• TABLE OF CONTENTS
HIDE
 Title Page
 Introduction
 Methods
 Results
 Discussion
 Conclusion
 Acknowledgement
 Reference






Group Title: Technical paper - Florida Sea Grant Program ;, no. 10
Title: Chemical attractants of the Florida spiny lobster, Panulirus argus
CITATION PAGE IMAGE ZOOMABLE
Full Citation
STANDARD VIEW MARC VIEW
Permanent Link: http://ufdc.ufl.edu/UF00072274/00001
 Material Information
Title: Chemical attractants of the Florida spiny lobster, Panulirus argus
Series Title: Technical paper - Florida Sea Grant College Program no. 10
Physical Description: 28 p. : ill. ; 28 cm.
Language: English
Creator: Ache, Barry W ( Barry Walter ), 1943-
Johnson, Bruce R. ( Bruce Randall ), 1942-
Clark, Edward ( joint author )
Publisher: State University System of Florida, Sea Grant College Program
Place of Publication: Gainesville
Publication Date: 1978
 Subjects
Subject: Spiny lobsters -- Florida   ( lcsh )
Genre: government publication (state, provincial, terriorial, dependent)   ( marcgt )
bibliography   ( marcgt )
non-fiction   ( marcgt )
 Notes
Bibliography: Bibliography: p. 26-28.
Statement of Responsibility: by Barry W. Ache, Bruce R. Johnson and Edward Clark.
General Note: "October 1978."
Funding: Technical paper (Florida Sea Grant College) ;
 Record Information
Bibliographic ID: UF00072274
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved, Board of Trustees of the University of Florida
Resource Identifier: aleph - 000990240
oclc - 04741025
notis - AEW7152

Table of Contents
    Title Page
        Title Page 1
        Title Page 2
    Introduction
        Page 1
        Page 2
    Methods
        Page 3
        Animals
            Page 3
        Physiological assay
            Page 4
            Page 5
        Behavioral assay
            Page 6
            Page 7
            Page 8
        Releasant assay
            Page 9
            Page 10
        Field tests
            Page 11
    Results
        Page 12
        Physiological assay
            Page 12
            Page 13
        Behavioral assay
            Page 14
            Page 15
            Page 16
            Page 17
        Releasant assay
            Page 18
        Field assay
            Page 18
            Page 19
            Page 20
    Discussion
        Page 21
        Page 22
        Page 23
    Conclusion
        Page 24
    Acknowledgement
        Page 25
    Reference
        Page 26
        Page 27
        Page 28
Full Text













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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