Gustatory sensitivity of the external taste buds of

Oreochromis niloticus L. to amino acids

S Y Yacoob, K Anraku, T Marui*, T Matsuoka, G Kawamura & M Vazquez Archdale

Laboratory of Fishing Technology, Faculty of Fisheries, Kagoshima University, Shimoarata 4-50-20,

Kagoshima 890-0056, Japan

*Present address: Department of Oral Physiology, Ohu University School of Dentistry, 31-1 Misumido, Tomita,

Koriyama, Fukushima Prefecture, Japan 963-8611

Correspondence: S Y Yacoob, Laboratory of Fishing Technology, Faculty of Fisheries, Kagoshima University, Shimoarata 4-50-20,

Kagoshima 890-0056, Japan

Abstract

The gustatory sensitivity of the Nile tilapia,

Oreochromis niloticus L., to different amino acids

was studied using an electrophysiological approach.

The electrical responses were recorded from a

branch of the facial nerve innervating the external

taste buds of the upper lip. The relative stimulatory

effectiveness (RSE) of nine amino acids and betaine

were determined at a concentration of 1 mM and all

of them elicited neural responses. This species

responded well to the neutral, basic and acidic

amino acids. The most stimulatory amino acids

were L-histidine, L-arginine, L-serine, L-methionine

and L-glutamine; L-proline and betaine were the

least stimulatory. The results of this study suggest

that the Nile tilapia has high external gustatory

sensitivity to some amino acids as a physiological

adaptation to search effectively for their sources.

The effect of the pH, ranging from 4.0 to 9.0, on the

RSE of three neutral amino acids and arti®cial pond

water (APW) was also studied. The RSE increased

below pH 6.0 and was relatively unaffected from 7.0

to 9.0, indicating that acidi®ed stimulants are

highly stimulatory in this species. Nile tilapia did

not discriminate the pH of APW as effectively as

some of the species studied earlier.

Keywords: Nile tilapia, gustatory responses, amino

acids, pH, electrophysiology

Introduction

In aquaculture, an ef®cient diet should meet not

only the nutritional requirements of ®sh, but also its

chemosensory capabilities. These physiological abil-

ities may not be altered by learning and are genetic

in origin (Kasumyan 1997; Hara, Carolsfeld &

Kitamura 1999). In many species, especially in

non-visual feeders, it is their chemical senses, most

probably the gustatory sense, which determine

whether a particular food is located and consumed

(Gerking 1994). In ®shes, the external and internal

taste buds innervated by different cranial nerves

(facial, glossopharyngeal and vagal nerves) mediate

the food search behaviour and palatability respec-

tively (Marui & Caprio 1992). Amino acids are

well-known chemical cues for food resources in

many species (Mackie & Mitchell 1985). The

gustatory sensitivity to amino acids has been

studied electrophysiologically in the common carp,

Cyprinus carpio L. (Marui, Harada & Kasahara

1983a), salmonids (Marui, Evans, Zielinski & Hara

1983b; Hara et al. 1999) and in several other ®sh

(Caprio 1975; Yoshii, Kamo, Kurihara & Kobatake

1979; Goh & Tamura 1980a; Ishida & Hidaka

1987), so far. Among the species of tilapia, only the

red belly tilapia, Tilapia zillii (Gervais), has been

studied earlier (Johnsen, Zhou & Adams 1990).

However, no work has been carried out on the

species Oreochromis niloticus L.

Some of the amino acids found to be stimulatory

through electrophysiological studies were also

# 2001 Blackwell Science Ltd 217

Aquaculture Research, 2001, 32, 217±222

effective in eliciting behavioural responses, in the

red belly tilapia (Johnsen & Adams 1986; Johnsen

et al. 1990), common carp (Marui et al. 1983a;

Kasumyan & Morsi 1996), and red sea bream,

Chrysophyrys major (Temminck & Schlegal) (Goh &

Tamura 1980a,b). However questionable the pre-

dictions of behavioural responses from electrical

recordings may be (Gerking 1994), such systemic

studies provide a spectrum of sensitive amino acids

out of which the attractants, stimulants and

deterrents can be identi®ed by behavioural studies

(Kasumyan 1997).

Nile tilapia is cultured under varied systems,

including human waste-fed water bodies or in the

ef¯uents of sewage treatment plants (Edwards

1988; Khalil & Hussein 1997). Because environ-

mental contaminants are known to affect the

chemoreceptors (Klaprat, Evans & Hara 1992), it

would be very valuable to know if the adverse water

quality parameters have any effect on the gustatory

senses of this species. But before such studies are

carried out, a more basic understanding of the

sensory capabilities of this species is required. Thus,

the objective of this study was to determine the

relative stimulatory effectiveness of some amino

acids on the external taste buds of O. niloticus by the

electrophysiological method. The pH of the stimuli is

another factor affecting the gustatory responses in

some species (Kiyohara, Yamashita & Harada 1981;

Marui et al. 1983a,b). Hence we also tested the effect

of pH on the gustatory responses in this species.

Materials and methods

Chemicals

The relative stimulatory effectiveness (RSE) of nine

amino acids and betaine, listed in Table 1, were

tested at a concentration of 1 mM. The amino acids

selected for this study consisted of neutral, acidic

and basic amino acids, known to be stimulatory

across several species. The stock solutions of reagent

grade L-amino acids (Sigma Chemical Co., Tokyo,

Japan) were prepared in distilled water biweekly,

stored in a refrigerator and diluted in arti®cial pond

water (APW; 0.3 mM NaCl, 0.2 mM KCL and 0.2 mM

CaCl2 in deionized water) daily for testing. The RSE

of amino acids were tested at pH 7.0 and the effect of

pH on gustatory responses was tested from 4.0 to

9.0. The pH adjustments were made using either

NaOH or HCl.

Fish maintenance

The experimental species O. niloticus (total length:

163 6 13 mm) were purchased from a commercial

tilapia farm in Ibusuki town of Kagoshima

Prefecture, Japan. The ®sh were maintained in

100 L aquaria (temperature 20 °C and pH 7.2) and

fed with commercial tilapia pellets. The ®sh were

acclimatized in the aquarium for at least 1 week

before the experiments.

Recording procedure

Just before the experiments, the ®sh were immobi-

lized by an intramuscular (i.m.) injection of

gallamine triethiodide (0.3 mg kg±1) after being

lightly anaesthetized with tricaine-methane sulpho-

nate (1:20 000 dilution). They were then wrapped

in wet tissue paper and clamped on a plastic plate

placed in a ¯ow through plastic tray. Clean and

aerated tap water perfused the gills throughout the

experiments. This was carried out through a pair of

¯exible silicone tubes, which came down from an

overhead tank and were inserted into each

operculum. Thus, the lips remained completely

unaffected by the ¯ow of perfusing water during

the recordings. During the experiments, supplemen-

tal doses of gallamine triethiodide were administered

using a syringe, whenever muscular movements

were observed.

Table 1 Relative stimulatory effectiveness (RSE) of

amino acids in Oreochromis niloticus L.

Amino acids (abbreviations) RSE (%)*

L-histidine (His) 161.5 6 17.1

L-arginine (Arg) 157.3 6 22.5

L-serine (Ser) 154.9 6 18.0

L-methionine (Met) 154.6 6 13.2

L-glutamine (Gln) 144.9 6 6.6

L-glutamic acid (Glu) 110.2 6 17.9

L-tryptophan (Trp) 102.6 6 21.4

L-alanine (Ala) 100.0 6 6.3

L-proline (Pro) 51.7 6 6.9

Betaine (Bet) 44.6 6 12.6

Data are means 6 standard deviations.

* Responses are expressed as percentage of response to

standard L-alanine.

218 # 2001 Blackwell Science Ltd, Aquaculture Research, 32, 217±222

Gustatory responses of O. niloticus to amino acids S Y Yacoob et al. Aquaculture Research, 2001, 32, 217±222

Gustatory responses were recorded from a branch

of the facial (VII) ± trigeminal (V) complex

innervating the gustatory receptors of the anterior

part of the oral cavity and upper lip. The left eye was

surgically removed and the nerve bundle running

across the bottom of the eye socket was gently

exposed from the surrounding tissues, cut centrally,

the membrane enveloping it was removed, and

dissected into smaller bundles. The peripheral end of

one of these nerve twigs was hooked on to a bipolar

silver electrode and covered with liquid paraf®n to

prevent drying. The multiunit electrical activity was

ampli®ed (Bioelectric ampli®er MEG-1200, Nihon

Khoden, Tokyo, Japan), monitored in an oscillo-

scope (Memory Scope VC23, Nihon Khoden, Tokyo,

Japan) and recorded by a data recorder (TEAC RD-

135T, TEAC Corp., Tokyo, Japan) in digital audio-

tapes for later analyses. The analogue data were

digitized by an A/D converter (MacLab/4S; AD

Instruments Pty. Ltd., Castle Hill, Australia), and

analysed by Chart v 3.4.6 software. The response

magnitude was measured in mm at the peak of the

integrated responses (time constant 0.6 s) from the

base-line level, meaning the response magnitudes

expressed were relative. The response to each amino

acid was measured in seven ®shes, three times in

each ®sh, and expressed as a percentage of the

averaged response for standard L-alanine. In each

®sh, the response to standard L-alanine was often

measured during the experiments and the mean

response was obtained.

Method of stimulation

The stimulating apparatus (Fig. 1) consisted of an

APW container and a disposable pipette for the

stimulant, each connected to one of the inlets of a

three-way electric valve (Takasago Clean Valve

MTV-3 -M6, Takasago Electric, Inc., Nagoya,

Japan). A constant ¯ow (8 mL min±1) of APW from

the outlet of the valve was directed gently over the

upper lip of the ®sh, through an over-¯ow system

and a thin tube connected to a capillary at its end.

For stimulation, 1 mL of stimulant was drawn into

the pipette and the electric valve was activated,

which instantly replaced the APW with stimulant.

The over-¯ow system ensured that the ¯ow rate

over the receptor ®eld was not affected by the

application of stimulant. The dilution ratio of stimuli

at the receptor region was determined to be 50% by

a dye test. The temperatures of the stimulant

solutions were adjusted to that of the APW before

application. The pipette was washed several times to

avoid contamination from successive applications of

different amino acids. An interstimulus interval of

240 s was given, by which time complete recovery

of responses after successive stimulant applications

was observed. During the interstimulus interval,

APW bathed the receptor ®eld constantly.

Results

The external taste receptors of the upper lip were

sensitive to mechanical stimulation. Appreciable

mechanical responses were observed when the

APW was dropped over the receptive ®eld.

However, using the constant ¯ow stimulation

system described above, the possible mechanical

responses due to the stimulant application were

eliminated. Thus, only the tested chemicals elicited

neural responses, but not the APW. The typical

integrated gustatory responses for the amino acids

and betaine are presented in Fig. 2. The responses

were phasic in nature and were quickly adapting

within 10±15 s.

The relative response magnitudes to each amino

acid, distributed normally, were averaged and

ranked in the order of their stimulatory effectiveness

(Table 1). The responses to seven out of nine amino

acids tested were greater than the response to the

standard L-alanine. In this species, both the basic

(L-arginine) and acidic (L-glutamic acid) amino

acids were stimulatory. Among the neutral amino

acids, L-histidine, L-serine, L-methionine and

Figure 1 Schematic diagram of the experimental set-up

for recording the gustatory responses of Oreochromis

niloticus L. APW, arti®cial pond water; P, pipette for

stimulant; V, electric valve.

# 2001 Blackwell Science Ltd, Aquaculture Research, 32, 217±222 219

Aquaculture Research, 2001, 32, 217±222 Gustatory responses of O. niloticus to amino acids S Y Yacoob et al.

L-glutamine were the most stimulatory. The re-

sponse to L-tryptophan was as high as that for

L-alanine. L-proline and betaine were the least

stimulatory among the chemicals tested.

The relative stimulatory effectiveness of APW and

three neutral amino acids L-alanine, L-histidine and

L-serine at various pH values are shown in Fig. 3.

The responses to these amino acids increased

sharply when the pH was reduced below 6.0 and

were relatively unaffected between 7.0 and 9.0. The

APW itself became stimulatory when its pH was

either increased or decreased from 7.0. However,

the maximum response to APW, which occurred at

pH 4.0, was only about 6% of response to the

standard L-alanine.

Discussion

In this study, the gustatory responses of external

taste buds located over the upper lip were recorded.

The features of the neural responses were similar to

those of other receptive areas such as the palate of

salmonids (Marui et al. 1983b; Hara et al. 1999) and

the lower lip of carp (Marui et al. 1983a). They were

relatively fast adapting and phasic responses.

However, the phasic portions of these integrated

responses to amino acids in this study were slightly

slower than those obtained in some of the earlier

studies (Caprio 1975; Yoshii et al. 1979; Kiyohara

et al. 1981; Marui et al. 1983a, b). This may be due

to the differences in the nature of perfusing water,

stimulus volume and dilution ratio of stimuli, used

in this study.

The gustatory effectiveness of basic or acidic

amino acids is strongly dependent on species (Marui

et al. 1983a). For example, acidic amino acids are

not effective gustatory stimuli in the Japanese eel,

Anguilla japonica (Temminck & Schlegal), whereas

the basic amino acids are not stimulatory in the

common carp and the rabbit ®sh, Siganus fuscescens

(Houttuyn) (Table 2). In the Nile tilapia, both acidic

and basic amino acids were stimulatory. Other

species such as channel cat®sh, Ictalurus punctatus

(Ra®nesque), and the tiger ®sh, Therapon oxyrhyncus

(Temminck & Schlegal), also responded to both

acidic and basic amino acids. However, their

response to glutamic acid was not as high as in

tilapia or rabbit ®sh (Table 2).

In general, stimulatory nature of neutral amino

acids is common across several species (Marui et al.

1983b; Marui & Caprio 1992). However, their

relative stimulatory effectiveness varies among

species, as mentioned earlier. The signi®cant ®nding

of this study is that the sensitivity of this species to

L-histidine, L-methionine and L-tryptophan is high-

er than for many of the species studied earlier

(Table 2). The gustatory sensitivity and speci®city of

®shes are known to develop evolutionarily and

ontogenetically in relation to their feeding habits

(Goh & Tamura 1980a,b; Johnsen & Adams 1986;

Ishida & Hidaka 1987; Johnsen et al. 1990; Hara,

Kitada & Evans 1994). In nature, the Nile tilapia

Figure 2 Typical integrated

gustatory responses recorded from

the facial (VII) ± trigeminal (V)

nerve complex to amino acids. The

arrow marks indicate the onset of

stimulus. Thick bars in the

horizontal axis indicate 5 s.

Figure 3 Effect of pH on the gustatory responses to

amino acids and APW. The relative response magnitudes

are expressed as percentage response to 1 mM L-alanine

at pH 7.0.

220 # 2001 Blackwell Science Ltd, Aquaculture Research, 32, 217±222

Gustatory responses of O. niloticus to amino acids S Y Yacoob et al. Aquaculture Research, 2001, 32, 217±222

feeds mainly on aquatic macrophytes and phyto-

plankton (Khallaf & Alne-na-ei 1987; Dempster,

Beveridge & Baird 1993). They can also derive their

nourishment from the detrital aggregate (Chapman

& Fernando 1994). It is interesting to note that the

proportions of L-histidine, L-methionine and

L-tryptophan in these dietary items are relatively

low (Muztar, Slinger & Burton 1978; Bowen 1980;

Ahlgren, Gustafsson & Boberg 1992) and that they

are among the essential amino acids required for the

growth of this species (Santiago & Lovell 1988).

Further, the bio-availability (enzymatically hydro-

lysable fractions) of L-histidine from detritus, and L-

histidine and L-methionine from plankton is also

relatively low (Dauwe, Middleburg, Van Rijswijk,

Sinke, Herman & Heip 1999). Because the function

of extra-oral taste buds is to perceive the food at a

distance (Marui & Caprio 1992), the high gustatory

sensitivity of this species to L-histidine, L-methio-

nine and L-tryptophan is clearly an advantage for

searching those food packets or items enriched with

these limiting nutrients that are required for normal

growth.

Acidic solutions were highly stimulatory in the

gustatory system of this species. Similar results have

also been observed in the rainbow trout,

Oncorynchus mykiss (Walbaum), and common carp

(Marui et al. 1983a,b). However, the responses to

APW below pH 6.0 were very low when compared

with the responses of carp or rainbow trout to the

tap water at these pH levels. As for the high

response to amino acids at low pH, it has been

shown that acidic substances enhance feeding in

the red belly tilapia (Adams, Johnsen & Hong-Qi

1988), which may be affected by the intra-oral taste

buds. Whereas the effects of pH of the stimuli on the

extra-oral taste buds tested in this study, may

mediate some changes in the food searching

behaviour rather than in the palatability.

The signi®cance of the high sensitivity of the

extra-oral taste buds to some amino acids and the

increase in effectiveness of amino acids at low pH, in

the food searching behaviour needs to be studied

further. Complementary behavioural studies will

have potential implications in the diet formulation

in the view of increasing the attractiveness of the

diets, especially when cheap and unpalatable

protein sources are used.

Acknowledgment

This study was supported by the Ministry of

Education, Science, Sports and Culture, Japan.

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Gustatory responses of O. niloticus to amino acids S Y Yacoob et al. Aquaculture Research, 2001, 32, 217±222