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The influence of ploidy on saltwater adaptation, acute stressresponse and immune function following seawater transfer in

non-smolting rainbow trout

J.F. Taylor *, M.P. Needham, B.P. North, A. Morgan, K. Thompson, H. Migaud

Institute of Aquaculture, University of Stirling, Stirling, Scotland FK9 4LA, UK

Received 14 September 2006; revised 23 February 2007; accepted 24 February 2007Available online 2 March 2007

Abstract

We investigated the effect of ploidy on osmoregulatory, stress and immune responses in non-smolting rainbow trout during saltwateradaptation. Sibling groups of diploid and triploid trout were acclimated in freshwater (FW) and then subjected to abrupt transfer to fullstrength (35 ppt) saltwater (SW) or back to FW. Fish were sampled pre-stress, and 1, 3, 6, 12, 24, 48, 72 and 168 h post-stress. Overallmortality in SW was less than 5% in either ploidy, with no mortality in FW. Significant elevations in plasma osmolality and gill ATPasewere observed within 13 h of SW transfer, but retuned to basal levels within 72 h indicative of rapid saltwater adaptation and did notdiffer between ploidy. Furthermore, FWSW transfer also caused significant and sustained elevations in total blood haemoglobin,plasma IGF-I, cortisol, glucose, total white blood cell counts, increased plasma but decreased mucus lysozyme, and enhanced head kid-ney macrophage respiratory burst activity. Conversely, FWFW transfer evoked more transient and less elevated responses, more typicalof primary and secondary responses to a single stressor. We conclude that the more elevated levels in these parameters are a function ofsaltwater adaptation as well as the generic stress response, and that this did not differ between ploidy. Strong positive correlations were

found between plasma IGF-I and cortisol, and with osmolality, glucose and WBC, while a negative correlation was found with plasmalysozyme irrespective of ploidy. Overall, the current results suggest that triploidy does not affect the ability of non-smolting trout to adaptto full strength seawater under optimum conditions, and that the osmotic and stress response to such transfer is similar to diploids. 2007 Elsevier Inc. All rights reserved.

Keywords: Triploid; Sea water challenge; Osmoregulation; Stress; Immune function; Rainbow trout

1. Introduction

Rainbow trout (Oncorhynchus mykiss) is one of the mostimportant aquaculture species worldwide, with a growing

trend towards the production of large (3 kg+) sea-growntrout since this improves growth and flesh quality. A signif-icant problem encountered when growing large trout is ahigh incidence of pre-harvest maturation, which is associ-ated with the diversion of energy into gonadal recrudes-cence, leading to reductions in somatic growth and fleshquality, as well as increased susceptibility to disease

(Bromage et al., 2001). Unlike the Atlantic salmon (Salmosalar) industry which uses photoperiod application exten-sively during grow-out to resolve this problem, such tech-niques have not been successful with trout to date. In this

respect, artificial induction of triploidy may offer a poten-tial solution to the problem.Morphologically, triploids are similar to diploids but

differ in three fundamental ways; being generally more het-erozygous, having larger but fewer cells due to the extrachromosomal set, and gonadal development is generallydisrupted resulting in sterility (Benfey, 1999), the latterbeing the trait of greatest interest to industry. However,there is significant scepticism within the industry regardingthe use of triploids due to reports on inferior performance,morphological abnormalities, reduced immune function,

0016-6480/$ - see front matter 2007 Elsevier Inc. All rights reserved.

doi:10.1016/j.ygcen.2007.02.029

* Corresponding author. Fax: +44 01768 472133.E-mail address: [email protected](J.F. Taylor).

www.elsevier.com/locate/ygcen

General and Comparative Endocrinology 152 (2007) 314325
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and higher mortalities. Within the literature there is stilldebate between fish and ploidy status regarding their phys-iology, and the mechanisms responsible for the apparentperformance differences are poorly understood. Further-more, high rates of mortality have been observed particu-larly under sub-optimal conditions such as hypoxia or

high temperature (Ojolick et al., 1995; Cotter et al., 2000;Hyndman et al., 2003b). Impaired ability to maintain suffi-cient oxygen supplies during and after stress may relate tothe cellular nature of triploidy and generally lower bloodhaemoglobin concentrations (Benfey and Sutterlin, 1984;Sadler et al., 2000a).

Rainbow trout have been shown to undergo a typicalstress response when transferred from fresh to saltwaterenvironments (Hegab and Hanke, 1986; Liebert andShreck, 2006). It is well known that both acute and chronicstress can increase susceptibility of fish to disease (Schreck,1997; Wendelaar Bonga, 1997). A major factor responsiblefor this is an alteration in both the number and composi-

tion of circulating leucocytes (Barton and Iwama, 1991).Acute stress often results in a decrease in circulating totalleukocyte concentrations (Benfey and Biron, 2000). Stressalso affects the phagocytic and/or respiratory burst activityof spleen, head kidney and plasma leucocytes (Thompsonet al., 1993; Pulsford et al., 1994; Vazzana et al., 2002; Lie-bert and Shreck, 2006). In addition, the response in plasmalysozyme activity following acute stress has been shown tobe both enhanced as well as suppressed depending on thetype, intensity and duration of the stressor (Fevoldenet al., 2003). However, despite considerable research intostress and immune response to environmental challenge,

differences in salinity tolerance between diploid and trip-loid trout remain unexplored.

The initial period following seawater transfer is an extre-mely stressful event for anadromous fish which must com-pensate for the effects of changing environmental salinity,and alter the morphology and physiology of the ionic-osmotic regulatory apparatus in order to maintain homo-eostasis (McCormick, 2001; McCormick and Bradshaw,2006). In salmonids, this involves an increase in numberand size of chloride cells in the gill and intestine, bothmajor sites of active ionic transport, characterised by highconcentrations of mitochondria and Na+, K+, ATPaseactivity (McCormmick, 1995). In fish, marked elevationsin numerous endocrine hormones including cortisol,growth hormone (GH) and insulin-like growth factor-I(IGF-I) have been documented which subsequentlyincrease Na+, K+, ATPase activity and enhance saltwatertolerance (McCormick, 2001). This process occurs priorto seawater transfer during smoltification which pre-adaptssmolts to life in the sea in the case of anadromous salmo-nids (Handeland et al., 2004). Conversely, non-smoltingstrains of rainbow trout are not required to undergo anypre-adaptation to seawater (Jackson, 1981; Alexis et al.,1984). The presence of salt alone appears to result in anincrease in chloride cell size as well as Na+, K+, ATPase

activity (Salaman and Eddy, 1987). In this respect, it has

been suggested that the cellular characteristics of triploidsmay result in a reduction in the capacity for osmoregula-tion, associated with a smaller surface area for active iontransport (Sadler et al., 2000a). Furthermore, increasedincidence of gill deformity and reduced cell surfacearea:volume ratios in triploid fish may significantly lower

the capacity to utilise oxygen in the marine environment,and subsequently reduce survival (Sadler et al., 2000b;Cal et al., 2006).

To date the ability of triploid trout to adapt to seawatertransfer has received very little attention with regards toionic-osmotic physiology or the stress response in relationto their diploid conspecifics. Therefore the objectives ofthe current study were to (a) characterise the adaptiveresponse of a non-smolting rainbow trout to abrupt seawa-ter transfer, (b) to compare this response between siblingdiploids and triploids and (c) use these results to clarifywhether or not the increased mortality of triploids duringseawater transfer is due to an inability to adapt. This was

achieved by studying a number of indicators of osmoregu-latory ability as well as primary and secondary stress andimmune parameters.

2. Materials and methods

2.1. Fish and facilities

All-female rainbow trout eggs of a non-smolting strain were obtainedfrom Barony College (Dumfries, Scotland, UK) in January 2005. Bothdiploids and triploids originated from the same parent broodstock(1 male:2 females) to eliminate any genotype interaction. Triploidy wasinduced by applying a hydrostatic pressure shock of 9500 psi, for 5 min,

200

C min post-fertilisation. Erythrocyte major axis length was used toverify triploidy status prior to experimentation. Verification was con-firmed with a mean axis length of 35.50 0.13 lm in control diploidsand 54.50 0.24 lm in triploids (p < 0.001). Hatching (April 2005) andpre-experimental rearing took place at the Niall Bromage FreshwaterResearch Laboratory (NBFRL, Stirling, Scotland, UK, 57N). Duringthe rearing period fish (1000 per ploidy) were ongrown in 25 m3 circulartanks (one per ploidy) within a flow through system under ambient lightand temperature and were fed on a commercial pelleted feed (Trouw, Elite45) according to manufacturers feeding tables.

2.2. Experimental design and sampling procedure

Two weeks prior to each experimentation, fish from the common pop-ulation were anesthetised using 2-phenoxyethanol (1:10,000 Sigma Chem-

icals, UK) and hand graded into two batches of 80 fish (one per ploidy)and held in separate 1 m3 FW tanks. Initial mean weight and length were191.1 9.3 g and 256.4 4.0 mm, respectively, and was maintained for alltrials. Stocking density during acclimation was approximately 16 kg m3

and tanks received constant aeration with mechanical/biological filtration,with 20% water changes made every other day using de-chlorinated tapwater. Fish were left to acclimate for a period of 2 weeks prior to experi-mental saltwater (SW) or freshwater (FW) challenge during which timethey were fed three times weekly to excess on a commercial pelleted feed(Trouw, Elite 45).

Following the acclimation period, fish were subjected to abrupt trans-fer from either FW to full strength (35 ppt) SW, or FW to FW (used todiscriminate between handling and SW induced responses). FWSW chal-lenge was carried out in duplicate with replicates 1 and 2 taking placebetween 1017th and 24th April1st May 2006, respectively. Unfortu-

nately, due to tank and system limitations the FWFW trial could not

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be replicated, with the trial carried out from 17 to 24th April. For eachtrial a baseline sample (0 h) was taken (n= 6) from each ploidy prior totransfer. During transfer, fish were rapidly netted into the experimentaltanks within 5 min from initial disturbance for each ploidy. Two tanks(one per ploidy) were assigned to each time point during the trials; 1 h,3 h, 6 h, 12 h, 24 h, 48 h, 72 h and 7 days post-transfer. Time points wereallocated different tanks to ensure the responses measured were due to theexperimental challenge and not the effect of repeated disturbance, in par-ticular with regards to cortisol. Each tank was stocked with 10 fish so that10 diploids and 10 triploids were assigned to every time point (stockingdensity of approximately 6 kg m3). Fish were allocated in block designso that diploids and triploids for each time point were situated next toone another. The SW trial was carried out using a fully recirculating par-allel line tank (0.5 m3) system. During acclimation in FW and SW chal-lenge water temperature was maintained at 10 1 C and totalammonia (TAN) remained below 3 mg ml1 with dissolved oxygen>7 mg l1. The FW control trial was carried out using a flow-through tank(0.5 m3) system. During acclimation and throughout the FW trial temper-atures were maintained at 8 1 C and dissolved oxygen >7 mg l1.

At each time point six diploid and six triploid fish were netted fromtheir respective two tanks and instantly killed with a lethal dose of 2-phen-oxyethanol (1:100; Sigma Chemicals, UK). Fish were immediately bledfrom the caudal vein with heparinised syringes (total volume of approxi-mately 23 ml) and sub-samples (200 ll) of whole blood were placed asideon ice for haematological analysis. The remaining blood was centrifuged(1200gfor 15 min) and the plasma was aliquoted for plasma analyses ofcortisol, IGF-I, glucose, osmolality and lysozyme activity and stored at

70 C prior to analysis. In each case, all fish were bled within 35 minin order to minimise the affects of sampling stress (i.e. disturbance and net-ting). Cutaneous mucus was collected from the entire body surface of eachusing the back of a sterile scalpel blade. Mucus was washed in an ammo-nium bicarbonate buffer (100 mM) and centrifuged (2500gfor 5 min) afterwhich the supernatant was removed and stored at 70 C prior to analysisfor mucus lysozyme activity. Gill biopsies were performed on the secondgill arch and 34 filament tips were stored in a sucrose, sodium EDTAand imizadole (SEI) buffer at 70 C prior to determination of gill Na+,K+, ATPase activity. At 0 h, 24 h, 72 h and day 7 the anterior kidneywas aseptically removed and forced through a 100 lm nylon mesh intoheparinised Leibovitz-15 medium (Sigma, Aldrich, UK) and the resultantmacrophage suspensions were immediately assayed for respiratory burstactivity.

2.3. Sample analyses

Total white (WBC) and red blood cell (RBC) counts were taken fromwhole blood diluted in phosphate buffered saline (WBC, 1:100; RBC,1:1000). Counts were made under an Olympus CH light microscope usinga Neubauer haemocytometer. Haematocrit (Hct, expressed as % of packedRBC volume) was measured on a micro-haematocrit reader after centri-fuging whole blood (5000gfor 5 min) in heparinised micro-haematocrittubes. Total blood haemoglobin concentration (Hb) was measured byDrabkins colorimetric assay using a commercially available kit and stan-

dard (Sigma, Aldrich, UK). From the previous parameters mean corpus-cular volume (MCV), mean corpuscular haemoglobin (MCH) and meancorpuscular haemoglobin concentration (MCHC) were obtained usingthe following formulas (Perruzi et al., 2005): MCV (fL) = Hct/RBC(106 ll1); MCH (pg) = [Hb (g dl1) 10]/RBC (106 ll1); MCHC(g dl1) = [Hb (g dl1) 10]/Hct.

Plasma osmolality were measured on sub-samples using a micro-osmometer (Advanced Instruments, Massachusetts, US) in order to deter-mine the time course of osmotic disturbance. Gill Na+, K+, ATPase wasdetermined using the method outlined by McCormick (1993) which isbased on the measurement of NADH oxidation by the ouabain sensitivehydrolysis of ATP and expressed as lmol mg prot1 h1.

Plasma cortisol concentrations were determined using the radioimmu-noassay method described by Ellis et al. (2004) and adaptated byNorthet al. (2006). The tritiated label was supplied by Amersham Biotech

(UK) and a sheep anti-cortisol antibody from Diagnostic Scotland

(UK). Intra- and inter-coefficients of variation were 2% and 11%, respec-tively. Minimum sensitivity was 12 pg tube1. Total plasma IGF-I wasmeasured using Gropep Ltd. Fish RIA kits following acid:ethanol extrac-tion. Extraction involved the incubation of 40 ll of plasma with 160 llacid:ethanol mix (62.5 ml 2 M HCl:437.5 ml 100% ethanol) for 30 min atroom temperature. The supernatant was neutralised with 80ll of0.855 M Tris. Samples were centrifuged at 10,000gfor 10 min at 4 C.The resultant supernatant was collected and 50 ll assayed in triplicateaccording to the Gropep kit protocol. The detection limit was0.15 ng ml1, with intra- and inter-assay coefficient of variation of 4.4%(n = 10) and 13.9% (n= 10), respectively. Plasma glucose concentrationswere assayed using a colorometric method (Sigma, Aldrich, UK). Plasmasamples were pipetted into a 96-well plate, in quadruplicate, along withTrinder reagent. Change in absorbance (505 nm), as a result of the glucosedependant production of quinoneimine dye, was measured after 5 min inan ELISA microplate reader (Dynex, UK). Plasma and mucus lysozymeactivities were assayed using a 96-well plate method adapted from Ellis(1990). Samples of plasma/mucus were added to a suspension of Micro-coccus lysodeiticus in 0.04 M sodium phosphate buffer (pH 5.8). Thechange in absorbance (540 nm), dependant on the rate of lysis, was mea-sured after 1 and 5 min.

Respiratory burst activity was measured by the rate of reduction ofnitro blue tetrazolium (NBT) into blue formazan which is dependant onthe rate of superoxide production during respiratory burst (Secombes,1990). Samples (200 ll) of head kidney suspension were pipetted into a96-well plate in six replicates and incubated to form monolayers. Phorbolmyristate (PMA) was used to stimulate respiratory burst in half of thesereplicates and the difference in absorbance (605 nm) between unstimulatedand stimulated cells was used to determine the rate of activity. The meannumber of macrophages per sample was calculated by lysing a sub-sampleof cells and counting the number of nuclei as described for WBC and RBCcounts. Values are expressed as NBT reduction (OD 605 nm) per 10 5 cells.

Finally, cumulative mortality was recorded throughout the period ofthe trials.

2.4. Statistical analyses

Data were checked for normality and homogeneity of variance byKolmogornovSmirnov and Bartletts F test. Where appropriate logtransformations where applied to normalise the data. A three-way nestedANOVA was also applied in order to calculate the overall effects of time,ploidy, and treatment (SW or FW) on all parameters measured. Tukeyspost hoc tests were applied to identify where significant differencesoccurred. Preliminary analyses prior to regression analysis were performedto ensure normality, linearity and homoscedasticity. Relationshipsbetween IGF-I, cortisol, osmolality, glucose and total white blood cellcounts (WBC) were tested using Pearson product moment correlationcoefficient and simple linear regression. All statistical analyses were carriedout on Minitab (v14). Significant differences were determined at p 6 0.05.All results are presented as means SEM with saltwater and freshwatertrials referred to by the abbreviations SW and FW, respectively. No signif-icant differences were seen between replicates with the exception of head

kidney macrophage respiratory burst activity.

3. Results

3.1. Growth and mortality

Throughout each trial weight, length and conditionfactor did not differ significantly between ploidy orbetween trials. During SW challenge total mortality was2.5% (n = 2) and 5% (n = 4) in triploids and diploids,respectively, with mortalities occurring at 72 h post-trans-fer. No mortalities were recorded throughout the

FWFW control trial.

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3.2. Haematology

Haematocrit (Hct) remained constant over time in bothdiploids (31.3 2.0% PCV) and triploids (32.0 1.5%PCV) and did not differ between FWSW and FWFW.

RBC concentration also remained constant over time in

both diploids and triploids and did not differ between FWSW and FWFW trials. However, triploids had signifi-cantly lower RBC concentrations than diploids (6.5 0.7vs. 9.2 0.7 109 cells ml1).

Triploids had significantly lower WBC concentrationsthroughout the FWSW trial but not during the FWFWtrial except at 1 and 72 h post-transfer (Fig. 1a and b).Resting WBC concentrations were significantly elevatedwithin 1 h of seawater transfer and remained so until 3and 12 h post-transfer in triploid and diploids, respectively(Fig. 1a). Thereafter levels declined and remained relativelysimilar to pre-transfer concentrations. WBC concentrationremained constant throughout the FW trial (Fig. 1b). In

SW, WBC levels were significantly higher in diploids than

in FW between 1 and 72 h whereas in triploids this wasonly observed at 3, 6 and 72 h.

Ploidy did not affect total blood haemoglobin (Hb) ineither trial (Fig. 1c and d). Hb increased significantly by12 h post-SW transfer, remained constant and significantlyhigher than pre-transfer levels until 72 h, before returning

to pre-transfer concentrations by day 7 (Fig. 1c). No signif-icant differences in Hb were apparent in FW betweenploidy over time, although levels fluctuated (Fig. 1d). Meancorpuscular volume (MCV) remained constant in both tri-als irrespective of time or salinity, with triploids having asignificantly higher MCV than diploids (SW: 2n34.2 3.2 fL vs. 3n 52.0 2.5 fL; FW: 2n 36.4 1.3 fLvs. 3n 50.3 1.6 fL, p < 0.0001). Mean corpuscular Hb(MCH) also remained constant in both trials irrespectiveof time or salinity, with diploids having a significantlylower MCH than triploids (SW: 2n 11.3 0.4 pg vs. 3n16.3 0.6 pg; FW: 2n 11.4 0.3 pg vs. 3n 15.7 0.6 pg,p< 0.0001). Mean corpuscular Hb concentration (MCHC)

increased significantly post-SW transfer in each ploidy

0 6 12 18 24 30 36 42 48 54 60 66 72 168

0 6 12 18 24 30 36 42 48 54 60 66 72 168

WBC(107ml-1)

0

1

2

3

4

52n FW-SW

3n FW-SW

0 6 12 18 24 30 36 42 48 54 60 66 72 168

0

1

2

3

4

5

2n FW-FW

3n FW-FW

a

b

a

b

a*a*a*

a*a

a

a

b

ab*

b*

bb b

b

b

Time (h)

0 6 12 18 24 30 36 42 48 54 60 66 72 168

MCHC(gdL-1)

0

20

25

30

35

40

45

50

55

Time (h)

0 6 12 18 24 30 36 42 48 54 60 66 72 168

0 6 12 18 24 30 36 42 48 54 60 66 72 168

Hbconc.(gdL-1)

0

8

9

10

11

12

13

14** *

**

*

*

*

**

* *

Fig. 1. Time course changes in total white blood cell concentration (WBC, a and b), total blood haemoglobin (Hb, c and d) and mean corpuscularhaemoglobin concentration (MCHC, e and f) in diploid (black) and triploid (white) rainbow trout transferred from FW to SW (circles) or FW to FW(squares). Values are expressed as means SEM (n= 6 fish/ploidy/tank). Significant differences between resting (0 h) and post-transfer (1168 h) values

are indicated by *. Significant differences between ploidies at a given time point are indicated by different superscripts.


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(Fig. 1e). No significant differences between ploidy wereobserved. Diploids reached a significantly higher MCHCthan pre-transfer levels at 12 h, while triploids achieved thisby 72 h. In general both ploidy showed a general increasein MCHC from 12 h onwards, with highest MCHCobserved on day 7 in both ploidy. Conversely, no signifi-

cant differences in MCHC were observed as a factor of timeor ploidy in FW (Fig. 1f).

3.3. Salt balance

SW transfer resulted in rapid and significant elevationsin plasma osmolality. However, there were no ploidy differ-ences in SW at any time point (Fig. 2a). Plasma osmolalitywas significantly elevated above pre-transfer levels from 1to 3 h in triploids and diploids, respectively. Levelsremained significantly elevated until 48 h, thereafter return-ing to basal. In FW, handling and transfer caused a brief

disturbance in plasma osmolality of triploids which was

significantly elevated over pre-transfer levels, and higherthan diploids but returned to basal by 3 h (Fig. 2b). Dip-loids did not show change in osmolality at this time point.

SW transfer induced an immediate marked increase ingill Na+, K+, ATPase activity in both diploids and triploidsfrom

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remained elevated above basal levels until 12 and 24 h post-transfer in diploid and triploid fish, respectively (Fig. 3a).Thereafter levels declined to basal and remained constant.FWFW transfer in trial 2 did not induce any change inplasma IGF-I over time in either ploidy, although bothshowed slightly elevated levels 3 h post handling (Fig. 3b).

SW transfer induced a significant elevation in plasmacortisol in both diploids and triploids which only returnedto basal levels (

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Triploids had significantly higher levels of activity than dip-loids after 12 h in SW, but at no other point were ploidy dif-ferences observed. Plasma lysozyme activity did not changeduring the FW trial in either ploidy (Fig. 4b). Lysozymeactivity in both triploids and diploids in the SW trial was sig-nificantly elevated above those in the FW trial from 12 honwards in triploids and 24 h onwards in diploids.

There was no significant effect of ploidy on mucus lyso-zyme activities in either trial, although triploids generallyhad lower resting levels (Fig. 4c and d). A depression inmucus lysozyme activity was observed within 3 h of trans-fer to SW in both ploidies although not significant due tohigh variation (Fig. 4c). Activities at the final time pointwere significantly lower than resting level in diploids.FWFW transfer did not affect mucus lysozyme activity(Fig. 4d). No significant differences were seen between theSW and FW trials at any time point.

Replicate differences for respiratory burst were observed

between the two SW trials which is believed to be due to

poor adhesion of macrophages to the ELISA plate in theassay of replicate 2 as indicated by low nuclei counts fol-lowing lysis. As such, replicate 2 was discarded from anal-ysis (Table 1). However, triploid macrophages generallydisplayed a 50% greater respiratory burst activity than dip-loids in both saltwater and freshwater trials although thisdifference was only significant at 24 h in replicate 1 ( Table1). Both diploids and triploid macrophages also displayedan increased activity by approximately 50% within 24 hpost-SW transfer. Activity peaked at 72 h in both ploi-dies returning to basal levels by the end of the experiment.Conversely, FWFW transfer did not appear to stimulateactivity.

A strong positive correlation was found between plasmaIGF-I and plasma cortisol levels in the saltwater trials irre-spective of ploidy (Fig. 5a). Conversely, no such relation-ship was found in the freshwater trial (Fig. 5b). In bothploidy, strong positive correlations were found between

plasma cortisol and osmolality, WBC and glucose follow-

0 6 12 18 24 30 36 42 48 54 60 66 72 168

Plasmalysozymeactivity(unitsmin-1m

l-1)

0

500

1000

1500

2000

2500

3000

2n FW-SW

3n FW-SW

0 6 12 18 24 30 36 42 48 54 60 66 72 168

0

500

1000

1500

2000

2500

3000

2n FW-FW

3n FW-FW

Time (h)

0 6 12 18 24 30 36 42 48 54 60 66 72 168

Mucuslysozymeactivity(unitsmin

-1m

l-1)

0

100

200

300

400

500

600

Time (h)

0 6 12 18 24 30 36 42 48 54 60 66 72 168

0

100

200

300

400

500

600

*

*

*

* *

* *

*

a*

b

*

Fig. 4. Time course changes in plasma lysozyme (a and b) and mucus lysozyme activity (c and d) in diploid (black) and triploid (white) rainbow trouttransferred from FW to SW (circles) or FW to FW (squares). Values are expressed as means SEM ( n = 6 fish/ploidy/tank). Significant differencesbetween resting (0 h) and post-transfer (1168 h) values are indicated by *. Significant differences between ploidies at a given time point are indicated bydifferent superscripts.


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ing FWSW transfer (Table 2). However, this relationshipwas only found between cortisol and osmolality followingFWFW transfer. In contrast, a strong negative relation-ship was found between cortisol and plasma lysozyme inboth ploidies following FWSW transfer, but was notfound in the FWFW trial. Similarly, strong positive corre-lations were found between plasma IGF-I and osmolality,WBC and glucose in both FWSW and FWFW trials.A strong negative relationship was also found between

IGF-I and plasma lysozyme in both ploidies followingFWSW transfer, but not in the FWFW trial.

4. Discussion

Few studies have investigated osmoregulation and stressresponses in non-smolting salmonids transferred to seawa-ter (Jackson, 1981; Alexis et al., 1984; Hegab and Hanke,1986; Besner and Pelletier, 1991; Almendras et al., 1993;

Table 1Respiratory burst activity (per 105 cells) determined by NBT reduction in the presence of PMA by head kidney leukocytes prior- (0 h) and 24, 72, and168 h post-transfer to seawater and freshwater in diploid and triploid rainbow trout

Treatment Ploidy Time (h)

0 24 72 168

FWSW Diploid 0.54 0.13a 1.04 0.25a 1.57 0.44ab 0.47 0.14b

Triploid 1.97 0.81

a

3.31 0.97

b

3.92 1.31

b

1.95 0.67

b

FWFW Diploid 0.27 0.08a 0.22 0.04a 0.16 0.02a 0.25 0.04a

Triploid 0.43 0.09a 0.97 0.13a 0.50 0.10a 0.27 0.06a

Values given are means SEM (n = 6 fish/ploidy/tank). Significant differences between ploidies at a given time point are indicated by different letters.

Cortisol (ng ml-1

) Cortisol (ng ml-1

)

0 20 40 60 80 100 120 140 160

IGF-I

(ngml-1)

IGF-I

(ngml-1)

0

50

100

150

200

250

300

350

y = 1.95x - 7.78

r2= 0.71

p

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Liebert and Shreck, 2006). Furthermore, comparisons ofsuch studies are made difficult by the diversity of species,salinities and speed of transfer tested. A limited amountof work has focused on triploids (Biron and Benfey,1994; Benfey and Biron, 2000; Sadler et al., 2000a,2000b;Hyndman et al., 2003a,2003b) and to our knowledge no

studies regarding saltwater adaptation and immune func-tion in triploid rainbow trout have been published to date.This work is thus the first to address within the same studythe influence of ploidy on osmoregulatory, stress andimmune responses in a non-smolting rainbow trout strainfollowing salt water challenge.

Our findings clearly show non-smolting rainbow troutwere able to adapt to full strength seawater within 1 weekregardless of ploidy. SW transfer in the current studyresulted in an immediate elevation in plasma osmolality.Since these values returned to basal within 1 week, didnot exceed the lethal limit for trout (>420 mOsm kg1,Alexis et al., 1984) and were accompanied by virtually no

mortality, it can be concluded that fish had successfullyadapted to seawater and entered a regulatory period. Thetimeframe to regain osmotic balance described in thisexperiment is similar to that of earlier studies with smallerrainbow trout (1330 g) albeit they were exposed to lowersalinities (2330 ppt) (Bath and Eddy, 1979; Jackson,1981; Alexis et al., 1984). Specifically, the current studyhas shown that abrupt transfer to SW transfer withoutthe use of intermediate salinities as previously studied inbrook trout (Salvelinus fontinalis) ploidy variants (Dumaset al., 1995) is tolerated both in triploid and diploid rain-bow trout.

Our study also induced a marked elevation in gill Na+

,K+, ATPase activity from

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regulatory hormone in teleost fish associated with increas-ing ATPase activity and plasma glucose, through increasedliver gluconeogenesis, which subsequently provides a sub-strate for the greater energetic demands of SW adaptation(McCormick, 1996, 2001; Liebert and Shreck, 2006). Thiswould certainly explain the positive correlation between

glucose and cortisol found in the FWSW trial but not inthe FWFW trial. Therefore, in the current study cortisoland glucose are undoubtedly performing a dual role duringthe adaptive phase following SW transfer.

Seawater transfer also resulted in an immediate dou-bling in total WBC concentrations of both diploid and trip-loid trout thus inferring a potentially enhanced cellularimmune capacity. This is in contrast to the generallyaccepted view that acute stressors result in a depressionin WBC concentration (Schreck, 1997; Benfey and Biron,2000) due to the immunosuppressive effects of cortisol.However, Pulsford et al. (1994) also reported increasedWBC concentrations in dab (Limanda limanda) following

an acute stressor, which was largely due to increases inperipheral phagocyte concentration. In this respect, it hasbeen suggested that cortisol protects trout leucocytesagainst the immediate affects of stress by maintaining phag-ocytic index (Narnaware and Baker, 1996). Thus, thestrong positive correlation we observed between cortisoland WBC in the FWSW trials could support such a the-ory. Unfortunately, as no differential leukocyte countswere made within our study we cannot derive which celltype may have been responsible for this increase and thiscertainly warrants further study.

An increase in peripheral and head kidney WBC concen-

trations has also been observed within 2 weeks of SWtransfer in diploid Atlantic salmon smolts (Pettersenet al., 2003). It may be possible that increased WBC con-centrations are a specific reaction to SW transfer which isindependent of the generic stress response. This may alsobe due to increases in circulating GH observed during sea-water transfer (McCormick, 2001) since this hormone hasbeen suggested to prevent the immunosuppressive activityof cortisol (Yada et al., 2004). Certainly since we demon-strated a significant positive correlation between plasmaIGF-I and total WBC, we could postulate that IGF-Imay be involved in promoting some of the effects of GHat the cellular level, although this remains to be investi-gated. Clearly, the high degree of positive correlationbetween both plasma cortisol and IGF-I in the FWSW tri-als, and subsequently with WBC counts, necessitates theindividual role of each hormone in these various responsesto be elucidated. Furthermore the interaction betweenstress and IGF-I should be given greater consideration.We clearly showed an elevation in IGF-I following FWSW transfer but also a minor elevation within 1 h ofFWFW transfer. Similarly, McCormick et al. (1998)showed elevated IGF-I within 4 h of an acute stress, whileLiebert and Shreck (2006) observed no change in IGF-Iafter an acute stressor and saltwater (25 ppt) transfer which

may in part relate to the role of IGFBPs. Of note, the lack

of correlation between IGF-I and cortisol in the FWFWtrials suggest that, although handling clearly evoked atypical stress elevation in cortisol, it is not in itselfdirectly driving the increase in IGF-I.

At present, although both ploidies increased their WBCconcentrations this was most pronounced in diploids.

However, given the morphological differences between dip-loid and triploid cells it is impossible to accurately infer agreater cellular immune capacity in diploids at this time.One of the major effects of ploidy seen in the current studywas the apparent greater respiratory burst activity (approx-imately 50% higher) of head kidney macrophages in trip-loids. Although phagocytic activity was examined duringthis study, results were not reported due to a problem ofpoor macrophage adhesion to the slides with the assay.However, this should undoubtedly be addressed in furtherstudies as this is a major indicator of innate immunitywhich may be influenced by ploidy status. It may thereforebe suggested that the greater respiratory burst observed in

triploid head kidney macrophages, and possibly peripheralleucocytes, may allow triploid fish to compensate for lowerWBC concentrations by having a higher killing capacity.This is clearly an interesting avenue for further study andhas only recently been investigated in turbot (Budinoet al., 2006).

In the current experiment increased levels in plasmalysozyme activity stimulated by seawater are in agreementwith previous studies in rainbow trout exposed to dilute(12 ppt) seawater (Yada et al., 2001). Notably, we alsofound a strong negative correlation between lysozymeactivity and cortisol or IGF-I which contradicts current

opinion (Fevolden et al., 2003) but are in accordance withNorth et al. (2006). To date few reports exist, although it iswidely accepted that corticosteroids are known to bepotent immunosuppressants. In terms of mucus lysozyme,SW transfer did not appear to statistically affect activitywhen compared to controls in freshwater. However, a morepronounced general depression in activity was seen whencompared to basal levels before transfer, which concurswith observations of Fast et al. (2002) in rainbow trout,coho salmon (Oncorhynchus kisutch) and Atlantic salmon.

In summary, on transfer to full strength seawater, non-smolting rainbow trout undergo an initial adaptive phaselasting up to 72 h before ionic-osmotic balance is re-estab-lished. This is accompanied with marked increases in ATP-ase activity which is more intense and short lived than thoseexhibited by salmon smolts. This process appears to beaccompanied by a certain amount of physiological stressas indicated by elevated cortisol and glucose, although thelevel of stress can not be directly inferred due to the dualrole of these factors in ionic-osmotic regulation. Seawatertransfer also increased blood leukocyte concentrations,respiratory burst and plasma lysozyme activity butappeared to decrease mucus lysozyme activity. This mightsuggest a generally improved defence mechanism againstpathogens during this physiologically stressful time. We

also showed these responses do not differ markedly between


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diploid and triploid trout of the same strain and parentalorigin. On the basis of our results it may be concluded thatan inability to adapt to changing salinity is not the sole rea-son behind the high rates of triploid mortalities reportedduring seawater transfer. It is more likely the result of multi-ple stressors (handling, temperature, pollution etc.) occur-

ring alongside changing salinity.

Acknowledgments

The authors like to extend their thanks to all the techni-cal staff at the NBFRL and Institute of Aquaculture forFish Maintenance during these trials. A special thanks goesto Dr. Iain Berrill for assistance with the ATPase assay.This project was funded by the Institute of Aquaculture,University of Stirling.

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