactant (Triton X-100) in this expt.
Expt.
No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Date
5/76
5/76
5/76
6/76
6/76
6/76
6/76
6/76
6/76
7/76
5/78
5/78
6/78
6/78
Catch.as
% of
Control
11.7%
1.7%
7.9%
33.3%
16.5%
87.8%
33.3%
39.7%
27.8%
24.2%
14.3%
11.5%
55.6%
57.1%
DISCUSSION
The discussion will interpret the results relative to the development
of an artificial attractant for the spiny lobster fishery. Implications of
the results towards understanding basic chemosensory organization in lobsters
has appeared elsewhere (Johnson and Ache, 1978) and will be considered further.
in a forthcoming publication (Clark and Ache, In preparation).
The physiological assay, in retrospect, provided little additional defini-
tion of potentially attractive compounds than did the initial selection of
compounds to be assayed physiologically from the published literature. The
10 most attractive compounds in the behavioral assay (Table 4) spanned 80% of
the range of physiological activity (Table 1). More formally, calculation of
the variance between the rank order of compounds based on attractiveness (Table 4)
and the rank order of these same compounds based on stimulatory ability for
antennular chemoreceptors (Table 1) shows no correlation in the paired rank
orderings at the 0.05 confidence level. A likely reason for the apparent lack
of power of the physiological assay is that any one substance was only tested
on too small a percentage of the receptor population. It is known that P. argus
lateral antennular filaments contain over 100,000 aesthetasc-type chemoreceptors
per filament (Laverack and Ardill, 1965). It also now appears the receptor
population is not homogeneous relative to the response spectra of individual
receptors (Fuzessery et al., 1978). This problem would be minimized by using a
whole-organ assay as the electroantennagram, but this technique has not been
applied successfully to marine organisms in our laboratory to date. It is also
possible that resolution among chemostimulants, particularly those of similar
composition, is a higher-order function of lobsters requiring at least partial
integration of the primary sensory information. If this is .true, a higher-order
physiological assay, e.g. heart rate or some other parameter associated with
arousal, would be more appropriate as an attractant screen than assaying
primary receptor activity. Physiological assays have the potential to
efficiently screen behaviorally-active chemical stimuli, but further work
is in order to define a system with stronger behavioral correlation than the
one used in the present study.
Some of the single compounds that were maximally attractive to the spiny
lobster also ranked high in other behavioral studies rank ordering stimulatory
compounds for decapod crustaceans and, as such, may be suitable attractants for
decapods other than the spiny lobster. Table 7 summarizes the appropriate data.
One other study ranked citric acid among the more stimulatory chemicals;
glycine was ranked among the more stimulatory chemicals for four other decapod
species. Caution is in order, however, for these studies, except Carr and
Gurin (1975), initially select substances for testing based on the same sources,
i.e., the published literature. They do not "work down" from a complete, complex
food source to identify all the stimulatory compounds. For example, in
Palaemonetes pugio, glycine, taurine and glutamic acid are the most stimulatory
"off the shelf"-type components of human serum that elicit feeding behavior,
but other compounds, higher molecular weight proteins, are more potent sti-
mulants of feeding behavior (Carr and Gurin, 1975). It appears each species
should be assayed independently to determine its maximum sensitivity until further
knowledge of crustacean chemostimulants allows more rigorous generalization.
The above discussion leads to the question of mixtures. It may be limiting
to focus on the "best" single compound if mixtures of substances are far more
attractive than any single substance. The idea of mixtures being more potent
stimulants than single compounds is supported by studies on Carcinus maenas
Table 7 Comparison of single compounds found maximally stimulatory in
eliciting food-finding (*) or feeding (**) behavior in decapod
crustaceans. Each study tested a different number and array of
compounds from which those listed were scored as most stimulatory.
Panulirus1 Palaemonetes2 Petrolisthes3 Penaeus4 Homarus5 Cancer6
Citric acid X X
Ascorbic acid X
Succinic acid X X
Glycine X X X X X
TMA X
Betaine X X
Taurine X X
Glutamic acid X X X X
1 Table 4
2 Carr and Gurin, 1975**
3 Hartman and Hartman, 1977**
4 Hindley, 1975*
5 Mcleese, 1970*
6 Allen, et al., 1975*
(Shelton and Mackie, 1971) and Homarus gammarus (Mackie, 1973) that show none
of the components of synthetic mixtures of chemicals based on the composition of
natural foods are as attractive as the complete mixtures. Similarly, stimulation
of feeding in Palaemonetes pugio by human serum can only be fully accounted for
by the combined action of 6 types of serum proteins and 37 low molecular weght
constituents (Carr and Gurin, 1975). The results of the present study, however,
show that selected single compounds, i;.e., citric acid, approach the attractiveness
of complex mixtures, i.e., shrimp extract, when compared on an equal weight per
unit volume basis. They further show that a single compound, citric acid, can
be more attractive than a mixture of four individually attractive compounds,
Mixture B, again when compared on an equal weight per unit volume basis. One
can conclude for the spiny lobster, at least, that single compounds can effectively
substitute for mixtures as artificial attractants. Assuming a positive dose/response
curve characterizes citric acid over its field-effective concentration range as
it does over the concentration range tested herein, rate-of-release might be a
suitable variable with which to even increase the catch effect of a single
substance as citric acid.
None of the substances tested was a "super-attractant", i.e., none greatly
exceeded the potency of a natural stimulus as shrimp extract. It remains to be
tested if highly stimulatory single compounds as citric acid could potentiate
or enhance the potency of more complex natural stimulants as sh-imp extract
when combined with the latter and appropriately released.
The field trials were inconclusive, as noted in Results, which precludes
a meaningful economic analysis of the use of artificial attractants and its
potential impact on the fishery. The field trials do provide a useful base
on which to design future field tests by indicating lower limits of the number
of trap-efforts required to offset the large variability inherent in these trials.
The use of natural sources of "artificial" attractants rather than commercially
prepared controlled-release vehicles is worthy of immediate further evaluation
for, if successful, it could be incorporated into the fishery directly without
the relatively large (estimated $15,000) initial cost to develop and pilot
manufacture a sufficient number of controlled-release devices for adequate
field testing.
CONCLUSION
Overall, the results indicate that simple organic compounds ar" effective
attractants for the spiny lobster. Of particular interest to the S. Florida
lobster fishery is that the strongest attractant defined by this study, citric
acid, is readily available in the form of dried citrus pulp as a byproduct of
the Florida citrus industry and, as such, must be considered a potentially
useful source of trap bait for the fishery. Deployment of artificial attractants
as citric acid using controlled-release methodology remains an interesting
concept, but requires further testing to evaluate its potential application to
the marine environment.
ACKNOWLEDGEMENTS
We would like to thank Drs. William Richards and Herman Kumpf of the
Southeast Fisheries Center, NOAA, Miami, FL, for generously providing the
research space and running seawater facilities required for the behavioral
assays. 'Dr. Agis Kydonieus of the Herculite Protective Fabrics Corporation,
New York,. NY, provided, at no cost to the project, the Hercon dispensers, for
which we are most appreciative. We also thankfully acknowledge the services
of Mr. Mal Rowand, Rowand Fisheries, Ft. Lauderdale, FL, and Ms. Pam Reeder
of our laboratory, which allowed field trials to be part of the project.
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