Gender- and season-dependent relationships between testosterone, oestradiol and immune functions in...

Gender- and season-dependent relationships between

testosterone, oestradiol and immune functions in

wild roach

A. VAINIKKA*†, E. I. JOKINEN*, R. KORTET‡ AND

J. TASKINEN§

*Department of Biological and Environmental Science, University of Jyvaskyla, P. O.Box 35 (YAC341), FIN-40 014, University of Jyvaskyla, Finland, ‡Neurobiology,Physiology & Behavior, University of California, Davis, One Shields Avenue, Davis,CA 95 616, U.S.A. and §Institute of Applied Biotechnology, University of Kuopio,

P. O. Box 1627, FIN-70 211 Kuopio, Finland

(Received 19 April 2003, Accepted 27 October 2003)

Plasma testosterone and 17�-oestradiol concentrations, differential leukocyte counts and

proportion of dead Rhipidocotyle campanula gill parasites (parasite resistance) were determined

five times during a year in two populations of roach Rutilus rutilus and analysed for seasonal

and gender differences. In addition to the above immune variables, plasma Immunoglobulin M

(IgM) concentration, chemiluminescence and migration differential of head kidney phagocytes,

size of the spleen, haematocrit and total leukocyte count were correlated with sex hormones for

each population, sampling time and sex separately, using condition factor as a partial correlate.

There were no clear gender differences in the determined immune variables. Both testosterone

and oestradiol concentrations were lowest after the spawning in June. Oestradiol was higher in

females than in males, but testosterone was present in equal concentrations in both sexes.

Statistically significant correlations between sex hormones and immune variables were rare and

mainly without any patterns with respect to population, sex or sampling date. The

meta-analysis on individual correlations, however, revealed a positive relationship of plasma

testosterone concentration to chemiluminescence of head kidney phagocytes, plasma IgM

concentration and the proportion of dead R. campanula to all R. campanula on the gills. In

the meta-analysis, the concentration of oestradiol was not found to correlate with any of the

studied immune variables. # 2004 The Fisheries Society of the British Isles

Key words: cyprinids; immune system; seasonality; sex steroids.

INTRODUCTION

Limited resources challenge animals to balance allocation between maintenance(including immune defence), reproduction and growth (Roff, 1992; Stearns,1992). Possible trade-offs between immune defence and reproduction could bemediated via sex steroids (Folstad & Karter, 1992; Sheldon & Verhulst, 1996).Studies on mammals suggest that androgens are generally immunosuppressive

†Author to whom correspondence should be addressed. Tel.: þ358 14 260 4219; fax: þ358 14 260 2321;

email: [email protected]

Journal of Fish Biology (2004) 64, 227–240

doi:10.1046/j.1095-8649.2004.00306.x,availableonlineathttp://www.blackwell-synergy.com

227# 2004TheFisheries Society of theBritish Isles

and oestrogens immunoenhancing (Ahmed et al., 1985; Grossman, 1985). Therelationships between sex steroids and immune functions have been widelyexamined in mammals and birds (Saino et al., 1994; Chao et al., 1996;Hasselquist et al., 1999), but studies on fishes have been largely confined to invitro effects of testosterone (Slater & Schreck, 1993, 1997, 1998; Slater et al.,1995), although in vivo data are increasing (Hou et al., 1999a, b; Law et al.,2001; Watanuki et al., 2002). Oestradiol has been shown to increase suscept-ibility to parasites and to inhibit some immune functions in fishes (Wang &Belosevic, 1994; Watanuki et al., 2002).Males are probably more susceptible to parasites and diseases in comparison to

females due to immunosuppressive androgens (Ahmed et al., 1985; Zuk, 1990;Møller et al., 1998) or due to different behavioural mating tactics, which mayexpose males to parasites (Tinsley & Jackson, 1988; Reimchen & Nosil, 2001).Gender differences in immune functions or parasitism have been reported ininsects (Kurtz et al., 2000), in mammals (Grossman, 1985; Zuk, 1990), in birds(Møller et al., 1998), in toads (Tinsley & Jackson, 1988) and in fishes (Collazoset al., 1994; Reimchen & Nosil, 2001; Kortet et al., 2003a).Kortet et al. (2003b) studied seasonal changes in immunological and physio-

logical variables of the cyprinid, roach Rutilus rutilus (L.) from the presentstudy populations, through a year. They found that males had a larger spleenand higher haematocrit than females. There were no gender differences inrespiratory burst and migration ability (both chemotaxis and spontaneousmigration) of head kidney phagocytes, but plasma immunoglobulin M (IgM)concentrations were marginally higher among males in one of the two popula-tions (Kortet et al., 2003b). As a continuation of that study, plasma testosteroneand 17�-oestradiol concentrations, differential leukocyte counts and proportionof dead Rhipidocotyle campanula Dujardin (Digenea, Bucephalidae) gillparasites were determined and analysed for gender and seasonal differencesand their correlations with testosterone and oestradiol. In addition to thesevariables, the correlations between plasma IgM concentration, chemilumines-cence and migration differential (longest chemotactic migration distance minuslongest spontaneous migration distance) of head kidney phagocytes, size of thespleen, haematocrit and total leukocyte count with respect to testosterone andoestradiol were also analysed. If both high hormone concentrations and goodimmune functions were condition-dependent, the actual correlations betweensex hormones and immune functions would be hidden (van Noordwijk & deJong, 1986). Therefore, condition factor was used as a partial correlate in thecorrelation analyses.Roach was chosen for the study because it is a common and widely distributed

fish which breeds in intensive ‘leks’ (Wedekind, 1996) and develops testosterone-induced breeding tubercles as sexual ornaments (Wiley & Collette, 1970). Thebreeding tubercles of roach are shown to indicate parasite resistance (Taskinen &Kortet, 2002) and parasite load (Wedekind, 1992). Roach is iteroparous, itundertakes spawning migrations (Mills, 1991), and methods for immunologicalresearch on this species are well developed (Aaltonen et al., 1994; Salo et al.,1998).The aims of the present study were to test the immunosuppressivity of

testosterone, the key assumption of immunocompetence handicap hypothesis(Folstad & Karter, 1992) using correlational field data, and to find general

228 A. VAINIKKA ET AL .

# 2004TheFisheries Society of theBritish Isles, Journal of FishBiology 2004, 64, 227–240

gender differences in immune variables. The hypotheses of this study are thatmales have lower measures of immune function than females, and that anegative correlation is found between plasma testosterone concentration andimmune functions.

MATERIALS AND METHODS

FISH

Mature male and female roach were collected by angling from Lakes Kuljunlahti(64�370 N; 24�250 E) and Jyvasjarvi (62�140 N; 25�460 E) in 1999. Lake Jyvasjarvi is coveredby ice from November to late April, whereas Lake Kuljunlahti is an ice-free recipient ofheated waste water and has an average annual temperature c. 10� higher than that of LakeJyvasjarvi (Kortet et al., 2003b). From each lake, five samples of c.15 male and 15 femaleroach were taken from the same site, sampling dates being timed in accordance with thereproductive cycle of the fish (Table I); spawning takes place 2 weeks earlier in LakeKuljunlahti than in Lake Jyvasjarvi (Kortet et al., 2003b).Fish were reared in accordance with the Guidelines for the Use of Animals in Research

(Anon., 1996) under licence from the Central Finland Regional Environmental Center(permission LS-2/99). After capture, fish were immediately anaesthetized using 0�01%MS-222 (Sigma Chemicals Co., U.S.A.), transported to the laboratory in aerated lakewater and processed within 2 h of capture. The mean� S.D. total length (LT) (cm) andmass (M) (g) of fish (n¼ 302) were 160�0� 19�8mm and 57�5� 26�0 g, respectively(Table I). Condition factor, K, was calculated for each population and sex separatelyusing the equation, K ¼ 105 �MCL�b

T where MC is the carcass mass and b is the slope ofa regression of log10 (MC) on log10 (LT) of the population and particular sex (Bolger &Connolly, 1989).

IMMUNE AND HORMONE ASSAYS

Following measurements of fish msss and LT, a blood sample was taken from thecaudal vein using heparinized syringes, and fish were killed and dissected for analysis ofspleen and gonad masses, sex, and directed migration as chemotactic differential andrespiratory burst (chemiluminescence) of head kidney phagocytes (neutrophils and

TABLE I. Sampling dates, sample sizes and fish sizes during 1999. Period 1, winter; 2,before spawning in spring; 3, after spawning in early summer; 4, late summer; 5, autumn

Lake Sampling time Males LT� S.D. (mm) Females LT� S.D. (mm)

Jyvasjarvi (1) 5–6 February 15 135�3� 8�8 15 140�7� 9�8(2) 10–14 May 14 165�1� 22�8 17 165�9� 24�3(3) 3–4 June 11 151�5� 13�3 16 170�3� 18�5(4) 17–18 August 16 155�5� 45�6 15 179�1� 16�0(5) 19–20 October 15 155�3� 16�0 18 154�6� 18�2

Kuljunlahti (1) 11–12 February 15 162�8� 12�3 17 177�0� 18�0(2) 28–29 April 13 146�1� 13�3 17 159�8� 15�1(3) 9–10 June 9 151�2� 11�1 16 171�1� 22�8(4) 24–25 August 15 156�1� 16�5 17 167�8� 20�4(5) 25–26 October 16 156�3� 15�6 16 156�4� 9�4

SEX STEROIDS AND IMMUNE FUNCTION IN ROACH 229


monocytes). The ability of phagocytes to produce oxygen radicals and to move towardspathogens are key measures of their ability to kill pathogens (mostly bacteria, protozoaand cells infected with viruses) in the blood stream and tissues (Secombes & Fletcher,1992). The spleen produces antibodies and is involved in the clearance of pathogens andforeign particles from the blood stream (Dalmo et al., 1997). Concentrations of plasmaimmunoglobulins usually indicate the ability of a fish to respond to pathogens which ithas encountered before (Manning, 1994). The total and differential counts of lympho-cytes are indices of current health status (Wester et al., 1994).

Haematocrit was measured immediately after blood sampling (12 000 g, 5min, HeraeusBiofuge A) (Kortet et al., 2003b), and whole blood was stained for enumeration of whiteblood cells (Shaw, 1930) in a cell chamber using light microscopy. The plasma was thenseparated and frozen at �80� C prior to determination of IgM and sex hormone con-centrations. Differential white blood cell counts (proportions of lymphocytes, macro-phages, granulocytes and thrombocytes) were examined from blood smears stained witha haematological stain (Diff-Quick, Baxter Diagnostic AG, Germany), and cell typeswere counted as cells per 200 white blood cells using light microscopy (�400). The IgMconcentration was determined using an enzyme-linked immunosorbent assay (ELISA)for roach IgM as described by Aaltonen et al. (1994).

The head kidney was homogenized through a nylon net of mesh-size: 80 wires per25�4mm (1 inch) (Salo et al., 1998) and the homogenate layered on two-step Percoll-gradients (Pharmacia LKB Biotechnology AB, Sweden). After centrifugation (400 g,30min) the cells were collected from 1�070 to 1�090 g cm�3 interphase, washed twiceand viable cells counted using trypan blue exclusion in a haemocytometer to adjust thecell concentration to 105 cellsml�1. The production of reactive oxygen species, as ameasure of respiratory burst of phagocytes (mostly granulocytes), was assayed usingthe phorbol-12-myristate 13-acetate (PMA)-stimulated, luminol-enhanced chemilumines-cence method (Scott & Klesius, 1981; Salo et al., 1998). Chemiluminescence wasmeasured at 25� C using a model 1250-Wallac luminometer. Peak chemiluminescence(mV) was expressed as mV10�6 granulocytes.

Migration of head kidney phagocytes was assessed using a modified under-agarosetechnique (Nelson et al., 1975) as described by Salo et al. (1998). Casein (100mgml�1,Sigma Chemical Co., U.S.A.) was used as a chemoattractant, the cells were allowed tomigrate in a humidified environment at 25� C for 3 h and then fixed overnight withmethanol. The agarose was carefully removed and the slides with migrated cells wereair-dried and stained with a haematological stain (Diff-Quick, Baxter Diagnostic AG,Germany). The distance (mm) that the leading front of cells migrated from the margin ofthe well towards the well containing casein and the distance the least migrated cellsmoved spontaneously away from the well were measured under a microscope equippedwith an ocular micrometer. The difference between spontaneous and directed migration(chemotactic differential) was used in the analyses. The proportion of dead R. campanulaparasites among all R. campanula individuals in the gills was assessed as described inTaskinen & Kortet (2002). The proportion was not defined if the total number ofparasites was less than three (Kortet et al., 2003b).

Plasma hormone concentrations were determined using RIA-based kits according tothe manufacturer’s instructions (TESTO-CTK, DiaSorin, Italy, for testosterone andESTR-CTK-4, DiaSorin, Italy, for 17�-oestradiol). The sample volume for the testoster-one analysis, however, was decreased to 30ml and for the oestradiol analysis to 10 mlfollowing preliminary tests on roach plasma (Kortet et al., 2003a).

STATISTICAL ANALYSES

Natural logarithm transformation was used for testosterone, oestradiol, white bloodcell count, chemiluminescence and migration differential of head kidney phagocytes andplasma IgM concentrations in order to meet the assumptions of ANOVA. Square rootarcsine transformation (Zar, 1999) was used for the relative proportions of blooddifferential white blood cell counts, proportion of dead R. campanula parasites of allR. campanula parasites and haematocrit. One-way ANCOVA with LT as a covariate was



used to analyse for effects of fish sex (random factor), population and sampling time(fixed factors) on the concentration of testosterone and oestradiol, proportion of deadR. campanula parasites, differential white blood cell counts and migration differential ofhead kidney phagocytes. Simple contrasts were used to locate pair-wise differencesbetween sampling periods. Random effects of gender in differential white blood cellcounts were explored with LT as a covariate within sampling times and populationsusing one-way ANCOVAs. Bonferroni correction was used to adjust significance level formultiple tests. Correlations of testosterone and oestradiol with immune measures werestudied using Pearson correlation using K as a partial correlate for each sampling time,population and sex separately. Overall 95% CI estimate for the correlation coefficientbetween the studied variable and a hormone was calculated using meta-analysis onindividual periodical correlation coefficients as described by Hedges & Olkin (1985).

RESULTS

SEX HORMONES

Testosterone concentration varied over time (ANCOVA, sampling period,d.f.¼ 4 and 3�71, P¼ 0�002), being lowest after spawning in June [Fig. 1(a)],but there were no differences between sexes or populations in testosteroneconcentration (ANCOVA, population, d.f.¼ 1 and 0�50, P¼ 0�519; sex,d.f.¼ 1 and 1�05, P¼ 0�803). The interaction terms were not statistically sig-nificant indicating that there were no sex and population-dependent variationsin seasonal patterns of testosterone. Oestradiol concentration also varied tem-porally (ANCOVA, sampling period, d.f.¼ 4 and 3�71, P¼ 0�010) [Fig. 1(b)],and females had higher oestradiol concentrations than males (ANCOVA, sex,d.f.¼ 1 and 2�25, P< 0�001). The significant interaction sex� sampling period(d.f.¼ 4 and 4�06, P¼ 0�039) indicated different seasonal oestradiol patterns inmales and females. In females the mean oestradiol concentration decreased fromFebruary to April to May whereas no change took place among males duringthat period [Fig. 1(b)].

IMMUNE VARIABLES

In differential leukocyte counts, the proportion of lymphocytes did not varybetween sexes, populations or sampling periods (ANCOVA, all terms, P> 0�05)(Fig. 2). Sampling period and the interaction sex� population were significantfactors explaining variation in the proportion of thrombocytes (ANCOVA,sampling period, d.f.¼ 4 and 4�16, P¼ 0�013, population� sex, d.f.¼ 1 and4�09, P¼ 0�046). Females had relatively more thrombocytes in February inLake Jyvasjarvi [Fig. 2(a)]. The proportion of granulocytes was affected bypopulation and by the interactions population� sampling period and sex�sampling period (ANCOVA, population, d.f.¼ 1 and 1�00, P¼ 0�028, popula-tion� sampling period, d.f.¼ 4 and 5�26, P¼ 0�002, sex� sampling period,d.f.¼ 4 and 4�17, P¼ 0�022). The interactions population� sampling periodand sex� sampling period were significant factors affecting the variation inmacrophage proportions (ANCOVA, population� sampling time, d.f.¼ 4 and4�81, P¼ 0�018, sex� sampling time, d.f.¼ 1 and 5�42, P¼ 0�009). Males had ahigher proportion of macrophages than females in June after spawning in LakeJyvasjarvi [Fig. 2(a)].



The variation in the proportion of dead R. campanula of all R. campanulaparasites on the gills was explained by LT (ANCOVA, d.f.¼ 1 and 222,P< 0�001) only, increasing by size of the fish. There were no statisticallysignificant gender differences in migration differential of head kidney phago-cytes (ANCOVA, sex and interaction terms, P> 0�05), but there were differ-ences between the populations (ANCOVA, d.f.¼ 1 and 0�81, P¼ 0�027) and

5

4

3

2

1

0

5

4

3

2

1

0

Tes

tost

eron

e co

nce

ntr

atio

n (

ng

ml–1

)O

estr

adio

l con

cen

trat

ion

(n

g m

l–1)

(a)

(b)

A

A

A

A

A

B

C

ba a ba ba b

AB

C

B

b

a

c

a b

a b

***

***

*** ***

*

n = 28 34 25 31 18 32 29 32 30 32February April to May June August October

n = 28 33 26 32 19 31 29 32 24 32February April to May June August October

Sampling period

**

FIG. 1. Mean� S.E. (a) testosterone, and (b) oestradiol concentrations in roach plasma during the year

(&, females; *, males). Same letters (a, b and c for males and A, B and C for females) are used to

indicate sampling dates, which do not differ statistically from each other within sex (simple

contrasts, P> 0�05). *, Significant gender differences (one-way-ANCOVA with fish total length

as a covariate: ***, P< 0�001; **, 0�001<P< 0�01; *, 0�01<P< 0�05). Populations were pooled

since ANCOVA did not show differences between the populations at any sampling period.



sampling periods (ANCOVA, d.f.¼ 4 and 4�08, P¼ 0�011). The seasonality andgender differences of chemiluminescence of head kidney phagocytes and plasmaIgM concentration were reported by Kortet et al. (2003b).

CORRELATIONS BETWEEN SEX HORMONES AND IMMUNEVARIABLES

Only a few statistically significant correlations were found between plasmatestosterone concentration and immune variables within the periodical samples.Meta-analysis on the periodical samples indicated overall positive relationshipsof plasma testosterone to chemiluminescence of head kidney phagocytes, plasmaIgM concentration and to the proportion of dead R. campanula gill parasites ofall R. campanula (Table II). The statistically significant correlations betweenimmune functions and oestradiol were also quite rare, and mainly positive,although somewhat higher in numbers than in the case of testosterone(Table II). Meta-analysis on the periodical samples, however, could not revealany statistically significant relationships between plasma oestradiol concentra-tion and the immune variables studied (Table II). In addition to the correlations

100

90

* *

**

80

70

60

50

40M FFebruary

M FApril to May

April to May

M FJune

M FAugust

M FOctober

100

90

80

70

60

50

40

February June August OctoberM FM FM FM FM F

(a)

(b)

Dif

fere

nti

al w

hite

blo

od c

ell c

oun

ts (

% o

f to

tal l

euko

cyte

cou

nt)

FIG. 2. Differential white blood cell counts as percentage of particular cell type count of total leukocytes

count in roach from (a) Lake Jyvasjarvi and (b) Lake Kuljunlahti. (See Table I for exact sampling

times). Statistically significant gender differences within periods: *, 0�01<P< 0�05. , unidentified

cells; &, macrophages; , granulocytes; &, thrombocytes; &, lymphocytes. M, males; F, females.



TABLEII.Partialcorrelationcoefficients

(r)betweentestosterone,

oestradiol,chem

iluminescence

ofheadkidney

phagocytes,

plasm

aIgM

concentration,proportion

ofdead

Rhipidocotyle

campanula

parasites,

totalwhite

blood

cell

count,

proportion

ofgranulocytesand

proportionoflymphocytes.

Theconditionfactorwasusedaspartialcorrelate.Proportionofgranulocytesandlymphocytesweresquare

rootarcsine-transform

ed(Zar,1999)andother

variableswereln-transform

ed.(See

TableIforexact

sample

sizesandsamplingdates).***,

P<0� 001;**,0� 001<P<0� 01;*,0� 01<P<0� 05.Bonferroni-adjusted

alpha-level

forthetestsis

0� 0025,whichis

reached

only

bytests

indicatedby***.95%

CIofoverallrwascalculatedbymeta-analysisonindividualcorrelationcoefficients

(Hedges

&Olkin,1985)

Lake

Chem

iluminescence

IgM

concentration

Parasite

killing

Horm

one

Sex

Period

12

34

51

23

45

12

34

5

TestosteroneM

J�0� 10

�0� 26

0� 37

0� 04

0� 14

0� 44

0� 49�0� 22

0� 19

0� 40

0� 57

0� 36

0� 50

0� 28

�0� 37

MK

0� 89***

0� 32

�0� 12

0� 29

�0� 17

�0� 10

�0� 03

0� 41

0� 34

0� 27

–�0� 05

0� 36

�0� 01

�0� 20

FJ

0� 16

�0� 38

�0� 31

�0� 23

0� 48

0� 61*�0� 30

0� 44

0� 33

�0� 06

0� 22

0� 36

�0� 25

�0� 03

0� 18

FK

0� 34

0� 02

0� 14

0� 26

0� 05

0� 04

0� 24

0� 47

0� 08

0� 61*

0� 11

0� 70*

0� 45

0� 05

�0� 12

Overallr

95%

CIforris0� 00–0� 25

95%


95%


Oestradiol

MJ

�0� 61*

�0� 47

0� 23

�0� 26

0� 26

0� 36

0� 27�0� 07�0� 26

0� 19

0� 60

0� 06

�0� 17

�0� 47

0� 18

MK

�0� 61*

0� 26

�0� 59

0� 16

0� 14

�0� 34

�0� 20

0� 41�0� 04

0� 11

–�0� 14

�0� 04

0� 07

�0� 11

FJ

0� 23

0� 05

0� 06

�0� 06

0� 70**

0� 40

0� 48

0� 16

0� 25

0� 64**

0� 61*

0� 55

�0� 09

�0� 10

0� 29

FK

0� 42

�0� 71**�0� 37

0� 34

�0� 05

�0� 10

�0� 41

0� 18

0� 29

�0� 14

0� 36

0� 46

0� 44

�0� 27

�0� 07

Overallr

risNS

risNS

95%


234 A . VAINIKKA ET AL .


TABLEII.Continued

overleaf

Lake

Whitebloodcells

Granulocytes

Lymphocytes

Horm

one

Sex

Period

12

34

51

23

45

12

34

5

TestosteroneM

J0� 18

�0� 34

0� 09

�0� 31

�0� 14

0� 24

0� 13�0� 29

0� 19

�0� 07

0� 26

0� 53

�0� 29

�0� 13

�0� 20

MK

�0� 40

0� 16

0� 13

�0� 22

�0� 18

0� 05

0� 07�0� 24

0� 08

�0� 06

�0� 17

0� 38

0� 27

0� 00

�0� 09

FJ

0� 11

�0� 12

�0� 26

�0� 38

0� 16

0� 40

0� 33�0� 45�0� 29

0� 35

�0� 15

�0� 07

0� 10

0� 39

�0� 32

FK

0� 17

0� 13

0� 15

0� 02

�0� 02

�0� 36

0� 04�0� 20

0� 06

�0� 03

�0� 27

�0� 31

0� 36

0� 59*�0� 12

Overallr

risNS

risNS

risNS

Oestradiol

MJ

�0� 42

�0� 32

�0� 27

�0� 08

0� 22

0� 59*�0� 06�0� 30�0� 27

�0� 50

�0� 08

�0� 23

�0� 31

0� 02

0� 15

MK

0� 31

0� 16

0� 13

0� 44

0� 15

0� 03

�0� 21

0� 13

0� 20

�0� 28

0� 47

0� 00

0� 79*

0� 00

0� 49

FJ

0� 15

�0� 09

0� 77***�0� 48

0� 13

0� 59*�0� 02�0� 22�0� 63*

0� 75***�0� 22

�0� 28

0� 53

0� 13

�0� 52*

FK

0� 20

0� 01

0� 32

0� 64**�0� 14

�0� 14

�0� 22

0� 09

0� 26

0� 02

0� 12

�0� 22

�0� 18

0� 37

0� 26

Overallr

risNS

risNS

risNS

S EX STEROIDS AND IMMUNE FUNCTION IN ROACH 235


in Table II, no other statistically significant correlations between the concentra-tions of sex steroids and the immune measures were found (P> 0�05).

DISCUSSION

The main mammalian androgen, testosterone, is known to suppress an arrayof immune functions in mammals (Grossman, 1985). In fishes, Slater et al.(1995) characterized androgen receptors responsive to testosterone in rainbowtrout Oncorhynchus mykiss (Walbaum) leukocytes, and suggested that they arean important link in the androgen-mediated immunosuppression. Hou et al.(1999a) found that testosterone implantation suppressed plasma IgM levels injuvenile rainbow trout. Hou et al. (1999b) also showed that sex steroids aredose-dependently able to reduce the number of rainbow trout lymphocytes andto decrease their ability to produce antibodies. Testosterone was shown tokill chinook salmon Oncorhynchus tshawytscha (Walbaum) leukocytes in vitro(Slater & Schreck, 1997) and to suppress the formation of plague-forming cellsfrom anterior kidney leukocytes (Slater & Schreck, 1993). Moreover, theresponsiveness of rainbow trout leukocytes to androgens was shown to varyseasonally (Slater & Schreck, 1998). Buchmann (1997) reported an increase inGyrodactylus derjavini Mikailov parasite load after the administration oftestosterone in rainbow trout. Law et al. (2001), however, found no effect oftestosterone on leukocyte phagocytosis with hybrid tilapia Oreochromis niloticus(L). x O. aureus (Steindachner) or common carp Cyprinus carpio L. Thus, itseems that the effects of testosterone on different immune functions varybetween taxa, season and methodology (Saino et al., 1994; Chao et al., 1996;Hasselquist et al., 1999). It is also possible that high in vivo testosterone con-centrations lead to increased 11-ketotestosterone or increased corticosteroidconcetrations, and some of the effects of testosterone may thus be indirect.The current study did not find any indication of testosterone-related immuno-

suppression. In fact, only positive correlations between testosterone andimmune functions were found. It is possible that there is a difference betweencyprinids and salmonids in the response of the immune system to testosterone,which is suggested also by the in vitro study of Law et al. (2001). Kortet et al.(2003a) showed that high testosterone concentration was related to high epi-dermal papillomatosis incidence, but the concentration of 11-ketotestosteronewas correlated positively with testosterone, and no causality was shown. It isclear that more studies in cyprinids are needed to address this issue. The lack ofnegative correlations between testosterone and immune functions in the currentstudy was unlikely to have been affected by the condition of individual fish (vanNoordwijk & de Jong, 1986), since the condition factor was used as a partialcorrelate in the analyses. The condition factor, however, is a rather roughmeasure and may not always indicate the real condition of biological import-ance. The correlational results were analogous to current results even whenanalysed using LT, instead of fish condition, as a partial correlate or withoutany partial correlate (unpubl. data).Law et al. (2001) reported that oestradiol suppressed phagocytotic activity of

peripheral blood leukocytes in vitro in common carp and inO. niloticus x O. aureus.Watanuki et al. (2002) studied the effects of oestradiol, 11-ketotestosterone,



progesterone and cortisol on the functions of phagocytotic cells in vivo in commoncarp, and found that all the steroids studied suppressed phagocytosis, superoxideanion and nitric oxide production in a dose-dependent manner. The results of thepresent study are partly consistent with these studies, since the only noteworthyindications of a negative relationship between oestradiol and immune variableswere the marginally significant individual negative correlations with chemilumines-cence of head kidney phagocytes in winter and spring. This conclusion, however,was not supported by the meta-analysis of individual correlations over the seasons.The measures of immune defence used in this study were indirect and can be

regarded as indices of both health and actual immunocompetence. Phagocytesform the major cell-mediated nonspecific defence mechanism in fishes(Secombes & Fletcher, 1992), and chemiluminescence and chemotaxis areoften used to assay the ability of phagocytes to kill pathogens (Collazos et al.,1995; Salo et al., 1998). For example, Hakoyama et al. (2001) found that thelower nitroblue tetrazolium (NBT, analogous to chemiluminescence) immunereaction in gynogenetic Carassius auratus (L). was related to their lower resist-ance against parasites (Metagonnimus sp., Trematoda). The spleen is involved inhaematopoiesis, the clearance of pathogens and other foreign particles from theblood stream and antibody-production (Manning, 1994; Dalmo et al., 1997),and a large spleen is thought to indicate good condition and the ability torespond to infection (Wester et al., 1994). Concentrations of plasma immuno-globulins increase with infection or exposure to parasites, but low concentra-tions may be indicative of a weakened immune system (Manning, 1994). Thetotal count of lymphocytes is an index used to assess health status (Wester et al.,1994) and differential counts of leukocytes are used to reveal possible infections,because the proportion of phagocytes usually increases with infection (Westeret al., 1994). Plasma cortisol concentration is shown to increase rapidly afteracute stress, but confounding changes in the immunological variables studiedwere unlikely to have occurred in the time-scale of fish processing (the capturedroach were immediately anaesthetized and processed within 2 h) (Pickering et al.,1982).The annual sex hormone pattern of roach was related to breeding in May,

when the highest level of testosterone was observed in both sexes. Oestradiolpeaked in August, both in males and females, and there was a subsequent minordecline until spawning. Seasonal variations in sex hormone levels were analo-gous to previously described patterns in fishes (Aida, 1988; Rinchard et al.,1997; Tveiten et al., 1998; Garnier et al., 1999; Kestemont et al., 1999). Theequal concentration of testosterone in males and females contrasts with obser-vations of Arctic charr Salvelinus alpinus (L.) and fathead minnow Pimephalespromelas Rafinesque, where females have lower concentrations of testosterone(Tveiten et al., 1998; Kahl et al., 2001).In several vertebrate species, males have a weaker immune system than

females (Zuk, 1990; Møller et al., 1998). Male-biased gender differences inparasitism and disease have been reported in fishes (Reimchen & Nosil, 2001;Kortet et al., 2002), but data concerning immune variables of fishes are scarce(Collazos et al., 1994; Kortet et al., 2003b). In this study, no clear genderdifferences in immune and health variables were found. Using the currentdata, Kortet et al. (2003b) showed that male roach have a larger spleen, higher



haematocrit and marginally higher concentration of plasma IgM than femalesthroughout the year. Thus, it seems that males have higher values of someimmunological and physiological measures than females in roach. This does notnecessarily indicate, however, that roach males are more immunocompetentthan females.The results of the present study suggest that testosterone may not have

immunosuppressive role in roach, and thus there is a difference between cyp-rinids and salmonids in this respect. In the future, experimental manipulationsof testosterone and the other androgen, 11-ketotestosterone concentrationsshould be conducted in vivo to reveal their possible immunomodulatory effectsin cyprinids. Finally, the biological significance of indirect immune measures(correlation with actual parasite resistance) should be emphasized.

The study was conducted in the Biological Interactions graduate school of the FinnishMinistry of Education (AV, RK). We thank R. Steel for financial support of the studyand M. Kajava for organization of the laboratory facilities near Lake Kuljunlahti. Wealso thank The Biological Society of Finland Vanamo and Emil Aaltonen Foundationfor grants to RK. We thank M. Jobling, J. Pirhonen, E.T. Valtonen, and T. Sinisalo forhelpful suggestions on the manuscript and R. Jones, who kindly corrected the English.

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