Parasitol Res (2008) 103:979–982 DOI 10.1007/s00436-008-1044-y
SHORT COMMUNICATION
Effect of tricaine methanesulfonate on survival and reproduction of the fish ectoparasite Ichthyophthirius multifiliis De-Hai Xu & Craig A. Shoemaker & Phillip H. Klesius
Received: 12 October 2007 / Accepted: 19 May 2008 / Published online: 16 June 2008 # Springer-Verlag 2008
Abstract The fish extoparasite Ichthyophthirius multifiliis was subjected to tricaine methanesulfonate (TM) exposure while fish were anesthetized for parasite collection. No information is available on the effects of TM exposure to I. multifiliis. This study evaluated the effects of TM on the survival and reproduction of I. multifiliis. Significant differences were not observed in survival and reproduction of tomonts collected from fish anesthetized using unbuffered or buffered TM or in control fish not exposed to TM. Results of the current study demonstrated that TM had no adverse effect on I. multifiliis survival and reproduction when used at concentrations and exposure times required for fish anesthetization.
Introduction The parasite Ichthyophthirius multifiliis infects most species of freshwater fish worldwide and can result in heavy economic losses for aquaculture. Ichthyophthirius has three developmental stages, an infective theront, a parasitic trophont, and a reproductive tomont (Hines and Spira 1974). Theronts and trophonts are commonly used to conduct efficacy and toxicity tests for screening chemotherapeutants against I. multifiliis especially in in vitro tests (Buchmann et al. 2003). The parasites are also frequently used in immunization and vaccination studies as antigens, in immobilization or enzyme-linked immunosorbent assay for evaluation of immune response, and in challenge trials D.-H. Xu (*) : C. A. Shoemaker : P. H. Klesius USDA, Agricultural Research Service, Aquatic Animal Health Research Laboratory, 990 Wire Road, Auburn, AL 36832–4352, USA e-mail:
[email protected]
for determining protective immunity (Dickerson et al. 1981; Xu and Klesius 2003). Most of these studies have one step in common, harvesting trophonts from fish infested with I. multifiliis. Two major methods are commonly used to harvest trophonts from I. multifiliis-infested fish: (1) scraping fish skin to dislodge trophonts (Everett et al. 2002) and (2) allowing trophonts to leave live or killed fish over a period of time (Cross and Matthews 1993). Tricaine methanesulfonate (TM), commonly known as MS-222, is a widely used anesthetic and euthanasia agent for fish to minimize pain and distress due to handling (Welker et al. 2007). For euthanasia, TM is commonly used unbuffered (Callahan and Noga 2002), but typically buffered when used as an anesthetic to avoid acid pH due to the formation of methanesulfonic acid (Smith et al. 1999). In a study to investigate the effect of tricaine on protozoan survival and infection, Callahan and Noga (2002) reported that tricaine caused rapid detachment and mortality of Ichthyobodo from fish skin. When fish are anesthetized or euthanized with TM, I. multifiliis trophonts in skin and gill of fish contact the anesthetic compound. No information is available on the effect of TM exposure on I. multifiliis, even though TM is commonly used to anesthetize fish before harvesting trophonts from fish infected with I. multifiliis (Burkart et al. 1990; Xu and Klesius 2003). The objective of the current study was to determine the effect of varying concentrations of buffered or unbuffered TM on the survival and reproduction of I. multifiliis.
Materials and methods Channel catfish were reared at USDA-ARS Aquatic Animal Health Research Laboratory, Auburn, Alabama. Experimental fish were supplied with flowing dechlorinated water
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between 22°C and 25°C. Ichthyophthirius was isolated from an infected channel catfish, Ictalurus punctatus (Rafinesque), from a fish pond in Alabama and maintained by continuous serial passages through channel catfish as previously described (Xu and Klesius 2003). In this study, the parasite collected from catfish infected with visible I. multifiliis (white spots) is defined as trophont. The trophont then becomes a tomont after attachment to substrate. Unbuffered TM solutions were made by dissolving TM (Argent Chemical Laboratories, Redmond, Washington) in dechlorinated tank water (103 mg/l total hardness, 34 mg/l alkalinity, pH 7.01). For buffered TM solutions, an equivalent volume of unbuffered TM stock solution (600 mg/l) was buffered with 500 mg/l sodium bicarbonate and further diluted with tank water. The concentrations for both unbuffered and buffered TM solutions were 300, 150, 50, and 0 mg TM per liter. The pH of unbuffered and buffered TM solutions was measured using Pinnacle/ Corning 540 pH meter (Corning Incorporated, Corning, NY, USA). The anesthetized catfish were rinsed in tank water and the skin gently scraped to dislodge the parasites. Skin scrapings containing trophonts were poured through a sieve with an opening of 425 μm (Dual MFG Co., Chicago, IL, USA) to retain sloughed fish skin and mucus. Trophonts were distributed to each well of 24-well tissue culture plate and allowed to attach. After discarding the water in each well to remove contaminating mucus, 1 ml of tank water was added to each well. The numbers of tomonts in each well were counted under low magnification (40×). The plates with tomonts were incubated for 18 h at 24°C. After counting dead tomonts, theronts in each well were enumerated in five 10-μl samples of theront solution with the aid of a Sedgewick-Rafter counting cell (Graticules Limited, Tonbridge, England). The mortality and reproduction of tomonts were determined for each well. The tomont reproduction was expressed as number of theronts released by each tomont, calculated by total theronts/live tomonts. To determine survival and reproduction of tomonts collected from anesthetized fish, two channel catfish heavily infected with maturing trophonts in each of three groups were exposed to: (1) unbuffered TM at 150 mg/l, (2)
buffered TM at 150 mg/l, or (3) water lacking TM for 2–3 min. Trophonts were collected from fish in each group, and approximately 450 trophonts were distributed to each well of 24-well plate. Four wells were used for each treatment, and the trial was repeated once. After attachment of trophonts to wells and incubated at 24°C overnight, the mortality and reproduction of tomonts were determined as described above. Approximately 300 trophonts were distributed to each well of 24-well plate to determine the effect of TM concentrations on the survival and reproduction of tomonts. After discarding the water in each well, 1 ml unbuffered or buffered TM solutions at the concentration of 300, 150, 50, or 0 mg/l TM were added to wells. Three wells were used for each treatment and each concentration, and the trial was repeated once. The mortality and reproduction of tomonts in groups with different TM concentrations were evaluated. To determine the effect of water pH on Ichthyophthirius tomont survival and reproduction, approximately 150–200 trophonts were distributed to each well of 24-well plate and allowed to attach. Tank water was adjusted pH to 7, 6, 5.5, or 5 with 0.1 N hydrochloric acid (HCl). After discarding the water in each well, 1 ml water with pH 7.0, 6.0, 5.5, or 5.0 were added to each well with attached trophonts. Four wells were used for each pH, and the trial was repeated once. The mortality and reproduction of tomonts were determined as previously described. All data analysis was performed with SAS software (SAS Institute 1989). Tomont survival and reproduction were compared with Duncan multiple range tests, and probabilities of 0.05 or less were considered statistically significant.
Results and discussion The pH significantly decreased from 6.93±0.04 to 5.11± 0.08 (mean±SD, N=3) as the TM concentrations increased from 50 to 300 mg/l (p0.05).
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Table 2 Effect of TM at different concentrations on Ichthyophthirius tomont survival Concentration (mg/l)
0 50 150 300
Buffered TM
Unbuffered TM
Number
Dead
Survival (%)
Number
Dead
Survival (%)
– 294±127 307±136 254±89
– 19±10 33±17 52±26
– 95.1±1.2aA 90.8±3.8aA 82.7±3.2bA
290±106 315±100 375±156 315±136
6±3 4±1 25±6 315±136
98.5±4a 98.9±1aA 90.1±2bA 0cB
Values are means±SD (N=6). Within a column, mean survival rates followed by the different lower case letter are significantly different (p0.05). There was also no significant difference in reproduction of tomonts collected from fish that received various anesthetization treatments. Each tomont divided and released approximately 320–382 viable theronts. TM at high concentration, buffered or unbuffered, significantly reduced tomont survival (Table 2). All tomonts were dead overnight in 300 mg/ml unbuffered TM solution. The survival of tomonts in 300 mg/l buffered TM solution (82.7%) was significantly lower than survival in 50 mg/l buffered TM (95.1%), even though pH was similar after buffering. Numbers of theronts released from each live tomont showed no significant difference (p>0.05) between tomonts exposed to different concentrations of TM. No statistical difference was noted on reproduction between tomonts exposed to unbuffered and buffered TM at the same concentration, except 300 mg/l TM in which all tomonts died and no theronts were released (Table 3). No statistical difference was noted on tomont survival and reproduction in water with pH ranging from 7 to 5 (p> 0.05). In water with pH 5.0, tomont survival and reproduction were 98.2% and 386 theronts/per tomont, respectively, comparable with tomont survival (96.3%) and reproduction (399 theronts/per tomont) in water with pH 7.0.
At prolonged exposure, high concentrations of TM significantly reduced tomont survival regardless whether of the solutions were buffered or not. The unbuffered 300 mg/l TM solution had a pH of 5.1, which was significantly lower than pH in tank water or 50 mg/l unbuffered TM solution. However, changing only the pH in water from 7 to 5 did not influence tomont survival and reproduction in this study. This indicates that other chemical property adversely affects I. multifiliis survival when used at high concentration, especially when the exposure water is not buffered and leads to acidic condition. In a study of TM on the mortality of protozoan, Callahan and Noga (2002) demonstrated that unbuffered TM concentrations as low as 50 mg/l significantly affect the motility of Ichthyobodo necator. Their results also suggest that the mortality of the parasite is not directly caused by the drop in pH but rather by a decrease in the unionized form of TM. The recommended anesthetic dose of TM for most fish is 50–250 mg/l (Callahan and Noga 2002). Channel catfish with a total length 5–15 cm reached full anesthesia in 2 min at concentrations of 100–120 mg/l. In a recent study, Welker et al. (2007) found that a TM concentration of 90 mg/l was optimal for anesthetization of juvenile channel catfish to promote ease of handling and reduce stress. When TM was used to anesthetize or euthanize fish infected with I. multifiliis to harvest the parasite, trophonts on fish were subjected to only a short exposure of TM, usually less than 10 min. The short duration of exposure to buffered or unbuffered TM showed no statistical difference on tomont
Table 3 Effect of TM on Ichthyophthirius theronts released from each tomont TM
Buffered TM
Unbuffered TM
Concentration (mg/l)
Live tomonts
Theronts per tomont
Live tomonts
Theronts per tomont
0 50 150 300
– 276±117 275±123 285±104
– 393±113aA 505±138aA 483±133aA
285±104 312±101 351±150 0
473±149a 456±176aA 486±153aA 0bB
Values are means±SD (N=6). Within a column, mean theronts released from each tomont followed by the different lower case letter are significantly different (p
