EVALUATION OF IMMOBILIZING METHODS FOR THE CHINESE MITTEN CRAB, ERIOCHEIR SINENSIS (MILNE-EDWARDS)

Grzegorz Jan Hajek, Marcin Choczewski, Robert Dziaman, Bernard Kłyszejko
Division of Fish Physiology, West Pomeranian University of Technology, Szczecin, Poland

ABSTRACT

This study investigated the efficacy of eight chemicals administered via bath and cooling to immobilize the Chinese mitten crab. Isobutyl alcohol, isoflurane, 2-phenoxyethanol, Propiscin, at the concentration of 20 mLL^-1 and MS-222 at the concentration of 1000 mgL^-1 had no anaesthetic effect on crabs for 30 min of exposure. Clove oil was only partially effective at the concentration of 20 mLL^-1.The best results were obtained when chloroform (1.5 mLL^-1) and diethyl ether (10 mLL^-1) were used, causing anaesthesia of all crabs within 30 min. The shortest induction time (about 5 min) and rapid recovery (about 1 min) was obtained by cooling. From the tested methods the usage of chloroform anaesthesia and cooling seem to be most suitable in the Chinese mitten crab.

Key words: anaesthesia, Eriocheir sinensis, immobilizati, mitten crab.

INTRODUCTION

Invasive species disturb original ecological balance, drawing special attention of ecologists and scientists of other disciplines. One of such invaders in Europe and North America is the Chinese mitten crab Eriocheir sinensis Milne-Edwards, 1854. The area of its origin is Eastern Asia, specially the Yellow Sea region [2]. Since it was first recorded in the German river Aller in 1912, it has colonized several costal regions, rivers and lakes in Europe. The conquest of North America began in 1965 [12]. In Poland it was first recorded in 1928 [7]. Nowadays, being regularly met in the Baltic Sea and the Oder River, it gains the position of the biggest Polish crustacean [4].

Anyone trying to take a close look at a mitten crab admits that it is much more difficult for handling than, for example, a crayfish. It has strong claws, sharp edges of the carapace and legs that are used to counteract the hand grab. Crabs can also move fast on the ground outside the water, literally escaping from the researcher. Therefore, because of its defense potential and mobility, for some procedures, harmless immobilization would be helpful. Another reason for developing an immobilization method is reducing stress in the examined animals.

One of the commonly used methods of immobilizing animals is anaesthesia. Anaesthetics used in aquaculture are intended to limit stress reactions of fish, facilitating various handling procedures, such as weighing, sorting, collection of spawning material, tagging, or veterinary treatment [16,15]. The most widely used anaesthetics include MS-222 (tricaine methanesulphonate), benzocaine, 2-phenoxyethanol, etomidate, metomidate, quinaldine, quinaldine sulphate and clove oil. The most popular way of drug administration is preparing a bath solution.

The usage of anaesthetics in crustaceans is much less examined and practiced. From a sparse documentation it can be concluded that the agents commonly used in fish (except the clove oil) are rather ineffective in crustaceans [1,5,6,10,13]. The best effect in lobster Homerus americanus (Milne-Edwards) was obtained when isobutyl alcohol, methyl pentynol [5] or eugenol [18] were used. Lidocaine-HCl and ketamine-HCl were efficacious for crayfish Orconectes virilis (Hagen) [1], while clove oil and diethyl ether took effect in Cherax destructor (Clark) [10]. Considering crabs, for Cancer pagurus L. and Carcinus maenas L. xylazine-HCl and procaine-HCl produced the best results [13]. Also Gardner [6] recommended xylazine-HCl and ketamine-HCl injections and a clove oil bath for the Australian giant crab Pseudocarcinus gigas (Lamarck). Morgan et al. [11] tested clove oil in three crab species and found it effective but the sensitivity to the agent was much different among the species. Clove oil was also suitable for prawns [3,14] and for Gammarus sp. [17].

The aim of this short investigation was to evaluate the anaesthetic properties of eight chemicals administered in a bath and cooling for the Chinese mitten crab, E. sinensis.

MATERIAL AND METHODS

This study involved 103 mitten crabs having an average weight of 185.8 ± 55.5 g (mean ±SD), 50 males and 53 females, collected by commercial fishers from Lake Dąbie on November 2007 and 2008. Crabs were acclimated in a 1000-L common tank with 3 m² bottom, partly covered with black PVC foil, in aerated tap water, at the temperature of 20 ± 1°C, fed with commercial fish food. Two days before the experiment the crabs were transferred to a 100-L aquarium and not fed.

The induction and recovery were carried out in two aquaria with aerated tap water at 20 ± 1°C. After the experiments the crabs were transferred to a 100-L tank and observed for 10 days.

Chemicals and concentrations used:

• Aerrane (isoflurane) – Baxter S.A., Lessines, Belgium – 20 mLL^-1,

• chloroform – CHEMPUR, Piekary Śląskie, Poland – 20; 10; 5; 2.5; 1.5; 1; 0.5 mLL^-1,

• clove oil – Avicenna-Oil, Wrocław, Poland  – 20 mLL^-1,

• diethyl ether – Merck, Darmstadt, Germany – 20; 10; 7.5; 5 mLL^-1,

• isobutyl alcohol (2-methyl-1-propanol) – Polskie Odczynniki Chemiczne S.A., Gliwice, Poland  20 mLL^-1,

• MS-222 – ICN Biomedicals Inc., Aurora, Ohio, USA  – 1000 mgL^-1,

• Propiscin (etomidate 0.2%) – IRS-ZPiIR, Żabieniec, Poland – 20mLL^-1,

• 2-phenoxyethanol – ICN Biomedicals Inc., Aurora, Ohio, USA – 20 mLL^-1.

Before being added to the experimental tank, clove oil was first dissolved with 95% ethanol at the ratio of 1:2 (clove oil : ethanol) and then diluted by shaking with a small amount of water. All anaesthetic solutions were made 30 min before the introduction of the crabs.

Criteria for assessing anaesthesia and recovery
Behavior and response (such as moving, taking a defensive posture or an ability to use claws) to visual and tactile stimuli were assessed. Crabs were considered anaesthetized when they became immobilized and lost the ability to right themselves when placed on their back [11]. Through immobilization we mean a loss of ability to walk and defend but not complete paralysis. Regaining the right body position was considered as the moment of recovery.

Experiment 1
The crabs in groups of five were exposed to each concentration for 30 minutes, then they were transferred to a recovery tank and observed until they regained a full movement activity.  The time of induction and recovery was measured and changes in the behaviour noted. Each agent was tested starting from the concentration of 20 mLL^-1 for liquid chemicals and 1000 mgL^-1 for MS-222. If the concentration was effective, the procedure was continued with the smaller one. From 5 to 7 crabs were used at each concentration.

Experiment 2
As an alternative method to chemical anaesthesia, cooling was tested. Eight crabs (individually) were transferred from a tank with the water of 14°C

RESULTS AND DISCUSSION

Experiment 1
Isobutyl alcohol, isoflurane, 2-phenoxyethanol, Propiscin at the concentration of 20 mLL^-1 and MS-222 at the concentration of 1000 mgL^-1 had no anaesthetic effect on crabs for 30 min of the exposure.

Clove oil at the concentration of 20 mLL^-1 resulted in anaesthesia of one small female (98 g), which recovered in 5 min. Two other crabs were slightly sedated (sluggish) displaying weaker reactivity. Two did not seem to be effected.

Only immersion in chloroform and diethyl ether solutions resulted in complete anaesthesia of all the crabs. The detailed data are presented in the Table 1. The smallest effective concentration of chloroform was 1.5 mLL^-1 and of diethyl ether – 10 mLL^-1.

Table 1. Percentage of Eriocheir sinensis (Milne-Edwards) anaesthetized during 30 min exposition to chloroform, diethyl ether and clove oil; range of induction and recovery time (mean ± SD)

Concentration mLL-1	Chloroform induction time	Recovery time	Ether induction time	Recovery time	Clove oil induction time	Recovery time
20	100%, n = 5 1–16 min	40 min 48s ± 26 min 32s	100%, n = 5 15–23 min	58 min 36s ± 29 min 57s	20%, n = 5 30 min	5 min 7s
10	100%, n = 5 11–28 min	49 min 36s ± 39 min 6s	100%, n = 7 19–27 min	30 min 48s ± 22 min 39s	–	–
7.5	–	–	43%, n = 7 30 min	7 min 5s ± 5 min 1s	–	–
5	100%, n = 5 12–28 min	41 min 36s ± 41 min 38s	20%, n = 5 30 min	10 min 15s	–	–
2.5	100%, n = 7 14–28 min	21 min 48s ± 8 min 55s	–	–	–	–
1.5	100%, n = 5 11–13 min	38 min 58s ± 16 min 50s	–	–	–	–
1	86%, n = 7 15–27 min	23 min 48s ± 10 min 11s	–	–	–	–
0.5	40%, n = 5 28 min	4 min 30s ± 42s	–	–	–	–

The anaesthesia process with ether, chloroform and clove oil had a generally similar run and could be described by following phases.

1. Agitation I – vigorous movement activity in the first minutes after being introduced to an anaesthetic solution.

2. Sedation – calming down. Sometimes all the crabs stayed still for several seconds, then started to walk slowly with breaks in activity.

3. Agitation II – increase in movement activity after approximately 10 min of exposure. In ether and chloroform crabs often began to turn vertically – with the mouth raised to the water surface. They stayed in this position or turned on their backs. The weakened reactivity to stimuli was displayed. This second agitation in a clove oil solution was much weaker.

4. General anaesthesia – almost total loss of activity, akinesia. Crabs stayed in various body positions, moving slowly with the legs and aprons, not responding to stimuli.

When put on their backs in the recovery tank, crabs kept motionless for several minutes often with deflexed aprons. Then they started to move and turn back to their normal position. At this stage the reactivity to tactile stimuli was very low. After that they started to walk a little and after being taken out of water they were still very weak, slow and helpless. Regaining the proper condition took about twice as much time as presented recovery time.

Phases of  recovery:

1. Motionlessness.

2. Appendages movement.

3. Regaining the right position.

At diethyl ether and chloroform concentrations, the outflows of not clotting, yellow-brown fluid from the mouth region was observed. It referred to all concentrations except chloroform 0.5 mLL^-1 but not to all the crabs. The outflows appeared at the end of the exposure or during the first two recovery phases.

One female was found dead four days after the experiment with chloroform at the concentration of 2.5 mLL^-1. It might be connected with the fact that during the exposition it pinched and chipped its apron. It was the only case of self-destructive and probably unconscious behavior. The rest of the crabs recovered and behaved normally during a 10-day observation period.

While preparing the anaesthetic solutions valid differences between diethyl ether and chloroform were observed. Ether, as much more volatile, did not cumulate on the bottom but its odour was so intense that it was necessary to cover the tank. Chloroform cumulated on the bottom of the tank but it was much less odorous, not causing any discomfort to the researcher.

Experiment 2
Cooling caused immobilization of all the crabs in 5min 35s ± 1min 19s. Recovery took 1 min 25s ± 29s. In most cases, regaining the right body position was sudden, not proceeded by any limb movement. In one case, the leakage of brown fluid during the induction was observed. All the treated crabs recovered and behaved normally during a 10-day observation period.

DISCUSSION

From all the tested agents only diethyl ether and chloroform appeared to be effective anaesthetics for mitten crabs. The smallest effective concentration of chloroform was 1.5 mLL^-1, while diethyl ether had almost 10 times weaker potency. Although such a simple method of anaesthetic bath praparation may not be ideal for agents of sparing solubility in water, it is practical and indicates chloroform's superiority over ether, strengthened by the fact, that diethyl ether is highly flammable.

Kurup [9] found these agents ineffective or lethal in two crab species – Petrolisthes cinctipes (Randall) and Hemigrapsus nudus (Dana). Gardner [6] used chloroform (1.25 and 2.5 mLL^-1) and managed to immobilize the giant crab, but because of long-term recovery (even to 24 hours) found the agent unusable. Similar conclusion was reached by McRae et al. [10] while anesthetizing crayfish Cherax destructor. Treating it with ether (20 mLL^-1), they received immobilization in 110 min and recovery of 180 min. According to Kleinholz [8], chloroform was the most efficient agent in Astacus trowbridgei (Stimpson) causing anaesthesia at the concentration of 1.6 mLL^-1 in 3 min. Our results and the cited literature indicate that the mitten crab is highly sensitive to anaesthetic properties of diethyl ether and specially chloroform, which could have a practical use.

A considerably less positive result was obtained when using clove oil. The concentration of 20 mLL^-1 was not potent enough to immobilize crabs in 30 min. Comparing our results to the papers of Gardner [6] and Morgan et al. [11], it can be concluded that mitten crab is less susceptible to clove oil anaesthesia than other species.

The results obtained from the trial with cooling can also be considered promising. The induction time was very short and the recovery rapid.

Our results are just the first step to establish the proper method of mitten crab immobilization. Further investigation on the effects of this methods on crab's physiology should be conducted.

CONCLUSIONS

The results of this study revealed that out of 8 tested agents, chloroform is the most potent anaesthetic for the mitten crab. Further investigation of the effects of chloroform anaesthesia and cooling on E. sinensis seems to be reasonable.

REFERENCES

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2. Chu K.H., Ho H.Y., Li C.P., Chan T.Y., 2003. Molecular phylogenetics of the mitten crab species in Eriocheir, sensu lato (Brachyura: Grapsidae). J. Crustacean Biol. 23(3), 738–746.

3. Coyle S.D., Dasgupta S., Tidwell J.H., Beavers T., Bright L.A., Yasharian D.K., 2005. Comparative efficacy of anesthetics for the freshwater prawn, Macrobrachium rosenbergii. J. World Aquacult. Soc. 36, 282–290.

4. Czerniejewski P., Wawrzyniak W., 2006. Body weight, condition, and carapace width and length in the Chinese mitten crab (Eriocheir sinensis H. Milne-Edwards, 1853) collected from the Szczecin Lagoon (NW Poland) in spring and autumn 2001. Oceanologia 48(2), 275–285.

5. Foley D.M., Stewart J.E., Holley A.R., 1966. Isobutyl alcohol and methyl pentynol as general anesthetics for the lobster, Homarus americanus Milne-Edwards. Can. J. Zool. 44, 141–143.

6. Gardner C., 1997. Options of humanely immobilizing and killing crabs. J. Shellfish Res. 16, 219–224.

7. Grabda E., 1973. Krab wełnistoszczypcy, Eriocheir sinensis Milne-Edwards, 1853 w Polsce [The Chinese mitten crab, Eriocheir sinensis Milne-Edwards, 1853 in Poland]. Prz. Zool. 17, 46–49 [in Polish].

8. Kleinholz L.H., 1947. A method for removal of the sinus gland from the eyestalks of crustaceans. Biol. Bull. 93, 52–55.

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11. Morgan J., Cargill C., Groot E., 2001. The efficacy of clove oil as an anesthetic for decapod crustaceans. Bull. Aquac. Assoc. Can. 101(3), 27–31.

12. Nepszy S.J., Leach J.H., 1973. First records of the Chinese mitten crab, Eriocheir sinensis, (Crustacea: Brachyura) from North America. J. Fish. Res. Board Can. 30, 1909–1910.

13. Oswald R.L., 1977. Immobilization of decapod crustacea for experimental procedures. J. Mar. Biol. Ass. U.K. 57, 715–721.

14. M., Marmari G.H., Mehrabi M.R., 2004. Acute toxicity and anesthetic effects of clove oil in Penaeus semisulcatus under various water quality conditions. Aquac. Int. 12 (4–5), 457–466.

15. Stoskopf M., 1993. Anaesthesia. in: Aquaculture for Veterinarians (ed. by L. Brown), 161–167. Pergamon Press, Oxford.

16. Summerfelt R.C., Smith L.S., 1990. Anesthesia, surgery, and related techniques. in: Methods for Fish Biology (ed. by C.B. Schreck & P.B. Moyle), 213–272. Am. Fish. Soc., Bethesda, Maryland.

17. Venarsky M.P., Wilhelm F.M., 2006. Use of clove oil to anaesthetize freshwater amphipods. Hydrobiologia 568, 425–432.

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Accepted for print: 3.06.2009

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Grzegorz Jan Hajek
Division of Fish Physiology,
West Pomeranian University of Technology, Szczecin, Poland
Kazimierza Królewicza 4, 71-550 Szczecin, Poland
email: gjh@fish.ar.szczecin.pl

Marcin Choczewski
Division of Fish Physiology,
West Pomeranian University of Technology, Szczecin, Poland
Kazimierza Królewicza 4, 71-550 Szczecin, Poland

Robert Dziaman
Division of Fish Physiology,
West Pomeranian University of Technology, Szczecin, Poland
Kazimierza Królewicza 4, 71-550 Szczecin, Poland

Bernard Kłyszejko
Division of Fish Physiology,
West Pomeranian University of Technology, Szczecin, Poland
Kazimierza Królewicza 4, 71-550 Szczecin, Poland

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