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Pharmacokinetics and Tissue Distribution ofThiamphenicol and Florfenicol in Pacific White ShrimpLitopenaeus vannamei in Freshwater following OralAdministrationWenhong Fang a , Guolie Li a b , Shuai Zhou a , Xincang Li a , Linlin Hu a & Junfang Zhou aa East China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, KeyLaboratory of Marine and Estuarine Fisheries Resources and Ecology, Ministry of Agriculture,300 Jungong Road, Shanghai, 200090, Chinab Nanchong Supervision and Test Center for Agricultural Products Quality, 137 Nongke Alley,Nanchong, Sichuan Province, 637000, ChinaVersion of record first published: 12 Mar 2013.

To cite this article: Wenhong Fang , Guolie Li , Shuai Zhou , Xincang Li , Linlin Hu & Junfang Zhou (2013): Pharmacokineticsand Tissue Distribution of Thiamphenicol and Florfenicol in Pacific White Shrimp Litopenaeus vannamei in Freshwaterfollowing Oral Administration, Journal of Aquatic Animal Health, 25:2, 83-89

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Journal of Aquatic Animal Health 25:83–89, 2013C© American Fisheries Society 2013ISSN: 0899-7659 print / 1548-8667 onlineDOI: 10.1080/08997659.2012.754799

ARTICLE

Pharmacokinetics and Tissue Distribution of Thiamphenicoland Florfenicol in Pacific White Shrimp Litopenaeusvannamei in Freshwater following Oral Administration

Wenhong Fang*East China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences,Key Laboratory of Marine and Estuarine Fisheries Resources and Ecology, Ministry of Agriculture,300 Jungong Road, Shanghai 200090, China

Guolie LiEast China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences,Key Laboratory of Marine and Estuarine Fisheries Resources and Ecology,Ministry of Agriculture, 300 Jungong Road, Shanghai 200090,China; and Nanchong Supervision and Test Center for Agricultural Products Quality,137 Nongke Alley, Nanchong, Sichuan Province 637000, China

Shuai Zhou, Xincang Li, Linlin Hu, and Junfang ZhouEast China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences,Key Laboratory of Marine and Estuarine Fisheries Resources and Ecology, Ministry of Agriculture,300 Jungong Road, Shanghai 200090, China

AbstractThis study evaluated the pharmacokinetic disposition of thiamphenicol (THA) and florfenicol (FLR) after oral

administration of each at a single dose of 10 mg/kg body weight in Pacific white shrimp Litopenaeus vannamei held infreshwater at 25.0 ± 1.0◦C. The THA and FLR concentrations in the hemolymph, muscle, and hepatopancreas weredetermined by HPLC. The profiles of hemolymph THA and FLR concentrations versus time were best described bya two-compartment open pharmacokinetic model with first-order absorption. The peak concentration (Cmax), peaktime (Tmax), absorption half-life (t1/2ka) and elimination half-life (t1/2β) of THA in hemolymph were 7.96 µg/mL, 2 h,0.666 h, and 10.659 h, respectively. The corresponding values for FLR were 5.53 µg/mL, 2 h, 1.069 h, and 17.360 h,respectively. After oral administration, THA and FLR were rapidly absorbed in white shrimp and THA in hemolymphwas absorbed and eliminated more quickly than FLR. The parameters in muscle and hepatopancreas were calculatedby a noncompartment model based on statistical moment theory. The Cmax, area under the concentration–time curve(AUC0-t), mean residue time (MRT0-t), and half-life (t1/2z) in muscle were 2.98 µg/g, 29.10 mg/kg·h, 9.77 h, and 6.84 h,respectively. The corresponding values for FLR were 1.91 µg/g, 15.97 mg/kg·h, 19.40 h, and 18.32 h, respectively. Inmuscle THA was eliminated more quickly than FLR. The peak concentrations of THA and FLR in the hepatopancreaswere 204.25 and 164.22 µg/g, respectively, and the values for AUC0-t were 1,337.74 and 871.73 mg/kg·h, respectively,which were much higher than those in hemolymph and muscle. The in vitro protein-binding value of THA (28.38%)was lower than that of FLR (37.91%), which might be related to the finding that THA in Pacific white shrimp wasabsorbed and eliminated more quickly than FLR.

*Corresponding author: [email protected] July 21, 2012; accepted November 23, 2012

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The Pacific white shrimp Litopenaeus vannamei has becomethe most important cultivated shrimp in China owing to its fastgrowth rate and strong adaptability. This shrimp is popularlyfarmed not only in seawater but also in freshwater (BOF 2011).Given its euryhaline characteristics, L. vannamei has becomethe main aquatic animal cultured in brackish water and fresh-water regions in recent years; in 2010, there were 615,010 tonsof shrimp produced in freshwater ponds in China. Recently, dis-eases caused by Vibrio bacteria have become a serious problemcausing economic losses for shrimp farming (Wang and Yang2005; Zhou et al. 2012).

Thiamphenicol (THA) and its fluorinated derivative, florfeni-col (FLR), are broad-spectrum antibiotics belonging to a part ofthe chloramphenicol family of drugs. Although the mechanismsof action of THA and FLR are similar to that of chlorampheni-col, THA and FLR do not exert the potential fatal side effect ofdose-unrelated aplastic anemia seen in humans (Dowling 2006).Because chloramphenicol is prohibited for use in aquaculture,THA and FLR are expected to be used as alternatives for antibi-otic treatment of diseases.

Owing to their broad antibacterial spectrum and high po-tency, THA and FLR are commonly used to control susceptiblebacterial diseases in poultry (Switala et al. 2007) and fish (vande Riet et al. 2003; Burridge et al. 2010) farming. They are effec-tive in the treatment of pseudotuberculosis in Yellowtail Seriolaquinqueradiata (also known as Buri) (Yasunaga and Yasumoto1988), edwardsiellosis in Channel Catfish Ictalurus punctatus(McGinnis et al. 2003), vibriosis in Goldfish Carassius auratus(Fukui et al. 1987), and furunculosis in Atlantic Salmon Salmosalar (Inglis et al. 1991). Both compounds also have potentialuse in the treatment of vibriosis and necrotizing hepatopancre-atitis infections in farm-raised shrimps. The pharmacokineticcharacteristics of THA and FLR in shrimp need to be under-stood to determine the optimal dose regimens for achieving andmaintaining therapeutic drug levels as well as predicting theresidue withdrawal time in edible tissues of treated shrimp.

Previous THA or FLR pharmacokinetic studies have beenperformed mainly on finfishes, including Sea Bass Dicentrar-chus labrax (also known as European Bass Morone labrax)(Castells et al. 2000; Malvisi et al. 2002), Gilthead SeabreamSparus aurata (Malvisi et al. 2002), Olive Flounder Paralichthysolivaceus (Lim et al. 2011), Red Pacu Piaractus brachypomus(Lewbart et al. 2005), Korean Catfish Silurus asotus (Park et al.2006), and Crucian Carp Carassius auratus cuvieri (Zhao et al.2011). The results show that THA and FLR are absorbed rapidly,distributed extensively, and eliminated rapidly. Although thecharacteristics of THA and FLR have been studied in a varietyof vertebrate species, little is known about the pharmacokineticsof THA and FLR in crustaceans, particularly in shrimp.

The objective of the present study was to investigate the phar-macokinetics and tissue distribution of THA and FLR in Pacificwhite shrimp held in freshwater after a single oral administrationat 25.0 ± 1.0◦C.

METHODSChemicals.—Standards for THA and FLR were purchased

from Sigma (St. Louis, Missouri). The THA and FLR rawmaterials were supplied by Zhangjiagang Hengsheng Phar-maceutical, Zhangjiagang, China. The active ingredients weredetermined by HPLC and found to exceed 98.0%. The or-ganic solvents used—methanol, ethyl acetate, acetonitrile, andn-hexane—were of HPLC grade (Tedia Company, Fairfield,Ohio). All other chemicals were analytical grade.

Experimental animals and aquarium conditions.—HealthyPacific white shrimp (∼9.1–12.3 g) were obtained from a shrimpfarm in suburban Fengxian, Shanghai, China. The shrimp werekept in 120-L fiberglass tanks (6 individuals/tank) containingfreshwater. Tap water was dechlorinated by a purifier (AC/KDF-150BSE, Shanghai Canature Environmental Products, Shang-hai, China). Water was recirculated, aeration was kept con-stant, and the temperature was maintained at 25.0 ± 1.0◦C.The shrimp were fed daily with pellet feed at approximately2–3% of their average body weight. The remnants, excretions,and moults were promptly removed. The shrimp were accli-mated for 10 d and analyzed to confirm the absence of THAand FLR before experiments. Prior to drug administration, theshrimp were not fed for 48 h.

Oral administration.—The THA (or FLR) was mixed in aslurry of food. The slurry contained 2 mg/mL of THA (or FLR).The single oral dose was 10 mg THA (or FLR)/kg body weight(BW). Thus, the dosing feed was gavaged at a single dose of5 mL/kg BW, which corresponded to a final dose of 10 mg/kgBW. The slurry was forced into the stomach of the shrimp usinga 1-mL syringe fitted with a blunt 12-gauge needle. Only thoseindividuals that did not regurgitate were used in the experiment.

Sample collection.—The sampling times were 0.25, 0.5, 1, 2,4, 6, 9, 12, 24, and 48 h, and 5 and 8 d after dosing. Six shrimpwere sacrificed at each time and tissues were sampled.

Hemolymph was collected from the pericardial cavity us-ing 1-mL syringes, placed in vials containing ammonium ox-alate (0.001 g) as an anticoagulant, and mixed. The collectedhemolymph was centrifuged at 850 × g (Hitachi CF16RXIIwith type 49 rotor) for 10 min at room temperature. Plasma wasremoved and placed in 1.5-mL microcentrifuge vials. Muscleand hepatopancreas tissues were also collected and placed inindividually marked plastic bags. The samples were frozen andstored at −80◦C prior to extraction.

Sample analysis.—The content of THA and FLR in plasmaand tissues was measured by HPLC (Switala et al. 2007) withmodification. The HPLC system used was an Agilent 1100 se-ries consisting of a double pump, an auto-injector, a columntemperature tank, and an ultraviolet light detector. The columnwas a Zorbax SB C-18 (4.6 × 150 mm). The mobile phase con-sisted of acetonitrile : deionized water (16:84, v/v). The mobilephase was filtered through a 0.45-µm Millipore filter and son-icated for 10 min before use. The injection volume was 10 µLand the flow rate was 1.0 mL/min. The column temperature was

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PHARMACOKINETICS OF THIAMPHENICOL AND FLORFENICOL IN SHRIMP 85

controlled at 40◦C and the wavelength of the detector was set at225 nm.

Plasma (500 µL) was placed in a 2-mL centrifuge tube and1 mL of ethyl acetate was added as an extractant. The mix-ture was then vortexed for 5 min and centrifuged at 9,500 ×g (Hitachi CF16RXII with type 49 rotor) for 5 min. The super-natant was transferred to a clean centrifuge tube and the residuewas re-extracted twice using the same procedure as above. Thecombined supernatants were evaporated to dryness using a vor-tex evaporator. The resulting residue was dissolved in 1.0 mLof mobile phase and then filtered through 0.45-µm disposablesyringe filters prior to analysis by the HPLC system.

Muscle samples (1.0 g) were thawed and triturated using amortar and pestle. Hepatopancreas samples (0.7–1.0 g) wereweighed after thawing and triturated with a glass rod in acentrifuge tube. About 1.0 mL of 0.02 M H3PO4 was addedto the muscle and hepatopancreas samples, which were thenvortexed for 2 min. After adding 4 mL of ethyl acetate, thesample was vortexed for 5 min and centrifuged at 8,300 × g(Hitachi CF16RXII with type 44 rotor) for 5 min. The super-natant was transferred to a clean centrifuge tube and the residuewas re-extracted twice using the above procedure. The com-bined supernatants were evaporated to dryness using a vortexevaporator. The resulting residue was dissolved in 1.0 mL ofmobile phase and 2 mL of n-hexane. The mixture was shakenvigorously and then transferred into a 5-mL centrifuge tube. Af-ter centrifugation at 4,150 × g (Hitachi CF16RXII with type 44rotor) for 5 min, the bottom-layer liquid was filtered through a0.45-µm disposable syringe filter prior to analysis by the HPLCsystem.

The calibration standards for THA and FLR were in therange of 0.05–10 µg/mL. The limits of quantification (LOQs)of THA and FLR were 0.05 µg/mL for plasma, 0.05 µg/g forhepatopancreas, and 0.05 µg/g for muscle. The recoveries for thevarious THA concentrations assayed were 86–97% in plasmaand 80–92% in tissues. The recoveries for the various FLRconcentrations assayed were 88–95% in plasma and 78–93% intissues.

Plasma-protein binding.—The ultrafiltration method wasused to determine the plasma-protein binding values. Untreatedhemolymph from 30 shrimp was collected and mixed by vor-texing. Plasma was obtained after centrifugation at 2,400 × g(Hitachi CF16RXII with type 49 rotor) for 5 min. About 500 µLof plasma was placed in a 2-mL centrifuge tube, and THA orFLR was added to achieve a final concentration of 0.5 µg/mL.The sample was then mixed and incubated for 4 h at 28◦C.About 300 µL of this sample was then added to each centrifugalultrafiltration tube (Microncon YM-10, 10 kDa) and was cen-trifuged at 13,700 × g (Hitachi CF16RXII with type 49 rotor)for 60 min at room temperature. The ultrafiltration filtrate fromthe plasma sample preparation was determined by HPLC.

Pharmacokinetic analysis.—The concentration–time pro-files of the plasma and tissues were analyzed accordingto compartmental and noncompartmental modeling analy-

ses, respectively. All compartmental and noncompartmentalmodelings were performed using Drug and Statistics Softwareversion 2.1.1 (Mathematical Pharmacology Professional Com-mittee of China).

RESULTS

Pharmacokinetics of THA and FLR after OralAdministration

The plasma concentrations of THA and FLR after oral ad-ministration at a dose of 10 mg/kg are shown in Figure 1. Thepeak concentrations of THA and FLR in plasma were 7.96 and5.53 µg/mL, respectively; both occurred 2 h after administrationand were followed by a rapid decrease until 12 h postgavage. Theplasma concentrations of FLR and THA could be best describedby a two-compartment open model with first-order absorption.The pharmacokinetic parameters derived from the compartmentmodel are shown in Table 1. The distribution and eliminationhalf-lives (t 1/2α and t1/2β), total body clearance (CLs), and ap-parent volume of distribution (Vd) of THA were found to be2.488 h, 10.659 h, 227.1 mL·kg−1·h−1, and 0.942 L/kg, respec-tively. The corresponding FLR values were 1.364 h, 17.360 h,242.0 mL·kg−1·h−1, and 0.887 L/kg, respectively. The plasma-protein binding values of THA and FLR were 28.38 ± 1.26%(mean ± SD) and 37.91 ± 7.61%, respectively. These val-ues were not high, and the value of THA was lower than thatof FLR.

Tissue Distribution and EliminationThe tissue concentration–time curves of THA and FLR in

muscle and hepatopancreas after oral administration are shownin Figure 2 and Figure 3, respectively. The peak concentrationsof THA and FLR in muscle were 2.98 and 1.91 µg/g, respec-tively. Both occurred 2 h after administration and were followedby a rapid decrease until 12 h postgavage; THA and FLR were

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FIGURE 1. Profiles of thiamphenicol (THA) and florfenicol (FLR) concen-trations in hemolymph of Pacific white shrimp versus time after oral gavage(n = 6).

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TABLE 1. Pharmacokinetic parameters of thiamphenicol and florfenicol inhemolymph from Pacific white shrimp based on the compartmental model (n =6). Cmax = maximum plasma concentration, Tmax = time when the maximumconcentration was reached, t1/2α = distribution half-life of the drug, t1/2β =elimination half-life of the drug, t1/2ka = absorption half-life of the drug, CLs =total body clearance of the drug, AUC0-t = area under the concentration–timecurve from zero to time, AUC0-∞ = area under the concentration–time curvefrom zero to infinity, Vd = apparent volume of distribution, Vss = apparentvolume of distribution at steady state, tlag = lag time before absorption.

Parameter Units Thiamphenicol Florfenicol

Cmax µg/mL 7.96 ± 1.61 5.53 ± 1.14Tmax h 2.0 2.0t1/2α h 2.488 1.364t1/2β h 10.659 17.360t1/2ka h 0.666 1.069CLs mL·kg−1·h−1 227.1 242.0AUC0-t mg/L·h 43.70 38.94AUC0-∞ mg /L·h 43.96 41.27Vd L/kg 0.942 0.887Vss L/kg 1.547 4.381tlag h 0.161 0.033

quickly cleared from muscle, and levels of 0.023 and 0.040 µg/gwere measured at 2 d, respectively.

Although the trends of the concentration–time curve weresimilar, the concentrations of THA and FLR in the hepatopan-creas were much higher than those in muscle and hemolymph.The THA and FLR peak concentrations in hepatopancreas tis-sue were 204.25 and 164.22 µg/g, respectively, which occurredat 1 and 0.5 h after dosing, respectively.

The behaviors of THA and FLR in tissues after oral ad-ministration were analyzed according to the statistical momenttheory. The pharmacokinetic parameters were calculated andare listed in Table 2. For muscle, the estimated AUC0-t valueswere 29.10 and 15.97 mg/kg·h for THA and FLR, respectively.

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FIGURE 2. Profiles of thiamphenicol (THA) and florfenicol (FLR) concen-trations in muscle tissue of Pacific white shrimp versus time after oral gavage(n = 6).

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FIGURE 3. Profiles of thiamphenicol (THA) and florfenicol (FLR) concen-trations in hepatopancreas tissue of Pacific white shrimp versus time after oralgavage (n = 6).

The elimination half-lives (t1/2z) were 6.84 and 18.32 h forTHA and FLR, respectively. For hepatopancreas, the estimatedAUC0-t values were 1,337.74 and 871.73 mg/kg·h for THA andFLR, respectively, and the t1/2z values were 31.29 and 41.38 hfor THA and FLR, respectively.

DISCUSSIONOverall, the pharmacokinetic properties of THA and FLR

in Pacific white shrimp were similar, and their plasmaconcentration–time curves could both be well described by atwo-compartment open model with first-order absorption afteroral administration.

After oral administration, THA and FLR were rapidly ab-sorbed in white shrimp. The mean Cmax values (7.96 and5.53 µg/mL for THA and FLR, respectively) peaked at 2.0 h.The Tmax value for THA in white shrimp was lower than thatin Sea Bass (Castells et al. 2000). The Tmax value for FLR inwhite shrimp was lower than those reported for Atlantic Salmon(Horsberg et al. 1996), Atlantic Cod Gadus morhua (Samuelsenet al. 2003), a hybrid tilapia Oreochromis niloticus × O. aureus(Feng et al. 2008), and Korean Catfish (Park et al. 2006), butnot for Crucian Carp (Zhao et al. 2011). In the present study, therapid oral absorption in white shrimp could be attributed to theshrimp’s open circulatory system and active excretion of excesswater for osmoregulatory purposes in freshwater.

The volume of distribution at steady state (Vss) is an accurateindicator of drug diffusion in body tissues. The Vss of 1.547 L/kgfor THA in Pacific white shrimp was lower than that for FLR (Vssof 4.381 L/kg). However, these values are higher than the valuesfor FLR reported in Korean Catfish (Park et al. 2006), AtlanticSalmon (Horsberg et al. 1996), and Atlantic Cod (Samuelsenet al. 2003). The Vss values of THA and FLR determined in thepresent study revealed that the drugs were well distributed inthe body organs and tissues of white shrimp.

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TABLE 2. Pharmacokinetic parameters based on the statistical moment theory of thiamphenicol and florfenicol in muscle and hepatopancreas from Pacific whiteshrimp after a single oral administration (n = 6). Cmax = maximum plasma concentration, Tmax = time when the maximum concentration was reached, AUC0-t =area under the concentration–time curve from zero to time, AUC0-∞ = area under the concentration–time curve from zero to infinity, MRT0-t = mean residue timeof drug in body from zero to time, MRT0-∞ = mean residue time of drug in body from zero to infinity, t1/2z = half-life of the drug, CLz = total body clearance ofthe drug.

Muscle Hepatopancreas

Parameter Units Thiamphenicol Florfenicol Thiamphenicol Florfenicol

Cmax mg/kg 2.98 ± 0.53 1.91 ± 0.18 204.25 ± 33.41 164.22 ± 25.64Tmax h 2.0 2.0 1.0 0.5AUC0-t mg/kg·h 29.10 15.97 1,337.74 871.73AUC0-∞ mg/kg·h 29.33 16.04 1,381.60 902.24MRT0-t h 9.77 19.40 27.71 38.43MRT0-∞ h 10.14 19.94 34.82 45.64t1/2z h 6.84 18.32 31.29 41.38CLz kg·h−1·kg−1 0.341 0.624 0.007 0.011

The t1/2z and CLz are important pharmacokinetic parametersthat describe how quickly a drug is eliminated from the body.After oral administration, the t1/2z values for THA and FLRwere similar to those reported in Korean Catfish (Park et al.2006), hybrid tilapia (Feng and Jia 2009), and Atlantic Salmon(Horsberg et al. 1996), but were lower than those in AtlanticCod (Samuelsen et al. 2003) and Olive Flounder (Lim et al.2011). However, the CLz values for THA and FLR in Pacificwhite shrimp were much higher than those reported in KoreanCatfish (Park et al. 2006), hybrid tilapia (Feng and Jia 2009),and Atlantic Salmon (Horsberg et al. 1996). In addition to thedifferences in how temperature and salinity affect the elimina-tion of the drug (Samuelsen et al. 2003; Park et al. 2006), thisresult can also be well explained by the differences betweencrustaceans and fishes in anatomical volumes, plasma proteins,and tissue binding of the drug (Øie and Tozer 1979; Barron et al.1988).

Plasma-protein binding determines the amount of drug thatis in free form and available for tissue distribution as well asits therapeutic action. In the present study, the plasma-proteinbinding values of THA and FLR were 28.38% ± 1.26 and 37.91± 7.61%, respectively. This low protein binding suggests thatalmost 70% of THA and 60% of FLR in the hemolymph arefree and available for treating systemic infections. The plasma-protein binding of THA was lower than that of FLR, whichperhaps explains why the distribution of THA in the plasma,muscle, and hepatopancreas of shrimp is wider than that ofFLR, as well as why THA is eliminated faster than FLR fromthe shrimp tissues.

In the present study, the drug concentrations in hepatopan-creas were significantly higher than those in hemolymph andmuscle. The highest mean concentration ratios of hepatopan-creas : hemolymph and hepatopancreas : muscle for THA were25.6 and 68.5, respectively, and for FLR were 29.7 and 86.0,respectively. The AUC0-t ratios of hepatopancreas : hemolymph

and hepatopancreas : muscle for THA were 30.57 and 45.97,respectively, and for FLR were 22.39 and 54.58, respectively.These results indicate that only a small amount of the drugs pen-etrated the systemic circulation and other tissues before leavingthe hepatopancreas. A large amount of the drugs were greatlyreduced in hepatopancreas after oral administration, which iscalled the “first-pass effect.” This phenomenon was also ob-served in other crustaceans, such as norfloxacin in L. van-namei (Fang et al. 2004), sulfadimethoxine in American lobsterHomarus americanus (James and Barron 1988), ormetoprim inthe shrimp, Penaeus vannamei (Park et al. 1995), triclopyr in thecrayfish, Orconectes propinquus (Barron et al. 1991), and oxyte-tracycline in Pacific white shrimp (Faroongsarng et al. 2007).These studies showed that the drug concentration in hepatopan-creas tissue was 101–102 times higher than that in other tissues.In crustaceans, there is substantial evidence that the hepatopan-creas plays a role not only in metabolism and elimination butalso in absorption (Verri et al. 2001; Faroongsarng et al. 2007).After oral administration, the drug is delivered from the shrimpstomach to the digestive gland, where both the drug uptake intoand elimination from the body take place competitively. In thepresent study, the times to peak concentrations of THA and FLRin the hepatopancreas (1 and 0.5 h, respectively) were faster thanthose in hemolymph and muscle (2.0 h), further illustrating thatthe drugs were absorbed from the stomach to the hepatopancreasof the shrimp.

Vibriosis is an important disease affecting the shrimp in-dustry in China and other countries (Roque et al. 2001; Wangand Yang 2005; Zhou et al. 2012). Despite many reports onshrimp vibriosis, pharmacodynamic data for vibrios is very lim-ited. Roque et al. (2001) determined the in vitro susceptibilityto florfenicol of 98 strains of Vibrio spp. isolated from shrimpin northwestern Mexico; the mean minimum inhibitory con-centration was 1.79 µg/mL with a range of 0.25–8.0 µg/mL.Zhou et al. (2012) reported a specific bacterial pathogen, Vibrio

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harveyi HLB0905 from “white tail” shrimp, which was gener-ally accompanied by mass mortalities. Later we examined thesensitivity of this microorganism to THA and FLR, and foundMIC values of 0.25 µg/mL for THA and 1.0 µg/mL for FLR.Shojaee Aliabadi and Lees (2000) suggested that for a bacterio-static drug like florfenicol, an optional dosage regimen shouldmaintain concentrations at the size of infection in excess ofMIC90 for the entire medication period. On the basis of theresults of this study, it is reasonable to assume that a dose of10 mg/kg given orally at 12-h intervals for THA and 8-h in-tervals for FLR should be appropriate for control of “bacterialwhite tail disease” of Pacific white shrimp. For the treatmentof infected shrimp by other bacterial species, speculation abouteffective dose regimens for THA or FLR in shrimp requires thecombination of pharmacokinetics and pathogen sensitivity. Thedose size and dosing interval could be adapted accordingly toachieve better therapeutic efficacy.

In conclusion, the pharmacokinetic profiles of THA and FLRwere shown, and the rapid absorption, extensive distribution,and quick elimination of the drugs were also demonstrated. InPacific white shrimp THA was distributed more extensively andeliminated more quickly than FLR. The pharmacokinetic datarevealed that THA and FLR could be used to treat bacterialinfections in this species.

ACKNOWLEDGMENTSThis work was supported financially by the Special Fund for

Agro-scientific Research in the Public Interest (200803012 and201203085).

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This article was downloaded by: [Department Of Fisheries]On: 08 April 2013, At: 19:32Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House,37-41 Mortimer Street, London W1T 3JH, UK


Clupeid Response to Stressors: The Influence ofEnvironmental Factors on Thiaminase ExpressionJ. M. Lepak a , C. E. Kraft b & M. J. Vanni ca Colorado Parks and Wildlife, 317 West Prospect Road, Fort Collins, Colorado, 80526, USAb Department of Natural Resources, Cornell University, 206D Fernow Hall, Ithaca, New York,14853, USAc Department of Zoology, Miami University, 188 Pearson Hall, Oxford, Ohio, 45056, USAVersion of record first published: 05 Apr 2013.

To cite this article: J. M. Lepak , C. E. Kraft & M. J. Vanni (2013): Clupeid Response to Stressors: The Influence ofEnvironmental Factors on Thiaminase Expression, Journal of Aquatic Animal Health, 25:2, 90-97








ARTICLE

Clupeid Response to Stressors: The Influenceof Environmental Factors on Thiaminase Expression

J. M. Lepak*Colorado Parks and Wildlife, 317 West Prospect Road, Fort Collins, Colorado 80526, USA

C. E. KraftDepartment of Natural Resources, Cornell University, 206D Fernow Hall, Ithaca, New York 14853, USA

M. J. VanniDepartment of Zoology, Miami University, 188 Pearson Hall, Oxford, Ohio 45056, USA

AbstractOver the past five decades, a reproductive failure related to thiamine deficiency, referred to as thiamine deficiency

complex (TDC), has been observed in valuable salmonine fishes in the Great Lakes and Finger Lakes in North Americaand the Baltic Sea in Europe. The cause of TDC has been linked to the consumption of clupeid fish, which contain highlevels of a thiamine-destroying enzyme called thiaminase I (hereafter referred to as “thiaminase”). High activities ofthiaminase have been reported from clupeids such as Alewife Alosa pseudoharengus, Gizzard Shad Dorosoma cepedi-anum and Atlantic (Baltic) Herring Clupea harengus, but no consistent explanation has accounted for the wide rangeof observed variation in levels of thiaminase in clupeids. Chronic stress can suppress the immune systems of Alewifeand other fishes, thereby reducing the number of circulating white blood cells available to suppress bacteria. Becausethe presence of thiaminase has been associated with thiaminolytic bacteria isolated from Alewife viscera, we hypoth-esized that stressful conditions, which can potentially limit clupeid immune response or alter internal physiologicalconditions, could allow for thiaminase to be produced more efficiently by bacteria or thiaminolytic bacteria could pro-liferate, or both events could occur, resulting in a subsequent increase in thiaminolytic activity. In this study, Alewivesand Gizzard Shad were exposed to severe winter temperatures and low food availability, respectively, in replicatedpond experiments to evaluate the influence of stressful conditions on clupeid thiaminase activity. Though responses incirculating white blood cell counts and metrics of fish condition indicated that experimental treatments affected theseclupeids, these effects were not related to increased thiaminase activity. The only significant treatment effect on clupeidthiaminase was an increase in mean thiaminase activity in Gizzard Shad from ponds where only high quality energysources were available. These data indicate that variability in clupeid thiaminase may be related to diet composition.

Reproductive failure in piscivorous salmonine fishes hasbeen observed over the past five decades in the Laurentian GreatLakes and Finger Lakes in North America and the Baltic Seain Europe. This phenomenon was first observed in the brood-stocks of salmonine fish in North American hatcheries duringthe 1960s (McDonald et al. 1998), and reproductive failure inBaltic Sea salmon became prevalent in the 1970s (Hanssonet al. 2001). Mortality of Coho Salmon Oncorhynchus kisutchfry that exhibited similar symptoms was first observed in wild

*Corresponding author: [email protected] June 19, 2012; accepted January 15, 2013

fish in 1967 (Johnson and Pecor 1969). During the next sev-eral decades managers and researchers working in the Lauren-tian Great Lakes and Finger Lakes of New York recognizedthat other salmonine fishes, including Lake Trout Salvelinusnamaycush, Atlantic Salmon Salmo salar, Chinook SalmonO. tshawytscha, Rainbow Trout O. mykiss, and Brown TroutS. trutta, suffered from a similar reproductive failure (Fisheret al. 1995; Marcquenski and Brown 1997; McDonald et al.1998).

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CLUPEID RESPONSE TO STRESSORS 91

In North America the syndrome associated with this typeof reproductive failure has been referred to as thiamine de-ficiency complex (TDC), which has been linked to thiaminedeficiency (Fitzsimons et al. 1999). During the mid-1990s itbecame increasingly evident that thiamine-deficient salmonidswere susceptible to TDC (Fisher et al. 1996; Fitzsimons et al.1999). Thiamine (vitamin B1) is an essential vitamin necessaryfor the conversion of carbohydrates and lipids into energy and isubiquitous for sustaining organisms across all kingdoms of life.Offspring of salmonine fishes susceptible to TDC die shortlyafter hatching, but fry from identical egg sources survive andexhibit normal behavior when treated with thiamine (Fitzsimons1995; Fisher et al. 1996).

Clupeid fishes, including Alewife Alosa pseudoharengus,Gizzard Shad Dorosoma cepedianum, and Atlantic (Baltic) Her-ring Clupea harengus, have been routinely observed to containhigh activity levels of thiaminase I, a thiamine-destroying en-zyme (Wistbacka et al. 2002; Tillitt et al. 2005). A large bodyof field and laboratory work conducted during the past twodecades supports a plausible hypothesis for an association be-tween a prey base composed of a large biomass of clupeidscontaining thiaminase I and the recruitment failures often expe-rienced by piscivorous salmonines. For example, observationsof sac-fry mortality in Atlantic Salmon (Norrgren et al. 1993;Bengtsson et al. 1994; Karlsson et al. 1996) and sea-run BrownTrout (Soivio 1996) in the Baltic Sea were attributed to the con-sumption of clupeid prey containing thiaminase I. Although twothiamine-degrading enzymes have been described (thiaminase Iand II; see Discussion for more about thiaminase II), thiaminaseI has been the specific thiaminase linked to reproductive failurein fish; therefore, all subsequent references to thiaminase in thispaper specifically refer to thiaminase I.

Thiaminase activity in Alewives has been attributed to thi-aminase positive bacteria Paenibacillus thiaminolyticus isolatedfrom Alewife viscera (Honeyfield et al. 2002), though Richteret al. (2012) reported that this bacteria is not the primary sourceof thiaminase activity. Thus, there is some debate over whetherthe ultimate source of thiaminase in fish is the fish themselves(Riley and Evans 2008; Richter et al. 2012), thiaminolytic bac-teria (Honeyfield et al. 2002), other sources, or a combina-tion of these sources. Nevertheless, the presence of thiami-nase in Alewives is believed to be a primary factor responsiblefor TDC in predators of the Alewife (Fitzsimons et al. 1999;Honeyfield et al. 2005a) even though thiamine deficiency hasnot been observed within clupeids containing high thiaminaseactivity (Wistbacka et al. 2002; Tillitt et al. 2005). Laboratoryexperiments have induced TDC in Lake Trout by altering di-etary levels of thiaminase, using feral Alewives containing thi-aminase and bacterial sources of thiaminase (Honeyfield et al.2005b). Although the ultimate source or sources of thiaminasecontributing to TDC are unknown in natural systems (Richteret al. 2012), available evidence suggests that clupeid or bacte-rial thiaminase, or both, play a key role in the development ofsalmonine thiamine deficiency.

Both TDC in salmonines and thiaminase activity in clupeidsfluctuate widely, and a mechanistic understanding of the pro-cesses influencing thiaminase activity in clupeids remains un-known (Wistbacka et al. 2002; Brown et al. 2005; Fitzsimonset al. 2005; Tillitt et al. 2005; Ikonen 2006). However, if inter-nal microbial populations are a source of thiaminase activity inthe Alewife and other clupeids, changes in physiological con-ditions (e.g., those resulting from environmental or other postu-lated sources of stress) that influence the growth characteristicsof bacteria can be expected to affect thiaminase activity (Tillittet al. 2005). For example, increases in Alewife thiaminase ac-tivity have been observed in association with decreases in watertemperature in natural systems and transport by truck and subse-quent holding in captivity (Tillitt et al. 2005; Lepak et al. 2008;J. Fitzsimons, Fisheries and Oceans Canada, personal commu-nication), and with immune system challenge (Wistbacka et al.2009). These increases in thiaminase activity may be related tophysiological stress, though the mechanism behind the observedincreases remains unknown.

Despite the fundamental role of clupeids in producing TDCin valuable apex predators including sport fishes, little is knownabout the factors responsible for inducing thiaminase productionin clupeids. Although research has focused mainly on Alewifeand Baltic Herring, other clupeid fishes can contain thiami-nase in high concentrations. For example, Gizzard Shad havehigh levels of thiaminase activity (means from 15,000 to 30,000pmol thiamine·g−1·min−1: Tillitt et al. 2005; Honeyfield et al.2008) relative to that for Alewife (on the order of 5,000 pmolthiamine·g−1·min−1: Tillitt et al. 2005). Given the tendency ofGizzard Shad to dominate biomass and their wide distributionin eastern U.S. waters (Bachmann et al. 1996; Hale et al. 2008),thiaminase in Gizzard Shad has the potential to negatively affectapex predator populations, the diets of which are dominated bythese prey fish. Therefore, we designed a set of experiments inreplicated pond systems to evaluate the influence of stressfulconditions on the thiaminase activity in Alewives and GizzardShad. Given the potential association between thiaminolyticbacteria and thiaminase in Alewife (or the production of thi-aminase by fish themselves), we expected that environmentalstressors that can influence physiological characteristics of clu-peids could also influence their thiaminase activity. Increasedstress in clupeids could increase thiaminase activity through anindirect mechanism whereby thiaminase is produced more effi-ciently by thiaminolytic bacteria or the bacteria proliferate be-cause of alterations in clupeid immune system function, or both,or thiaminase activity is increased directly by the induction ofphysiological changes in clupeids that result in the increasedproduction of thiaminase. Severe winter temperatures and lowfood availability were selected as treatments in this set of experi-ments. These treatments were selected because Alewives can besensitive to low temperatures (Colby 1973) and Gizzard Shad, inmore oligotrophic systems (i.e., where food availability is low),tend to have low densities, growth rates, and condition factors(Bachmann et al. 1996; Hale et al. 2008). We hypothesized that

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these treatments would result in stressful conditions, ultimatelyleading to an increase in clupeid thiaminase activity.

METHODSAlewife pond experiment.—Alewives were collected from

Waneta Lake, Schuyler County, NewYork, on the evening of26 October 2004 and transported by truck to the Cornell Ex-perimental Pond Facility in Ithaca, New York. Alewives (120–140 mm) were stocked at a density of 160 individuals intoeach of four fishless ponds (640 Alewives total) on the morn-ing of 27 October 2004. Each individual pond was approxi-mately 1,800 m3 with a maximum depth of approximately 2 m.Contrasting winter temperatures were produced by maintain-ing ice-free conditions (using an aeration system described inLepak and Kraft 2008) in two of the four study ponds from 11December 2004 to 5 January 2005. On 21 April 2005 each pondwas drawn down to a depth of approximately 1.0 m. A bagseine approximately 40 m long and 1.2 m tall was used to col-lect the remaining Alewives from the ponds. Alewives (10 fromeach pond) were collected for thiaminase analysis and analy-ses of circulating white blood cells. The remaining survivingAlewives from each pond were used for wet–dry weight anal-ysis as an indicator of fish condition. Alewives collected forthiaminase analyses were flash frozen and stored at −80◦C untilanalysis. Thiaminase analyses were conducted at the CanadaCentre for Inland Waters, Burlington, Ontario, using the proce-dure described by Zajicek et al. (2005). Mixed-model analysis(pond as a random effect and treatment as a fixed effect usingthe PROC MIX procedure) and ANOVA were conducted usingSAS (SAS 2010) to test for the effect of treatment on Alewifethiaminase and circulating white blood cell counts, and pondtemperatures, respectively.

Gizzard Shad pond experiment.—This experiment wasconducted at the experimental pond facility at the EcologyResearch Center at Miami University, Oxford, Ohio. Replicateponds (approximately 800 m3 with a maximum depth of ap-proximately 2.5 m) lined with heavy-duty plastic (covering anysediment present) were used to evaluate the influence of nutrientand sediment additions on pond chemistry and Gizzard Shad.Gizzard Shad from Acton Lake, Ohio, (source population)were stocked within the ponds. The treatments, each with threereplicates, were: no nutrient or sediment addition (no additions),nutrient addition (+N), sediment addition (+S), and addition ofnutrients and sediment (+N+S). The experiment was initiallydesigned to evaluate the influence of agricultural sediment andnutrient inputs on Gizzard Shad (Pilati et al. 2009). However,a thiaminase component was added to evaluate the influence ofstressful conditions associated with the lack of nutrients and sed-iments (a potential food source for Gizzard Shad: Babler et al.2011) in ponds with no nutrient or sediment additions, relativeto ponds in which nutrients, sediments, or both were added.

Details of the experimental set up and treatment conditionsare available in Pilati et al. (2009). Briefly, all nutrienttreatments (+N and +N+S) received weekly additions of

ammonium nitrate (NH4NO3, to provide N) and sodiumphosphate (NaPO4·H2O, to provide P) at loading rates of150 µg N/L and 15 µg P/L in pond water per week. Theseloading rates, designed to stimulate phytoplankton production,are similar to those in Acton Lake (Vanni et al. 2001). The+S and +N+S treatments first received a ∼2–3 cm layer ofsediments, and then weekly additions of sediments (0.06 m3,∼70 kg) dispersed evenly by means of a pump. Sediments wereobtained near the inflows to Acton Lake, and hence presumablywere composed mostly of allochthonous material. Weeklyinputs mimicked the long-term sedimentation rate in ActonLake (Renwick et al. 2005).

Ponds were filled on 29 May 2004 with water from an olig-otrophic supply pond (6.8 µg chlorophyll/L) and each exper-imental pond was subsequently inoculated with 400 L of wa-ter from Acton Lake to supply plankton for colonization. On10 June 2004, all ponds were stocked with 100 Gizzard Shadindividuals, at a biomass of ∼175 kg wet mass/ha. Two size-classes of Gizzard Shad were stocked, juveniles (120–140 mm,40 fish/pond) and adults (180–200 mm, 60 fish/pond). Theexperiment lasted 11 weeks, and on 8 September 2004, theponds were drained and all adult Gizzard Shad were collected,weighed and measured, and Fulton’s condition factor, K (anindicator of condition; K = W/L3 × 100,000, where W is themass in grams and L is the length in mm: Williams 2000), wascalculated.

Adult Gizzard Shad (three from each individual pond, ninein total for each treatment) were collected and frozen on dry icefor thiaminase analysis at two different times; the first groupof fish were collected and sent to the laboratory for analysiswithin 5 d of collection, and the second batch was evaluatedafter storage at −20◦C for approximately 9 months. Thiami-nase analyses were conducted at the Canada Centre for InlandWaters, Burlington, Ontario, using the procedure described byZajicek et al. (2005). ANOVA and ANCOVA were conductedusing SAS (SAS 2010) to test for the effect of treatment onthiaminase activity and condition (Fulton’s K), the relationshipbetween thiaminase activity and condition (Fulton’s K), and Nand P excretion rates of in Gizzard Shad. However, due to thelimited amount of data available for Gizzard Shad thiaminase, aMonte Carlo randomization simulation using 10,000 iterationswas conducted using SAS (SAS 2010) to assess the validity ofthe relationship between thiaminase activity and condition inGizzard Shad. Fulton’s K was based on mean values for adultshad collected from each of the four treatments after the com-pletion of the experiment.

RESULTS

Alewife Pond ExperimentMean thiaminase activity in Alewives was not influenced

by the treatments in this experiment. The treatments resultedin the fish being exposed for approximately 2 months (mid-December to mid-February) during which time the water

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FIGURE 1. Thiaminase activities (mean ± SE) (pmol thiamine·g−1·min−1,denoted as pmol·g−1·min−1) in Alewives from the source population (open bar,Waneta Lake) and control and treatment experimental ponds (black bars). Thenumber of fish tested (N) to establish each mean is shown at the base of theindividual bars.

temperatures in the treatment ponds (temperature was 1.2 ±0.6◦C, mean ± SD; below the lower lethal limit for Alewivesobserved in tank experiments by Colby 1973) were significantlycolder (ANOVA: F3 = 442.4, P < 0.01) than temperatures inthe control ponds (temperature was 3.6 ± 0.9◦C; temperatureswhere Alewives are routinely sampled in the Great Lakes). De-spite significant differences in blood cell composition (approx-imately 22,000 lymphocytes/µL in fish from cold ponds versus36,000 lymphocytes/µL in fish from control ponds) indicatingthere was stress associated with the experimental treatments (seeLepak and Kraft 2008), the mixed-model analysis that comparedcontrol and treatment groups showed no relationship betweenthiaminase activity and the prolonged exposure to cold temper-atures (mixed-model: F20, 20 = 0.37, P = 0.55; see Figure 1).Mean water content of Alewives after the experiment was within2% of the water content (70%) of the source population ofAlewives from Waneta Lake (see Lepak and Kraft 2008), whichwas within the range of other Alewives previously measured infreshwater systems (Hartman and Brandt 1995), and did not dif-fer significantly by treatment (Lepak and Kraft 2008). Similarly,mean thiaminase activity in the fish (approximately 5,200 pmolthiamine·g−1·min−1; Figure 1) after the experiment was withinthe range of thiaminase activities observed for wild-caughtAlewife populations in freshwater systems (range, 1,700–7,000pmol thiamine·g−1·min−1: Tillitt et al. 2005).

Gizzard Shad Pond ExperimentGizzard Shad from the second batch of samples sent for thi-

aminase analysis lost a consistent amount of thiaminase whilein storage (ANCOVA: F1 = 28.42, P < 0.01). A multiplecontrast comparison showed that least-squares means ± SE(accounting for the effect of batch) for thiaminase activities

FIGURE 2. Least-squares means ± SE (accounting for the effect of batch)for thiaminase activity in Gizzard Shad (pmol thiamine·g−1·min−1, denotedas pmol·g−1·min−1) from the source population (open bar, Acton Lake) andthe various treatments for the Gizzard Shad pond experiment (black bars). Thenumber of fish tested (N) to establish each mean is shown at the base of theindividual bars. The asterisk (*) denotes mean thiaminase activity that wassignificantly higher relative to other experimental ponds. Note the difference inscale of the y-axis relative to Figure 1.

in Gizzard Shad were significantly higher in fish from pondsthat were treated with nutrients (approximately 30,000 ± 1,800pmol thiamine·g−1·min−1) relative to ponds with no nutrient orsediment additions (22,000 ± 2,500 pmol thiamine·g−1·min−1)and those that had sediments (24,000 ± 1,900 pmol thiamine·g−1·min−1) or nutrients plus sediments (22,000 ± 2,100 pmolthiamine·g−1·min−1) added (ANCOVA: F3 = 4.31, P = 0.01;Figure 2).

Chlorophyll (µg/L), primary productivity (mg C·m−2·d−1),total phosphorus (µg P/L), and suspended solids (mg/L) weresignificantly lower in ponds with no nutrient or sediment ad-ditions relative to all other ponds (Pilati et al. 2009). AdultGizzard Shad condition (Fulton’s K) was significantly influ-enced by treatment (ANOVA: F3 = 87.60, P < 0.01). Specif-ically, Fulton’s K was lowest in ponds with no nutrient orsediment additions and significantly higher in the +S and +N+Streatments; K in the +N treatment was intermediate and not sig-nificantly different from the other treatments (Table 1). Thus,we observed the lowest K in control fish and the highest K infish from the +N+S treatment, while fish with an intermediateK (+N treatment) had the highest thiaminase activities. Be-cause thiaminase activity and condition factor were influencedby treatment, we conducted a comparison to evaluate whetherthiaminase activity was related to condition in Gizzard Shad.

TABLE 1. Gizzard Shad Fulton’s K (mean ± SD) by treatment.

Treatment Mean ± SD Fulton’s KNo additions 0.703 ± 0.071Nutrient 0.795 ± 0.085Sediment 0.807 ± 0.073Nutrients and sediments 0.838 ± 0.069

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An ANCOVA (F1 = 3.17, P = 0.09) showed that the relation-ship was not significant at the level of α = 0.05. These resultswere confirmed using a Monte Carlo randomization simulationshowing that the relationship was significant at the level of α =0.10 (an F-value of 3.04 was required to confirm this) but not atα = 0.05.

Importantly, excretion of P and N in Gizzard Shad was signif-icantly higher (approximately 1.5–3 times higher) in fish fromthe ponds where nutrients were added relative to all other ponds(Pilati et al. 2009). Further, analyses of bulk sediment compo-sition indicated that P, N, and organic C were the highest inponds where nutrients were added relative to all other ponds(Pilati et al. 2009). The combination of these two observationsindicates that the food available to Gizzard Shad in the pondswhere nutrients were added was of higher quality relative tofood in the other ponds. The thiaminase activities in fish fromthese ponds (those where nutrients alone were added) were sig-nificantly higher (30,000 ± 1,800 pmol thiamine·g−1·min−1,mean ± SE) than those from the other ponds (Figure 2).

DISCUSSIONThe results observed in this study did not support the hypoth-

esis that prolonged cold temperatures and low food availabilityproduced an increase in clupeid thiaminase activity. Followingthe experiments, the thiaminase activity found in Alewives andGizzard Shad held in pond systems was within the range ofthe original source populations. The only significant treatmenteffect on clupeid thiaminase was an observed increase in meanthiaminase activity in Gizzard Shad from the ponds where nu-trients alone were added.

Alewives and Gizzard Shad held under conditions that werehypothesized to increase thiaminase activities (prolonged coldtemperatures and low food availability) responded to experi-mental treatments in other ways. Alewives held in prolongedcold temperatures had significantly lower counts of circulatinglymphocytes relative to Alewives held in warmer water tem-peratures (Lepak and Kraft 2008). Gizzard Shad that were heldin ponds where nutrient and sediment additions were absenthad significantly lower Fulton’s K values and weighed less thanGizzard Shad held in ponds where nutrients, sediments, or bothwere added. These results were expected; however, no evidencewas found that linked these responses to thiaminase activity inclupeids. Arguably this is evidence, in the case of the Alewife,that there is a disconnect between the immune response andthiaminolytic bacteria, or between thiaminolytic bacteria andthiaminase activity in Alewives.

Increases in mean thiaminase activities have been observedin Alewives held in captivity (replicated tanks) and provideda high quality diet in the form of commercial pellet food thatwas heat treated, thereby denaturing any thiaminase found inthe feed (e.g., Lepak et al. 2008). Alewives maintained un-der laboratory conditions that were analyzed by Lepak et al.(2008) had thiaminase activities on the order of two- to three-

fold higher than thiaminase activities in wild-caught Alewives(ranging from 1,700 to 7,000 pmol thiamine·g−1·min−1 in thewild: Tillitt et al. 2005; Lepak et al. 2008), but while in captivitythese Alewives were not exposed to any known external sourcesof thiaminase (e.g., cyanobacteria, zooplankton, or other dietarysources). The clupeids in the current study were held in pondswhere natural food sources were available instead of being pro-vided heat-treated commercial pellet food. Again, the results ofthe pond study reported here support the laboratory experimentfindings of Lepak et al. (2008) in that the mean thiaminase ac-tivities of Gizzard Shad with access to relatively high qualityforage were elevated relative to those without access to highquality forage.

Gizzard Shad from the +N ponds were provided with highquality forage, which resulted in elevated P and N excretionrates and bulk sediment composition relative to the other ponds(Pilati et al. 2009). These shad had a mean thiaminase activitythat was significantly higher than that for fish from the otherponds, supporting the notion that a high quality (high nutrient)diet was consumed by shad in ponds where only nutrients wereadded (Pilati et al. 2009). Gizzard Shad held in ponds wheresediments were added were probably relying, at least to someextent, on these lower quality inputs for energy, experiencing a“dilution” effect on food quality (Heinrichs 1982; Mundahl andWissing 1987; Higgins et al. 2006). Recent data from severalOhio reservoirs suggests that Gizzard Shad rely on energy de-rived from both phytodetritus and terrestrial detritus, but theirbiomass is higher in reservoirs where phytodetritus is the dom-inant energy source (Babler et al. 2011). Together these resultssuggest that food quality was highest in the +N ponds, whereGizzard Shad thiaminase activity was also highest.

The one consistent finding across the set of experiments de-scribed in this study and from similar data from tank experimentscollected by Lepak et al. (2008) is that clupeids that had highquality diets available to them had higher thiaminase activitiesrelative to those that did not. The observed mean water content,Fulton’s K, and thiaminase activity of pond-reared Alewives andGizzard Shad in this study were within the range of wild-caughtfish (Hartman and Brandt 1995; Tillitt et al. 2005; Pilati et al.2009). However, the mean water content values of Alewives heldin the laboratory by Lepak et al. (2008) were lower than anyother previously reported values (Hartman and Brandt 1995),yet thiaminase activities were consistently higher than for anyAlewives previously evaluated from natural freshwater systems(Tillitt et al. 2005).

Although the isolation of thiaminase-positive bacteria fromAlewife viscera has been described as the potential source of clu-peid thiaminase (Honeyfield et al. 2002), the sources of observedthiaminase activity in Alewives remain uncertain (Richter et al.2012). Bacterial communities and their gene expression withinfish viscera are altered by different feeding regimes and fishcondition (Šyvokienė and Mickėnienė 1999). The results of ourexperiments suggest that clupeids feeding on high quality foodsources sustain high levels of thiaminase activity that could be

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fostered by internal conditions in clupeids that affect bacteriallyproduced thiaminase. Our results do not discount the possi-bility that clupeid physiology may be influenced by feedingconditions, and if clupeids produce thiaminase, feeding condi-tions could influence thiaminase activity. However, we specu-late that bacteria in clupeids react similarly to bacteria in otherorganisms. For example, thiaminolytic bacteria can proliferatein the gastrointestinal tracts of ruminants under certain feed-ing regimes (increased carbohydrate consumption), ultimatelyresulting in thiamine deficiency (Brent 1976). We note otherobservations in the animal husbandry literature regarding theincrease in thiaminase production in the presence of high qual-ity feed when a sudden change in rumen pH occurred (Brentand Bartley 1984; Zinn et al. 1987). The gastrointestinal tractof fish provides a similar environment with abundant thiamineduring periods of active feeding on high quality forage, and thetransition between the stomach and intestine provides a locationwhere pH can rapidly change from 7, mimicking con-ditions found in ruminants suffering from thiamine deficiency.

Although thiaminases have been generally considered in thefisheries literature for their role in degrading available thiamine,one form of thiaminase (thiaminase II, now referred to as tran-scriptional enhancer A or “TenA”) can function in thiaminesynthesis by salvaging pyrimidine from ring-opened thiamine(Toms et al. 2005; Jenkins et al. 2007). Jenkins et al. (2007)and Bettendorff (2007) suggest that the primary function of thi-aminase II is associated with thiamine biosynthesis by bacteria,rather than destruction. In light of these findings, Soriano et al.(2008) proposed that the evolution of thiamine-degrading activ-ity by thiaminase I might be linked to a salvage pathway thatrecycles degraded forms of thiamine.

Thiamine degradation occurs under environmental condi-tions in which pH > 7 (Maier and Metzler 1957); therefore,fish gastrointestinal tracts with pH > 7 could be conduciveto thiaminase-producing bacteria capable of utilizing degradedforms of thiamine. In our study, Gizzard Shad from environ-ments where high quality forage was available were observedto have the highest levels of thiaminase activity. Similarly, weobtained results in a laboratory study in which we found thatAlewives held in captivity and fed high quality diets in the formof commercial pellet feed had greater thiaminase activities thanthose previously reported (Lepak et al. 2008). We suggest thatconsumption of high quality food sources could have led to theproduction of thiamine degradation products that are salvagedfor thiamine synthesis by bacteria, fostering conditions that trig-ger the production of bacterial thiaminase. Thus, if thiaminolyticbacteria are the primary source of thiaminase in clupeids (notclupeids themselves), it is possible that clupeid feeding con-ditions could result in altered expression of thiaminase or theproliferation of thiaminolytic bacterial populations, or both.

Progress in understanding TDC in valuable fisheries will re-quire a better understanding of several phenomena stemmingfrom the observation that thiaminase has been primarily foundin the visceral tissues of fish and only small amounts of thiami-

nase have been detected in muscle tissue (Fujita 1954). First,we need to determine the primary sources of fish thiaminaseactivity. Then, we need to determine the environmental factorsthat regulate and affect production of thiaminase by bacteriaor fish themselves, or both. Finally, we need a model fish sys-tem in which thiaminase activity can be manipulated in orderto document the conditions that affect thiaminase activity inthese organisms. If thiaminase is produced primarily by bacteriawithin fish, we will need a broader understanding of the vari-eties and abundance of thiaminase I-producing bacteria presentin specific environmental conditions, (e.g., within fish gastroin-testinal tracts). Thus, the characterization of internal clupeidmicrobial communities and their expression of thiaminase (orlack thereof) will provide important insights into thiaminaseresearch, help determine the ultimate sources of thiaminase infish, and help focus future research.

Understanding the dynamics of thiaminase expression in clu-peids will aid ongoing efforts to reestablish sustainable, natu-rally reproducing salmonine communities in the Great Lakes andmaintain naturally reproducing salmonine communities aroundthe world. Given the dependency of salmonines on clupeidsas forage, the ongoing spread of clupeids across the UnitedStates and Canada, and the threat of thiamine deficiency asso-ciated with clupeids to salmonine natural reproduction, it willbe important for scientists, managers, and stakeholders to rec-ognize and acknowledge the importance of these prey fish. Forexample, further introductions of Alewives carry serious, yetunpredictable, implications related to salmonine communitiesbecause of variation in Alewife population size and thiaminasecontent. Continuing efforts to evaluate clupeid thiaminase vari-ability and the use of whole-system manipulations and otherinnovative techniques will provide applicable results that couldlead to the remediation of the negative impacts that clupeidshave on salmonines and possibly other predators.

ACKNOWLEDGMENTSWe thank Tom Brooking, Lars Rudstam, Robert Johnson,

Paul Bowser, Richard DeFrancisco, Joanne Messick, SteveLamb, and William Ridge for valuable insights and equip-ment at the onset of this project. We thank Lisa Brown forconducting thiaminase analyses and Jennifer Sun for conduct-ing white blood cell differential counts. Jennifer Sun, LaurenGallaspy, Jillian Cohen, Geoffrey Steinhart, Dana Warren, Ja-son Robinson, Daniel Josephson, Peter Stevens, Beth Boisvert,Hannah Shayler, and the Cornell Pond Facility staff providedtechnical and logistic support. Madeleine Mineau, AlexandraDenby, Summer Rayne Oakes, Nathan Smith, Tara Bushnoe,Thomas Bell, Mark Dettling, Jeremiah Dietrich, Geofrey Eck-erlin, Michael Estrich, Taylor McLean, Ned Place, Kirk Smith,and Theodore Treska provided support in the field. RichardDeFrancisco and Joanne Messick provided laboratory materi-als and training. New York Sea Grant provided funding forthe Alewife tank and pond experiments under project number

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96 LEPAK ET AL.

R/FBF-15. We are grateful to the staff of the Ecology ResearchCenter at Miami University for support during the pond ex-periment. Gizzard Shad pond experiments were supported by aNational Research Initiative (NRI) grant (OHOR-2003-01756)from the U.S. Department of Agriculture, and a Summer Work-shop grant (Department of Zoology, Miami University).

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Effect of Dietary Herbal Supplements on SomePhysiological Conditions of Sea Bass DicentrarchuslabraxSevdan Yılmaz a , Sebahattin Ergün a & Ekrem Şanver Çelik ba Department of Aquaculture, Faculty of Marine Sciences and Technology, Çanakkale OnsekizMart University, Çanakkale, Turkeyb Department of Basic Science, Faculty of Marine Sciences and Technology, ÇanakkaleOnsekiz Mart University, Çanakkale, TurkeyVersion of record first published: 05 Apr 2013.

To cite this article: Sevdan Yılmaz , Sebahattin Ergün & Ekrem Şanver Çelik (2013): Effect of Dietary Herbal Supplements onSome Physiological Conditions of Sea Bass Dicentrarchus labrax , Journal of Aquatic Animal Health, 25:2, 98-103








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Effect of Dietary Herbal Supplements on Some PhysiologicalConditions of Sea Bass Dicentrarchus labrax

Sevdan Yılmaz* and Sebahattin ErgünDepartment of Aquaculture, Faculty of Marine Sciences and Technology,Çanakkale Onsekiz Mart University, Çanakkale, Turkey

Ekrem Şanver ÇelikDepartment of Basic Science, Faculty of Marine Sciences and Technology,Çanakkale Onsekiz Mart University, Çanakkale, Turkey

AbstractThis study was conducted in order to investigate the effects of

dietary thyme Thymus vulgaris, rosemary Rosmarinus officinalis,and fenugreek Trigonella foenum graecum as feed additives on totalliver fat levels and biometric indices of Sea Bass Dicentrarchuslabrax. Four isonitrogenous (48% crude protein) and isocaloric(21 kJ/g) diets were formulated to contain 0% (control), or 1% ofthyme, rosemary, or fenugreek. In a 45-d feeding trial, 12 fiberglasstanks (140 L) were each stocked with 17 fish (20.43 ± 0.03 g).Herbal supplemented diets

pharmacokinetics and tissue distribution of thiamphenicol and … · 2013. 6. 20. ·...

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