12
ISSN 00329452, Journal of Ichthyology, 2010, Vol. 50, No. 8, pp. 682–693. © Pleiades Publishing, Ltd., 2010. Original Russian Text © A.O. Kasumyan, S.S. Sidorov, 2010, published in Voprosy Ikhtiologii, 2010, Vol. 50, No. 5, pp. 708–720. 682 Concepts of the functional properties of the gusta tory system in fish have been long based mainly on results of electrophysiological studies (Sorensen and Caprio, 1998). In recent years, this knowledge has been considerably extended thanks to the use of behavioral methods, which made it possible to estab lish several new important specific features of taste reception in fish. For instance, comparison of spectra of substances that have repellent, attractive, or indif ferent taste properties for fish detected a high species specificity of taste preferences and their difference in closely related species and in fish with similar mode of life and feeding (Kasumyan, 1997; Kasumyan and Døving, 2003). It was shown that taste preferences are similar in fish of different generations and do not depend on the individual feeding experience of an individual, its population, or sex (Kasumyan and Morsi, 1997; Nikolaeva and Kasumyan, 2000; Fokina and Kasumyan, 2003; Kasumyan and Sidorov, 2005a). The attitude of fish to the taste of substances is weakly subjected to the effect of environmental abiotic factors (salinity, temperature); it is retained at the stimulation of feeding stimulation by smells but changes at an increase of the feeding motivation of fish caused by feeding cessation (Kasumyan et al., 1993, 2009; Kasumyan and Mikhailova, 2010; Kasumyan and Sidorov, 2010). The available data on the functional characteristics of the gustatory system in fish, including taste prefer ences, in most cases concern its intraoral component. However, many benthoseating fish or fish feeding in the twilight or night time, besides taste receptors located in the oral cavity have external taste buds (Atema, 1971; Kapoor et al., 1975; Gomahr et al., 1992). In some fish, they cover the whole body, and their total number reaches several hundred thousand (Finger et al., 1991). Maximum density of external taste buds is observed at lips and barbels, at the lower surface of the head and the anterior part of the body, and at gill covers and paired fins, i.e., at sites with which fish with a great probability can touch a food item during food search (Gomahr et al., 1992; Devitsina and Kazhdaev, 1992; McCormick, 1993; Booth, 1996; Lombarte and Aquirre, 1997). Func tional characteristics of the external gustatory system have been poorly studied and only in a limited number of species. It was shown, in particular, that, according to its sensitivity and the range of spectrum, it exceeds the intraoral gustatory system but differs in a less pro nounced species specificity of the composition of effective stimuli (Caprio et al., 1993; Kasumyan, 1999; Jafari Shamushaki et al., 2008). Stone loach Barbatula barbatula belongs to a spe cies having developed external gustatory system Taste Preferences and Behavior of Testing Gustatory Qualities of Food in Stone Loach Barbatula barbatula (Balitoridae, Cypriniformes) A. O. Kasumyan and S. S. Sidorov Moscow State University, Moscow, 119899 Russia email: [email protected] Received March 22, 2010 Abstract—Extraoral and intraoral taste preferences of stone loach Barbatula barbatula to 21 free amino acid (Lisomers) and 4 classic taste substances were established. It was found that most amino acids (19), as well as citric acid and calcium chloride are suppressants, i.e., significantly decrease grasping of artificial pellets. Such action is most typical of cysteine, glutamine, asparagine, and citric acids. The number of deterrent stim uli among the used substances is smaller than that of suppressants. Only aquatic chironomid extract has pos itive gustatory properties. The remaining substances have no pronounced taste properties. Touching of a pel let by barbels is an obligatory element of behavior of testing by fish of pellet properties always preceding grasp ing. It was found that the subsequent development of feeding behavior of fish proceeds according to one of the possible behavioral stereotypes of testing by fish of taste properties of food items. The revealed behavioral response stereotypes differ in the number of manipulations performed by fish with a food item (rejections and repeated graspings) and in the duration of its retention. Stereotype selection is determined by the extraoral taste attractiveness of the food item for fish. DOI: 10.1134/S0032945210080138 Key words: chemoreception, gustatory system, taste preferences, feeding behavior, food testing behavior, behavioral stereotypes, amino acids, classic taste substances.

Taste preferences and behavior of testing gustatory qualities of food in stone loach Barbatula barbatula (Balitoridae, Cypriniformes)

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ISSN 0032�9452, Journal of Ichthyology, 2010, Vol. 50, No. 8, pp. 682–693. © Pleiades Publishing, Ltd., 2010.Original Russian Text © A.O. Kasumyan, S.S. Sidorov, 2010, published in Voprosy Ikhtiologii, 2010, Vol. 50, No. 5, pp. 708–720.

682

Concepts of the functional properties of the gusta�tory system in fish have been long based mainly onresults of electrophysiological studies (Sorensen andCaprio, 1998). In recent years, this knowledge hasbeen considerably extended thanks to the use ofbehavioral methods, which made it possible to estab�lish several new important specific features of tastereception in fish. For instance, comparison of spectraof substances that have repellent, attractive, or indif�ferent taste properties for fish detected a high speciesspecificity of taste preferences and their difference inclosely related species and in fish with similar mode oflife and feeding (Kasumyan, 1997; Kasumyan andDøving, 2003). It was shown that taste preferences aresimilar in fish of different generations and do notdepend on the individual feeding experience of anindividual, its population, or sex (Kasumyan andMorsi, 1997; Nikolaeva and Kasumyan, 2000; Fokinaand Kasumyan, 2003; Kasumyan and Sidorov, 2005a).The attitude of fish to the taste of substances is weaklysubjected to the effect of environmental abiotic factors(salinity, temperature); it is retained at the stimulationof feeding stimulation by smells but changes at anincrease of the feeding motivation of fish caused byfeeding cessation (Kasumyan et al., 1993, 2009;Kasumyan and Mikhailova, 2010; Kasumyan andSidorov, 2010).

The available data on the functional characteristicsof the gustatory system in fish, including taste prefer�ences, in most cases concern its intraoral component.However, many benthos�eating fish or fish feeding inthe twilight or night time, besides taste receptorslocated in the oral cavity have external taste buds(Atema, 1971; Kapoor et al., 1975; Gomahr et al.,1992). In some fish, they cover the whole body, andtheir total number reaches several hundred thousand(Finger et al., 1991). Maximum density of externaltaste buds is observed at lips and barbels, at the lowersurface of the head and the anterior part of the body,and at gill covers and paired fins, i.e., at sites withwhich fish with a great probability can touch a fooditem during food search (Gomahr et al., 1992;Devitsina and Kazhdaev, 1992; McCormick, 1993;Booth, 1996; Lombarte and Aquirre, 1997). Func�tional characteristics of the external gustatory systemhave been poorly studied and only in a limited numberof species. It was shown, in particular, that, accordingto its sensitivity and the range of spectrum, it exceedsthe intraoral gustatory system but differs in a less pro�nounced species specificity of the composition ofeffective stimuli (Caprio et al., 1993; Kasumyan, 1999;Jafari Shamushaki et al., 2008).

Stone loach Barbatula barbatula belongs to a spe�cies having developed external gustatory system

Taste Preferences and Behavior of Testing Gustatory Qualities of Food in Stone Loach Barbatula barbatula

(Balitoridae, Cypriniformes)A. O. Kasumyan and S. S. Sidorov

Moscow State University, Moscow, 119899 Russiae�mail: [email protected]

Received March 22, 2010

Abstract—Extraoral and intraoral taste preferences of stone loach Barbatula barbatula to 21 free amino acid(L�isomers) and 4 classic taste substances were established. It was found that most amino acids (19), as wellas citric acid and calcium chloride are suppressants, i.e., significantly decrease grasping of artificial pellets.Such action is most typical of cysteine, glutamine, asparagine, and citric acids. The number of deterrent stim�uli among the used substances is smaller than that of suppressants. Only aquatic chironomid extract has pos�itive gustatory properties. The remaining substances have no pronounced taste properties. Touching of a pel�let by barbels is an obligatory element of behavior of testing by fish of pellet properties always preceding grasp�ing. It was found that the subsequent development of feeding behavior of fish proceeds according to one ofthe possible behavioral stereotypes of testing by fish of taste properties of food items. The revealed behavioralresponse stereotypes differ in the number of manipulations performed by fish with a food item (rejections andrepeated graspings) and in the duration of its retention. Stereotype selection is determined by the extraoraltaste attractiveness of the food item for fish.

DOI: 10.1134/S0032945210080138

Key words: chemoreception, gustatory system, taste preferences, feeding behavior, food testing behavior,behavioral stereotypes, amino acids, classic taste substances.

JOURNAL OF ICHTHYOLOGY Vol. 50 No. 8 2010

TASTE PREFERENCES AND BEHAVIOR OF TESTING GUSTATORY QUALITIES 683

(Ducros, 1954). It is considered that, thanks to exter�nal taste reception, these fish retain capacity for dis�tant food search according to chemical signals thatdistribute in water after experimentally induced acuteanosmia (Kasumyan and Marusov, 2005). This spe�cific feature makes the stone loach a convenient objectfor the study of different aspects of taste reception.

The purpose of this study was to compare tastepreferences of the stone loach provided by externaland oral taste receptors, as well as to study behaviorrelated to taste testing by fish of qualities of food items.Such behavior mediated by external taste receptorsremains completely unstudied.

MATERIAL AND METHODS

Experiments were performed on mature individu�als of the stone loach (body length 65–90 mm) caughtin the Tarusa River (Kaluga oblast) and brought to theDepartment of Ichthyology of Moscow State Univer�sity. Prior to experiments, fish were kept during severalmonths in a common aquarium at a water temperatureof 18–19°C.

Several days prior to experiments, fish were placedsingly in small aquariums with a volume of 5 l. Water inaquariums was aerated using microcompressors; somepart of water was replaced by fresh water daily; groundin aquariums was absent. Each aquarium was coveredwith a nontransparent cover with an opening in thecenter to introduce food or pellets. Fish were fed onlive larvae of chironomids at the end of the day. Afterone–two days of acclimatization to new conditions,fish were taught to approach the anterior part of theaquarium. For this purpose, live chironomid larvae

and later agar–agar pellets (2%) containing extract oflarval chironomids (175 g/l) were delivered item byitem in the anterior part of the aquarium with an inter�val of 5–15 min.

After training, experiments were started duringwhich one agar–agar pellet was introduced into theaquarium with fish and then during 1–2 min the num�ber of graspings of the pellet, the duration of retainingit at the first seizing and totally during the experiment,as well as consumption (whether the delivered pelletwas eaten or not towards the end of the experiment)were visually recorded. In most experiments, visual,without precise recording, assessment of the fre�quency of touchings of the pellet and the duration oftactile contact with it of fish (qualitative estimate) wasperformed. The accurate number of touchings of thepellet was calculated only in experiments in which fishafter one or several touchings did not grasp the pellet.Experiments in which no touching by fish of the pelletwas observed were not counted. The pellet that was leftunconsumed was removed from the aquarium. A totalof 27 types of pellets were used: control pellets that didnot contain any taste substances, pellets with anextract of chironomid larvae, pellets with free aminoacids (21 amino acids, L�stereoisomers), and with fourclassic taste substances (a list of substances and usedconcentrations see in Tables 1 and 2). Experimentswith different types of pellets were performed in ran�dom sequence.

All pellets, including control, were of bright redcolor formed by Ponceau 4R stain (5 µM) that wasintroduced into agar–agar solution (2%, Reanal) dur�ing its preparation. Pellets from agar–agar gel were cutout using a tube from stainless steel directly before the

Table 1. Taste responses (M ± m) of the stone loach Barbatula barbatula to pellets with an extract of chironomid larvae and withclassic taste substances

Stimulant Concentra�tion, M (%)

Pro

port

ion

of e

xper

imen

ts w

ith

pelle

t gra

spin

g, %

Number of

graspings1

Pellet con�sumption, %

Tast

e at

trac

tive

ness

inde

x

Numberof pellet graspings

in the exper�iment

Duration of pelletretention, s

Number of exper�

imentsafter first grasping

throughout the experi�

ment

Saccharose 0.29 (10.0) 90.9 ± 3.1 1.5 ± 0.2 46.3 ± 5.6 10.5 1.8 ± 0.1 3.5 ± 0.3 6.2 ± 0.4 88

Potassium chloride

1.73 (10.0) 77.3 ± 4.5* 1.5 ± 0.2 29.4 ± 5.6 –12.1 1.8 ± 0.1 2.9 ± 0.3 5.4 ± 0.6 88

Calcium chloride

0.9 (10.0) 73.9 ± 4.7** 1.5 ± 0.2 18.5 ± 4.8* –33.9 1.7 ± 0.1* 2.4 ± 0.3* 3.4 ± 0.4*** 88

Citric acid 0.26 (5.0) 45.4 ± 5.3*** 1.1 ± 0.1* 0*** –100 1.2 ± 0.1*** 0.8 ± 0.1*** 0.9 ± 0.1*** 88

Chironomid extract

175.0 100** – 94.3 ± 2.4*** 43.1 1.6 ± 0.1*** 5.8 ± 0.4*** 7.9 ± 0.4*** 88

Control – 90.9 ± 3.1 1.5 ± 0.2 37.5 ± 5.4 – 2.1 ± 0.1 3.5 ± 0.3 5.8 ± 0.4 88

Note: Here and in Table 2, concentration of chironomid extract is expressed in g/l; 1—number of touchings in experiments that did not ter�minate with pellet grasping is provided; M ± m is average value of the index and its error; differences from the control are significantat p: * <0.05, ** <0.01, and *** <0.001.

684

JOURNAL OF ICHTHYOLOGY Vol. 50 No. 8 2010

KASUMYAN, SIDOROV

Tabl

e 2.

Tas

te r

espo

nses

(M

± m

) of

the

ston

e lo

ach

Bar

batu

la b

arba

tula

to p

elle

ts w

ith

an e

xtra

ct o

f chi

rono

mid

larv

ae a

nd fr

ee a

min

o ac

ids

(L�s

tere

oiso

mer

s)

Sti

mul

ant

Con

cent

ra�

tion

, M

Pro

port

ion

of e

xper

i�m

ents

wit

h pe

llet

gras

ping

, %

Num

ber

of to

uchi

ngs

Pel

let

cons

umpt

ion,

%Ta

ste

attr

acti

ve�

ness

inde

xN

umbe

rof

gra

spin

gs

Dur

atio

n of

pel

let r

eten

tion

, sN

umbe

r of

exp

eri�

men

tsaf

ter

firs

tgr

aspi

ngth

roug

hout

the

expe

rim

ent

Ala

nine

0.1

85.4

± 4

.82.

0 ±

0.2

**34

.0 ±

7.0

11.7

1.9 ±

0.1

2.8 ±

0.3

4.4 ±

0.4

**55

Pro

line

0.1

61.8

± 6

.61.

6 ±

0.2

20.6

± 7

.0–

13.3

1.5 ±

0.1

2.1 ±

0.3

2.8 ±

0.4

55

Arg

inin

e0.

154

.5 ±

6.8

**1.

5 ±

0.1

10.0

± 5

.6–

45.7

1.5 ±

0.1

1.4 ±

0.2

*2.

5 ±

0.3

55

His

tidi

ne0.

147

.2 ±

6.8

***

1.4 ±

0.1

11.6

± 6

.4–

39.7

1.3 ±

0.1

1.8 ±

0.3

2.4 ±

0.4

55

Gly

cine

0.1

45.4

± 6

.8**

*1.

8 ±

0.2

12.0

± 6

.6–

38.3

1.7 ±

0.2

1.9 ±

0.2

2.7 ±

0.4

55

Nor

valin

e0.

143

.6 ±

6.7

***

1.6 ±

0.1

4.2 ±

4.2

*–

73.0

1.5 ±

0.2

1.7 ±

0.4

2.2 ±

0.4

55

Thr

eoni

ne0.

140

.0 ±

6.7

***

1.4 ±

0.1

4.5 ±

4.5

*–

71.3

1.3 ±

0.1

1.5 ±

0.3

1.8 ±

0.3

*55

Ser

ine

0.1

36.4

± 6

.5**

*1.

4 ±

0.1

19.0

± 8

.8–

17.2

1.5 ±

0.2

2.1 ±

0.3

2.7 ±

0.4

55

Met

hion

ine

0.1

36.4

± 6

.5**

*1.

4 ±

0.1

10.0

± 6

.8–

45.7

1.5 ±

0.2

1.7 ±

0.3

2.5 ±

0.4

55

Lys

ine

0.1

27.2

± 6

.1**

*1.

4 ±

0.1

20.0

± 1

0.7

–13

.21.

5 ±

0.2

1.8 ±

0.3

2.7 ±

0.6

55

Asp

arag

ine

0.1

27.2

± 6

.1**

*1.

4 ±

0.1

6.7 ±

6.7

–60

.11.

3 ±

0.1

1.1 ±

0.2

*1.

5 ±

0.4

**55

Glu

tam

ine

0.1

23.6

± 5

.8**

*1.

3 ±

0.1

0*–

100

1.5 ±

0.2

1.3 ±

0.3

2.3 ±

0.5

55

Valin

e0.

121

.8 ±

5.6

***

1.3 ±

0.1

8.3 ±

8.3

–52

.81.

3 ±

0.3

1.1 ±

0.1

1.5 ±

0.4

*55

Phe

nyla

nine

0.1

16.3

± 5

.0**

*1.

3 ±

0.1

0–

100

1.1 ±

0.4

1.2 ±

0.3

1.3 ±

0.3

*55

Cys

tein

e0.

11.

8 ±

1.8

***

1.1 ±

0.0

**0

–10

01.

00.

40.

455

Leu

cine

0.01

36.4

± 6

.5**

*1.

8 ±

0.1

*5.

0 ±

5.0

*–

68.6

1.2 ±

0.1

*1.

4 ±

0.3

1.6 ±

0.3

**55

Trip

toph

an0.

0140

.0 ±

6.7

***

1.3 ±

0.1

4.5 ±

4.5

*–

71.3

1.1 ±

0.1

*1.

1 ±

0.2

*1.

3 ±

0.3

***

55

Isol

euci

ne0.

0121

.8 ±

5.6

***

1.5 ±

0.1

0*–

100

1.1 ±

0.1

0.9 ±

0.2

*1.

1 ±

0.3

**55

Asp

arti

c ac

id0.

019.

1 ±

3.9

***

1.6 ±

0.1

0–

100

1.6 ±

0.6

2.5 ±

1.2

3.3 ±

1.3

55

Glu

tam

ic a

cid

0.01

5.5 ±

3.1

***

1.3 ±

0.1

0–

100

1.0 ±

0.0

0.8 ±

0.1

0.8 ±

0.1

*55

Tyro

sine

0.00

121

.8 ±

5.6

***

1.3 ±

0.1

0*–

100

1.1 ±

0.1

0.8 ±

0.1

*0.

8 ±

0.1

**55

Chi

rono

mid

ext

ract

175

99.4

± 0

.1**

*3

91.5

± 2

.3**

*54

.61.

3 ±

0.1

**4.

7 ±

0.3

***

5.6 ±

0.3

***

154

Con

trol

–76

.1 ±

4.6

1.3 ±

0.1

127

.9 ±

5.4

–1.

6 ±

0.1

2.1 ±

0.2

3.1 ±

0.3

88

JOURNAL OF ICHTHYOLOGY Vol. 50 No. 8 2010

TASTE PREFERENCES AND BEHAVIOR OF TESTING GUSTATORY QUALITIES 685

experiment. Pellets had a length of 4.0 mm and adiameter of 1.35 mm. The procedure of preparingagar–agar gel and conditions of its storage were pro�vided in more detail previously (Kasumyan andSidorov, 1994; Kasumyan and Morsi, 1996).

In the experiment, 11 fish were used, on which twoseries of experiments were performed: 528 with classictaste substances and 1397 with free amino acids. Inboth series, experiments with control pellets and pel�lets containing an extract of chironomid larvae werealso performed. The quantitative criteria of taste pref�erence of substances contained in the pellet included:extraoral taste attractiveness—the proportion (%) ofexperiments in which pellets were grasped from thenumber of experiments with touching the pellet—andintraoral attractiveness—the proportion of experi�ments in which the pellet was consumed from thenumber of experiments in which the pellet wasgrasped. We also calculated the index of taste attrac�tiveness according to the formula: Indpal = (R –C)/(R + C) × 100, where R is the number of swallowedpellets with the substance, %, and C is the number ofswallowed control pellets, % (Kasumyan and Morsi,1996).

Statistical analysis of results was performed usingχ2 criterion and Student’s t�test. The relationshipbetween individual characters was assessed usingSpearman’s rank coefficient of correlation (rs).

RESULTS

Background Behavior and Response to the Pellet

Experimental fish are inactive; most of the timethey usually spend on the bottom of the aquarium nearthe back or other walls. Sporadically fish begin to swimenergetically on the bottom or over the bottom and,less frequently, in the middle water layers and along thewalls of the aquarium, or exhibit search for food, dur�ing which, on inclining head to the bottom and slidingover it with barbels and the lower surface of the head,rapidly and chaotically move in search for food. Suchsearch is drastically intensified during feeding whenseveral live chironomid larvae are introduced in theaquarium that are grasped by fish mainly from the bot�tom. The transition of fish from immobility to swim�ming is usually drastic and impulsive.

The introduction into aquarium of agar–agar pel�let does not cause in fish the intensification of foodsearch activity. Therefore, during experiments, pelletswere usually introduced at the moment when the fishapproached the front wall of the aquarium orremained here for some time. Frequently fish did notnotice the introduced pellet and did not respond to it(such deliveries were not counted). Fish responded tothe pellet only in the case when it descended to thebottom of the aquarium several centimeters from theirhead. After a short period of waiting (several seconds)during which fish remained immobile, it made an

energetic dart with forward�directed barbels so thatthe pellet often turned to be between them near themouth. Having closely approached the pellet andtouching it by its barbels and sometimes with the upperor lower surface of the head, fish could also locate bar�bels on the pellet or embrace it with them and remainin such position during several seconds or swim withthe granule a small distance after which they lose it.Touching the pellet was an element always precedinggrasping, after which the pellet turned out to be in theoral cavity of the fish. However, touching did notalways lead to grasping, and after a sufficiently longcontact or retaining the pellet with barbels or afterrepeated touchings, fish could reject from grasping thepellet and move away. Rapid rejections from seizingwere observed when short�time light touching withbarbels led to a drastic moving aside of the fish and tothe loss of any interest in the pellet (Tables 1, 2).Sometimes cases were observed when fish grasped thepellet not from the bottom but in the water columnduring the slow descent of the pellet downwards nearthe fish. However, in such cases, fish also did not graspthe pellet without a preliminary touching it with itsbarbels.

After grasping, typical small and frequent chewingmovements with jaws were usually observed, afterwhich fish either swallowed the pellet or rejected it.The rejected pellet or its parts fish could immediatelygrasp repeatedly to several times during the experi�ment. Swallowing the pellet was judged from the nor�malization of the rhythm of the jaw and opercularmovements. If the experiment terminated with a rejec�tion from consumption, the fish lost interest in thepellet, moved to another part of the aquarium, and didnot return to the pellet. The duration of the experi�ment did not exceed 2–3 min on average.

Taste Preferences and Pellet Testing Behavior

Classic taste substances, except saccharose, inhib�ited pellet grasping by fish. Citric acid had the stron�gest negative effect; the effect of calcium chloride andpotassium chloride was less pronounced. Singletouching of the pellet containing citric acid caused adrastic moving aside and return of fish; repeatedgraspings of such pellets were observed extremelyrarely. Grasping pellets with citric acid occurred onlyin 45.4% of experiments, in which at least one touch�ing of the pellet was recorded. Not one grasped pelletwith citric acid was consumed by fish. Fish consumedpellets with calcium chloride almost two times lessthan control pellets, whereas potassium chloride andsaccharose did not produce such an effect (Table 1).Pellets with citric acid and potassium chloride werenot only less repeatedly grasped by fish in the experi�ment but also were retained by them in the oral cavityfor a smaller amount of time. This was especially typi�cal of pellets with citric acid that were rejected by fishafter the first grasping and for all time of the experi�

686

JOURNAL OF ICHTHYOLOGY Vol. 50 No. 8 2010

KASUMYAN, SIDOROV

ment, on average, by 3.9 and 6.4 times more rapidlythan the control.

Most free amino acids (19 of 21) significantlydecreased grasping of pellets by fish. The strongestaction was observed in cysteine, glutamic and asparticacids, pellets with which fish grasped extremely rarelyafter touching: of 55 experiments performed with eachtype of these pellets, grasping was observed only in 1,4, and 5 experiments. Brief (fragments of a second)touching of the pellet containing cysteine by barbelscaused in fish an instantaneous response resembling aresponse to a burn—a drastic movement aside oftenreplaced by a vehement swimming over the aquarium.Responses of fish to touching pellets with glutamicand aspartic acids were similar, but less intense; some�times fish touched these pellets repeatedly. The nega�tive response to pellets with phenylanine, tyrosine,isoleucine, valine, asparagine, threonine, triptophan,and norvaline was weaker but observed during mosttouchings, despite the fact that in some experiments arelatively prolonged touching of these pellets with bar�bels was recorded (to 2–3 s). The longest contacts withthe pellet (to tens of seconds, sometimes more than1 min) were observed in experiments with glutamine,methionine, lysine, tyrosine, asparagine, serine, andhistidine. Although, pellets were not always grasped inexperiments with these amino acids. Touching pelletscontaining leucine by barbels were short whereas fishcould embrace pellets with alanine with their barbelsand retain them rather long. Similar in duration con�tacts with the pellet were in experiments with proline,arginine, and glycine.

Unlike most amino acids, alanine and proline didnot significantly differ from the control in the propor�tion of experiments that terminated in pellet grasping.More willingly than in control experiments, fish aftertouching grasped only pellets with an extract of chi�ronomid larvae. This positive effect of the extract,close to 100%, was observed in both series of experi�ments: in a series with classic taste substances, all88 experiments terminated with grasping pellets con�taining an extract of chironomids; in a series withamino acids, 153 experiments of 154 terminated withgrasping of pellets (Tables 1, 2). The extract with chi�ronomid larvae was also a single taste stimulus that sig�nificantly increased consumption of pellets. Werevealed no amino acids having the same action. Mostamino acids (14) did not exert a significant impact onpellet consumption by fish; the remaining amino acids(7) led to a significant decrease in pellet consumption.Pellets with 3 of 7 deterrent amino acids (glutamine,isoleucine, and tyrosine) were rejected by fish in 100%of experiments. Pellets with cysteine, glutamic, andaspartic acids and phenylanine were also not con�sumed by fish in any experiments; however, no signif�icant difference with respect to control was revealed(Table 2).

Many amino acids that decrease pellet consump�tion caused a decrease in the number of repeated

graspings and reduced the duration of pellet retentionin the oral cavity. Such action was the strongest in cys�teine in a single experiment when the pellet wasgrasped: having rejected the pellet after a record shortretention in the mouth, the fish rejected from itsrepeated grasping. The same action occurred for theglutamic acid, slightly weaker than that of cysteine butconsiderably stronger than of the remaining aminoacids. Fish retained pellets with an extract of chirono�mid larvae and with alanine (total retention) for alonger period than the control after the first graspingand totally throughout the experiment (Table 2). Cor�relation analysis revealed the presence of significantpositive relationship between all main parameters oftaste response of fish to pellets (Table 3).

To elucidate additional specific features of exhibi�tion by fish of testing behavior, we performed compar�ison of experiments that terminated with pellet con�sumption and of experiments that terminated withrejection by fish from its consumption. Such compar�ison detected a considerable difference of these groupsof experiments in parameters of taste response. Theduration of retaining pellets by fish after the first grasp�ing and totally throughout the experiment was thestrongest difference. These indices in experimentswith consumption in all types of pellets were higher, asa rule, by 3–4 times and more (Table 4). Differences inthe number of repeated pellet graspings in experimentsthat terminated with pellet consumption and in exper�iments that terminated with rejection by fish fromconsumption are less pronounced and exhibited fornot all types of pellets. On the whole, repeated grasp�ings took place approximately 1.5 times more fre�quently in experiments in which pellets were eaten byfish.

DISCUSSION

Stone loach is a typical inhabitant of small rapidrivers and shallow waters of oligotrophic lakes with asandy or pebbly bottom and sites covered with aquaticvegetation (Spanovskaya, 1971; Fischer, 2000; Nunnet al., 2007). In feeding, bottom organisms prevail, toa less degree, detritus and epibioses (Spanovskaya,1971; Michel and Oberdorff, 1995). Stone loach feedsmainly during twilight and night hours of the day,searching for food not with the help of vision but usingother sensory systems (Bateson, 1890; Evans, 1940;Street and Hart, 1985). Low vision development is evi�denced by a weak response of following by the stoneloach of moving visual reference points (optomotorresponse) (Pavlov, 1970, 1979). Low visual capacitiesexplain the absence of response of the stone loach inour experiments to the delivered bright red pellets, ifthey occurred not near the fish but at a small distancefrom it. Trout Salmo trutta, arctic char Salvelinus alpi�nus, minnow Phoxinus phoxinus, roach Rutilus rutilus,smelts Gasterosteus aculeatus and Pungitius pungitius,and many other fish that possess a more developed

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TASTE PREFERENCES AND BEHAVIOR OF TESTING GUSTATORY QUALITIES 687

visual reception and another feeding strategy andmode of life respond to such pellets by a rapid and pre�cise dart (Kasumyan and Sidorov, 1994; 2005a;Kasumyan and Sidorov, 1995; Kasumyan and Morsi,1996; Kasumyan and Nikolaeva, 2002; Kasumyan andMikhailova, 2005; Mikhailova and Kasumyan, 2006).

Bad vision is compensated in the stone loach by agood development of chemosensory systems (olfac�tory and gustatory) that allow it to orientate and find asource of food chemical signals distributing in water.The loss of one of these two systems only partiallyblocks such capacity (Kasumyan and Marusov, 2005),whereas, in most other fish, anosmia and an acuteperiod lead to a complete loss of receptivity to solu�tions of substances or to food extracts causing in thenorm a search behavior (Kasumyan and Kazhdaev,1993a; Kasumyan and Marusov, 2002, 2005).Undoubtedly, partial retention of orientation andsearch for a source of food chemical signals is providedby external taste buds present in the stone loach, as inchannel cats Ictalurus (Ducros, 1954; Atema, 1971;Kasumyan and Marusov, 2005). In many fish leading,as the stone loach, a bottom mode of life and feedingon benthos, external taste buds are especially numer�ous and cover considerable sites of the body (Kapooret al., 1975; Gomahr et al., 1992). Maximum densityof external taste buds in such fish is observed at thefront and lower surface of the head and at barbels, ifpresent (Atema, 1971; Ovalle and Shinn, 1977; Devit�sina and Kazhdaev, 1992; McCormick, 1993; Booth,1996; Kasumyan and Devitsina, 1997; Lombarte andAquirre, 1997; Harvey and Batty, 1998, 2002; Fox,1999; Aquirre and Lombarte, 2000; Hansen et al.,2002; Devitsina, 2006; Zhang et al., 2006). As in allstudied fish, taste buds in the stone loach are present

also in the oral cavity, on the palate (Ducros, 1954;Kapoor et al., 1975; Jakubowski and Whitear, 1990).

There are no published data on the characteristicsof the taste system of the stone loach. Our results showthat the taste reception in fish of this species is welldeveloped and allows them to distinguish food itemswith different taste qualities. Such ability is providedby external and intraoral taste receptors successivelyinvolved in the process of testing properties of thefound fish. The obligatory element of testing is touch�ing of the pellet by barbels. Only in this case, fish cangrasp it or rapidly reject and pass to a new search cycle.The same pattern of food testing occurs also in otherfish, for instance, in Acipenseridae and Siluridae hav�ing weak vision or in fish using barbels for examiningsubstrate during food search, as is done by Mullidaeand Gadidae. (Andriashev, 1944a, 1944b; Pavlov,1959, 1962; Bardach and Case, 1965; Brawn, 1969;Pavlov et al., 1970; Kasumyan et al., 1991; Kasumyanand Kazhdaev, 1993b; Kasumyan, 1999). Food is nec�essarily touched with the rostrum and only thengrasped by several fish having no barbels, for instance,ruffe Gymnocephalus cernuus (Kasumyan et al., 2003).Since on the barbels and on the head of such fish thereis a great number of taste buds, it seems obvious thatthe main destination of the contact with a food item isto obtain preliminary information on the taste proper�ties of the found prey. This information is of greatimportance. It determines the way along which feed�ing behavior will develop further: touching will leadeither to grasping the item if it has positive propertiesmediated by the external taste reception or it will ter�minate with rejection from grasping and, as a result,cessation of feeding behavior if the object does nothave taste characteristics essential for the fish.

Table 3. The value of Spearman coefficient of correlation between parameters of taste response in the stone loach Barbatula bar�batula

Taste response parameters Numberof touchings1

Pelletconsumption

Numberof graspings

Duration of pellet retention, s

after first grasping throughoutthe experiment

Proportion of experimentswith pellet grasping

Number of touchings1

Pellet consumption

Number of graspings

Duration of pellet retentionafter first grasping

Note: Above the line is for 21 amino acids and below the line is for 21 amino acids and 4 classic taste substances together. For the remainingdesignations, see Table 1.

0.62**0.53**������������� 0.76***

0.76***��������������� 0.57**

0.68***��������������� 0.62**

0.66***��������������� 0.61**

0.68***���������������

0.50*0.53**������������� 0.67***

0.67***��������������� 0.71***

0.72***��������������� 0.69***

0.69***���������������

0.61**0.74***��������������� 0.68***

0.78***��������������� 0.71***

0.81***���������������

0.87***0.90***��������������� 0.94***

0.95****������������������

0.96***0.98***���������������

688

JOURNAL OF ICHTHYOLOGY Vol. 50 No. 8 2010

KASUMYAN, SIDOROV

Table 4. Parameters of taste response (M ± m) of the stone loach Barbatula barbatula in experiments terminating with pelletconsumption (above the line) or rejection (under the line)

Stimulant Concentration, M Numberof graspings

Duration of pellet retention, sNumber

of experimentsafter first grasping throughoutthe experiment

Classic taste substances

Saccharose 0.29

Potassium chloride 1.73

Calcium chloride 0.9

Citric acid 0.26

Chironomid extract 175

Control

Amino acids

Alanine 0.1

Proline 0.1

Arginine 0.1

Histidine 0.1

Glycine 0.1

Norvaline 0.1

Threonine 0.1

Serine 0.1

Methionine 0.1

Lysine 0.1

Asparagine 0.1

Glutamine 0.1

Valine 0.1

2.0 0.1±

1.6 0.1*±������������������� 4.6 0.5±

2.5 0.2***±������������������������ 8.9 0.6±

3.9 0.3***±������������������������ 37

43����

2.7 0.2±

1.5 0.1***±������������������������ 4.7 1.0±

2.2 0.2***±������������������������ 10.6 1.2±

3.3 0.3***±������������������������ 20

48����

2.3 0.5±

1.5 0.1*±������������������� 5.1 1.2±

1.7 0.1***±������������������������ 8.1 0.9±

2.4 0.2***±������������������������ 12

53����

–1.2 0.1±����������������� –

0.8 0.1±����������������� –

0.9 0.1±����������������� 0

40����

1.7 0.2±

2.4 0.4±����������������� 6.0 0.4±

2.3 0.6*±������������������� 8.0 0.4±

5.8 0.5±����������������� 83

5����

2.4 0.2±

2.0 0.2±����������������� 5.3 0.7±

2.4 0.2***±������������������������ 8.6 0.6±

4.2 0.3***±������������������������ 30

50����

1.9 0.1±

1.9 0.1±����������������� 4.9 0.6±

1.7 0.2***±������������������������ 7.0 0.6±

3.1 0.3***±������������������������ 16

31����

1.7 0.3±

1.4 0.1±����������������� 4.1 0.9±

1.6 0.3±����������������� 5.3 0.7±

2.1 0.3***±������������������������ 7

27����

1.3 0.3±

1.5 0.1±����������������� 2.9 1.0±

1.3 0.2±����������������� 4.8 0.1±

2.3 0.4±����������������� 3

27����

2.0 0.6±

1.2 0.1±����������������� 4.3 1.3±

1.5 0.2±����������������� 6.3 0.3±

1.9 0.3±����������������� 3

23����

4.0 0.6±

1.4 0.2±����������������� 2.6 0.6±

1.8 0.3±����������������� 6.1 0.8±

2.2 0.4±����������������� 3

22����

1.01.5 0.2±����������������� 6.4

1.5 0.4±����������������� 6.4

2.0 0.4±����������������� 1

23����

1.01.3 0.1±����������������� 5.6

1.3 0.2±����������������� 5.6

1.6 0.2±����������������� 1

21����

2.0 0.7±

1.4 0.2±����������������� 3.5 1.1±

1.8 0.3±����������������� 5.1 0.3±

2.2 0.4±����������������� 4

17����

1.5 0.5±

1.5 0.2±����������������� 5.3 0.5±

1.3 0.2±����������������� 5.7 0.1±

2.2 0.4±����������������� 2

18����

2.7 0.9±

1.3 0.1±����������������� 2.7 0.6±

1.6 0.3±����������������� 4.6 1.6±

2.2 0.5±����������������� 3

12����

2.01.2 0.1±����������������� 4.0

0.9 0.1±����������������� 5.8

1.2 0.2±����������������� 1

14����

–1.5 0.2±����������������� –

1.3 0.2±����������������� –

2.3 0.5±����������������� 0

13����

4.01.1 0.1±����������������� 1.8

1.0 0.1±����������������� 4.8

1.2 0.1±����������������� 1

11����

JOURNAL OF ICHTHYOLOGY Vol. 50 No. 8 2010

TASTE PREFERENCES AND BEHAVIOR OF TESTING GUSTATORY QUALITIES 689

In the process of testing and formation of a deci�sion either to grasp or to ignore the pellet, stoneloaches perform one or several touchings with barbels.According to our observations (without preciserecording), repeated touchings occurred more fre�quently in experiments using pellets containing extractof chironomids, leucine, aspartic and glutamic acids,alanine, proline, arginine, and glycine, whereas nor�valine, threonine, asparagine, valine, and some otheramino acids did not cause such an effect. Touchingsdid not always lead to grasping. Fish repeatedlytouched pellets containing cysteine extremely rarely,and grasping occurred only in one experiment of 55.The touching of pellet with cysteine, in most cases,resembled a burn because of an instantaneous drasticdart of fish aside and movement away. The action ofcitric acid was similar, but slightly weaker. One cannotexclude that such a response can be provided not bythe taste system but by the general chemical sensemediating defense responses to hazardous or damag�ing chemical substances (Parker, 1912; Whitear, 1992).The contact of fish with some pellets was frequentlymore prolonged (histidine, glutamine, methionine,

lysine, and serine) than with other (cysteine, citricacid, norvaline, triptophan, threonine, asparagine,valine, etc.). Our observations do not give grounds toconsider that the duration of tactile contact with thepellet or repeated touchings are signs showing thatsuch behavior with a great probability will lead to pel�let grasping. Revealing and specifying these relationsrequire special studies with a quantitative recording.However, the performed observations allow us to statethat the number of repeated touchings of the pelletsand the duration of contact with them depend on thesubstance that is present in them, which, in turn, indi�cates that touchings allow fish to obtain informationon the taste properties of the found objects.

We counted the number of pellet touchings only inexperiments in which no grasping of pellets wasobserved. The results of these calculations indicatethat usually one to two touchings occur during anexperiment; in some cases, three to four. Approxi�mately the same number of times, stone loaches rejectand repeatedly grasp pellets in the course of the exper�iment (Tables 1, 2). The frequency of manifestation ofrepeated touchings and graspings correlate between

Table 4. (Contd.)

Stimulant Concentration, M Numberof graspings

Duration of pellet retention, sNumber

of experimentsafter first grasping throughoutthe experiment

Phenylanine 0.1

Cysteine 0.1

Leucine 0.01

Triptophan 0.01

Isoleucine 0.01

Aspartic acid 0.01

Glutamic acid 0.01

Tyrosine 0.001

Chironomid extract 175

Control –

Note: Differences between experiments that terminated with pellet consumption or rejection from consumption are significant at p: * <0.05,** <0.01, and *** <0.001. For remaining designations, see Table 1.

–1.1 0.4±����������������� –

1.2 0.3±����������������� –

1.3 0.3±����������������� 0

9��

–1.0 0.0±����������������� –

0.4 0.4±����������������� –

0.4 0.4±����������������� 0

1��

1.01.2 0.1±����������������� 6.8

1.1 0.1±����������������� 6.8

1.4 0.2±����������������� 1

19����

1.01.1 0.1±����������������� 4.8

1.0 0.2±����������������� 4.8

1.1 0.2±����������������� 1

21����

–1.1 0.1±����������������� –

0.9 0.2±����������������� –

1.0 0.3±����������������� 0

12����

–1.6 0.6±����������������� –

2.5 1.2±����������������� –

3.3 1.3±����������������� 0

5��

–1.0 0.0±����������������� –

0.8 0.2±����������������� –

0.8 0.2±����������������� 0

4��

–1.1 0.1±����������������� –

0.8 0.1±����������������� –

0.8 0.1±����������������� 0

12����

1.3 0.1±

2.0 0.3***±������������������������ 4.9 0.2±

2.4 0.4***±������������������������ 5.5 0.2±

4.8 0.5±����������������� 140

13�������

2.1 0.3±

1.4 0.1**±���������������������� 3.7 0.4±

1.5 0.2***±������������������������ 5.4 0.3±

2.2 0.3***±������������������������ 19

48����

690

JOURNAL OF ICHTHYOLOGY Vol. 50 No. 8 2010

KASUMYAN, SIDOROV

themselves (rs = 0.64, p < 0.01; amino acids) and arerelated to the taste attractiveness of pellets (rs = 0.50,p < 0.05 and rs = 0.59, p < 0.01, respectively; aminoacids). Apparently, such manipulations (repeatedtouchings and repeated graspings) are important ele�ments of behavior related to testing by fish of proper�ties of the food item. Such actions during feeding areperformed by many species (Ware, 1972; Ibrahim andHuntingford, 1992; Ellis and Gibson, 1997). Rejec�tions and repeated graspings of the prey are to a greaterdegree typical of fish dwelling in stagnant or low�activewaters and are less typical of rheophylic fish(Kasumyan and Prokopova, 2001; Kasumyan andSidorov, 2005a).

Substances stimulating grasping called insitants(Kasumyan and Døving, 2003) were not found in thestone loach. Most used substances according to theiraction turned out to be suppressants, i.e., substancescausing an increase in the number of rejections fromgrasping food items (three classic taste substances offour and 19 amino acids of 21). We found no sub�stances that increase the consumption of grasped pel�lets (taste stimulants). According to taste qualitiesmediated by intraoral taste receptors, the used sub�stances were for the stone loach either deterrents, i.e.,decreasing consumption, or indifferent stimuli. Onlypellets containing extract of chironomids—a complexmixture of diverse groups of substances—had tasteattractiveness. Pellets with this natural taste stimuluswere willingly grasped and consumed by the stoneloach, the smell (olfactory) stimulation of this process,as was shown previously, is absent (Kasumyan andMorsi, 1996; Kasumyan and Sidorov, 2005a, 2005b).The absence or an extremely low number of aminoacids with an attractive taste was previously shown forsome fish—arctic flounder Liopsetta glacialis, grasscarp Ctenophyryngodon idella, starred sturgeon Aci�penser stellatus, arctic char Salvelinus alpinus erythri�nus, ruffe Gymnocephalus cernuus, and some others(Kasumyan and Sidorov, 1995; Kasumyan andNikolaeva, 2002; Kasumyan and Døving, 2003;Kasumyan et al., 2003). The taste attractiveness offood items consumed by these fish is apparentlyformed not by free amino acids but by other types ofchemical compounds, for instance, amines, nucle�otides, and nucleosides. (Carr et al., 1996).

Cysteine that is a strong deterrent for the stoneloach has an attractive taste for benthophages of thecarp Cyprinus carpio and tench Tinca tinca and anindifferent taste for goldfish Carassius auratus, min�now, and roach. Tyrosine and glutamine have a repel�lent taste for the stone loach but an indifferent orattractive taste for most aforementioned cyprinids(Kasumyan and Morsi, 1996; Kasumyan and Prokop�ova, 2001; Kasumyan and Nikolaeva, 2002;Kasumyan and Marusov, 2003). One can find manyexamples of noncoincidence or diametrically oppositeattitude to the taste of the same substance in the stoneloach and other fish species close in systematics

(which together with the stone loach belong to theorder Cypriniformes) or similar in the mode of lifeand the type of feeding (benthophages). They sup�port once again a high species specificity of tastepreferences of fish (Kasumyan, 1997; Kasumyan andDøving, 2003).

Comparison of the range and correspondence ofextra� and intraoral taste spectra of fish is of interest.In acipenserid species (Russian sturgeon A. gueldens�taedtii, Siberian sturgeon A. baerii, A. stellatus, andPersian sturgeon A. persicus), extraoral taste spectraare considerably wider than intraoral taste spectra andconsist of amino acids positive by their action(Kasumyan et al., 1992; Kasumyan, 1999; Shamushakiet al., 2008). Such ratio of the composition and widthof the spectra and their high correlation found in aci�penserids are considered an important functionaladaptation. Thanks to this, fish at the stage of prelim�inary assessment select (grasp) a large number ofpotentially suitable food items but consume only thoseof them that at the final stage of testing pass through anarrow�oriented intraoral sensory control (Pavlov andKasumyan, 1998; Kasumyan, 2002).

In the stone loach, the situation is opposite to thatdetected in acipenserids: in taste spectra, there are nosubstances of positive effect, and suppressant aminoacids (extraoral reception) are more numerous (>twotimes) than deterrent amino acids (intraoral recep�tion). How can these results be interpreted? In ouropinion, they clearly show that objects with low(repellent) taste properties are subjected to a strictrejection already at the stage of preliminary food test�ing. Rejection from grasping, early elimination ofobjects that can be rejected subsequently, at the stageof intraoral testing, on the whole, optimize the feedingprocess, decreasing nonproductive time expenditures.Between extraoral and intraoral spectra in the stoneloach a significant positive correlation is observed.This means that those objects that at the stage of pre�liminary taste control are most frequently ignored byfish, in the case of grasping, will be with a high proba�bility rejected at the stage of intraoral assessment. Fishwill more frequently consume those grasped objectswhose negative extraoral taste properties are less pro�nounced. Such strategy of selecting objects will pro�vide widening of the food spectrum of fish during star�vation. It was established that the spectrum of deter�rent stimuli at food deprivation decreases (Kasumyanand Sidorov, 2010). It is quite probable that duringstarvation, the spectrum of not only deterrent stimuli,but also of suppressants decreases, which increases thediversity of the grasped objects, first of all, due to thosein which negative extraoral taste properties are small.On the whole, the data obtained on the stone loach donot contradict conclusions made previously from theresults of studies on acipenserids and supplement con�cepts of those sensory mechanisms that form the basisof the selective choice of food items by fish.

JOURNAL OF ICHTHYOLOGY Vol. 50 No. 8 2010

TASTE PREFERENCES AND BEHAVIOR OF TESTING GUSTATORY QUALITIES 691

The decision to swallow the grasped prey or toreject it in the simplest variant is formed as a result ofa chain of reflectory events that includes stimulusreception by intraoral receptors and transfer of theobtained information as an electric impulse to tastecenters. After decoding and analysis, the signal ispassed to secondary neurons, and then to motoneu�rons innerving the respective muscles of the oral andpharyngeal zones (Finger, 2008). It is considered thatmost of the time required for this cycle is spent on pro�cesses passing in the nerve centers (Halpern, 1986).Events related to signal reception require, as isassumed, considerably less time and can be limited infish to fractions of a second (Kasumyan and Prokop�ova, 2001; Mikhailova, 2009). The informationobtained in the course of the first testing apparently atits very beginning is sufficient to form a decisionwhether the object is suitable or unsuitable. This isindicated by the fact that already from this momentfish behavior begins to develop according to one of twovariants. If the decision is positive, then, before beingswallowed, the object is long tested (retained) by fish;it can be rejected and again grasped for repeated con�trol. When a negative decision is taken, the object isretained for a shorter time and less frequently sub�jected to repeated testings. The differences betweenthese two variants are especially considerable accord�ing to duration of retaining the object and are mani�fested apparently for all types of pellets. Small samplesizes for experiments terminating with pellet con�sumption do not allow us to speak about it with greaterassurance. New data supporting the validity of thissuggestion give grounds to consider that there are twodifferent behavioral stereotypes of testing taste proper�ties of food items in fish (stone loach). It remainsunknown whether such stereotypes exist in other spe�cies, how they differ in fish of a different mode of lifeand feeding strategy, and how stereotypes are resistantto different external factors and changes in the innerstate of fish.

ACKNOWLEDGMENTS

We are grateful to E.A. Marusov for constructiveremarks on the manuscript, E.A. Marusov andV.Ya. Pushkar’ for help in catching and keeping fish,and to L.S. Alekseeva for help in the layout of themanuscript.

The study was supported by the Russian Founda�tion for Basic Research (project no. 07�04�00793), the“Universities of Russia” Program (projectno. 2.1.1.3267), the “Leading Scientific Schools”Program (NSh�3231.2010.4), and the “Scientific andScientific–Pedagogical Personnel of Innovation Rus�sia for 2009–2013” Federal Central Program (StateContract no. 02.740.11.0280).

REFERENCES

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