Effects of dietary l-tryptophan on the agonistic behavior,growth, and survival of freshwater crayfish Astacusleptodactylus Eschscholtz

Muzaffer Mustafa Harlıoglu • Ayse Gul Harlıoglu •

Serpil Mise Yonar • Tuba Cakmak Duran

Received: 10 May 2013 / Accepted: 16 September 2013 / Published online: 22 September 2013� Springer Science+Business Media Dordrecht 2013

Abstract In this study, the effects of dietary tryptophan (a precursor of 5-hydroxytryp-

tamine, 5-HT, serotonin) on the agonistic behavior, growth, and survival of freshwater

crayfish were investigated. For this aim, a control diet (D1) and three experimental diets

(D2, D3, and D4) were prepared. D1 contained no additional tryptophan (TRP), but D2,

D3, and D4 diets were supplemented with TRP at 0.50, 0.75, and 1.00 % in dry diet,

respectively. The control contained 0.33 % TRP. Results revealed that higher supplemental

dietary TRP levels caused a significant decrease in the aggressive behavior (P \ 0.05), but

an increase in the calmness of crayfish. In addition, the results showed that 5-HT levels in

the hemolymph before the fight (after 15 days of feeding; resting) were significantly

different (P \ 0.05) between D1 and D4. There was a significant difference (P \ 0.05) in

the 5-HT level of hemolymph between the crayfish fed control and D4 after 15 days of

feeding. However, after the fight, 5-HT concentration was significantly higher (P \ 0.05)

in TRP-supplemented A. leptodactylus as compared with the control. The findings of this

study showed that supplemental dietary TRP caused a significant increase (P \ 0.05) in the

growth and survival rate of A. leptodactylus. The crayfish fed D4 had the best survival rate

at the end of the experiment (91.60 % in August). The findings of this study also showed

that difference in survival rate between the crayfish fed control and D2 in June, July, and

August was not significant (P [ 0.05). In addition to these, supplemental dietary TRP gave

rise to significantly higher specific growth rate (SGR) values in June and July (P \ 0.05).

For example, in June, it was 0.38 for the crayfish fed D4, 0.28 for the crayfish fed D3, 0.18

for the crayfish fed D2, and 0.13 for the crayfish fed control. However, in August, the

highest SGR (0.56) was obtained from the crayfish fed control. The results also showed

that the SGRs of females were lower than the males in June, July, and August (P \ 0.05).

In conclusion, this research shows that the aggressive behavior of A. leptodactylus can be

suppressed, and rearing efficiency (i.e., increased growth and high survival rate) of this

species can be improved by increasing TRP levels to 1.00 % in its diet.

M. M. Harlıoglu (&) � A. G. Harlıoglu � S. Mise Yonar � T. Cakmak DuranFisheries Faculty, Fırat University, 23119 Elazig, Turkeye-mail: mharlioglu@firat.edu.tr; mharlioglu1@hotmail.com

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Aquacult Int (2014) 22:733–748DOI 10.1007/s10499-013-9702-1

Keywords Crayfish � Tryptophan � Diet � Agonistic behavior � Survival � Growth

Introduction

Agonistic behavior and cannibalism are significant problems in crayfish culture as with

most crustaceans. Grovest (1985) stated that cannibalism is one of the most important

factors affecting the survival rate of crayfish. It can be decreased, but not entirely elimi-

nated, by providing shelter, additional food, and lower stock densities. However, because

crayfish have cannibalistic behavior, they are still cannibalistic under ideal conditions of

shelters, food items, and stock density (Jover et al. 1999; Vergara et al. 2003; Claudia et al.

2004).

Aggressive behavior of crayfish is important especially during molting. During this

sensitive period, their shell is very soft and they are vulnerable to predation attacks

(Lowery 1988). For this reason, in intensive culture of crayfish, the main problems are their

belligerent and combative behavior, and cannibalism features. Thus, high mortality rates

due to cannibalism were reported in juvenile stages in intensive culture experiments with

crayfish (Wickins and Lee 2002).

Some environmental factors, such as light regime, shelter number, stock density, and size

heterogeneity, have been adjusted to prevent cannibalism in crustaceans (Perez et al. 1997;

Wickins and Lee 2002). In addition, a very limited number of reports have been published on

the effects of dietary TRP on the reduction in aggressiveness behavior between individuals of

some farmed fish (i.e., Oncorhynchus mykiss, Aequidens pulcher, Apteronotus leptorhyn-

chus, Epinephelus coioides, Gadus morhua) (Munro 1986; Maler and Ellis 1987) and crab

(Scylla serrata) (Laranja et al. 2010) species. In addition, Peeke et al. (2000) studied the

effects of injected 5-HT in a lobster (Homarus americanus) species.

Furthermore, only a few studies published on the aggression control and mitigate

agonistic behavior in crayfish. For example, the effects of serotonin in fighting Astacus

astacus were investigated by Huber and Delago (1998); the behavioral effects of serotonin

and serotonin agonists in Procambarus clarkii and Orconectes rusticus were investigated

by Tierney and Mangiamele (2001); the behavioral effects of serotonin and serotonin

agonists in P. clarkii and O. rusticus were researched by Tierney et al. (2004); and the

dynamic neurochemical properties in the agonistic behavior of Orconectes rusticus were

investigated by Panksepp et al. (2003). These publications concluded that serotonin

influences aggressive behavior in crustaceans.

Astacus leptodactylus is the native freshwater crayfish species in Turkey. It has eco-

nomic importance and is widely distributed in lakes, ponds, and rivers throughout the

country. Turkey was the largest supplier of this species to Western Europe from 1970 until

1986. The peak production of this species was reached in the early 1980s, with over 5,000

tons being exported in 1984. However, as a result of a fungal disease (Aphanomyces astaci

Schikora), the harvest of A. leptodactylus was diminished severely in most populations in

the country after 1985 (Koksal 1988). So, crayfish catch was forbidden between 1985 and

1990. In 1991, the crayfish yield was only 320 tons (Harlıoglu 2004, 2009). Recent

investigations showed that some populations (i.e., Iznik, Hirfanlı, Egirdir) of A. lepto-

dactylus are still carry A. astaci (Kokko et al. 2012; Svoboda et al. 2012).

Production and farming of aquatic organisms have gained increasing importance in

recent years. For example, the production and culture of sea and freshwater fish in Turkey

have been started approximately 10–15-year ago. Trouts, carps, sea bream, sea bass, and

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mussels are the cultured species (Harlıoglu 2011a,b). On the other hand, the production and

culture of A. leptodactylus by semi-intensive and intensive stocking methods are not

carried out in Turkey, and crayfish production is obtained from the wild populations by

catching. However, to increase crayfish production, many uncontrolled crayfish stockings

were carried out into lakes in Turkey. At present, crayfish production of Turkey is only

10–15 % of the crayfish production obtained in 1980s (before the observation of A. astaci).

This situation confirms that crayfish populations have not recovered for nearly 30 years.

So, they need professional supports to have a full recovery (Harlıoglu 2008). Therefore, it

is clear that production of juveniles under controlled conditions, rearing of juveniles until

determined size for stocking, and stocking of juveniles into freshwater resources come first

in order to increase crayfish production of Turkey.

The aim of this study is to increase rearing efficiency (i.e., survival and growth) of A.

leptodactylus by reducing agonistic and cannibalistic behavior by adding TRP to the diet of

this species. In the literature, no studies have been published on the reduction in aggres-

siveness to increase production rate of A. leptodactylus or any other crayfish species by

supplementing TRP (or any other chemical) to their diet. Therefore, this study also clarifies

the role of supplementary TRP on crayfish growth and aggression.

Materials and methods

Experimental crayfish

The crayfish samples used in the present study were obtained from the Keban Dam Lake

population of A. leptodactylus and were stocked into concrete tanks (7 m 9 4 m 9 0.5 m

or 16 m 9 4 m 9 1 m).

A total of 32 males and 32 females (mean total length = 80 mm (SD = ± 0.9) and

mean body weight = 20 g (SD = ± 1.2) for both sex) in the intermolt stage were used to

observe the effects of dietary TRP on the aggressive behavior and the level of 5-HT

concentration in the hemolymph of A. leptodactylus.

In addition, 336 males and 336 females (mean total length = 80 mm (SD = ± 1.1) and

mean body weight = 17 g (SD = ± 1.3) for both sex) were used to observe the effects of

dietary TRP on the growth and survival rates of A. leptodactylus.

Diet preparation

Four diets were prepared to investigate the effect of different levels of dietary TRP on the

agonistic behavior, growth, and survival of A. leptodactylus. For this aim, one control (D1)

and three experimental diets (D2, D3, and D4) were prepared (Table 1). The practical

control diet used in the present study was modified from Harlıoglu et al. (2012). The basal

diet with 35 % crude protein (CP) has a calculated TRP level of 0.33 % dry diet and

3,600 kcal/kg gross energy. The calculated TRP was based on the TRP content of the

different ingredients used such as casein (1.21 %), gelatin (0.01 %), wheat starch (0.17 %),

and wheat bran (0.25 %) (National Research Council (NRC) 1993). Graded levels of

dietary L-TRP (Merck, Inc., Philippines) were added to the basal diet. By calculation, the

TRP level of the different experimental diets used is 0.33 % (control), 0.5, 0.75, and 1 %

of dry diet.

The crude protein content was analyzed by Kjeldahl’s method; gross energy was cal-

culated based on physiological fuel values of 9 kcal/g for lipid and 4 kcal/g for protein and

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carbohydrate; dry matter after the sample was dried at 105 �C for 6 h; ash content after

24 h at 550 �C in the furnace; and lipid was analyzed by an ether extraction method

(AOAC 1997).

Casein, gelatin, dextrin, wheat starch, a-cellulose, vitamin–mineral mixture, dicalcium

phosphate, sodium phosphate, antioxidant, and sunflower oil were donated by a local food

producer firm (Urun Veren Su Urunleri, Turizm, Medikal, Hayvancılık, Yem Sanayi

Limited Cooperation, Elazıg, Turkey).

The ingredients for each diet were finely grounded (1–4 mm) and then thoroughly

mixed before adding water. Food additives (dicalcium phosphate, sodium phosphate,

antioxidant, vitamin, and minerals) were dissolved in water at 20 �C. This mixture was

added to each homogenated experimental ingredients at 1:1 rate and was cold pelleted

using a laboratory pellet mill, air-dried at 60 �C for up to 12 h, and then was stored in a

refrigerator at 4 �C until further use.

Amount of food to feed crayfish was calculated following equation (National Research

Council (NRC) 1987), and calculated amount of food was offered in four separate meals:

Daily food amount to feed g food=dayð Þ¼ water temperature �C=10ð Þ x crayfish weight=100ð Þ:

During the trial, dissolved oxygen, pH, and water temperature were measured daily.

Ammonia, iron, copper, alkalinity, hardness, calcium, and water flow were measured twice

a week. Mean dissolved oxygen was 7.8 ± 0.7 mg/l; mean ammonia, iron, and copper

content were less than 0.001 mg/l (for each parameter); mean calcium was 42.4 ± 1.8 mg/

l; mean alkalinity was 214.7 ± 3.2 mg CaCO3/l; mean hardness was 33 ± 2 FS8; mean pH

Table 1 Composition of the experimental diets (modified from Harlıoglu et al. 2012)

Food ingredients Experimental diets (%)

D1 (control) Diet D2 Diet D3 Diet D4

Casein (83 % crude protein) 36 36 36 36

Gelatin (83 % crude protein) 6.2 6.2 6.2 6.2

Dextrin 18 18 18 18

Wheat starch 12 12 12 12

Wheat bran 17.62 17.45 17.2 16.95

Sunflower oil 8 8 8 8

Dicalcium phosphate 1.00 1.00 1.00 1.00

Sodium phosphate 0.40 0.40 0.40 0.40

Antioxidanta 0.10 0.10 0.10 0.10

Vitamin mixtureb 0.50 0.50 0.50 0.50

Mineral mixturec 0.18 0.18 0.18 0.18

L-tryptophan – 0.17 0.42 0.67

Calculated L-tryptophan 0.33 0.50 0.75 1

a Antioxidan (mg/kg): Butylated hydroxsytoluene 12.5b Vitamin mixture (IU or mg/kg): vitamin A 2,000,000 IU, vitamin D3 200,000 IU, vitamin E 20,000 IU,vitamin K 3,000 mg, vitamin B1 1,000 mg, vitamin B2 3,000 mg, Niacin 30,000 mg, Calcium D-Pantoth-enat 10,000 mg, vitamin B6 2,000 mg, vitamin B12 4 mg, folic acid 600 mg, D-Biotin 200 mg, colinchloride 100,000 mg, vitamin C 60,000 mgc Mineral mixture (mg/kg dry diet): Mn 80, Fe 35, Zn 50, Cu 5, I 2, Co 0.4, Se 0.15

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was 7.9 ± 0.4 (APHA 1985). Mean water temperature was 19.45 ± 0.84 �C in June,

23.50 ± 0.70 �C in July, and 22.35 ± 0.44 �C in August.

Effects of dietary TRP on the aggressive behavior of A. leptodactylus

Crayfish from the holding tanks were individually stocked in plastic containers

(20 9 22 9 15 cm) and were provided with 2l water and moderate aeration.

Crayfish were set up individually to avoid social contact from conspecifics. Isolation

will increase the crayfish’s aggressiveness and will also standardize their experience prior

to meeting (Laranja et al. 2010). They were divided into four groups based on the four

treatment diets (Control-D1, D2, D3, and D4).

Sixteen replicates per treatment (eight males and eight females) were used to provide

enough crayfish during the fight experiment and hemolymph sampling (16 9 4 = 64 in

total) to observe the effects of dietary TRP on the aggressive behavior of A. leptodactylus.

Crayfish were fed four times daily at 800, 1200, 1600, and 2000 hours for one month before

they were used in the fight experiment. Excess feeds were siphoned out from the bottom of the

plastic containers, and 60–70 % of water was replaced daily prior to feeding. Natural photo-

period was applied during the studies. The water temperature ranged from 13 to 15 �C.

After 1 month, size-matched crayfish were placed for fighting in an aquarium

(20 9 20 9 30 cm) divided equally into two compartments with a removable layer. For

nearly 5 mins, crayfish were in the aquarium before the divider was removed. The divider

was taken out when the fight was about to begin, and calmness, fights, and agonistic

behavior of crayfish were recorded for 1 hour by use of a video camera. In addition,

photographs of important moments during the test were taken by a photo camera (Sony,

HDR-CX115E). Later, frequency of calmness, attacks, number of retreats, and intensity of

attacks were analyzed in the laboratory by the use of laptop software (Asus K53S Windows

7 Home Basic).

As meeting of crayfish progressed, the intensity of the calmness and attacks was scored

according to the following criteria (modified from Hubert et al. 1997; Laranja et al. 2010):

(1) Calmness—both crayfish are stationary, walking slowly or do not attack other

crayfish when walking, do not strike/attack the opponent using its claws at all, staying far

away from other crayfish,

(2) Attacking—crayfish strikes/attacks the opponent using its claws,

(3) Claw lock—crayfish grabs and locks the opponent using its claws,

(4) Strike and rip—crayfish makes unrestrained use of claws resulting to ripping or

tearing off an opponent’s appendages.

The rate (%) of a behavior was determined by how long a behavior occurred in 1 hour.

Water in the aquarium was replaced with new water prior to the next fight to avoid the

presence of chemical signals from previous pair of crayfish samples which may influence

the behavior and performance of the succeeding pair of crayfish (Moore and Bergman

2005; Laranja et al. 2010).

Effects of dietary TRP on the level of 5-HT concentration in the hemolymph of A.

leptodactylus

To investigate the effects of TRP added to diets on the level of 5-HT concentration in

crayfish hemolymph, the hemolymph samples of crayfish fed diets (D1, D2, D3, and D4)

were examined. For this aim, hemolymph samples were taken from rested crayfish at the

15th and 30th days of feeding, and after crayfish fought.

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At least 100 ll volume of hemolymph was sampled from the heart or at the base of the

walking legs of the crayfish using a sterile BD PrecisionGlideTM 1 ml syringe (26 G �;

0.45 9 13 mm). To prevent the oxidation of 5-HT, the hemolymph samples of crayfish

were maintained in an Eppendorf tube containing 1 % sodium citrate as anticoagulant (1:1)

and 1 % ascorbic acid solution (1:10). The hemolymph was centrifuged at 1,600 rpm for

10 min, and the resultant plasma was kept at -70 �C until needed. The quantification of

5-HT was carried out using the HPLC (Shimadzu, Kit: Immuchorm, mg/dl) by the Bio-

chemistry Laboratory of Fırat University, Elazıg, TURKEY.

Effects of dietary TRP on the growth and survival rates of A. leptodactylus

Crayfish were reared for 3 months in summer (June, July, and August in which the most

frequent moltings occur) in 2 9 2 9 0.5 m concrete tanks with a stocking density of 14

ind/m2 (1 male; 1 female) (56 crayfish/tank). Crayfish were fed the experimental diets

containing different TRP levels in four rations daily (800, 1200, 1600, and 2000 hours).

Studies were applied in three replicates, and a total of 12 concrete tanks were used.

Survival rate and SGR of crayfish fed experimental diets during the 3 months were

assessed monthly at the end of the 3 months. SGR was calculated with the following

formula (Reynolds 2002)

SGR ¼ ðln Wtð Þ � ln WIÞ� �

X 100=T,

where Wt = weight at time t (g), WI = initial weight (g), and T = time (days).

Sixty plastic pipes (15 cm in length and 4 cm in diameter) were provided as shelters for

the crayfish in each tank. Supplemental water flow was 1.5 l/s per 1 square meter of tank.

Results

Effects of dietary TRP on the aggressive behavior of A. leptodactylus

The results revealed that higher supplemental dietary TRP levels caused an increase in the

calmness of crayfish and caused a significant decrease in the aggressive behavior of

crayfish (two-way MANOVA, P \ 0.05).

It was found that the males and females fed control diet (D1) were significantly more

aggressive than the males and females fed D2, D3, and D4 (One-way ANOVA, P \ 0.05).

It was also found that although the aggression of males and females fed D2 and D3 was

similar, the males and females fed D2 were significantly more aggressive than those of

crayfish fed D4 (two-way MANOVA, Tukey test, P \ 0.05) (Figs. 1, 2, 3, 4).

Similarly, as regarding the intensity (number, in percentage) of ‘‘claw lock’’ and ‘‘strike

and rip,’’ the males and females fed D1 were significantly (two-way MANOVA, P \ 0.05)

more than the males and females fed D2, D3, and D4. Moreover, the results showed that

although the intensities of ‘‘claw lock’’ and ‘‘strike and rip’’ in the males and females fed

D2 and D3 were similar, the intensities of ‘‘claw lock’’ and ‘‘strike and rip’’ in the males

and females fed D2 were significantly higher than crayfish fed D4 (two-way MANOVA,

P \ 0.05) (Figs. 1, 2 3, 4).

In addition, it was found that there was a significant difference in the calmness of

crayfish. For example, the males and females fed D2, D3, and D4 were significantly more

calm in comparison with the males and females fed D1 (two-way MANOVA, P \ 0.05).

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On the other hand, the results showed that there was a significant difference between the

males and females in the aggressiveness in the crayfish fed D1, D2, D3, and D4 (two-way

MANOVA, P \ 0.05). The aggressiveness of females was lower than the males.

Effects of dietary TRP on the level of 5-HT concentration in the hemolymph of A.

leptodactylus

The results showed that an increase in TRP level of diets caused an increase in the 5-HT

level of crayfish hemolymph. However, the results showed that 5-HT levels in the

hemolymph before the fight (after 15 days of feeding, in resting crayfish) were

Fig. 1 a Rate (%) of calmness during 1 h performed by the males fed D1 and the females fed D2, D3, andD4 b Rate (%) of attacking during 1 h performed by the males fed D1 and the females fed D2, D3 and D4c Rate (%) of claw lock during 1 h performed by the males fed D1 and the females fed D2, D3, and D4d Rate (%) of strike and rip during 1 h performed by the males fed D1 and the females fed D2, D3, andD4 ± standard error. Asterisk (*) indicates significant difference (P \ 0.05) from the control by two-wayMANOVA test

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significantly different (P \ 0.05) between D1 and D4. In addition, there was a significant

difference (One-way MANOVA P \ 0.05) in the 5-HT level of hemolymph between the

crayfish fed control and D4 after 15 days of feeding (Fig. 5).

Furthermore, the results showed that following the competition test carried out after

30 days of feeding, there was a reduction in the 5-HT level of hemolymph in the control

group, but an increase in the TRP level of diets caused a significant increase (One-way

MANOVA, P \ 0.05) in the 5-HT level of hemolymph in the crayfish fed D2, D3, and D4

(Figs. 6, 7).

The results also showed that there was not a significant difference in the 5-HT level of

hemolymph between the males and females in the crayfish fed D1, D2, D3, or D4 (One-

way MANOVA, P [ 0.05).

Fig. 2 a Rate (%) of calmness during 1 h performed by the males fed D1 and the males fed D2, D3, and D4b Rate (%) of attacking during 1 h performed by the males fed D1 and the males fed D2, D3, and D4 c Rate(%) of claw lock during 1 h performed by the males fed D1 and the males fed D2, D3, and D4 d Rate (%) ofstrike and rip during 1 h performed by the males fed D1 and the males fed D2, D3, and D4 ± standard error.Asterisk (*) indicates significant difference (P \ 0.05) from the control by two-way MANOVA test

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Effects of dietary TRP on the survival rates and growth of A. leptodactylus

Survival rates

The survival rates (%) of the crayfish fed control (D1), D2, D3, and D4 in June, July, and

August are given in Table 2.

The best survival rate was obtained from the crayfish fed D4. It was 91.60 % in August.

That of the crayfish fed control, D2, and D3 was 78.89, 81.25, and 85.85, respectively, in

August.

Fig. 3 a Rate (%) of calmness during 1 h performed by the females fed D1 and the females fed D2, D3, andD4 b Rate (%) of attacking during 1 h performed by the females fed D1 and the females fed D2, D3, and D4c Rate (%) of claw lock during 1 h performed by the females fed D1 and the females fed D2, D3, and D4d Rate (%) of strike and rip during 1 h performed by the females fed D1 and the females fed D2, D3, andD4 ± standard error. Asterisk (*) indicates significant difference (P \ 0.05) from the control by two-wayMANOVA test

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There was not a significant difference in the survival rate between the crayfish fed

control and D2 in June, July, and August (two-way ANOVA, P [ 0.05). However, the

survival rate of crayfish fed D4 was significantly higher than the crayfish fed control and

D2 (two-way ANOVA, P \ 0.05).

In addition, the increase in survival rate between D2 and D3, and between D3 and D4

was not significant (two-way ANOVA, P [ 0.05).

The results also showed that the survival rate of females was lower than the males in

June, July, and August (One-way MANOVA, P \ 0.05).

Fig. 4 a Rate (%) of calmness during 1 h performed by the females fed D1 and the males fed D2, D3, andD4 b Rate (%) of attacking during 1 h performed by the females fed D1 and the males fed D2, D3, and D4c Rate (%) of claw lock during 1 h performed by the females fed D1 and the males fed D2, D3, and D4d Rate (%) of strike and rip during 1 h performed by the females fed D1 and the males fed D2, D3, andD4 ± standard error. Asterisk (*) indicates significant difference (P \ 0.05) from the control by two-wayMANOVA test

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Fig. 5 5-HT level of crayfish hemolymph after 15 days of feeding. Values are mean ± standard error. a,b

Values with different letters are significantly different (P \ 0.05) by one-way MANOVA test

Fig. 6 5-HT level of crayfish hemolymph after 30 days of feeding. Values are mean ± standard error. a,b

Values with different letters are significantly different (P \ 0.05) by one-way MANOVA test

Fig. 7 5-HT level of crayfish hemolymph after 30 days of feeding. Values are mean ± standard error.a,b,c,d Values with different letters are significantly different (P \ 0.05) by one-way MANOVA test

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Growth

The SGRs of the crayfish fed control (D1), D2, D3, and D4 in June, July, and August were

given in Table 3.

In June and July, the increased levels of TRP in diets (D4, D3, and D2) caused a

significant increase in SGR of crayfish (two-way ANOVA, P \ 0.05). For example, in

June, it was 0.38 for the crayfish fed D4, 0.28 for the crayfish fed D3, 0.18 for the crayfish

fed D2, and 0.13 for the crayfish fed control. Those of July was 0.24 for D4, 0.21 for D3,

0.16 for D2, and 0.13 for control, respectively.

However, in August, the highest SGR (0.56) was obtained from the crayfish fed control.

It was 0.50 for the crayfish fed D2, 0.39 for the crayfish fed D3, and 0.48 for the crayfish

fed D4.

The results also showed that the SGRs of females were lower than the males in June,

July, and August (One-way MANOVA, P \ 0.05).

Discussion and conclusion

Crayfish must molt or shed its hard external shell (exoskeleton) to increase in size as with

all crustaceans. Thus, this growth process includes periodic molting interspersed with inter-

molt periods (Lowery 1988; Aiken 1992). Molt-associated cannibalism is a major cause of

mortality in cultured crustaceans (Wickins and Lee 2002). For example, in a study on the

growth, mortality, and molting rate of noble crayfish, Astacus astacus juveniles in aqua-

culture experiments, Taugbøl and Skurdal (1992) found that cannibalism caused 68–90 %

of mortality.

Cannibalism in crustaceans was decreased somewhat by reducing stocking density,

stocking similar size individuals, increasing food availability and shelters, and maintaining

Table 2 The survival rates (%) of the crayfish fed control (D1), D2, D3, and D4 in June, July, and August

June July August

Control (D1) Female 65.47 ± 5.02 70.91 ± 7.21 76.92 ± 5.36

Male 72.61 ± 5.88 77.05 ± 6.47 80.85 ± 4.86

Mean 69.04 ± 5.45x,a 73.98 ± 6.84x,y,a 78.89 ± 5.11y,a

D2 Female 64.28 ± 7.54 75.93 ± 5.11 80.49 ± 5,02

Male 75.00 ± 6.72 79.37 ± 5.43 82.00 ± 4.32

Mean 69.64 ± 7.13x,a 77.65 ± 5,27x,y,a 81.25 ± 4.67y,a

D3 Female 78.57 ± 6.33 83.33 ± 5.79 83.64 ± 4.43

Male 92.85 ± 8.55 85.90 ± 5.23 88.06 ± 5.01

Mean 85.71 ± 7.44x,b 84.62 ± 5.51x,b 85.85 ± 4.72x,a,b

D4 Female 88.09 ± 5.82 82.43 ± 8.32 88.52 ± 6.49

Male 95.23 ± 8.12 93.75 ± 7.64 94.60 ± 6.17

Mean 91.66 ± 6.97x,b 88.09 ± 7.98x,b 91.60 ± 6.33x,b

Values are mean ± standard errorx,y The groups in the same rows with different letters are statistically significant (P \ 0.05) by two-wayANOVA testa,b The groups in the same columns with different letters are statistically significant (P \ 0.05) by two-wayANOVA test

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individuals in separate containers. For example, Jover et al. (1999) found lower cannibalism

rate in P. clarkii for wider than 70 cm2/individual stocking density. In addition, survival rate

was higher when they were stocked at 250 cm2/individual (Jover et al. 1999). Similarly,

lower cannibalism rate was observed by decreasing stocking density in Cherax quadrica-

rinatus by Vergara et al. (2003) and in Procambarus llamasi by Claudia et al. (2004).

However, except our study and a study by Laranja et al. (2010) on the effects of dietary

TRP on the agonistic behavior, growth, and survival of juvenile mud crab Scylla serrata,

no studies have been carried out on reduction in cannibalism in crustaceans by supple-

menting TRP or any other chemical to their diet. Laranja et al. (2010) investigated the

effects of feeding formulated diet with different TRP levels (0.32 % as control, 0.5, 0.75,

and 1 % of dry diet) on the agonistic behavior, growth, and survival of juvenile mud crab.

Laranja et al. (2010) stocked crab samples individually and fed the experimental diets

for 4 weeks before they were set to a 1-hour fight experiment. A video camera was used to

record fights, and the aggressiveness of the crabs was quantified. In addition, they sampled

hemolymph after 15 and 30 days of feeding (when crayfish rest) and right after the fight to

measure circulating 5-HT concentration. At the end of the experiments, Laranja et al.

(2010) found that higher TRP levels suppressed the aggressiveness of mud crab in a dose-

dependent manner. They also found that the intensity and frequency of attacks were both

significantly lower (P \ 0.05) in those given diets containing 0.75 and 1 % TRP compared

with control. Furthermore, the results of Laranja et al. (2010) showed that 5-HT levels in

the hemolymph before the fight (after 15 and 30 days, when crayfish rest) were not signif-

icantly different between treatments, but after the fight, 5-HT concentration was significantly

higher in TRP-supplemented mud crabs compared with control (0.5 % = P \ 0.05; 0.75 %

and 1 % = P \ 0.01).

Similar to Laranja et al.’s (2010) findings, supplemental dietary TRP gave rise to an

increase in the calmness of A. leptodactylus and caused a significant diminish in the

Table 3 SGR of the crayfish fed control (D1), D2, D3, and D4 in June, July, and August

June July August

Control (D1) Female 0.1 ± 0.03 0.03 ± 0.01 0.45 ± 0.03

Male 0.16 ± 0.01 0.22 ± 0.01 0.67 ± 0.05

Mean 0.13 ± 0.02x,a 0.13 ± 0.01x,a 0.56 ± 0.04y,a

D2 Female 0.1 ± 0.01 0.06 ± 0.01 0.38 ± 0.04

Male 0.26 ± 0.04 0.25 ± 0.03 0.61 ± 0.05

Mean 0.18 ± 0.03x,b 0.16 ± 0.02y,b 0.50 ± 0.05z,b

D3 Female 0.16 ± 0.02 0.12 ± 0.01 0.32 ± 0.04

Male 0.40 ± 0.02 0.29 ± 0.02 0.45 ± 0.03

Mean 0.28 ± 0.02x,c 0.21 ± 0.02y,c 0.39 ± 0.04z,c

D4 Female 0.23 ± 0.01 0.19 ± 0.01 0.32 ± 0.03

Male 0.53 ± 0.02 0.29 ± 0.01 0.64 ± 0.04

Mean 0.38 ± 0.02x,d 0.24 ± 0.01y,d 0.48 ± 0.03z,b

Values are mean ± standard errorx,y,z The groups in the same rows with different letters are statistically significant (P \ 0.05) by two-wayANOVA testa,b,c The groups in the same columns with different letters are statistically significant (P \ 0.05) by two-wayANOVA test

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aggressive behavior of this species in the present study. Our study showed that an increase

in dietary TRP caused an increase in the 5-HT level in crayfish hemolymph. However,

there was not a significant difference in the 5-HT level of hemolymph between the crayfish

fed control, D2, and D3 after the 15 days of feeding, and the difference in the 5-HT level of

hemolymph between the crayfish fed control and D4 after 15 days of feeding was sig-

nificantly different. In addition, the results revealed that following the competition test

carried out after 30 days of feeding, there was a reduction in the 5-HT level of hemolymph

in the control group, but an increase in the TRP level of diets caused a significant increase

in the 5-HT level of hemolymph in the crayfish fed D2, D3, and D4.

The calming effect of TRP was observed in a research on the effect of exogenous TRP

on the cannibalism, survival, and growth of fish species, Epinephelus coioides by Hseu

et al. (2003). They found that the cannibalism rate was significantly decreased after feeding

formulated diets containing 0.75 to 1.5 % TRP. In addition to Hseu et al. (2003)’s study,

calming effect of dietary TRP was also observed in rainbow trout (Oncorhynchus mykiss)

after feeding formulated diets containing 8.75 and 74.3 mg/g TRP in dry diet by Winberg

et al. (2001) and in Atlantic cod (Gadus morhua) after feeding 2.8 % TRP by Hoglund

et al. (2005). These observations suggest that the ability of higher TRP to suppress

aggression is due to the increased activity of 5-HT in the brain and affects the locomotor

activity in these species. These findings were also supported by Tierney and Mangiamele

(2001) who found that increasing level of circulating 5-HT through injection reduces the

level of aggression in crayfish Procambarus clarkii. Therefore, it was stated that circu-

lating 5-HT influences aggressive behavior in crustaceans.

The present study showed that inclusion of TRP to the diet of A. leptodactylus caused

significantly higher survival rates. For example, in August, the survival rate of A. lepto-

dactylus fed control was 78.89 %, while that of A. leptodactylus fed D4 was 91.6 %. In

addition, the survival rate of crayfish fed D4 was significantly higher than the crayfish fed

control and D2 (P \ 0.05). In a similar manner, Laranja et al. (2010) also obtained higher

survival in TRP-supplemented mud crabs (0.5 = 35 %, 0.75 = 33.33 %, 1 = 35 %)

compared with control (18.33 %) after the survival experiments. This higher survival rate

was attributed to the inclusion of TRP to the diet of this species.

In the present study, the results revealed that the increased levels of TRP in diets caused

an important increase in SGR of A. leptodactylus in June and July. However, Laranja et al.

(2010) found opposite results to our study. They found that the daily growth gain, relative

growth rate, and specific growth rate in S. serrata were reduced in TRP-supplemented

groups than with the control group.

The optimum TRP demand of A. leptodactylus has not been investigated, but related

studies on some other crustaceans like shrimp Penaeus monodon revealed an optimum

TRP requirement of 0.2 % dry diet or 0.5 % protein (Millamena et al. 1999). Likewise, a

0.8 TRP (in % protein) in shrimp diet was recommended by Akiyama et al. (1991). In the

present study, basal diet had a TRP level of 0.33 in % dry diet or 0.94 in % protein and

was very close to the above-suggested TRP levels for shrimps.

In conclusion, the inclusion of TRP to the diet of A. leptodactylus significantly increased

the circulating 5-HT and significantly suppressed the agonistic behavior of this species.

Thus, the survival of A. leptodactylus was enhanced. Therefore, diminishing cannibalism

may cause better survival, higher growth rate, and more successful A. leptodactylus

rearing.

Acknowledgments This study was carried out as a part of a research Project, ‘‘An investigation on theeffects of dietary l-tryptophan on the agonistic behavior, growth, and survival of freshwater crayfish Astacus

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leptodactylus Eschscholtz’’ supported by the Scientific and Technological Research Council of TURKEY(TUBITAK-TOVAG, Project No: 111O418).

References

Aiken DE, Waddy SL (1992) The growth process in crayfish. Rev Aquat Sci 6(3,4):335–381Akiyama DM, Dominy WG, Lawrence AL, (1991) Penaeid shrimp nutrition for the commercial feed

industry revised. In: Akiyama, DM, Tan RKH (Eds.) Proceedings of the aquaculture feed processingand nutrition workshop, Thailand and Indonesia Sept. 19–25. American Soybean Association, Sin-gapore, pp. 80–98

American Public Healt Association (APHA) (1985) Standart methods for the examination of water andwastewater, 16th. edn. 1268 pp. Washington

AOAC (1997) Official methods of analysis of association of official analytical chemists, 16th edn. AOAC,Arlington, p 1298

Claudia CO, Miguel RS, Angela ONM, Yurrita PJG (2004) Effect of density and sex ratio on gonaddevelopment and spawning in the crayfish P. llamasi. Aquaculture 236:331–339

Grovest RE, (1985) The crayfish: ıts nature and nurture fishing new boks Ltd. England, pp:72Harlıoglu MM (2004) The present situation of freshwater crayfish, Astacus leptodactylus (Eschscholtz,

1823) in Turkey. Aquaculture 230:181–187Harlıoglu MM (2008) The harvest of the freshwater crayfish Astacus leptodactylus Eschscholtz in Turkey:

harvest history, impact of crayfish plague, and present distribution of harvested populations, Aquacult.Int 16:351–360

Harlıoglu MM (2009) A comparison of the growth and survival of two freshwater crayfish species, Astacusleptodactylus Eschscholtz and Pacifastacus leniusculus (Dana) under different temperature and densityregimes. Aquacult Int 17:31–43

Harlıoglu AG (2011a) Present status of fisheries in Turkey. Rev Fish Biol Fisheries 21:667–680Harlıoglu, A.G., 2011b. Problems and solutions in the aquaculture sector in Turkey, World Aquaculture,

42(4):15–16, 69–71Harlıoglu MM, Koprucu K, Harlıoglu AG, Yılmaz O, Aydın S, Mise Yonar S, Cakmak Duran T, Ozcan S

(2012) The effects of dietary n-3 series fatty acid on the fatty acid composition, cholesterol and fat-soluble vitamins of pleopadal eggs and stage 1 juveniles in a freshwater crayfish, Astacus leptodactylus(Eschscholtz). Aquaculture 356–357:310–316

Hoglund E, Bakke MJ, Øverli Ø, Winberg S, Nilsson GE (2005) Suppression of aggressive behaviour injuvenile Atlantic cod (Gadus morhua) by l-tryptophan supplementation. Aquaculture 249:525–531

Hseu J.R., Lu F.I., Su H.M., Wang L.S., Tsai C.L., Hwang P.P., 2003. Effect of exogenous tryptophan oncannibalism, survival and growth in juvenile grouper, Epinephelus coioides. Aquaculture, 1–12

Huber R, Delago A (1998) Serotonin alters decisions to withdraw in fighting crayfish, Astacus astacus: themotivational concept revisited. J Comp Physiol 182A:573–583

Hubert, R., Smith, K., Delago, A., Isaksson, K., Kravitz, E., 1997. Serotonin and Aggressiveness Motivationin Crustaceans: Altering the Decision to Retreat. Proceedings of the National Academy of ScienceUSA 5939–5942

Jover M, Carmona JF, Del Rıo MC, Solee M (1999) Effect of feeding cooked-extruded diets, containingdifferent levels of protein, lipid and carbohydrate on gowth of red swamp crayfish (P. clarkii).Aquaculture 178:127–137

Kokko H, Koistinen L, Harlioglu MM, Makkonen J, Aydın H, Jussila J (2012) Recovering Turkish narrowclawed crayfish (Astacus leptodactylus) populations carry Aphanomyces astaci. Knowl ManagtAquatic Ecosyst 404:12

Koksal G (1988) Astacus leptodactylus in Europe. In: Holdich DM, Lowery RS (eds) Freshwater Crayfish:Biology. Croom Helm, London and Timber Press, Oregon, Management and Exploitation, pp 365–400

Laranja JLQ, Quinitio ET, Catacutan Mae R, Relicardo MC (2010) Effects of dietary l-tryptophan on theagonistic behavior, growth and survival of juvenile mud crab Scylla serrata. Aquaculture 310(1–2):84–90

Lowery, R.S., 1988. Growth, moulting and reproduction, In: : Holdich D.M., Lowery R.S. (Eds.), Fresh-water Crayfish: Biology, Management and Exploitation, Croom Helm. London and Timber Press,Oregon, pp: 83–114

Maler L, Ellis WG (1987) Inter-male aggressive signals in weakly electric fish are modulated by mono-amines. Behav Brain Res 25:75–81

Millamena OM, Teruel MB, Kanazawa A, Teshima S (1999) Quantitative dietary requirements of postlarvaltiger shrimp, Penaeus monodon, for histidine, isoleucine, leucine, phenylalanine and tryptophan.Aquaculture 179:169–179

Aquacult Int (2014) 22:733–748 747

123

Moore PA, Bergman DA (2005) The smell of success and failure: the role of intrinsic and extrinsic chemicalsignals on the social behavior of crayfish. Integr Comp Biol 45:650–657

Munro AD (1986) Effects of melatonin, serotonin, and naloxone on aggression in isolated cichlid fish(Aequiidens pulcher). J Pineal Res 3:257–262

National Research Council (NRC) (1987) Predicting feed intake of food-producing animals subcommitteeon feed ıntake committee on animal nutrition board on agriculture. National Academy Press, Wash-ington, D.C

National Research Council (NRC), 1993. Nutrient Requirements of Fish. National Research Council (NRC),Washington, D.C. National Academy Press. 114 p

Panksepp JB, Yue Z, Drerup C, Huber R (2003) Amine neurochemistry and aggression in crayfish. MicroscRes Tech 60:360–368

Peeke HVS, Blank GS, Figler MH, Chang ES (2000) Effects of exogenous serotonin on a motor behaviorand shelter competition in juvenile lobster (Homarus americanus). J Comp Physiol A 186:575–582

Perez JR, Carral JM, Celada M, Saez-Royuela M, Sierra A (1997) Current status of astaciculture productionand commercial situation of crayfish in Europe. Aquacult Europe 22(1):6–13

Reynolds, J.R., 2002. Growth and reproduction, In: Holdich D.M. (Ed.) Biology of Freshwater Crayfish.Lowa State University Press

Svoboda J, Kozubıkova E, Kozak P, Kouba A, Bahadir Koca S, Diler O, Diler I, Policar T, Petrusek A(2012) PCR detection of the crayfish plague pathogen in narrow-clawed crayfish inhabiting LakeEgirdir in Turkey. Dis Aquat Organ 98:255–259

Taugbøl T, Skurdal J (1992) Growth, mortality and molting rate of noble crayfish, Astacus astacus L.,juveniles in aquaculture experiments. Aquacult Fisher Manage 23:411–420

Tierney AJ, Mangiamele LA (2001) Effects of serotonin and serotonin analogs on posture and agonisticbehavior in crayfish. J Comp Physiol A 187:757–767

Tierney AJ, Greenlaw MA, Dams-O’Connor K, Aig SD, Perna AM (2004) Behavioral effects of serotoninand serotonin agonists in two crayfish species, Procambarus clarkii and Orconectes rusticus. CompBiochem Physiol A 139:495–502

Vergara MPH, Rouse DB, Novoa MAO, Davis DA (2003) Effects of dietary lipid level and source on gowthand proximate composition of juvenile redclaw (C. quadricarinatus) reared under semi-intensiveculture conditions. Aquaculture 223:107–115

Wickins, J.F., Lee, D.O’C., 2002. Crustacean Farming, Ranching and Culture. Blackwell Science, pp: 446Winberg S, Øverli Ø, Lepage O (2001) Suppression of aggression in rainbow trout (Oncorhynchus mykiss)

by dietary L-tryptophan. J Exp Biol 204(22):3867–3876

748 Aquacult Int (2014) 22:733–748

123