REACTION OF TUNAS
TO STIMULI, 1952-53

Marine Biological Laboratory

' LIBRARY

JUN2 7 IS55
WOODS HOLE, MASS.

SPECIAL SCIENTIFIC REPORT-FISHERIES Na 130

UNITED STATES DEPARTMENT OF THE INTERIOR
FISH AND WILDLIFE SERVICE

Explanatoiy IToto

The series embodies results of investigations, usually of
restricted scope, intended to aid or direct management or utilization
practices and as i!;uides for administrative or legislative action. It
is issued in limited quantities for the official use of Federal, State
or cooperating Agencies and in processed form for economy ajid to avoid
delay in publication.

United States Department of the Interior, Douglas McKay, Secretary
Fish and Wildlife Service, John L. Farley, Director

REACTION OF TUNA TO STIMULI, 1952-53

Part I: Response of tuna to chemical stimuli, by Albert L. Tester,

P. B. van Weel, and John J. Navighton
Part II: Response of tuna to visual and visual-chemiical stimuli, by

Sidney C. Hsiao and Albert L. Tester

Special Scientific Report: Fisheries No. 130

Note:--This report is also Contributions Nos. 47 and
48 of the Hawaii Marine Laboratory,
University of Hawaii

WASHINGTON: MARCH 1955

Accepted for publication June 1954

CONTENTS

Page
Part I: Response of tuna to chemical stimuli 1

Fishingc 2

Establishment of tunas in captivity 3

Concrete taink 3

Pond Noo 5 4

Feeding 5

Methods and procedures in testing , 7

Concrete tank 7

Pond Noo 5 11

The response 13

Problems in testing 16

Weather 16

Power failure 17

Erratic behavior of the fish 17

Measurement of response 18

Variation in response. 24

Source and preparation of test substauices 27

Materials c 27

Prepeuration 28

Response to simple extracts of tuna and other fish 30

Flesh extracts of tuna 30

Flesh extracts of other fish 32

Page

Extracts of tissues other than flesh 32

Cajinery by-products: preservation. , 33

Response to chemical compounds and miscellaneous

materials 34

Attempts at isolation and identification of the

attractant(s) 37

Summary and discussion 57

General 57

Nature of the attractive substance 58

Preservation of the attractant 59

Possibility of conditioning of the fish 59

Literature cited,. 62

Part II: Response of tuna to visual and visual-chemical

stimuli 63

Methods 63

Response under control and experimental conditions 67

Pattern of response 67

School entrances 70

Percentage of Tinme in Area 70

Fish-seconds in area 71

Speed of swimming , 72

Feeding activitv 73

Response to lures of different colors 74

SuiTii-nary 76

Appendix: A summary of experiments on chemical stimulation

conducted in tank and pond 77

ILLUSTRATIONS

Figure Page

o
1. Temperature ( C„), chlorinitv (p„ po m,, )(, and

mortality (number of fish dying) in Pond NOc 5

over the period from September 1^ 1952 to

June 30, 1953 6

Ze Diagram of the concrete tank showing the arrange-
ment for introducing the test materialSj the
attraction area^ and the obse rvation booth o 8

3o Number of passes and time (seconds) out of

successive 2-minute periods for three tank
experiments - NoSo 57, 58^ and 59 c 10

4. Diagram of Pond NOo 5 showing the arrangement
for introducing test materials ^ the attraction
area, and the observation tower. ...„ 12

5„ Number of passes and time (seconds) out of
successive 3-minute periods in six pond
experiments - Nos, 136 to 141, inclusive 14

6o Number of passes and time (seconds) out of

successive S^tninute periods in seven pond
experiments - NoSo 94 to 100, inclusive^ 20

7o Time (seconds) out of successive 3-minute periods
in pond experiment NOo 112^ in which a
gradient of stock extract of skipjack flesh was
established for 48 minutes o . 31

Bo Fractionation of stock extract of skipjack flesh:
procedure euid results in experiments No8„ 83
to 86, and 88 40

9o Fractionation of an alcohol extract of skipjack
flesh? procedure and results in experiments
Nos. 178, 179, 181, and 182 44

lOo Fractionation of aji alcohol extract of skipjack
fleshs procedure and results in expe riments
Nosc200 to 202, and 204 to 206 45

11. Fractionation of alcohol extracts of skipjack flesh:
procedure and results in experiments NoSa 215,
216, 218p and 223 to 225 47

Figure Page

i2„ Fractionation of an alcohol extract of bigeye

flesh: procedure and results in experiments

Nos„ 26-1, 262, 266 to 269. 272, and 277 49

13. Fractionation of a first alcohol extract of bigeye

flesh: procedure and results in experinnents

Nos. 273 to 276, and 278.. 51

14. Fractionation of a second alcohol extract of

bigeye flesh: procedure and results in

experiments NoSc 280 to 283 52

15. Fractionation of a third alcohol extract of

bigeye flesh: procedure and results in

experiments NoSo 285 to 287, 54

16. Fractionation of a second alcohol extract of

bigeye flesh: procedure and results in

experiments NoSo 298 to 303 . , 56

17. Diagram of mechanical device for lowering

lures into water: AB - crosspiece; LL' ~

lures; U - supporting post; F - fulcrum;

C - notch; P - plate to cover notch; W -

weight; C - cord ......,, 64

1 8„ Diagram of transcribing^recording device: A -
lever operated by toggle switch (E) for re-
cording "passes"; B - lever operated by
dial (D) for recording nuinber of fish in area;
C - timer; F - kymograph „ 66

19o Typical kymograms: A - under control conditions;
B - when stinnulated by lures; C - when
stimulated by lures plus extract 68

Fronti8piece--Tuna Study Pond at Coconut Island, Oahu

PART I

RESPONSE OF TUNA TO CHEMICAL STIMULI

BY

Albert L„ Testerp P., Bo van Weelj and
John Jo Naughton
University of Hawaii

In 1951„ exploratory studies of the response of tuna and
other fish to chemicalj, visual^ auditory, and electrical stimuli were
conducted by Po Bo van Weel„ So Co HsiaO;, !„ Miyake„ and Ao Lo
Tester (Special Scientific Reports Fisheries Noo 9l> of the faculty of
the University of Hawaii under contract (Nool^fw-.l 3 33 1) with the
U„ So Department of the luteTior, Fish and Wildlife Service^ Pacific
Oceanic Fishery InvestigationSo It was hoped that these studies not
only would contribute to an understanding of tuna behavior but also
would provide an indication of ways of attracting tuna to within the
reach of a fishing vessel at seao

Of the various 1951 studies;, the most promising was in
the field of chemoreceptiono Van Weel fl952) found that little tunny
(Euthynnus yaitoll and yellowfin (Neothunnus macropterus) held in cap-
tivity responded to cleaXj, almost colorless extracts of tuna fleshy

A new l=.year contract (Noo I6fw.= 18564) for a further study
of the reactions of tuna to chem.ic;al stimuli became effective June 1,
1952a Efforts were to be made to duplicate and extend van Weel's ob-
servations „ to identify chennicilly the attractive 8ubstance(8), and to
make observations on the reaction of the fish to combined chemical
and visual stimulio

This report deals with the 1952-53 results in the field of
chemoreception and includes a brief account of the success attained in
fishing and establishing the tuhas in captivityo

Notes Contribution No. 47 of the Hawaii Marine Laboratory,
University of Hawaii,

The tunas used in the study were caught and transported to
Coconut Island by Mr. Lester Zukeran, skip^ier of the University of
Hawaii research vessel Salpa. He was ably assisted by Mr. Stanley
Kitagawa and Mr. Royden Ikeda, students at the University. In the
ponds, the tunas were fed and otherwise cared for by Mr. Charles
Ncdiamoto, assistant to the Hawaii Marine Laboratory, who also took
water temperatures and samples of water for chlorinity determination.

At times, Mr. Austin Pritchard and Miss Shirley Trefz,
graduate students in nnarine zoology, assisted in pond testing,
Mr. Carl Swanholnn, student in chemistry, assisted in preparing and
analyzing test materials.

Samples of chemicals were provided gratis by E, I.
DuPont de Nemours and Company through Mr. Raoul Pantaleoni, by
Van Ameringen-Haebler, Inc. , through Dr. Ernst T. Theimer, by
P. R. Dreyer, Inc. , through Mr. George H. Zirkel, and by Sindar
Corporation through Mr, R, E. Horsey,

Mr. Fritz Jermann of Hawaiian Tuna Packers, Ltd. ,
spent r^any hours preparing materials for testing and assisted in
many other ways. Certain materials were also forwarded by Mr. Pete
Sunderland of B. C. Packers, Ltd.

To all of these individuals, to the companies they repre-
sented, and to others who assisted in various ways we extend our
sincere thanks. We are also indebted to Mr. O. E. Sette, Director,
and other members of the stciff of the Pacific Oceanic Fishery Inves-
tigations, U, S. Fish and Wildlife Service, for advice, assistance,
and encouragement.

FISHING

As in the previous year (Tester 1952), tuna were caught
by trolling with the 46-foot Salpa, using two lines suspended fronn
each of two poles fastened to the mast amidships, and sometimes
using one or two additional lines fastened to the stern. The ship
operated three mornings a week off Kaneohe Bay during the period
June 6 to August 31, 1952. As shown in table 1, the catch consisted
oi 63 skipjack (Katsuwonus ijelamis), 66 kawakawa or little tunny
(Euthynnus affinis), 1/33 yellowfin (Neothunnus nnacropterus ), 6 dol-
phin (Coryphaenus hippurus), and 2 wahoo (Acanthocybium solandri).
The catch ; er hour (all species) was low in June (1. 70) but higher in

1/ Euthynnus yaito oi yrevious veuoi'ts (Tester 1952: van Weel 1952).

2

July (Zo 36) and August (Zo40)o The Ceitch per hour for the three
months (2o 17) was less than that for the same three months in 1951
(3o20)o

For tunny and yellowfiiHe the two species of tuna which we
have established in captivityp the catch per hour was higher in 1952
(Go 84 and 0o42) than in 1951 (0„40 and 0,31) for a comparable period.
For skipjack, however, the catch per hour in 1952 (OoSl) weis con-
siderably less than that in 1951 (l<,48)o

Fishing was not continued after August 1, 1952, (except
for incidental trips) as the tank and pond were stocked with sufficient
fish for our purposeso

Table lo--The number of fish caught^ fishing hours, and
catch per hour for the 1952 fishing operations
of the Salpa, with comparable data for 1951

Catch

per
hour

40

23-1/2

lo70

79

33-1/2

2,36

51

21-1/4

2.40

70

78-1/4

2. 17

17

67-3/4

3o20

ESTABLISHMENT OF TUNAS IN
CAPTIVITY

Profiting by last year's experience, attempts were made
to establish only yellowfin and tunny in the concrete tank and in
Pond No o 5^ which were used successfully in 1951 (Tester 1952)o
The tank and pond are located on Coconut Islando

Concrete Teink

The concrete taJik, which nneasures 35x11x4 feet and
is supplied with running seawater by punnps, was modified slightly
by rounding the corners with sheets of Masonite and painting the
walls whiteo Despite these improvements only 2 out of 1 1 yellowfin

and 2 out of 13 tunny were established, the former but a short
period of time.

Nine yellowfin (3 to 6 pounds) died within 1 or 2 days after
introduction to the tank, without starting to feed. One, introduced on
June 30 (4 pounds), started feeding on July 4 but was caught and
stolen by poachers during the same evening. Another, introduced
on July 14 (5-1/2 pounds), started feeding on July 22 but stopped on
July 26, In obvious difficulty, it was removed from the tank on
July 30 and was found to be blind, with bubbles in the eyeballs.

Eleven tunny {1 to 4 pounds) died within a few days after
introduction to the tank. Of these, two started feeding but after 12
days becanne blind; one died and the other, in obvious difficulty, was
removed from the tajik. Two tunny, introduced respectively on
June 16 and August 15, 1952, became established and lived throughout
the winter. They were still alive on June 30, 1953. Both weighed
about 1 pound when established and grew to about 4 pounds during the
year 2/.

Pond No, 5

This pond, described in detail by Tester (1952), is about
360 feet long and 75 feet wide. The south side is shallow over its
eritire length, averaging about 3 feet in depth at high water. The
north side includes a deep trough, which averages about 10 feet in
depths A slow circulation is effected by tidal action through screened
gates at both ends. Both yellowfin and tunny were established in this
pond in the sumimer of 1951 but died during the fall, following wet,
stormy weather.

Between July 23 and August 27, 1952, 13 yellowfin (5 to
10 pounds) were placed in this pond. Of these, five started feeding
and became established. Four of the 5 disappeared from the pond
during the first 2 weeks in September--it is suspected that they were
caught by poachers, although they may have died "naturally. " In any

2/ One of these jumped from the tank and died on July 19, 1953. It
was partially eaten (by rats or cats?) when discovered, and was
not weighed. The other started ramming the walls of the tank
and died on July 22, 1953, A film over the eyes indicated partial
blindness, which nnay have contributed to its death when alone in
the tank. It weighed 3-3/4 pounds at death.

event, the carcasses were not fotindo The fifth yellowfin died on
September 16, 1952o An additional yellowfin (10 pounds) was placed
in the pond on October 28, but it died 5 days l^ter without starting to
feedo

Of 15 tunny (1 to l^l/Z pounds) introduced to Pond NOo 5
between July 25 and August 25, 1952, 13 started to feed and became
established, despite the fact that several were tagged fthe tags later
became detached)o The remaining 2, which were tagged„ died within
2 days. The 13 established tunny were present until October 16, 1952,
following which 6 died 3 betv^een October 16 and 20^ 2 between
October 21 and 27, and 1 between October 30 and 31, The remaining
population of 7 fish was maintained until December 5^ 1952, when 1
more diedo The 6 remaining fish persisted until January l6j 1953o
One died on January 16^ 2 between January 17 and 20, and 1 between
January 22 and 23^ 1953„ The 2 tunny which remained survived the
winter and spring and are still living at the time of writing, more theia
1 year after they were introducedo During this period they have in-
creased in weight from about 1 to 4 pounds o

The reason for the mortalities of tunny reported above is
uncertaino The temperature and chlorinity in Pond NOo 5 are shown
in figure U The six destths in October coincided with the beginning of
a steep downward trend in temperature and a slight downward trend
in chlorinity, both of which were associated with the incidence of
rainy weathero However, the four deaths in Janu:jry cannot be related
to changes in temperature and salinityo Moreover, the two remaining
fish survived through February and Marchp when considerable rainy
weather was encountered and when the chlorinity in the pond reached
a low for the year (15o834 p„pomo on March 11, J.953)o

Although comparable temperature aind chlorinity data for
the previous year (when all fish died following wet stormy weather)
are not available, in generalp the winter weather in 1952-'5 3 was nnuch
better than in 1951-52, with markedly fewer "kona" storms (south to
southeast winds with rain)o This may have contributed to the success
in maintaining two of the fish throughout the winter „ During the one
bad storm of the 1952-53 season on October 13 and 14, mortality may
have been reduced by tying the Salpa to the dock near the outer gates
and flushing fresh water from the pond with the wash of the propeller„

In both the tank and pond the tunas were fed during the
late afternoon of Tuesday;, Thursday, Saturday, and occasionally
Sunday, of each weeko Their food consisted mostly of tuna flesh

Jl.

\ H

H 1 H

NUMBER DYING

H h

TEMPERATURE

-I h

1 1 1 1 \ 1 1-

SEPT. OCT. NOV. DEC JAN FEB MAR APR MAY JUNE

1952 1953

Fig. 1. --Temperature ( CI, chlorinity (p. p. m. ), and mortality (number offish
dying) in Pond No. 5 os'er rhe period from September 1, 1952 to June 30, 1953.

(skipjack, yellowfin, or bigeye) or mar lin- -whichever was available- -
plus such other scrap fish as came to hando In general they were fed
at the rate of about one-half pound of food per fish per feeding, which
is probably much less than their normal requiremento This schedule
was maintained in order that the fish would be in a constant state of
hunger on Tuesdays^ Thursdays^ and Saturdays when tests were usu-
ally conducted. Van Weel (1952) showed that this precaution was
necessaryo

Two departures from this diet are worthy of notCo Firstly,
the tunny established in the tank on June 16^ 1952, was kept on a non=
tuna diet, consisting of squid, anchovy^ and other fish, until about
the middle of July when certain tests had been completed. Secondly,
following April 25^ 1953, the two tunny in the pond were fed on im-
ported (frozen) squid exclusivelyo

METHODS AND PROCEDURES IN TESTING

Concrete Tajik

Tests were run in the concrete tank 2 or 3 times a week
from July 1 to August 20, 1952, and occasionally thereafter through-
out the remainder of the summer, the fall, and the wintero At first
the methods were similar to those used by van Weel (1952)o The ob-
server stood by the railings at the side of the tank near the south end
until the fish had become used to his presence and had resumed a
steady slow circling^ Several successive measurements and counts
were made of U) the time (in seconds t for 10 passes past the observ-
er, with the fish swimming either up or down the length of the tank,
and ^b) the number out of 10 passes made in the half of the tank ad-
jacent to the observero Materials were then siphoned into the tank
using a rubber hose, and the timing and counting were resumedo Ex-
citement induced when the fish sensed the material was indicated by
a decrease in the time of 10 passes (increase in rate of swimming)
and attraction was indicated by an increase in the number of passes
along the near side of the tanko

Often during attempts to establish control conditions the
fish would remain excited for long periods of time by the presence of
the observer, whom it could see; during both control and test periods
it might also respond to the observer's movements while tirming, in-
troducing the materials^ or recording the results o This difficulty was
largely overcome on July 23, 1952, (following Test 131 by enclosing
the upper railed section of the tank with tarpaper and building an ob-
servation booth (figo 2), The three-ply front of the booth overhung
the tank at its upper edge to increase the range of visiono It contained

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DO a

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tv'O rectangular 6 x 10 -inch observation windows the size of which
could be varied to a narrow horizontal slit by means of sliding doors.
Later, in August, the observation windows were equipped with one-
way glass o As nearly as could be determined, the fish were gener-
ally unaware of the presence of the observer as long as strict silence
was maintainedo Occasionally, however, before the glass was used
it seemed as if the fish were aware of the small part of the observer's
face which was visible through the small slit-like aperture.

By means of a 3/8=inch centrifugal pump driven by a
1/30 ho p. near-silent electric motor, water was sucked from the
surface of the tank at one side near the center eind was injected into
the southern or "downstream" one-third in a continuous stream dur-
ing both control and test periods. When desired, the test material
was introduced into this stream from within the booth through a glass
T connection in the rubber tubingc

The method of measuring the reaction was modified from:
that of van Weel (1952). Each test consisted usually of five succes-
sive 2-minute control periods, followed by at least five successive
2- minute test periodSo For each period, the time spent by the fish in
the downstream one-third of the tank, marked off with string, was
measured by an electric clock operated by a push switch, and the
number of loops (half»circles) nnade by each fish in this "attraction"
area was recorded on a hand counter. By means of fluorescein dye
it was found that a liquid quickly spread throughout the downstream
ore-third of the tank, remained there for about 10 minutes, and then
gradually dispersed up the tank along the far wallo If attracted by
the material, the fish would be expected to spend a longer period of
tiine in the attraction area as compared with control conditions. If
excited by the nnaterial the fish would be expected to swim more
rapidly and to circle more times in the attraction area than during
control conditions. If a fish entered, turned, and left the area, the
count would be one loopo If the fish entered, made a complete circle
within the area, and then left, the count would be three loops. If
two fish performed: in this way, either together or independently, the
counts would be doubled. Later the method of counting was modified
to conform with that in the pond (see below)o

The tests in the tank were conducted for the most part on
one established tunny during the sunnmerj aund on two established
tunny thereaitero Unestablished fish did not react to the test sub-
stances auid were ignored in timing and countingo A typical series of
reaction graphs is shown in figure 3.

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Pond No, 5

When weather and other conditions pernxitted, testa were
run in Pond Noo 5 two or three times a weekp starting in August 1952
and continuing throughout the fallj winterj, and spring. The general
arrangement for testing is illustrated in figure 4o

By means of a 2 -inch pump driven by a 3-ho Po electric
motor p water was sucked from outside the "seaward" gates at the
western end of the pond, led through a 2-l/2--inch rubber hose along
the concrete gate supports to a point near the center of the deep
trough in the pond, and then injected into the pond about 5 feet below
the surface (from the high tide mark) through an L^shaped piece of
2=inch galvanized pipeo Test material was introduced into the flow
through a funnel, the base of which was attached to a piece of 3/4-
inch galvanized pipe by a rubber hosCo The 3/4'=inch pipe was brazed
at an acute angle into a hole in the "down" section of the L-shaped
pipes creating a suctions The funnel was located within a small
house erected above the seaward gates„ thus effectively screening
the operator when waiting to introduce test liquidso

The pumpe which was rather noisy and which created a
strong visible flow of water for about 20 feet into the pond, was kept
running for sonne time before as well as during both control and test
periods to accustom the fish to both the noise and the currento After
tracing the path of the current with fluorescein dyej, an "attraction"
area was marked off with two pieces of cord stretched across the
pondo one 39 feet and the other 78 feet from the seaward gateso The
dye indicated that liquids introduced into the flow would reach the
near edge of this area within about 1 minute;, would spread throughout
most of the area within about 3 or 4 minutes^ and would for the most
part rennain In the area for ain additional 10 minutes before appreci-
ably dispersing downstream across the far cordo

To observe the fish, a wooden tower 20 feet high was
built opposite the center of the attraction area on the north side of
the pond, A platform and seat on top of the tower was reached by a
ladder. As the material was introduced when the fish were starting
to return from the far end of the pondj after pouring the nnaterial
down the funnel the observer was able to move quickly from the small
house to the tower^ climb the ladder, and sit on the platform before
the fish approached the attraction area. It is reasonably certain
that he was not noticed by the fish either when moving to the platform
or when seated on it. Timing ajid counting were conducted from the
platfornric Observation was improved by wearing polaroid sun
glasses,

11

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Each experiment, usually included five 3-minute control
periods and five 3- minute test periodSo During each period the time
spent by the tunny school or schools in the attraction area was meas-
ured on an electric clock operated by a push switcho The switch was
depressed when the first fish of a school entered the area and was
held down until the last fish lefto A fish swimming by itself was ig-
nored; often it was a partially=blind or otherwise incapacitated indi-
vidual which was unable to keep pace with the otherSo In addition to
timing the fish, each "pass" of each fish in either the upstream or
downstream direction was recorded on a hand countero ThuSj if
three fish swam through the areap the count would be 3o If they en-
tered the area, loopedp and left on the same side as they entered, the
count would be 60 If they entered the oreao desciibed a connplete
circle within the area aJid then left on the opposite side to which they
entered, the count would be 9o Time spent in the area was accumu-
lated on the clock and the number of passes was accumulated on the
covinter. Both were recorded at the end of each 3-nniinute periodo
When the tunny population was large (6 to 13 fish), two observers
were required on the towerp one of whom clinnbed down and introduced
the test material between control and test periods „ a-nd returned when
the fish were at the far end of the pond to avoid being seeno When the
fish were excited during the early part of a reaction it waa difficult
to record all the movementSj even with two observers; in some cases
the counts were nninimal estimateSo As in the case of the tank, the
difference between control and test periods in average time spent by
the fish in the attraction area was used as a measure of attraction
and the difference in count was used as a nneasure of the excitement
induced by the test substances A typical reaction graph is shown in
figure 5o

In both tank «md pond, in addition to the quantitative
mea^flure of reaction discussed above, the observer also indicated
the strength of the response in one of several "observational"
categories ( - to XXXX). These will be discussed in a later section,

THE RESPONSE

A summary of data on 356 experiments which were con-
ducted in the tank and pond at intervals over the period July l^ 1952,
to May 19, 1953, is given in the appendix a* the end of this reporto
The experiments will be referred to by numbeTj eogo Nos„ 168, 170,
etCo The results will be discussed in detail in later sections^ For
the present, attention will be directed at a description of a typical
response in the tank and the pondo

When aJi attractive substance, such as an extract of tuna
flesh, was introduced into the attraction area of the tank, the

13

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material was usually sensed by the tunny in its first pass into the
area,, Usually the fish would become excited immediatelyo It would
increase its swimming ratCj, often quickly circling the tank« On re-
turning to the area it would swim rapidly in snnall circles „ splashing
and swirling at the surfaceo At timies the fish would flex its dorsal
fin and snap with its moutho Occasionally it would root uneaten food
from the bottom and "pl^y" with it Calthough not eating it cts far as
could be observed^. This excitement would usually be prono\inced in
the first and second periods (4 nninutes from, start) but woxild grad-
ually lessen^ until after the fifth period (10 minutes from staxtj only
a more rapid rate of swinnming would be notedo If the reaction con-
tinued after the fifth period^ it would usually take place outside of
the attraction area along the far wall„ where the material had been
cariried by the slowp counterclockwise current in the tanko

When an attractive substance was introduced into the
pondp a reaction would taJce place innmediately if the tunny happened
to be near the point of injectioHo Usually^ however „ the material
was introduced when the fish were starting to return from the up-
stream end of the pond. A reaction usually ensued during the first
3-minute period^ as soon as the fish entered the attraction areas
When several tunny were present^ they would immediately depart
from a linearp follow<= the -leader type of schooling and fan out across
the pondj darting into and out of the attraction area at or near the
surface;, frequently breaking the surface with their backs and creat-
ing a "bow Wave" as they swanio Often individuals would make short
circles in the areSp splashing and biting at bubblesp dead leaves^ or
other objects which happened to be floating on the surface. This ac-
tivity usually lasted for two periods (6 minutes from start) and often
persisted with lessening tempo for five periods {lb minutes from
start* or more. When darting into and out of the area„ the point at
v/hich they turned back seemed to coincide roughly with the disper-
sion front of the matertalo the position of which could be inferred
from experiments conducted with fluorescein dyeo Usually the re-
action was over within 15 minutes and normal swimming up and down
the pond was resumedo Thus a typical strong reaction was indicated
by (a) increased rale of swimmings (b) surfacingp (cH breaking up of
usual school formation; a "f,anning out" of the schoolp often followed
by independent circling of individuals^ (d) return to area after passing
through the front of the substance^ (e) feeding activity? splashing,
lungingj and snapping at objects on the surface,,

There was considerable variation in both the pattern and
strength of the reactiono Sometimes the fish reacted to the material
for but a short time within earh period^ in the meantime racing the
entire length of the pond ajid quickly returning„ When only two tunny

15

were present the reaction was similar to that described in the fore-
going paragraph, but was less violent and less spectacular. Even
with a larger number of fish, it could vary in its intensity from the
very strong reaction described above to a slight sensing of the mate-
rial - a mere swerving in the area as the fish passed through. Often
only one or two (e, g. a and d above) of the five reaction components
were evident. Also, of course, many of the substances which were
tested gave no noticeable or measurable response,

PROBLEMS IN TESTING

From the foregoing account of the methods, procedures,
and response one might assume that precise, clear-cut conclusions
could be drawn from each test. This was not the case. There were
several uncontrolled variables which affected the results and which
nnade it difficult, and often impossible, to obtain reasonably consist-
ent and comparable data.

Weather

In the tank, when the sky was overcast the fish were
difficult to see and hence to time and count. Several experiments
v/ere conducted under unfavorable weather conditions, thus introduc-
ing an additional source of error in the quantitative data. Testing
during rain squalls was usually avoided, for the rain rippled the sur-
face and made observation very difficult. Occasionally, however,
rain occurred while experinnents were in progress, thus not only
changing the conditions for observation of the fish during test as com-
pared with control periods, but also providing a distraction which
might affect the reaction of the fish.

These same difficulties were encountered to even a
greater extent in the pond, where the water was deep>er, and the fish
were difficult to see from the tower when swimming deep. Occasion-
ally, visibility was greatly reduced by southeast "kona" winds, which
not only created a turbidity but also, blowing lengthwise down the
pond, created ripples on the surface. Often, to avoid wasting a whole
day, experiments were conducted under such conditions. Although
the resulting data were not strictly comparable with those obtained
during fair weather, a positive response during test periods could
usually be detected as, when reacting, the fish tended to swim at the
surface where they could be seen even under unfavorable weather
conditions.

16

Power Failare

During a Z-week p>eriod In November 1952(, and occasion-
ally at other timesp the power supply to the inland and/or to the pumps
failedo Rather than waste valuable testing timcp pond experinnents
were conducted without the use of the pump„ Test material was in-
troduced by throwing it to the sxirface from the top of the tower when
the fish were distant from the attraction area. It was noted that a
material which was usually attractive to the fish was not sensed as
soon as when introduced by the punnpo Apparently it formed a layer
on the surface under which the fish could swim without being aware
of its presenceo When it was finally sensedj, it produced a reaction
which was stronger than usual^ probably because it was more con-
centrated than when introduced through the flow from the pump.
Again, the data would not be strictly comparable with those obtained
when the material was introduced in a normal fashiono

Erratic Behavior of the Fish

In the tank under ideal control conditions the fish would
make full circles, keeping close to the walls and swimming at a
steady ratCo They would thus spend about one-third of their time in
the attraction area and each would average about 3 or 4 loops in a
2-mlnute periodo Howeverj ideal control conditions were not often
encounteredo Usually experiments were run when inspection of the
control data indicated fair regularity in movement and thus moderate
variance in the data, even though the times and counts might differ
considerably from the "idealo " A* timeSo particularly when one
tunny was the sole occupant of the tanks '* would circle for long
periods in either the upstream o* downstream end of the tank, giving
either zero or ma^tinnal times and counts o At times it would circle
for several successive minutes at one end ^gund would then repeat the
performance at the other endo At other tinaes it would swim back
and forth along one wallp entering the attraction airea either occasion<>-
ally or not at all. When behavior patterns such as these were en-
countereds testing was usually abandonedo with consequent loss of
tinneo Occasionally^ in desperationp the material was iniroduced
undei such conditions, in the hope that the fish would sense it and
show some response even though the data would not be comparable
with those obtained with nnore nearly normal behavior. When two
fish were present in the tanko even if one was not yet established^
the pattern of movennent was more uniform and usually yielded con-
trol conditions which did not depart excessively from the ideal.

In the pondj the laTge black yellowfin were more con-
spicnous and swam more slowly than the small blue-^green tunny,

17

Although their reactions were noted, they were not measured because
of the unsuitable swinnnning behavior. At times they would circle slow-
ly for hours at one end of the pond; at other times they would circle
for long periods at one end, move along one wall to the other end, amd
repeat the performance; only rarely would they move regularly up amd
down the length of the pond.

Under ideal control conditions, the tunny would swim up
arid down the pond in one or two schools at a leisurely but regular
pace, occasionally pausing to circle or "play" for a few seconds at
the ends„ A trip down the pond ajid back usually required about 3
minutes. When joined by the yellowfin, sometimes the faster tunny
ar.d sometimes the slower yellowfin acted as pacennakers, thus vary-
ing the swimming speed. At times, either of their own volition or
attracted by the yellowfin, the tunny would spend long periods of time,
sometimes an hour or nnore, at one end of the pond„ When this oc-
curred, experiments could not be conducted for the test material
might be diluted below the threshold for response before the fish en-
tered the attraction area. Several experiments were spoiled when,
following uniform control conditions and the introduction of the test
substance, the fish failed to return to the area within the required
time. Even if they entered the area for the first time during the
second or third test period, the quantitative data would not be com-
parable with those obtained during normal behavior.

Measurement of Response

From the sources of variation discussed so far - weather,
variation in testing procedure, and erratic behavior of the fish during
eit;her control or test conditions - the difficulty of obtaining consistent
quantitative data will be apparent. Moreover, the timing and counting
did not measure the more obvious characteristics of a connplex posi-
tive reaction - surfacing, splashing, and feeding activity. On one
occasion the response was recorded on motion picture film, a method
which might lead to a better quantitative measure. However, this
method was not feasible in regular testing because of the great varia-
tion in visibility of the fish (depending on weather, turbidity of the
water, height of the tide, and depth of swimnning of the fish) and also
because of the prohibitive cost of the thousands of feet of filnn that
would be necessary to record the many experiments which were con-
ducted,

A series of pond experiments (Nos. 94 to 100, inclusive)
has been chosen to illustrate the nature of the data and the problenns
of statistical amalysis and interpretation under average conditions of
observation and behavior of the fish. These seven experiments were
undertaken in succession throughout the day of August 26, 1952, four

18

in the morning and three in the afternoono The sky was overcast, with
a few rain squalls in the nnorringo Twelve tunny and four yellowfin
were present, but only the former were timed and counted. The na-
ture of the test substances is not pertinent to this discussion.

The basic data are shown graphically in figure 6,

It will be noted immediately that there is a high correla-
tion between the times and number of passes for successive test
periods (r = 0, 874; P less than OoOr)^ Both variates reflect the re-
actions alnnost equally well m this particular series. It will also be
noted that in all experiments except No, 98j the times and counts dur»
ing the test periods are generally higher than during the preceding
control periodSo

Analysis of variance was employed to determine if there
were significant differences between the means of control and test
periods (equivalent to a "t" test) for each separate experiment. The
results are shown in table 2o It may be concluded that there is a
statistically significant response to the test substances of experiments
Nos, 95, 97, 99, and 100 (P less than 0,05 or OoOUo

In experiment Noo 98 the average time and count during
the test periods were both slightly less thdn during the preceding con-
trol periods. Obviously^ however, this was due to unusual activity
during the controls. To overcome this difficulty a more mfornnative
method of analyzing the data is to group all control periods for all
experiments as Tqp determine the mean square between means for
experiment Tq to Ty, determine the pooled nnean square within ex-
periments, and use this last to determine a least significant difference
(fiducial interval) of a treatment mean for comparing not only Tj with
Tqj T2 with Tq, etCj, but also for comparing T^ with T^t etc. The
analysis, for time data only^ is as follows"

Source of

variation

Degrees
freedonn

of j Sum of
^jsguares

Mean
square

F

Between treatments

7

16„157

2,308

5,0**

Individual periods

62

28,671

462

** P less than 0„0I

19

r 1 1 1 1 1 1 1 1 1 1

no. 100

CONTROL RESIDUE

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20

The fiducial interval of a treatment nnean nnay be calcu-
lated as follows (Snedecor 1948s p^ 221);

- + 2„0 |/~462_
\' "?„ 14

= + 16

The mean for the controls is 36,2 seconds; those for the
experiments are included m table 2o Again, the means for only four
experiments, NoSo 95, 97, 99j and 100, as before, differ significant-
ly (by 32 seconds or more) from the mean for the controls. There
are no significant differences between the means for any of the seven
experimentSo This latter conclusion is also evident from a further
analysis of the variance of the combined results:

Treatment vs. control
Treatment nneans
Individual periods

The above analysis, based on quantitative data, has
demonstrated a significant response in four of the seven experiments.
Observation, however, showed a response m all seven, nannely, ob-
\'ious sensing of the material connbined with one or more of the several
components of the reaction^ as described previously. Only in No. 98
was there any doubt; even here, however, there was an obvious change
in pace and surfacing of the fish during the first and second test
periods o The data and their analyses do not always reflect a response
which is obvious and "significant" to the observer o Other experiments
could be selected in which the discrepancies between the data and the
observations are even more pronounced.

The difficulties outlined above were recognized early.
Nevertheless tinning and counting were continued: they did measure
certain phases of the reaction and have been used in some quantitative

21

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22

comparisons; in addition they assisted in maintaining standard tests,,
Generally, however, reliance was placed on the observer's judge-
nneni of a response as gained from visual observation^ At the close
of each experiment, the strength of a response was recorded in one of
several categories (or a fraction thereof I which are designated as
follows;

Designation

Numerical
equivalent

Description

0

0

No noticeable response.

(X)

Oo

5

An apparent sensing of the material; of
doubtful biological significance.

X

1

A weak but positive response, usually
including few of the components
(surfacing, speeding, etc,)

XX

2

A moderate positive response, includ-
ing most components o

XXX

3

A strong positive response, including
all components o

XXXX

4

A very strong positive response, in-
cluding all components; great

excitemento

Often a reaction would be indicated by X(X^ or XX{X),
with numerical equivalents of 1-1/2 and 2-1/2 respectively.

Although admittedly subjective and not well-suited to
statistical analysis, the foregoing measure is simpler and at times
more nneaningful than that dependent on the tinning and counting
alone. The two observers, by working together, attempted to
standardize their judgennent of the strength of a reactionc The ob-
served reactions, measured in the above categories^ are included
m table 2 for the series of experiments analyzed in the preceding
paragraphs. They may be compared with the summed time and count
differences in the second to last column of the table. There is gen-
eral agreement between the two measures in that the stronger ob-
served reactions (XX and XXX) are associated with the greater
differences between control and test quantitative d«ita. However,
there are also notable discrepancies as^ for exannpie, in compeiring

23

NOo 94 and No, 96; although the observed reaction is judged the
same, X, the quantitative data diverge widely (although not signi-
ficantly according to statistical tests)„ As will be indicated later,
the observer's judgement of the strength of a reaction tended to
change with time and with decreased reaxtivity of the fish. Thus
a reaction which might be classed as X in August would likely be
classed as XX or XXX in ApriL

Variation in Response

The strength of the response to an attractive substance,
whether measured by the quantitative data or estimated by visual
observation, varied greatly from test to test both within and be-
tween daySo Throughout the mass of data there are numerous
examples of this„ For exannple in experiments NoSc 67, 72, 80,
97, 106, 111, 117b and 173, which were all conducted by one ob-
server using the same material (250 ml„ stock extract), the res-
ponse varied from 1 to 3 (X to XXX) on the observational scale.

At times it was suspected that the variation in response
was associated with either the frequency of testing or the accunnu-
lation of test products. These factors could readily influence the
results obtained with fish in the tank, for it had both a small
volume and a small r<»te of flow of salt water. Thus, following
four expe rinnents in which relatively large quantities of test sub-
stances were used (Nos, 59 to 62|, there was no response to a
relatively large volume (1, 000 ml,) of stock extract (No, 63) in the
fifth experiment of the day, -Afte r a rest period of 3 days in which
no tests were conducted, much snnaller volumes (250 ml, ) of stock
extract gave strong responses (Nos, 81 and 82),, In the pond, at
times there appeared to be a gradual increase in activrity under con-
trol conditions and a gradual decrease in response to attractive
substances when several experiments were conducted in succession
during a day (fig, 5). This may be attributed to several possible
causes including chance or random variation, an increasing con-
centration of materials in the pondj, or aji increasing threshold of
response under repeated stmnul^itiono When a response was pro-
tracted beyond the ordinary tmne limit, usually the observer
would wait for 15 to 30 minutes before starting the next experiment
to allow the fish to resume normal control activity. To sonne
extent this would overcome the trends which are noted above.

In April 1953 a series of experiments (No, 313 ^t_se q, )
was undertaken to determine if there were differences in response
to materials maintained at three different hydrogen ion concentra-
tions (Ti"-pH 2, T2 = -pH 7, T3»-pH 10), but also to investigate

24

the variation in response within and between daySo Both the
experimental plan and the observational data are included in
table 3o Two tunny were present in the pond throughout the 3-week
periods Various difficulties were experienced which prevented the
completion of all experiments in the series ei'her erratic behavior
of the fish or other difficulties as listed m the footnotes to table 3«

The great variation in response is apparent from the data
of table 3„ For Tj^ and T2 it ranged from 1/2 to 4„ and for T3 from
1/2 to 2»l/2„ A superficial analysis of that portion of the data com-
prising complete blocks (ao nio onlyl showed that none of the main
effects (order of testings daySp and treatments) was significanto
Order of testing contributed the largest me^n squares days the next
largestj and treatment the smallest „ The last was less than the
residual mean squares In these and other series of experiments
often it has been noted that the first experiment of the day gives the
strongest response; the fish are suddenly aroused from a quiescent
condition and give a strong response relative to both the preceding
control periods and the succeeding test periodSo This contributes
to the variation between means for order of testing (respectively
2o4j, lo3, and 1<,3 for the observational data above)o Variation in
reactivity from day to day^ apart from erratic behavior, has been
noted repeatedly by both observers, and is believed real despite
the non~ significance of the data aboveo The reason is obscure--it
is unlikely that it is related to the state of hunger of the fish; it has
been suspected at times that it is related to the weather, tides k or
turbidity, but these factors cannot be adequately investigated with
the present datac

There seenaed to be a decrease in the response and an
increase in its variability between the summer of 1951 and the
spring of 1953o Thus, in six pond experiments conducted with the
same material (each equivalent to about 25 go of tuna flesh) during
August, the mean difference between timing for test and control
periods was 32o6 seconds and the range was lie 2 to 52^2 secondSo
In the test series conducted in April, in which the material was
equivalent to about 350 g^ of tuna flesh (14 times as much as before)
treated in a different manner but one which did not significantly de-
crease its attractive properties, the mean difference between the
timing for test and control periods was 7o 1 seconds with a range of
- 13o0 to 6la8 seconds. The decreased response was reflected to
only a slight extent in the observational dat-i which dveraged lo9 for
August and lc7 for April; apparently there was a change in the ob-
server's judgement of the absolute strength of o reaction. The
reason for the decrease in response and the increase in its varia-
bility may be related to a decrease m the pond population from 1 3 to

25

X

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2 tunnyo This decrease in the number of fish would, of course,
decrease the absolute values of the quantitative data^ particularly
the counts,, but it would not necessarily change the difference between
control and test datao Apparently 13 fish gave d much greater
response than 2 fish to the seume amount of materialj, suggesting a
"group" effect- "=a contagious excitemento On the other hand, the
decrease in response may have been related to decrease in the
"condition" of the fish and a declining interest in food substances
during the winter monthSo

SOURCE AND PREPARATION OF TEST SUBSTANCES

Materials

The materials which were used are listed for each
experiment in the appendix at the end of this reporto

Because of its ready availajjility and also because it had
given a positive response in experiments conducted by v^n Weel
(19521, most of the preparations consisted of extracts of tuna flesh,
including skipjack, tunnyj yellowfin, and bigeyCo In addition, ex-
tracts of trina skin, blood, liver, ovaries, testes, and unsegregated
viscera were usedo

A few tests were conducted with extracts prepared from
"white ■= fleshed" fish (as opposed to the red«fleshed tunas) when
materials were available. They included "aholehole" (Kuhlia
sandvicensis) flesh, jack (Caranxl flesh and blood, barracuda
^§£^Y£££B^ .^!l£j^^£H^ J -l^s^o ^nd wahoo (Acanthocybiurn s^lgj;^dri)
fleslio

The tuna and other fish used in these preparations were
either fresh-caught or fresh -frozeno They were obtained from our
own fishing operations, from the Pacific Oceanic Fishery
Investigations, or from the Hawaiian Ttrna Packers, Ltdo

Beef blood obtained from a local slaughter house was
used in comparison with fish bloodo

Tuna cannery byproducts, including fish meal, stick-
water (effluent), and various preserved digests of skipjack viscera
made for other purposes by Mr, F^ Jermann oi Hdwaiian Tuna
Packers, Ltdo , were testedo A herring reduction pl^ant byproduct,
"condensed herring solubles" prepared by Bo Co Packers, Ltdo ,
Vancouver- Canada^ was also usedo

27

As time permitted, tests were run on a variety of
organic chemicals purchased from Nutritional Biochemicals
Corporation and Eastman Kodak Company„ A variety of aromatics
used in perfume manufacture and food flavoring were supplied by
courtesy of E„ !„ DuPont de Nenaours and Company, Van Ameringen-
Haeblerj Inc„g P, Ro Dreyer, Inc., and Sindar Corporation. Mis-
cellaneous substances which were tested included alleged commercial
fish attractants and pineapple juice „

Preparation

It is not feasible to describe in detail the method of
preparation of each substance which was tested. This has been sum-
marized for each experiment and has been included in the appendix.
General techniques and procedures, only, will be discussed here.
Where necessaryj, details of special preparations will be included in
later sections,,

Simple extracts of fish flesh, skin, gonads, liver, etc.
were prepared as follows; a quantity of material (usually about 100 g, )
was weighed, cut into small pieces, placed in a Waring blendor with
about 200 ml, of sea or tap water per 100 g. of material, and reduced
to a homogeneous suspension of finely-divided particles over a

5-minute period. The material was then diluted to about 1 liter with

o o

water and was placed in a refrigerator (at about 1 or 2 C„ ) for sev-
eral hour So The mixture would be used as such, or it would be sepa-
rated into liquid and solid components by either filtering or centrifu-
ging before testing „

A "stock extract" of skipjack flesh was prepared in bulk
by grinding 9 pounds of flesh in a nneat grinder, placing this in a
nnilk can of 10 gallons capacity, adding tap or sea water to nearly
fill the can, and placingthe can and contents in the refrigerator.
The solid anaterial would settle from the liquid in a few hours. The
clear, amber to cherry-red liquid (the exact color depending on the
proportion of light to dark red meat in the sample) could be decanted
from the surface,, It was calculated that 1 liter of liquid contained
the extract from about 100 g^ of flesh. The stock extract could be
maintained without putrefaction for 2 or 3 -/eeks in the refrigerator
if the temperature was held just at or above the freezing point. The
stock extract was used frequently as a standard in testing other sub-
stances.

In most of the experinnents conducted during the winter
and springs 95-percent ethyl alcohol was used in the preliminary ex-
traction of ground tuna flesh. The alcohol extract was annber to

28

cherry-red in color as in the case of the water extracto Alcohol
served as both an extr acting oiid a preserving agent; refrigeration of
the extract was not necessaryp although it was used occasionallyo

More connplex procedures were used in attennpts to
isolate and identify the attractive substancefs) in extracts of tuna
flesh and viscerao These included fractionation of the extract into two
or several components to determinen on pond testingo which still re-
tained the attractive propertieSo In some preparations „ a first sepa-
ration into "protein" and "non-protein" factions was achieved by
precipitation of the proteins <ind similar complex substanceSo Pre-
cipitation was accomplished in various waySj eogoo heatingo boilingj
saturation with sodium chloride or "salting out", adding tannic and/or
phosphotungstic acidp adding the salts of heavy metals such as lead,
barium^ etCoa as well as adding other chemicals to remove those used
in precipitationo In other preparations the extract was separated into
two fractions by dialysis to determine whether the attractant was com-
prised of a small molecule which would pass through a dialyzing mem-
branes, or a large molecule which would not pass through a membraneo
In still others the extract was divided into a "fatty" and a "non-fatty"
fraction, and each was tested in the pond to determine if the attract-
ant was a "fatty" or a "non«fatty" substanceo Fractionation was
accomplished by rennoving the fatty acids and similar compounds by
extraction with petroleum ether and/or acetone,. Other procedures
were aimed at determining whether the attractive substance consisted
of a positive or negative lono In these, fractionation was achieved by
passing the extract through anion and/or cation exchange resin columns
gjving two portions 5 that adsorbed by the column (subsequently removed
by elution or washing) and that not adsorbed by the column (the fil^-
trate)o In addition, columns of activated charcoal and activated alumina
were used to see if they would adsorb the attractive substanceo

In many of the preparations, several of the foregoing
fractionation procedures were used in succession, each successive
portion being tested and being either accepted or rejected for further
fractionation depending on whether pond tests indicated that it did or
did not include the attractive substanceo Several of the procedures
were used even though they did not remove the attractive substance;
they did remove non^active substances and thus constituted a step in
the purification of the attractanto The attractant, when purified as
completely as possible by the successive removal of non attractive
substances, was then subject to basic chemical tests in an attempt to
determine its basic components and chemical structures

Miscellaneous chemical compounds were prepared for
testing bv dissolving in tap water or in weak alcohol solutions; or in

29

the case of fatty or oily substances, by dispersing in water with a
dispersing agent "Tergitol". If the latter was not effective, the
mixture was forced through an emulsifying apparatus to insure its
complete dispersion.

RESPONSE TO SIMPLE EXTRACTS OF TUNA AND OTHER FISH

Flesh Extracts of Tuna

There are nnany experinnents to show that in both the tank
ajid the pond the tunny (and to a lesser extent, the yellowfin) gave a
positive response to clear, aqueous extracts of skipjack flesh (Nos„ 1,
8, 10, 13, 20, 50, 54, 64, 65, 66, 67, 72, 80, 81, 82, 87, 93, 97,
106, 111, 112, 117, 118, 122, 128, 135, 173, 199, 207), yellowfin
flesh (Nos, 14, 18, 26), tunny flesh (No, 27) and bigeye flesh
(NoSo 239, 252, 259), To these might be added a large nxxnttber of ex-
periments using either water or alcohol for extraction in which the
materials were subjected to further treatment and which in many cases
gave a positive response. In only a few experiments with simple ex-
tracts were negative results obtained when positive results were
expected. In tank experiment No. 16, the fish had been fed 2 hours
previously and were not responsive to skipjack flesh extract. In tank
experiment No. 63 there was no response, due probably, as has been
mentioned previously, to the dulling produced by an accumulation of
test products in the tank with too frequent testing. In pond experiment
No. 336, the fish failed to enter the attraction area during the 15-minute
test period. In a few experiments, of course, the response was un-
certain or slight (X)„

The nature of JJie response to extracts of tuna flesh has
already been described. It is a complex reaction, consisting prima-
rily of an urge to feed, which is manifested by speeding, surfacing,
circling, snapping at objects, together with a return to the area of
stimulation.

It is presumed that in tuna flesh there is present a
substance (or substances) which, when extracted and presented to the
tunny, can be perceived through the fish's sense of smell or taste.
The substance which, when smelled or tasted, promotes the urge to
feed is henceforth called the "attractant, " Unfortunately, as already
pointed out, the response varies greatly in strength or manifestation
between similar experiments. It appears to be influenced by many
uncontrolled and perhaps uncontrollable factors.

One experiment (No. 112) is described in detail, for it
differed from the others in method and purpose. Three liters of stock

30

extract was introduced into the pond, with the pump in operation, by
slowly siphoning it into the funnel over a 48-minute periods This es-
tablished a gradient of attractant from the outlet to beyond the attrac-
tion area. Timing, only^ was attempted and this consisted of the time
out of 3 minutes spent in the first 1/lOth of the pondp i„eo the area
between the seaward gates and the first string i^figo 4), not including
the shallows. The results are shown in figure 7,

ISO
160
140

2 '20

z

S 100

(A

80

z

60

? 40

20

0

CONTROL

3 LITERS STOCK EXTRACT
SIPHONED IN

TEST PERIODS

Fig. 7, --Time (seconds) out of successive 3-minute periods in pond experiment
No, 112, in which a gradient of stock extract of skipjack flesh was established
for 48 minutes.

31

The material was sensed very quickly by the tunny and produced a
typical response, with all components, in the vicinity of the outlet.
The response was maintained during the entire time necessary to
siphon in the material A few fish would leave the area and dash down
the pond, but they would invariably return to the area of greatest con-
centration. After all the material had been siphoned in, the response
quickly decreased until finally, after about 18 minutes, normal
swimming was resumed.

Flesh Extracts of Other Fish

Positive responses were obtained not only to extracts of
tuna flesh, but also to extracts of aholehole flesh (Nos. 49, 99, and
105), jach or ulua flesh (No. 96), and barracuda flesh (No. 98)„ There
was no response to wahoo or ono flesh (No. 6), but the fish were not
behaving well in this early tank experiments It is evident that an at-
tractant is present in at least some of the white-fleshed fish, as well
as in the red-fleshed tuna. We gained the impression that the response
was generally less in these flesh extracts than in those from tuna, but
this cannot be proved because of thefew experiments conducted with
the former, variation in the quantity of material used, and the great
variability in the strength of the response. In most of the compar-
isons the response appeared less (compare Nos. 49 and 50, 96 and 97,
97 and 98, 105 and 106) but in one it was greater (97 and 99). Van Weel
(1952) obtained only a weak response to white-fleshed anchovy or nehu
(Stolephorus purpureus) extract.

Extracts of Tissues Other than Flesh

Strong responses, ranging from XXXX to X, were obtained
with extracts of unsegregated skipjack viscera treated in several dif-
ferent ways (Nos. 137, 139, 141, 145, 146, 219, 220, 221, 236, 264,
279, 352, 353, 356)„

Excellent responses were also obtained with extracts of
skipjack blood (Nos. 43, 70, 95), yellowfin blood (No. 23), and tunny
blood (No, 30). Jack or ulua blood (No. 94) gave a positive though
weaker response. In contrast to these results beef blood, either whole
(Nos, 55 and 74) or the plasma portion (No. 6l), was not attractive.
In fact, whole beef blood, of a deep red color, was avoided by the tunny
in both the tank and the pond experiments- -a repellent effect which was
probably visual in origin.

Excellent responses were obtained with extracts of yellow-
fin liver (No, 25) and tunny liver (No, 28),

32

Extracts of skipjack ovaries (No„ 3) and yellowfin testes
(NoSo 4p 17) were not attractiveo However 5 whole extracts were used
eind the fish may have been frightened by the murky cloud which they
produced in the water,,

Unfiltered extracts of yellowfin skin (NOo 24) and tunny
skin (Noo 29) also formed a dark cloud in the water and were more
repellent than attractive^ An almost clear centrifuged extract of skip^
jack skin (Noo 48^ gave a weak positive responseo

It appears that the attractantis present not only in tuna
flesh;, but also m other parts of the fisho Again„ however^ it is
difficult to come to any firm conclusion as to the relative strength of
the response 5 and thus the relative concentration of the attractant in
the various partSo In general the viscera extracts appeared to be at
least as attractive as the flesh extractSp and possibly slightly more
attractivCo In two series of esjperinnents designed to compare ex^
tracts of bloody liver, skin, and flesh (NoSc 23 to 26„ Nos, 27 to 30),
extracts of blood and liver appeared to be more attractive than ex=
tracts of flesho and extracts of skin appeared to be less attractiveo

C anne ryJBygr oductsj Preservation

Good (XX) responses were obtained with extracts of tuna
fish mealp both boiled Wo, 119^ and not boiled (No,, 120) during ex-
traction^ Excellent responses jXXX> were obtained with tuna sticks
water both by itself (NOo 217) or mixed with fluorescein dye to trace
its dispersion m the pond (Noo 222'o

"Condensed herring solubles^ " a product derived from the
stickwater of a herring reduction plant (B, C„ Packers^ Ltdo ) was not
sensed by the fish (Nos, 328 and 329U

Several experiments were run on skipjack viscera
preparations submitted by Hawaiian Tuna Packers, Ltdo 0 with excel-
lent results (XX to XXXXt, In all preparations the viscera from the
cannery were first ground in a meat grindero In some they were pre =
served in 2- or 3-percent sulphur dioxide and self-digested^ produc*
ing a particulate portion which settled to the bottom of the container
and a supernatant dirk brown liquid (NoSo 219g 221^ 290^ etCc,)o In
others, the ground \nscera were steamed at 20 pounds pressure and
preserved in 3 -percent phosphoric acid; again producing a solid and
a supernatant yellow liquid 'No, ZZO)^, A ground viscera preparation
preserved in 2-percent sodium bisulphite ^nd digested with pepsin
gave little evidence of attraction in three tests (NoSo 258^ 260^ and
263II0 Apparently loss of attraction was due to something associated

33

with pepsin digestion rather than to the use of sodium bisulphite, for
this preservative was used successfully in other preparations
(Nos. 264, 279, etc, ),

The above results, indicating that the attractive qualities
are not lost when the materials are preserved in chemicals, are in
accord with those obtained with our own preparations which were
tested at an earlier date. Skipjack viscera extracted with water and
treated with 2-percent sulphur dioxide (Nos. 140, 141), with 2-percent
sulphuric acid (No. 144), or with 2-percent phosphoric acid (No, 145)
were still attractive to the fish even after being kept at room tempera-
ture for several days,

RESPONSE TO CHEMICAL COMPOUNDS
AND MISCELLANEOUS MATERIALS

As time pernnitted, a variety of chemical compounds
ranging from amino acids to proteins and including vitamins and vari-
ous miscellaneous aromatics were tested as follows:

Arginine (No, 32, 103)

1-Asparagine (No, 102)

Creatine (No, 109)

1 -Glutamic acid (No. 191)

Glycyl-glycine (No, 127)

1-Histidine monohydrochloride (No, 192)

dl-Isoleucine (No, 34)

1-Leucine (No, 44)

Methionine (No, 157)

dl-Norleucine (No, 45)

dl- Phenylalanine (No, 31)

dl-Serine (Nos, 51, 56, 101, 104, 108)

1-Taurine (No. 42)

34

Acetyl tryptophane (NOo 176)

1-Tyrosine vNo. 33)

dl- Valine (No„ 41)

Isoeugenol (No„ 37)

Nucleic acid (No„ 110)

Histamine diphosphate (NOo 198)

Adenosine (NOo 242)

Adenosine triphosphate, disodium salt
(Noso 243, 254, 271)

Adenosine diphosphate, bariunn salt
(NoSo 245, 255, 270)

Adenylic acid (Noo 244)

Trinnethylamine oxide (Noo 188)

Animal lecithin (NOo 129)

Albumen (Noo 62)

Peptone "I" (No„ 189)

Vitamin B (No„ 166)

Acetyl choline chloride (No^ 192)

Pregnenolone (No<, 132)

Ambergris (NOo 131)

Anethol (NoSo 22, 71)

Phenyl acetic acid (No,, 251)

Alpha ionone (No„ 158)

Alamask (No,, 172)

35

Furfuryl mercaptan (Nos. 231, 241)

Coumarin (No„ 249)

Methyl anthrcinilate (No. 250)

Meat flavor (No. 253)

Pineapple juice (No, 168)

Eosin B (No. 36)

Methylene Blue (No. 38)

None of the above substances gave a positive response
which could be verified. In some, however, there appeared to be a
sensing of the substance as will be discussed below.

Among the amino acids, serine appeared to give a weak
positive response in one tank experiment (No. 51) ajid a very weak
positive response in another (No. 56). In three pond experiments,
there appeared to be a sensing of the material in one (No, 101) but not
in two others (Nos. 104 and 108), On the sanne day on which the fish
appeared to sense the serine in the pond (No. 101) they also appeared
to sense asparagine (No. 102) and arginine (No. 103), suggesting that
they were in a particularly responsive condition. On another occasion
they appeared to sense methionine (No. 157). Apparently the fish were
aware of the presence of the substajices but the slight reaction seemed
to be one of curiosity rather than attraction: certainly it lacked the
features of a typical response.

This same sensing of material was noted in the case of
certain strong-smelling aromatics, e.g., anethol (No, 22), "meat
flavor" (No. 253), and particularly furfuryl mercaptan (Nos. 231 and
241). The first substance, which has a licorice smell, is the active
ingredient of oil of anise, a reputed fish attractant; the second is a
strong, spicy, pleasant-smelling artificial flavor; the third is a- very
foj.1- smelling chemical of great potency. A similar response was
noted with odorless vitamins, B (No. 166) and acetyl choline chlo-
ride (No. 192), and also with ordinary pineapple juice (No. 168).
Again, however, the response seemed to be one of curiosity.

The series of experiments with adenosine, adenosine conn-
pounds, and adenylic acid was conducted in an attempt to determine if
the attractive substance was one which is present in mammalian mus-
cle. That it might be was suggested by chemical tests of the attract-
ant (see later) which showed the presence of phosphorus and the amide

36

linko The substances were dissolved m sea water immedia'ely before
testing to avoid hydrolysiSo The barium salt of adenylic diphosphate
was dissolved in 0„ 1 N hydrochloric acid and the barium was precipi-
tated with 6N sulphuric acid to have it present as free adenosine
diphosphate acidi, rather than as a barium salt (there had been indica =
tions that barium tended to destroy the attractive material^o I* was
presumed that the pH of seawater would assure the proper molecular
form of the sodium salt of adenosine triphosphateo First tests indica =
ted a sensing and an attraction of the tunny to both adenosine triphos*
phate (Noo 243) and diphosphate (Noo 2451 but not to adenosine (NOo
242) nor to adenylic acid (Noo 2445o However^ later tests with both
the triphosphate JNoSo 254 and 271) and the diphosphate (NoSo 255 and
270) gave negative resultSo

The two dyeSj, eosin red (fNoo 36) and methylene blue
(Noo 38);, were visually repellent to the fisho This sanne reaction^ an
avoidance^ was occasionally noted with strong solutions of the green~
ish fluorescein:,

In addition to the substances listed above^ two alleged fish
attractants were tested^ one known as "Slik" which smelled of cod
liver oil;, and the other an unnanned "fish lure" which snnelled of tuna
or herring viscera extracto There was a slight sensing of the first
(Noo 174) and a weak but positive reaction to the second 'NOo 349)o

ATTEMPTS AT TSOLATTON AND IDENTIFICATION
OF THE ATTRACTANT (S<

The attractantCs) was initially segregated from the flesh
or other parts of the fish by virtue of its solubility in water,, ThuSj, on
centrifugingj the aqueous extract of finely-^divided tuna, flesh formed
a clear amber to reddish colored solution which usually caused a
strong positive response fNos, 1^ Sj, lOj, and numerous other experi=
ments in both tank and pond)o The residue from centrifuging was
either attractive fNOu 15) or not (NOo 2)^ depending on either the
thoroughness of the extraction or the part played by vision in the case
of the murky mixture o

In early tank experimentSj boiling of tuna flesh with water,
thus precipitating the proteins and similar compounds^ appeared to
remove or destroy the attractanto The clear filtrate from the boiled
flesh failed to elici' a response 'NoSo 5^ 9^ 11„ 12t, 211 as was also found
previously by van Weel » 19521, However j, as will be discussed later^
this was not the case with boiled extract of flesho

DialysiS;, using a cellophane tube (K-S/S inch diameter),,
was next tried in an attempt to segregate the attractantp using clear

37

aqueous extracts of skipjack flesh (several experiments) or tunny
blood (one experiment). In all cases the dialyzate gave a positive
response when tested (Nos„ 39, 46, 53, 57, 90, 118, 123), showing
that the attractant consists (in part, at least) of a small molecule,
rather than a large molecule such as a protein. In no case, however,
was complete separation attained by dialysis. In most of the experi-
ments the residue (portion remaining in the tube) gave a stronger
response than the dialyzate (compare Nos, 39 and 40; 52 and 53; 57
and 58; 90 and 91) despite several changes of the distilled water sur-
rounding the tube.

Precipitation of the proteins by dilute (10-percent) hydro-
chloric acid was next attempted. After the centrifugate of an aqueous
extract of skipjack flesh was treated with acid and centrifuged, both
the neutralized centrifugate (No. 68) and the neutralized residue
(Noo 69) gave positive responses, showing incomplete separation of
the attractant.

As the above results indicated that the attractant was not
a protein, a series of pond experiments was undertaken in which the
proteins were precipitated by heatings On heating the centrifuged
aqueous extract of skipjack flesh to 69° C. , a precipitate appeared.
When this was removed by centrifuging, the centrifugate was still
attractive (No^ 76), When the centrifuged aqueous extract was boiled
for 30 minutes, resulting in a heavy precipitate, both the filtrate in
one experiment (No. 77) and the centrifugate in another (No. 78) were
still attractive to the fish. However, separation was not complete,
as is shown by the weaker positive responses obtained with the washed
residue from centrifuging (compare Nos, 75 and 76; Nos. 78 and 79),

It is uncertain why the filtered extract of boiled tuna flesh
failed to give a positive response in the earlier tank experiments
NoSo 5, 9, 11, 12, and 21)o Although the behavior of the fish in the
tank was not too satisfactory, it seems certain that had a response
occurred, it would have been noted or measured in one of the five
experimentSo It is unlikely that the attractant was destroyed by
boiling in view of the results reported in the preceding paragraphs and
others to be presented later. There remains the possibility that the
attractant was precipitated with the soluble proteins or otherwise
adsorbed with the insoluble proteins during the process of boiling the
flesh; the residue, which may have contained the attractant, was not
tested in the five experinnents conducted with the boiled flesh.

Chemical tests were run on the clear centrifugate obtained
after boiling of the extract. The Biuret test was positive, showing
the presence of the amide link. The Xanthoproteic test wa,s positive,

38

indicating the presence of the benzene ringo No positive test for
protein was given on layering the solution over concentrated nitric
acido The lead test for sxilphur was negativeo

A series of ejtperiments was conducted to compare the
effects of precipitation by boiling and precipitation by salting out of
the proteins on the attractiveness of the filtrateo The procedure a:n.d
results are depicted m figure 80 Clear stock extract was boiled for
15 minutes and centrifuged; the centrifugate was divided into three
equal partSp the first of which was tested (NOo 85)0 the second of which
was saturated with sodium chloride;, centrifuged^ and both centrifugate
(Noso 8 3, 86) and residue (NOo 88) tested^ and the third of which was
boiled for an additional hour^ centrifuged^ and the centrifugate tested
(Noo 84>o Strong responses were obtained with all tests except that
with the residue (No„ 88)0 The attractant was not removed with pre-
cipitation in either boiling or saltmgo Apparently fractionation was
complete when both boiling amd salting were used (compare NoSo 83
and 86 with No^ 88^0

The boiling ar.d salting procedure was carried one step
farther in two additional experiments „ The centrtfugate„ after boiling
and salting, was bo>led to a residue. When re-dissolved in seawater,
a positive response was obtained iNoSo HSj 121)o

The possibility of precipitating or destroying the attract
tive substance by heat after it had been segregated in a relatively
pure (protein-free) state by dialysis was next investigatedo One liter
of the dialyzate from a stock extract was boiled to 10 mlo and tested^
producing a very strong response (NOo 118)o The residue from dialy<=
SI8 was boiled to a gummy residue;, producing weaker but positive
responses with both the 95 percent alcohol soluble portions and the
alcohol insoluble portions (NoSo 114^ 115^ II6II0 In a second serieSj
the dialyzate was boiled to dryness and a strong positive response was
obtained with the portion of the residue which was soluble in absolute
ethyl alcohol (No, IZJK The solubility in absolute alcohol was con-
firmed m later experiments WoSo 201^ ?8Ho

Thus the attractant is a substance comprised of a small
molecule which is (at leasts partly) soluble in absolute ethyl alcohol.
This property rules out most of the known amino acids (except nnethio-
nine and proline>o However^ that part of the attractant or attractants
was insoluble in alcohol yet soluble in water is indicated by several
experiments in which the alcohol^^insolubleo water -^ soluble portion
gave a positive response (NoSo 115p 116, 124^ I83\o

Because of the time required for dialysis and the
incomplete sepciration that was achieved^ attention was directed at

39

Stock extract, clear
1,800 ml

Boiled 15 mino , centrifuged

Centrifugate

600 ml.

No. 85
XX

600 ml.

Saturated with sodium
chloride, centrifuged

Centrifugate

Residue

No, 88

600 ml.

Boiled I hour,
centrifuged

Centrifugate

No« 84
XXX

Figure 8, --Fractionation of stock extract of skipjack flesh; pro^
cedure and results in experiments Nos, 83 to 86, and
88.

40

the possibility of adsorption on various mediae In one experiment^
stock extract was concentrated by boilings filtered^ boiled with
activated caibon^ filtered;, evaporated to a gumnny residue^ re-^
dissolved and tested^ giving a positive response iNOo 125;)o In
anotherp the same procedure was followed except that after boiling
with activated carbon the filtr-ste was treated with activated aluminaj
again with a positive response (Noo 130'o Similar results were
obtained with anion fNoo 133) and cation (Noo 134) exchange resinso
Apparently the attractant was not fcompletely) adsorbed on any of
these media.0

At this time (September 1952j the chemical work was
undertaken at the University of Hawaii rather than the Hawaii Marine
Laboratory; the 'estSo of courscp were continued on the fish in Pond
Noo 5 at Coconut Islando To obviate the need for refrigeration and to
facilitate condensation^ extracts were prepared with 95=percemt ethyl
alcohol instead of watero *The actiial strength of the alcohol in the
extract was less than 95='percenta depending on the quantity of water
in the flesh and the relative quantities of flesh and alcohol which were
used; but it was always strong enough to act as a preservative at room
temperature o Numerous e>:periment6 showed that this alcohol extract
promoied a strong posiSrive response fNoSo 147p lb3s I8O5 235^ etCa)^
whereas there was no response to alcohol alone ilNoo 19)o However„
complete extraction of the attractive substance with alcohol was
difficult to attairio In two experiments^ the flesh was treated with 95='
percent alcohol for severad hours and the alcohol, containing some of
the attractantj, was filtered o£i and used for further fractionationo
Tlie flesh was agdin treated with alcoholg and the alcohol filtered off
and usedo The flesh was then treated with water and the filtrate was
testedo It still gave a strong response (NoSo 164 and 169) showing that
the attractant had not been connpletely removed by the previous two
alcohol extractionso To see if complete separation could be achieved
with alcohol extractionp flesh was extracted twice with 95-^percent
alcohol as fiboveo It was then further extracted with alcohol for 14
hours in the Soxhlet appaxatuSo The alcohol waS then removedp and
the flesh was extracted for ah. additional 12 hours with distilled watero
The cen'rlfuged water extrac*. gave only a slight response (NOo 17\
indicating that almost complete extraction of the attractant had been
achieved by the previous alcohol treatmentSo

Further attention was directed at fracMonjition of the
alcohol extract by use of various adsorption medi^. To improve the
technique;, columns of the adsorbing media were piep.ij-edc through
which the extract was allowed to filter slowly,, Two portions were
obtained, the filtrate or material which filtered ♦hrough the column
without being adsorbed^ and the eluate or portion which w^s adsorbed

41

on the column and which was eluted or washed from it with various
solutions such as water, weak acids, weak bases, alcohol, acetone,
etCo

In a series of experiments with columns of activated
alumina, zeolite, and activated carbon (Nos, 149 to 154) positive
responses were obtained from both the filtrate and (water) eluate of
the alcohol extract of skipjack flesh. However, the attractant appeared
to be adsorbed most on activated alumina (No. 150) and least on acti-
vated carbon (No„ 153)o Adsorption on activated alunnina was obtained
m two additional experiments (Nos„ 156 and 165), but complete separa-
tion of the attractant was not achieved (Nos, 155 and l62)o Attempts
at fractionation with alumina were abandoned in favor of anion and
cation exchange columns c

Small white crystals which appeared in the (acetone)
eluate from the activated carbon column, when dissolved in water,
appeared to cause a positive response (No„ 159), Further prepara-
tions gave no response (No, 167) and a doubtful response (No. 171),
It was concluded that the positive responses were apparent rather than
real and that the white crystals were not attractive. The attractive
substance, for the most part, appeared in the filtrate from the carbon
columno Thus either mixing or boiling activated carbon with the ex-
tract or paissing the extract through a carbon column would remove
certain inactive substances and constitute a stage of purification.
This procedure was followed in some of the later preparations.

Another step in purification was the removal of fatty acids
and related compounds which were soluble in petroleum ether or
acetone. Van Weel (1952) found that the attractant was not soluble in
petroleum ether. This finding was verified (Nos. 18, 142, 175, 177,
eTc„)„ It was also found that the attractant was not removed by ex-
traction with acetone (No, 142).

Still another step in purification was the removal of
inactive proteins by precipitation with tannic acid (No. 215), lead
acetate (No, 197; Nos, 200 to 206; etc.), or barium acetate (Nos, 211,
212, 225) and the removal of, purines and similar compounds as silver
salts (Nos, 183, 184) or with phosphotungstic acid (No^ 193, etc.).

Some of the above steps in purification either preceded or
succeeded fractionation by adsorption on anion or cation exchange
resins. Mciny different experiments, employing one to several suc-
cessive stages of purification, were conducted. Two typical series
only will be discussed to illustrate the procedure and results. Others
may be followed from data given in the Appendix.

42

The firsto a relatively simple series designed to determine
whether the charcoal-purifiedp "fat-free" attractant was adsorbed on
ion exchamge resm columns^ is illustrated in figure 9o An alcohol e3E=
tract of skipjack flesh was passed through a column of activated
charcoal; the filtrate was evaporated to dryness -ind extracted with
petroleum ether and the residue was dissolved m alcohol and divided
into two portiox.So One portion was passed through ol Duolite C = 3
cation exchange resin column and the filtrate collected and tested
(Noo 178|; the column was then eluted with 5<=percent sulphuric acid
and the eluate was collected and tested |Noo ISljo The other portion
was passed through a Duolite A<=3 anion exchange resin column, and
the filtrate wa.s collected and tested »NOo 179); the column was then
eluted with 5«percent soditon hydroxide and the eluate was collected
and tested (Noo 182'jo In this particular aeries all tests were positives
but the eluate from the anion column {Noe, 182j) appeared to give the
strongest responsca

The above results indicated that a further investigation of
the adsorption on an anion colunnn would be profitable^ particularly to
see (a) if complete adsorption of the attra^ctant could be attained, (b)
to see if the adsorbed material waa completely removed by a water
eludte, or if additional adsorbed material could be recovered with
either a weak base or a weak acid and Ic) to examine the solubility of
the attractant in absolute alcoholo The series is illustrated in figure
lOo A clear alcohol extract of tuna flesh was treated with lead
acetate to remove proteinSp filteredj and the filtrate was extracted
with petroleum ether; it was then treated with hydrogen sulphide gas to
precipitate the lead and excess gas was removed in vacuunio The fil-
trate was passed twice through a Duolite A->3 anion exchange resin
column and the filtrate collected and tested (No,, 200)o The column was
then eluteds first with water, next with a weak acid, and finally with
a weak bases The water eluate was evaporated to dryness and dissolved
in absolute alcohol {No», 201); the residue w^s dissolved in water (NOb
20Z)a The base eluate was similarly treated, giving an alcohol soluble
portion (Noc 204; and an alcohol-insoluble!, water-soluble portion
(NOo 205)o A positive response was obtained only with the alcohol
soluble por'ion of the water eluatCo In this series it appears that com-
plete separation of the attractant was achieved by use of the anion ex=.
change column and that the adsorbed attractant was completely removed
by the water eluate a

In these and other expetiments conducted during the autumn,
it appeared that the attractant, purified to a varying extent, was not
adsorbed on a cation exchange column^ for positive responses were
obtained with the filtrate (No&o 142^ 178, 216;, 225 > rather than the
eluateo Only two tests were performed with the latter, however, one

43

Skipjack flesh 250 g, , clear alcohol extract

Passed through activated carbon column

Filtrate evaporated to dryness and extracted
with petroleum ether

Residue dissolved in alcohol

Pass
Duolite

CO

ed through
C-3 cation
lumn

i !
Filtrate 5% H,SO .

1
eluate
1

1

No„ 178 No. 181

X

(X)

Passed through

Duolite A- 3 anion

colunnn

Filtrate

5% NaOH
1

1

eluate

1

No, 179

1

No, 182

X

XX

Figure 9, --Fractionation of an alcohol extract of skipjack flesh:
procedure and results in experiments Nos, 178, 179,
181. and 182,

44

Skipjack fleshj 350? go „ clear alcohol extract

Treated with lead aceta&eo filtered

Filtrate treated with petroleum ether

Hydrogen sulphide addedj, filtered,, excess gas removed in

vacuum

Filtrate through Duolite Ac»3 anion nolumno twice

Filtri

Lte

1

Water
eluate

1

Acid
eluate
neutralized

i

Base
eluate

No, 200

Evaporated to
dryness

1

No, 206

r

Evaporated to
dryness

1

Alcohol solu- Alcohol inaolu- Alcohol solu- Alcohol insolu"
ble part ble part; ble part ble part;

I water soluable I water soluble

Noo 201

No, 202

No, 204

No„ 205

XX

Figure 10, --Fractionation of an alcohol extract of skipjack flesh;
procedure and results in experiments Nos, 200 to
202„ and 204 to 206,

45

giving no response (No, 143) and the second giving a slight response
{No„ 181), More attention was directed to the anion exchange colunnn,
for it seemed to adsorb the attractant in several experiments as shown
by moderate to strong (X and XX) positive responses with a water,
dilute sodium hydroxide, or dilute ammonium hydroxide elutant (Nos,
182, 185, 193, 194, 201, 208, 213), Up to this stage of the study, the
eluate failed to give a response in only two tests (No, 196 and 223) in
which a response was expected on the basis of the preceding results.
The filtrate, on the other hand, gave either a weak positive response
or no response (NoSo 179, 200)„

It was clear, however, that the attractant was held very
"loosely" on the anion exchange columnp appearing nnostly in the
first portion of the water or weak base eluate (compare Nos. 185,
186, 187; Nos, 208, 209, and 210; 213,214; 246, 247 and 248), The
weak affinity (possibly not a true ion exchange) explains the failure of
complete separation in some of the above experiments, and may have
contributed to certain inconsistencies to be reported later.

Chemical tests were run on the attractant in its purest
form yet attamedj, namely, that portion of the water eluate from the
anion excheinge column which was soluble in absolute ethyl alcohol
(NOo 201)„ Both the Biuret and Ninhydr in tests were positive, show-
ing the presence of the ainide link„ The Xanthoproteic reaction was
positive, showing the presence of the benzene ring, A test for phos-
phorus was positive, but a test for sulphur was negative.

In subsequent experiments which were run from the latter
part of December on through the winter, the results became erratic.
There was difficulty in obtaining meaningful and consistent results
with the various chemicals used in purification, and also with the
anion and cation exchange colunnns used in fractionation. For ex-
ample, when barium acetate was substituted for lead acetate to pre-
cipitate the proteins and the filtrate was treated with sulphuric acid
rather than hydrogen sulphide gas to precipitate the barium as a
sulphate, no response was obtained (No, 211) , Also there was no
response for the solution that resulted when the protein precipitate
of Noo 211 was dissolved with nitric acid and the barium precipitated
as a sulphate and filtered off (No. 212), The loss of activity, if real,
may have been due to either destruction or CO precipitation of the
attractant by the barium ion or by the sulphuric or nitric acid. That
the barium ion was not responsible is shown by the series of experi-
ments depicted in figure 11, An alcohol extract of skipjack flesh was
acidified and treated with phosphotungstic acid to precipitate the
purines. Barium acetate (in one experiment) and barium chloride (in
another) was used to precipitate the phosphotungstic acid and the

46

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h

a<

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43

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47

proteins. There was a good response when barium acetate was used,
the barium ion was removed by passage of the material through a
strong cation exchange column (Amberlite IR-120), and the filtrate
was tested (No. 216, 225). On the other^hand, strangely enough there
was no response with exactly the same treatment, but using barium
chloride (No, 218). When the material treated with phosphotungstic
acid was passed through a strong cation exchange column (Amberlite
IR-120), the filtrate was passed through a strong anion exchange
column (Amberlite lRA-400), and the eluate was tested (No, 223), there
was no response, although one was expected judging by the results of
previous tests o However, a weak response was obtained with the final
filtrate from a aeries of precipitations involving the use of both the
barium ion and sulphuric acid (No. 215), following the method of
Suzuki et ale (1909).

Inconsistent or negative results were also obtained in
attempting to repeat the various purification and fractionation
techniques discussed above on a self-digested extract of ground skip-
jack viscera, preserved with sulphur dioxide, supplied by Hawaiian
Tuna Packers, Ltd, The extract itself promoted a strong response in
some experiments (No. 219), but only weak responses in others (Nos.
229, 230). It could be cooked under pressure (No. 236) or extracted
with alcohol (No. 235) without loss of its attractive properties, but it
could not be carried successfully through the various precipitation nor
ion adsorption processes (Nos. 232, 233, 234, 237, 238).

As skipjack flesh was no longer available, attention was
directed at a study of extracts of bigeye tuna flesh. Simple alcohol
extracts gave good responses (Nos. 239, 240)^ However, weaJc or
negative responses were oDbtained when the purified materials were
passed through ion exchange columns (Nos. 246, 247, 248, 256, 257,
265).

The negative results in these tests may have been due
either to loss or destruction of the attractant at some stage of frac-
tionation, or they may have been due to the moribund condition of the
fish prior to a January mortality. To examine the former possi-
bility a series of experiments with extracts of bigeye flesh was under-
tciken as illustrated in figure 12. The filtrate from alcohol extrac-
tion was treated with lead acetate to remove the proteins, and
filtered; the filtrate was treated with sodium chloride to precipitate
the lead as lead chloride, and was tested (No. 266) with an excellent
response. The filtrate from the sodium chloride treatment, when
boiled, gave only a slight response. Some loss may have been asso-
ciated with boiling after treatment with lead, although a repeat of the
procedure by heating on a water bath, rather than boiling, gave a
very strong response in a later experiment (No, 273). When part of

48

Bigeye fleshj 1168 go alcohol extract

1

Filtrate

Lead acetate added; filtered

Filtrate

Treated with sodium
chloride

r

i_

No„ Z66
XXX

Filtrate 5
boiled

No„ 267
(X)

Passed through strong cation
exchanger

Filtrate

About pH 3 Adjusted to Passed through
I pH 5 to 7 strong anion

exchanger

Noo 268 Noo 269 j j

Filtrate Eluate

(repeat)

i

'repeat)

1

1
pH 2

J

1
pH 7

i

NOo 277

J

No, 272

„

X

NOo 261 NOo IhZ

Figure 12o ■= --Fractionation of an alcohol extract of bigeye flesh;

procedure and results in experiments Nob- 261 j, 262,
266 to 269p 272„ and 277o

49

the filtrate from the lead treatment was put through a strong cation
exchange colunnn to remove the lead, andithe unadsorbed portion, or
filtrate, was tested, there was no positive response (No„ 268),
When the pH of this material was adjusted from about 3 to near
neutral, there was attraction (No, 269). When a further portion of
the unadsorbed material from the cation exchange column was put
through a strong anion exchange column and the adsorbed (eluate)
and the unadsorbed (filtrate) portions tested, there was no response
to either one (Nos, 26l and 262); the pH was unknown, but was pro-
bably basic. In a repeat experiment with the unadsorbed material
from the cation exchange column, adjustment to pH 7 gave a response
(No. 272), but adjustment to pH 2 gave no response (No. 277), There
was the possibility that loss of attraction was related to pH,

In another series of experiments designed to check the
stage at which loss occurred and the effect of strong and weak ex-
changers, bigeye flesh after extraction with alcohol was again treat-
ed with lead acetate, filtered, treated with sodium chloride (fig, 13),
and divided into aliquots such that each test contained the treated ex-
tract from 400 g„ of flesh. One portion gave an excellent response
(No, 273) even after heating (see above), A second portion was
passed through a weakly acidic (Duolite C-3) cation exchanger and
the filtrate was tested, with a weak positive response (No, 278).
A third portion was passed through a strongly acidic (Amber lite
IR~120) cation exchanger and the filtrate was tested, with negative
results (No„ 274), Fourth and fifth portions were treated as above,
but the filtrate from the strong cation exchanger was passed through
a strongly basic (Amberlite IRA-400) anion exchanger (No, 275) and
through a weakly basic (Duolite A- 3) anion exchcinger (No, 276), and
the eluates tested, in each case with negative results. The only
noticeable reaction with materials fractionated with ion exchangers
was with the filtrate from the wecikly acidic column. Again, the neg-
ative results may have been due to the unresponsive condition of the
fish (although they reacted well in No. 273) or to the rather extreme
changes in pH which occurred in passage through the ion exchangers
--from about 7 to 3 with the cation exchangers and from about 3 to
12 with the anion exchangers.

Another series, designed to check the stage at which
loss of the attractive substance occurred and the effect of pH, is
depicted in figure 14. Yellowfin flesh was extracted with alcohol,
the filtrate was acidified with hydrochloric acid to precipitate the
lead as lead chloride (rather than as sodium chloride, as before),
and filtered. One portion of the filtrate gave a good reaction when
tested (No, 280). A second portion of the filtrate was evaporated
to dryness over a water bath and dissolved in absolute ethyl alcohol,

50

Bigeye fleshy ZOOO go , 1st alcohol extract
filtrate

Lead acetate added; filtered

Sodium chloride added to filtrate; filtered

Filtrate

Heated on Through weak
water bath cation exchange
I column

No, 273

Filtrate

Through strong cation
exchange colunnn

Filtrate

XXX

NOo Z78
X

NOo 274 Through strong Through weak
anion e><change anion exchange
column column

Eluate

j

No„ 275

Eluate

!

Noo 276

Figure 13o --Fractionation of a first alcohol extract of bigeye
flesh; procedure and results in experiments
NoSo 273 to 276„ and 278o

51

Bigeye flesh, 2000 g. , 2nd alcohol extract,
filtrate

Lead acetate added, filtered

Filtrate acidified with hydrochloric acid,
filtered

Filtrate

No. 280 Evaporated
to dryness
XX I

Dissolved in
absolute
alcohol

No. 281

XX

Adjusted to pH 9

Passed through weak anion
exchange column

Eluate

No'. 282

Filtrate

No, 283

XX

Figure 14. --Fractionation of a second alcohol extract of bigeye
flesh: procedure and results in experinnents
Nos. 280 to 283.

52

again giving a good response when tested ,Noo »:81»o A third portion
was adjusted to pH 9 with axnmonium hydroKide;, passed through a
weakly basic jDuolite A-3* ajiion exchange column^ and the adsorbed
material was eluted with weak ammonium hydroxide and testedj, giv =
ing a positive but weak response (NOo ^82io The unadsorbed iiltratep
however^ gave a stronger response |Noo 283)o Apparently there was
only partial separation of the active substance on the anion exchangerc
The fact that the results were generally positivCo as compared with
the negative results of the previous serieSj, is surprising as they
were based on a second and presum.-ibly weaker alcohol extraction of
the same material as was used previouslyo The main differences
were the precipitatfon of lead with hydrochloric acid rather than
sodium chloride and the elimination of the cation exchanger „ neither
of which should have led to the differences o They may have been
caused by either aberrant fish behavior or slight differences in
techniqueo

In a third alcohol e3etraction of the same tuna flesh as
used previously »ifig<, 15% the filtrate was treated with lead acetate^
filter edj the filtrate was treated with hydrochloric acidp filter edp the
filtrate was adjusted to pH 7„ passed through a strong ; Amberlite
IR-120) cation exchangerj, and the filtrate of pH Z was tested with
negative results (No, 285, „ When the filtrate from the cation ex-^
changer was adjusted to pH 8 with ammonium hydro>ide„ there was
a positive response ?NOo 286'o When the materisil adsorbed on the
cation exchanger was eluted with weak acetic acid and tested^ it
gave an even stronger response (NOo Z87>o Again^ the results are
puzzlingo On prior testSo the attractant had not been adsorbed (to
any great extents, at least) on the cation exchanger o For the materi-=
al that was not adsorbedp the attractive qualities seemed to be lost
at a low pHo

At this time it was suspected that the variability and
inconsistency of the results might hdive some relationship to the pH
of the materials -ind the length of time »^hey were subjec 'o extremtrS
of pHo In passing through cation and Anion exchangers;, and in other
treatments which were usedp pH s ranging from 2 to 12 were cGn\ =
monly induced for varying periods of timeo

Several preliminary experiments with skipjack v.scera
preparations (NoSo 288^ 289, 290; No&. 295, 296„ 297? and bigeye
flesh preparations (Nos, 292^, 29 5^ 294'i m which e>tracts exposed
to pH's of 2^ 7j and 10 were compaiedj gave inconsistent results
but suggested that the response might be less at the two extremes;
the mean tobservationalj scores for the three sets of experiments
listed above were UO^ l,8j and 1„ 5 for pH-s of 2p 7, and 10^
respectively;,

53

Bigeye flesh, 2000 g. , 3rd alcohol extract,
filtrate

Lead acetate added, filtered

Filtrate acidified with hydrochloric acid,
filtered

Filtrate adjusted to pH 7

Passed through strong cation exchange
column

Eluate

No

287

Filtrate

pH 2 pH

XX

No. 285 No, 286
X

Figure 15. --Fractionation of a third alcohol extract of bigeye
flesh: procedure and results in experiments
Noso 285 to 287.

54

A series of experiments was designed to tes' the hy^
pothesis that the phosphate ionp assumed to be an integral part of
the attractanto was made available at a high or low pH through hy =
drolysiSo and that this was then precipitated ((and losti as magne-
sium ammonium phosphate by reaction with the seawater when the
test substance was introducedo Bigeye flesh was extracted with
alcohol and the filtrate was adjusted to pH 3 with hydrochloric acid
to make the phosphate available for later removalo The filtrate was
divided into sut parts,^ each treated differently as shown in figure
160 Since there was a strong response to that part subjected to the
most drastic method of removing the phosphate (No,; 303)o nannelyj
adjusting to pH l^, boiling 15 nnmuteSj adding Oo ^ go magnesium
chloride a adjusting to pH 9„ standing overnightj and then filtering
and testings, the hypothesis that attraction is associated with labile
or easily removed phosphate was discarded as untenablco

Finallyj, a series of experiments was designed to in-
vestigate the variability of the data iwith only ',wo tunny left in the
pond)j the effects of pH, and the effects of time lag between pre-
paration of the material and testingo This serieSj conducted over
a 3=>week period in April 1952 (No. 313 et s^e^o )» has already been
discussed from the point of view of variability of the datao As may
be Calculated from table 3 fusing eight complete replicates! the
mean (observational; scores were lo8„ i„ \^ and lo3 at pH s of 2,
1, and lOj, respec tivelyo Although the results are suggestive of
decreased response at the two extremes of pHp the differences wheaa
tested with either the observational or the quantitative data are not
significant statisticallyo Moreover^ the relative superiority of the
acid and base ajfe reversed as compared with the preliminary re-
sults given in a preceding series with skipjack viscera extracto

The preparations were made on a Saturday and were
tested in two replicates on the following Tuesday or Thursday by
one obseiverj and agaJi". in two more replicites on the following
Saturday by a second observer^ Thus the material tested by one
observer was prepared for a shorter period of time than that tested
by the second observero The datSi, although incomplete for reasons
discussed previously;, yield 12 paired comparisons which may be
segregated as follows according to time duration and pH;

1

"Tr-— — -

pH 2
pH 7
pH 10

Shorter time
(Observer 1^

Longer time
(Observer 2)

2ol
lo4
lo3

lo7
2ol
lo2

Number of
comparisons

55

"2 ffi

<

■d

1— 1

0)

■M

m

ffi

0

a

Tl

<C

09

o o

o

CO

O
CO

■O vu '-'

X

t3 H *

« OJ Id 0)

?> N ;S :2 T3

« -^ :S -S ^

O

• . X

o ""
Z

4)

0)
U

o

u

CO

(4

o

h

CO

•M

ii

0

0)

4J

.-1

00

0

o^

^

(M

0

u

•

r-l

§

-H

^

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m

u

0)

^

w

V

(d

s

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^<

0

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15

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56

The data suggest that a* pH c the shorter the period be-
tween preparation and testing the greater the responseo Agdin^ how-
everj the difference is not statistically significanto Moreover^ an
even greater difference in the opposite' direction is present at the
neutral pH 1 ,, AIsOj of course^ any^ diiierences due to time may be
confounded with possible differences between observers o

The experiment provided no proof that differences in
either pH or in tiine lag at various pH s caused differences m re-
ponseo Rather^ it emphasized the great vairiability of the response
--a factor which doubtless contributed mostly to the erratic results
encountered after the nniddle of December 1952o In view of this^
further attempts at isolaiion and identification of the attractant were
abandonedo

SUMMARY AND DISCUSSION

General

The present investigation^ conducted from June 1„ 1952
to May 31i, 195 3j, is a study of the response oi captive tunny to ex-
tracts of fish flesh, viscerap etCo and to certaon chemicals in solu-
tion or suspensioHo

The tunaSj caught by trolling^ were transported to shore
and established in a small concrete tank and a large pond at the
Hawaii Marine LaboratorVp Coconut Island^ OahUo For the most
partj they were fed strips of tuna flesh 3 or 4 times a week andj on
this dietp more than tripled their weight during the course oi the yearc
The two tunny estiblished in the t^nk during the summ<=r of 19^2 sur-
vived until the following summero The population o^ 1 3 tunny^ estab-
lished in the pondj waS decimated in two periods of mortality during
the fall and winter; two fish survived to the following sunnmero

Test substances were introduced into the tarik or pond
through a continuous stream of water supplied by an electric pump.
When and where possible;, precautions were taken to avoid distrac
tion of the fish by e>tr ^r-eous audio or visual stimullo

When certain test substances such as clear aqueous or
alcohol extracts of tuna flesh were introduced into the tank or pondg
a response was obtainedo This took the form of a feeding reaction
and included one or all of the following components^ speeding or
acceleration of the swimming ratCp a return to the area of distribu-
tion of the substanceo surfacing^, fanning-out and eventually ^ break <»
down of school fcrmationo circltngi, splashing^ and bitmg at incident-
al objects on the surface of the wa+ero

S7

Although a positive response was obvious to the observer ^
it was extremely difficult to measure quantitatively because of its
complexity and also at times because of erratic behaviour of the fish„
Two quantitative measures were used, namely (1) the time out of 2-
(tank) or 3« (pond) minute periods spent by the fish in an "attraction"
area in which the test substance was dispersed, and (2) the number
of "passes" (movements of each fish in one of two directions) in the
attraction areao These measurements were usually made during five
control periods (before introducing a substance) and during five test
periods (after introducing a substance),, A roughly quantitative
(observational) measure of the strength of a response in one of five
categories (- to XXXX) was also recorded for each experiment^ With
either measure, the response to the sanne test substance was ex =
tremely variable in both manifestation and strength, producing appar-
ently inconsistent results in many of the replicated experinnentSo

It is postulated that in tuna flesh there is present a
substance (or substances), herein called the attractant, which is
perceived by the tunny through its sense of smell or taste and which
promotes the urge to feed.

The attractant was present in the flesh, viscera^, and
blood of several species of tuna and also in the flesh and blood of
certain white-fleshed fish. It was not present in beef bloodo It was
found in tuna cannery byproducts such as viscera preparations,
stickwater, and fish mealo

Some 40 chemical compounds were tested as time
permitted, including certain amino acids, vitamins, aromatics,
proteins, etc„ In some, notably certain amino acids, vitannins, and
aromatics, there appeared to be a sensing of the dissolved or sus-
pended compound but the results could not be duplicated. In no case
did the response include all ol the components of a typical reaction to
a tuna flesh extracts

Nature of the Attractive Substance

Much of the work was devoted to attempts at
purification, fractionation, and identification of the unknown attract-
ant. Its properties are summarized in the paragraph which follows;

The attractive substance is soluble in water and alcohol
but not in petroleum ether or acetone. It is not destroyed by cold or
by heat (although it may be partly precipitated on boiling an extract
which contains it)„ It is not precipitated by sodium chloride, tannic
acid, lead acetate, phosphotungstic acid, or other substances which

58

remove proteins and purineso It will pass through a dialyzing

membrane (in partj, at leastJo It is not adsorbed fto any l&rge extent)
on activated caybono It may be loosely adsorbed on colunnns oi ac-
tivated Alumina or ion exchange resuiSo Of the la^tterp it appeiirs to
be held to a greater extent on an cinion exchanger thjtn on a cation
exchanger^ although the results are not consistento It is affected
slighrlyp if at all^ by radical changes in hydrogen ion concentration^
In its purest form yet att.ainedj chemical teste showed the presence
of phosphorus (but not sulphur% the amide linltp and the benzene ringo

It is not ye*: possible to identity the attractive 8ubstance(8)
from its chemical properties o it does not appear to be an amino acid,
a fatty acid or lipoidj a pur-ne^ or a protemo In many of its proper-
ties it resembles Vitamin Bi^o but this substance^ while sensed by
the fisht, did not promote a typical positive response when testedo

Preservation of the Attractant

As sea testing of the extracts was contemplatedu
considerable interest centered on methods of preservationo Aqueous
extracts of flesh ^nd viscera could be kept, for 2 or 3 weeks at tem=
peratures at or just above the freezing point without excessive
putreiiction and loss of aotivityo Aqueougi extracts could be preserved
indefinitely in a Z^percent solution of sulphur dioxide gaSj, sulphuric
acidj, phosphoric acidp or sodiiim bisvilphlte; apparently these chemi-
cal preservatives were not repul&ive to the fisho Alcohol extracts
could be kept indefinite lyo Both water and alcohol extracts could be
boiled to drynesSp producing a. dark brown^ gummy residue which
could be kept without deterioration lor long periods of time^ p>aJticu=
larly if suspended on sodium chloride crystals; when the gum.my sub-
stance was re^dissolved in water it was still attractive to the fisho

Pos sibility of Conditioningjof^the_ Fish

The question arises as to what extent the typical response
to extracts of tunc flesh and viscera is promoted by a conditioning of
the tunny to life in captivity andp particul^irryo to the type oi food fed
and the method of feedingo

The life of the tuna in the tank and pond differs greatly
from that in the seao The fishes' movements a-re linnited by the walls
and they fall into a pattern which is often precise and regular -much
different from that of the open seao Not only are their movements
limited horizontally but they are also limited vertically by the
shallowness of the water o

S9

To survive in the pond or tank the tuna must become
conditioned to feeding on inert, dead rather than motile, living food.
After beconming conditioned to feeding on food such as strips of aku
flesh, the fish appeared to have little interest in live food such as
small baitfish (Stolephorus and Pranesus), which at times were
present in the pond in fair abundance. On rare occasions they were
observed to pursue the baitfish, which usually schooled near the sea-
ward gates of the pond, but they would give up when the baitfish re-
ceded to a position close to the gates. They were not observed to
actually capture any, and usually they ignored them. On the other
hand, the tunas would alnnost invariably circle and snap at objects
other than food which were thrown or fell accidently on the water
surface (leaves, pieces of cellophane, etc.). This response was
probably conditioned by the method of feeding. When a person ap-
peared at the edge of the pond with a bucket of food, the fish would
often see him and gather nearby, waiting for the food to be thrown to
them. When it was thrown, they would follow its path and take it
immediately it landed on the surface.

It is possible that they were conditioned to being fed at
regular intervals of time (usually 4 p.m. every second day), although
they would respond to the presence of both persons and food at any
time except immediately after being fed.

As most of the extracts which were used were not visible
to the fish, there is no doubt that the response was through the fish's
sense of snnell or taste rather than through its vision. However, there
is the question as to whether the fish responded to skipjack flesh ex-
tracts because they were being fed pieces of skipjack, or closely-
related tunas, as a regular diet. There is evidence that the response
was not conditioned by the type of food which was fed.

Before and during the early experinnents in the tank (to
July 18, 1952) the one established tunny was fed a non-tuna diet
(squid and baitfish), yet it still responded to extracts of skipjack
flesh (NoSo 1, 8, 10) which it had not tasted while in captivity.
Following April 25, 1953, the two tunny in the pond were fed exclu-
sively on squid, yet eilmost 1 month later. May 19, 1953, they still
responded to extracts prepared from skipjack viscera and yellowfin
flesh (Nos, 350 to 356).

The fact that jpositive responses were obtained to extracts
of fish other than tuna- -aholehole (Nos. 49, 88, 105), jack (No. 96),
and barracuda (No, 98) --while the tunny were still being fed skipjack
is also evidence that the response was not conditioned by the species
of fish which was being used as food. This, of course, is also

60

illustrated by responses to extracts of tunny and yellowfin while the
fish were being fed on skipjack^ and responses to extracts of skip-
jack while the fish were being fed yellowiin or bigeye tunao

It may be concluded that the response of the tunny m
captivity IS not directly conditioned to the species of food wh^ch was
fedo However^ there still remains the possibility that the tunny
formed a mental association between feeding and the smell or taste
of the dead food^ which was cut up or otherwise macerated and
exuded juices of similar composition to the extractSo An association
such as this would not necessarily be present in the case of wild fish
feeding on whole living organisms at sea, ThiSj, or some other
subtile type of conditioning to life in the pond^ may have contributed
to the response.

The part played by conditioning can be ascertained only
by testing the extracts on schools of fish at seao Certain materials
for sea testing have already been prepared and pond-tested (Nos, Z64,
279, 29I0 304 to ilZc 350 to 356'jo The results of sea tests of these
and other preparations will be discussed in a later reporto For the
present it may be stated that the preliminary sea tests were largely
negative o

61

LITERATURE CITED

SUZUKI, U. , K. JOSfflMURA, M. JAMAKAWA, and Y. IRIE

It
1909. Uber die Extrakfivstof^e des Fischfleisches. Hoppe-
Scyler's Zeitschrift fur Physiologische Chemie
62{1)- 1-35,

TESTER, A. L.

1952o Establishing tuna and other pelagic fishes in ponds
and tanks. U. S, Fish and Wildlife Service, Spec.
Sci, Rept. : Fisheries No. 71. 20 pp.

VAN WEEL, Po B.

1952. Reaction of tuna and other fish to 8timuli-1951:

Part II - observations on the chemoreception of tuna.
U, S. Fish and Wildlife Service, Spec, Sci. Rept.:
Fisheries No. 91: 8-36.

62

PART 11

RESPONSE OF TUNA TO VISUAL
AND VISUAL -CHEMICAL STIMULI

BY

Sidney C. Hsiao and Albert L, Tester
University of Hawaii

During September 1952 a few experiments were con-
ducted on a population of 12 little tunny 1/ (Euthynnus yaitoli established
in Pond Noo 5 at Coconut islands Oahuj, to determine {1} whether visual
lures would promote a tropistic response^ fZi whether the response
was heightened when combined with chemical stimulip io e^^ an ex-
tract of tuna fleshj, and (V whether the response varied with lures of
different colors o It was hoped that the results would be< of interest
and value in devising lures which, either alone or in combination
with extracts^ would assist in attracting tuna to the stern of fishing
vessels at sea,

METHODS

The lures consisted of 2-inch sections of l/2»inch rubber
tubing of different colors which were suspended from a cross bar
attached to the tip of a 25=foot bamboo polej pivoted near the point
of balance ffigo 17)o By means of a cordj, a counterweight^ and a
System' of pulleys, two lures^ one suspended from each end of the
cross bar^ could be momentarily dipped into the water^ When an
experiment was in progress the lures were lo^wered about once every
2 secondso

The lure array was fastened near the base of a 20^foot
observation tower and could be manipulated from the platform on top.
Observations of the fish were made when they entered the 39 x 75-foot
attraction area {the same area that was used in chemical stimulation
studies) marked off by two pieces of cord stretching across the pond.

Note; Contribution NOo 48 of the Hawaii Marine Laboratory,
University of Hawaii, Honolulu, To Ho

\/ The activity of one "sick" tunny was not recordedo

63

Fig, 17. --Diagram of mechanical device for lowering lures into water: AB - cross-
piece: LL' - lures: U - supporting post: F - fulcrum; C - notch; P - plate to cover
notch; W - weight; C - cord.

64

All experiments were conducted while the pump at the seaward or
western end of the pond was operatingo This projected i strong
current of water imo the first 1/ 10T,h of the pond atA a weaker cur-
rent into the second l/10*hp the attraction area. In some experi-
mentSp the lures only were used; in others^ stock extracts of tuna
flesh were introduced through the pump flow at the same time as the
lures were presentedo A detailed description of the pond^ pumpp
stock extract^ normal behavior of the fish^ etCoj is given in Part I
of this report by Tester^ van Weel^ ond Naughtonp and the general
arraungement for testing is shown in figure 4o

In a typical experiment the pump was switched on and the
observers on top of the tower waited vintil the fish were behaving in
a normal fashionj io e<, „ cruising slowly back and forth along the
length of the pond in one or two schools o The activity of the fish was
observed and recorded during a 15-minute control period ^the data
could be divided into 3-mmute intervalsjo The lures were then dipped
momentarily into the water once every I seconds and the activity of
the fish was observed and recorded for a 15 -minute experimental
periodo The colored lures were then reversed in position (switching
left to right and right to left)) and the observations were repeatedo
After two or more 15»minute experiments with lureSj stock extract
was addedp the lures were dipped into the water „ and observations of
activity were made and recorded for another experimental period.
The lures would then be switchedj, or changedp dJid the experiment
repeated,, In some of the early tests (9/3 and 9/5? the control and/or
exppirimental periods were of shorier duration ^9 or 12 mmutesJo

The behavior of the tunny;, particularly during expert^
mental conditions „ was exceedingly complexo It was difficult to
measure the activity with the usual device5==.electrically controlled
time clockp stop watch;, and mechanic al counter=--and to record the
data with pencil and paper, even when two observers were working
together on top of the towero A system of recording the behavior was
worked out which consisted eventually of (1) the use of a tape recorder
to give a running verbal description of the activity of the fishj and
(2) the transcription of this information on the smoked paper of a
kymograph drum by operating a series of levers to which styli were
attachedo The transcribing apparatus is illustrated in figure 18„ It
consists of a kymogrdph iF'^, a Franz kymograph timer 'C- with two
recording pointSp a lever 'B) and stylus attached by a cord to the
axle of a telephone dial (D; so that the length of the stroke on the
drum 18 proportional to the number dialed^ and a lever (A) and stylus
attached by a rigid rod to a toggle key (Ei which can be flipped up or
down from a neutral position to make an up or down stroke on the
smoked paper. The time is recorded by Cp the rumber of fish in

65

n

S E

.a £

> 3

u o
o o

(J 0)

5 D

S ^

•"

J3

XI

fi

«l

11)

h

0

tlf

K

o

66

the area is recorded by Bj, and the number of passes at one fupl or
the other (down) of the two lures is recorded by A„ Typical record^
ings are shown in figure 19o

In addition to the above „ the speed of the tunny was
determined in several trials under control and experinnental condi=
tions by timing the leader of the school as it passed through the
attraction area;, using a stop watcho

RESPONSE UNDER CONTROL AND
EXPERIMENTAL CONDITIONS

The pattern of activity of the fish was observed under
control and experimental conditions and is discussed below,, The
activity was measured quantitatively in several different waySp each
of which will be presented and discussed in the sections to followo

Pattern of Response

During control periods the tunnyj, usually in one school
or in two schools of unequal size;, swam slowly back and forth along
most of the length of the pond^ looping in deeper water near the ends^
or often pausing to "play" m shallower water at the ends before
looping,

A kymograph record of observations during a 15-minute
control period in one experiment is shown in figure 19Ao It is dis-
cussed in detail to illustrate the interpretation of the kymogramo
The first histogram shows that 22 seconds after the startp a school
of 12 tunny entered the field from the easto After spending 24 seconds
m the fieldp they left by the sime side as they came m^ and swam to
the eastern end of the pondo After 165 seconds the school returned
and swam through the field m 10 secondSf as shown by the second
histogramo The school looped in the western 1/lOth of the pond
during a 9-'Second interval and re-entered the area from the western
side. As shown by the third histogram, they sped through the field
in 8 secondSo After 147 seconds 'he schools re-entered the area
from the eastp stretched out in linear formation^ with the leader
entering the field about 6 seconds ahead of the 12*h funny (fourth
histogram! „ After leaving the fieldp the school spent 84 seconds in
the western 1/lOth of the pondj, during which time they circled in
the shallow southwestern corner near the pump. After passing
through the field for a fifth timei, the tunny stayed in the eastern part
of the pond for a long period- =about 4 minutes- before returning to
pass through the area fsixth histogram)o When they entered the area,
2 turned back, but 10 passed through to spend 60 seconds m the

67

M U

a. *>
H 5

"J

68

western 1/lOth of the pond. On turning back they sped eastward
through the field in 12 seconds (seventh histogram,, The two tunny
which separated from the school earlier circled slowly outside the
eastern border of the fieldp and 15 seconds after the nriajor school
left these two fish re-^entered the area and swam inside for 18
seconds (eighth histogram)o

When a pair of colored lures was lowered repeatedly
into the water inside the attraction area^ one or more tunny might
be moving westerly towards the ^rea and near enough to see the
moving objects. In this case they would usually swim toward the
lures and try to take thema It often happened that when one or two
tunny started to bite^ the others near them would become active and
circle back to the lureSo On the other hadj, if the tunny were at the
far end of the pond„ the lures might be lowered repeatedly for a
long time without any response from the fish until they approached
the attraction areau When taking after the lureSj, the speed of move*
nnent was not noticeably increased compared with that during the
control periods

Figure 19B shows the reaction of the tunny in an experi-
ment in which two lures, one red and the other blacky were presented„
They entered the field 17 times in groups of different sizes^ In the
third 3-minute interval,, attraction and excitement is indicated by the
close spacing of the histograms. The symbols "C" and "O" in the fig-
ure-ir.dic ate looping andcirclingp respectively^ inside the area, Fronn
the strokes indicating the response to the lures (upper line), it will
be seen that they attennpted to "take" the red lure 8 times and the
black lure 6 timeSo

When a stock extract of tuna flesh was introduced
through the stream of water at the western end of the pond the tun-"
ny would become greatly excited as soon as they sensed itj Usually
the extract was propelled Into the attraction area about 1 minute
after its introductiono The fish would dash about with increased
speedp biting at floating objects such as leaves and sticks, as well
as running for the lures and fighting among themselveSo There was
a clearly visible feeding reactiono But after 10 to 15 minutes the
response would gradually disappear and they would assume a pattern
sinnilar to that seen when lures alone were used,^

In figure 19C a kymograph record of the response to
lures and extract is seen. The number of passes at the lures is
indicated by numbers rather than by strokes. The great excitement
when extract was added is evident from the increased frequency of
the histograms,, together with their varied shapeo The fish took the

69

lures 18 times in the first 3-minute interval, 9 times in the second,
11 times in the third, and 3 times in the fourth.

These and other kymograph records are analyzed more
extensively in the sections which follow.

School Entrances

The activity under various conditions of stiinulation may
be measured by the number of schools (of varying size) entering the
attraction area during a 15-minute period. The results of the ex-
periments are summarized in table 4. In this, and other tables to
follow, the data for September 5 and 9 have been adjusted to a 15-
minute periodc The results for similar experiments conducted on
the same day have been averaged.

It will be seen that on each day schools entered the
attraction area more frequently (as compared with control conditions)
when a pair of colored lures was dipped into the water, and still more
frequently when lures were used together with extract. The grand
mean number of entrances was 6=7 for controls, 14„ 6 for lures, amd
21.6 for lures plus extract. Obviously the differences between the
means are statistically significant.

Table 4, ==Summary of data on the number of
schools entering the attraction area
under various conditions of stimulation

Condition

Control

Lures

Lures plus
extract

9/3

38,25

9/5

10 I 5
19,25 13

2U25

Date

9/8
9

22,33

9/10

3

12,5
14,00

9/12

7
16
16,75

9/17

6
12
17

Mean

6,7
14. 6
21.6

Percentage of Time in Area

The percentage or relative time spent by the fish in the
attraction area is summarized in table 5 for the various conditions
of stimulation. On the average the time spent in the area was 12, 0

70

percent during control conditions^ Z4o 5 percent when two lures were
usedp and 29o 5 percent when the lures plus extract were usedc A
rough test (using "t'M of signiiicajice of the difference of the paired
observations (neglecting the varying intrinsic accuracy of the mean
determinations for each day^s experiments" indicates a significant
difference between control and lures {mean difference^ 13,8 percent;
P < OoODj but not between lures and lures plus extract (mean
difference^ 4o 0; P - 0„2)o

Table 5, ^-Summary of data on percentage time
spent in the iTtraction area under
various conditions of stimulation

Fish^seeonds in Area

The summation of the product of the number of fish and
time spent in the area is probably a better measure of activity than
the two measures discussed aboveo This is equivalent to summing
the areas under the histograms in the kymograph records^ and may
be expressed in fish= seconds per I5-niinute periods The results are
given for individual experiments in table 6o

In comparison with the controlSj, it will be observed that
the number of fish^seconds is generally greater when the lures are
usedp and slightly greater still when the lures are combined with
extracto The grand means for controlp lures^ and lures plus ex-
tract are respectively 114,3p 352o8j and 364o8 fish-sec ondSo In
experiments with lures .^alonej which were repeated in succession on
the same d<ayp the measure tends to fluctuate rather widelvp showing
neither a consistent increase not decrease in responseo In experi>=
ments with lures plus extractj the measure tends to decrease with
repeated stimulation;, indicating a dulling of the response There are
five comparisons of controlp lureSp and lures plus extract involving

71

Table 6, --Number of fish-seconds spent in the
attraction area under various condi-
tions of stimulation

Date

Condition

Control

Lures

Lures plus
extract

9/3

47

(1) 253

(1) 522

(2) 640

(2) 107

(3) 101

9/5

100

(1) 683

(1) 371

9/8

120

_

(1) 657

(2) 310

(3) 345

9/10

34

(1) 646

(1) 380

(2) 165

(2) 167

9/12

135

(1) 453

(1) 504

(2) 319

(3) 292

(4) 289

9/15

124

(1) 207

(2) 168

(3) 115

~

9/17

240

(1) 80

(1) 635

(2) 370

(2) 209

initial experiments, indicated as "(1)" in table 6, These yield the
following comparable means: 111„2, 423,0, and 482,4 fish^seconds,
respectively„ Obviously, the means for both lures alone and for
lures plus extract are both significantly greater than that for controls „
Although the mean for lures plus extract is greater than that for lures,
the statistical significance of the difference cannot be established with
the present data.

Speed of Swimnning

Another measure of activity is speed of swimming. This
was determined by timing the leader of the school as it passed be-
tween the two lines marking the boundary of the attraction area.

72

Sources of error include the effect of parallax in determining when
the fish is exactly below the lines, deviation from a path exactly
perpendicular to the lines ^ and deviation from a hbrizontal plane.
However f, variation induced by these sources of error is small com«
pared with the variability from trial to triaL The results of obser-
vations under control and experimental conditions are summarized
in table 7o

Table 7o "-Summary of data on speed of swim-
ming under various experimental
conditions

Condition

Number of

Mean time and

Mean speed

observations

standard error

(feet /second)

Control

12

18,42 + U60

2„12

Lures

IZ

15,08 + 0„90

2o59

Lures plus

20

lOo 15 + 0„63

3,84

extract

The mean speed of swimming increased beyond that of
the controls when the tunny were stimulated by lures^ and it in-
creased still more when they were under the combined stimulus of
lures ajid extractc The mean difference between control and lures is
not significant statistically (P ~ O0O8L However, that between lures
and lures plus extract^ and also that between control and lures plus
extract are both highly significant fP < 0, 01 in each case)„ The in-
creased rate of swimming of the tunn/ when stimulated by the extract
was obvious to the observer. The actual mean swimming rate under
these conditions (3„84 feet per second) is equivalent to 2,6 nniles per
hour. Of course^ bursts of speed several tinnes this magnitude were
frequently observed,

j'eeding Activity

Feeding activity may be measured by the number of
passes (bitings or attempted bitingst at the lures both when used
alone and when used in conjunction with extract.

Table 8 shows the nnean number of passes at the lures
per IS'-minute period on 5 successive days The grand means for
lures and for lures plus extract are respectively 14 and 40 passes

73

per 15-minute period. The increased feeding response under the
additional stimulation of extract is apparent without further statis-
tical analysis. The data are not suitable for isolating and testing
the component of variance associated with daily variation in
response;

Table G - Mean number of passes at lures

under two experimental conditions

RESPONSE TO LURES OF DIFFERENT COLORS

Each lure of a pair differed from its mate in color.
The following combinations were tried: white-red; white-black;
white- silver; red=black; and silver = black„ As already explained, the
positions of the lures were interchanged between experiments o The
number of passes at each lure per 15-minute period was determined
when lures were used alone and when they were used in combination
with extracto The results are summarized in table 9.

From the table it will be observed that the white lures
received either the same or, more usually, a greater number of
passes than the colored lures (either tested alone or with extract)
when paired with red, black, or silver lures. Similarly, the red
lure seemed superior to the black in nunnber of passes. Howeverj
none of the ratios p either individually or grouped according to simi-
lar experiments r differ significantly from a 1:1 ratio when tested
with Chi'Square- From the pooled data for white versus all other
colored lures with which it was paired there is evidence of the superi-
ority of white. The ratio of pooled values for white vs, color is
155;122 which, when compared with the hypothetical 138,5tl38„5„

74

yields an adjusted Chi-square o£ 3,7 with one degree of freedom
(P = 0<,06), which could be regarded as evidence for rejecting the
hypothetical equality ratio. Although there is this evidence of a
significantly greater number of passes at the white as compared with
the colored lureS;, the superiority of the white lure is slight. Knowing
that the tunny will make a pass at any inanimate object which appears
on the surface,, including not only lures but also leaves^ stickSj pieces
of paper, etCo ;, the slight superiority of the white lure may perhaps be
attributed to its greater visibilityo There is no assurance that this
superiority would be manifested in the sea;, where conditions of sea
water transparency and background color would be much different
from those in the pond.

Table 9== -Number of passes at paired lures

of different colors under two experi^
mental conditions

White -red

White-black

White -silver
Red-black

Silver -black

(1)

5=5

(1) 31=27

12)

10-8

(2) 7-4

(1)

8^5

(1) 10^9

(2)

4-4

(2) 12-7

(3) 7-2

(4) 21 = 19

(5) 6-5

(6) 15-10

(1) 8.8

fl)

(2)

3^3

9=9

(1) 1U8

(1) 11 = 9

(1) 23=20

(2) 16-13

(3) 9^6

(1) 12-12

75

SUMMARY

The activity of 12 little tunny in a large pond was observed
during September 1952 under control conditions (no stimulation), when
a pair of lures was dipped into the water once every 2 seconds, and
when the lures were used together with stock extract of tuna flesh.

Observations were made in an "attraction area" which
could be viewed from the top of a 20-foot tower. Activity was re-
corded verbally on a tape recorder, and later transcribed to a kymo-
graph drum„ The kymograms furnished quantitative data on activityo

As compared with control conditions, schools entered
the area more frequently when stimulated with the lures, and still
more frequently when stimulated with lures plus extract.

As compared with control conditions, the fish spent more
time in the area when stimulated with lures, and still more time when
stimulated with lures plus extract. However, the difference in rela-
tive time between the two experimental conditions was not statisti-
cally significants

When activity was expressed as number of fish-seconds
per unit time in the area, the results were similar to the foregoing.
When lures and extract were used together, there was a dulling of
the response with repeated testing.

The use of both lures and extract increased the rate of
swimming from an average of 2, 12 feet per second for controls to
an average of 3,84 feet per second.

The fish made many more passes at the lures when
stimulated with extract^ showing a heightened feeding response.

Although, in general, the fish nnade more passes at
white as compared with colored lures, the superiority of the white
lures was slight. It may have been associated with greater visibility
rather than color preference. There is no assurance that white
lures would be superior to colored lures in the open sea.

76

APPENDIX

A summary of experiments on chemical stimulation
conducted in tank (T) and pond (P) by van Weal (vW), Tester (T),
or Naughton (N), over the period July 1, 1952 to May 19, 1953,
with reaction classified in categories ranging from "-" to "XXXX'

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