Äîêóìåíò âçÿò èç êýøà ïîèñêîâîé ìàøèíû. Àäðåñ îðèãèíàëüíîãî äîêóìåíòà : http://vertebrata.bio.msu.ru/Ots_IMMUNE.PDF Äàòà èçìåíåíèÿ: Mon Feb 8 19:41:49 2010 Äàòà èíäåêñèðîâàíèÿ: Mon Oct 1 19:24:34 2012 Êîäèðîâêà:

doi 10.109 8 / rsp b.20 01.163 6

Im m u n e c h a l l e n ge a f f e c t s b a sa l m e t a bol i c act i v i t y i n w i n t e r i n g gr e at t i t s
I nd rek O ts1, A nvar B. Kerimov2 , Elena V. Ivan k i na 3, Tatyan a A . Ilyi na ¬ and Peete r Horak1*
1In 2 3

2

stitute of Zoology and Hydrobiol ogy, T u Universit y, Vanemuise 4 6, T u 51014, Estonia art art Dep artment of Verteb rate Zoology, Biological Faculty, Mosco w State Universit y, Mosco w 119 8 9 9, Russia Zvenigoro d Biological Station of Mosco w State Universit y, Post Box Shikhovo, Mosco w Region 143 0 92, Russi a The cost s of exp loiting an organis m's immune function are exp ected to form the basi s of many life- histor y trade- o¡ s. Howeve r, there has been debate about whether such cost s can be p aid in energetic and nutritional terms. We addressed this question in a study of wintering, free-living, male great tits by injecting them with a novel, non-p athogenic antige n (sheep red bloo d cells) and measuring the changes in their basal metabolic rate s and variou s condition indices subsequent to immune challenge. The exp eriment showed that activatio n of the immune syste m altere d the metabolic activit y and p ro¢le of immune cells in birds during the week subsequent to antige n injection : individuals mounting an immune resp onse had nearly 9% higher basal metabolic rates , 8% lower p lasm a albumin level s and 37% higher heterop hileto-ly mp hocyte ratios (leucocytic stres s indices) than sham- injecte d control birds. They also lost nearly 3% (0.5 g) of their body mass subsequent to the immune challenge. Individual s that mounted stronge r antibody resp onse s lost more mass during the immune challenge. These results suggest that energetic exp enditures to immune re sp onse may have a non-trivial imp act up on an individual's condition. Keywords : immune challenge ; basal metabolic rates ; Parus major ; heterop hile-to-ly mp hocyte ratio ; mass loss the energetic cost of this immune resp onse was considered to be very low. Hence, the assu mp tion of energetic an d nutritiona l cost s of mountin g an immune resp onse has been questioned. Howeve r, the generalit y of such a conclusion await s further research . Consequently, the ai m of this study was to asses s the energetic and nutritional cost s of mounting an immune challenge against a novel antige n in free-living, w intering great tits (Parus major). The great tit is a smal l (ca. 19 g), short-live d p asser ine that has been rep eatedly use d as a model organism in research into life-history evolution , not least in the context of ecologica l immunolog y (see e.g. Norris & Evan s 200 0). We studie d the cost s of mountin g an immune resp onse in wintering, male great tits by challenging them with sheep red blood cells ( SRBC s) as a novel, multigenic antigen. A ntibody resp onse against SRBCs involve s both T and B lymp hocyte s (e.g. Munns & Lamont 1991) and the magnitude of the resp onse can be easily assesse d by a haemagglutin ation assay. We exp ected the energetic cost of immune challenge to be reveale d by increase d basal metaboli c rate s ( BMRs) as comp ared to control (saline- injected) individuals. The nutritiona l statu s of individuals was estimate d on the basi s of seru m p rotein concentrations an d p ro¢les. A decreas e in tota l seru m p rotein concentration accomp anies almost al l disease s but is a p articularly p rominent symp tom of malnutritio n and in fection, p articularl y when cause d by a decline in albumin level (e. g. Kawai 1973; C oles 1997). A decreas e in serum albumi n content during in¸ammation is usually accomp anied by a simultaneou s increase in globulin concentration, mainly caused by the globulin s belonging to the b- and g-fractions. The g- globulin fraction of seru m includes most of the know n antibodies involve d in the immune re sp onse to p rotozoan , bacteria l 1175
© 20 01 The Royal Society

1 . INTRODUCTIO N

Over the p ast few decades, the role of p arasite s in the shap ing of life-histor y p attern s has been increasingly recognize d by evolutionar y animal ecologist s (see Sheldon & Verhulst 199 6; Lochmille r & Deerenberg 20 00; Norris & Evans 20 00 ). Several studie s have suggeste d that immune function is directly involve d in the mechanisms causin g evolution and maintenanc e of ornamental trait s (e. g. Hamilto n & Zuk 1982; Westneat & Birkhead 1998), the generatio n of rep roductive cost s (e. g. Gustafsson et al. 1994; Mæller 1997) and driving p op ulation cycle s (e.g. Lochmiller 1996). In order to be involve d in life- history trade- o¡ s, the immune function has to be costly (e.g. Sheldon & Verhulst 1996; Owens & Wilson 1999 ). While the p resence of such cost s ha s been supp orte d in studie s on di¡erent organism s (see reference s above), a debate ha s emerge d about the currencies in which the cost s of using and maintaining an immune function are to be p aid ( Ròberg et al. 199 8; Westneat & Birkhead 199 8). The traditiona l view of animal ecologists , namely that the cost s involve d in life-history trade- o ¡s are basically energetic , has recently been challenge d by evidence suggestin g that the energetic demand s required for maintenanc e of immune function ( Klasing 1998; but see Lochmiller & Deerenberg 200 0 ) an d for mountin g an immune resp onse against sp eci¢c foreign antigen s ( Svensson et al. 1998) are negligible. The latter study showed that, in cap tive blue tits (Paru s caeruleu s), coldstresse d individuals had lower immune re sp onses to a dip htheria ^ tetanus vaccine than control birds, but that this could not be exp laine d by energy limitatio n because
*

Author for corresp ondenc e (horak@ut.e e).

Proc. R. Soc. Lon d. B (20 01) 26 8, 1175^1181 Rece ived 4 Octobe r 20 0 0 Accep ted 19 Februar y 20 01


1176

I. Ots and other s

Costs of immune resp onse in great tits
s ign i¢c ant e¡ect on the var iable s e xam ined ( p ^ 0.3^0.7). An alyse s wer e p e r for med wit h t he SAS GLM p roce du re ( SAS In stit ute 19 85) us ing t yp e III sums of s qu are s, th ereby e nabl ing us to ac count for all the e¡ects of t he i ndep endent var iable s s i mult aneously. Cor resp onde nce of res iduals to a nor m al di str ibut ion was ch ecked wit h t he SAS UNIVARIATE p ro ce dure ( Shap ir o ^Wi lk's W- te st) and i n no case was t he as sump t ion of nor mal ity violat ed. Man n ^W hit ne y U- t ests were us ed when c omp ar ing group ave rage s of non- nor mally dist r ibut ed t rait s. A ll s ign i¢cance l evels r efer to t wo- t ailed t est s. Subs crip ts u sed in c on ne cti on wit h test st at ist ics re fer to de grees of fr ee dom. The value s ar e means ( § s.d.). Th e study was p er formed in accordance wit h the law s of the Rus s ian Fe derat ion r egar ding t he c ap t ur ing and h olding of wild an im als and all individual s were r el eased aft er t he exp e r ime nt.

an d viral infections, while the b- globulin fraction contain s the acute-p hase p roteins , including ¢brinogen (e. g. Coles 1997). In both acute disease an d chronic infection or in¸amm ation, disease d individuals revea l a higher tota l globulin concentratio n and lowe r albumin to globuli n ratio s than healthy individuals. In addition, we registere d the changes in body mass , BMR and blood immune cell concentrations during the exp eriment and looke d for correlation s between these vari ables and the magnitude of the immune resp onse.
2 . METHODS

(a) Study site and researc h p rotocol
The study was p e r for med b et ween 8 Febr u ary and 2 March 20 0 0 at t he Z ven igoro d Biologic al Stat ion of Mos c ow Un iver s ity (5584 4' N, 36851' E ), which comp ri ses two sm all vil lage s s urr ounded by m ixed and c on ifer ous forest an d is locat ed 70 km we st of Mos c ow. The daily ambie nt temp erat ure du r ing t he stu dy p e r iod r anged fr om 717.5 to 3.5 8C w ith the mean t emp e rature bei ng 73.0 8C. Th e weat he r was relat ively m i ld as c omp are d to t he s eas on's aver age ( 77.2 8C f or t he p rev iou s ¢ve ye ar s). Great t its were cap tu red w it h m i st nets and sp r ing t rap s and kep t indo or s in ind ividual cages supp lied wit h wat er, mealworm s and su n¸ower se e ds unt il n ight when th eir b ody m as ses and BMR s were rec orded. In t he morn ing followin g the ¢r st c ap t ure, blood s amp les we re take n f rom the birds ( for measu r ing leuc oc yt e count s). The bir ds we re t hen ran domly as s igned to i mmu ne challenge ( injecti on w ith s usp en s ion of SR BCs) and c ont r ol ( inject ion w ith is otonic s al ine) t r eatme nts. Th ere af ter, t hey were releas ed. Henc e, t he birds p er formed t he ir nor m al act ivit ie s in a natur al env iron ment du r ing the exp e r iment. A ltogeth er, 42 m ale gr eat t its were c ap t ured and as s igned to c ont rol and ant ige n inject ions at ¢rst c ap t ure. Twent y- ¢ve of t h ese birds ( ten contr ol and 15 exp er i mental s) wer e subs eque nt ly recap tur ed aft er s ix to t en days (mean ^ 7.4 § 1.5 days). A ll of t hese individu als could b e us ed for e st im at ing t he e¡ect of i mmu ne challen ge on body mass change, wh ile 24 in div idu al s cou ld b e used for exam in ing change s in t h eir leuc oc yt e p ro¢les. The samp le s i ze was s maller (21 b ird s) f or e st im at ing ch an ges in BMR b ec ause fou r b irds did not sleep dur ing t he meas urement of t he ir BMRs dur ing eit her t he ¢r st or se cond c ap t ure and , h ence, we re exclude d f rom t he analyse s. Cont r ol and exp er i mental b irds at ¢r st cap tu re did not di¡er s igni¢c ant ly in r esp ect of t he ir BMR s (3.9 § 0.3 ver sus 4.0 § 0.4 m l O2 g71 h71 for c ont rol s and exp e r imentals, r esp e ct ive ly) (t8,11 ^ 0.89 and p ^ 0.4), b ody m as ses (18.6 § 1.1 ver sus 18.8 § 0.7 g for c ont rols and exp e r ime nt al s, resp e ct ive ly) (t9,14 ^ 0.52 and p ^ 0.6) or h et erop hi le- tolymp ho cyte ( H :L) r atio s (1.4 § 0.6 ver sus 1.5 § 0.8 for c ont rol s and exp e r ime ntals, resp ectively) (z8,14 ^ 0.17 and p ^ 0.9). Ne it her did the c ont rol and exp er i mental bir ds di¡er in r esp ect of date of in itial cap t ure ( medi an 8 Febr uary ve rs us 9 Febr uary for cont rols an d e xp er i ment al s, resp e ct ive ly) (t14,9 ^ 1.05 and p ^ 0.3) or t he int er val b etween cap tu res (7.3 § 1.7 vers us 7.4 § 1.4 day s for c ont r ol s and exp er imentals, resp ect ively) (t9,14 ^ 0.16 and p ^ 0.9). In or der to acc ou nt for inte r individual di¡erences i n in it ial t rait value s, t he e¡ect of i mmu ne challenge on change s in tr ait value s was examin ed in ANCOVA s including each t rait 's valu e at ¢rst c ap t ure as a c ovar iat e (s ee e.g. Me r ilë & Wiggin s 19 97). A ll m ode ls were tested f or p os sible conf oun ding e ¡e cts of age, t er r itor ial status, date and b et ween- c ap t ure int er val, all of wh ich (e xcep t t he e¡ect of dat e on BMR) tur ned out to have no
Proc. R. Soc. Lon d. B (20 01)

(b) Measuremen t of BMRs, immune resp onses and condition indices
The metab ol ic r at es of t he gr eat tits wer e de te rm i ned as t he r at e of oxyge n c on sump t ion by e ach re st ing b ird at n ight ( bet ween 23.0 0 and 03.0 0 ) u s ing a modi¢ed Kalabukh ov closed s yst em ( D oln ik & G avr i lov 1979; Gavr ilov 1979). Each b ird was p lac ed in a s mall cage and kep t in t he dark wit hout fo od at 22 ^ 25 8C for at least 4 h. The c age s and birds wer e t hen p lace d in 3- l glas s metab ol ism chamb ers and kep t in t he dark in an incubator at 25.5^2 6 8C. One h ou r was al lowe d f or adap tat ion and t he rm al st abil izat ion. The chamb er was t hen sealed and oxyge n c on sump t ion was meas ured by me asur ing t he ¸ow of p ure O2. CO2 in t he chamb er was r em oved u sing KOH. O xygen c on sump t ion was me asured ove r 10 - m in int er val s f or 6 0 ^ 70 m in. The averag e of t he se 10 - m in i nte rvals was us ed as an e st imat e of individual oxyge n c on sump t ion. The temp erat ure in t he chamb er was me asured us ing a merc ury t herm ometer. Bi rds we re we ighed aft er re moval f rom a chambe r. Oxygen cons ump t ion value s wer e c or rect ed to standard temp eratu re an d p res sure ( D ep ocas & Hart 1957). BMR s were exp res s ed as t he amount of oxyge n con sumed p er gr am of b ody mass p er hour (m l O2 g71 h71) (s ee also e.g. R æskaft et al. 19 8 6; S chi eltz & Murp hy 19 95). The use of ¢ve resp i rome te rs and t wo se r ies of me asure me nts e nable d us to e st im ate t he BMR s of up to ten bi rds p er night . The ¢r st t r ial of BMR me asureme nts started at 23.0 0 h ( i.e. 6 h aft er sun set) and t he sec ond t r ial ( for anot her ¢ve birds) start ed at 02.0 0. In t he mor n ing aft er ¢r st c ap t ure, followin g the me asureme nt of t heir BMR s, exp er i me ntal b irds wer e inj ected wit h 50 ml s usp en s ion of 4 0% SR BCs in i sotonic sal ine into t heir p e ctor ali s mus cles, wh i le t he s ham- op er ated c ont r ol b irds were inject ed wit h t he same amount of s aline. Pr ior to inject ion, SR BCs were double was he d and re susp en ded in sal ine in or der to ach ieve t he de s ir ed c oncent rat ion. Bloo d s amp les (ca. 150 ml) t hat were to b e us ed for as s es s ment of im mune resp ons e and p la sma p rotei ns we re obt ai ne d s ix to t en day s after in it ial s amp li ng. Serum was s ep arate d by c ent r i fugat ion at 30 0 0 r.p . m. for 10 m in and p re se r ved at 720 8C u nt il analys is . Im mune resp ons e ( SR BC antib ody t it re) was measu red us ing a h aem agglut i n at ion as s ay ( Wegmann & Sm ith ies 19 6 6; Lawl er & Re dig 19 8 4) in 96 - we ll m icrop lat es. A 12.5 ml aliquot of s er um was adde d to 87.5 ml of s al ine in t he ¢rst wel l of a p late and se r ially di luted us ing 50 ml of saline (0.5, 0.25, etc.). Then 50 ml of 1% susp en s ion of SR BCs in sali ne was adde d to al l samp le s. The m ic rop late s were inc ubate d at 37 8C for 1 h. Tit r e was sc or ed a s t he inve rse of t he di lut ion t hat contained s u¤c ient ant ib odie s to haem agglut inat e SR BCs ( hence, t he h igher t he t itr e, t he st ronger t he im mune


Cost s of immune resp onse in great tits

I. Ot s and others 1177

Table 1. E¡ec t of immune challeng e with SRBC s ( treatment ) on subsequen t change s in BMR, bod y mass and the H:L ratio
( For the d ir e ct i on of ch an ges s ee ¢gur e 1. R2 ^ 0.75 for B MR, R2 ^ 0. 41 f or bod y m a ss a nd R2 ^ 0.7 7 for t he H :L r ati o. ) change in BMR e¡ect t r eat m en t in i t ial va lu e d ate Fd. 12. 1 32. 5 11. 3
f.

change i n b ody mass p Fd.
f.

ch ange in t he H :L r at io Fd.
f.

p 0 .024 0 .010 Æ

p 0. 007 5 0. 001 Æ

1 , 17 1 , 17 1 , 17

0. 003 5 0. 001 0. 004

5.91 , 8.01 , Æ

22 22

9. 01 , 2 1 63. 1 1 ,2 1 Æ

0.15 0.05 h- 1)

(a) 0.00 change in body mass (g)

(b)

0.00 - 0.15 - 0.30 - 0.45 - 0.60 - 0.75 - 0.90 10 control 15 SRBC - 1.05

( c)

change in BMR (mlO2 g

- 0.15 - 0.25 - 0.35 - 0.45 9 con trol 12 SR BC

- 0.30

- 0.45

- 0.60

change in H/L ratio

- 0.05

- 0.15

-1

9 con tro l

15 SR BC

F igu re 1. Comp ar is on of ch an ge s in B MR, b od y m ass an d t he H :L ra tio b e tw een i mm un e- ch al len ged ( S R BC- i nj e c te d) and c ont r ol ( sa li ne- in je c t ed) gr e at t it s. L eas t- s qu are m e ans a nd s .e .s de r iv ed f r om t he m o de ls i n t able 1. Th e nu m b ers u n der ba rs d en ote s amp le s i ze s. resp on se). The r est of the ser um was u sed for de te rm inat ion of p rote in c oncent rat ions by st an dar d agar os e gel e lect rop hor es is (s ee Ot s et al. 19 9 8 for detai ls). Leuc ocyte p ro¢les and c oncent rat ions we re e sti mat ed from t h in bloo d sme ars t hat we re p rep are d i n t he m or nin gs foll owin g t he me asure me nt of BMR s. We c oncent rat ed on t wo le uco cytic c ondit ion indice s : t he total leuc ocyte c ount ( white blood c ell s, WBCs), the e le vat ion of which i s charact er ist ic of in¸am matory p roce s ses in resp ons e to m icr obial and mac rop ara sit e in fections (e.g. D ein 19 86) and t he H :L r at io, wh ich i s widely used as a st ress in dex in p oult ry (e.g. G ross & Siege l 19 83; Maxwel l 19 93) but wh ich is als o used in studies of wil d ani mal s (e.g. Hor ak et ¬ al. 19 9 8). In order to c ount leuc ocyte s, a drop of bloo d wa s sm eared on t wo individually m arked m ic roscop e sl ide s, ai r dr i ed, ¢xe d i n absolut e me thanol and st ai ned wit h azur e- e os in. The p rop or tions of d i¡ere nt typ e s of leucoc yt e were as se s s ed on t he b as is of an examin at ion of a tot al of 10 0 leuc oc ytes under ¸10 0 0 m agni ¢c at ion un der oi l im mer s ion. Est imates of WBC s wer e obt ain ed by c ount ing t he number of leuc ocytes p e r ca. 10 0 0 0 er yt hrocyte s. The rep eat abil itie s of leuco cyte c ou nts obtained u si ng t h is me thod are h igh and si gn i¢cant , a s s hown by Ot s et al. (19 9 8). 3 . RESULTS

The BMR decrease d with calendar date ove r the course of the study p er iod (r ^ 70.41, p ^ 0.00 8 and n ^ 42). In order to account for this e¡ect, we included
Proc. R. Soc. Lon d. B (20 01)

the date of cap ture as a covari ate when analysin g the e¡ect of immune challenge up on the change in individual BMR values. The BMR decreased between the ¢rst and second cap ture among control birds, whereas there was no change among immune- challenged male s (table 1 an d ¢gure 1a). A s a result, immune- challenged birds had on averag e an 8.6 % h igher BMR at second cap ture than control birds (4.0 § 0.2 versu s 3.7 § 0.3 ml O 2 g71 h71 for exp erimenta l an d control birds, resp ectively) (t11,8 ^ 2.80 and p ^ 0.011) Birds injected with SRBCs on averag e lost 0.5 g (2.8% ) of body mas s between ep isode s of cap ture, while the mas s of control birds did not change (¢gure 1b and table 1). The H:L ratio s of control birds decreased more than those of immune- challenge d individuals between the ¢rst and second cap tures (¢gure 1c and table 1). This resulted in 37% higher H:L ratio s in immune- challenged birds at secon d cap ture (1.11 § 0.48) as comp are d to control s (0.70 § 0.48). Antigen injection had no e¡ect up on the change in tota l leucocyt e count (F1,21 ^ 0.37 and p ^ 0.5). Immune- challenge d birds had signi ¢cantly lower total seru m p rotei n (21.9 § 1.9 versus 24.1 § 1.9 g l71) (t13,9 ^ 2.63 and p ^ 0.015) and albumin (17.8 § 1.6 versu s 19.4 § 1.9 g l71) (t13,9 ^ 2.30 and p ^ 0.031) level s at second cap ture, while albumin to globuli n ratios did not di¡er between exp erimenta l categorie s (4.5 § 1.1 versus 4.3 § 0.9) (t13,9 ^ 0.45 and p ^ 0.7). Hence, immune challenge resulted in a relative increase in metabolic rate s and


1178

I. Ots and other s

Costs of immune resp onse in great tits

0.2 0.0 - 0.2 body mass change (g) - 0.4 - 0.6 - 0.8 - 1.0 - 1.2 - 1.4 -1 1 observation 2 observations

0.6 h) change in BMR (mlO2 g

-1 -1

0.3

0.0

- 0.3 - 0.6 - 0.9

0

1

2 3 SRBC titre

4

5

- 1.5

- 1.0

- 0.5 0.0 0.5 body mass change (g)

1.0

F ig ure 2. R e l ati on s hip b e t we en S R BC t i tre an d bod y m ass c h ange ( m ass i n s ec ond cap t u re m inus m a ss i n ¢rst c ap t ur e) i n m ale gr e at t i ts ( r ^ 70.63 , p ^ 0. 027 an d n ^ 15) .

F igu re 3. R e la ti on sh ip b e t we en b od y m ass c han s e c ond c ap tu re m in us m ass in ¢r st cap t ur e) an d B MR ( B MR in s ec o nd c ap tu re m in us BM R i n ¢ i n m ale gr e at t its ( rS ^ 70. 45, p ^ 0.04 1 an d n ^

ge ( m ass i n c han ge in r st c ap t u re) 2 1) .

stres s indices and loss of body mass and p lasma nutrient content , while the p lasma globulin content did not increase. The birds that mounte d a stronge r immune resp onse against SRBCs lost more mass (¢gure 2), while the antibody titre did not correlate signi ¢cantly w ith change in BM R (rS ^ 70.13, p ^ 0.7 and n ^ 12). Body mas s loss was accomp anied by an increase in metabolic rat e (¢gure 3) an d the H :L rati o (¢gure 4).
4 . DI SCUSSIO N

Our exp eriment clearly demonstrate d the energetic and p hysiologica l imp act of immune challenge in wintering, male great tits. Birds mounting antibody resp onses against the foreign antige n had nearly 9% higher metabolic rates, 37% higher H:L ratios and 8% lower p lasma albumi n level s than control birds an d they also lost nearly 3% (0.5 g) of their b ody mas s subsequent to the immune challenge. This means that non-p athogenic immune challenge (i.e. mere activatio n of the immune system) can a¡ect basal metabolis m and change immune cell p ro¢les in adult birds. Furthermore, the cost of exp loitin g an immune syste m is suggeste d by the result that individuals that mounte d stronge r antibody resp onse s against SRBCs lost more mas s during the immune resp onse (¢gure 2). T the authors ' knowledge, o this is the ¢rst study to show such an e¡ect in a w ild bird sp ecies. A study in red grous e (Lagop us lagop us scoticu s) found a 16% increas e in metaboli c rate follow ing a truly p athogenic challenge with nematode s ( Delahay et al. 1995). Studies in chicken s ( Klasing et al. 19 87; but see Hen ken & Brandsma 1982) an d quails ( Fair et al. 1999) have show n that SRBC -induced immune challenge can supp ress growth and reduce p lasma p rotei n content. The reaso n for the latte r is decreased p rotei n synthesis in the muscle and an increase in the use of amino acid s in the live r for the synthesi s of acute-p hase p lasm a globulin s ( Klasing & Austic 1984). Such a mechanism could
Proc. R. Soc. Lon d. B (20 01)

exp lain the lower seru m albumin and tota l p rotei n concentrations of the exp erimental birds in ou r study. In addition, the low level s of p lasm a albumin in the immune- challenged gre at tits might have resulted from reduced food intake. Such SRBC -induced anorexi a has been demon str ated in chicken s ( Klasing & Austic 19 84); howeve r, the functional exp lanation s for this p henomenon are still obscure (see Kyriazaki s et al. 1998). The 37 % higher H :L rati o of the exp erimental versu s control birds in ou r study suggest s that injection w ith SRBCs a¡ecte d the concentration s of di¡erent immune cells in the p er ip heral blood of gre at tits. Elevate d H:L ratio s in studies of p oultr y have traditionally been regarde d as general symp toms of almost any kind of stres s (cause d by injuries, infectious diseases , starvatio n or the administratio n of glucocorticoids ) (e. g. Gros s & Siegel 19 83; Gray et al. 1989; Maxwel l 1993; Dhabha r et al. 1995). H :L ratios have also been show n to increase in resp onse to exp erimentally increase d rep roductive e¡ort ( Horak ¬ et al. 1998) and p hytohaemagglutinin-induce d immune challenge ( Horak et al. 20 00 ) in great tits. In the p resent ¬ study, the increase in the H :L rati o between the events of cap ture was associate d with a decreas e in body mass (¢gure 4), suggestin g that stres s level s increase d in p aralle l with deterioratio n of body condition. The increase in the H:L ratio in resp onse to antige n injection could be p ossibly ascribe d to corticosterone-induce d redistributio n of lymp hocyte s to secondar y lymp hoid tissues ( Dhabha r et al. 1995; Trout et al. 1996) and simultaneous migratio n of heterop hils (which p articip ate in the p hagocytosi s of antigen) into the p er ip heral blood in the course of an in ¸ammator y resp onse ( Klasing & Austic 19 84). Unlike Fair et al. (1999) in the study of growin g quails, we faile d to detect an increas e in the total leucocyte count in re sp onse to SRBC injection. Considering the lack of e¡ect of antige n injection on p er ip heral immune cell p roliferation and immunoglobulin p roduction, the cost s of mounting an immune


Cost s of immune resp onse in great tits

I. Ots and others 1179

1.0 0.5 change in H/L ratio 0.0 - 0.5 - 1.0 - 1.5 - 2.0 - 1.5 - 1.0 - 0. 5 0.0 0. 5 body mass change (g) 1. 0

F igu re 4. R e lat i on s hip b e tw een b ody m ass c han ge ( ma ss in s e cond c ap t u re m in us m as s in ¢r st c ap t ur e) an d ch an ge in t he H :L r at io ( H :L r at io in s ec ond cap t u re m in us H:L r at io in ¢rst c ap t ur e) in m ale gr e at t its ( rS ^ 70.4 6, p ^ 0 .025 a nd n ^ 24) .

resp onse in our study were unlikely to have resu lte d from the reallocatio n of limited energ y, p rotei n or other nutrient s for the p roductio n of immune cells and molecules. However, while the quantitativ e need for nutrients for the p roliferation of leucocyte s and p roductio n of antibodies may be ver y small ( Klasing 1998), there app ear to be a number of indirect cost s associate d with systemi c cytokine mediated in¸ammator y an d acute-p hase resp onses, anorexia, cellular metabolic rate s and cellular^ molecular turnove r ( Klasin g & Leshchinsky 199 9; Lochmiller & Deerenberg 200 0 ) which may lead to increase d energy turnove r and metabolis m in the p roces s of up - regu latio n of the immune system . Given that such metabolic cost s could be readily detected in ou r study desp ite the modest samp le siz e and relatively mild weathe r conditions, the contention of Lochmiller & Deerenberg (20 00 ) that the cost s of up regulation of the immune syste m may p ush an animal beyond the minimal level s of b odily reserve s to survive should p robabl y be given seriou s attention. Notabl y, increase d metabolis m was accomp anie d by a considerable loss of body mass in our great tits (¢gure 3), suggestin g that energetic exp enditures to an immune resp onse may indeed have a non-trivia l imp act up on a host's condition. Our results on the e¡ect of antige n injection on BMR were of comp arable magnitude to thos e found by Svensson et al. (1998) in a relate d sp ecies, i.e. the blue tit. Svensson et al. (1998) calculate d that the upp er limit of the energetic cost s of an immune resp onse may range up to 8 or 13% of BMR, but these di¡erences (in a samp le size comp arabl e to that of the current study) turned out to be insigni¢cant. Howeve r, in contras t to the great tits in ou r study that lost mas s in resp onse to antige n injection, their immune- challenged blue tit s had signi ¢cantly higher body masse s than the control birds during the secondar y immune resp onse. We can see two (not mutually exclusive) exp lanation s why the immune
Proc. R. Soc. Lon d. B (20 01)

resp onse in ou r study was accomp anied by reduced body mass but not in blue tits. First, the antige n use d for immunizing blue tits (a dip htheria ^ tetanus vaccine) might have induced a lower extent of in¸ammator y resp onse than the SRBCs use d in our study. Since the immune re sp onse against SRBC s involve s both B and T lymp hocyte s and antigen s that induce T- ly mp hocyt e resp onses are thought to induce greate r level s of in¸ammator y cytokine s than those that induce immunoglobuli n resp onse s ( Klasing & Leshchinsky 1999), the resp ons e to SRBCs may app ear energeticall y more costly than the re sp onse to dip htheria ^ tetanus vaccine. Second, the blue tits in the exp eriment of Svensso n et al. (19 98) were kep t in aviarie s where they had access to foo d ad libitum, which evidently enabled them to retain an energy balance throughout the exp eriment. In contrast , the immune- challenged great tits in our study were release d to natural conditions, which means that they were not dep rived of the behaviourally mediate d cost s of ¢nding and defending food sources , socia l interaction s and anti-p redato r vigilance . Evidently, such behavioural cost s might have been su¤cient to boost the energetic cost s of mountin g an immune resp onse, which could remain undetecte d in laborator y conditions. Hence, ou r study illustrate s the necessity of measuring the cost s of using the immune syste m under natura l conditions. Both the p resent study an d that of Svensson et al. (199 8) raise the question of the biologica l signi¢cance of elevation of the BMR in resp onse to immune challenge. A lthough smal l p asserines can work at a level of daily energ y exp enditure ( DEE ) that equals fou r time s the BMR under extreme conditions (e. g. Stevenso n & Bryant 20 00 ), the optimal DEE :BMR rati o for p asserin e birds is considered to be equal to 2.5 ( Dolni k 1982, 1996). This rati o is ver y close to that of non-territorial , w intering, male great tits ( DEE ^ 2.7 BMR) in Zvenigoro d ( Nagy et al. 1999). At ambient te mp eratures similar to those of the p resent study, the DEE of the magnitude of 2.5 time s the BMR was found to be ver y close to existenc e metabolism, which includes exp enditures of ca. 1.8 time s the BMR for basal metabolis m and thermoregulatio n ( Gavrilov et al. 1996). This means that wintering great tit s normally sp end energ y at a magnitude of only 0.7^0.9 time s the BMR for activitie s such as foragin g and ¸ocking behaviou r, anti-p redato r vigilance , etc. This p rop ortio n of energ y becomes stil l smaller with a decreas e in ambient temp erature. In this context , an extr a increase in BMR of 8^ 13% due to activatio n of the immune syste m can be considered a serious challenge to the energy budget of an animal. The question of whether this challenge is serious enough to comp romis e investment s in other vital function s of an organis m remain s to be solve d in future studie s measurin g bot h the BMR an d ¢eld metabolic rate s of immune- challenged individuals. In conclusion, our study shows that the traditiona l energetic and nutritiona l currencies in which the cost s of life-histor y trade- o ¡s are thought to emerg e may also be relevant for research in ecological immunolog y. This of course does not mean that alternativ e cost s of immune resp onse s (such as immunop atholog y or free radical p roduction) will be unimp ortant . Rathe r, our results suggest the need for careful consideration of al l di¡erent exp enditures relate d to activatio n of the immune system.


1180

I. Ots and other s

Cost s of immune resp onse in great tits
Hor ak, P., Ots, I., T ge lman n, L. & Mæller, A. P. 20 0 0 He alth ¬ e imp act of p hytohaemagglut in in- induce d i m mune chal lenge on gr eat t it nest lin gs. Can. J. Zool. 78, 9 05^910 Kawai , T. 1973 Clinical asp ects of the p lasma p roteins. T o : Igaku oky Shoin L t d. K l as ing, K. C. 19 9 8 Nut r it ional m odulat ion of r es istance to infe ct ious dis eas es. Poultry Sci. 77, 1119^ 1125. K l as ing, K. C. & Aust ic, R. E. 198 4 Change s in p las ma, t i ss ue and ur inary n it rogen met abolit es due to in¸am mator y challe nge. Proc. Soc. Exp. Biol. Med. 176, 276^284. K l as ing, K. C. & L es hchi ns ky, T. V 19 9 9 Funct ions, co st s, and . ben e¢t s of t he i mmu ne system du r ing develop me nt. In Proceedings of the 2 2nd Inte rnational Ornithological Congress, 16^22 August 199 8 ( ed. N. Adam s & R. Slotow), pp . 2817^2835 . Joh anne sbur g : B ird li fe S out h A f r ica. K l as ing, K. C., Laur in, D. E., Peng, R. K. & Fr y, D. 1987 Immu nologically me diated gr owt h dep re s si on in ch icks : in¸uence of feed int ake, c ort ic oste rone and int erleuk in-1. J. Nutr. 117, 1629^ 1637. Kyr iazaki s, I., T lkamp , B. J. & Hutch ings, M. R. 19 9 8 T ward s o o a f unct ional exp lanat ion for t he oc cur rence of anorex i a dur ing p aras itic infect ion s. Anim. Behav. 56, 265^274. L awler, E. M. & Red ig, P. T. 19 8 4 The ant ibody resp onses to sheep red blood ce lls of t he red- tai led hawk and gr eat- hor ned owl . Devel. Comp. Immunol. 8, 733^738. L ochm i ller, R. L. 19 9 6 Im munoc omp e tence and ani mal p op ulat ion regu lati on. Oikos 76, 59 4^ 6 02. L ochm i ller, R. L. & D ee renbe rg, C. 20 0 0 Trade - o¡s in evolu t ionar y im munology : ju st what is t he cost of i mmu nity ? Oikos 8 8, 87^9 8. Maxwel l , M. H. 19 93 Avian bloo d leuc oc yt e resp on ses to st re s s. World. Poultry Sci. J. 49, 34^43. Mer i lë, J. & Wiggin s , D. A. 19 97 Mas s l os s in br eed ing blue t it s: t he role of energe tic st r es s. J. Anim. Ecol. 66, 452^4 6 0. Mæller, A. P. 19 97 Paras it i sm and t he evolut ion of h ost l ife hi stor y. In Host^p arasite evolution. General p rincip les and avian models (e d. D. H. Clayto n & J. Moore .), pp . 105^ 127. Oxford Un iver s ity Pres s. Mun n s, P. L. & L amont, S. J. 19 91 E¡ects of age and i mmu ni sat ion inter val on t he anam nes tic resp on se to T- c el l- dep ende nt and T- cell- indep endent ant igen s in chicke n s. Poultry Sci. 70, 2371^2374. Nag y, K. A., Gavr i lov, V M. , Ker imov, A. B. & Ivan kin a, E. V . . 19 9 9 Re lations hip s bet we en ¢eld met abolic rate, bas al metab ol ic rate and te rr itor iality in p as se r ine s. In Proceedings of the 22nd International Ornithological Congress, 16^2 2 August 19 9 8 (e d. N. Adams & R. Slotow), pp. 39 0^4 0 0. Johan nesburg : Bir dl ife Sout h Af r ic a. Norr is, K. & Evan s , M. R . 20 0 0 E cologica l im munology : l ife hi story tr ade- o¡ s an d im mune de fense in bir ds. Behav. Ecol. 11, 19^26. Ot s, I., Muru mëg i, A. & Horak, P. 19 9 8 Hem atologica l h ealt h ¬ st ate indices of rep roduc ing gr eat t its : meth odolog y and sources of natur al var iat ion. Funct. Ecol. 12, 70 0^707. O we n s, I. P. F. & Wil s on, K. 19 9 9 Immu noc omp ete nce: a neglected li fe h istory t rait or c on sp icuous red h err ing ? T ends r Ecol. Evol. 14, 170^172. Ròb er g, L., Grahn, M., Has se lqu ist, D. & Sven s son, E. 19 9 8 On the adap t ive s ign i¢canc e of st res s - induced i m munosupp re ss ion. Proc. R. Soc. Lond. B 265, 1637^ 16 41. R æskaft, E., Jër vi , T. , B akke n , M., Be ch, C. & Reine rts en, R. E. 19 86 The relat ion ship b et ween s ocial stat us and r est ing metab ol ic rate in great t its Parus major and p ied ¸ycatcher s Ficedula hyp oleuca. Anim. Behav. 34, 838 ^8 42. SAS In st itute 19 85 SAS user 's guide, v. 6. Cary, NC : SAS Inst itut e. Schie lt z, P. C. & Mu rp hy, M. E. 19 95 D iu rn al var iat ion in oxyge n c on s ump t ion by m olt ing and non molt ing sp arrows. Comp. Biochem. Physiol. A 112, 265^272.

We th ank Helen Vel lau for cou nt ing leuc oc yt es, L aur i Pe il for p er form i ng h aem agglutin atio n a ss ays and V lad im ir G r inkov and Nikolay For mozov f or as s ist ance in t rap p ing bird s. Two anonymou s referees p rovide d c on str uct ive c r it ici sm on t he m anu scr ip t. Unit ed L aborator ies , C lin icu m of t he Tar tu Univers ity, k indly p rovide d facil it ies for s er um analys e s. The st udy was ¢nanciall y s upp orted by Estonian Science Foundat ion grant n o. 3551 ( to P. H.) and Ru ss ian Fund of Basic Re s earch grant s (nos 0 0- 04 - 4 878 4 and 0 0-15- 97735 ). REFERENCES C oles , B. H. 1997 Avian medicine and surgery. Oxford, UK: Blackwel l S cie nc e. D ei n, J. 198 6 Hem atolog y. In Clinical avian medicine (e d. G. J. Har r i son & W. R. Har r ison.), pp . 174^191. London : Saunde rs C o. D el ahay, R. J. , Sp eakman, J. R. & Mos s, R. 1995 The en erget ic cons equenc es of p aras it ism Æ e¡ects of a deve lop ing i nfe ct ion of T ichostrongylus tenuis ( Ne matoda) on red grous e (Lagop us r lagop us scoticus) ener gy- b alanc e, body- weight an d condit ion. Parasitology 110, 473^4 82. D ep oca s, F. & Hart, J. S. 1957 Use of t he Paul ing oxyge n analyz e r for meas urement of oxyge n c on sump t ion of an im als in op en- ci rcu it s yst ems and in a s hort lag, clos e d- ci rcu it app aratu s. J. App l. Physiol. 10, 38 8^392. D habhar, F. S., Mil ler, A. H., Mc Ewen , B. S. & Sp encer, R. L. 19 95 E ¡ects of st ress on i m mune ce ll di st r ibution: dynam ic s an d hor monal mechan is m s. J. Immunol. 154, 5511^ 5527. D oln ik,V R. 19 82 Met hods of t i me and e nergy budget s st udy. In . . Time and energy budgets in free- living birds (e d. V R. D olni k), pp. 3^37. Le ningr ad : Nau ka. ( In Rus s ian w ith E ngl ish sum mary.) D oln ik, V R. 199 6 Pas se r ine b irds as e nerget ic p he nomen on . . Z. Obshch. Biol. 57, 533^56 6 ( in Rus s ian wit h E ngl ish s um mar y). D oln ik, V R. & Gavr i lov, V M. 1979 Bioen er get ics of molt in . . t he cha¤nch (Fringilla coelebs). Auk 2, 253 ^26 4. Fai r, J. M. , Hans en, E. S. & R icklef s, R. E. 19 9 9 G rowt h, develop mental stab ility and im mune resp on se in juven ile Jap anes e quails (Coturnix coturnix jap onica). Proc. R. Soc. Lond. B 266, 1735^ 1742. G avr i lov, V M. 1979 B ioene rgetics in l arge p as se r ine sp e cies. I. . Re st i ng and ex iste nce met aboli sm. Zool. Z. 5 8, 530^5 41 ( in Rus s ian wit h English sum mar y). G avr i lov, V M. , Ker im ov, A. B., G olubeva , T. B., Ivank ina , . E. V , Ilyin a, T. A., Kare lin, D. V & Kolyaskin , V V 19 9 6 . . .. Energe tics, morp hologica l and p hys iologic a l hete roge neity an d p op u lat ion str uct ure in bir ds. I. Energet ic s, m op hologic a l an d p hysi ologica l hete rogen eity an d p op ulat ion st r uct ure in great t its in Mosc ow region. Ornithologia 27, 34^73 ( in Rus s ian wit h Engli sh s umm ar y). G ray, H. G., Paradi s, T. J. & Chang, P. W. 19 89 Phys iologica l e¡ects of ACTH and hyd rocort is one in layin g h ens. Poultry Sci. 6 8, 1710^ 1713. G ros s, W. B. & Sie gel, H. S. 19 83 Evaluat io n of t he hete rop hil/ lymp hocyte r atio as a mea sure of st ress in chicken s. Avian Dis. 27, 972^ 979 . G ustafs s on, L., Nor dling, D., A nder s son, M. S. , Sheldon, B. C. & Q varn st rím , A. 19 9 4 Infecti ous di se ase s, rep roduct ive e¡ort and the co st of rep roduct ion in bir ds. Phil. T ans. R. Soc. r Lond. B 346, 323^331. Ham ilton, W. D. & Zuk, M. 1982 Her it able t r ue ¢t ne ss and br ight bir ds : a r ole of p ara sit es. Science 218 , 38 4^387. Hen ke n, A. M. & B rands m a, H. A. 19 82 The e ¡e ct of e nvir onment al temp eratu re on i m mune resp ons e and metab ol ism of t he youn g ch icken. Poultry Sci. 61, 1667^ 1673. Hor ak, P., Ots, I. & Mu ru mëgi, A. 19 9 8 Hem atologica l healt h ¬ st ate indices of r ep roduc ing gre at t its : a resp ons e to br ood s ize man ip ulati on. Funct. Ecol. 12, 750^756.

Proc. R. Soc. Lon d. B (20 01)


Costs of immune resp onse in great tits
She ldon, B. C. & Verhu lst, S. 19 9 6 Ec ologica l im munology : co st ly p aras ite defences and t r ade- o¡ s in e volut ionar y ec olog y. T ends r Ecol. Evol. 11, 317^321. St eve ns on, I. R. & Br yant, D. M. 20 0 0 Cl i mate ch an ge and c on str ai nts on bree ding. Nature 4 06, 36 6^ 367 Sven s son, E., Ròb erg, L., Koch, C. & Ha ss elqu ist, D. 19 98 E ne rget ic str es s, im munosupp r es s ion and t he c osts of an antib ody resp on se. Funct. Ecol. 12, 912^ 919. Trout , J. M., Mas haly, M. M. & Siegel, H. S. 199 6 Change s in blood and sp leen lymp hoc yt e p op ulat ions follow in g antige n challenge in i mm at ure male chicken s. Br. Poultry Sci. 37, 819^827.

I. Ots and other s 1181

We gman n, T. G. & Sm it h ies, O. 19 6 6 A s imp le he magglut inat ion system requ ir ing sm all am ount s of r ed cells and antibodies. T ansfusion 6, 67 ^73. r We st neat, D. F. & Bi rk head, T. R. 19 9 8 Alt ernat ive hyp ot hes es l in king t he im mune s ystem an d m ate ch oic e f or goo d gene s. Proc. R. Soc. Lond. B 265, 10 65^ 1073.

A s t h is p ap e r excee ds t he ma xi mum length norm ally p er m it te d, t he aut hor s have agre ed to contr ibute to p roduct ion c ost s.

Proc. R. Soc. Lon d. B (20 01)