Taste Preferences and Feeding Behavior in Ninespined … · 2015-10-01 · vae once a day after the...

ISSN 0032�9452, Journal of Ichthyology, 2015, Vol. 55, No. 5, pp. 679–701. © Pleiades Publishing, Ltd., 2015.Original Russian Text © E.S. Mikhailova, A.O. Kasumyan, 2015, published in Voprosy Ikhtiologii, 2015, Vol. 55, No. 5, pp. 541–564.

679

INTRODUCTION

The studies performed in recent years demonstratethat free amino acids, carboxylic acids, salts, sugars,and many other chemical compounds in the foodorganisms (Carr et al., 1996) have important gustatoryproperties for fish. Some of these substances are highlypalatable and increase consumption of food by fish,others have an aversive taste and cause rejection offood, and yet others have an indifferent taste and donot influence the process of feeding (Kasumyan andDøving, 2003). Gustatory spectra is the stable species�specific feature; they differ in their range and compo�sition between various fish species, including thosebeing closely related or having a similar mode of life(Kasumyan, 1997). Since the consumption of food byfish depends on the gustatory properties of the latter(Mackie, 1982), differences in feeding preferencesmay diverge coexisting fish based on their food objectsand decrease the competition for food between them.

The taste preferences of fish are little influenced byvarious external factors and retain upon qualitativeand quantitative changes in fish diet (Kasumyan andMorsi, 1997; Kasumyan and Sidorov, 2010b) or after

sharp changes of the water salinity (Mikhailova andKasumyan, 2010). The gustatory spectra of femalesand males are the same in fish having noticeable gen�der differences based on the feeding intensity and dailytime spent on this form of life activity (Poecilia reticu�lata guppies) (Nikolaeva and Kasumyan, 2000). Agreat similarity has been demonstrated for the gusta�tory spectra of fish individuals belonging to the samespecies but to the different morphobiological forms(Kasumyan and Mikhailova, 2007) or different popu�lations (Kasumyan and Sidorov, 2005; Kasumyan andMikhailova, 2014). However, data on the absence orweak population differences have been obtained basedon the limited number of samples and demand furtherverification. The population differences in the feedingbehavior have been even more insufficiently studied(Kasumyan and Mikhailova, 2014).

The aims of this work are to compare the taste pref�erences to various classes of substances and the feedingbehavior observed when testing food on the nine�spined stickeback Pungitius pungitius from geographi�cally distant and isolated populations.

Taste Preferences and Feeding Behavior in Nine�spined Stickleback (Pungitius pungitius) in Three Geographically Distant Populations

E. S. Mikhailova and A. O. KasumyanMoscow State University, Moscow, 119991 Russia

e�mail: [email protected] June 23, 2014

Abstract—Taste preferences of classical taste substances (10% each sodium chloride, calcium chloride, andsucrose; and 5% citric acid), and 21 free amino acids (L�isomers, 0.1–0.001 M) and feeding behavior of thenine�spined stickleback Pungitius pungitius belonging to the populations of the Moskva River (Caspian Seabasin), Lake Mashinnoe (White Sea, Kandalaksha Gulf), and Bol’shaya River (Western Kamchatka, OkhotskSea) are determined. It is proven that the spectra of taste preferences are similar in different stickleback pop�ulations. For sticklebacks in all three populations, citric, aspartic, and glutamic acids are mostly palatable.The maximum number of palatable amino acids (16) is found in sticklebacks from the Moskva River. Stick�lebacks from the White and Okhotsk seas prefer the lower number of amino acids, 10 and 4, respectively.Deterrent amino acids are not found. The gustatory amino acid spectra of sticklebacks from different popu�lations are positively correlated (p < 0.05). Sticklebacks from the Sea of Okhotsk relatively rare taste theoffered food objects again (agar–agar pellets with the studied taste substances). Such behavior is more com�mon in sticklebacks from the White Sea, and those from the Moskva River have an intermediary position. Themanipulation activity in the compared fish is associated with the hydrology of the water bodies. In nine�spined sticklebacks from all three populations, the actions performed upon intra�oral testing of a food objectfollow two alternative behavioral stereotypes. The stereotypes of swallowing and of rejection of food differslightly between the populations and depend on the palatability of the offered object.

DOI: 10.1134/S0032945215050094

Keywords: nine�spined stickleback Pungitius pungitius, populations, chemoreception, taste, taste preferences,feeding behavior, stereotypes in food testing, amino acids

680

JOURNAL OF ICHTHYOLOGY Vol. 55 No. 5 2015

MIKHAILOVA, KASUMYAN

MATERIALS AND METHODS

Experiments were performed on adult specimensof nine�spined sticklebacks with total length (TL)5.5–7.5 cm. Fish were caught using a mesh net in threegeographically distant natural water bodies (Fig. 1): inthe small Khimka Stream (Pokrovskoe�Glebovo For�est Park, Moscow), Lake Mashinnoe (Chkalovskii Vil�lage, Loukhskii district, Republic of Karelia, Kan�dalaksha Gulf, White Sea), and near the banks in theBol’shaya River (Western Kamchatka, Okhotsk Sea

basin)1. Experiments on sticklebacks from the

Okhotsk Sea and Moskva River were carried out at theDepartment of Ichthyology, Moscow State University,and at the White Sea Biological Station, Moscow StateUniversity, on those from the White Sea. Juvenilesfrom the population in the Okhotsk Sea after deliveryin Moscow were raised for 8–9 months at the temper�ature of 18–21°С upon reaching sexual maturity.

1 We did not differentiate between nine�spined sticklebacks withincomplete and complete series of bone plates, which werefound together in the Bol’shaya River and other water bodies ofthe Kamchatka Peninsula (Pichugin, 2014). Some researchersassociate these fish with different subspecies—P. p. pungitiusand P. p. sinensis (Berg, 1949; Nikol’skii, 1956; Wootton,1976)—or forms (Sheiko and Fedorov, 2000), while others withdifferent species (Zyuganov, 1991; Dolganov and Kravchenko,2011) or consider them as a complex species (Nelson, 2009).

A methodology allowing us to estimate the feedingpreferences of fish mediated by intra�oral taste recep�tion was used to perform the study. The methodologywas developed and used to reveal the gustatory attrac�tiveness of various substances for many fish species,including nine� and three�spined (Gasterosteusaculeatus) sticklebacks (Mikhailova and Kasumyan,2006). Sticklebacks were kept individually in aquaria(5 L) in which all walls, except the font one, wereopaque for visual isolation of neighboring specimens.The food and agar–agar pellets were introducedthrough an opening in the cover. The aquaria had noground; half of the water in aquarium was periodicallyreplaced with fresh water, and aeration was enabled bymicrocompressors. The temperature of water was 15–18°С. The fish were fed with living Chironomidae lar�vae once a day after the end of the trials .

Following a short period of acclimation (2–3days), the fish learned to grasp individual Chironomi�dae larvae and then agar–agar pellets containing anwater extract of Chironomidae larvae (at a concentra�tion of 175 g/L). On the first day, the fish did not showany fear as the experimenter approached their aquar�ium, and immediately grasped individual Chironomi�dae larvae. On the second day, the same was observedwith agar–agar pellets containing the extract of Chi�ronomidae larvae. After several days, the fish kept per�

Fig. 1. Range ( ) and sites of capture (�) of nine�spined stickleback (Pungitius pungitius): (1) Khimka Stream, Borisovo�GlebovoForest Park, Moscow; (2) Lake Mashinnoe, Chkalovskii village, White Sea basin; and (3) Bol’shaya River, Kamchatka Peninsula.

1 23


TASTE PREFERENCES AND FEEDING BEHAVIOR IN NINE�SPINED STICKLEBACK 681

manently in the central part of the aquarium under theopening in the cover immediately grasped the offeredpellets in 100% of cases.

Each trial was started with introduction into theaquarium of one agar–agar pellet and was completedby swallowing or final rejection by the fish of the pelletafter one or several subsequent grasps and rejections.The moment of swallowing of the grasped pellet wasdetermined visually by termination of characteristicmovements of jaws and restoration in the fish of usualrhythm of respiratory movements of opercula. Con�clusion on the final refusal of the fish from consump�tion of the pellet was made by loss of interest and with�drawal of the fish from the pellet or by orientation ofthe fish to another direction from the pellet, often tothe opposite side. In the cases when the fish did notgrasp the offered pellet during 1 min, the pellet wasremoved from the aquarium and the trial was notcounted. The pellets with tested substances and thecontrol pellets were offered in random sequence andwere alternated with offering of pellets containing theextract of Chironomidae larvae. The interval betweenthe trials was less than 10–15 min. In the course ofeach trial, the total number of grasps, duration ofkeeping of the pellet in the mouth at the first graspingand during the whole period of the trial , as well asconsumption of pellets were counted (whether theoffered pellet was swallowed or not by the end of thetrial). In trials in which the fish destroyed the pellet butdid not swallow it or swallowed less than half of thedestroyed pellet, we considered that the pellet was notconsumed. The nonconsumed pellet was removedfrom the aquarium immediately after the end of trial .

A total of 27 types of pellets were used in trials : thecontrol ones, without any taste substances; the pelletswith the extract of Chironomidae larvae; the pelletswith one of 25 tested substances (four classical tastesubstances, 21 free amino acids, L�isomers; The sub�stances and their concentration in pellets are shown intables). The pellets with classical taste substances andfree amino acids were tested during individual experi�mental series, which also included trials with the con�trol pellets.

All pellets, including the control ones, were col�ored bright red with the Ponceau 4R (5 µM) dye addedto the agar–agar gel (2%, Reanal) upon preparation.

Pellets were cut out of agar–agar gel with a stainlesssteel tube immediately before the trial . The pelletswere 4.0 mm in length and 1.35 mm in diameter. Theprocedure for preparation of agar–agar gel and itsstorage conditions were described in our previous pub�lications (Kasumyan and Sidorov, 1994a; Kasumyanand Morsi, 1996).

A total of 3718, 3944, and 1880 trials were carriedout on 22, 17, and eight nine�spined sticklebacks fromthe Moskva River, White Sea, and Okhotsk Sea. Forquantitative evaluation of taste preferences, the indexof gustatory attraction was calculated by the equation:Indpal = [(R – C)/(R + C)] × 100, where R is the numberof swallowed pellets with the substance, %, and C is thenumber of swallowed control pellets, % (Kasumyanand Morsi, 1996).

Statistical analysis of the results was made using χ2

test, Mann–Whitney U�test, and Spearman rank cor�relation coefficient (rs).

RESULTS

Feeding Behavior

After distribution into separate aquaria, the nine�spined sticklebacks of all three groups were habituatedand, already after several days, stayed in the center ofthe aquarium under the opening in the cover andgrasped the offered pellets in almost 100% of cases.The pellet was grasped almost immediately after intro�duction. In most trials on the sticklebacks from theMoskva River and White Sea, repetitive rapid rejec�tions and repeated grasps of the pellet were observed.Such actions were less common for the sticklebacksfrom the Okhotsk Sea: in almost 70% of all trials withthe fish from this population, the pellets were swal�lowed or finally rejected after the only one grasp. Theproportion of such trial on the sticklebacks from theMoskva River and White Sea was two and three timeslower. The maximum number of repeated grasps of thepellet by the fish from all populations was 20–22(Table 1). Only after these manipulations the fisheither swallowed or rejected the pellet, canceledrepeated grasps, and moved away. Even at the highnumber of repeated grasps and rejections, testing ofthe pellet lasted for not more than 1 min.

Table 1. Share of experiments with different number of grasps of the pellet by nine�spined sticklebacks (Pungitius pungitius)from different populations, % of the total number of trials

Popula�tion

Number of grasps

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

Moskva River

35.52 18.72 14.60 10.59 6.69 4.53 3.19 2.09 1.60 0.90 0.51 0.42 0.21 0.21 0.02 0.08 0.05 0.05 – – – 0.02

White Sea 20.20 19.00 19.60 14.60 8.90 5.50 5.80 2.09 1.40 0.89 0.89 0.38 0.28 0.20 0.09 0.05 0.03 0.05 – 0.05 – –

Kamchat�ka

69.80 14.83 6.72 2.84 2.20 0.98 0.87 0.62 0.26 0.30 0.19 0.19 0.05 – 0.05 0.05 – – – – – 0.05

682



Classical taste substances. Citric acid was a highlypalatable substance for all three populations. Thesticklebacks from the Moskva River, White Sea, andOkhotsk Sea consumed about half of the offered pel�lets with citric acid (57.6, 48.2, and 37.3%, respec�tively), which is many times higher than the consump�tion of control pellets; along with that, Indpal of citricacid had even more similar values (83.1, 78.2, 88.3%)(Tables 2–4). Sodium chloride and sucrose were indif�ferent stimuli for all fish. The gustatory attractivenessof calcium chloride (CaCl2) was different for the stick�lebacks of the compared populations. For those fromthe Moskva River, this substance had an attractivetaste and increased the consumption of pellets bymore than three times compared to the control ones.For the sticklebacks from the Okhotsk Sea, CaCl2 hadan indifferent taste. For those from the White Sea, ithad a aversive taste. However, both these fish com�pletely refused the pellets of this type, i.e., rejectedthem in all trials (75 and 170, respectively).

Repeated grasps and rejections of the pellets withclassical taste substances were typical for the behaviorof the sticklebacks from all three populations. Thisbehavior was mostly expressed in fish from the popu�lations in the Moskva River and White Sea and to alesser degree in those from the Okhotsk Sea. Suchbehavior was stimulated by the pellets with citric acidoffered to the nine�spined sticklebacks of the WhiteSea population (р < 0.001). The number of repeatedgrasps was significantly lower for the nine�spinedstickleback population from the Moskva River as forthe pellets with citric acid (р < 0.05) and calcium chlo�ride (р < 0.001). Other substances did not have a sig�nificant effect on fish of these populations. Inclusionof any of the classical substances in the pellet resultedin a significant decrease in the number of repeatedgrasps.

In the sticklebacks from all populations, the pres�ence of citric acid caused a significant increase in thetime during which the pellet was kept in the mouthcavity after the first approbation by the fish and duringthe whole trial (р < 0.001) (Tables 2–4).

Free amino acids were divided into two groupsbased on the gustatory properties for the sticklebacksfrom all populations: stimulating the consumption ofpellets and indifferent ones. The highest number ofamino acids with an attractive taste was revealed in thesticklebacks from the Moskva River (16) and the lowernumber in those from the White and Okhotsk seas(10 and 4, respectively). All four amino acids palatablefor the sticklebacks from the Okhotsk Sea (cysteine,alanine, aspartic acid, and glutamic acid) are includedin those being highly palatable for the sticklebacksfrom two other compared populations (Tables 2–4).The number of amino acids with an indifferent tastewas 5, 11, and 17, respectively, for the sticklebacksfrom the Moskva River, White Sea, and Okhotsk Sea.Deterrent amino acids were not found.

The number of amino acids that changed themanipulation activity of fish (repeated grasps of thepellet throughout the trial) and the sign of this effectvaried widely among different populations of stickle�backs. In the sticklebacks from the Moskva River, foursuch amino acids were found with three of them(glutamine, cysteine, proline) having the most attrac�tive taste. All four amino acids significantly reducedthe number of manipulations with the pellets. In thesticklebacks from the White Sea, 14 amino acidschanged the frequency of repeated grasps, all of themintensified the manipulative activity. This groupincluded almost all amino acids, which were palatableto the White Sea fish (9 of 10), and some indifferent ofthem. In the sticklebacks from the Okhotsk Sea, noamino acids, including those of highly attractive ones,changed the frequency of repeated grasps of the pelletsby fish. This parameter of taste response was approxi�mately 2.0–2.5 times lower in fish from the OkhotskSea than in other sticklebacks. Overall, the parameterof response in the fish from all populations varied rel�atively insignificantly (Tables 2–4).

Many amino acids changed significantly the dura�tion of testing of the pellets by the fish. In all cases,such influence was manifested in longer retention ofthe pellet in the mouth cavity of the fish both after thefirst grasp and throughout the whole trial. For thesticklebacks from the Moskva River, this effect wasproduced by 17 and 18 amino acids, seven and 15amino acids for the White Sea sticklebacks, and fiveand five for the Okhotsk Sea sticklebacks. The effect ofsome amino acids was manifested in much (three tofive and more times) longer retention of the pellet bythe fish (Tables 2–4).

The extract of Chionomidae larvae. The waterextract of Chironomidae larvae significantly increasedthe consumption of pellets by the fish from all popula�tions. The stimulating effect produced by the extract ofchironomids is mostly expressed in the populationfrom the Moskva River, where it is higher than thatproduced by citric acid and highly palatable aminoacids. The stickleback from the White and, especially,Okhotsk seas show less taste preferences to the extractof chironomids: the pellets containing the extract werethree and six times less consumed by them than by thesticklebacks from the Moskva River population. Fishfrom the White and Okhotsk seas also consumed sig�nificantly more pellets with citric acid and moreattractive amino acid than with the extract. Despitethese differences, the index of taste attractiveness ofthe extract of Chironomidae larvae is similar for thesticklebacks in all three populations (Tables 2–4).

The pellets with the extract of Chironomidae larvaewere repeatedly grasped by the stickleback from theMoskva River almost two times less frequently thanthe control ones, but the sticklebacks from the Whiteand Okhotsk seas significantly more frequently dem�onstrated these manipulations with the pellets. Theduration of pellet retention with the extract by fish of



Tabl

e 2.

Tas

te r

espo

nse

s (M

± m

) of

nin

e�sp

ined

sti

ckle

back

(P

ungi

tius

pun

giti

us)

from

th

e M

oskv

a R

iver

pop

ulat

ion

to

pell

ets

wit

h t

este

d su

bsta

nce

s

Sub

stan

ceC

once

ntr

atio

n,

M (

%)

Con

sum

ptio

n o

f pel

lets

, %

Inde

x of

tas

te

attr

acti

ven

ess,

%N

umbe

r of

gra

sps

Pel

let

rete

nti

on t

ime,

sec

Num

ber

of t

rial

saf

ter

the

firs

t gr

asp

thro

ugh

out

the

tria

l

Cla

ssic

al t

aste

sub

stan

ces

Cit

ric

acid

0.26

(5)

57.6

± 4

.3**

*83

.13.

4 ±

0.2

*7.

1 ±

0.6

***

14.5

± 0

.8**

*13

2

Cal

cium

ch

lori

de0.

9 (1

0)15

.2 ±

3.1

**48

.33.

8 ±

0.2

2.8

± 0

.27.

5 ±

0.4

132

Sod

ium

ch

lori

de1.

73 (

10)

10.6

± 2

.733

.33.

1 ±

0.2

***

2.9

± 0

.36.

2 ±

0.4

132

Suc

rose

0.29

(10

)7.

6 ±

2.3

17.2

4.6

± 0

.33.

6 ±

0.2

***

9.0

± 0

.6**

132

Ch

iron

omid

ae la

rvae

ext

ract

12.7

79

.2 ±

3.3

***

87.5

2.6

± 0

.2**

*8.

3 ±

0.3

***

12.7

± 0

.5**

*15

4

Con

trol

–5.

3 ±

2.0

–4.

3 ±

0.2

2.6

± 0

.26.

7 ±

0.4

132

Fre

e am

ino

acid

s (L

�ste

reoi

som

ers)

Glu

tam

ine

0.1

72.7

± 3

.9**

*77

.81.

8 ±

0.1

***

9.0

± 0

.6**

*12

.2 ±

0.7

***

132

Cys

tein

e0.

167

.4 ±

4.1

***

76.2

2.4

± 0

.2**

9.0

± 0

.7**

*13

.0 ±

0.7

***

132

Ala

nin

e0.

166

.7 ±

4.1

***

76.0

2.5

± 0

.26.

7 ±

0.4

***

11.1

± 0

.6**

*13

2

Pro

lin

e0.

147

.7 ±

4.4

***

68.0

2.3

± 0

.2**

6.3

± 0

.5**

*10

.3 ±

0.7

***

132

His

tidi

ne

0.1

44.7

± 4

.3**

*66

.22.

4 ±

0.2

5.7

± 0

.5**

*9.

7 ±

0.6

***

132

Ser

ine

0.1

34.1

± 4

.1**

*57

.93.

1 ±

0.2

4.8

± 0

.4**

*11

.5 ±

0.8

***

132

Gly

cin

e0.

132

.6 ±

4.1

***

56.4

2.9

± 0

.24.

1 ±

0.3

***

8.8

± 0

.6**

132

Arg

inin

e0.

130

.3 ±

4.0

***

53.8

2.9

± 0

.24.

9 ±

0.4

***

9.7

± 0

.7**

*13

2

Nor

vali

ne

0.1

30.3

± 4

.0**

*53

.82.

6 ±

0.2

6.0

± 0

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*10

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0.8

***

132

Lys

ine

0.1

28.8

± 4

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*52

.03.

5 ±

0.2

5.2

± 0

.5**

*10

.9 ±

0.8

***

132

Ph

enyl

alan

ine

0.1

25.8

± 3

.8**

*47

.92.

5 ±

0.2

*4.

4 ±

0.4

***

8.2

± 0

.7*

132

Met

hio

nin

e0.

120

.5 ±

3.5

**38

.33.

0 ±

0.2

3.6

± 0

.4*

9.1

± 0

.9*

132

Asp

arag

ine

0.1

17.4

± 3

.3*

31.3

3.3

± 0

.23.

0 ±

0.3

8.5

± 0

.7*

132

Vali

ne

0.1

15.2

± 3

.125

.13.

2 ±

0.2

3.4

± 0

.3*

8.4

± 0

.7*

132

Th

reon

ine

0.1

9.1

± 2

.50

3.1

± 0

.22.

7 ±

0.2

8.2

± 1

.013

2

Asp

arti

c ac

id0.

0137

.9 ±

4.2

***

61.3

3.2

± 0

.35.

2 ±

0.4

***

10.4

± 0

.8**

*13

2

Leu

cin

e0.

0125

.0 ±

3.8

***

46.6

3.2

± 0

.24.

4 ±

0.4

***

9.7

± 0

.8**

*13

2

Glu

tam

ic a

cid

0.01

20.5

± 3

.5**

38.5

3.4

± 0

.23.

4 ±

0.3

8.2

± 0

.6*

132

Isol

euci

ne

0.01

16.7

± 3

.329

.52.

8 ±

0.2

4.0

± 0

.4**

8.0

± 0

.713

2

Try

ptop

han

0.01

13.6

± 3

.019

.83.

3 ±

0.2

3.6

± 0

.3*

8.6

± 0

.3*

132

Tyr

osin

e0.

001

9.8

± 2

.63.

73.

0 ±

0.2

3.0

± 0

.37.

0 ±

0.7

132

Con

trol

–9.

1 ±

2.5

–3.

1 ±

0.2

2.7

± 0

.26.

3 ±

0.6

132

Her

e an

d in

Tab

les

3–7,

th

e co

nce

ntr

atio

n o

f C

hir

onom

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exp

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f a

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met

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nd

its

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p: *

p <

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* p

< 0

.01,

***

p <

0.0

01.

684



Tabl

e 3.

Tas

te r

espo

nse

s (M

± m

) of

nin

e�sp

ined

sti

ckle

back

(P

ungi

tius

pun

giti

us)

from

th

e W

hit

e S

ea p

opul

atio

n t

o pe

llet

s w

ith

tes

ted

subs

tan

ces

Sub

stan

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once

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M (

%)

Con

sum

ptio

n

of p

elle

ts,

%In

dex

of t

aste

at

trac

tive

nes

s, %

Num

ber

of g

rasp

sP

elle

t re

ten

tion

tim

e, s

Num

ber

of t

rial

saf

ter

the

firs

t gr

asp

thro

ugh

out

the

tria

l

Cla

ssic

al t

aste

sub

stan

ces

Cit

ric

acid

0.26

(5)

48.2

± 3

.8**

*78

.24.

9 ±

0.3

***

5.0

± 0

.5**

*12

.9 ±

0.8

***

170

Cal

cium

ch

lori

de0.

9 (1

0)0*

**–

100

3.7

± 0

.21.

5 ±

0.1

***

4.5

± 0

.317

0

Sod

ium

ch

lori

de1.

73 (

10)

3.5

± 1

.4–

25.5

3.5

± 0

.21.

7 ±

0.1

5.3

± 0

.317

0

Suc

rose

0.29

(10

)2.

4 ±

1.2

–42

.23.

0 ±

0.2

1.9

± 0

.14.

5 ±

0.3

170

Ch

iron

omid

ae la

rvae

ext

ract

75.0

(10

)24

.5 ±

4.3

***

61.2

6.5

± 0

.5**

*2.

3 ±

0.2

**12

.0 ±

0.9

***

102

Con

trol

–5.

9 ±

1.8

–3.

5 ±

0.2

1.9

± 0

.15.

2 ±

0.3

170

Fre

e am

ino

acid

s (L

�ste

reoi

som

ers)

Glu

tam

ine

0.1

13.2

± 2

.9**

*4.

6 ±

0.3

***

3.2

± 0

.3**

*11

.3 ±

0.9

***

136

Cys

tein

e0.

166

.9 ±

4.0

***

3.7

± 0

.3**

*10

.7 ±

0.9

***

21.3

± 1

.1**

*13

6

Ala

nin

e0.

111

.8 ±

2.8

***

3.3

± 0

.2**

4.3

± 0

.4**

*9.

9 ±

0.7

***

136

Pro

lin

e0.

15.

1 ±

1.9

**3.

3 ±

0.2

**2.

4 ±

0.3

6.5

± 0

.7**

*13

6

His

tidi

ne

0.1

03.

3 ±

0.1

**1.

8 ±

0.1

4.7

± 0

.3*

136

Ser

ine

0.1

3.7

± 1

.6*

3.0

± 0

.22.

0 ±

0.1

5.1

± 0

.4*

136

Gly

cin

e0.

10

3.0

± 0

.21.

9 ±

0.1

4.8

± 0

.3*

136

Arg

inin

e0.

12.

9 ±

1.5

*4.

0 ±

0.2

***

1.9

± 0

.16.

5 ±

0.5

***

136

Nor

vali

ne

0.1

0.7

± 0

.72.

8 ±

0.1

2.2

± 0

.15.

0 ±

0.3

*13

6

Lys

ine

0.1

10.3

± 2

.6**

*3.

2 ±

0.1

**2.

4 ±

0.3

7.0

± 0

.8**

*13

6

Ph

enyl

alan

ine

0.1

02.

7 ±

0.1

1.7

± 0

.1*

3.5

± 0

.113

6

Met

hio

nin

e0.

10.

7 ±

0.7

3.2

± 0

.1**

1.9

± 0

.14.

8 ±

0.3

*13

6

Asp

arag

ine

0.1

2.2

± 1

.33.

7 ±

0.2

**1.

6 ±

0.1

*4.

9 ±

0.4

136

Vali

ne

0.1

3.7

± 1

.6*

3.5

± 0

.2**

*2.

1 ±

0.1

6.0

± 0

.4**

*13

6

Th

reon

ine

0.1

02.

8 ±

0.1

1.9

± 0

.14.

3 ±

0.2

136

Asp

arti

c ac

id0.

0149

.3 ±

4.3

***

3.9

± 0

.3**

*5.

9 ±

0.5

***

14.2

± 0

.8**

*13

6

Leu

cin

e0.

010

3.1

± 0

.1*

1.8

± 0

.14.

5 ±

0.2

136

Glu

tam

ic a

cid

0.01

47.8

± 4

.3**

*4.

2 ±

0.3

***

5.2

± 0

.5**

*14

.6 ±

0.9

***

136

Isol

euci

ne

0.01

03.

2 ±

0.2

*2.

1 ±

0.1

5.2

± 0

.3**

136

Try

ptop

han

0.01

0.7

± 0

.72.

4 ±

0.1

1.8

± 0

.13.

9 ±

0.2

136

Tyr

osin

e0.

001

0.7

± 0

.72.

8 ±

0.1

1.8

± 0

.14.

2 ±

0.2

136

Con

trol

–0

2.7

± 0

.11.

9 ±

0.1

4.0

± 0

.213

6



Tabl

e 4.

Tas

te r

espo

nse

s (M

± m

) of

nin

e�sp

ined

sti

ckle

back

(P

ungi

tius

pun

giti

us)

from

th

e O

khot

sk S

ea p

opul

atio

n t

o pe

llet

s w

ith

tes

ted

subs

tan

ces

Sub

stan

ceC

once

ntr

a�ti

on,

M (

%)

Con

sum

ptio

n

of p

elle

ts,

%In

dex

of t

aste

at

trac

tive

nes

s, %

Num

ber

of g

rasp

sP

elle

t re

ten

tion

tim

e, s

Num

ber

of t

rial

saf

ter

the

firs

t gr

asp

th

roug

hou

t th

e tr

ial

Cla

ssic

al t

aste

sub

stan

ces

Cit

ric

acid

0.26

(5)

37.3

± 5

.6**

*88

.31.

6 ±

0.1

*4.

2 ±

0.5

***

6.7

± 0

.8**

*75

Cal

cium

ch

lori

de0.

9 (1

0)0

–10

01.

4 ±

0.1

**1.

3 ±

0.1

1.7

± 0

.2**

*75

Sod

ium

ch

lori

de1.

73 (

10)

8.0

± 3

.255

.31.

6 ±

0.1

*1.

7 ±

0.2

2.7

± 0

.375

Suc

rose

0.29

(10

)0

–10

01.

5 ±

0.1

*1.

3 ±

0.1

2.0

± 0

.2*

75

Ch

iron

omid

ae la

rvae

ext

ract

75.0

(10

)13

.3 ±

4.0

*70

.52.

9 ±

0.3

**1.

8 ±

0.2

4.0

± 0

.4*

75

Con

trol

–2.

3 ±

1.9

–2.

1 ±

0.2

1.7

± 0

.22.

9 ±

0.3

75

Fre

e am

ino

acid

s (L

�ste

reoi

som

ers)

Glu

tam

ine

0.1

3.1

± 2

.20

1.7

± 0

.11.

3 ±

0.1

2.0

± 0

.265

Cys

tein

e0.

166

.2 ±

5.9

***

91.0

1.5

± 0

.28.

9 ±

0.8

***

10.8

± 1

.2**

*65

Ala

nin

e0.

116

.9 ±

4.7

**69

.02.

6 ±

0.5

2.1

± 0

.2**

*5.

3 ±

1.0

**65

Pro

lin

e0.

110

.8 ±

3.9

55.4

1.6

± 0

.22.

3 ±

0.4

**3.

4 ±

0.6

*65

His

tidi

ne

0.1

3.1

± 2

.20

1.9

± 0

.21.

3 ±

0.1

2.7

± 0

.565

Ser

ine

0.1

0–

100

1.6

± 0

.21.

2 ±

0.1

1.6

± 0

.165

Gly

cin

e0.

13.

1 ±

2.2

01.

5 ±

0.1

1.4

± 0

.11.

9 ±

0.2

65

Arg

inin

e0.

13.

1 ±

2.2

01.

7 ±

0.2

1.3

± 0

.12.

3 ±

0.4

65

Nor

vali

ne

0.1

0–

100

1.5

± 0

.11.

6 ±

0.5

2.0

± 0

.565

Lys

ine

0.1

0–

100

1.6

± 0

.21.

2 ±

0.1

1.9

± 0

.265

Ph

enyl

alan

ine

0.1

0–

100

1.4

± 0

.11.

2 ±

0.1

1.6

± 0

.165

Met

hio

nin

e0.

10

–10

01.

4 ±

0.1

1.3

± 0

.11.

7 ±

0.2

65

Asp

arag

ine

0.1

6.2

± 3

.033

.31.

6 ±

0.2

1.2

± 0

.11.

9 ±

0.2

65

Vali

ne

0.1

0–

100

1.7

± 0

.21.

2 ±

0.1

1.9

± 0

.265

Th

reon

ine

0.1

0–

100

1.6

± 0

.21.

3 ±

0.1

1.8

± 0

.265

Asp

arti

c ac

id0.

0116

.9 ±

4.7

**69

.02.

3 ±

0.3

2.1

± 0

.2**

*4.

3 ±

0.7

**65

Leu

cin

e0.

010

–10

01.

6 ±

0.2

1.3

± 0

.11.

8 ±

0.2

65

Glu

tam

ic a

cid

0.01

15.4

± 4

.5*

66.5

2.1

± 0

.32.

1 ±

0.3

**5.

1 ±

1.2

*65

Isol

euci

ne

0.01

4.6

± 2

.619

.51.

3 ±

0.1

1.5

± 0

.22.

0 ±

0.3

65

Try

ptop

han

0.01

0–

100

1.8

± 0

.31.

1 ±

0.1

1.8

± 0

.265

Tyr

osin

e0.

001

0–

100

1.4

± 0

.11.

4 ±

0.1

1.9

± 0

.265

Con

trol

–3.

1 ±

2.2

–1.

7 ±

0.3

1.2

± 0

.12.

1 ±

0.4

65

686



all populations was higher than by the control ones.This effect is mostly expressed in sticklebacks from theMoskva River (Tables 2–4).

Correlations between the parameters of behavioralresponse. In order to analyze the relations betweendifferent parameters of the behavioral response of fishfrom different populations and between different pop�ulations based on the parameters of behavioralresponse, data obtained when testing the pellets withamino acids were used. Sticklebacks from all popula�tions had the closest positive relations between theconsumption of pellets and their retention after thefirst grasp and in the course of the entire trial, as wellas the relation between the latter two parameters: thecorresponding correlation coefficients vary from 0.8 to1.0. In the sticklebacks from the Moskva River and theWhite Sea, relations between the consumption and thenumber of repeated grasps, between grasps and thepellet retention time after the first grasp are significantbut opposite in nature. In sticklebacks from theOkhotsk Sea, these relations are not significant, as wellas the relation between number of grasps and the pelletretention time throughout the trial. The relationbetween the last two parameters does not reach a sig�nificant level in sticklebacks from the Moskva River as

well, whereas it is highly significant and positive insticklebacks from the White Sea (Fig. 2).

When comparing fish from different populations, itwas found that they are similar in consumption of thepellets with amino acids: the correlation coefficients(p < 0.05) were significant and positive in all variants ofpairwise comparison. The reliable relations are absentwhen comparing the three populations based on thenumber of grasps of the pellet but highly significantand positive in most cases when comparing fish basedon the duration of keeping of the pellet after the firstgrasp and the duration of keeping of the pellet in thecourse of the trial (Fig. 3).

Behavior of testing of the pellets during consump�tion and rejection. The comparison of the behavior offish in the trials , which ended by swallowing of thepellet (SP trials ) or rejection of it (RP trials ), demon�strated that the pellet is tested differently in these twocases. The differences are manifested to a lesser degreewhen comparing the number of grasps of the pellet,especially in sticklebacks from the Moskva River, inwhich the SP trials in four cases revealed a significantlyhigher value of this parameter and in other four casesit was significantly lower. In sticklebacks from theWhite Sea, it was in six of seven cases and in those from

Consumption

Grasps

(а)

0.93***

–0.

60**

0.86*** –0.62**

0.87

***

–0.32

t

T

Consumption

Grasps

(b)

0.77***

0.70

**

0.87*** 0.53*

0.88

***

0.83***

t

T

Consumption

Grasps

(c)

0.97***

0.18

0.98*** –0.01

0.93

***

0.33

t

T

Fig. 2. Spearman’s correlation coefficient between the parameters of taste response to pellets with free amino acids calculated intotal for all trials on nine�spined stickleback (Pungitius pungitius) from the (a) Moskva River (b) White Sea, and (c) Okhotsk Sea.The duration of pellet retention: (t) after the first grasp; (T) throughout the trial; differences are significant at p: * < 0.05, ** < 0.01,*** < 0.001.



the Okhotsk Sea in all eight cases of the revealed sig�nificant differences that the number of grasps of thepellet in the SP trials was higher than in the RP trials.The differences are even more expressed when com�paring the SP and RP trials on the duration of keepingof the pellet after the first grasp and totally in thecourse of the trial : these values were always higher inthe SP trials with the difference of up to four to fiveand more times (Tables 5–7).

When estimating the connection between the reac�tion of fish in the SP and RP trials , the correlation wassignificant only in two of nine cases (Table 8). Theindependence of the samplings of the SP and RP trialsis underlined by the results of correlation analysis ofthe connections between different parameters of theresponse, which showed that these relationships differin nature or strength in many cases. This also fullyapplies to the relations between the parameters ofresponse and consumption of the pellets: they do notcoincide or have the opposite sign in the SP and RPtrials , such as in the White Sea stickleback. The natureand strength of these relations among different fishpopulations usually do not coincide (Fig. 4). Duringthe correlation comparison of fish from different pop�

ulations based on different parameters of response,significant correlations are found extremely rarely inboth SP and RP trials (Fig. 5).

The dependence of fish behavior in the SP and RPtrials on the taste attractiveness of substance in pelletswas evaluated using regression analysis, which demon�strated that these dependencies are similar in fish fromdifferent populations. In general, judging by theregression coefficient, such parameters as the numberof grasps of the pellet and, especially, the duration ofkeeping the pellet in the mouth cavity more depend onthe palatability of the offered substance in the SP trialsthan in the RP trials (Figs. 6–8).

DISCUSSION

Intrapopulation similarities in taste preferences.Population peculiarities of the gustatory system havebeen still insufficiently covered with regard tochemoreception in fish. Electrophysiological studiesreveal either similar (Goh and Tamura, 1980) or dif�ferent (Hara et al., 1999) gustatory spectra among rep�resentatives of different populations. Using the behav�ioral method, significant similarities were demon�

Moskva River

White Sea Kamchatka

0.53

*

0.48*

0.50*

(а)

Moskva River

White Sea Kamchatka

–0.

22

0.220.19

(b)

Moskva River

White Sea Kamchatka

0.60

*** 0.27

0.68***

(d)

Moskva River

White Sea Kamchatka

0.54

**

0.45*

0.63**

(c)

Fig. 3. Spearman’s correlation coefficient between different parameters of taste response to pellets with free amino acids calcu�lated for all trials on nine�spined stickleback (Pungitius pungitius) from the Moskva River, White Sea, Okhotsk Sea: (a) consump�tion, (b) number of grasps, (c) pellet retention time after first grasp, and (d) pellet retention time throughout the trial; see Fig. 2for designations.

688



Table 5. Taste responses (M ± m) of nine�spined stickleback (Pungitius pungitius) from the Moskva River population in thetrials that ended with consumption (above the line) or rejection of pellets (below the line)

Substance Concentration, M (%)

Number of grasps

Pellet retention time, s Number of trials after the first grasp throughout the trial

Classical taste substances

Citric acid 0.26 (5)

Calcium chloride 0.9 (10)

Sodium chloride 1.73 (10)

Sucrose 0.29 (10)

Chironomidae larvae ex�tract 175

Control –

Free amino acids (L�stereoisomers)

Glutamine 0.1

Cysteine 0.1

Alanine 0.1

Proline 0.1

Histidine 0.1

Serine 0.1

Glycine 0.1

Arginine 0.1

Norvaline 0.1

Lysine 0.1

Phenylalanine 0.1

Methionine 0.1

Asparagine 0.1

3.2 0.4±

3.7 0.3*±�� 10.1 0.8±

3.0 0.5***±�� 19.5 0.9±

8.0 1.0***±�� 75

57��

3.2 0.4±

4.0 0.2±�� 6.1 1.0±

2.3 0.2***±�� 13.8 0.8±

6.5 0.4***±�� 19

112��

2.8 0.4±

3.2 0.2±�� 8.3 1.3±

2.3 0.2***±�� 14.6 1.0±

5.3 0.3***±�� 14

118��

7.7 1.0±

4.4 0.3**±�� 7.7 0.7±

3.2 0.2***±�� 23.2 3.5±

7.8 0.5***±�� 10

122��

2.0 0.2±

5.2 0.8***±�� 9.4 0.4±

4.0 0.5***±�� 12.9 0.5±

11.7 1.8*±�� 122

32��

7.6 1.4±

4.1 0.2*±�� 3.8 1.5±

2.5 0.2±�� 15.7 1.3±

6.2 0.4***±�� 7

125��

1.6 0.1±

2.6 0.4**±�� 11.2 0.7±

3.2 0.4***±�� 14.2 0.7±

6.8 1.4***±�� 96

36��

1.9 0.2±

3.3 0.4***±�� 12.2 0.8±

2.2 0.3***±�� 16.5 0.7±

5.6 0.7***±�� 89

43��

2.5 0.2±

2.5 0.3±�� 8.3 0.4±

2.5 0.3***±�� 13.7 0.5±

5.9 1.0***±�� 88

44��

2.3 0.3±

2.3 0.2±�� 10.4 0.8±

2.4 0.3***±�� 15.9 0.7±

5.0 0.7***±�� 64

68��

2.2 0.2±

2.6 0.2±�� 9.8 0.8±

2.3 0.2***±�� 14.7 0.7±

5.7 0.6***±�� 59

73��

4.2 0.5±

2.5 0.2**±�� 8.1 0.9±

3.1 0.3***±�� 20.6 1.1±

7.8 1.0***±�� 45

87��

2.9 0.3±

2.9 0.2±�� 7.0 0.7±

2.7 0.2***±�� 14.2 1.0±

6.2 0.5***±�� 43

89��

3.0 0.6±

2.9 0.2±�� 10.2 0.9±

2.7 0.2***±�� 17.5 1.2±

6.3 0.6***±�� 40

92��

2.6 0.3±

2.6 0.2±�� 11.3 1.0±

3.7 0.4***±�� 19.5 1.3±

7.0 0.7***±�� 40

92��

2.6 0.5±

3.4 0.3*±�� 10.2 0.9±

3.0 0.4***±�� 17.6 1.2±

8.1 0.9***±�� 38

94��

2.1 0.2±

2.6 0.2±�� 9.4 0.9±

2.6 0.3***±�� 15.3 0.8±

5.7 0.7***±�� 34

98��

3.9 0.5±

2.8 0.2*±�� 7.5 1.1±

2.5 0.3***±�� 20.6 2.5±

6.1 0.8***±�� 27

105��

4.8 0.9±

3.0 0.2*±�� 6.0 1.1±

2.3 0.2***±�� 19.8 1.6±

6.1 0.5***±�� 23

109��



strated in the taste preferences of specimens fromdifferent populations, but such comparisons were per�formed only for two fish species (the brown troutSalmo trutta and the nine�spined stickleback(Kasumyan and Sidorov, 2005; Kasumyan andMikhailova, 2014). Young brown trout from the popu�lations inhabiting the White, Baltic, and Caspian seas,similarly to mature specimens of three�spined stickle�backs from the populations in the White, Baltic,North, and Okhotsk seas, do not have significant dif�ferences in their taste preferences. The substanceshaving the most attractive taste are the same for browntrout and three�spined sticklebacks from differentpopulations. The results of our investigation carriedout on the nine�spined stickleback fully confirm thisconclusion. Having compared the taste preferences ofnine�spined sticklebacks from three geographicallydistant populations, it was demonstrated that for allthese fish the most palatable were cysteine, citric acid,alanine, glutamic acid, and aspartic acid. Many sub�stances, such as sodium chloride, sucrose, isoleucine,tryptophan, tyrosine, threonine, have no (indifferent)taste for nine�spined sticklebacks from all popula�tions. Thus, they do not change consumption of pel�lets (Fig. 9).

Similarly to other behavioral methods, the evalua�tion of taste preferences and reaction of fish to the pel�let gives variability in results. For example, consump�tion of the control pellets by nine�spined sticklebacks

in two consecutive series of trial differs by almost twotimes (sticklebacks from the Moskva River), eventhough it remains at a low level in general (Tables 2–4).This variability of results was observed previously(Kasumyan and Nikolaeva, 2002; Kasumyan andSidorov, 2010a). It can be prompted by various exper�imentally intractable external and internal factors,which can trigger short�term shifts in the motivationalstate of experimental fish. This is reflected in the val�ues of the registered parameters, especially inresponses to indifferent or low�efficient stimuli. Thisis what explains relatively low correlation between thespectra of gustatory attractiveness of amino acids forsticklebacks from different populations, despite thefact that amino acids, which are mostly attractive forthem, are virtually the same (Fig. 9).

However, some differences—such as extraordinaryhigh palatability of glutamine and calcium chloride forsticklebacks from the White Sea and leucine for thosefrom the Moskva River—the most noticeable, couldprobably be caused by the population peculiarities offish. Probably, different range of spectrum of palatableamino acids is associated with population peculiaritiesand not variability in the responses observed duringbehavioral tests: their number is 16 in sticklebacksfrom the Moskva River, ten in the White Sea, and fourin the Okhotsk Sea. It cannot be excluded that the rel�atively wide spectrum of attractive amino acids insticklebacks from the Moskva River is triggered by a

Table 5. (Contd.)


Number of grasps

Pellet retention time, s Number of trials after the first grasp throughout the trial

Valine 0.1

Threonine 0.1

Aspartic acid 0.01

Leucine 0.01

Glutamic acid 0.01

Isoleucine 0.01

Tryptophan 0.01

Tyrosine 0.001

Control –

3.9 0.5±

3.1 0.2±�� 8.5 1.1±

2.6 0.2***±�� 21.0 1.9±

6.2 0.5***±�� 20

112��

6.5 1.1±

2.8 0.2***±�� 6.0 1.1±

2.4 0.2**±�� 31.1 1.1±

5.9 0.5***±�� 12

120��

2.1 0.3±

3.9 0.4***±�� 9.2 0.6±

2.8 0.4***±�� 15.0 1.3±

7.6 0.8***±�� 50

82��

4.1 0.6±

2.9 0.2±�� 9.5 1.0±

2.7 0.2***±�� 20.7 1.5±

6.1 0.6***±�� 32

99��

3.4 0.5±

3.5 0.2±�� 8.3 0.9±

2.1 0.2***±�� 16.4 1.3±

6.1 0.5***±�� 27

105��

3.6 0.7±

2.6 0.2±�� 11.6 1.7±

2.5 0.2***±�� 22.3 1.9±

5.1 0.4***±�� 22

110��

4.1 1.0±

3.2 0.2±�� 10.0 1.5±

2.5 0.2***±�� 21.7 2.8±

6.5 0.6***±�� 18

114��

4.5 1.0±

2.9 0.2±�� 9.2 1.4±

2.3 0.2***±�� 22.7 2.8±

5.3 0.5***±�� 13

119��

3.9 0.9±

3.0 0.2±�� 8.9 1.0±

2.1 0.2***±�� 19.3 3.2±

5.0 0.4***±�� 12

120��

690



Table 6. Taste responses (M ± m) of nine�spined stickleback (Pungitius pungitius) from the Okhotsk Sea population in thetrialsthat ended with consumption (above the line) or rejection of pellets (below the line)


Number of grasps

Pellet retention time, s Number of trialsafter the first grasp throughout the trial





Sucrose 0.29 (10)

Chironomidae larvae extract 175

Control –


Glutamine 0.1

Cysteine 0.1

Alanine 0.1

Proline 0.1

Histidine 0.1

Serine 0.1

Glycine 0.1

Arginine 0.1

Norvaline 0.1

Lysine 0.1

Phenylalanine 0.1

Methionine 0.1

Asparagine 0.1

2.0 0.2±

1.4 0.1*±�� 7.9 1.1±

2.0 0.2***±�� 13.4 1.3±

2.7 0.4***±�� 28

47��

–1.4 0.1±�� –

1.3 0.1±�� –

1.7 0.2±�� 0

75��

2.2 0.4±

1.6 0.1±�� 4.5 1.6±

1.4 0.1*±�� 8.1 0.7±

2.2 0.3***±�� 6

69��

–1.5 0.1±�� –

1.3 0.1±�� –

2.0 0.2±�� 0

75��

3.7 0.7±

2.8 0.3±�� 3.7 0.9±

1.5 0.1*±�� 8.9 1.4±

3.3 0.3***±�� 10

65��

3.0 2.0±

2.0 0.2±�� 7.5 3.1±

1.5 0.1*±�� 12.3 1.7±

2.7 0.3*±�� 2

73��

4.0 1.0±

1.6 0.1*±�� 3.7 0.7±

1.2 0.1*±�� 5.8 5.4±

1.9 0.2±�� 2

63��

1.5 0.2±

1.5 0.3±�� 11.9 0.8±

3.0 0.6***±�� 14.8 1.5±

3.1 0.6***±�� 43

22��

9.6 1.9±

1.2 0.1***±�� 2.8 0.6±

1.9 0.2±�� 20.3 2.8±

2.3 0.3***±�� 11

54��

3.4 0.9±

1.3 0.1*±�� 8.4 2.7±

1.5 0.1**±�� 16.1 0.9±

1.9 0.2***±�� 7

58��

5.0 1.0±

1.8 0.2*±�� 3.1 1.7±

1.2 0.1±�� 17.3 10.3±

2.3 0.4*±�� 2

63��

–1.6 0.2±�� –

1.2 0.1±�� –

1.6 0.1±�� 0

65��

5.0 2.0±

1.4 0.1*±�� 4.2 3.2±

1.3 0.1±�� 1.7 0.1±

9.5 3.3*±�� 2

63��

5.0 4.0±

1.6 0.2±�� 4.6 2.8±

1.2 0.1*±�� 16.0 8.6±

1.9 0.2*±�� 2

63��

–1.5 0.1±�� –

1.6 0.5±�� –

2.0 0.5±�� 0

65��

–1.7 0.2±�� –

1.2 0.1±�� –

1.9 0.2±�� 0

65��

–1.4 0.1±�� –

1.2 0.1±�� –

1.6 0.1±�� 0

65��

–1.4 0.1±�� –

1.3 0.1±�� –

1.7 0.2±�� 0

65��

5.0 1.2±

1.3 0.1**±�� 1.6 0.2±

1.2 0.1±�� 7.3 1.0±

1.6 0.1**±�� 4

61��



more intensive metabolism of fish in this most south�ern population among the studied ones, which inhab�its the basin of the Caspian Sea.

Interspecific differences in taste preferences. Com�parison of gustatory spectra in various fish species,including nine�spined sticklebacks from the WhiteSea, was considered in detail in the previous article(Kasumyan and Mikhailova, 2014). We performed thisanalysis for all populations of nine�spined sticklebacksunder study, which proved that significant correlationsare rarely found when comparing taste preferences ofdifferent species. They have a random character anddo not reflect similarities in the biology and systemat�ics of fish (Table 9). It is of interest to compare suchspecies as three� and nine�spined sticklebacks, whichare not only closely related, but also have similar modeof life, feeding, behavior, and often inhabit the samewater bodies (Hynes, 1950; Delbeek and Williams,1987; Maksimenkov and Tokranov, 1999, 2000). Com�parison of three populations of nine�spined stickle�backs and four populations of three�spined stickle�backs (trachurus) revealed significant positive correla�tions in ten of 12 cases, in 12 out of 15 cases withleiurus, which is in sharp contrast to the results ofinterspecific comparisons when such correlations werefound only in nine of 45 cases.

Decrease in the level of competition for food(Hynes, 1950) during sympathy between three� andnine�spined sticklebacks is achieved due to the use of

different biotopes (Mikhailova and Kasumyan, 2006;Kasumyan and Mikhailova, 2014). Nine�spined stick�lebacks adhere to aquatic vegetation, whereas three�spined sticklebacks prefer open areas of the water body(Hynes, 1950; Delbeek and Williams, 1987; Hart andGill, 1994; Hart, 2003). Upon this strategy, changes ingustatory spectra will not lead to any additional advan�tages for fish. In addition, retention of taste prefer�ences is important for fish feeding on the same objectsbut utilizing feeding resources of different biotopes.

Feeding behavior. More clear population differ�ences concern rather feeding behavior than taste pref�erences of the nine�spined stickleback: repeatedgrasps by fish when testing of the pellet. Such manip�ulations with the feeding object are more common forsticklebacks from the White Sea, which grasps pelletshaving an attractive taste approximately four to fiveand more times before swallowing or final rejection ofthe feed. Sticklebacks from the Okhotsk Sea performconsiderably fewer (by 2–3 times) repeated grasps ofpellets with an attractive taste, whereas those from theMoskva River occupy an intermediate position. Thesefish are arranged in the same order based on the num�ber of repeated grasps of the pellets with indifferenttaste (Tables 2–4).

The revealed peculiarities in behavior can be deter�mined by several main reasons. The first reason isassociated with the hydrology of water bodies, wherefish were sampled for further experiments. Stickle�

Table 6. (Contd.)


Number of grasps


Valine 0.1

Threonine 0.1

Aspartic acid 0.01

Leucine 0.01

Glutamic acid 0.01

Isoleucine 0.01

Tryptophan 0.01

Tyrosine 0.001

Control –

–1.7 0.2±�� –

1.2 0.1±�� –

1.9 0.2±�� 0

65��

–1.6 0.2±�� –

1.3 0.1±�� –

1.8 0.2±�� 0

65��

4.4 1.0±

1.8 0.3**±�� 4.3 0.8±

1.6 0.1***±�� 13.2 2.2±

2.5 0.3***±�� 11

54��

–1.6 0.2±�� –

1.3 0.1±�� –

1.8 0.2±�� 0

65��

6.5 1.2±

1.3 0.1***±�� 5.3 1.7±

1.5 0.1***±�� 22.8 4.6±

1.9 0.2***±�� 10

55��

4.0 1.7±

1.2 0.1±�� 5.3 4.1±

1.3 0.1±�� 11.9 3.8±

1.5 0.1**±�� 3

62��

–1.8 0.3±�� –

1.1 0.1±�� –

1.8 0.2±�� 0

65��

–1.4 0.1±�� –

1.4 0.1±�� –

1.9 0.2±�� 0

65��

11.5 0.5±

1.4 0.2***±�� 1.5 0.9±

1.1 0.1±�� 18.3 0.7±

1.5 0.1*±�� 2

63��

692



Table 7. Taste responses (M ± m) of nine�spined stickleback (Pungitius pungitius) from the White Sea population in the tri�als that ended with consumption (above the line) or rejection of pellets (below the line)


Number of grasps






Sucrose 0.29 (10)

Chironomidae larvae ex�tract 175

Control –


Glutamine 0.1

Cysteine 0.1

Alanine 0.1

Proline 0.1

Histidine 0.1

Serine 0.1

Glycine 0.1

Arginine 0.1

Norvaline 0.1

Lysine 0.1

Phenylalanine 0.1

Methionine 0.1

Asparagine 0.1

5.1 0.4±

4.8 0.3±�� 8.6 1.0±

1.7 0.1***±�� 24.6 1.0±

7.9 0.6***±�� 82

88��

–3.7 0.2±�� –

1.5 0.1±�� –

4.5 0.3±�� 0

170��

6.7 1.6±

3.3 0.2*±�� 4.1 1.3±

1.6 0.1***±�� 15.9 2.6±

4.9 0.3***±�� 6

164��

6.5 2.0±

3.0 0.2±�� 6.7 3.5±

1.7 0.1±�� 16.1 0.5±

4.3 0.2**±�� 4

166��

9.2 1.0±

5.6 0.5***±�� 3.1 0.5±

2.1 0.1**±�� 21.8 1.8±

8.8 0.7***±�� 25

77��

8.0 1.6±

3.2 0.2***±�� 2.5 0.4±

1.9 0.1±�� 12.6 1.8±

4.7 0.3***±�� 10

160��

7.7 0.9±

4.1 0.2***±�� 8.4 1.5±

2.4 0.1***±�� 32.0 3.6±

8.1 0.5***±�� 18

118��

3.2 0.3±

4.6 0.4**±�� 14.8 1.1±

2.6 0.5***±�� 26.8 1.1±

10.2 1.3***±�� 91

45��

4.1 0.8±

3.2 0.2±�� 14.8 2.2±

3.0 0.2***±�� 27.1 2.1±

7.6 0.5***±�� 16

120��

4.7 1.3±

3.2 0.2±�� 12.2 3.9±

1.9 0.1***±�� 34.3 3.0±

5.0 0.3***±�� 7

129��

–3.3 0.1±�� –

1.8 0.1±�� –

4.7 0.3±�� 0

136��

5.0 2.0±

2.9 0.2±�� 4.5 1.4±

1.9 0.1±�� 14.5 5.0±

4.7 0.3*±�� 5

131��

–3.0 0.2±�� –

1.9 0.1±�� –

4.8 0.3±�� 0

136��

8.8 1.0±

3.8 0.2**±�� 5.2 0.6±

1.8 0.1**±�� 31.6 3.8±

5.7 0.4***±�� 4

132��

8.02.7 0.1±�� 5.4

2.1 0.1±�� 21.4

4.9 0.3±�� 1

135��

5.4 0.6±

2.9 0.1***±�� 7.9 1.1±

1.8 0.2***±�� 31.6 3.2±

4.2 0.3***±�� 14

122��

–2.7 0.1±�� –

1.6 0.1±�� –

3.5 0.1±�� 0

136��

63.1 0.1±�� 2.2

1.9 0.1±�� 20

4.7 0.2±�� 1

135��

8.3 2.7±

3.2 0.2±�� 3.7 0.5±

1.6 0.1**±�� 31.0 3.5±

4.3 0.2***±�� 3

133��



backs from the White Sea were caught in a small iso�lated lake with highly transparent water and no cur�rents owing to the absence of tributaries. Under theseconditions, repeated grasps or rejection of the foodobject pose the minimum risk of losing it. Sticklebacksfrom the Okhotsk Sea belong to the population in theBol’shaya River, a mountain watercourse with contin�uous current even in the littoral zone, where young fishwere caught for further experiments. The probabilityof losing the prey because of the drift is considerablyhigher under these conditions if it is taken intoaccount that the nine�spined stickleback has a smallsize and low locomotor abilities. It increases evenmore with every rejection. The Khimka River, inwhich sticklebacks from the Moskva River werecaught, is a stream with transparent water and lowspeed of current. Under these conditions, although a

temporary rejection of the grasped prey increases theprobability of losing it, such chances are still lowerthan in a large river. In the previously studied three�spined sticklebacks, interpopulation differences in thefeeding behavior of fish from the White and Okhotskseas were not found (Kasumyan and Mikhailova,2010). Probably, the feeding behavior of the migratorythree�spined stickleback is less dependent on the cur�rent than that of nonmigratory forms as they movefaster (Tudorache et al., 2007).

Another reason for the existence of differencesbetween nine�spined sticklebacks based on the ten�dency to manipulations with pellets can be differentpopulation density in water bodies, where fish weresampled. It is known that sticklebacks, after graspingthe prey, do not test it repeatedly but rather swallow itimmediately if there are other fish in the nearby, which

Table 7. (Contd.)


Number of grasps


Valine 0.1

Threonine 0.1

Aspartic acid 0.01

Leucine 0.01

Glutamic acid 0.01

Isoleucine 0.01

Tryptophan 0.01

Tyrosine 0.001

Control –

7.8 1.2±

3.3 0.2***±�� 3.9 0.9±

2.0 0.1**±�� 22.6 5.0±

5.4 0.3***±�� 5

131��

–2.8 0.1±�� –

1.9 0.1±�� –

4.3 0.2±�� 0

136��

3.5 0.4±

4.2 0.4±�� 9.5 0.8±

2.3 0.3***±�� 20.3 0.9±

8.2 0.8***±�� 67

69��

–3.1 0.1±�� –

1.9 0.1±�� –

4.5 0.2±�� 0

136��

4.4 0.4±

4.1 0.4±�� 8.5 0.8±

2.1 0.2***±�� 22.2 0.9±

7.7 0.9***±�� 65

71��

–3.2 0.2±�� –

2.1 0.1±�� –

5.2 0.3±�� 0

136��

9.02.4 0.1±�� 2.6

1.8 0.1±�� 15.8

3.8 0.2±�� 1

135��

102.8 0.1±�� 1.2

1.8 0.1±�� 18.6

4.1 0.2±�� 1

135��

–2.7 0.1±�� –

1.9 0.1±�� –

4.0 0.2±�� 0

136��

Table 8. Spearman’s correlation coefficient between SP and RP experiments based on taste responses of nine�spined stick�leback (Pungitius pungitius) from different populations to pellets with free amino acids

ParametersPopulation

Moskva River Okhotsk Sea White Sea

Number of grasps 0.11 –0.19 –0.59*

Pellet retention time, s:

– after the first grasp 0.18 0.46 0.73**

– throughout the trial –0.08 –0.01 0.35

694



can intercept the food object (Gill and Hart, 1996a,1996b). Sticklebacks from the White Sea performingthe highest number of repeated grasps were caught inLake Mashinnoe, where the population density ofthese fish is very low. Sticklebacks from the OkhotskSea in the Bol’shaya River, which are not inclined tomanipulations with the pellet, belong to the dominantfish species and form dense aggregations during theperiod of spawning migrations (Bugaev, 1992). Stick�lebacks from the Moskva River occupies an intermedi�ate position based on the number of repeated graspsand density of fish in a small population in the KhimkaStream (Table 10). It is of interest that all nine�spinedsticklebacks keep the pellets with most attractive sub�stances for approximately 9–11 sec or 14–15 sec in thetrials, which ended by consumption of the pellet, i.e.,longer than the so�called “period of waiting for prey”(approximately 7 sec, determined for the three�spinedstickleback) (Tables 2–7)—the time during which thecompetitive fish stays near the fish with food in orderto intercept the prey (Gill and Hart, 1996a). It is quitepossible that both factors (hydrodynamical conditionsin the water body and population density) jointlydetermined the divergence of nine�spined sticklebackswith different origin based on the tendency to manip�

ulate with food objects. This feature in the behavior offish should be inherent, because all experimental fishwere kept under the same conditions, and those fromthe Okhotsk Sea were caught at the age of severalmonths and kept under the aquarium conditions formore than 6 months before experiments. To clarify thisissue, further investigations are needed, includingcomparison of the behavior manifested by fish, whichwere caught in geographically distant water bodieshaving similar hydrology and abundance of stickle�backs, or comparison of the behavior of fish fromneighboring water bodies of different types.

Another parameter of the response that we regis�tered, i.e., the duration of keeping of the pellet in themouth cavity after the first grasp, was similar in fishfrom different populations. It is clear from comparisonof the values of this parameter for the pellets with asimilar level of consumption, such as those containingcysteine, a highly attractive substance, and some otherelements (Tables 2–4). It is quite possible that the sim�ilarity is caused by the same reception of gustatorystimuli and processing of the obtained information inthe brain centers exhibited by nine�spined stickle�backs.

Consumption

Grasps

(а)

0.36

–0.

80**

* –0.83*** –0.56**

–0.

09

0.81***

t

T0.31

0.75

***

0.27

0.11 –0.16–

0.31

Consumption

Grasps

(b)

0.85***

–0.

83**

* 0.39–0.82***

0.83

***

0.15

t

T0.84***

0.51

0.52*

0.65** 0.51*

0.61

**

Consumption

Grasps

(c)

0.33

–0.

11

0.39 –0.60

0.50

*

0.39

t

T0.19

0.18

0.52*

0.45* –0.45*

–0.

37

Fig. 4. Spearman’s correlation coefficient between the parameters of taste response to pellets with free amino acids calculated fortrials ended with consumption (semi�bolded) and rejection in nine�spined stickleback (Pungitius pungitius) from the (a) MoskvaRiver (b) White Sea, and (c) Okhotsk Sea; see Fig. 2 for designations.



Moskva River

White Sea Kamchatka

0.54

* 0.30

0.10

(а)

0.42

–0.

05

–0.22

Moskva River

White Sea Kamchatka

0.17

–0.26

0.30

(b)

0.37

0.19

–0.07

Moskva River

White Sea Kamchatka

–0.

09

–0.21

0.57**

(c)

–0.26

0.21 0.22

Fig. 5. Spearman’s correlation coefficient between differ�ent parameters of taste response to pellets with free aminoacids calculated for trials ended with consumption (semi�bolded) and rejection in nine�spined stickleback (Pungitiuspungitius) from the Moskva River, White Sea, and Kam�chatka: (a) number of grasps, (b) duration of pellet reten�tion after first grasp, (c) duration of pellet retentionthroughout the trial; see Fig. 2 for designations.

16

14

12

10

8

4

2

0

Num

ber

of r

epea

ted

gras

ps o

f pel

lets

6

(а)

16

14

12

10

8

4

2

10090806040200 3010 50Consumption of pellets, %

6

(c)

70

16

14

12

10

8

4

2

0

6

(b)

Fig. 6. Dependence of the number of repeated grasps ofpellets with free amino acids on their total consumption bynine�spined stickleback (Pungitius pungitius) from differ�ent populations in trials ended with consumption (–�–)and rejection (��) of the pellet: (a) the Moskva Riverpopulation (consumption of the pellet— y = 4.8 – 0.048x,rs = –0.80, p < 0.001); rejection— y = 3.0 – 0.004x, rs =–0.31, p > 0.05); (b) the White Sea population (consump�tion of the pellet— y = 7.5 – 0.072x, rs = –0.83, p < 0.001;rejection— y = 3.0 + 0.024x, rs = 0.61 p < 0.01) ), (c) theOkhotsk Sea population (consumption of the pellet— y =5.4 – 0.042x, rs = –0.11, p > 0.05; rejection—y = 1.5 –0.002x, rs = –0.28, p > 0.05).

Stereotypes of intra�oral testing of food. Actionsperformed by fish upon intra�oral testing of the foodobject follow two alternative behavioral stereotypes.One of them ends with swallowing of the food object,another leads to final rejection of food. Both these ste�reotypes are manifested with regard to any food,regardless of taste, and, therefore, they can be consid�ered as stable behavioral characteristics. The stereo�

696



16

14

12

10

8

4

2

0

Pel

let

rete

nti

on t

ime

afte

r th

e fi

rst

gras

p, s

6

(а)

16

14

12

10

8

4

2


6

(c)

70

16

14

12

10

8

4

2

0

6

(b)

Fig. 7. Dependence of duration of retention after the firstgrasp of pellets with free amino acids on their total con�sumption by nine�spined stickleback (Pungitius pungitius)from different populations in trials ended with consump�tion (–�–) and rejection (��) of the pellet: (a) the MoskvaRiver population (consumption of the pellet— y = 8.0 +0.039x, rs = 0.36, p > 0.05; rejection— y = 2.5 + 0.003x,rs = 0.27, p > 0.05); (b) the White Sea population (con�sumption of the pellet— y = 5.1 + 0.127x, rs = 0.85, p <0.001; rejection— y = 1.9 + 0.009x, rs = 0.52, p < 0.05), (c)the Okhotsk Sea population (consumption of the pellet—y = 3.4 + 0.122x, rs = 0.33, p > 0.05; rejection— y = 1.2 +0.026x, rs = 0.52, p < 0.05); for designations see Fig. 6.

Pel

let

rete

nti

on t

ime

thro

ugh

out

the

tria

l, s

40

35

30

25

20

10

5


15

(c)

70

40

35

30

25

20

10

5

15

(b)

40

35

30

25

20

10

5

15

(а)

Fig. 8. Dependence duration of retention throughout thetrial of pellets with free amino acids on their total con�sumption by nine�spined stickleback (Pungitius pungitius)from different populations in trials ended with consump�tion and rejection of the pellet: (a) the Moskva River pop�ulation (consumption of the pellet—y = 23.5 – 0.153x,rs = –0.83, p < 0.001; rejection— y = 6.2 + 0.003x, rs =0.11, p > 0.05), (b) the White Sea population (consump�tion of the pellet— y = 24.6 + 0.005x, rs = 0.39, p > 0.05;rejection— y = 4.7 + 0.079x, rs = 0.65, p < 0.01), (c) theOkhotsk Sea population (consumption of the pellet— y =12.2 + 0.088x, rs = 0.39, p > 0.05; rejection— y = 2.2 +0.013x, rs = 0.45, p < 0.05); see Fig. 6 for designations.



types of consumption or rejection of food have theirown peculiarities for different fish species. Thus, thecommon carp Cyprinus carpio, rainbow trout Onco�rhynchus mykiss, and roach Rutilus rutilus perform arelatively small number of grasps of the food objectbefore swallowing it, whereas the stone loach Barbat�ula barbatula and three�spined stickleback subject it tonumerous repeated grasps then upon rejection of theobject. However, all these species manifesting the ste�reotype of consumption spend more time for intra�oral testing of the object than upon rejection of thefood (Kasumyan and Sidorov, 2010a, 2010b, 2012;Kasumyan and Tin’kova, 2013; Kasumyan andMikhailova, 2014). It is assumed that this featuredevelops owing to the need of fish to more accuratelyestimate the gustatory properties of food before swal�lowing it.

In the nine� and three�spined sticklebacks of allthree populations, the behavioral stereotypes of intra�oral testing of food are well manifested. The behaviorof fish during the SP and RP trials on the same pelletsis different (Table 8); the strength and nature of rela�tions between different parameters of the responseupon intra� and inter�group correlation analysis of theSP and RP trials did not agree (Figs. 4, 5). All thisunderlines the isolation of patterns of reactions mani�fested by fish when consuming and rejecting the pelletand justifies the appropriateness of singling out twodifferent stereotypes.

Similarly to all fish species that were studied previ�ously, nine�spined sticklebacks spend much more timeon keeping the pellet in the mouth during the processof consumption than when rejecting it. The duration of

testing of the pellet in the SP and RP trials differs betweensticklebacks by 4–5 and more times (Tables 5–7). As thetaste attractiveness of pellets increases, these differ�ences become more pronounced, which is clearly seenfrom the distribution of dots in the figures demonstrat�ing the dependence of the duration of keeping of pel�lets on their total consumption, as well as follows fromthe parameters of regression equations (Figs. 7, 8). Inother words, the higher the gustatory attractiveness ofpellets, the more considerable differences are observedbetween the SP and RP trials based on the pelletsretention time. The differences become more pro�nounced owing to increase in the duration of testing ofpellets over the SP trials , whereas this parameterchanges insignificantly with increase in the attractive�ness of pellets in the RP trials . Upon longer keeping ofthe pellet after the first grasp (2–3 s) or total duration(5 s), the probability of consumption of the pelletincreases. These values are slightly lower in the three�spined stickleback (Kasumyan and Mikhailova, 2014).It is considered that much of this time is spent on anal�ysis of the information about gustatory qualities offood in the brain centers and on formation of the ade�quate locomotor response (Halpern, 1986).

Differences between two stereotypes based on thenumber of repeated testings of the object are poorlyexpressed in the nine�spined stickleback. In general,it, similarly to the three�spined stickleback and stoneloach, performs such actions more often upon con�sumption of the object than when refusing it. It is hardto assume what can cause this similarity in food testingby nine� and three�spined sticklebacks and stoneloach, as well as difference from the behavior of the

(c)

10080

40

017151385 72 11

(а)

Co

nsu

mp

tio

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Fig. 9. Consumption of pellets with classical taste substances and Chironomidae larvae extract (1–6) and free amino acids (7–28) by specimens of nine�spined stickleback (Pungitius pungitius) from different populations: (a) Moskva River, (b) White Sea, (c)Okhotsk Sea. Tested substances: (1) citric acid, (2) calcium chloride, (3) sodium chloride, (4) sucrose, (5) Chironomidae larvaeextract, (6) control relative to classical substances, (7) glutamine, (8) cysteine, (9) alanine, (10) proline, (11) histidine, (12)serine, (13) glycine, (14) arginine, (15) norvaline, (16) lysine, (17) phenylalanine, (18) methionine, (19) asparagine, (23) leucine,(24) glutamic acid, (25) isoleucine, (26) tryptophan, (27) tyrosine, (28) control relative to free amino acids; see Fig. 2 for otherdesignations.

698



common carp, rainbow trout, and roach, all perform�ing fewer grasps of food before swallowing than duringrejection of it. Probably, further investigations willclarify this issue. As in the case with the duration oftesting, the attractiveness of pellets influences the

number of grasps of the object by nine�spined stickle�backs, but this dependence has an opposite sign and isexpressed only in the SP trials .

Interpopulation differences in manifestation of thestereotypes of testing of food in the previously studied

Table 9. Spearman’s correlation coefficient between nine�spined sticklebacks (Pungitius pungitius) and other fish specieswith respect to taste preferences to amino acids

Species, populationPopulation of nine�spined stickleback

Source of informationMoskva River Okhotsk Sea White Sea

Common carp Cyprinus carpio 0.49* 0.75*** 0.48* Kasumyan and Morsi, 1996

Tench Tinca tinca 0.75*** 0.44* 0.67*** Kasumyan and Prokopova, 2001

Gold fish Carassius auratus gibelio –0.36 –0.09 –0.22 Kasumyan and Nikolaeva, 2002

Roach Rutilus rutilus –0.01 –0.46* –0.02 Ibid

European minnow Phoxinus phoxinus 0.19 0.06 0.24 Kasumyan and Marusov, 2003

Siberian sturgeon Acipenser baerii –0.34 –0.06 –0.38 Kasumyan and Sidorov, 1994a

Russian sturgeon A. gueldenstaedtii 0.35 0.09 0.05 Ibid

Persian sturgeon A. persicus 0.57** 0.34 0.25 Jafari Shamushaki et al., 2008

Stellate sturgeon A. stellatus –0.08 0.21 0.28 Kasumyan and Sidorov, 1994a

Arctic flounder Liopsetta glacialis 0.16 0.01 –0.05 Kasumyan and Nikolaeva, 2002

Arctic char Salvelinus alpinus erythrinus 0.17 0.38 0.34 Kasumyan and Sidorov, 1995

Lake char S. namaycush 0.09 0.30 0.24 Kasumyan and Sidorov, 2001

Brown trout Salmo trutta, Caspian Sea popu�lation

0.20 0.22 0.04 Kasumyan and Sidorov, 1994a

Chum salmon Oncorhynchus keta –0.40 –0.30 –0.04 Kasumyan and Sidorov, 1992

Guppy Poecilia reticulata 0.46* 0.28 0.28 Kasumyan and Nikolaeva, 1997

Three�spined stickleback Gasterosteus ac�uleatus, trachurus:

– Baltic Sea 0.59** 0.56** Kasumyan and Mikhailova, 2014

– Norvegia 0.54** 0.59** Ibid

– Kamchatka 0.61** 0.68*** ''

– White Sea 0.41 0.48* Kasumyan and Mikhailova, 2010

Three�spined stickleback G. aculeatus, leiurus 0.20 0.63** 0.53* Kasumyan and Mikhailova, 2007

For Siberian sturgeon, correlation coefficients were calculated according to taste responses to 19 free amino acids (without norvalineand cysteine), according to taste responses to 20 amino acids (without norvaline) for Persian sturgeon, and according to taste respons�es to 21 free amino acids for remaining species; differences are significant at p: * <0.05, ** <0.01, *** <0.001.

Table 10. Mean values of the parameters of taste responses in nine�spined sticklebacks (Pungitius pungitius) from differentpopulations to pellets containing free amino acids (in total for all trials)

Population

Parameters of taste response

consumption of pellets, % number of grasps Pellet retention time, sec

after the first grasp throughout the trial

Moskva River 31.8 2.88 4.88 9.63

Okhotsk Sea 7.1 1.69 1.82 2.84

White Sea 10.5 3.68 2.89 7.25



three�spined sticklebacks were slightly expressed(Kasumyan and Mikhailova, 2014). In the nine�spined stickleback, they are more pronounced mainlydue to differences in the absolute values of responseparameters. Nine�spined sticklebacks from theOkhotsk Sea mostly stand out. Its stereotypes arecharacterized by the lower number of grasps andshorter time of keeping of the object in the mouth cav�ity than those from other populations. The nine�spined stickleback from different populations havesimilar stereotypes of rejection owing to the absence ofdependence of parameters of the response on theattractiveness of pellets. Data on sticklebacks from theMoskva River clearly demonstrate it, in which the gus�tatory attractiveness of pellet types under study wasdivided more evenly in the range of possible values ofthis parameter. In the stickleback from the White Seaand, especially, Okhotsk Sea, the consumption ofalmost all pellets was limited to a narrow range (0–20%).

CONCLUSIONS

Many fish have wide area, in which populations orforms either partially or completely isolated from eachother are formed. As fish inhabit water bodies, whicheither belong to different basins, zoogeographicalprovinces, and climatic zones or are related to geo�graphically distant zones, it results in their divergencebased on feeding owing to differences in the composi�tion of available food (Rikardsen et al., 2000, 2004;Barrientos et al., 2006; Chuwen et al., 2007; Gerryet al., 2012). These differences may occur during sym�patry or colonization of different biotopes by fish(Yamaoka et al., 2003; Bertrand et al., 2008). Theduration of existence of populations is often sufficientfor manifestation of differences between them basedon morphology, physiology, and behavior (Bergstromand Reimchen, 2002; Bell, 2005; Rafferty and Bough�man, 2006; Tudorache et al., 2007; Bell et al., 2010).The performed investigation demonstrates that suchshort�cycle fish as the nine�spined stickleback havegenetically inherent population differences in thefeeding behavior and stereotypes of intra�oral testingof food. However, the taste preferences of fish fromdifferent populations at a distance of many thousandkilometers and historically isolated over a long periodof time change insignificantly, and the majority of sub�stances with attractive taste are the same. Similarity ofthe gustatory spectra of these fish and stimuli havingthe highest gustatory attractiveness for them empha�sizes the stability of taste preferences and their low sus�ceptibility to the influence of external factors. Theobtained results make it possible to suggest that foodorganisms, which are selectively consumed by fishfrom different populations, can have a similar gusta�tory profile despite their taxonomic differences.

ACKNOWLEDGMENTS

We thankful to E.A. Marusov (Moscow State Uni�versity), S.S. Sidorov (Moscow State University), andP.S. Sidorov (All�Russia Research Institute of Fisher�ies and Oceanography) for their help during the sam�pling of fish. Also, we thankful to E.A. Pivovarov(Moscow State University), who captured and trans�ported juveniles sticklebacks from the Okhotsk Sea toMoscow.

Capturing, transportation, and keeping of fish, aswell as experimental part of the work was supported bythe Russian Foundation for Basic Research, projectno. 13�04�00711 and the Program “Leading ScientificSchools,” contract no. NSh�2666.2014.4. Processingof the initial data, analysis of the results, and prepara�tion of the manuscript were supported by the RussianScience Foundation, grant no. 14�50�00029.

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Translated by N. Shulaev

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