Physiology & Behavior 140 (2015) 111–117

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Taste preferences and taste thresholds to classical taste substances in the carnivorous fish, kutum Rutilus frisii kutum (Teleostei: Cyprinidae) Sheyda Goli a, Valiollah Jafari a, Rassol Ghorbani a, Alexander Kasumyan b,⁎ a b

Department of Fisheries, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Golestan, Iran Department of Ichthyology, Faculty of Biology, Moscow State University, Leninskie Gory, Moscow 119992, Russia

H I G H L I G H T S • • • • •

Palatability of classical taste substances is assessed for kutum, the teleost fish. Threshold concentration for citric acid, the most palatable substance, is 0.01 M. Kutum differentiates pellets' taste if citric acid concentration differs twice. Kutum retains palatable pellets in the mouth for much longer time than other pellets. Carnivorous kutum and omnivorous roach have opposite taste preferences.

a r t i c l e

i n f o

Article history: Received 18 April 2014 Received in revised form 5 December 2014 Accepted 8 December 2014 Available online 10 December 2014 Keywords: Taste Taste preferences Threshold concentration Feeding behavior Teleost fish

a b s t r a c t The objective of this study was to compare the taste preferences in the closely related sympatric fish species with different feeding patterns. For this purpose, palatability for four classical taste substances was evaluated for carnivorous kutum Rutilus frisii kutum and the results were compared with the taste preferences of the omnivorous roach Rutilus rutilus which had been studied earlier. In addition, the threshold concentration and the dose– response relationship of the most palatable tastants were evaluated and the ability of kutum to differentiate food with tastants in different concentrations was estimated. It was found that citric acid significantly increases the agar gel pellet consumption within the range of concentrations from 0.01 M to 0.52 M; the pellets with a concentration of 0.026 M were the most palatable. The pellet consumption is significantly different if the concentration of citric acid in the pellets differs more than two times. The absolute threshold concentration is 0.01 M, or 2.74 μg of citric acid per pellet. Sucrose and NaCl have deterrent taste at the highest concentrations tested (0.29 and 1.73 M, respectively). Both substances are palatable at 10 times lower concentrations and become indifferent after further gradual decrease in their concentration. CaCl2 decreases the pellets consumption at 0.9 M but is an indifferent tastant at lower concentrations (0.45, 0.09 and 0.045 M). The number of rejections and repeated grasps of a food pellet is fewness and is not related to the pellet's palatability, while the retention time of pellet in the oral cavity positively and highly correlates with the pellet's palatability. Kutum have opposite taste preferences for most substances tested in comparison with the roach. It indicates that the taste preferences mediated by the oral taste receptors are different in closely related sympatric fish displayed diet divergences. © 2014 Elsevier Inc. All rights reserved.

1. Introduction All sensory systems are involved in the regulation of feeding behavior in fish and the final steps of this complex behavior are based on the function of the gustatory system [1]. The taste bud is the end organ of the gustatory system in all vertebrates. In fish, taste buds are numerous and cover the oral cavity, and in many species they are also distributed on the fish body surface [2]. The extraoral taste buds mediate the food

⁎ Corresponding author. E-mail address: [email protected] (A. Kasumyan).

http://dx.doi.org/10.1016/j.physbeh.2014.12.022 0031-9384/© 2014 Elsevier Inc. All rights reserved.

grasping behavior while the stimulation of the oral taste buds releases the food swallowing reflex and food rejections [3]. Using the standard bioassay method developed to study taste preferences in fish it was found that some substances are highly effective taste stimuli and increase the consumption of flavored food items but other substances make food unpalatable for fish. Comparison of the responses of intact and anosmic specimens do not reveal any significant differences in flavored food pellets' consumption [4,5]. Taste preferences have evident similarity among conspecifics [6]. The taste responsivity to free amino acids and classical taste substances is the same in males and females of the guppy Poecilia reticulata and in both the brown trout Salmo trutta and the nine-spined stickleback Pungitius

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pungitius individuals from different geographically isolated populations [7–9]. The dependence of taste preferences upon feeding experience is weak, as it has been shown in the grass carp Ctenopharyngodon idella juveniles reared on different diets for several months [10]. In contrast, the taste preferences seem to be highly species specific in fish. It was found that the same substance dramatically increases the food consumption in one species of fish and has the opposite effect on other fish [6]. As an example, amino acid L-cysteine and citric acid are highly palatable for brown trout S. trutta but evoke strong deterrent response in chum salmon Oncorhynchus keta, which belongs to the same family Salmonidae [8,11]. The species specificity in taste preferences still remains not clear especially in closely related fish. The comparison of several acipenserids (Russian sturgeon Acipenser gueldenstaedtii, Siberian sturgeon Acipenser baerii, stellate sturgeon Acipenser stellatus and Persian sturgeon Acipenser persicus) revealed that the oral taste preferences are highly specific among these fish species, but the specificity of extraoral taste preferences are less pronounced [12,13]. However, oral taste spectra of threespined stickleback Gasterosteus aculeatus and ninespined stickleback P. pungitius (both sticklebacks belong to family Gasterosteidae) are similar and the list of highly palatable amino acids coincide in both species [14]. The taste spectra in closely related fish having different feeding ecology were not compared. The threshold concentrations estimated by electrophysiological recordings from the taste nerves are between 10−6 and 10−9 M for the most potent substances, and the responses increase sharply with a logarithmic increase in their concentrations [15]. The threshold concentrations determined by behavioral assay are usually in the range of 10−2–10−4 M [4,16,17]. It was supposed that there might be several reasons for the discrepancy between the threshold concentrations obtained by behavioral and by electrophysiological methods [6]. Behavioral data concerning differential gustatory sensitivity in fish as well as dose–response relationships have not been obtained. The aim of the present study was to investigate the taste preferences in the closely related sympatric fish species displayed differences in diet. For this purpose, palatability for four classical taste substances was evaluated for kutum Rutilus frisii kutum which is a carnivorous fish. The results were compared with the taste preferences of the omnivorous roach Rutilus rutilus which had been studied earlier using the same behavioral method [18]. The behavioral method was used also for evaluation of the threshold concentration and the dose–response relationship of citric acid, the most effective taste substances in kutum. In addition, the ability of kutum to differentiate artificial food pellets containing citric acid and other classical taste substances in different concentrations was estimated. 2. Materials and methods 2.1. Fish maintenance The study was carried out on 20 specimens of the 4–5 month old kutum juveniles with the 5.9 ± 0.8 cm1 total length (TL) and 1.5 ± 0.5 g2 body weight reared at the Sijaval fish farm (Turkmen seaport, Golestan province, Iran). The fish were transported to the facilities of the Shahid Fazli Aquaculture Research Station of the Gorgan University of Agricultural Sciences and Natural Resources and were maintained for one month in the 100 l aquaria. The fish were fed manually once a day until an apparent satiation with pellets (0.8 mm diameter; brown color) of the commercial formulated diet containing 56% protein, 18% lipid and 10.4% ash (BioMar Group, Denmark; http://www.biomar. com/). Three weeks before the trials fishes (n = 20) were placed individually in separate 5-liter aquaria with fresh tap water (water temperature 22–23 °C; oxygen level 7.2–7.8 mg·L−1; pH 7.3–7.4; salinity 1 2

The standard error of the mean. The standard error of the mean.

0.009–0.011 g·L−1; total hardness 270–300 mg·L−1; electrical conductivity 30–35 μS·cm−1; 16 L:8D photoperiod). Aquaria were equipped with an air pump and did not contain gravel or other material on the bottom. The back and side walls of aquaria were opaque for visual isolation of fish. During the first 2–3 days, the fish were fed daily with thawed freshly frozen Chironomidae larvae which were offered to the fish one by one. After several days, when the fish have been adapted to the new conditions and feeding procedure, they were trained for 3–5 days to take the artificial agar pellets flavored with Chironomidae larvae extract, 175 g·L−1. Flavored pellets were given to the fish one by one with an interval of 1–2 min. In the course of the training and experiment the fish were fed ad libitum with Chironomidae larvae once a day after the completion of daily session in order to ensure that they have the similar feeding motivation at the beginning of a new session the next day. A part of water in aquaria was replaced daily with fresh water. Observations and video recordings were made through the transparent frontal wall of the aquaria. 2.2. Taste stimuli Sucrose (0.29; 0.145; 0.029; 0.0145; 0.0029 M), sodium chloride (1.73; 0.173; 0.0865; 0.0173 M), calcium chloride (0.9; 0.45; 0.09; 0.045 M) and citric acid (0.52; 0.26; 0.182; 0.13; 0.026; 0.01; 0.0052; 0.0026 M) and the fresh Chironomidae larvae water extract (175 g·L−1; g in wet weight) were used as taste stimuli. Classical taste substances are used often for various studies of taste physiology and taste-evoked behavior in animals and man [19–25]. The concentrations of these substances are relatively high in the hemolymph of a variety of potential food organisms for fish: citrate and sodium chloride concentrations can reach over 3 × 10−2 M and 0.5 M respectively [26,27]. The chemical substances were produced by Merck KGaA (Germany). The Chironomidae larvae were purchased from a local aquarium fish shop. The Chironomidae larvae were homogenized in a ceramic mortar and the obtained homogenate was mixed with distilled water in the proportion of 175 g·L−1 and then was filtered through a cotton cloth after extraction for 5 min at 18 °C. 2.3. Preparation of pellets The pellets were prepared from 2% agar gel. After dissolving agar (Merck KGaA) in boiled distilled water, a dye (Cr2O3,3 0.35%; Merck KGaA) and water solution of one of the four classical substances were added into the agar solution. The water extract of Chironomidae larvae was added to the cooled (50 °C) agar solution. The agar solution was then mixed and poured into a Petri dish. The blank pellets with the dye only were used as control. The agar gels which contained chemical substances and Chironomidae larvae extract were kept at +5 °C for up to 7 and up to 3–4 days, respectively. The cylindrical pellets with a diameter of 1.1 mm and a length of 1.5 mm were cut from the cold gel with a stainless steel tube just before each trial. In total, 23 types of agar pellets differing by substance and their concentration in the agar matrix were used for trials. 2.4. Experimental procedure Twenty fish (each fish in individual aquarium) were divided on 5 groups by 4 fish in each. Each fish in a group received the same type of pellets during the day; next day the pellets of another type were offered to each fish in a group. The fish in different groups received pellets with different substances. The blank pellets were used every 5 days. Thus, within 5 days each fish from 20 fish used received 5 different types of the pellets including the control ones. Different types of the 3 Chromium (III) oxide is inert and indigestible green color marker that a commonly used for studies in fish feeding and digestion.

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pellets were used in random order for different groups of fish. On the following 5 days the next 5 types of pellets were tested. The same type of the pellets was offered to the same fish 2 times only with a time interval 20–25 days. Each trial was started when the first pellet was introduced into an aquarium and was ended after 60 s. During the trial, the pellets of the same type were offered one by one to each fish in a group. The next pellet was offered when the previous one had been swallowed or refused by fish, usually after several repeated grasps. If the fish did not grasp the first offered pellet during 30 s the trial was canceled. All trials were recorded with the video camera Panasonic SDRH250GS-S and several behaviors were scored from the video recording in each trial: i) number of the pellets offered to fish, ii) total number of grasps made by fish during trial, iii) the pellet retention time for each grasp, and iv) number of offered pellets swallowed by fish. The retention time was registered with an accuracy of 0.01 s using a hand stop digital watch “Hanhart Stopstar” (Germany). The moment when the pellet was swallowed was determined by the abrupt termination of the characteristic chewing movements of the fish jaws and the restoration of normal movements of the gill covers. The pellets that were not swallowed were removed from the aquarium immediately after the end of the trial. The experimental period lasted 53 days. In total, 488 trials were performed using the pellets containing the four classical taste substances at different concentrations, 73 trials with the pellets contained the Chironomidae larvae extract and 149 trials were performed with the control pellets. 2.5. Data analysis Statistical analyses were performed using Chi-square and Mann– Whitney tests, t-test, Kruskal–Wallis ANOVA and Spearman rank correlation coefficient (rs). The pellets' consumption was calculated as a ratio of the number of pellets swallowed to the number of pellets offered. For the estimation of taste preference the index of palatability was calculated following the ratio: Ind pal = [(R − C) / (R + C)] × 100, where R and C are the means of consumption of the flavored pellets and of the non-flavored pellets (control) correspondingly [4]. 3. Results Kutum juveniles are agile fish in aquarium conditions. They show both high motor activity and high feeding motivation. The trained fish grasps the offered agar or feed pellets almost immediately in many cases, usually during the first 2–3 s after the pellet was dropped into the water. Agar pellets which were finally refused after one or several repeated grasps sunk to the bottom and did not provoke any interest in the fish. Such pellets were not grasped by fish later. Those trials in which the offered agar pellet did not evoke any response in the fish and was not grasped in the first 30 s were not taken into account. However, in the next trial any offered pellet, agar or feed, was usually grasped by the fish almost immediately. Usually, fish performed 1–2 repeated grasps of the offered pellet during a trial before swallowing or finally refusing it. The mean number of grasps per a pellet varied from 1.02 to 1.44. The mean number of offered pellets and the mean number of grasps per trial (60 s) varied in the ranges 3.0–9.4 and 4.3–9.7, respectively. The most palatable pellets flavored with the Chironomidae larvae extract were swallowed after the first grasp in almost all trials (Table 1). The more pellets were offered the more grasps were performed by the fish during the trials (rs = 0.86***) and the fewer grasps were performed for each offered pellet (rs = − 45*). The rate of grasps was not related to the acceptance ratio and to the retention time both after the first grasp and during the whole trial. The fish retained the pellet in the mouth for 1.0–6.8 s after the first grasp and for 4.0–28.8 s during the whole trial. The retention time both after the first grasp and during the whole trial strongly

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depended on the pellet palatability. These two fish behaviors significantly and positively correlated with the pellets' acceptance ratio and as well as each other (Table 2). 3.1. Palatability of substances The number of pellets swallowed strongly depends on both the substance and its concentration in the agar gel matrix (Table 1). Citric acid significantly increased the pellet consumption within the wide range of concentrations from 0.01 M to 0.52 M. The highest palatability was observed for the concentration of 0.026 M but the effect was two times smaller than that of the Chironomidae larvae extract; the latter increased the pellets consumption up to 98.7%. Citric acid at the lowest concentrations, 0.0026 and 0.0052 M, was ineffective and had no significant effect on the pellets' consumption in relation to control. Calcium chloride, sodium chloride and sucrose at the highest concentrations (0.9, 1.73 and 0.29 M, respectively) significantly decreased the pellets consumption and their acceptance value was 2.6, 3.5 and 6.9 times less than that of the blank pellets (Table 1). 3.2. Threshold concentration and dose–response relationship A significant difference in the consumption of pellets with different concentrations of citric acid (p b 0.01) and of sucrose (p b 0.05) was found (Kruskal–Wallis test). The palatability of pellets increased slightly with diminishing concentration of citric acid, and reached its maximum at 0.026 M and then decreased when the concentration was lowered further down to 0.0026 M. The threshold concentration was 0.01 M for citric acid. The lower concentration of citric acid, 0.0052 M, had no effect on the pellets consumption but still significantly increased the pellet retention time after the first grasp and also during the whole trial (Table 1). The number of pellets swallowed is significantly different (Chi-square test) if the concentration of citric acid changes in 4–5 times (0.13 M versus 0.026 M and 0.01 M versus 0.0026 M; p b 0.001 for both pairs) or even less − 2.6–2 times (0.026 M versus 0.01 M and 0.01 M versus 0.0052 M; p b 0.01 and p b 0.001, respectively). It is noteworthy that there was no significant difference in the pellets acceptances between the four highest concentrations of citric acid (0.52 M, 0.26 M, 0.182 M, and 0.13 M) (Table 3). The dose–response relationship was evident for sucrose and sodium chloride also (Table 4). The consumption of pellets containing sucrose increased abruptly with decreasing the concentration from 0.29 M to 0.145 M. Sucrose at a concentration of 0.029 M was highly palatable and significantly increased the number of pellets swallowed (p b 0.001). Further decreasing of sucrose concentration down to 0.029 M did not change the pellets consumption in relation to control (Table 4). The consumption of pellets shifted dramatically with the decreasing of sodium chloride concentration also. Sodium chloride loses aversive taste but becomes palatable with decreasing its concentration from 1.73 M to 0.173 M. Sodium chloride had no effect in lower concentrations, 0.0865 M and 0.0173 M (Table 1). 4. Discussion 4.1. Taste preferences The present study shows that the classical taste substances which are considered to be sweet, sour, bitter and salty for man are tastants for kutum too. These substances are perceived as different and evoke different taste responses. Citric acid is a taste stimulant for kutum and increases the pellet consumption in the range of concentrations from 0.52 M to 0.01 M. Sucrose, sodium chloride and calcium chloride are taste deterrents at the highest concentrations tested (0.29 M, 1.73 M and 0.9 M, respectively) and significantly decrease the pellet consumption in kutum. Taste preferences to citric acid, sucrose, sodium chloride and calcium chloride are different in many fish species [6]. Among the species

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Table 1 Taste responses of kutum Rutilus frisii kutum to agar pellets containing classical taste substances and the Chironomidae larvae water extract. Data are presented as mean ± SEM. Substance, concentration (M)

Number of pellets offered

Number of pellet grasps

Pellets consumption, %

Index of palatability

Pellet retention time, s: First grasp

All grasps

Number of trials

Citric acid 0.52 0.26 0.182 0.13 0.026 0.01 0.0052 0.0026

57 192 56 124 129 156 118 60

82 228 75 147 150 175 140 85

28.1 ± 4.6** 21.9 ± 2.3** 30.4 ± 6.7*** 26.6 ± 5.2*** 52.7 ± 8.7*** 35.3 ± 8.4*** 13.6 ± 6.2 8.3 ± 3.6

34.1 22.7 37.6 31.7 58.5 43.8 0.7 −24.9

6.8 ± 2.1*** 5.4 ± 0.7*** 6.1 ± 1.7* 3.0 ± 0.6 3.7 ± 0.8 3.1 ± 0.4*** 2.0 ± 0.3* 1.7 ± 0.6

17.9 ± 3.3* 28.8 ± 3.2*** 18.5 ± 4.0* 19.1 ± 3.6 18.6 ± 2.7*** 16.3 ± 1.8*** 16.4 ± 1.4*** 10.6 ± 2.0

19 46 16 32 26 30 20 13

Calcium chloride 0.9 0.45 0.09 0.045

133 90 85 97

164 109 109 125

5.3 ± 3.9* 18.9 ± 7.4 21.2 ± 6 9.3 ± 4.9

−44.5 15.6 21.1 −19.5

1.4 ± 0.2 1.7 ± 0.6 2.2 ± 0.3*** 1.6 ± 0.3

18.4 ± 2.4*** 10.4 ± 2.2 13.8 ± 2.0* 11.9 ± 2.0

20 20 18 18

Sodium chloride 1.73 0.173 0.0865 0.0173

231 83 97 78

269 96 121 93

3.9 ± 2.1*** 24.1 ± 10* 16.5 ± 7.1 9.0 ± 3

−55.9 27.2 8.9 −21.1

1.9 ± 0.2*** 1.9 ± 0.4 2.1 ± 0.6 1.0 ± 0.2

18.0 ± 1.6*** 9.8 ± 2.2 9.8 ± 2.1 4.0 ± 0.8***

37 18 20 20

Sucrose 0.29 0.145 0.029 0.0145 0.0029 Chironomidae, 175a Control

247 92 98 122 96 689 819

326 117 134 139 118 706** 1046

2.0 ± 1.7*** 18.5 ± 8.6 33.7 ± 12.4*** 18.9 ± 5.2 15.6 ± 6.6 98.7 ± 4.4*** 13.8 ± 2.2

−74.7 14.6 41.9 15.6 −6.1 75.5 –

1.6 ± 0.3 1.1 ± 0.3* 2.4 ± 0.5* 1.8 ± 0.3* 1.1 ± 0.3* 4.1 ± 0.1*** 1.4 ± 0.1

13.6 ± 1.6** 6.7 ± 1.6* 14.3 ± 2.0** 13.4 ± 2.0* 7.5 ± 1.7 24.1 ± 0.1*** 8.6 ± 0.5

37 20 19 20 19 73 149

*, **, *** — differences from the control are significant at p b 0.05, p b 0.01 and p b 0.001, respectively (Chi-square and Mann–Whitney tests for “Pellets consumption” and “Pellet retention time” respectively). a The concentration of the Chironomidae larvae water extract is given in g·L−1.

belonging to the genus Rutilus the taste preferences to classical taste substances have been studied for the roach only [18]. Kutum and roach have different taste preferences. In contrast to kutum, both sucrose at 0.29 M and sodium chloride at 1.73 M are palatable for roach, however citric acid at 0.26 M is a highly potent taste deterrent. Calcium chloride is an indifferent taste substance for roach but decreases significantly the pellets' consumption in kutum (Fig. 1). This comparison supports the results obtained earlier which showed that the taste preferences mediated by the oral taste receptors are highly specific in fish and are different even in closely related species [13]. Species specificity in taste preferences is highly important to selective feeding and to decreasing the feeding competition between sympatric fish. Roach and kutum inhabit the same waters in the Caspian Sea basin but these fish are different in their biology and feeding ecology. In rivers, the kutum larvae and fry feed on phyto- and zooplankton, insect larvae and oligochaetes while in later life stages after downstream migration into the sea kutum becomes carnivorous and feed mainly on mollusks, crabs and other benthic invertebrates and some small fish like gobiids [28–31]. Roach is an omnivorous fish but filamentous algae are pronounced components of the roach diet and dominate in roach feeding during late summer and autumn [32–38].

There is no clear concept explaining a fish's taste preference to some substances and the rejection of the others. The taste preference to sucrose seems to be related to fish diets. Usually sucrose is an indifferent taste substance to carnivorous fish but a highly palatable one to herbivorous fish [6]. For example, sucrose has attractive taste not only to roach but also to some other fish like guppy that partly feed on various aquatic plants, and to grass carp C. idella which is an obligate herbivorous fish in adult age [4,18]. At the same concentration (0.26 M) sucrose has indifferent taste to many carnivorous fish like the brown trout, Arctic char Salvelinus alpinus and some others [8,39]. Fish at different life stages can, due to different food choice, be stimulated by different compounds. Older fish have a wider spectrum of effective taste stimuli than the larvae but the taste preferences do not change dramatically with fish development [40,41]. It should be stressed that both kutum and roach had the same life stage when their taste preferences were studied. 4.2. Absolute and differential taste preference thresholds and dose–response relationship The minimal concentration needed to provoke a positive or negative response of a fish towards a given taste substance is an important

Table 2 The Spearman rank correlation coefficients between parameters of kutum Rutilus frisii kutum response to agar pellets containing classical taste substances. Number of pellet grasps

Number of pellets offered (per a trial) Number of pellet grasps Number of pellet grasps (per a pellet) Pellets consumption Retention time for the first grasp

0.57**

Number of pellet grasps (per a pellet)

−0.29 −0.28

*, **, *** — correlations are significant at p b 0.05, p b 0.01 and p b 0.001 respectively (t-test).

Pellets consumption

−0.30 −0.25 −0.25

Retention time: First grasp

All grasps

−0.26 −0.11 −0.10 0.76***

0.09 0.23 −0.23 0.43* 0.77***

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Table 3 The differences in the consumption of pellets with different concentrations of citric acid for kutum Rutilus frisii kutum (Chi-square test).

0.52 M 0.26 M 0.182 M 0.13 M 0.026 M 0.01 0.0052

0.26 M

0.182 M

0.13 M

0.026 M

0.01

0.0052

0.0026

–

– –

– – –

** *** * ***

– ** – – **

* – ** * *** ***

** * ** ** *** *** –

*, **, *** — differences are significant at p b 0.05, p b 0.01 and p b 0.001, respectively.

characteristic of the taste perception. Using the pellets containing different amounts of a substance it was found that the threshold concentration for citric acid is 10−2 M for the gustatory system of kutum. Citrate ion concentrations in many common food organisms for marine and freshwater fish are more than 10−3 M [26]. It was found also that citrate ions enhance gustatory neural responses in fish and mammals to substances like free amino acids. The citrate taste-enhancing effect is likely based on the binding of calcium ions at the surface of taste receptor cell microvilli [42,43]. It was suggested that the citrate taste-enhancing effect probably occur naturally during consummatory feeding behavior in fish like the carnivorous largemouth bass Micropterus salmoides. It was supposed also that the enhancing effect may be based on acting of tricarboxylic citrate ions as a calcium chelator at the surface of the taste receptor cell [43]. The threshold concentration for the effective taste substances is usually around 10−2–10−4 M in many fish species studied [6,16,19,44]. For citric acid, the threshold concentration that significantly increases pellet consumption was slightly lower in common carp Cyprinus carpio (5 · 10− 3 M) and tilapia Tilapia zillii (10− 3–10− 2 M) but somewhat higher in Siberian sturgeon A. baerii and stellate sturgeon A. stellatus (5 · 10−2 M) [4,19,45]. In mammals, the threshold concentration for citric acid is in the same range — (2–5) · 10−3 M in mice and 1.7 · 10− 2 M in man [24,46]. The threshold concentration values depended on the method and parameter used for assessing the fish taste response. Based on the value of the pellet retention time it was found that the citric acid at a concentration lower than 10− 2 M (5.2 · 10−3 M) is effective for kutum taste receptors and shifts the behavioral taste response in relation to the control (Table 1). Electrophysiological methods give much lower threshold concentrations for fish gustatory system, 10−7–10−9 M for some free amino acids [47–50]. On the basis of the threshold concentration (10−2 M) and the volume of pellets used (1.425 mm3) it was calculated that the real amount of citric acid per pellet is 2.74 μg. This amount of citric acid is sufficient for increasing the pellets' consumption in kutum and is closely the

Fig. 1. The palatability index to citric acid 0.26 M (1), calcium chloride 0.9 M (2), sodium chloride 1.73 M (3) and sucrose 0.29 M (4) in kutum Rutilus frisii kutum and roach R. rutilus.

same with that of common carp (3.39 μg) [4]. It is necessary to note that only a small amount of a substance from the thin layer of pellet surface can bind to the taste receptors when fish tests a grasped food item. It means that the real amount of citric acid that is needed to make food palatable for kutum is much less than 2.74 μg. Pellet with sucrose and sodium chloride in a concentration sufficient for eliciting the aversive taste response in kutum (0.29 M and 1.73 M, respectively) contains 141.3 μg and 2465 μg of these substances. The ability of fish to discriminate neighboring concentrations of taste substances is a poorly studied physiological aspect of fish gustatory system. A clearly pronounced decrease in the taste substance effectiveness with increasing its concentration was observed in the behavioral studies of the taste preferences in common carp (citric acid) and European eel Anguilla anguilla (sodium chloride, quinine-hydrochloride, citric acid) [4,40]. According to electrophysiological studies the rate of dose– response function usually raises much faster for highly effective taste substances [51]. The present study shows that the dose–response relationship to citric acid in kutum has a form similar to a parabolic with positive skewness (0.61) and kurtosis (1.01) (Fig. 2). Kutum juveniles consume agar pellets differently if the citric acid concentration changes minimally 2 times – 0.01 M versus 0.0052 M (p b 0.001) or 2.5 times – 0.13 M versus 0.026 M (p b 0.001) and 0.026 M versus 0.01 M (p b 0.01) (Table 3). As it was shown earlier, man has an ability to discriminate by taste sodium chloride solutions between 0.1 and 0.3 M [52]. Rufous hummingbirds Selasphorus rufus distinguished sucrose solutions differing by only 1% at a concentration level of 20%, which

Table 4 The differences in the consumption of pellets with different concentrations of sucrose, sodium chloride or calcium chloride for kutum Rutilus frisii kutum (Chi-square test). Sucrose

Sucrose 0.29 M 0.145 M 0.029 M 0.0145 M

NaCl

0.145 M

0.029 M

0.0145 M

0.0029 M

***

*** *

*** – *

*** – **

NaCl 1.73 M 0.173 M 0.0865 M CaCl2 0.9 M 0.45 M 0.09 M *, **, *** — differences are significant at p b 0.05, p b 0.01 and p b 0.001, respectively.

CaCl2

0.173 M

0.0865 M

0.0173 M

***

*** ***

– *** ***

0.45 M

0.09 M

0.045 M

**

*** –

– – *

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S. Goli et al. / Physiology & Behavior 140 (2015) 111–117

Fig. 2. The dose–response relationship plot to citric acid in kutum Rutilus frisii kutum.

matches sucrose concentrations of hummingbird-pollinated flowers. When concentration levels were raised, the differences necessary to cause a preference increased in curvilinear fashion [53]. Almost the same results were obtained for three species of frugivorous tanagers, flame-crested tanager Tachyphonus cristatus, blue dacnis Dacnis cayana and green honeycreeper Chlorophanes spiza. These birds were able to distinguish between solid diets in which the sugar (sucrose and glucose) content differed by 1% (5% versus 6%). The preferences for a food containing 1% more sugar than the other food depended on the absolute amount of sugar in both foods. When birds were offered a choice between one food containing 12% sugar and the other 13%, birds showed no preference [54]. The results of our study are in accordance with this. Differences of citric acid at high concentration levels (0.52 M– 0.13 M) are not detectable for kutum in contrast to lower concentrations (b 0.026 M). It is noteworthy that there are some substances that not only lose the taste effectiveness and become indifferent with decreasing concentration but also acquire a diametrically opposite taste quality at a low concentration. It was shown for the first time that sucrose and sodium chloride at higher concentrations (0.29 M and 1.73 M) are taste deterrents for kutum and become the taste stimulants at concentrations 10 times lower (0.029 M and 0.173 M). Both substances lose their effectiveness after further gradual decrease in the concentrations. The similar dose–response relationship was found in some other animals also. There is a significant difference between concentrations for the low concentration pairs in sucrose, because more sucrose was consumed by the white-bellied sunbirds, Cinnyris talatala at the higher concentration, but in higher concentration more sucrose was consumed at the lower concentration [55]. Mice showed no particular preference for aqueous solutions of glutathione compared with distilled water in the 2–bottle preference test at low (0.1 and 0.3 mM) and high (30 mM) concentrations, but they preferred the solutions at concentrations ranging from 1 to 10 mM [56]. 4.3. Feeding behavior The time during which fish retain a food item in the mouth before swallowing or rejecting it, is especially of interest [3]. The retention time is spent for the implementation of several consecutive processes: i) reception of the taste substance; ii) transmission of the information in the form of an electrical spike through nerve fibers to the brain gustatory nuclei; iii) decoding of the incoming taste signal and activation of the motoneurons which in turn activate both the palatal and pharyngeal musculatures; and iv) development of the appropriate behavioral response, ingestion or rejection of the grasped food item. In kutum the shortest retention time was around 1.0 s. Most of this time is required for information processing in brain centers and development of behavioral response [57]. The pellets flavored with citric acid and the Chironomidae larvae extract were retained by kutum much for a longer time than all other types of pellets. A positive and highly significant correlation between

the pellets' palatability and the pellets' retention time is characteristic not only for kutum but for roach and for other fish species also [4,18, 58]. It may be supposed that a prolonged retention of palatable food item in the oral cavity allows a more precise assessment of the food before it is being swallowed. In kutum the mean of the pellet retention time varies between 1.0 and 6.8 s which is much longer than the behavioral taste response in human, from 0.4 to 0.8 s (the time between the application of the taste substance on the taste receptors and the time of response) [59]. Unlike the results obtained for human, there is a negative relationship in kutum between the retention time and the taste substance concentration which is typical for other fish also [4,60]. Fish usually swallow or reject the food item after several grasps. Such manipulations or repeated tests of a food item are typical for feeding behavior of fish [61,62]. The mean number of the grasps per pellet is not more than 1.4 in kutum which is less than in many other fish. In riverine fish like the European minnow Phoxinus phoxinus and brown trout, the mean number of the repeated grasps reached 2.1 and 2.6, respectively. Those fish which inhabit slowly moving waters, such as common carp, tench Tinca tinca and guppy, grasp pellets even more often, up to 4–5 times [4,8,18,60]. In kutum, the number of repeated grasps is not related to the pellets palatability as in many other fish. 4.4. Practical applications Patterns of taste preferences in fish are important for the practical use in both aquaculture and fisheries. Palatable substances could be used for improving the attractiveness of artificial feeds, baits and lures. Kutum belongs to fish having a high commercial value in fisheries and aquaculture in the countries around the Caspian Sea [63]. Therefore, the data on taste preferences in kutum may hold a practical significance for the development new feeds and technologies of cultivation. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgments We thank our colleagues Eugene Marusov, Goli Noori, Marziye Abolfathi and Ali Johari for the valuable comments on earlier versions of the manuscript. We also express thanks for Sergey Ostroumov and Andrey Golubev for their help with the English editing of the manuscript. The authors would also like to express their gratitude to the anonymous reviewer for the careful reading of the manuscript and for giving useful suggestions. The study was supported by the Gorgan University of Agricultural Sciences and Natural Resources and by the Russian Foundation for Basic Research (grant Nr. 13-04-00711). References [1] J. Atema, Chemical senses, chemical signals and feeding behaviour in fishes, in: J.E. Bardach, J.J. Magnuson, R.B. May, J.M. Reinhart (Eds.), Fish behaviour and its use in the capture and culture of fishes, ICLARM Conference Proceedings 5, International Center for Living Aquatic Resources Management, Manila, Philippines, 1980, pp. 57–101. [2] A. Gomahr, M. Palzenberger, K. Kotrschal, Density and distribution of external taste buds in cyprinids, Environ. Biol. Fish 33 (1992) 125–134, http://dx.doi.org/10.1007/ BF00002559. [3] T.E. Finger, Sorting food from stones: the vagal taste system in goldfish, Carassius auratus, J. Comp. Physiol. A 194 (2008) 135–143, http://dx.doi.org/10.1007/ s00359-007-0276-0. [4] A.O. Kasumyan, A.M. Morsi, Taste sensitivity of the carp Cyprinus carpio, to free amino acids and classic taste substances, J. Ichthyol. 36 (1996) 391–403. [5] A.O. Kasumyan, S.S. Sidorov, Effects of the long term anosmia combined with vision deprivation on the taste sensitivity and feeding behavior of the rainbow trout Parasalmo (= Oncorhynchus) mykiss, J. Ichthyol. 52 (2012) 109–119, http://dx.doi.org/10.1134/S0032945212010079. [6] A.O. Kasumyan, K.B. Døving, Taste preferences in fish, Fish Fisher. 4 (2003) 289–347, http://dx.doi.org/10.1046/j.1467-2979.2003.00121.x.

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