MASARYKOVA UNIVERZITA Přírodovědecká Fakulta

MASARYKOVA UNIVERZITA Přírodovědecká fakulta

Karolína Rohlenová

Imunoekologické studium vztahů mezi reprodukcí, imunitou a parazitismem u sladkovodních ryb

Dizertační práce

školitel: doc. RNDr. Andrea Vetešníková Šimková, Ph.D. Brno, 2011

Bibliografická identifikace

Jméno a příjmení autora: Karolína Rohlenová

Název disertační práce: Imunoekologické studium vztahů mezi reprodukcí, imunitou a parazitismem u sladkovodních ryb

Název disertační práce anglicky: Immunoecological study of the relationships among reproduction, immunity and parasitism in cyprinid fish

Studijní program: Biologie

Studijní obor (směr), kombinace oborů: Parazitologie

Školitel: doc. RNDr. Andrea Vetešníková Šimková, Ph.D.

Rok obhajoby: 2011

Klíčová slova v češtině: imunokompetence, parazitismus, sezónní změny, kompromis,

11-ketotestosteron, ploidie, stimulace, kaprovité ryby

Klíčová slova v angličtině: immunocompetence, parasitism, seasonal changes, trade-off, 11- ketotestosterone, ploidity level, stimulation, Cyprinidae

ABSTRAKT Životní funkce poikilotermních obratlovců jsou přímo ovlivňovány abiotickým prostředím, ve kterém se tyto organismy nacházejí; přičemž jejich fyziologický a imunitní systém může být současně ovlivňován parazitickými organismy. Mezi jednotlivými složkami fyziologického a imunitního systému potenciálního hostitelského organismu rovněž existuje velmi složitý systém interakcí. Předložená dizertační práce je věnována studiu těchto interakcí z několika různých úhlů pohledu.

V první části dizertační práce (studie 1) byly studovány sezónní změny vybraných fyziologických, imunitních parametrů a parasitismu u jelce tlouště (Leuciscus cephalus). Dále byly analyzovány potenciální vztahy mezi fyziologií, imunokompetencí a parazitismem. Sezónní změny významně ovlivnily dynamiku hlavních skupin mnohobuněčných cizopasníků (Monogenea, Acanthocephala a Cestoda). Statisticky významný vliv sezóny byl zaznamenán i pro fyziologické (gonado-somatický index, počet erytrocytů) a imunitní parametry (slezino-somatický index, oxidativní vzplanutí, počet leukocytů). Potvrzeny byly významné asociace mezi složkami imunity a fyziologíe hostitele, které podpořily hypotézu kompromisů v alokaci energie mezi životně důležitými složkami. Negativní vztah byl zaznamenán mezi kondičním faktorem a slezino- somatickým indexem a dále pak u samců vztah mezi gonado-somatickým indexem a imunitními parametry (slezino-somatický index a počet leukocytů). Získané výsledky naznačují význam interakcí parazitů s imunitním a fyziologickým systémem hostitele. Negativní vztah byl také zaznamenán mezi oxidativním vzplanutím a skupinou Monogenea. Navíc byla zjištěna pozitivní asociace mezi gonado-somatickým indexem a parazitickými skupinami Monogenea a Acanthocephala, která může být výsledkem imunosupresivní role steroidních hormonů, investice do nákladné reprodukce na úkor imunokompetence, či může naznačovat synchronizaci životních cyklů parazitů s reprodukčním cyklem hostitele.

Na tuto práci tematicky navazovala studie 3, jejímž cílem byla analýza vztahů mezi fyziologií, imunokompetencí a parazitismem u kapra obecného (Cyprinus carpio). Dalším cílem bylo analyzovat, zda je imunokompetence u ryb jako zástupců poikilotermních obratlovců větší mírou ovlivněna sezónními změnami nebo jsou změny imunokompetence spojené s parazitární infekcí. V této studii byla testována hypotéza imunosupresivního vlivu 11-ketotestosteronu. Získané výsledky ukázaly, že sezónní změny velmi významně ovlivňují složky fyziologického a imunitního systémů poikilotermních obratlovců a rovněž dynamiku parazitární infekce.

Vzájemné korelace mezi celkovou mírou investice do fyziologie a imunity byly zaznamenány rovněž po korekci pro efekt sezóny, co naznačuje potenciální existenci kompromisů mezi imunitou a fyziologií rybího hostitele. Při analýze jednotlivých fyziologických a imunitních parametrů vztah mezi imunitou a fyziologií potvrzen nebyl. Investice do jednotlivých složek fyziologického systému naznačují rozdělení na základě kompromisů, tj. negativní vztahy byly zaznamenány mezi kondičním a hepato-somatickým indexem na jedné straně a indexem gonado-somatickým na straně druhé. Tento výsledek naznačuje, že kondice ryb v průběhu reprodukce, kdy je energie přednostně akumulovaná do tkáně gonád, může klesat. Dále byla potvrzena imunosupresivní role 11-ketotestosteronu, tento steroidní hormon negativně ovlivňoval aktivitu komplementu. Výsledky studie také naznačují souvislost mezi rozmanitými životními strategiemi parazitů a jednotlivými složkami fyziologického a imunitního systému hostitele.

V rámci studie 2, která zahrnovala hodnocení reprodukčních investic u samců jelce tlouště (Leuciscus cephalus) v pre-reprodukčním, reprodukčním a post-reprodukčním období, bylo zjištěno, že energie je alokována mezi životně důležité složky (kondice, fyziologie a imunokompetence) na principu kompromisů. Tato energie je investována do životně důležitých složek v závislosti na aktuálních potřebách, které jsou v pre-reprodukčních, reprodukčních a post-reprodukčních periodách odlišné. V pre-reprodukčním období byl zaznamenán nejvyšší gonado-somatický a kondiční index. V následující reprodukční periodě byl zjištěn významný pokles obou těchto indexů. Tyto výsledky naznačují, že tření je pro ryby fyzicky velmi vyčerpávající a stresující období, a proto je tendencí ryb akumulovat jejich energetické rezervy. V reprodukční periodě byly zaznamenány nejvyšší hodnoty většiny analyzovaných imunitních parametrů (oxidativní vzplanutí, počet leukocytů a leukokrit). Statisticky významné rozdíly mezi pre-reprodukčním, reprodukčním a post-reprodukčním obdobím byly zaznamenány u tří skupin nejvíce početných skupin mnohobuněčných cizopasníků (Monogenea, Digenea a Cestoda). Abundance těchto skupin však dosahovala v reprodukčním období nižších hodnot, což spíše naznačuje, že zvýšené hodnoty imunitních parametrů zjištěné v tomto období byly pravděpodobně indukovány stresem spojeným s třením ryb, nebo byly asociovány s infekcí jiného původu než s mnohobuněčnými parazity. Na základě vztahů mezi kvalitou spermatokritu, exprese sekundárních pohlavních znaků (tj. třecích vyrážek) a parazitace byly v této studii nepřímo potvrzeny hypotézy „imunokompetenčního handicapu“ a „sperm protection“.

V souvislosti s hormonální stimulací a chromozomální manipulací spojenou s modifikací hladiny ploidie a způsobu reprodukce u lína obecného (Tinca tinca) (studie 4) byly studovány změny ve vybraných parametrech imunitního a fyziologického systému ryb. Hormonální stimulace za účelem indukce ovulace a spermiace vyvolala významné změny v koncentraci glukózy a hemoglobinu a rovněž dvou imunitních parametrů (oxidativní vzplanutí a koncentrace lysozymu), které mohou být spojeny s tzv. sekundární stresovou reakcí či jinak indukovány (např. zvýšenou teplotou, manipulací apod.). Ploidní stav, navozený chromozomální manipulací, se projevil především v rozdílných počtech krevních elementů. Statisticky významné rozdíly v počtu erytrocytů, leukocytů a fagocytů byly zaznamenány mezi amfimiktickými diploidními a indukovanými triploidními jedinci.

ABSTRACT

The life functions of poikilothermic vertebrates are directly influenced by the abiotic environment in which these organisms are found. Furthermore, physiological and immune system of poikilothermic vertebrates can be affected by parasites. The interactions between the individual components of immune and physiological systems of a potential host organism are very complicated. This Ph.D. thesis is focused on the study of that kind of interactions from different points of view.

The seasonal changes of certain physiological and immunological parameters and parasitism in chub (Leuciscus cephalus) were studied in the first part of the thesis (study 1). Moreover, in the study, the potential relations between physiology, immunocompetence and parasitism were analyzed. It was recorded that seasonal changes significantly influenced dynamics of the main groups of metazoan parasites (Monogenea, Acanthocephala a Cestoda). Furthermore, there was noted a statistically significant effect even for physiological (gonad-somatic index, number of erythrocytes) and immunological parameters (spleen-somatic index, respiratory burst, number of leukocytes). There were confirmed important associations between components of immunity and physiology of host organism which supported the existence of potential trade off in energy allocation between important life traits. In addition to a negative relation between condition factor and spleen-somatic index, there was also indicated a negative implication in males between gonado-somatic index and immunological parameters (spleen-somatic index and leukocyte count). The results suggest an important role of interactions of parasites with the immune and physiological system of the host. A negative correlation was also discovered between the respiratory burst and Monogenea group. Above all, a positive association of gonado-somatic index and parasite abundance of Monogenea and Acanthocephala was recorded, which can be considered either a result of immunosuppressive role of steroid hormones, investment in demanding reproduction at the expense of immunocompetence, or synchronisation of parasite life cycles with the reproduction cycle of the host.

The following study 3 was focused on the analysis of the relations between physiology, immunocompetence and parasitism of common carp (Caprinus carpio). As well as that, this part of study was concerned with the immunocompetence of poikilothermic vertebrates and its susceptibility to seasonal changes in relation to parasite infection. Furthermore, the hypothesis of immunosuppressive effect of 11-ketotestosterone was tested. According to obtained results,

seasonal changes significantly influence the components of physiological and immune systems of poikilothermic vertebrates, as well as dynamics of parasite infection. After a correction for seasonal effect, a correlation between a total investment into physiology and immunity was noted, which indicates a potential existence of compromises between immunity and physiology of fish host. When studying the individual immunological and physiological parameters, the relation between immunity and physiology was not found. The investments into the individual components of physiological system suggest the distribution based on trade-off, i. e. negative relations were recorded between condition and hepato-somatic index on the one hand and gonado-somatic index on the other. This suggests the decrease of fish condition during the reproduction process when the energy is preferentially accumulated into gonad tissues. Moreover, the immunosuppressive role of 11-ketotestosterone was confirmed. This steroid hormone negatively affected the activity of complement. On top of that, the results indicate a connection between different strategies of parasites and individual components of physiological and immune system of the host.

In terms of the study 2, which included the evaluation of reproduction investments in chub (Leuciscus cephalus) during its pre-reproduction, reproduction and post-reproduction periods, it was found that the principle of trade-off decides in which important life traits components (condition, physiology and immunocompetence) the energy is allocated. The energy investments into vitally important components depend on current needs which are in pre- reproduction, reproduction and post-reproduction periods different. During the pre- reproduction period, the highest gonado-somatic and condition index was noted. In the following reproduction period, there was recorded a significant decrease of both these indexes which indicates that spawning is physically very exhausting and stressful period, which is the reason why the fish tend to accumulate energetic reserves. During the reproduction period, the level of most analyzed immunological parameters (respiratory burst, leukocyte count and leukocrit) increased. There were noted statistically significant differences between pre- reproduction, reproduction and post-reproduction periods in three numerous groups of metazoan parasites (Monogenea, Digenea a Cestoda). Nevertheless, abundance of all the mentioned groups reached lower values during the reproduction period, which indicates that the increased immunological parameters in this period were either induced by a stress connected with spawning of the fish, or were associated with infection which resulted from a

different pathogen and not from metazoan parasites. Based on the spermatocrit quality, the expression of secondary sex characteristics (i.e. spawning tubercles), and parasitism, the hypotheses of “immunocompetence handicap” and “sperm protection” were indirectly confirmed in this study.

In connection with hormonal stimulation and chromosomal manipulation joined with modification of ploidy level and a way of reproduction in Tinca tinca (study 4), there were studied the changes in selected parameters of immunological and physiological systems of fish. The hormonal stimulation aimed to induction of ovulation and spermiation induced significant changes in glucose and haemoglobin concentration, as well as in two immunological parameters (respiratory burst and lysozyme concentration) that can be connected with so called secondary stress reaction or can be induced in a different way, such as increased temperature, manipulation etc. Ploidy state, induced by a chromosomal manipulation, revealed different blood elements, which statistically differed between amphimictic diploid and induced triploid individuals.

OBSAH

1 ÚVOD ...... 14 2 CÍLE PRÁCE ...... 16 3 LITERÁRNÍ PŘEHLED ...... 17 3.1 Vztah mezi rybím hostitelem a jeho prostředím ...... 18 3.1.1 Vztah imunitního systému ryb k sezóně a parazitismu ...... 18 3.1.1.1 Velikost sleziny ...... 19 3.1.1.2 Počet leukocytů, lymfocytů a leukokrit ...... 21 3.1.1.3 Oxidativní vzplanutí a počet fagocytů...... 22 3.1.1.4 Koncentrace protilátek...... 23 3.1.1.5 Aktivita komplementu ...... 24 3.1.2 Vztah fyziologického systému ryb k sezóně a parazitismu ...... 25 3.1.2.1 Fyziologie ryb: kondice, játra a gonády...... 26 3.1.2.2 Počet erytrocytů, hematokrit a koncentrace hemoglobinu ...... 27 3.2 Vztah mezi parazitární infekcí a sezónou ...... 29 3.2.1 Monogenea ...... 29 3.2.2 Crustacea ...... 30 3.2.3 Mollusca ...... 30 3.2.4 Acanthocephala ...... 30 3.2.5 Digenea ...... 31 3.2.6 Nematoda ...... 31 3.2.7 Cestoda ...... 32 3.3 Vztah fyziologického, imunitního systému ryb a parazitismu v souvislosti s hypotézami aplikovanými v imunoekologii ...... 33 3.3.1 Základní hypotézy ...... 33 3.3.2 Vztahy mezi reprodukcí, somatickou kondicí, imunokompetencí a parazitismem na principu kompromisu ...... 34 3.4 Fyziologie a imunita ve vztahu k ploidii a hladině hormonů ...... 36 3.4.1 Reprodukce ryb ...... 37 3.4.1.1 Úloha hormonů v reprodukčním procesu ...... 37 3.4.1.2 Techniky umělé reprodukce ...... 38 3.4.2 Imunitní systém ryb: vliv ploidie a hladiny hormonů ...... 41

3.4.2.1 Velikost sleziny ...... 41 3.4.2.2 Počet leukocytů, lymfocytů a leukokrit ...... 42 3.4.2.3 Oxidativní vzplanutí a počet fagocytů...... 43 3.4.2.4 Koncentrace protilátek...... 44 3.4.2.5 Aktivita komplementu ...... 44 3.4.2.6 Koncentrace lysozymu ...... 45 3.4.3 Fyziologický systém ryb: vliv ploidie a hladiny hormonů ...... 46 3.4.3.1 Fyziologie ryb: kondice, játra a gonády...... 46 3.4.3.2 Počet erytrocytů, hematokrit a koncentrace hemoglobinu ...... 48 3.4.3.3 Koncentrace glukózy ...... 49 4 MATERIÁL A METODIKA ...... 51 4.1 Studované modely, lokality výzkumu ...... 51 4.1.1 Jelec tloušť ...... 51 4.1.2 Kapr obecný ...... 51 4.1.3 Lín obecný ...... 52 4.2 Fixace a determinace parazitologického materiálu ...... 57 4.3 Zpracování výsledků ...... 57 4.4 Metodika analýzy hematologických parametrů ...... 58 4.4.1 Odběr krve ...... 58 4.4.2 Diferenciální rozpočet leukocytů ...... 60 4.4.3 Stanovení počtu erytrocytů a leukocytů ...... 65 4.4.4 Hematokrit a leukokrit ...... 67 4.4.5 Množství hemoglobinu ...... 67 4.5 Metodika analýzy vybraných fyziologických a imunologických parametrů ...... 69 4.5.1 Velikost těla a vnitřních orgánů ...... 69 4.5.2 Odvozené indexy ...... 69 4.5.3 Koncentrace glukózy ...... 70 4.5.4 Koncentrace 11-ketotestosteronu ...... 70 4.5.5 Aktivita komplementu ...... 70 4.5.6 Koncentrace lysozymu ...... 71 4.5.7 Oxidativní vzplanutí fagocytů ...... 72 4.5.8 Stanovení koncentrace IgM protilátek ...... 73

5 VÝSLEDKY ...... 74 5.1 Sezónní změny imunokompetence a parazitismu u jelce tlouště (Leuciscus cephalus), sladkovodní kaprovité ryby ...... 74 5.2 Je imunitní systém ryb skutečně ovlivňován parazity? Imunoekologická studie u kapra obecného (Cyprinus carpio) ...... 76 5.3 Ovlivňují reprodukční investice kaprovitých ryb jejich imunokompetenci a výskyt mnohobuněčných parazitů? ...... 77 5.4 Fyziologie a imunologie lína obecného (Tinca tinca): efekt hormonální stimulace a ploidie spojené s chromozomální manipulací ...... 79 6 ZÁVĚRY ...... 81 7 SEZNAM LITERATURY ...... 85 8 PUBLIKACE TVOŘÍCÍ DIZERTAČNÍ PRÁCI ...... 98 9 OSTATNÍ PUBLIKACE ......

Poděkování ...mojí školitelce, doc. RNDr. Andree Vetešníkové Šimkové, Ph.D., za zázemí, které mi poskytla, za finanční podporu a hlavně za odborné vedení a cenné rady, které mi vždy ochotně a pohotově udílela, a to i za ztížených podmínek, kterým čelila v průběhu její mateřské dovolené;

...paní doktorce Božence Koubkové, za její praktické rady a lidský přístup;

...Nadě Musilové, za to, že mi po celou dobu studia byla psychickou oporou a hlavně přítelkyní, která mě vždy nasměrovala tím správným směrem;

... všem z parazitologie, kteří se trpělivě podíleli na sběru dat, jenž se často protáhl do časných ranních hodin. Doufám, že spolupráce se mnou na nich nezanechala žádné trvalé následky a rádi mě občas uvidí ☺;

...celé mojí rodině, která mě vždy podporovala a upřímně držela palce;

....a v neposlední řadě pak mému manželovi, díky jehož lásce, pohodové povaze a trpělivosti jsem se mohla plnohodnotně koncentrovat na dokončení doktorandského studia. Také děkuji všemožným televizním pořadům, které mu místo mě poslední rok a půl dělaly po večerech společnost ☺.

ÚVOD

1 ÚVOD

Ryby mohou být považovány za perspektivní a poměrně snadno dostupný vědecký model pro studium složitých vztahů, ať už na úrovni jedince a jeho životního prostředí (tj. hostitel- prostředí), nebo vztahů mezi jednotlivými životními funkcemi na úrovni jednoho organizmu. Jedná se o zástupce nižších obratlovců, jejichž tělesná teplota je závislá na teplotě jejich okolí.

Mnohobuněční paraziti, kteří jsou rovněž předmětem výzkumu předkládané práce, jsou díky svým unikátním vlastnostem považováni za vhodný model pro sledování biologických či ekologických zákonitostí. Cizopasníci ryb jsou ovlivňováni dvěma typy prostředí, tzv. prostředím prvního řádu (organismem hostitele) a prostředím druhého řádu (prostředím hostitele). Parazit je definován jako organismus, který čerpá alespoň po určité období svého životního cyklu zdroje (nejčastěji živiny) na úkor živého hostitelského organismu. Fakt, že parazit je ve velmi úzkém fyzickém kontaktu se svým hostitelem, mu poskytuje příležitost k cíleným zásahům do fungování hostitelského organismu.

Studium fyziologického a imunitního systému ryb může být považováno za základní kámen či jako mezistupeň pro studium některých vztahů u dalších skupin nižších obratlovců, ale i pro studium vyšších obratlovců včetně člověka. Protože se zájem většiny vědeckých pracovišť v současnosti z pochopitelných důvodů zaměřuje především přímo na studium savčího fyziologického systému, mnohé základní otázky týkající se fyziologie včetně imunity ryb, zůstávají dosud nezodpovězeny.

I přesto, že v současné době existuje řada vědeckých prací, které se zabývají studiem vybraných vztahů v rámci systému „parazit-hostitel-prostředí“, většinou jsou studovány v laboratorních podmínkách. Počet studií, které by nám umožnily komplexnější pohled na rozmanitost vztahů v přirozených podmínkách, je stále limitován. Proto je v předkládané práci na tyto vztahy pohlíženo z několika různých úhlů. Jelikož se v poslední době dostává do popředí vědeckého zájmu i moderní „imuno-ekologický“ a „imuno-evoluční“ pohled, jsou vztahy mezi imunitou, fyziologii a parazitismem často diskutovány z hlediska potenciálních kompromisů („trade-offs“) mezi základními složkami životních strategií.

Předkládaná práce je tematicky rozdělena na 4 části. První tematickou část představuje studium sezónních vlivů a parazitismu na fyziologický a imunitní systém kaprovitých ryb. Druhá část je věnovaná problematice investic do životně důležitých funkcí ryb na principu alokace energie

ÚVOD

(reprodukce, imunitní obrana či celkový stav jedince). Poslední část je pak věnována vlivu hladiny hormonů a efektu ploidie, jako důsledku chromozomální manipulace na vybrané fyziologické a imunitní parametry ryb.

Předkládaná práce byla realizována na Oddělení parazitologie Ústavu botaniky a zoologie Přírodovědecké fakulty Masarykovy univerzity za finanční podpory Centra základního výzkumu (LC522), Výzkumného záměru (MSM 0021622416), projektů GAČR 524/04/1128 a 524/07/0188 a v neposlední řadě Programu rektora MU na podporu tvůrčí činnosti studentů.

CÍLE PRÁCE

2 CÍLE PRÁCE

1) Studium sezónních změn imunokompetence a odhad potenciálních asociací mezi fyziologií, imunitou a parazitismem u kaprovitých ryb, srovnání vlivu sezóny a parazitismu na vybrané imunitní a fyziologické parametry

2) Studium investic do kondice a imunity u kaprovitých ryb v období různých investic do reprodukce (před-reprodukční, reprodukční tj. tření a post-reprodukční období) a vztah k parazitární zátěži

3) Studium vlivu stimulace a ploidie, jako důsledku chromozomální manipulace, na vybrané parametry imunitního a fyziologického systému vybraného modelu kaprovité ryby

LITERÁRNÍ PŘEHLED

3 LITERÁRNÍ PŘEHLED

Vztahy mezi parazitem, hostitelem a jeho prostředím mohou být značně komplikované. Složitost těchto vztahů je schematicky naznačena na Obr. č. 1. Vztahům vybraných abiotických faktorů prostředí (A) a parazitární infekce (C) na složky fyziologie a imunity hostitele je věnovaná kapitola 3.1. Vztahy mezi abiotickým prostředím a parazitární infekcí (B) u hlavních skupin mnohobuněčných cizopasníků jsou komentovány v kapitole 3.2. Následuje kapitola věnovaná hypotézám aplikovaným v imunoekologii (kapitola 3.3) a na závěr jsou diskutovány vybrané vztahy v rámci fyziologického a imunitního systému v souvislosti s hladinou hormonů a ploidním stavem, jako důsledkem chromozomální manipulace (kapitola 3.4.).

Obr. 1: Vzájemné vztahy mezi hostitelem, jeho prostředím a parazitem

LITERÁRNÍ PŘEHLED

3.1 Vztah mezi rybím hostitelem a jeho prostředím

Ryba se jako potenciální hostitel nachází v prostředí rozmanitě působících abiotických či biotických faktorů. Mezi abiotické faktory patří především sezónnost, teplota vody a další faktory (rychlost vodního toku, stupeň kyselosti, znečištění apod.) (Lipský, 1998). Tyto „neživé“ faktory pak zcela zásadním způsobem ovlivňují nejen fyziologické procesy těchto poikilotermních živočichů, ale i životní cykly parazitů (viz kap. 3.2).

Biotické faktory prostředí jsou charakterizovány jako vzájemné interakce mezi živými organismy. Za biotický faktor, který působí na hostitele, může být tedy považován i parazit. Paraziti jsou schopni modifikovat různé vlastnosti svého hostitele, počínaje morfologií, regulací metabolismu a rozdělováním energie mezi jednotlivé životní funkce hostitele až po specifické zásahy, například do nervového systému za účelem změny chování hostitele. Cizopasník je v tomto úzkém soužití na svém hostiteli metabolicky závislý a kromě výše zmíněných účinků může hostiteli působit značné energetické ztráty (Ebert & Herre, 1996), poškozovat tkáně či orgány (Thompson et al., 2005), případně způsobit až úhyn silně infikovaných hostitelů (Esch & Fernández, 1993). Hlavní strategií parazita, v tomto nepříliš harmonickém soužití, je úspěšné rozmnožování, přežívání a perzistence parazita v hostiteli. Hostitel se naopak v tomto souboji snaží vynaložit veškeré úsilí k tomu, aby si vytvořil rezistenci proti parazitární infekci nebo alespoň do jisté míry negativní působení parazita toleroval (Wakelin, 1996).

3.1.1 Vztah imunitního systému ryb k sezóně a parazitismu

Vliv sezóny Ryby disponují podobnými mechanismy imunitní obrany jako savci, tj. nespecifickými (vrozenými) i specifickými (získanými) (Du Pasquier, 1993). Jejich imunitní systém se však markantně liší od savčího tím, že je velmi významně ovlivňován abiotickými faktory prostředí, z nichž za nejvýznamnější je považována teplota vody.

Ainsworth et al. (1991) ve své práci uvádí, že specifická větev imunity ryb je více náchylná na nižší teplotu vody než nespecifická. Navíc podle studie Le Morvan et al. (1998) dochází u ryb k tomu, že zatím co je specifická imunita při snížení teploty vody potlačena, nespecifická imunita má tendenci tento stav kompenzovat. Tento stav přetrvává do doby, než se specifická

LITERÁRNÍ PŘEHLED

část imunitního systému na změnu teploty adaptuje. Z těchto důvodů by mohla nespecifická imunita u poikilotermních obratlovců hrát významnější roli než specifická imunita.

Vliv parazitismu K nejčastějším fyziologickým změnám, jež jsou parazity indukovány, patří změny imunitní odpovědi hostitele. Parazitismus je v rámci všech živých organismů právem považován za vůbec nejúspěšnější životní strategii (Poulin & Morand, 2000). Tato úspěšnost je dána nejen obrovskou rozmanitostí druhů parazitů, ale především různými mechanismy, kterými jsou paraziti aktivně schopni vyhýbat se imunitnímu systému hostitele.

1) ukrývají se před účinky imunitního systému v tzv. „imunoprivilegovaných orgánech“ nebo se pomocí aktivit imunitního systému izolují (enkapsulují);

2) migrují tělem tak, aby nebyli imunitním systémem dostihnuti;

3) vyvíjejí se tak rychle, že imunitní systém nestačí na různá stádia parazitů reagovat imunitní odpovědí;

4) používají rozmanité mimikry nebo maskování;

5) působí imunomodulačně či dokonce imunosupresivně atd. (viz Sitja-Bobadilla, 2008).

Kromě těchto aktivních účinků parazita, může hrát parazit v interakcích s imunitním systémem hostitele i roli čistě pasivního agens, na jehož přítomnost reaguje hostitel specifickou imunitní odpovědí. Veškeré aktivity parazita by však měly být regulovány tak, aby výsledkem nebylo usmrcení hostitele. Imunita namířená proti parazitární infekci může být s lehkou nadsázkou přirovnávána ke stolní hře šachy. Tento souboj se však od klasické hry liší v tom, že je rozehrán už po milióny let (Cox, 1997).

3.1.1.1 Velikost sleziny

Kromě hematopoézy, hraje slezina kostnatých ryb rovněž důležitou úlohu i v imunitních reakcích. Tento sekundární lymfatický orgán se účastní jak tvorby protilátek, tak odstraňování patogenů a jiných cizorodých částic z krevního řečiště (Dalmo et al., 1997). Kromě toho, že velikost tohoto sekundárního lymfatického orgánu může hrát potenciálně významnou roli v imunitní obraně proti parazitům (Manning, 1994; Kortet et al., 2003a; Ottová et al., 2005), rovněž může poskytnout důležité informace o aktuálním stavu imunokompetence a o

LITERÁRNÍ PŘEHLED investicích do imunity (John, 1995; Morand & Poulin, 2000; Skarstein et al., 2001; Ottová et al., 2005) (viz kapitola 3.3).

Vliv sezóny Práce, jejichž cílem je studium sezónních změn velikosti sleziny (případně slezino-somatického indexu), nejsou početné a jejich výsledky nejsou příliš konzistentní. Zapata et al. (1992) zaznamenali, že v průběhu sezóny se u poikilotermních obratlovců mění jak struktura, tak funkčnost imunitního systému. Proto byla slezina u pstruha obecného (Salmo trutta) studována morfometricky (Alvarez et al., 1998). V této studii bylo zjištěno, že distribuce lymfocytů v tomto orgánu se v průběhu sezóny liší. Nejvíce lymfocytů ve slezině bylo zaznamenáno na jaře a na podzim, naopak v zimě a létě byl zjištěn jejich pokles.

Dále byly sezónní změny velikosti sleziny studovány u plotice obecné (Rutilus rutilus) (Kortet et al., 2003a) nebo u sivena severního (Salvelinus alpinus) (Skarstein et al., 2001). U obou druhů ryb byl zaznamenán pokles velikosti sleziny v průběhu reprodukce. Tento pokles velikosti sleziny v průběhu reprodukce může být vysvětlen na základě existence fyziologického „trade-off“, kdy může jedinec přednostně investovat dostupnou energii do reprodukce na úkor imunitní obrany (Kortet et al., 2003a; Piersma & Lindstrom, 1997) (viz kapitola 3.3).

Vliv parazitismu Vztah velikosti sleziny a parazitární infekce byl studován z pohledu ultrastrukturální přestavby tohoto imunitního orgánu. U jedinců kapra obecného (Cyprinus carpio), experimentálně infikovaných motolicí Sanguinicola inermis, byla zaznamenána statisticky významná redukce erytrocytů ve slezině a rovněž byla parazitismem negativně ovlivněna váha tohoto orgánu (Richards et al., 1994). Podobný závěr byl publikován ve studii plotice obecné (Rutilus rutilus), infikované plerocerkoidy tasemnice Ligula intestinalis (Taylor & Hoole, 1989). Opačný efekt parazitární infekce na tento lymfatický orgán byl pozorován ve studii Lefebvre et al. (2004), kde u úhoře říčního (Anguilla anguilla) bylo pozorováno zbytnění sleziny vlivem infekce způsobené hlísticí Anguillicola crassus. Tento efekt může být výsledkem: (1) patologické reakce na přítomnost parazita; (2) manipulace parazita za účelem zvýšení krevního metabolismu nebo (3) imunitní reakce hostitele.

LITERÁRNÍ PŘEHLED

3.1.1.2 Počet leukocytů, lymfocytů a leukokrit

Mezi důležité parametry, vypovídající o zdravotním stavu organismu, patří hodnoty bílého krevního obrazu. Pro účely dizertační práce byly hodnoty bílého krevního obrazu použity jako parametry odrážející se v celkové imunitní odpovědi, v kondici studovaného jedince a potenciální parazitární infekci. Ačkoliv byla shledána určitá paralela (funkční či morfologická) mezi některými typy savčích a rybích lymfocytů, již nyní je jisté, že mezi nimi existují zásadní rozdíly. Tyto odlišnosti jsou v současnosti předmětem intenzivního výzkumu. Jelikož počet recentních druhů kostnatých ryb se odhaduje na 24–30 tisíc, lze navíc předpokládat, že rozdíly budou i mezidruhové. Kromě druhové příslušnosti můžeme pro bílý krevní obraz ryb zaznamenat odlišnosti sezónního charakteru nebo vnitrodruhové rozdíly. Ty jsou dány teplotou vody, environmentálním stresem, pohlavními rozdíly nebo věkem jedinců (Lusková, 1997; Modrá et al., 1998).

Vliv sezóny Problematikou vlivu sezónních změn na hodnoty bílého krevního obrazu se detailně zabývala ve své práci Lusková (1997), která studovala kromě jiných krevních parametrů (viz níže) i dynamiku sezónních změn v počtu leukocytů a leukokrit u různých druhů ryb. U pstruha obecného (Salmo trutta), lipana podhorního (Thymallus thymalus) a ostroretky stěhovavé (Chondrostoma nasus) byla dynamika celkového počtu leukocytů analogická sezónním změnám teploty vody s výjimkou některých měsíců, kdy došlo k vychýlení z tohoto trendu. Navíc v této práci bylo zdůrazněno, že ačkoliv je leukokritová hodnota některými autory dokonce doporučena jako diagnosticky vhodný parametr (především pro jednoduchost jeho měření), vyznačuje se vysokou individuální variabilitou a statisticky málo významnou korelací s celkovým počtem leukocytů. Trend závislosti počtu leukocytů na teplotě vody, zaznamenaný v této studii, nebyl u leukokritu jednoznačně potvrzen. Sezónní dynamika změn počtu leukocytů byla dále studována u střevle americké (Pimephales promelas) (Thomas et al., 1999). V uvedené studii byl zaznamenán pozitivní vztah mezi počtem leukocytů a teplotou vody a navíc byly zjištěny zvyšující se hodnoty leukocytů v období reprodukce ryb.

Vliv parazitismu Hodnoty bílého krevního obrazu byly rovněž předmětem studia v souvislosti s parazitární infekcí. U pstruha obecného (Salmo trutta) byla po experimentální infekci tasemnicí Diphyllobotrium dentriticum pozorována lymfocytóza. Ačkoliv součástí analýz, v souvislosti

LITERÁRNÍ PŘEHLED s parazitární infekcí u pstruha obecného, nebylo stanovení hladiny protilátek, zmnožený počet lymfocytů byl pravděpodobně spojený se syntézou specifických protilátek proti D. dentriticum (Rahkonen & Pasternack, 1998). U pstruha duhového (Oncorhynchus mykiss), infikovaného stejným druhem parazita, byla pozorována imunita namířená proti plerocerkoidům již od 2. týdne po infekci. Konkrétně pak zvýšení počtu leukocytů bylo zaznamenáno 3-6 týdnů po infekci a přítomnost plně enkapsulovaných plerocerkoidů byla pozorována 6 týdnů po infekci (Sharp et al., 1992).

V další studii (Lamas et al., 1994) vedla infekce způsobená parazitem Myxobolus cerebralis (Myxozoa) u pstruha duhového (Oncorhynchus mykiss) ke snížení celkového počtu leukocytů a malých lymfocytů v periferní krvi. Lymfopénie, zaznamenaná u takto infikovaných ryb, pak může být výsledkem destrukčních mechanismů parazita nebo migrací lymfocytů z periferní krve do napadených orgánů a tkání.

3.1.1.3 Oxidativní vzplanutí a počet fagocytů

Fagocyty a jejich schopnost produkce reaktivních kyslíkových radikálů patří mezi hlavní mechanismy protektivní imunity namířené proti patogenům překonávající přirozené bariéry imunitního systému (Secombes, 1996).

Vliv sezóny Vztah teploty vody a oxidativního vzplanutí byl studován u různých druhů ryb (Scott et al., 1985; Angelidis et al., 1988; Ainsworth et al., 1991). Výsledky těchto studií však nejsou příliš konzistentní. Imunosupresivní efekt teploty vody na přirozenou složku imunity ryb byl zaznamenán u sumečka skvrnitého (Ictalurus punctatus) (Ainsworth et al., 1991) nebo lína obecného (Tinca tinca) (Collazos et al., 1995). V laboratorních podmínkách však tento vztah u pstruha duhového (Oncorhynchus mykiss) potvrzen nebyl (Nikoskelainen et al., 2004). Statisticky významné sezónní změny oxidativního vzplanutí byly dále pozorovány u dvou různých populací plotice obecné (Rutilus rutilus) (Kortet et al., 2003a). U samic jedné z těchto populací byly zaznamenány dočasně zvýšené hodnoty oxidativního vzplanutí v reprodukčním období. Tento výsledek naznačuje, že zatímco některé složky imunitní obrany mohou být v průběhu reprodukce potlačeny, jiné nemusí být reprodukcí ovlivněny. Nejvyšší hodnoty oxidativního vzplanutí u samců i samic obou sledovaných populací byly zjištěny v říjnu. Tyto

LITERÁRNÍ PŘEHLED výsledky by podle autorů studie mohly být spojeny s adaptací jedinců na nižší teplotu vody, která může vést k vyšším investicím do nespecifické imunity (Kortet et al., 2003a).

Vliv parazitismu Ve vztahu k parazitární infekci byl u ryb rovněž studován počet fagocytujících elementů či jejich aktivita (viz např. Alvarez-Pellitero, 2008). Častou reakcí na výskyt parazitózy pak bývá zvýšení počtu fagocytů a rovněž zvýšení aktivity jejich obranných mechanismů (oxidativní vzplanutí) za účelem eliminace parazita, což bylo potvrzeno experimentálně (např. Redondo et al., 2004; Sitja-Bobadilla et al., 2006).

V současné době jsou však známy i strategie parazitů, které zahrnují různé mechanismy vyhýbání se imunitní odpovědi hostitele. Mezi tyto mechanismy patří například schopnost potlačení či dokonce funkční poškození fagocytů. Mezi parazitární infekcí a počtem či funkcí fagocytů může proto existovat i negativní vztah. Nízká aktivita fagocytů měřená pomocí oxidativního vzplanutí byla například demonstrována u koljušky tříostné (Gasterosteus aculeatus), experimentálně infikované tasemnicí Schistocephalus solidus (Scharsack et al., 2004).

3.1.1.4 Koncentrace protilátek

Ačkoliv je v současné době již známo několik tříd imunoglobulinů u ryb, například IgD (Harding et al., 1990), IgZ (Danilová et al., 2005) nebo IgT (Hansen et al., 2005), za hlavní složku humorální imunity ryb jsou považovány protilátky typu IgM.

Vliv sezóny Sezónní dynamika koncentrace IgM protilátek byla v dosavadních publikacích diskutována z několika různých úhlů pohledů a rovněž s různými závěry. Sezónní změny v dynamice koncentrace IgM protilátek úměrné teplotě vody byly pozorovány u karase zlatého (Carassius auratus). U pstruha duhového (Oncorhynchus mykiss) byl zaznamenán pokles koncentrace IgM v zimě, což bylo přisuzováno nízké teplotě vody v tomto období (Sánchez et al., 1993). U kapra obecného (Cyprinus carpio) byly také zaznamenány nejvyšší titry IgM protilátek v období tření ryb. Tento výsledek je rovněž spojován s maturací gonád (Saha et al., 2002). V experimentální studii Suzuki et al. (1997), kdy byla hladina IgM sledována za konstantní teploty a za přirozené délky dne u pstruha duhového (Oncorhynchus mykiss), byl naopak zaznamenán imunosupresivní účinek steroidních hormonů. Saha et al. (2002) ve své práci naznačují, že

LITERÁRNÍ PŘEHLED diametrální odlišnosti hladin IgM protilátek zaznamenané především v období reprodukce, mohou souviset s druhovou rozdílností lososovitých a kaprovitých ryb.

Vliv parazitismu Vliv koncentrace IgM na parazitární infekci byl studován v práci Vainikka et al. (2009), kde byl zaznamenán pozitivní vztah mezi koncentrací IgM a množstvím mrtvých jedinců motolice Rhipidocotyle campanula u plotice obecné (Rutilus rutilus). Autoři této práce proto navrhli, že koncentrace IgM může být považována za imunitní parametr, vypovídající o schopnosti imunitního systému udržovat parazitární infekci pod kontrolou. Další publikované studie se však spíše soustřeďují na analýzu specifických protilátek než na stanovení celkové koncentrace IgM. Například u různých rybích hostitelů byla zaznamenána produkce specifických protilátek proti motolicím Cryptocotyle lingua a Diplostomum spathaceum (viz Alvarez-Pellitero, 2008). Specifické protilátky byly, například po experimentální infekci pstruha duhového (Oncorhynchus mykiss) plerocerkoidy Diphyllobothrium dendriticum, detekovány pět týdnů po infekci. Maximální hladina protilátek byla zaznamenána po 11-ti týdnech (Sharp et al., 1992).

3.1.1.5 Aktivita komplementu

Komplementová kaskáda je součástí fylogeneticky původních mechanismů přirozené imunitní obrany proti patogenní infekci. Komplement ryb je stejně jako u savců aktivován buď klasickou, nebo alternativní cestou. Recentně byly dokonce u kostnatých ryb objeveny i některé komponenty lektinové cesty aktivace komplementu (Kania et al., 2010). Její skutečná funkčnost však zatím nebyla potvrzena. V imunitní obraně proti patogenům dochází v rámci složek komplementového systému ke kooperaci při fagocytóze, zánětu a navíc k tvorbě tzv. lytických pórů. Tyto mechanismy zasahují jak do specifické (protilátky), tak do nespecifické části imunity (fagocyty) (Flajnik & Du Pasquier, 2004).

Vliv sezóny Bylo zjištěno, že i komplement, stejně jako ostatní imunitní složky, je ovlivňován teplotou vody. Navíc i ostatní fyziologické faktory, jako je pohlavní dozrávání (Roed et al., 1992) nebo příprava ryb na změnu sezóny (Hayman et al., 1992), mohou ovlivnit funkci komplementového systému. U ryb byl komplement studován v souvislosti s teplotou vody například u mořana zlatého (Sparus aurata) (Hernández & Tort, 2003). Se vzrůstající teplotou vody vzrůstala i aktivita komplementu, tj. nejnižší aktivita komplementu byla zaznamenána v lednu a naopak nejvyšší na

LITERÁRNÍ PŘEHLED začátku podzimu, kdy teplota vody dosahovala nejvyšších hodnot. Tento vztah autoři studie vysvětlují zvýšeným metabolismem poikilotermních živočichů při vyšší teplotě. Dále byl studován význam aktivity komplementu pro aklimatizaci pstruha duhového (Oncorhynchus mykiss) ve vztahu k teplotě vody. Čím vyšší byla teplota vody, při které byly ryby aklimatizovány, tím vyšší byla lytická schopnost jak celkové, tak alternativní cesty komplementu (Nikoskelainen et al., 2004).

Vliv parazitismu Komplementový systém může hrát velmi důležitou úlohu při imunitní obraně proti infekci vyvolané parazity. U pstruha duhového (Oncorhynchus mykiss) byl komplement nalezen v pokožce a séru, kde může být snadno aktivován tělním pokryvem ektoparazitických monogeneí bohatým na karbohydráty (Buchmann, 1998). Letální efekt komplementu byl rovněž prokázán proti Discocotyle sagittata (Monogenea) u pstruha obecného (Salmo trutta) a pstruha duhového (Oncorhynchus mykiss) (Rubio-Godoy et al., 2004) nebo Cryptobia salmositica (Kinetoplastea) u pstruha duhového (Oncorhynchus mykiss)(Mehta & Woo, 2002).

3.1.2 Vztah fyziologického systému ryb k sezóně a parazitismu

Vliv sezóny Kromě rozmanitě působících klimatických podmínek, ve kterých se volně žijící ryby nacházejí a které zcela zásadním způsobem zasahují do jejich fyziologických funkcí, se v různých sezónních obdobích navíc mění spektrum i množství dostupné potravy. Energie získaná z potravy je dle potřeb organismu akumulována do různých životně důležitých funkcí (např. Smith et al., 1990; Craig et al., 2000). Zásadní změny v akumulaci proteinů a lipidů lze pozorovat například v průběhu reprodukce. Během somatického růstu mimo reprodukci jsou proteiny a lipidy akumulovány, zatímco v průběhu reprodukce je energie v zásadě spotřebovávána (Jorgensen et al., 1997).

Vliv parazitismu Volně žijící jedinci se obvykle nacházejí v prostředí bohatém na patogenní organismy, včetně parazitů, které mají na fyziologické funkce svého hostitele většinou negativní vliv. Ačkoliv je v současnosti v odborné literatuře spíše kladen důraz na hodnocení imunitních procesů (viz kapitola 3.1.1) zaměřených proti konkrétním cizopasníkům, neměl by být opomenut zejména

LITERÁRNÍ PŘEHLED celkový stav vyšetřovaného jedince a imunologické analýzy by měly být doplněny i o měření aktuální kondice hostitele.

3.1.2.1 Fyziologie ryb: kondice, játra a gonády

Jako nepřímé ukazatele fyziologického stavu organismu mohou být využity následující indexy: kondiční, hepato-somatický a gonado-somatický. První dva jmenované indexy vypovídají o kondičním stavu jedince a třetí v pořadí vypovídá o reprodukční zralosti ryb (Busacker et al., 1990).

Vliv sezóny U samců koljušky tříostné (Gasterosteus aculeatus) bylo zjištěno, že mezi investicemi energie do gonád a do ostatních somatických tkání existuje negativní vztah. Tento vztah byl zjištěn především v období před rozmnožováním, kdy dochází k toku glykogenu a tuků do reprodukční tkáně na úkor celkového stavu jedince (Huntingford et al., 2001). Z pohledu imunoekologického mohou být tyto vztahy vysvětleny na základě teorie „trade-off“ (kompromisu) v alokaci energie. Podle této teorie je energie dle aktuálních potřeb investována do životně důležitých funkcí (Roff, 1992) (viz kapitola 3.3). V závislosti na ročním období se však tyto investice organismu mohou měnit, což může být spojeno se sezónními změnami fyziologických parametrů. Bylo například zjištěno, že játra ryb jsou schopna velmi rychle reagovat na změny environmentálních podmínek (Foster et al., 1993).

Vliv parazitismu Stav fyziologického systému, z pohledu akumulace energie v různých orgánech, může být posuzován i v souvislosti s parazitární infekcí. Negativní vliv parazitární infekce na kondici hostitele, tzv. fitness-redukující efekt (Ebert & Herre, 1996), je obecně uznávaným faktem u většiny skupin parazitů. Navíc bylo zjištěno, že parazitární infekce u různých druhů ryb měla prokazatelně negativní efekt na hepato-somatický index (Malek, 2001). Pozitivní vztah mezi gonado-somatickým indexem a početností Proteocephalus sagittus (Cestoda) byl zaznamenán u mřenky mramorované (Barbatula barbatula) (Šimková et al., 2005). Podle autorů této studie může být tento vztah indukován vzrůstající hladinou steroidních hormonů za účelem synchronizace životního cyklu parazita se začátkem reprodukce hostitele. Podobné výsledky, týkající se parazitární infekce tasemnic a reprodukce ryb, byly dokumentovány i u koljušky tříostné (Gasterosteus aculeatus) (Heins et al., 1999). Dále Radhakrishnan et al. (2010)

LITERÁRNÍ PŘEHLED zaznamenali negativní vliv infekce vyvolané parazitem Philometra cephalus (Nematoda) u cípala dlouhoploutého (Valamugil cunnesius) na kondiční, hepatosomatický i gonadosomatický index.

3.1.2.2 Počet erytrocytů, hematokrit a koncentrace hemoglobinu

Mezi parametry červeného krevního obrazu patří počet erytrocytů, hematokrit a koncentrace hemoglobinu. Od těchto základních ukazatelů lze dále odvodit střední objem erytrocytů (MCV), střední obsah hemoglobinu v erytrocytu (MCH) nebo střední koncentraci hemoglobinu (MCHC). Z funkčního hlediska indikují hodnoty základních parametrů červeného krevního obrazu schopnost vázat kyslík a uspokojit tak metabolické nároky všech živých organismů na tento nezbytně důležitý biogenní prvek (Houston, 1990).

Hodnoty ukazatelů červených krvinek mohou být ovlivněny celou řadou biotických i abiotických faktorů, podobně jako je to v případě bílých krvinek. Kromě mezidruhových odlišností v parametrech červeného krevního obrazu mohou být pozorovány rozdíly i vnitrodruhové. Pro tuto skutečnost existuje několik možných vysvětlení. Rozdíly mohou být podmíněny geneticky (Houston, 1990) či indukovány různými faktory působící v daném ekosystému nebo lokalitě (Vanvuren & Hattingh, 1978).

Vliv sezóny Okolní teplota, koncentrace kyslíku či fotoperioda jsou faktory, které mají přímý vliv na schopnost vázat kyslík na hemoglobin, což se následně odráží na koncentraci hemoglobinu i na počtu erytrocytů v krvi ryb (Houston, 1980). Koncentrace hemoglobinu je navíc úzce spojena s velikostí či stupněm zralosti erytrocytů, tj. nezralé erytrocyty obsahují méně hemoglobinu a naopak (Hardig & Hoglund, 1983). Rovněž je nutné mít na paměti, že stres vzniklý při manipulaci s analyzovanými jedinci může zásadně ovlivnit parametry červeného krevního obrazu (Aldrin et al., 1982).

McKnight (1966) uvádí ve své práci, že ze všech hematologických parametrů je sezónou nejvíce ovlivněn počet erytrocytů a hematokrit. Dynamikou sezónních změn hematologických parametrů u pstruha obecného (Salmo trutta), lipana podhorního (Thymallus thymalus) a ostroretky stěhovavé (Chondrostoma nasus) se podrobně zabývala Lusková (1997). Například u pstruha obecného (Salmo trutta) byly zaznamenány zvýšené hodnoty počtu erytrocytů v květnu, červenci, říjnu a prosinci. Hodnoty hematokritu a obsahu hemoglobinu také v průběhu roku oscilovaly. Dále byly zaznamenány statisticky vyšší hodnoty obou proměnných u samců.

LITERÁRNÍ PŘEHLED

Navíc se sezónní dynamika těchto parametrů lišila mezi všemi vyšetřovanými druhy. Autoři studie uvádí, že při hodnocení dynamiky změn v červeném krevním obraze bylo často obtížné detekovat určitou pravidelnost sezónních změn a zaznamenané sezónní výkyvy byly pravděpodobně způsobeny vlivem teploty vody, reprodukcí ryb a jejich vlastní biologií. Konkrétní příčina těchto sezónních oscilací však nebyla v této studii jednoznačně objasněna.

Vliv parazitismu Anémie, která může být diagnostikována na základě červeného krevního obrazu, je také často spojena s probíhající parazitární infekcí, poškozením krvetvorných orgánů či jinak navozeným stresem (Pravda & Svobodová, 2003). Bylo například zjištěno, že i velmi nízká intenzita infekce, způsobená druhem Sparicotyle chrysophrii (Monogenea), může u ryb vyvolat anémii. Taková infekce může následně vést k aktivaci hematopoézy a vyplavení nezralých, na hemoglobin chudých, červených krvinek do krevního řečiště (Sitja-Bobadilla & Alvarez-Pellitero, 2009). Další příčinou anémie mohou být bičíkovci z rodu Trypanosoma nebo Trypanoplasma (Kinetopastea), kteří se živí látkami obsaženými v krevní plazmě a tkáňových tekutinách. Při masivní infekci, která je typicky doprovázena anémií žaber, dochází až k úhynu infikovaných hostitelských jedinců. Dalším parazitem souvisejícím se změnami v krevním obrazu ryb je zástupce myxosporeí Sphaerospora renicola (Pravda & Svobodová, 2003). Tento parazit se složitým vývojovým cyklem může kromě poškození krvetvorné funkce ledvin poškozovat i jejich vylučovací funkci. Příčinou změn v červeném krevním obraze, které mají spíše sekundární charakter, může být motolice Sanguinicola inermis. Vlivem infekce může docházet například ke snížení respirační plochy žaber a následně k dušení plůdku (Pravda & Svobodová, 2003).

LITERÁRNÍ PŘEHLED

3.2 Vztah mezi parazitární infekcí a sezónou

Životní cyklus parazitů ryb je v přirozených podmínkách významně ovlivněn životním prostředím hostitele (Esch et al., 1990; Rohde et al., 1995; Kennedy, 1975). Sezónní změny působí na početnost, vývoj i samotný výskyt parazitů. U některých skupin parazitů (např. Acanthocephala, Digenea, Cestoda nebo Nematoda) mohou být navíc životní cykly závislé na mezihostiteli, jehož přítomnost může být rovněž ovlivněna sezónními změnami (např. Hanzelová & Gerdeaux, 2003). Klíčovou roli u těchto skupin parazitů navíc hraje i úspěšný přenos mezihostitele na hostitele definitivního.

3.2.1 Monogenea Početnost této skupiny ektoparazitů se mění v závislosti na sezónních změnách teploty vody, s nimiž úzce souvisí jak rozmnožovací cyklus, tak mortalita těchto parazitů (Kearn, 1994). Monogenea parazitují především na žábrách, ploutvích či pokožce ryb. Zvláště hojně jsou zastoupena u kaprovitých ryb a obecně se jedná o druhově i početně nejbohatší skupinu cizopasníků. Životní cyklus vejcorodých monogeneí zahrnuje uvolnění vajíčka do prostředí, líhnutí volně plovoucí obrvené larvy (onkomiracidia), která musí během svého krátkého života nalézt svého hostitele, na němž dospívá (Kearn, 1994). Významnou skupinou vejcorodých monogeneí je rod Dactylogyrus. Zatímco některé druhy rodu Dactylogyrus dosahují nejvyšší abundance v letních měsících spojených s nejvyšší teplotou vody (např. Koskivaara et al., 1991b), některé druhy naopak preferují nižší teplotu. Například Pojmańska (1995) ve své studii zaznamenala nejvyšší abundanci druhu Dactylogyrus lamellatus v zimních měsících, zatímco druh Dactylogyrus nobilis dosahoval nejvyšší hodnoty abundance na podzim a na jaře.

Mezi vejcorodá a navíc hematofágní monogenea patří zástupci čeleď Diplozoidae. Vývojový cyklus zahrnuje stádia: vajíčko, onkomiracídium, diporpa, juvenil a adult. Khotenovsky (1985) uvádí, že juvenilní spárovaní jedinci přežívají zimní období přichyceni na žábrách hostitelské ryby a na jaře pak dochází k rychlému vývoji jejich gonád a následné produkci vajíček (zejména v průběhu června a července). Sezónní dynamika druhu Paradiplozoon homoion byla sledována například u hrouzka obecného (Gobio gobio) (Pečínková et al., 2007). Nejnižší průměrná abundance byla pozorována od dubna do července, tj. v období, kdy jsou dospělí jedinci připraveni produkovat vajíčka nové generace.

LITERÁRNÍ PŘEHLED

Životní cyklus živorodých monogeneí rodu Gyrodactylus je charakteristický specifickým typem nepohlavního rozmnožování označovaným jako progenetická polyembryonie. V porovnání s vejcorodými druhy bývají živorodé druhy monogeneí považovány spíše za chladnomilnější. Sezónní dynamika rodu Gyrodactylus byla studována například u plotice obecné (Rutilus rutilus), kdy nejvyšší početnost byla pozorována na podzim (Koskivaara et al., 1991a).

3.2.2 Crustacea Jedním z nejběžnějších zástupců parazitických členovců u kaprovitých ryb je rod Argulus. Tento obligátní ektoparazit cizopasí na povrchu těla a ploutvích, kde se živí tkáňovým mokem svého hostitele. Někteří zástupci rodu Argulus jsou schopni tolerovat široké rozpětí teploty vody. Nejpočetnější evropský druh, Argulus foliaceus, dosahuje maximální početnosti především v pozdních letních měsících a také na podzim (Hakalahti et al., 2004; Harrison et al., 2006). Hakalahti & Valtonen (2003) zjistili, že nízká abundance druhu Argulus coregoni v zimním období je dána tím, že tento parazit přezimuje díky vajíčkům, která jsou nakladena na podzim. Dalším početným rodem parazitických korýšů je rod Ergasilus. Dospělé samičky se přichytávají na žaberní oblouky pomalu se pohybujících ryb (např. lín obecný), kde se živí epitelovými a slizovými buňkami. Reprodukce některých druhů rodu Ergasilus je zahájena v jarních měsících a zpravidla bývá ukončena na podzim. Konkrétně druh Ergasilus sieboldi dosahuje nejvyšší abundance v létě (Alston & Lewis, 1994).

3.2.3 Mollusca Další unikátní skupinou ektoparazitů napadajících žábry, ploutve a pokožku ryb, jsou parazitické larvy mlžů (Mollusca) neboli glochidie. Maximální intenzita glochidií na rybách bývá spojena s dobou, kdy je pro jejich uvolňování z mlžů optimální teplota a rovněž závisí na průběhu jejich metamorfózy. Dále je na teplotě vody závislá i celková délka parazitické fáze glochidií (Dudgeon & Morton, 1984). Například výskyt druhů glochidií rodu Anodonta byl pozorován na hostitelských rybách od listopadu do května, přičemž jejich prevalence v chladných měsících stoupala až ke 100 %. Na jaře byl pak pozorován rychlý pokles intenzity infekce a v červnu následovalo jejich vymizení (Dartnall & Walkey, 1979; Jansen, 1991; Jokela et al., 1991).

3.2.4 Acanthocephala Sezónní dynamika parazitární infekce způsobené zástupci rodu Acanthocephalus byla studována jak u mezihostitelských korýšů, tak u různých druhů ryb jako definitivních hostitelů (Ohtaka et al., 2002). Nejvyšší prevalence larválních stádií parazita u mezihostitele Asellus hilgendorfii i u

LITERÁRNÍ PŘEHLED definitivních hostitelů, tj. pstruha duhového (Oncorhynchus mykiss), hlavatky obecné (Hucho perryi), korušky východoasijské (Hypomesus nipponensis) a koljušky tříostné (Gasterosteus aculeatus), byla zaznamenána koncem zimy a na jaře. Kromě toho byla častější a těžší infekce spojena s vyšší denzitou mezihostitele na dané lokalitě. U druhu Pomphorhynchus laevis byla zaznamenána nejvyšší prevalence u mezihostitelského blešivce balkánského (Gammarus balcanicus) v pozdním létě a na podzim a následně pak na jaře u definitivního hostitele - střevle potoční (Phoxinus phoxinus) (Dudiňák & Špakulová, 2003).

3.2.5 Digenea Ukázalo se, že teplota vody významnou mírou zasahuje i do životního cyklu a následně i sezónní dynamiky infekce vyvolané motolicemi. Například uvolňování cerkárií z jejich prvního mezihostitele (plž) je úměrné zvyšující se teplotě vody (Chubb, 1979). Optimální teplota pro vývoj cerkárie Posthodiplostomum cuticola v prvním mezihostiteli je 10°C a jelikož vývoj v plži trvá čtyři až osm týdnů, první vlna infekce metacerkárií u druhého mezihostitele (ryby) může být očekávána začátkem léta. V přírodě je navíc intenzita infekce způsobená cerkáriemi závislá na hustotě kaprovitých ryb na dané lokalitě (Vladimirov, 1960). Sezónní dynamika této motolice byla dále studována u tří druhů juvenilních ryb (Ondračková et al., 2004). U plotice obecné (Rutilus rutilus), cejnka malého (Abramis bjoerkna) a perlína zlatobřichého (Scardinius erythrophthalmus) přibližně odpovídala intenzita infekce metacerkárií očekávanému sezónnímu trendu s nejvyššími hodnotami v podzimních měsících.

Sezónní dynamika motolic parazitujících u ryb, jako definitivních hostitelů, byla studována např. v práci Evans (1977). U plotice obecné (Rutilus rutilus) byl sledován sezónní výskyt Sphaerostomum bramae, kdy nejvyšší infekce byla zaznamenána na podzim a v zimě. V tomto období byli nalezeni hlavně juvenilní jedinci. Na jaře byl sledován pokles parazitární infekce spojený s dozráváním těchto parazitů. Kromě měnících se environmentálních podmínek byl ve zmíněné studii diskutován sezónní výskyt i z hlediska potravního chování plotice. Dále byla u plotice obecné sledována sezónní dynamika Sphaerostomum globiporum. Parazitární infekce tohoto druhu byla pozorována od října do začátku srpna. Maximální intenzita infekce byla v tomto období zaznamenána na jaře (Lebedeva, 2006).

3.2.6 Nematoda Mezi obecně nejpočetnější a nejrozsáhlejší skupinu živočichů patří hlístice. Dosud bylo popsáno téměř 20 tisíc druhů parazitujících v obratlovcích, přičemž mnoho dalších parazituje u

LITERÁRNÍ PŘEHLED bezobratlých nebo rostlin či žijí volným způsobem života. Jejich vývojové cykly mohou být přímé nebo zahrnují mezihostitele. Ve vývojovém cyklu hlístic velmi často figurují různá larvální stádia, která složitě migrují tělem hostitele (Volf et al., 2007). Mezi ekonomicky významné zástupce hlístic parazitující u ryb patří například druh Raphidascaris acus. Ryba figuruje ve vývojovém cyklu tohoto druhu hlístice jako mezihostitel. Sezónní dynamika této hlístice byla studována například u mřenky mramorované (Barbatula barbatula) (Koubková et al., 2004). Relativně vysoká prevalence (73-100 %) byla zaznamenaná v průběhu celého roku. Nejvyšší teplota vody byla zaznamenána v srpnu a následující měsíc byla zaznamenána nejvyšší abundance tohoto cizopasníka. Podle autorů je vývojový cyklus R. acus vázaný na teplotu vody a dále je synchronizován (v čase i prostoru) se sezónními cykly nejen mezihostitele i definitivního hostitele, kterým je nejčastěji štika (Esox lucius) nebo pstruh obecný (Salmo trutta). Sezónní změny v infekčních parametrech byly pozorovány i u dalších druhů hlístic. Například u druhu Philometra abdominalis bylo zaznamenáno, že sezónní reprodukční cykly této hlístice korelují s reprodukcí hostitele. Gravidní samice s pohyblivými larvami byly pozorovány pouze na konci května a na začátku června v dobu, kdy byla jejich hostitelem plotice obecná (Rutilus rutilus) v období tření (viz Moravec, 1994).

3.2.7 Cestoda Poslední neméně významnou skupinou parazitů kaprovitých ryb jsou tasemnice. Ryba může v jejich životním cyklu hrát úlohu definitivního hostitele nebo může být mezihostitelem. Příkladem parazita, který využívá rybu jako mezihostitele, může být larvocysta tasemnice Neogryporhynchus cheilancristrotus. Sezonní výskyt tohoto parazita byl studován u cejna sinného (Abramis ballerus). Nejvyšší prevalence byla zaznamenána na jaře a dále pak na přelomu podzimu a zimy (Pietrock & Scholz, 2000). Nižší infekci zaznamenanou v průběhu léta vysvětlují autoři kromě již výše zmíněných faktorů, tj. absencí prvního mezihostitele či vlivy prostředí druhého mezihostitele, případnou změnou v potravním chování ryb.

Ačkoliv byly donedávna v Evropě za ekonomicky nejvýznamnější považovány dospělci druhů tasemnic parazitujících na kaprovi obecném, Cyprinus carpio (např. Khawia sinensis, Bothriocephalus acheilognathi nebo Caryophyllaeus laticeps), v poslední době toto prvenství přebírá nově importovaný druh Atractolytocestus huronensis (např. Molnár et al., 2003; Oros et al., 2004). I přesto, že dosud není známo mnoho informací o sezónním výskytu tohoto druhu, Kappe et al. (2006) zaznamenali nejvyšší početnost této tasemnice na konci léta.

LITERÁRNÍ PŘEHLED

3.3 Vztah fyziologického, imunitního systému ryb a parazitismu v souvislosti s hypotézami aplikovanými v imunoekologii

3.3.1 Základní hypotézy Rozmanitost životních strategií je dána především existencí „trade-off“ (tj. kompromisů) mezi tzv. „life-history“ složkami. Podle této teorie disponuje každý organismus omezenými zdroji dostupné energie, které je v průběhu života nucen rozdělit mezi růst, reprodukci a přežití (Roff, 1992). V posledních letech je za důležitou životní složku považována imunita, a proto se do popředí zájmu imunoekologických studií dostává kompromis mezi reprodukcí a imunitou organismu. V průběhu rozmnožování, kdy je tendencí jedinců investovat do úspěšné reprodukce, mohou parametry imunitní obrany podléhat rychlým, ale dočasným změnám (Sheldon & Verhulst, 1996). Dále mohou být imunitní funkce, a to nejen v tomto období, modulovány hladinou pohlavních hormonů (Hillgarth et al., 1997).

Hypotéza imunokompetenčního handicapu byla formulována před téměř dvaceti lety (Folstad & Karter, 1992) a vychází z původní hypotézy handicapů (Zahavi, 1975; Zahavi, 1977). Je založena na mechanismu fungování imunitního systému ve vztahu k androgenům (zejména testosteronu) a roli těchto hormonů v sexuální selekci. Podle této hypotézy androgeny na jedné straně působí pozitivně na expresi pohlavních znaků a na druhé straně negativně na imunitní funkce. Proto samec, který nese nejrozvinutější sekundární pohlavní znak, má nejvyšší hladinu testosteronu, omezenou výkonnost imunitního systému a následně může být více náchylný vůči infekci. Takový handicap si však mohou dovolit jen ti nejzdatnější samci. Existuje řada studií, které hypotézu imunokompetenčního handicapu podporují, byť některé jen částečně např. (Skarstein & Folstad, 1996; Moller, 1997; Kurtz & Sauer, 1999; Kortet et al., 2003b).

Jelikož steroidní hormony u samců mají zároveň vliv na produkci spermatu, na hypotézu imunokompetenčního handicapu navázala tzv. „sperm protection“ hypotéza. Podle této hypotézy by mohla míra exprese sekundárních pohlavních znaků představovat potenciální investici ve smyslu kompromisu mezi kvalitou spermií a obranyschopností organismu (Kortet et al., 2004).

Hypotéza imunokompetenčního handicapu byla testována v rozsáhlé meta-analýze u ptáků, plazů a savců (Roberts et al., 2004). V této studii byl zpochybňován jak pozitivní vztah testosteronu a sexuální signalizace, tak i jeho negativní efekt na imunitní systém. Roberts et al.

LITERÁRNÍ PŘEHLED

(2004) však uvádí, že efekt testosteronu může být odlišný v rámci druhů obratlovců. Podle studií Braude et al. (1999) a Wedekind & Folstad (1994) může testosteron spíše působit na redistribuci imunitních funkcí nebo přerozdělení zdrojů.

3.3.2 Vztahy mezi reprodukcí, somatickou kondicí, imunokompetencí a parazitismem na principu kompromisu Reprodukce je obecně považována za fyzicky vyčerpávající a velmi stresující období (Bolger & Connolly, 1989). Navíc existuje předpoklad, že vlivem energetických nákladů spojených s reprodukcí může docházet k rychlým a dočasným změnám v imunokompetenci (Hillgarth et al., 1997; Folstad & Skarstein, 1997). Tyto změny mohou být spojeny s energetickým kompromisem mezi imunitou a reprodukcí nebo imunomodulační aktivitou pohlavních hormonů (Hillgarth et al., 1997). Vliv steroidních hormonů na vybrané parametry imunitního systému je podrobně diskutován v kapitole 3.4.

Velikost sleziny, která je považována za parametr vypovídající o aktuálním stavu imunokompetence, byla studována například v souvislosti s reprodukčními investicemi a parazitární infekcí. U samců sivena severního (Salvelinus alpinus) a plotice obecné (Rutilus rutilus) byla v průběhu reprodukce zaznamenána menší slezina, navíc byli jedinci v tomto období více parazitováni (Skarstein et al., 2001; Kortet et al., 2003a). Dále byl vztah mezi velikostí sleziny a parazitismem testován například v interspecifické studii kaprovitých ryb (Šimková et al., 2008). Ve zmíněné práci byl u samic zaznamenán pozitivní vztah mezi velikostí sleziny a abundancí ektoparazitů, který lze vysvětlit jako reakci tohoto imunitního orgánu na vyšší parazitární infekci a naopak negativní vztah může být výsledkem vyšších investic do imunity a následné rezistence vůči parazitismu (Skarstein et al., 2001).

Ottová et al. (2005) zaznamenali u cejna velkého (Abramis brama) v průběhu reprodukce negativní vztah mezi velikostí sleziny a kondicí. Tímto výsledkem byla podpořena existence potenciálního kompromisu („trade-off“) mezi investicemi do somatické kondice na úkor imunitních funkcí. Kondice ryb byla dále studována ve vztahu k intenzitě sexuální ornamentace, která může být považována za nepřímou míru intenzity do reprodukce. Slabý vztah mezi intenzitou exprese třecích vyrážek a kondičním faktorem u plotice obecné (Rutilus rutilus) zaznamenal Wedekind (1992). Taskinen & Kortet (2002) však tento vztah u stejného druhu nepotvrdili. Rovněž Ottová et al. (2005) nezaznamenali vztah mezi intenzitou exprese třecích vyrážek a kondice u cejna velkého (Abramis brama).

LITERÁRNÍ PŘEHLED

Úloha sexuální ornamentace v průběhu reprodukce byla studována v souvislosti s parazitární infekcí a imunokompetencí. U plotice obecné (Rutilus rutilus) byl zaznamenán pozitivní vztah mezi intenzitou exprese třecích vyrážek a parazitární infekcí způsobenou Myxobolus mülleri (Myxozoa). Podobné závěry přinesly studie u sivena severního (Salvelinus alpinus) a tří různých populací plotice obecné (Rutilus rutilus) (Skarstein & Folstad, 1996; Taskinen & Kortet, 2002). Uvedené studie částečně podporují hypotézu imunokompetenčního handicapu a rovněž Hamilton-Zuk hypotézu, podle níž může sexuální ornamentace signalizovat rezistenci vůči parazitární infekci (Hamilton & Zuk, 1982). U sivena severního (Salvelinus alpinus) byl navíc zaznamenán negativní vztah mezi sexuální ornamentací a imunitní odpovědí, měřenou pomocí počtu lymfocytů, kdežto u plotice obecné (Rutilus rutilus) korelace mezi ornamentací, imunitní odpovědí či kondicí zaznamenána nebyla. Dále byl v průběhu tření plotice obecné (Rutilus rutilus) zaznamenán pozitivní vztah mezi ornamentací, koncentrací androgenů a zvýšeným rizikem papilomatózy (Kortet et al., 2003b). Existují však studie, které vztah mezi sexuální ornamentací a parazitismem nepotvrdily (např. Ottová et al., 2005). Dále byla v průběhu reprodukce ryb studována intenzita infekce vyvolaná parazity rodu Gyrodactylus u plotice obecné (Rutilus rutilus) (Koskivaara et al., 1991a). Rovněž v této studii byla zaznamenaná nejvyšší abundance a prevalence parazitární infekce v období tření, což může signalizovat, že jedinci investují v tomto období přednostně do reprodukčních funkcí a jejich imunitní funkce mohou být oslabeny.

LITERÁRNÍ PŘEHLED

3.4 Fyziologie a imunita ve vztahu k ploidii a hladině hormonů

Kromě toho, že se hostitel nachází v prostředí měnících se abiotických a biotických podmínek (viz kapitoly 3.1 a 3.2), uvnitř jeho organismu existuje velmi složitý systém regulačních mechanismů. Do těchto mechanismů jsou zapojeny jak složky fyziologického, tak imunitního systému a jejich kooperací je zabezpečována rovnováha celého organismu. Pochopení rozmanitých vztahů, jako výsledku působení regulačních mechanismů, je mnohdy velmi komplikované. V následující kapitole budou analyzovány především vybrané vztahy mezi fyziologickým a imunitním systémem s ohledem na ploidii ryb (viz Obr. 2 „A“) a vliv hladiny hormonů (viz Obr. 2 „B“).

Obr. 2: Schematické znázornění vzájemných vztahů mezi imunitním a fyziologickým systémem potenciálního hostitele s ohledem na ploidní stav jedince a vliv hladiny hormonů

LITERÁRNÍ PŘEHLED

3.4.1 Reprodukce ryb

3.4.1.1 Úloha hormonů v reprodukčním procesu V reprodukčním procesu (ať už přirozeném nebo uměle řízeném), hrají hormony ryb stejně jako hormony vyšších obratlovců zcela zásadní úlohu. Gametogeneze a finální zrání gamet je regulováno kaskádou hormonů podél reprodukční osy „mozek-hypofýza-gonády“. Sekrece hypofyzárních gonadotropinů, folikulo-stimulačního (FSH) a luteinizačního (LH) hormonu, podléhá hormonálnímu řízení hypothalamu, ze kterého je vylučován gonadotropin-releasing hormon (GnRH) (Peter & Yu, 1997). Hladina GnRH je závislá na externích podnětech (např. teplota, proudění, vnitrodruhové interakce) a následně přímo působí na hladinu FSH i LH. Sekrece gonadotropinů je rovněž kontrolována řadou vnitřních i vnějších faktorů. U některých druhů ryb je inhibiční funkce přisuzována především dopaminu (Chang & Jobin, 1994). Kromě toho se však na regulaci gonadotropinů podílejí faktory související například s nutričním stavem organismu, stresem či podmínkami vnějšího prostředí.

Testikulární spermatogeneze a rovněž spermiace je u ryb stimulována hladinou LH i FSH. Tyto gonadotropiny stimulují sekreci steroidních hormonů a také dalších růstových faktorů, které následně působí na cílovou tkáň – především na gonády (viz Obr. 3). Za hlavní androgen je u kostnatých ryb považován 11-ketotestosteron. Jeho hladina je u samců několikanásobně vyšší než u samic a účastní se sexuálního chování, spermatogeneze či exprese sekundárních pohlavních znaků (viz Borg, 1994).

U samic je hlavní úloha při vitelogenezi přisuzována především FSH (u ryb se synchronním ovariálním vývojem), případně FSH v kombinaci s LH (u ryb s asynchronním ovariálním vývojem), které působí na produkci 17-beta estradiolu (E2). Kromě gonadotropinů a 17-beta estradiolu, hrají při vitelogenezi důležitou roli i další hormony (např. testosteron) a parakrinní faktory, které se mohou podílet na absorpci vitelogeninu rostoucími folikuly (Mylonas et al., 2010) (viz Obr. 3).

LITERÁRNÍ PŘEHLED

Obr. 3: Zjednodušené schematické znázornění reprodukční osy ryb - modifikováno podle studie Mylonas et al. (2010)

3.4.1.2 Techniky umělé reprodukce Hormonální stimulace Převážná většina akvakulturních ryb se rozmnožuje sezónně. Umělé rozmnožování je v akvakultuře využíváno především k dosažení pohlavní zralosti a k výtěru v očekávanou dobu roku synchronně u všech generačních ryb, které jsou k dispozici. Účelem je především zvýšení produktivity u některých druhů ryb. Navíc existují i další výhody spojené s řízenou reprodukcí, např. využití technik genetické selekce nebo hybridizace (viz níže). Zvýšení úspěšnosti tření lze v umělých podmínkách dosáhnout manipulací s environmentálními podmínkami jako je teplota vody, fotoperioda, salinita, objem či hloubka nádrže apod. (Zohar et al., 1989; Munro et al., 1990; Yaron, 1995). U některých druhů ryb je však možno dosáhnout reprodukce v umělých chovech pouze s využitím hormonů. Navíc ryby držené v umělých podmínkách mohou vykazovat nějaký typ reprodukční dysfunkce. Tyto dysfunkce mohou být výsledkem různých faktorů či jejich vzájemných kombinací (stresem indukovaným v zajetí, odlišnost od přirozených

LITERÁRNÍ PŘEHLED podmínek apod.). U samic se může jednat například o poruchy dozrávání oocytů a následné selhání ovulace a tření (např. Peter et al., 1993). U samců může docházet k produkci malého objemu mlíčí či produkce mlíčí nízké kvality (Billard, 1986).

V současnosti je k indukci tření využívána celá řada hormonů reprodukční osy „mozek-hypofýza- gonády“. Jedná se například o metody umělé reprodukce využívající gonadotropiny (GtH), syntetické gonadotropiny „releasing hormons“ (GnRH) nebo jejich agonisty (GnRHa), lidského choriového gonadotropinu (hCG), extraktů hypofýzy apod. (viz Zohar & Mylonas, 2001). Nejpoužívanějším způsobem hormonálně indukovaného výtěru u ryb jsou hypofyzární injekce. Hlavní výhodou je, že univerzálním donorem gonadotropinů (tzn. kapří hypofýzy) je naše nejběžněji chovaná ryba kapr obecný (Cyprinus carpio). Kapří hypofýzu lze použít k indukci ovulace či spermiace u řady druhů ryb (Kouřil et al., 1999).

Je prokázáno, že hormony, které jsou při rozmnožování uvolňovány („releasing hormons“, gonadotropiny, steroidní hormony a příslušné růstové faktory), mohou kromě jejich primárního účinku na reprodukci ovlivňovat i další životně důležité funkce. Tyto hormony se mohou účastnit například při rozvoji či modulaci imunitního systému a jejich výsledný účinek na imunitu může být pozitivního či negativního charakteru (Tanriverdi et al., 2003).

Chromozomové manipulace u ryb V podmínkách moderní akvakultury našly své místo i další techniky, jako například mezidruhová hybridizace či genomové manipulace, které jsou využívány především za účelem zvyšování kvality ekonomicky zajímavých ryb. Mezi chromozomové manipulace u ryb je řazena: a) indukce polyploidie; b) indukce gynogeneze; c) indukce androgeneze; d) zvrat pohlaví v kombinaci s předchozími postupy (Thorgaard, 1983; Ihssen et al., 1990; Benfey, 1999; Gomelsky, 2003). Tyto techniky byly v posledních desetiletích různou měrou zavedeny do komerčních chovů ryb.

Polyploidie, tj. zmnožení celých chromozómových sad v somatických buňkách jedince nad jejich běžnou diploidní úroveň, může vzniknout přirozeně nebo je výsledkem umělé reprodukce a manipulace s gametami. U vývojově starších ryb je spontánní polyploidie široce rozšířeným fenoménem, což není tak běžné u zástupců vyšších kostnatých ryb (Leggatt & Iwama, 2003). U některých druhů komerčně chovaných ryb je umělá indukce polyploidie v současné době více či méně rutinně zavedenou technikou (např. Donaldson, 1996; Gomelsky, 2003). Obecně je indukována za účelem zvýšení produktivity a dosažení sterility.

LITERÁRNÍ PŘEHLED

Nejčastěji se v podmínkách rybích akvakultur, pomocí fyzikálních nebo chemických zásahů do vývoje zygoty, cíleně indukují triploidní formy. Triploidní jedinci jsou geneticky sterilní a mají buď částečně, nebo kompletně redukované gonády. Umělá indukce triploidizace se provádí supresí sekundárního pólového tělíska krátce po oplození, přičemž vznikají tři sady chromozomů. K zadržení sekundárního pólového tělíska se využívá několik technik (např. tepelný šok, chladový šok, hydrostatický tlakový šok a působení chemických látek). V současnosti je nejčastěji využívanou technikou tepelný šok. Jedinci s odlišnou ploidii jsou pro své specifické vlastnosti předmětem výzkumu různých srovnávacích studií (např. Svobodová et al., 2001; Ballarin et al., 2004; Budino et al., 2006; Maxime, 2008).

Další technikou chromozomální manipulace využívanou v umělém chovu je tzv. indukce gynogeneze (např. Yamamoto, 1999). Takto vzniklá samičí populace vykazuje řadu specifických rysů (např. růst, přežití či morfologie), které ji dělají velmi atraktivní pro základní výzkum. Při gynogenezi se samčí pohlavní buňky přímo nepodílí na tvorbě nového genomu. Spermie slouží jako stimulační prostředek pro dokončení meiózy a začátku rýhování, nikoliv jako donor genetické informace (Thorgaard, 1983). Zdvojením genomu supresí sekundárního pólového tělíska dochází k tzv. meiotické gynogenezi. Pokud dojde k zamezení prvního mitotického dělení, jedná se o tzv. mitotickou gynogenezi. Principem umělé indukce gynogeneze je složitá technika, při níž je genom spermie inaktivován (pomocí rentgenového záření, gama záření nebo nejčastěji UV záření) takovým způsobem, aby nedošlo k potlačení schopnosti spermie proniknout do jikry a aktivovat ji (Nagy et al., 1978; Pipota & Linhart, 1986; Sumantadinata et al., 1990).

LITERÁRNÍ PŘEHLED

3.4.2 Imunitní systém ryb: vliv ploidie a hladiny hormonů Vliv ploidie Obecně triploidní jedinci disponují většími somatickými buňkami a ani buňky imunitního systému nejsou výjimkou. Tyto rozdíly ve velikostech buněčných komponent u jedinců odlišné ploidie by potenciálně mohly hrát důležitou úlohu i v jejich funkčnosti a tudíž odlišné rezistenci vůči infekci. Některé dosavadní práce poukazují na to, že rezistence vůči infekci může být u triploidních jedinců snížena (např. Dunham, 2004; Ozerov et al., 2010), avšak existuje také řada prací, které tuto myšlenku nepodporují (např. Yamamoto & Iida, 1995b).

Vliv hladiny hormonů Nenahraditelnou roli v těle mnohobuněčného organismu hrají hormony. Konkrétně hladina steroidních hormonů ovlivňuje u ryb kromě reprodukce další životně důležité funkce jako je například růst, metabolismus, osmoregulace a v neposlední řadě pak zasahuje i do funkce imunitního systému (viz např. Cuesta et al., 2007). Mezi komponentami imunitního a reprodukčního systému existuje významná spojitost. Například celá řada složek imunitního systému (lysozym, komplementový systém apod.) disponuje svými receptory i v tkáni gonád (viz Milla et al., 2011). V souvislosti s imunitními funkcemi kostnatých ryb byla především studována úloha androgenů a estrogenů, z nichž nejvíce pozornosti bylo věnováno 11-ketotestosteronu. Nyní je známo, že existuje i celá řada dalších molekul, které interferují s účinkem steroidních hormonů, např. endokrinní disruptory (viz např. Milla et al., 2011). Vliv steroidních hormonů v kontextu s hypotézami aplikovanými v imunoekologii byl také diskutován v kapitole 3.3.

3.4.2.1 Velikost sleziny

Vliv ploidie Předpokládá se, že celková velikost jednotlivých orgánů se mezi diploidními a triploidními jedinci neliší (Benfey, 1999). U triploidních jedinců je totiž celková velikost orgánů kompenzována, tj. jejich orgány jsou sice tvořeny většími buňkami, avšak jejich počet je redukován (Benfey, 1999). Určité rozdíly ve struktuře sleziny byly zaznamenány mezi některými diploidními a triploidními jedinci pstruha duhového (Oncorhynchus mykiss) (Okada, 1985). Velikost sleziny a další důležité indexy (např. gonado-somatický a hepato-somatický) byly studovány u lína obecného (Tinca tinca) (Buchtová et al., 2003). Při porovnání velikosti sleziny mezi indukovanými triploidními a amfimiktickýmí diploidními samicemi ve věkové kategorii L3+, byla u indukovaných triploidních samic zaznamenána signifikantně větší slezina. Ve stejné

LITERÁRNÍ PŘEHLED věkové kategorii byl zaznamenán podobný trend i u samců, avšak nebyl statisticky významný. Výsledky týkající se velikosti sleziny v souvislosti s ploidií ryb nebyly v této studii jednoznačně interpretovány. Je však známo, že váha vnitřních orgánů může být ovlivněna různými vnějšími i vnitřními faktory (věk, pohlaví, teplota vody, spektrum potravy, obsah kyslíku atd.) (Kouřil et al., 1978). Studie, které by se zaměřily na funkční rozdíly sleziny, jakožto důležitého imunitního orgánu u jedinců odlišné ploidie, zatím chybí.

Vliv hladiny hormonů V souvislosti s testováním hypotézy imunokompetenčního handicapu byl studován především vliv testosteronu či 11-ketotestosteronu na velikost sleziny, např. u lína obecného (Tinca tinca) (Vainikka et al., 2005), sivena severního (Salvelinus alpinus) (Skarstein et al., 2001), plotice obecné (Rutilus rutilus) (Kortet et al., 2003a) nebo koljušky tříostné (Gasterosteus aculeatus) (Kurtz et al., 2007). U všech druhů ryb byl zaznamenán pokles velikosti tohoto imunitního orgánu, což bylo přisuzováno většinou imunosupresivnímu efektu testosteronu. U lína obecného (Tinca tinca) byl tento negativní vztah, spíše než s hladinou steroidních hormonů, spojován s energetickým kompromisem (Vainikka et al., 2005). Navíc podle studie Lynshiang & Gupta (2000) může být testosteron příčinou zvýšení metabolismu, což může vést k poklesu hmotnosti těla nebo hmotnosti jednotlivých orgánů.

3.4.2.2 Počet leukocytů, lymfocytů a leukokrit

Vliv ploidie Na rozdíl od velikosti sleziny jsou hodnoty bílého krevního obrazu u ryb odlišné ploidie poměrně často studovaným imunitním parametrem (např. Ohtsu, 1993; Svobodová et al., 1998; Burrows et al., 2001; Svobodová et al., 2001). U lína obecného (Tinca tinca) byl zaznamenán snížený počet leukocytů u indukovaných triploidních jedinců (Svobodová et al., 1998). Podle autorů této studie to může naznačovat, že triploidní jedinci disponují nižší úrovní nespecifické imunity. V další studii (Svobodová et al., 2001) byli srovnáváni jedinci lína obecného stejného původu ve dvou po sobě následujících letech (L3 a L4). Mezi tříletými diploidy a triploidy nebyly shledány rozdíly ani v celkovém, ani v diferenciálním počtu leukocytů. Stejný trend byl zaznamenán u čtyřletých jedinců lína obecného u celkového počtu leukocytů a u většiny parametrů diferenciálního počtu leukocytů. Zaznamenán byl však statisticky vyšší počet neutrofilních metamyelocytů u diploidů a dále vyšší celkový počet granulocytů u triploidů. Rozdíly mezi tří- a

LITERÁRNÍ PŘEHLED

čtyřletou populací mohly být dány zhoršenou kondicí ryb, způsobenou předchozím stresem nebo lokální bakteriální infekcí kůže.

Vliv hladiny hormonů Ačkoliv je úloha androgenů či estrogenů v hematologické homeostázi považována některými autory spíše za kontroverzní, existují studie, které funkci steroidních hormonů jednoznačně interpretují. Například bylo zjištěno, že zvýšená hladina 17-beta estradiolu může indukovat proliferaci lymfocytů (Suzuki et al., 1996; Thilagam et al., 2009). Další studie však vedly k opačným závěrům (Suzuki et al., 1997; Hou et al., 1999; Cuesta et al., 2007). Mnohem více konzistentní jsou studie týkající se úlohy androgenů, které mají na proliferaci lymfocytů prokazatelně negativní efekt (Slater & Schreck, 1997; Saha et al., 2004; Ros et al., 2006). Podle Milla et al. (2011) je úloha steroidních hormonů a udržování imunitní homeostáze zcela zásadní především v období reprodukce.

3.4.2.3 Oxidativní vzplanutí a počet fagocytů

Vliv ploidie Oxidativní vzplanutí u ryb s odlišnou ploidií bylo studováno u kambaly velké (Psetta maxima) (Budino et al., 2006). Fagocytární aktivita vztažená na jednotlivé fagocyty byla u triploidů vyšší než u diploidních jedinců, což bylo vysvětleno tím, že fagocyty triploidů disponují větší povrchovou membránou a rovněž objemem, čímž je zvýšena jejich schopnost pohlcovat cizorodé částice. Pokud však byla fagocytární aktivita přepočítána na mikrolitr krve, statisticky významný rozdíl mezi diploidy a triploidy již zaznamenán nebyl. Podobné výsledky, týkající se fagocytární aktivity neutrofilů, byly zaznamenány i u pstruha duhového (Oncorhynchus mykiss) (Yamamoto & Iida, 1995a), tj. statisticky nevýznamné rozdíly ve fagocytární aktivitě mezi diploidy a triploidy. Tyto výsledky naznačují, že výsledná nespecifická imunita je u triploidních jedinců stejná jako u diploidních, a tudíž se ploidní stav nemusí nutně projevovat mírou náchylnosti k infekci.

Vliv hladiny hormonů Vliv steroidních hormonů na složky bílého krevního obrazu (viz výše) se může rovněž projevit i na aktivitě fagocytů. Zaznamenán byl negativní efekt estrogenů (Hou & Han, 2001; Yamaguchi et al., 2001) i androgenů (Yamaguchi et al., 2001; Watanuki et al., 2002) na fagocytární funkci ryb. Zdá se, že vliv těchto hormonů může být druhově specifický. Například u japonského druhu

LITERÁRNÍ PŘEHLED mořského okouna (Lateolabrax japonicus) došlo vlivem estradiolu ke zvýšení aktivity oxidativního vzplanutí (Thilagam et al., 2009). V souvislosti s hypotézou imunokompetenčního handicapu byl dále studován imunosupresivní efekt 11-tetotestosteronu u koljušky tříostné (Gasterosteus aculeatus) (Kurtz et al., 2007). V této studii byl potvrzen negativní vliv 11- ketotestosteronu na oxidativní vzplanutí a další složky nespecifické imunity.

3.4.2.4 Koncentrace protilátek

Vliv ploidie Recentně byla publikována studie zabývající se protilátkami u triploidních a diploidních ryb na úrovni genové exprese (Ching et al., 2010). Exprese genů pro IgM, MHC-II a β-actin u triploidních lososů (Oncorhynchus tshawytscha), v porovnání s diploidními jedinci, byla redukována. Autoři studie vysvětlují tento výsledek vyšší senzitivitou triploidních ryb na stres, která by teoreticky mohla negativně ovlivnit transkripci těchto genů. Studie, které by porovnávaly celkovou koncentraci protilátek u ryb s odlišnou ploidií však zcela chybí.

Vliv hladiny hormonů Podrobněji byl testován vliv steroidních hormonů na koncentraci protilátek, což bylo často studováno společně s proliferací lymfocytů (viz výše). Hladina 17-beta estradiolu tedy kromě indukce proliferace lymfocytů vedla ke zvýšené produkci IgM protilátek (Suzuki et al., 1996; Thilagam et al., 2009) nebo byl zaznamenán opačný efekt (Suzuki et al., 1997; Hou et al., 1999; Cuesta et al., 2007). Produkce IgM protilátek může být také negativně ovlivněna androgeny (Hou et al., 1999; Saha et al., 2004; Suzuki et al., 1997).

3.4.2.5 Aktivita komplementu

Vliv ploidie Yamamoto & Iida (1995a) a Budino et al. (2006) srovnávali aktivitu komplementu mezi diploidními a triploidními jedinci. Statisticky významné rozdíly ani v jedné studii nebyly prokázány. Hypotéza, že funkčnost imunitního systému triploidních ryb může být slabší než u ryb diploidních, byla podpořena v práci Langston et al. (2001) u lososa obecného (Atlantic salmon). Diploidním a triploidním jedincům byl intraperitoneálně injikován lipopolysacharidový roztok a následně byla testována aktivita alternativní cesty aktivace komplementu. U triploidních jedinců bylo zapotřebí delšího času potřebného k obnovení funkce komplementu,

LITERÁRNÍ PŘEHLED což naznačuje, že triploidní jedinci mohou být v porovnání s diploidními v určité nevýhodě z hlediska obrany proti bakteriální infekci.

Vliv hladiny hormonů Potenciálně významnou roli v aktivitě komplementu může hrát i hladina steroidních hormonů. Například u platýze bradavičnatého (Patichthys flesus) bylo zjištěno, že hladina estrogenů byla příčinou snížené exprese genů spojených s imunitní obranou. Další publikace se rovněž přiklánějí k názoru, že estrogeny mohou být důležitými modifikátory aktivity komplementového systému a obecně pak může být tvorba jednotlivých složek komplementu ryb (například C1 a C3) steroidními hormony potlačena (Cuesta et al., 2007; Moens et al., 2006; Williams et al., 2007).

3.4.2.6 Koncentrace lysozymu

Lysozym je považován za důležitou komponentu přirozené imunity ryb a indikátor rezistence. Jedná se o mukolytický enzym leukocytárního původu, který byl u ryb identifikován ve slizu, lymfatických tkáních, plazmě a dalších tělních tekutinách či tkáních. Bylo zjištěno, že aktivita lysozymu je závislá na pohlaví, stáří nebo velikosti jedinců, dále na sezóně, teplotě vody, pH, toxinech, infekci, případně dalších faktorech (stres apod.) (Saurabh & Sahoo, 2008).

Vliv ploidie Doposud byla koncentrace lysozymu studována pouze u diploidních a triploidních jedinců kambaly velké (Psetta maxima). Rozdíly v koncentraci lysozymu, analyzovaného v séru ryb odlišné ploidie, nebyly u tohoto druhy zaznamenány (Budino et al., 2006). Výsledky této studie naznačují, že se aktivita humorální složky, na rozdíl od buněčné složky přirozené imunity ryb, mezi jedinci odlišné ploidity zřejmě neliší.

Vliv hladiny hormonů Rovněž doposud neexistují studie popisující přímo vliv steroidních hormonů na koncentraci lysozymu. Specifická a nespecifická imunitní odpověď byla studována u lososa čavyča (Oncorhynchus tshawytscha) v průběhu jeho sladkovodní migrace a reprodukce (Maule et al., 1996). Ačkoliv byl zaznamenán pozitivní vztah mezi hladinou sérového lysozymu a koncentrací steroidních hormonů, ze studie jednoznačně nevyplynulo, zda se jedná o přímý vztah či korelaci s ročním obdobím.

LITERÁRNÍ PŘEHLED

3.4.3 Fyziologický systém ryb: vliv ploidie a hladiny hormonů

V současnosti jsou jedinci odlišné ploidie předmětem srovnávacích fyziologických studií (např. Suzuki et al., 1985; Johnson et al., 1986; Nakamura et al., 1989; Benfey, 1999; Felip et al., 2001; Tiwary et al., 2004). Kromě fyziologických ukazatelů uvedených v této kapitole, jsou v těchto studiích často hodnoceny základní životní složky tj. přežívání, růst či reprodukce jedinců s odlišnou ploidii.

3.4.3.1 Fyziologie ryb: kondice, játra a gonády

Vliv ploidie Původní hypotéza, týkající se vztahu mezi velikostí somatických buněk a celkové velikosti těla triploidních jedinců (Ihssen et al., 1990), byla vyvrácena (Benfey, 1999). Lepší růst triploidních jedinců v porovnání s diploidními je tedy odvozen spíše od jejich sterility než od velikosti jejich somatických buněk. Sterilita může mít tedy pozitivní vliv na růst, přežívání a lepší kvalitu svalové tkáně triploidů. Beaumont et al. (2003) ve své práci uvádí, že diploidní jedinci spotřebují část své energie na vývoj gamet, zatímco triploidní jedinci většinu energie investují do tělesného růstu. Na druhé straně však steroidní hormony u diploidních jedinců mohou svým anabolickým efektem kompenzovat tuto růstovou výhodu triploidů (Benfey, 1999). Příčinou zmíněné sterility triploidních jedinců je narušení párování chromozómů v meióze v důsledku přítomností tří sad homologních chromozómů. Následný vývoj gonád triploidních jedinců i jejich GSI se od diploidů významně liší. Jestliže jsou triploidi schopni produkce gamet, pak jsou jejich pohlavní buňky obvykle aneuploidní (Ihssen et al., 1990). Při makroskopickém hodnocení gonád lína obecného (Tinca tinca) zaznamenal Flajšhans (1997) zpomalený růst gonád u indukovaných triploidních samic a samců, kdežto gonády diploidních jedinců odpovídaly očekávanému stádiu v závislosti na ročním cyklu. Ovaria triploidních samic vykazovala ojedinělý výskyt oocytů v 3. - 4. stádiu a triploidní samci měli buď nezralou testikulární tkáň s buňkami v raném stádiu spermatogeneze, či ostrůvky zárodečné tkáně a spermatid s tukovými depozity kolem pojivové tkáně.

Flajšhans et al. (1993) dále publikovali studii, v níž byla testována užitkovost 4letých triploidních a diploidních jedinců lína obecného (Tinca tinca). Z práce vyplývá, že triploidní samice dosáhly o 13,5% vyšší tělesné hmotnosti než diploidní samice a triploidní samci o 23,7% než diploidní samci. Na druhé straně gonado-somatický index (GSI) triploidních samic dosáhl pouze 25,1% hodnoty GSI diploidních samic a GSI triploidních samců dosáhl 60% hodnot GSI diploidních samců. Hmotnostní indexy u diploidních a triploidních jedinců lína obecného byly dále

LITERÁRNÍ PŘEHLED studovány ve studii Buchtové et al. (2003), která podpořila závěry předchozí studie. U triploidních jedinců byly pak zaznamenány statisticky vyšší hodnoty růstových parametrů (celková délka i celková hmotnost) ve věkové kategorii L3 u obou pohlaví a ve věkové kategorii L3+ u samic. Navíc byly zaznamenány významně větší hmotnosti gonád a GSI u diploidních samic L3, a také GSI u diploidních samic L3+. Tato studie přinesla další důkaz o rychlejším somatickém růstu a vyšší hmotnosti triploidní populace lína ve srovnání s diploidy, a to u obou věkových kategorií. U kapra obecného (Cyprinus carpio) například pozitivní efekt sterility na růst triploidních jedinců zaznamenán nebyl a naopak byl u triploidů zaznamenán pomalejší růst (Cherfas et al., 1994).

Kromě již zmíněných fyziologických ukazatelů byla u ryb s odlišnou ploidií studována velikost jater vyjádřená pomocí hepato-somatického indexu (např. Johnson et al., 1986; Felip et al., 2001; Buchtová et al., 2003). U triploidních jedinců mořčáka evropského (Dicentrarchus labrax) byly zaznamenány významně nižší hodnoty hepato-somatického indexu v porovnání s jedinci diploidními. Nižší hodnoty hepato-somatického indexu, zaznamenané u triploidních jedinců, mohou souviset se sníženou syntézou některých látek např. vitelogeninu v tkáni jater, které se následně účastní vývoje gonád. Opačný efekt ploidie, tj. vyšší hodnoty hepato- somatického indexu u triploidních jedinců lína obecného (Tinca tinca) byl zjištěn ve studii Buchtové et al. (2003). Tento výsledek pravděpodobně souvisí s větším příjmem potravy a somatickým růstem triploidních jedinců.

Vliv hormonů Účinky steroidních hormonů na reprodukční systém ryb jsou tedy všeobecně známé (viz kapitola 3.4.1.1), stejně tak byla v předchozím textu již zmiňovaná jejich potenciální úloha v investicích do dalších životních složek na základě teorie kompromisu v alokaci energie (viz kapitola 3.3). Hladina steroidních hormonů byla studována také v souvislosti s příjmem potravy. Van den Heuvel (2008) ve své práci uvádí, že příjem potravy hraje u ryb důležitou úlohu nejen při ukládání energie do tělesného růstu a vývoje gonád, ale navíc se může odrazit i v produkci steroidních hormonů. Reprodukční cykly samců a samic v souvislosti s hepato-somatickým indexem byly studovány například u tresky obecné (Gadus morhua L.). Vývojová stádia gonád byly spojeny nejen s hladinou pohlavních hormonů, ale i s velikostí jater (Dahle et al., 2003). V průběhu časného vývoje gonád nebyly zaznamenány změny v hepato-somatickém indexu. Pokud byli porovnáváni jedinci s gonádami v terminálním stádiu zrání a následně jedinci po

LITERÁRNÍ PŘEHLED reprodukci, byl zaznamenán pokles hepato-somatického indexu. Redukce velikosti jater v období terminálního zrání gonád byla v této studii vysvětlena tím, že jedinci tresky v průběhu tření nepřijímají potravu, a proto v tomto období využívají energetické rezervy uložené v těle a tímto způsobem pokrývají energetické požadavky spojené se zráním gonád.

3.4.3.2 Počet erytrocytů, hematokrit a koncentrace hemoglobinu

Vliv ploidie Srovnáváním hematologických parametrů u triploidních a diploidních ryb se zabývala celá řada autorů (např. Benfey et al., 1984; Biron & Benfey, 1994; Nakamura et al., 1989; Suzuki et al., 1985; Svobodová et al., 2001). V literatuře jsou dobře popsány rozdíly mezi erytrocyty triploidů a diploidů. Erytrocyty triploidů disponují větším jádrem i celkovým objemem erytrocytů (MCV). Objemové proporce erytrocytů jsou u triploidů kompenzovány jejich počtem tak, že výsledná hematokritová hodnota je u triploidů a diploidů srovnatelná. Větší proporční rozměry erytrocytů triploidních ryb také úzce souvisí s větším obsahem hemoglobinu. Jak se však zdá, tak tento pozitivní vztah neplatí u všech druhů ryb. U pstruha duhového (Oncorhynchus mykiss) byl zaznamenán nižší obsah hemoglobinu u triploidních jedinců v porovnání s jedinci diploidními (Yamamoto & Iida, 1994). Obsah hemoglobinu u diploidních a triploidních jedinců sivena amerického (Salvelinus fontinalis) nebyl prokazatelně odlišný (Stillwell & Benfey, 1996).

Vliv hormonů Zdá se, že steroidní hormony mohou u ryb rovněž zasahovat do procesu erytropoézy. Pottinger & Pickering (1987) zjistili, že počet erytrocytů u samců pstruha obecného (Salmo trutta) pozitivně koreloval se sezónní dynamikou hladiny 11-ketotestosteronu. Stejný vztah byl potvrzen u samců i samic pstruha duhového (Oncorhynchus mykiss) (Van den Heuvel et al., 2008). U jedinců sivena severního (Salvelinus alpinus) byly naopak zaznamenány nejvyšší hodnoty červeného krevního obrazu (tj. hematokritu a koncentrace hemoglobinu) mimo období tření a v průběhu reprodukce byla naopak zaznamenána redukce MCHC (koncentrace hemoglobinu v erytrocytech) (Lecklin & Nikinmaa, 1998). Výsledky týkající se úlohy steroidních hormonů při hematopoéze nejsou zatím jednoznačné a jistě by měl být jejich efekt v budoucnu předmětem dalších studií.

LITERÁRNÍ PŘEHLED

3.4.3.3 Koncentrace glukózy Glukóza neboli krevní cukr hraje nezastupitelnou úlohu v bioenergetice živočichů, ryby nevyjímaje. Při energetickém metabolismu, kde hraje glukóza centrální úlohu, je transformována nejprve do chemické energie (ATP), která může být následně přeměněna na energii mechanickou (Lucas, 1996). Hladina glukózy je společně s hladinou kortizolu u ryb často používána jako jeden z nejběžnějších indikátorů stresu. Obecně jsou hlavní zásobárnou cukrů především játra a svaly, kde je energie uložena v podobě glykogenu (Busacker et al., 1990). Obvykle je hladina glukózy v krvi ryb nízká (Busacker et al., 1990). Při stresu jsou však energetické rezervy ze svalů a jater využívány a následně může být v krvi pozorován nárůst koncentrace glukózy (Hoar et al., 1992). Hladina glukózy může být ovlivněna celou řadou faktorů jako je například nutriční stav, roční období, stupeň vývoje jedince nebo může být druhově specifická apod. (viz Martínez-Porchas et al., 2009).

Vliv ploidie Původně se předpokládalo, že triploidní jedinci budou díky svým charakteristickým vlastnostem (velikost a počet somatických buněk, vývoj gonád apod.) na akutní stres reagovat odlišným způsobem. U lososovitých ryb bylo zjištěno, že rozsah a průběh fyziologické odezvy na akutní stres v důsledku držení v zajetí je u diploidů a triploidů podobný (Biron & Benfey, 1994; Benfey & Biron, 2000). Podle studie Benfey (1999) a Benfey & Biron (2000) se však triploidní jedinci hůře vyrovnávají s chronickým stresem (např. kvalita vody v nádržích), zejména pak pokud je stres dlouhodobý nebo opakovaný.

Vliv hormonů Hoar et al. (1992) uvádí, že hladina glukózy je u ryb závislá na sezónních vlivech spojených především s reprodukcí. Stresové stavy, kam reprodukce a s ní spojený vliv hormonů rozhodně patří, jsou spojeny s glykogenolýzou neboli rozpadem glykogenu v játrech a mobilizací glukózy. Navíc je hladina takto uvolněné glukózy při akutním stresu často spojena s hladinou kortizolu (Vijayan et al., 1997; Rotllant & Tort, 1997).

Ve studii Kubokawa et al. (1999) byla v průběhu tření zaznamenána pozitivní korelace mezi hladinou glukózy a testosteronu u samic i samců tilápie mosambické (Oreochromis mossambicus). U samic však zvyšující se hladina glukózy nebyla spojena s hladinou kortizolu. Podle autorů studie je odlišná odpověď na navozený stres v průběhu tření u samců a samic

LITERÁRNÍ PŘEHLED dána především různou hladinou testosteronu. U samců je v průběhu tření navozena tzv. akutní reakce, kdežto u samic ve stejném období již probíhá tzv. pozdní reakce na stres.

MATERIÁL A METODIKA

4 MATERIÁL A METODIKA

4.1 Studované modely, lokality výzkumu

4.1.1 Jelec tloušť

Jelec tloušť (Leuciscus cephalus) (Obr. 4 a) může být pro vědeckou činnost perspektivním modelovým objektem (Jurajda & Gelnar, 1998). Jedná se o dostupný a všeobecně rozšířený druh kaprovité ryby v tekoucích vodách, který je často velmi silně infikován a navíc disponuje širokým druhovým spektrem cizopasníků. Na vybrané lokalitě v katastrálním území Brno- Modřice (soutok Svitavy a Svratky) probíhal v souvislosti s řešením diplomové práce odlov jedinců jelce tlouště (Obr. 5 a 6). V roce 2004 byly provedeny čtyři sezónní odlovy (duben, červen, srpen, listopad), při nichž bylo celkem získáno 83 jedinců jelce tlouště (samci i samice). K odlovu na této lokalitě byl použit bateriový agregát typu LENA a podběrák. Data získaná z odlovů byla podkladem pro analýzu a zpracování studie č. 1. V roce 2005 byly realizovány tři odlovy spojené se studiem reprodukčních investic jelce tlouště (začátek května, konec května, konec června). Pro tuto studii bylo odloveno 90 samců a výstupem analýz ze získaných dat je studie č. 2.

4.1.2 Kapr obecný

Kapr obecný (Cyprinus carpio) (Obr. 4 b), zástupce čeledi kaprovité, patří na našem území k jednomu z hospodářsky nejdůležitějších druhů ryb. Je charakteristický značnou přizpůsobivostí k různým typům biotopů, proto se s ním můžeme setkat ve všech typech stojatých vod, včetně velkých údolních nádrží a ve větších tocích mimopstruhového charakteru. Jedná se o teplomilnou všežravou rybu zdržující se především u dna (Baruš et al., 1995). Z hlediska parazitární infekce je kapr rovněž parazitován širokým druhovým spektrem cizopasníků, pro které je charakteristická silná intenzita infekce. Z těchto důvodů může být považován za vhodný model pro imunoekologické studie. V průběhu let 2007 – 2008 bylo provedeno celkem 5 odlovů (červen, srpen, listopad, únor a duben). Odlovy probíhaly v pokusném hospodářství VÚRH JU ve Vodňanech (Obr. 7 a 8), kde byly ryby získány standardním výlovem s použitím podběráků. Každému jedinci byl implantován čip pro pozdější determinaci. Výstupem analýz získaných dat byla studie č. 3.

MATERIÁL A METODIKA

4.1.3 Lín obecný

Lín obecný (Tinca tinca) (Obr. 4 c) je kaprovitá ryba charakteristická vysokou odolností vůči nedostatku kyslíku, kyselosti a vysoké teplotě vody. Preferuje lokality s bahnitým nebo jílovitým dnem, zarostlé rákosem a dalšími vodními rostlinami (Baruš et al., 1995). Pro jeho reprodukční a další charakteristické vlastnosti je tento sladkovodní druh doporučovaný jako modelový organismus vhodný ke genetickým manipulacím (viz Flajšhans et al., 1995). Cizopasníci lína obecného jsou v porovnání s ostatními zástupci kaprovitých ryb charakterističtí především nízkou intenzitou a prevalencí parazitární infekce (Svobodová & Kolářová, 2004). V průběhu vybraného týdne v červnu 2008 bylo v genetickém rybářském centru VÚRH JU ve Vodňanech odloveno celkem 93 jedinců (Obr. 9 a 10). Do studie byli zahrnuti amfimiktičtí diploidní jedinci (22 samic, 16 samců), indukovaní triploidní jedinci (15 samic, 20 samců) a 20 meiotických gynogenetických samic. Přibližně polovina odlovených jedinců byla bezprostředně po odlovu analyzována. Druhá polovina ryb byla analyzována až po dvoudenní stimulaci ovulace a spermiace. Triploidie byla uměle navozena teplotním šokem (0-2°C, 35 minut) 2-5 minut po aktivaci gamet podle metodiky Flajšhanse (1993). Diploidní gynogenetická populace vznikla meiotickou gynogenezí podle metodiky Linharta et al. (1995). K inaktivaci genomu spermie bylo použito gama záření izotopu kobaltu (Co60). K zadržení sekundárního pólového tělíska oocytu při meióze II. byl použit chladový šok (0-2°C). K umělému výtěru byla použita metodika podle Rodiny et al. (2004) a Linharta et al. (2006). Jedinci byli chováni při teplotě 20-23°C. U samců byla jednorázově intramuskulárně aplikována injekce kapří hypofýzy (1.0 mg/kg) a u samic GnRH analog (D-Ala6, GnRH ProNHEt, Kobarelin, 5 μg/ kg). Výstupem analýz získaných dat byla studie č. 4.

MATERIÁL A METODIKA

a) Jelec tloušť (Leuciscus cephalus)

b) Kapr obecný (Cyprinus carpio)

c) Lín obecný (Tinca tinca)

Obr. 4: Studované druhy ryb (Holčík & Mihálik, 1971)

MATERIÁL A METODIKA

Obr. 5: Mapa studované lokality: Brno-Modřice, Soutok Svitavy a Svratky- studie č. 1 a 2

Obr. 6: Foto studované lokality: Brno-Modřice, Soutok Svitavy a Svratky- studie č. 1 a 2

MATERIÁL A METODIKA

Obr. 7: Mapa studované lokality: Vodňany, pokusné hospodářství VÚRH – studie č. 3

Obr. 8: Foto studované lokality: Vodňany, pokusné hospodářství VÚRH – studie č. 3

MATERIÁL A METODIKA

Obr. 9: Mapa studované lokality: VÚRH Vodňany, genetické rybářské centrum – studie č. 4

Obr. 10: Foto studované lokality: VÚRH Vodňany, genetické rybářské centrum – studie č. 4

MATERIÁL A METODIKA

4.2 Fixace a determinace parazitologického materiálu

Ryby byly vyšetřeny na přítomnost mnohobuněčních cizopasníků podle standardní metodiky Ergens & Lom (1970). Podle příslušnosti parazitů k jednotlivým taxonomickým skupinám byly k fixaci použity různé metodiky. Monogenea byla fixována tlakem s použití směsí glycerinu a amoniumpikrátu (GAP) v poměru 1:1 (Malmberg, 1970); Digenea, Cestoda, Acanthocephala, Mollusca, Hirudinea a Crustacea v 4% formaldehydu (Ergens & Lom, 1970) a Nematoda směsí glycerínu a 70% ethanolu (Moravec, 1994). Pro další determinaci byli navíc zástupci ze skupiny Digenea a Cestoda barveny železitým acetokarmínem (IAC), odvodněni vzestupnou alkoholovou řadou a nakonec montováni do kanadského balzámu (Ergens & Lom, 1970). Nematoda byla před determinací projasňována v roztoku glycerinu a vody a následně byla vložena do glycerinové želatiny (Moravec, 1994).

Determinace cizopasníků byla provedena pomocí světelného mikroskopu OLYMPUS BX 50 s fázovým kontrastem a DIC (diferenciační interferenční kontrast podle Nomarského) a pomocí počítačové analýzy obrazu Micro Image 4.0 for Windows. K determinaci mnohobuněčných cizopasníků byly použity dostupné monografie a klíče (Ergens & Lom, 1970; Kabata, 1979; Gussev, 1985; Khotenovsky, 1985; Scholz, 1989; Moravec, 1994; Niewiadomska, 2003). Pro popis parazitární infekce byly použity klasické epidemiologické parametry (tj. prevalence, intenzita infekce a abundance) definované v práci Bush et al. (1997).

4.3 Zpracování výsledků

Statistická data byla zpracována a vyhodnocena pomocí programu Statistica verze 7.1, 8.0 a 9.0. Konečná úprava grafů byla provedena v programu Adobe Illustrator CS3 13.0.1. K vytvoření schémat a obrázků použitých v této dizertační práci byl použit program Microsoft Office PowerPoint 2003. Fotografie byly zhotoveny fotoaparátem Olympus C-3030 ZOOM a upraveny v programu Adobe Photoshop 8.0.

MATERIÁL A METODIKA

4.4 Metodika analýzy hematologických parametrů

Vybrané hematologické parametry pro studii č. 3 nebo 4., tj. počet erytrocytů, leukocytů, hematokrit a koncentrace hemoglobinu, byly analyzovány na Výzkumném ústavu rybářském a hydrobiologickém Jihočeské univerzity ve Vodňanech. Analýza těchto parametrů byla provedena doc. Ing. Martinem Flajšhansem, Dr.rer.agr., MVDr. Veronikou Piačkovou, Ph.D. a Marií Pečenou.

4.4.1 Odběr krve

Pro eliminaci vlivu stresu na následně měřené hematologické parametry byl proveden odběr krve bezprostředně po odlovu ryb. Odběr krve pro studii č. 1 a č. 2. byl proveden podle metodiky Čítka et al. (1992), tj. punkcí srdce. Tato metodika byla k našim účelům modifikována (viz Obr. 11) a detailně byla již popsána v diplomové práci (Lamková, 2006). Pro studii č. 3 a č. 4 byla krev získána punkcí ocasní vény podle metodiky Svobodové et al. (1986) (viz Obr. 12). Odběr krve punkcí ocasní vény byl proveden pomocí heparinizované jehly (1,2 x 40 mm) a 5ml stříkačky. Získaná krev byla ihned přenesena do speciálních baniček určených ke stanovení počtu krevních elementů (viz kapitola 4.4.3), které byly předem heparinizovány. Takto připravené vzorky krve byly v terénu uloženy do přenosné chladničky s ledem a ihned po příjezdu do laboratoře byly zpracovány.

MATERIÁL A METODIKA

Obr. 11: Odběr krve metodou punkcí srdce

Obr. 12: Odběr krve metodou punkcí ocasní cévy

MATERIÁL A METODIKA

4.4.2 Diferenciální rozpočet leukocytů

Díky barvení krevních roztěrů jsme schopni stanovit procentuální zastoupení jak jednotlivých druhů bílých krvinek, tak jejich vývojových stádií. Zhotovení krevních nátěrů, jejich prohlížení a určení jednotlivých krevních elementů bylo provedeno podle metodiky Svobodové (1986) a Pravdy & Svobodové (2003) (viz Obr. 13-15). Pro stanovení krevního diferenciálu ve studiích č. 1 a 2 bylo použito panoptické barvení podle Pappenheima (May-Grünwald, Giemsa Romanovský a pufr pH 7,1). Tato metodika byla již detailně popsána v diplomové práci (Lamková, 2006). Krevní roztěry pro studii č. 3 a 4 byly barveny metodou tzv. rychlého barvení. K tomuto postupu byl použit komerčně dostupný barvící set Hemacolor®(Merck). Výsledné barvení jednotlivých typů krevních buněk je dokumentováno v Tab. 1 a znázorněno na Obr. 16 a 17. Výhodou barvení pomocí barvícího setu Hemacolor®, v porovnání s dříve používaným panoptickým, je jeho rychlost, jednoduchost, snadná dostupnost reagencií a hlavně standardizovaná kvalita. Jednotlivé druhy leukocytů a jejich procentuální zastoupení bylo stanovováno použitím světelného mikroskopu a imerzního objektivu (zvětšení: 10 x 100) na 200 krevních buňkách.

MATERIÁL A METODIKA

Obr. 13: Technika zhotovení krevního roztěru

Obr. 14: Správný (A, B) a nesprávný (C, D) krevní roztěr

MATERIÁL A METODIKA

Obr. 15: Meandrovitý způsob prohlížení krevního roztěru

Typ buňky Část buňky Barva - jádro červená až fialová lymfocyty cytoplazma modrá monocyty cytoplazma šedomodrá neutrofilní granulocyty granula světle fialová eosinofilní granulocyty granula cihlově červená až červenohnědá basofilní granulocyty granula tmavě fialová až černá trombocyty - fialová erytrocyty - načervenalá

Tab. 1: Rychlé barvení (Hemacolor®): výsledné barvení jednotlivých krevních elementů

MATERIÁL A METODIKA

Obr. 16: Schematické rozdělení krevních elementů na modelu kapra obecného (Cyprinus carpio) 63

MATERIÁL A METODIKA

Obr. 17: Vybrané krevní elementy v roztěru na modelu kapra obecného (Cyprinus carpio) 64

MATERIÁL A METODIKA

4.4.3 Stanovení počtu erytrocytů a leukocytů

Pro stanovení počtu krevních buněk byl použit roztok Nat-Herriku, který umožňuje počítání jak červených, tak bílých krevních elementů na rozdíl od dříve používaného Hayemova roztoku (stanovení počtu erytrocytů) a roztoku podle Procházky a Škrobáka (stanovení počtu leukocytů). Roztok Nat-Herriku je možné zhotovit dle návodu podle Pravdy & Svobodové (2003) nebo ho lze nechat odborně připravit ve specializované lékárně. Postup přípravy vzorků pro stanovení počtu erytrocytů a leukocytů byl proveden podle metodiky Svobodové (1986) a

Pravdy & Svobodové (2003). Vlastní počítání krevních elementů se provádí ve čtvercovité mřížce speciálně vybroušeného podložního sklíčka, tj. Bürkerově komůrce. Na této komůrce se erytrocyty počítají ve 20-ti obdélnících (plocha jednoho obdélníku je 0.2 mm2) a leukocyty v 50- ti čtvercích (4 mm2) při 200x zvětšení. Toto počítání se provádí v souladu s tzv. Bürkerovým pravidlem (viz Obr. 18). Dle tohoto pravidla počítáme všechny krvinky, jež se nacházejí uvnitř čtverců nebo obdélníků a dotýkají se jejich dvou stran (do „L“). Krvinky, které se dotýkají zbylých dvou stran, nepočítáme. Toto pravidlo zajišťuje zpřesnění a zvýšení objektivnosti počítané plochy.

Celkové množství erytrocytů napočítaných ve 20-ti obdélnících se dělí číslem 100, čímž získáme počet erytrocytů v T.l-1 (T-tera = 1012), kdežto celkový počet leukocytů v 50-ti čtvercích je počet leukocytů v G.l-1 (G-giga = 109).

MATERIÁL A METODIKA

Obr. 18: Stanovení celkového počtu leukocytů a erytrocytů na Bürkerově komůrce

MATERIÁL A METODIKA

4.4.4 Hematokrit a leukokrit Hodnota hematokritu/leukokritu udává objem erytrocytů/leukocytů k celkovému objemu krve (Svobodová et al., 1986; Pravda & Svobodová, 2003). Principem metody je usazování krevních elementů nesražené krve na základě jejich hustoty (URL1). Oddělení jednotlivých krevních elementů lze docílit jen dokonalým odstředěním, které se provádí v hematokritové odstředivce s použitím speciálních heparinizovaných kapilár. Postup přípravy vzorků byl proveden podle metodiky Svobodové (1986) a Pravdy & Svobodové (2003). Zaznamenanou výšku vrstvy erytrocytů/leukocytů je nutné přepočítat na celkový objem krve v kapiláře a vyjádřit v % (viz Obr. 19). Zjištěná procentuální hodnota erytrocytů/leukocytů se násobí koeficientem 0.01 a výsledná hodnota se udává v jednotkách l.l-1. Výška vrstvy leukocytů byla měřena s použitím světelného mikroskopu a okulárového mikrometru (zvětšení: 10 x 60).

Obr. 19: Centrifugovaná plná krev ve skleněné kapiláře

4.4.5 Množství hemoglobinu Metoda stanovení množství hemoglobinu je založena na oxidaci železnatých iontů hemoglobinu na ionty železité prostřednictvím kyanoželezitanu. Vzniklý methemoglobin reaguje s kyanidem za vzniku methemoglobinkyanátu (kyanomethemoglobin), který se měří spektrofotometricky (Van Kampen & Zijlstra, 1961). Jako transformační roztok, který uvolňuje hemoglobin z erytrocytů, byl použit roztok podle Van Kampena & Zijlstra (1961).

Postup přípravy vzorků: • do zkumavky odměříme 7 ml transformačního roztoku, ke kterému přidáme 25 µl heparinizované krve (50 U/ml ) a obsah ihned promícháme; • odečítání na spektrofotometru (Helios Unicam, USA) lze provést již po 3 minutách.

MATERIÁL A METODIKA

Vlastní měření vzorků bylo provedeno v 1cm kyvetě při vlnové délce 540 nm proti transformačnímu roztoku. Množství hemoglobinu bylo určeno z kalibrační křivky, která byla připravena běžným způsobem za použití kyanohemoglobinového standardu. Množství hemoglobinu se udává v g.l-1 (Svobodová et al., 1986).

MATERIÁL A METODIKA

4.5 Metodika analýzy vybraných fyziologických a imunologických parametrů

Vybrané fyziologické a imunitní parametry pro studie č. 3 a 4., tj. koncentrace glukózy, množství 11-ketotestosteronu, oxidativní vzplanutí, aktivita komplementu, koncentrace IgM protilátek a lysozymu, byly analyzovány na Oddělení fyziologie a imunologie živočichů, Ústav experimentální biologie, PřF MU. Analýza těchto parametrů byla provedena RNDr. Pavlem Hyršlem, Ph.D., Mgr. Soňou Tolárovou a Mgr. Liborem Vojtkem.

4.5.1 Velikost těla a vnitřních orgánů

Celková délka ryb (TL, mm) byla měřena od začátku rypce po konec ocasní ploutve, standardní délka (SL, mm) po bázi ocasní ploutve, tj. po konec ošupení. Stanovena byla hmotnost ryb (g) a vybraných vnitřních orgánů (g) - slezina, gonády a játra.

4.5.2 Odvozené indexy

Jelikož ryby mohou akumulovat svoji energii buď ve svalové tkáni, nebo v játrech (ve formě glykogenu), pro nepřímé stanovení stavu organismu mohou být využity tyto dva indexy – kondiční a hepato-somatický. Kondiční faktor (K), který předpokládá, že kondici jedince lze vyjádřit pomocí délko-hmotnostního vztahu (Bolger & Connolly, 1989) byl počítán na základě následujícího vzorce:

K = konstanta x hmotnost těla (g)/(standardní délka těla [cm])3

Ve studii č. 4 byla použita jeho modifikace označována jako Fultonův kondiční faktor (Anderson & Neumann, 1996):

KF = [hmotnost těla (g) / (celková délka [cm])3 ] × 100000

Dalším indexem, který je aplikován jako nepřímý ukazatel stavu organismu je hepato- somatický index (HSI) (Busacker et al., 1990). Pro zhodnocení aktuální reprodukční zralosti jedinců je používán gonado-somatický index (GSI). V imunologických studiích je často aplikován slezino-somatický index (SSI). Jednotlivé indexy (tj. HSI, GSI, SSI) lze vyjádřit pomocí následujících vztahů:

MATERIÁL A METODIKA

HSI = hmotnost jater (g)/hmotnost těla (g) × 100

GSI = hmotnost gonád (g)/hmotnost těla (g) × 100

SSI = hmotnost sleziny (g)/hmotnost těla (g) × 100

4.5.3 Koncentrace glukózy Ke stanovení hladiny glukózy v krevní plazmě byl použit komerčně dostupný kit (Glu L 1000, PLIVA-Lachema, Česká republika). Krevní plazma byla oddělena od ostatních složek heparinizované krve centrifugací při 1500 x rpm/10 min. Následně bylo 10 µl vzorku smícháno s 1 ml činidla a 200 µl tohoto roztoku bylo měřeno dle instrukcí výrobce v duplikátech na 96-ti jamkové destičce pomocí ELISA readeru při vlnové délce 500 nm (Tecan Sunrise, USA). Výsledná koncentrace (mmol/l) byla stanovena pomocí standardního roztoku glukózy.

4.5.4 Koncentrace 11-ketotestosteronu

Hladina 11-ketotestosteronu byla analyzována v krevní plazmě samců. K analýze byl použit komerčně dostupný kit založený na kompetitivním EIA testu (Cayman Chemical, Estonia). Koncentrace 11-ketotestosteronu ve vzorcích byla stanovována ve dvou různých ředěních (50x, 1000x) a analýza probíhala v duplikátech na 96-ti jamkové destičce pomocí ELISA readeru při vlnové délce 420 nm (Tecan Sunrise, USA). Výsledná koncentrace 11-ketotestosteronu (pg.ml−1) byla přepočtena podle standardu dle instrukcí výrobce.

4.5.5 Aktivita komplementu

Pro stanovení celkové bakteriolytické aktivity komplementu byla použita krevní plazma a analýza byla provedena na základě modifikovaných metodik Virta et al. (1997) a Nikoskelainen et al. (2002). Podstatou této metody je rekombinantní G- bakterie E. coli (kmen K12luxAmp), do jejíž genomu byly vloženy geny kódující enzym luciferázu a jeho substrát luciferin. Tento kmen bakterií byl získán z Department of Biochemistry and Food Chemistry, Turku University z Finska a je charakteristický rezistencí vůči ampicilinu a schopností vlastní bioluminiscence. Tato metoda pak využívá principy bioluminiscence na základě následující reakce:

MATERIÁL A METODIKA

ATP + D – luciferin + O2 → ADP +PPi + oxyluciferin + H2O

Zmíněná reakce je katalyzována enzymem luciferázou a navíc se vyznačuje velkou spotřebou ATP, který může být produkován pouze živými buňkami (Lehtinen et al., 2003). Proto viabilita buněk odpovídá intenzitě bioluminiscence, což je základním předpokladem této metody (Nikoskelainen et al., 2002). V praxi je pak výsledkem reakce světelná emise, kterou je možné změřit luminometrem. K našim účelům byl použit luminometr typu LM01-T (Immunotech, Česká republika). Získané hodnoty luminiscence (RLU = Relative Luminiscence Unit), které korelují s viabilitou bakterií, byly převedeny na procenta viability. Z výsledné křivky viability bakterií byl odečten čas potřebný k usmrcení 50% bakterií. Detaily metodiky byly publikovány v práci Buchtíkové et al. (2011).

4.5.6 Koncentrace lysozymu

Množství lysozymu v kožním mukusu bylo stanoveno in vitro metodou radiální difúze v agaróze. Tato metoda využívá bakterii Micrococcus luteus (CCM 169), která je citlivá na působení lysozymu. Stěr pokožky byl proveden pomocí tupé strany skalpelu a dále byl vzorek nasát do injekční stříkačky a přenesen do mikrozkumavky. Pro přípravu vzorků byla použitá metodika uvedená v publikaci Poisot et al. (2009). Ke kalibraci byl použit roztok lysozymu (E. C. 3.2.1.17, Sigma) o množství 2, 5, 10, 15 a 20 mg/ml. Inkubace probíhala ve vlhké komůrce při laboratorní teplotě po dobu 24 hodin. Odečet průměru difúzní zóny (lýza bakteriálních stěn) byl proveden měřítkem IDP – SEVAC (viz Obr. 20). Množství lysozymu ve vzorku bylo přepočítáno podle kalibrační křivky na mg/ml mukusu.

MATERIÁL A METODIKA

Obr. 20: Schematické znázornění agarózového gelu s difúzními zónami vzniklými působením lysozymu

4.5.7 Oxidativní vzplanutí fagocytů

Při fagocytárním ději dochází ke zvýšení metabolické aktivity fagocytů po stimulaci jejich povrchové membrány. V dizertační práci bylo jako stimulans použito opsonizovaného zymozanu (OZP). Díky opsoninům, které jsou navázány na zymozanové částice, se OZP váže na komplementové a imunoglobulinové receptory, což vede ke spuštění kaskády reakcí fagocytárního děje spojené s aktivací klíčového enzymu oxidativního vzplanutí- NADPH oxidázy, degranulaci specifických a azurofilních granul a tvorbě fagolysozómu. Takto aktivované fagocyty uvolňují při tomto ději určitá světelná kvanta (chemiluminiscence, CL), které je možno po zesílení luminofory (luminol, lucigenin) změřit na citlivých přístrojích, tzv. luminometrech (URL2). Postup přípravy a analýzy vzorků byl publikován v práci Poisot et al. (2009) a Buchtíková et al. (2011). Při konečném hodnocení aktivity fagocytů je za nejdůležitější ukazatel oxidativního vzplanutí považována plocha píku pod naměřenou křivkou, hodnota píku (vrchol CL odpovědi) a

MATERIÁL A METODIKA

čas potřebný k jeho dosažení. Tyto hodnoty jsou transformovány na počet fagocytů, získaný z analýz celkového počtu bílých krvinek a z krevního diferenciálu (viz kapitola 4.4.2 a 4.4.3).

4.5.8 Stanovení koncentrace IgM protilátek

Pro celkové stanovení množství protilátek v rybí plazmě lze použít metodu precipitace síranem zinečnatým (McEwan et al., 1970). K našim účelům byl použit komerčně dostupný kit (Bio-Rad,

USA). Principem zmíněné metody je specifická dehydratace proteinů plazmy 0.7 mM ZnSO4 x

7H2O (pH = 5.8), následně vysrážení těchto proteinů ze vzorku a konečně jejich oddělení centrifugací. K 1.5 ml 0.7 mM ZnSO4 x 7H2O bylo přidáno 25 µl vzorku krevní plazmy, následovala inkubace při laboratorní teplotě 2 hodiny a centrifugace 6000 rpm / 15 min. Celkové množství IgM (v g/l) bylo stanoveno na základě rozdílu mezi celkovým obsahem proteinů před centrifugací a proteinů obsažených v supernatantu po centrifugaci. Absorbance byla měřena při 700 nm ELISA readerem (Tecan Sunrise, USA). Ačkoliv v současné době existuje pro stanovení protilátek celá řada modernějších technik (např. ELISA), výhodou této metody je především její jednoduchost a spolehlivost.

VÝSLEDKY

5 VÝSLEDKY

5.1 Sezónní změny imunokompetence a parazitismu u jelce tlouště (Leuciscus cephalus), sladkovodní kaprovité ryby

Cílem studie č. 1 (článek A) byla (1) analýza sezónních změn vybraných fyziologických, imunitních parametrů a parazitární infekce a (2) odhad potenciální míry imunokompetence a parazitace u jelce tlouště (Leuciscus cephalus). Vybrané fyziologické a imunitní parametry a parazitární infekce byly analyzovány v průběhu čtyř sezónních odlovů (duben, červen, srpen a listopad). Dále byly testovány vzájemné interakce mezi imunokompetencí, fyziologií a parazitismem.

V této studii byly zaznamenány statisticky významné sezónní rozdíly ve velikosti sleziny. Nejvyšší investice do velikosti sleziny byly zjištěny v dubnu, tj. po přezimování ryb a dále v srpnu, tj. v období po reprodukci ryb spojeném s nízkými hodnotami gonado-somatického indexu. Navíc byly zjištěny statisticky významné sezónní rozdíly v počtu erytrocytů, leukocytů, fagocytů a v aktivitě fagocytů, měřené pomocí oxidativního vzplanutí. Naopak sezóna neměla vliv na složení jednotlivých typů leukocytů, tj. monocytů, lymfocytů a neutrofilních granulocytů. V průběhu sezóny se však měnil poměr různých typů neutrofilních granulocytů, tj. myelocytů, metamyelocytů, tyček a segmentů. Změny v proporcionálním zastoupení jednotlivých vývojových stádií neutrofilních granulocytů mohou být buď výsledkem přímého působení sezónních změn, nebo mohou být nepřímo spojeny se sezónní dynamikou parazitární infekce či infekce jiného původu. Nejvyšší diverzita parazitických společenstev byla zaznamenaná v dubnu a červnu a naopak nejnižší v listopadu. Při porovnání početnosti mnohobuněčných parazitů na úrovni infraspolečenstva byly zaznamenány nejvyšší hodnoty abundance v dubnu a červnu pro Monogenea, v dubnu a listopadu pro Acantocephala a rovněž v dubnu pro Cestoda. Sezónní dynamika celkové abundance ektoparazitických Monogeneí přibližně odpovídala očekávanému sezónnímu trendu vázaného na teplotu vody. Sezónní dynamika ostatních skupin mnohobuněčných cizopasníků byla pravděpodobně ovlivněna kromě sezónních změn v teplotě vody i přítomností mezihostitele.

Analyzovány byly vzájemné korelace mezi měřenými parametry imunity, fyziologie a parazitismu. Zaznamenán byl pozitivní vztah mezi skupinou Monogenea a oxidativním vzplanutím. V průběhu celé studie byla skupina Monogenea nejpočetnější a druhově nejbohatší

VÝSLEDKY skupinou mnohobuněčných cizopasníků, což se pravděpodobně odrazilo na zaznamenané zvýšené aktivitě fagocytů. Vyšší investice do kondice a velikosti gonád ryb byly spojeny s poklesem investic do imunitních funkcí (velikost sleziny a počet leukocytů), což bylo především dokumentováno u samců. Tento vztah může naznačovat existenci kompromisu („trade-off“) mezi investicemi do somatické kondice a reprodukce na úkor imunitních funkcí. Dále byla zaznamenaná pozitivní korelace mezi velikostí gonád a parazitární infekcí (Monogenea, Acanthocephala a Cestoda). Tento vztah může být výsledkem synchronizace vývojového cyklu parazitů s reprodukcí hostitele nebo mohou být imunitní funkce v průběhu reprodukce potlačeny zvýšenou hladinou steroidních hormonů.

VÝSLEDKY

5.2 Je imunitní systém ryb skutečně ovlivňován parazity? Imunoekologická studie u kapra obecného (Cyprinus carpio)

V rámci studie č. 3 (článek C) proběhlo v průběhu jednoho roku celkem pět sezónních odlovů kapra obecného (Cyprinus carpio) z chovného rybníku. Jednotlivé odlovy byly odlišné sezónností abiotických a biotických faktorů. Cílem práce bylo analyzovat, zda je imunokompetence kapra obecného (Cyprinus carpio) větší mírou ovlivněna sezónními změnami způsobenými především teplotou vody nebo jsou změny imunokompetence spojené s parazitární infekcí. Dalším cílem bylo analyzovat potenciální investice mezi fyziologií a imunokompetencí kapra obecného v návaznosti na hypotézu kompromisu v rozdělení energie („trade-off“) a hypotézu imunokompetenčního handicapu, zejména imunosupresivní efekt 11-ketotestosteronu.

V této studii bylo potvrzeno, že sezónní změny zásadní měrou ovlivňují fyziologický a imunitní systém hostitele a rovněž se významně podílejí na změnách parazitární infekce. Bylo zjištěno, že jedinci kapra obecného, více infikovaní zástupci třídy Monogenea, měli nižší kondici (vyjádřenou pomocí kondičního faktoru). Zmíněná skupina parazitů byla po celou dobu studie druhově i početně nejbohatší. Počet i aktivita fagocytů stanovovaná pomocí oxidativního vzplanutí byla negativně asociována s parazitární infekcí vyvolanou zástupci třídy Cestoda. Hostitelé, více infikovaní zástupci třídy Digenea, měli vyšší hodnoty sleziny vyjádřené pomocí slezino- somatického indexu.

Korelační analýza potvrdila vztahy mezi celkovou mírou fyziologie, imunity a parazitismu po korekci pro efekt sezóny. I když nebyl zjištěn přímý vztah založený na principu kompromisu mezi jednotlivými ukazateli fyziologie a imunity ryb, bylo zjištěno, že alokace energie mezi životně důležité funkce (především reprodukci a kondici) by mohla být řízena sezónními změnami. Analýza vlivu 11-ketotestosteronu na imunitní parametry byla vázaná na reprodukční období ryb. Zaznamenán byl negativní vztah mezi koncentrací 11-ketotestosteronu a aktivitou komplementu, který potvrzuje imunosupresivní roli 11-ketotestosteronu. Zaznamenané výsledky naznačují, že rozmanité životní strategie parazitů ovlivňují různé složky fyziologie a imunity hostitele i přes to, že jsou tyto funkce silně ovlivňovány sezónními změnami.

VÝSLEDKY

5.3 Ovlivňují reprodukční investice kaprovitých ryb jejich imunokompetenci a výskyt mnohobuněčných parazitů?

Cílem studie č. 2 (článek B) byla analýza investic samců jelce tlouště (Leuciscus cephalus) do imunokompetence v průběhu tří period lišících se ve vztahu k reprodukci (tj. pre-reprodukční, reprodukční a post-reprodukční). Dále bylo v práci testováno, zda investice do reprodukce mají vztah k vyšší vnímavosti hostitele k parazitární infekci, nižší kondici či imunokompetenci. V návaznosti na hypotézu imunokompetenčního handicapu a „sperm protection“ hypotézu byly analyzovány vztahy mezi expresí sekundárních pohlavních znaků, kvalitou spermií a parazitární infekcí.

Z výsledků studie vyplývá, že investice v průběhu reprodukce hrají u ryb důležitou úlohu a odrážejí se následně v alokaci energie do kondice a imunity. Tyto investice byly odlišné v periodách pre-reprodukční, reprodukční a post-reprodukční. Vyšší hodnoty imunitních parametrů (oxidativního vzplanutí, počtu leukocytů a leukokritu), zaznamenané především v průběhu reprodukce (tj. v období tření), však nebyly v této periodě odrazem probíhající parazitární infekce, způsobené mnohobuněčnými parazity. Tento stav byl pravděpodobně známkou stresu vzniklého v průběhu reprodukce nebo byla reakce imunitního systému iniciovaná infekcí jiného původu (protozoální, bakteriální či virové). V průběhu tří period s odlišným vztahem k reprodukci nebyly zaznamenány změny v počtu leukocytů a neutrofilů, avšak rozdíly byly zjištěny ve spektru vývojových stádií neutrofilních granulocytů. V pre- reprodukční periodě byl zaznamenán nejvyšší gonado-somatický index, který v reprodukční periodě prudce klesl. Obdobná dynamika změn byla zjištěna i pro kondiční faktor. Reprodukční perioda ryb je považována za energeticky velmi nákladné období, a jak vyplývá z těchto výsledků, samci akumulují dostupné zdroje energie před samotnou reprodukcí. Následně je pak tato energie v průběhu reprodukční fáze spotřebována či přerozdělena do jiných životních funkcí.

Statisticky významné změny byly zaznamenány i v dynamice parazitární infekce. Jedinci byli nejvíce infikovaní zástupci třídy Monogenea v pre-reprodukční a post-reprodukční periodě. V porovnání s pre-reprodukční periodou byla v průběhu reprodukční periody rovněž zaznamenaná nižší infekce způsobená zástupci třídy Cestoda. Maximální početnost zástupců třídy Digenea byla zaznamenána rovněž v pre-reprodukční periodě.

VÝSLEDKY

Výsledky analýz částečně potvrzují hypotézy imunokompetenčního handicapu i „sperm protection“ hypotézy. Samci s vyšší kvalitou spermatokritu disponovali větší slezinou a navíc byli parazitováni vyšším počtem motolic Metorchis xanthosomus. Dále byl zaznamenán pozitivní vztah mezi počtem třecích vyrážek a početností motolice Sphaerostomum bramae.

VÝSLEDKY

5.4 Fyziologie a imunologie lína obecného (Tinca tinca): efekt hormonální stimulace a ploidie spojené s chromozomální manipulací

V rámci studie č. 4 (článek D) proběhl v rámcí jednoho týdne odlov jedinců lína obecného (Tinca tinca). Studována byla fyziologie a imunita tří populací: amfimiktické diploidní, indukované triploidní a celosamičí meiotické gynogenetické. Dále byl testován vliv hormonální stimulace na fyziologické a imunitní parametry.

Hormonální stimulace měla statisticky významný vliv na oxidativní vzplanutí, koncentraci lysozymu, glukózy a hemoglobinu. Kromě koncentrace glukózy byly u všech ostatních zmíněných parametrů po stimulaci zaznamenány statisticky významně vyšší hodnoty. Tyto rozdíly byly pravděpodobně spojeny se stresem či manipulací u skupiny stimulovaných ryb, u nichž byla za účelem indukce spermiace a ovulace použita teplotní a hormonální stimulace. Vyšší hodnoty ostatních zmíněných parametrů, zaznamenané po stimulaci, byly pravděpodobně spojeny s tzv. sekundární reakcí na stres, případně se jednalo o reakci na zvýšenou teplotu vody, ve které byly stimulovaní jedinci drženi.

Dále byly zaznamenány statisticky významné rozdíly mezi jedinci odlišné ploidie. Diploidní jedinci byli v porovnání s triploidními v lepší somatické kondici (měřené pomocí kondičního faktoru). Tímto výsledkem tedy nebyla podpořena hypotéza lepšího růstu triploidních jedinců. Naopak se zdá, že pomalejší růst triploidních jedinců může být spojen s jejich nižší konkurenceschopností při potravní kompetici či jejich vyšší náchylnosti ke stresu. Dále trend vyšších hodnot 11-ketotestosteronu, zaznamenaných u diploidních samců, nebyl spojen s odlišnou velikostí gonád. U samic byl zjištěn významný vliv populace na slezino-somatický index a zaznamenán byl trend populačního efektu pro hepato-somatický. Nejnižší hodnoty obou indexů byly zaznamenány pro amfimiktické diploidní samice. Počty většiny stanovovaných krevních elementů korespondovaly s očekávaným trendem tj. nižší počet erytrocytů, leukocytů a fagocytů byl zaznamenaný u indukovaných triploidních jedinců. V porovnání s diploidními jedinci, by zaznamenané nižší počty krevních elementů u triploidních jedinců měly být kompenzovány jejich větší velikostí a případně i vyšší aktivitou přepočítanou na jednotlivé buněčné elementy. Procentuální zastoupení jednotlivých typů leukocytů, které byly určeny pomocí krevního diferenciálu, vykazovaly normální hodnoty odpovídající zdravým jedincům.

VÝSLEDKY

Výjimkou byla skupina nestimulovaných amfimiktických diploidních a indukovaných triploidních samců, kde byl zjištěn nižší poměr lymfocytů a naopak vyšší procento neutrofilů a monocytů. Takový výsledek může být například známkou probíhající infekce.

ZÁVĚRY

6 ZÁVĚRY

První část dizertační práce (Studie č. 1 a 3) je věnována především studiu sezónních změn imunokompetence a hodnocení asociací mezi fyziologií, imunitou a parazitismem u dvou druhů kaprovitých ryb (Leuciscus cephalus a Cyprinus carpio).

Dosažené výsledky lze sumarizovat následovně:

• Sezónní vlivy zásadní měrou zasahují do fungování imunitního i fyziologického systému poikilotermních obratlovců a rovněž se významně ovlivňují intenzitu parazitární infekce vyvolané mnohobuněčnými cizopasníky.

• Předpoklad kompromisů mezi životně důležitými složkami lze aplikovat k vysvětlení některých vztahů mezi vybranými parametry imunitního a fyziologického systému. Přímý vztah imunity a fyziologie však potvrzen nebyl.

• Alokace energie mezi fyziologii a imunitu je pravděpodobně zprostředkována nebo usnadněna vlivem sezóny na jednotlivé parametry.

• Potvrzen byl imunosupresivní efekt steroidních hormonů, získané výsledky naznačují efekt steroidních hormonů jenom na určité složky imunitního systému.

• I přes velmi silný vliv sezónních změn, parazitismus ovlivňuje fyziologické a imunitní funkce napadených hostitelů.

• Mnohobuněční parazité využívají v průběhu infekce rozmanitých životních strategií a zasahují tak do různých částí fyziologického a imunitního systému.

• Skupina Monogenea patřila k nejvíce početně i druhově zastoupené skupině parazitů. Při vysoké infekci mohou kromě imunitních funkcí ovlivňovat i celkovou kondici rybího hostitele.

ZÁVĚRY

Další část dizertační práce (Studie 2) byla věnována studiu imunokompetence a parazitismu ve vztahu k reprodukčním investicím u samců jelce tlouště (Leuciscus cephalus).

Výsledky této studie lze shrnout následovně:

• V průběhu reprodukce ryb je energie alokována mezi kondici, imunitu a reprodukci.

• Investice mezi životně důležité složky (kondice, fyziologie a imunita) se mění v závislosti na reprodukční investici, která je odlišná v období pre-reprodukčním, reprodukčním a post-reprodukčním.

• Vysoké hodnoty imunitních parametrů zaznamenané v reprodukční periodě nejsou spojeny s parazitární infekcí způsobené mnohobuněčnými cizopasníky.

• Nepřímo byla potvrzena hypotéza imunokompletenčního handicapu a „sperm protection“ hypotéza.

ZÁVĚRY

Poslední část (Studie 4) byla zaměřena na analýzu vlivu hormonální stimulace a ploidie spojené s chromozomální manipulací na vybrané parametry fyziologického a imunitního systému lína obecného (Tinca tinca).

Dosažené výsledky lze shrnout následovně:

• Stimulace i ploidie ryb signifikantně ovlivnila některé parametry fyziologického a imunitního systému ryb.

• Při stimulaci ryb se spíše než samotný vliv hormonů pravděpodobně jednalo o změny navozené stresem spojeným s manipulací při stimulaci.

• Zaznamenané nižší hodnoty glukózy u stimulovaných jedinců jsou pravděpodobně spojeny se sníženou vnímavostí ryb na opakovaný či dlouhodobější stres.

• Zvýšené hodnoty některých parametrů fyziologického a imunitního systému, zaznamenané po stimulaci, jsou dány buď sekundární reakcí na stres či teplotou vody.

• Zjištěné výsledky, týkající se nižší somatické kondice u triploidních jedinců, nepotvrdily hypotézu lepšího růstu triploidů.

• Statisticky významné rozdíly mezi jedinci s odlišnou ploidií byly zjištěny především pro počty krevních elementů. Vyšší počty krevních elementů byly zaznamenány u diploidních jedinců.

• Při porovnání procentuálního zastoupení jednotlivých typů leukocytů u různých skupin ryb nebyly zjištěny výrazné rozdíly. Výjimkou byla skupina nestimulovaných amfimiktických diploidních a indukovaných triploidních samců, kde hodnoty některých parametrů krevního diferenciálu vykazovaly výraznější odchylku od hodnot zaznamenaných pro ostatní skupiny ryb.

ZÁVĚRY

Závěrem dizertační práce lze konstatovat, že předložené výsledky této práce přispívají k objasnění obecných vztahů mezi rybím hostitelem, jeho abiotickým prostředím a parazitární infekcí. Rovněž práce přináší nové poznatky v oblasti fyziologie a imunity na úrovni analyzovaných hostitelských druhů v přirozených podmínkách a v podmínkách akvakultur. Výsledky práce jednoznačně potvrzují složitost systému hostitel-parazit a odhalují nutnost dalších komplexních analýz v oblasti imunoekologie a evoluční imunologie. Budoucí vědecký výzkum by měl proto směrovat k verifikaci obecné platnosti vztahů zaznamenaných v předložené dizertační práci na různých modelech kaprovitých ryb.

SEZNAM LITERATURY

7 SEZNAM LITERATURY Ainsworth AJ, Dexiang C, Waterstrat PR, Greenway T (1991) Effect of temperature on the immune system of channel catfish (Ictalurus punctatus)—I. Leucocyte distribution and phagocyte function in the anterior kidney at 10°C. Comparative Biochemistry and Physiology a-Physiology, 100, 907-912. Aldrin JF, Messager JL, Baudin Laurencin F (1982) La biochimie clinique en aquaculture. Interet et perspective. Acres Colloquenational, 291-326. (ve francouzštině) Alston S, Lewis JW (1994) The ergasilid parasites (Copepoda: Poecilostomatoida) of British freshwater fish. In: Parasitic Diseases of Fish (ed. by Pike AW, Lewis JW). Samara Publishing, Dyfed, pp. 171–188. Alvarez F, Razquin BE, Villena AJ, Zapata AG (1998) Seasonal changes in the lymphoid organs of wild brown trout, Salmo trutta L: A morphometrical study. Veterinary Immunology and Immunopathology, 64, 267-278. Alvarez-Pellitero P (2008) Fish immunity and parasite infections: from innate immunity to immunoprophylactic prospects. Veterinary Immunology and Immunopathology, 126, 171-198. Anderson RO, Neumann RM (1996) Length, weight, and associated structural indices. In: Fisheries Techniques, 2nd edition. American Fisheries Society, Bethesda. Angelidis P, Baudinlaurencin F, Youinou P (1988) Effects of temperature on chemiluminescence of phagocytes from sea bass, Dicentrarchus labrax L. Journal of Fish Diseases, 11, 281-288. Ballarin L, Dall'Oro M, Bertotto D, Libertini A, Francescon A, Barbaro A (2004) Haematological parameters in Umbrina cirrosa (Teleostei, Sciaenidae): a comparison between diploid and triploid specimens. Comparative Biochemistry and Physiology a-Molecular & Integrative Physiology, 138, 45-51. Baruš V, Oliva O, kol. a (1995) Mihulovci a ryby (2), Fauna ČR a SR, Academia, Praha. Beaumont A, Boudry P, Hoare K (2003) Biotechnology and Genetics in Fisheries and Aquaculture, Blackwell Publishers. Benfey TJ (1999) The physiology and behavior of triploid fishes. Reviews in Fisheries Sciences, 7, 39–67. Benfey TJ, Biron M (2000) Acute stress response in triploid rainbow trout (Oncorhynchus mykiss) and brook trout (Salvelinus fontinalis). Aquaculture, 184, 167-176. Benfey TJ, Sutterlin AM, Thompson RJ (1984) Use of erythrocyte measurements to identify triploid salmonids. Canadian Journal of Fisheries and Aquatic Sciences, 41, 980-984. Billard R (1986) Spermatogenesis and spermatology of some teleost fish species. Reproduction Nutrition Development, 26, 877-920. Biron M, Benfey TJ (1994) Cortisol, glucose and hematocrit changes during acute stress, cohort sampling, and the diel cycle in diploid and triploid brook trout (Salvelinus fontinalis Mitchill). Fish Physiology and Biochemistry, 13, 153-160. Bolger T, Connolly PL (1989) The selection of suitable indexes for the measurement and analysis of fish condition. Journal of Fish Biology, 34, 171-182. Borg B (1994) Androgens in teleost fishes. Comparative Biochemistry and Physiology C- Pharmacology Toxicology & Endocrinology, 109, 219-245. Braude S, Tang-Martinez Z, Taylor GT (1999) Stress, testosterone, and the immunoredistribution hypothesis. Behavioral Ecology, 10, 345-350.

SEZNAM LITERATURY

Budino B, Cal RM, Piazzon MC, Lamas J (2006) The activity of several components of the innate immune system in diploid and triploid turbot. Comparative Biochemistry and Physiology a-Molecular & Integrative Physiology, 145, 108-113. Buchmann K (1998) Binding and lethal effect of complement from Oncorhynchus mykiss on Gyrodactylus derjavini (Platyhelminthes: Monogenea). Diseases of Aquatic Organisms, 32, 195-200. Buchtíková S, Šimková A, Rohlenová K, Flajšhans M, Lojek A, Lilius EM, Hyršl P (2011) The seasonal changes in innate immunity of the common carp (Cyprinus carpio). Aquaculture, 318, 169-175. Buchtová H, Svobodová Z, Flajšhans M, Vorlová L (2003) Analysis of slaughtering value of diploid and triploid population of tench (Tinca tinca, Linnaeus 1758). Czech Journal of Animal Science, 48, 285-294. Burrows A, Fletcher T, Manning M (2001) Haematology of the turbot, Psetta maxima (L.): ultrastructural, cytochemical and morphological properties of peripheral blood leucocytes. Journal of Applied Ichthyology, 17, 77–84. Busacker GP, Adelman IR, Goolish EM (1990) Methods for fish biology, American Fisheries Society, Maryland. Bush AO, Lafferty KD, Lotz JM, Shostak AW (1997) Parasitology meets ecology on its own terms: Margolis et al revisited. Journal of Parasitology, 83, 575-583. Collazos ME, Barriga C, Ortega E (1995) Seasonal variations in the immune system of the cyprinid Tinca tinca. Phagocytic function. Comparative Immunology Microbiology and Infectious Diseases, 18, 105-113. Cox FEG (1997) Designer vaccines for parasitic diseases. International Journal for Parasitology, 27, 1147-1157. Craig SR, MacKenzie DS, Jones G, Gatlin DM (2000) Seasonal changes in the reproductive condition and body composition of free-ranging red drum, Sciaenops ocellatus. Aquaculture, 190, 89-102. Cuesta A, Vargas-Chacoff L, Garcia-Lopez A, Arjona FJ, Martinez-Rodriguez G, Meseguer J, Mancera JM, Esteban MA (2007) Effect of sex-steroid hormones, testosterone and estradiol, on humoral immune parameters of gilthead seabream. Fish & Shellfish Immunology, 23, 693-700. Dahle R, Taranger GL, Karlsen O, Kjesbu OS, Norberg B (2003) Gonadal development and associated changes in liver size and sexual steroids during the reproductive cycle of captive male and female Atlantic cod (Gadus morhua L.). Comparative Biochemistry and Physiology a-Molecular & Integrative Physiology, 136, 641-653. Dalmo RA, Ingebrigtsen K, Bogwald J (1997) Non-specific defence mechanisms in fish, with particular reference to the reticuloendothelial system (RES). Journal of Fish Diseases, 20, 241-273. Danilová N, Bussmann J, Jekosch K, Steiner LA (2005) The immunoglobulin heavy-chain locus in zebrafish: identification and expression of a previously unknown isotype, immunoglobulin Z. Nature Immunology, 6, 295-302. Dartnall HJG, Walkey M (1979) Distribution of glochidia of the Swan mussel Anodonta cygnea (Mollusca) on the Three-spined stickleback Gasterosteus aculeatus (Pisces). Journal of Zoology, 189, 31-37. Donaldson EM (1996) Manipulation of reproduction in farmed fish. Animal Reproduction Science, 42, 381-392.

SEZNAM LITERATURY

Du Pasquier L (1993) Evolution of the immune system, Raven Press, New York. Dudgeon D, Morton B (1984) Site selection and attachment duration of Anodonta woodiana (Bivalvia: Unionacea) glochidia on fish hosts. Journal of Zoology, 204, 355-362. Dudiňák V, Špakulová M (2003) The life cycle and seasonal changes in the occurrence of Pomphorhynchus laevis (Palaeacanthocephala, Pomphorhynchidae) in a small isolated lake. Parasite-Journal De La Societe Francaise De Parasitologie, 10, 257-262. Dunham R (2004) Aquaculture and fisheries biotechnology: Genetic approaches, CAB International, Cambridge. Ebert D, Herre EA (1996) The evolution of parasitic diseases. Parasitology Today, 12, 96-101. Ergens R, Lom J (1970) Původci parasitárních nemocí ryb, Academia, Praha. Esch G, Fernández J (1993) A functional biology of parasitism, Chapman & Hall, London. Esch GW, Bush AO, Aho JM (1990) Parasite communities: patterns and progresses, Chapman and Hall, London. Evans NA (1977) The occurrence of Sphaerostoma bramae (Digenea: Allocreadiidae) in the roach from the Worcester-Birmingham canal. Journal of Helminthology, 51, 189–196. Felip A, Piferrer F, Carrillo M, Zanuy S (2001) Comparison of the gonadal development and plasma levels of sex steroid hormones in diploid and triploid sea bass, Dicentrarchus labrax L. Journal of Experimental Zoology, 290, 384-395. Flajnik MF, Du Pasquier L (2004) Evolution of innate and adaptive immunity: can we draw a line? Trends in Immunology, 25, 640-644. Flajšhans M (1997) Reproduction sterility caused by spontaneous triploidy in tench (Tinca tinca). Polish Archives of Hydrobiology, 44, 39-45. Flajšhans M, Linhart O, Kvasnička P (1993) Genetic studies of tench (Tinca tinca L) - induced triploidy and tetraploidy and 1st performance data. Aquaculture, 113, 301-312. Flajšhans M, Linhart O, Kvasnička P (1995) Tench, Tinca tinca (Linnaeus, 1758): a model for chromosomal manipulation studies. Polish Archives of Hydrobiology, 42, 123–131. Folstad I, Karter AJ (1992) Parasites, bright males, and the immunocompetence handicap. American Naturalist, 139, 603-622. Folstad I, Skarstein F (1997) Is male germ line control creating avenues for female choice? Behavioral Ecology, 8, 109-112. Foster AR, Houlihan DF, Hall SJ (1993) Effects of nutritional regime on correlates of growth rate in juvenile Atlantic cod (Gadus morhua): comparison of morphological and biochemical measurements. Canadian Journal of Fisheries and Aquatic Sciences, 50, 502-512. Gomelsky B (2003) Chromosome set manipulation and sex control in common carp: a review. Aquatic Living Resources, 16, 408-415. Gussev A (1985) Opredeliteľ parazitov presnovodnych ryb fauny SSSR, Tom 2. Parazitičeskije mnogokljetočnyje, Nauka, Leningrad. (v ruštině) Hakalahti T, Pasternak AF, Valtonen ET (2004) Seasonal dynamics of egg laying and egg-laying strategy of the ectoparasite Argulus coregoni (Crustacea: Branchiura). Parasitology, 128, 655-660. Hakalahti T, Valtonen ET (2003) Population structure and recruitment of the ectoparasite Argulus coregoni Thorell (Crustacea: Branchiura) on a fish farm. Parasitology, 127, 79- 85. Hamilton WD, Zuk M (1982) Heritable true fitness and bright birds: a role for parasites. Science, 218, 384-387.

SEZNAM LITERATURY

Hansen JD, Landis ED, Phillips RB (2005) Discovery of a unique Ig heavy-chain isotype (IgT) in rainbow trout: Implications for a distinctive B cell developmental pathway in teleost fish. Proceedings of the National Academy of Sciences of the United States of America, 102, 6919-6924. Hanzelová V, Gerdeaux D (2003) Seasonal occurrence of the tapeworm Proteocephalus longicollis and its transmission from copepod intermediate host to fish. Parasitology Research, 91, 130-136. Hardig J, Hoglund LB (1983) On accuracy in estimating fish blood variables. Comparative Biochemistry and Physiology a-Physiology, 75, 35-40. Harding FA, Amemiya CT, Litman RT, Cohen N, Litman GW (1990) Two distinct immunoglobulin heavy chain isotypes in a primitive, cartilaginous fish, Raja erinacea. Nucleic Acids Research, 18, 6369-6376. Harrison AJ, Gault NFS, Dick JTA (2006) Seasonal and vertical patterns of egg-laying by the freshwater fish louse Argulus foliaceus (Crustacea: Branchiura). Diseases of Aquatic Organisms, 68, 167-173. Hayman JB, Levine RP, Lobb CJ (1992) Complement deficiencies in channel catfish (Ictalurus punctatus) associated with temperature and seasonal mortality. Fish & Shellfish Immunology, 2, 183–192. Heins DC, Singer SS, Baker JA (1999) Virulence of the cestode Schistocephalus solidus and reproduction in infected threespine stickleback, Gasterosteus aculeatus. Canadian Journal of Zoology-Revue Canadienne De Zoologie, 77, 1967-1974. Hernández A, Tort L (2003) Annual variation of complement, lysozyme and haemagglutinin levels in serum of the gilthead sea bream Sparus aurata. Fish & Shellfish Immunology, 15, 479-481. Hillgarth N, Ramenofsky M, Wingfield J (1997) Testosterone and sexual selection. Behavioral Ecology, 8, 108-109. Hoar WS, Randall DJ, Conte PF (1992) Fish Physiology: The Cardiovascular System, Academic Press, London. Holčík J, Mihálik J (1971) Sladkovodní ryby, Svoboda, Praha. Hou Y, Han XD (2001) Effects of estradiol-17beta on immunocompetence in rainbow trout. Acta Zool Sin, 47, 285–291. Hou Y, Suzuki Y, Aida K (1999) Effects of steroids on the antibody producing activity of lymphocytes in rainbow trout. Fisheries Science, 65, 850-855. Houston AH (1980) Components of the hematological response of fishes to environmental temperature change. In: Environmental physiology of fishes (ed by Ali MA). Plenum. Houston AH (1990) Methods for fish biology, American Fisheries Society, Maryland. Huntingford FA, Chellappa S, Taylor AC, Strang RHC (2001) Energy reserves and reproductive investment in male three-spined sticklebacks, Gasterosteus aculeatus. Ecology of Freshwater Fish, 10, 111-117. Chang JP, Jobin RM (1994) Regulation of gonadotropin release in vertebrates: a comparison of GnRH mechanisms of action. In: Perspectives in Comparative Endocrinology (ed. by Davey KG, Peter RE, Tobe SS). National Research Council of Canada, Ottawa, pp. 41– 51. Cherfas NB, Gomelsky B, Bendom N, Peretz Y, Hulata G (1994) Assessment of triploid common carp (Cyprinus carpio L) for culture. Aquaculture, 127, 11-18.

SEZNAM LITERATURY

Ching B, Jamieson S, Heath JW, Heath DD, Hubberstey A (2010) Transcriptional differences between triploid and diploid Chinook salmon (Oncorhynchus tshawytscha) during live Vibrio anguillarum challenge. Heredity, 104, 224-234. Chubb JC (1979) Seasonal occurrence of helminths in freshwater fishes. Part II. Trematoda. Advances in parasitology, 17, 141–313. Ihssen PE, McKay LR, McMillan I, Phillips RB (1990) Ploidy manipulation and gynogenesis in fishes: Cytogenetic and fisheries applications. Transactions of the American Fisheries Society, 119, 698-717. Jansen WA (1991) Seasonal prevalence, intensity of infestation, and distribution of glochidia of Anodonta grandis simpsoniana Lea on yellow perch, Perca flavescens. Canadian Journal of Zoology-Revue Canadienne De Zoologie, 69, 964-972. John JL (1995) Parasites and the avian spleen - helminths. Biological Journal of the Linnean Society, 54, 87-106. Johnson OW, Dickhoff WW, Utter FM (1986) Comparative growth and development of diploid and triploid coho salmon, Oncorhynchus kisutch. Aquaculture, 57, 329-336. Jokela J, Valtonen ET, Lappalainen M (1991) Development of glochidia of Anodonta piscinalis and their infection of fish in a small lake in Northern Finland. Archiv Fur Hydrobiologie, 120, 345-355. Jorgensen EH, Johansen SJS, Jobling M (1997) Seasonal patterns of growth, lipid deposition and lipid depletion in anadromous Arctic charr. Journal of Fish Biology, 51, 312-326. Jurajda P, Gelnar M (1998) Jelec tloušť – modelový druh tekoucích vod. Sborník referátů III. české ichtyologické konference, Vodňany: 6.-7. květen 1998, 61-68. Kabata Z (1979) Parasitic Copepoda of Brithis fishes, Ray Society, London. Kania PW, Sorensen RR, Koch C, Brandt J, Kliem A, Vitved L, Hansen S, Skjodt K (2010) Evolutionary conservation of mannan-binding lectin (MBL) in bony fish: Identification, characterization and expression analysis of three bona fide collectin homologues of MBL in the rainbow trout (Onchorhynchus mykiss). Fish & Shellfish Immunology, 29, 910-920. Kappe A, Seifert T, El-Nobi G, Brauer G (2006) Occurrence of Atractolytocestus huronensis (Cestoda, Caryophyllaeidae) in German pond-farmed common carp Cyprinus carpio. Diseases of Aquatic Organisms, 70, 255-259. Kearn GC (1994) Evolutionary expansion of the Monogenea. International Journal for Parasitology, 24, 1227-1271. Kennedy CR (1975) Ecological animal parasitology, Blackwell Scientific Publications, Oxford. Khotenovsky IA (1985) Monogenea, Nauka, Leningrad. (v ruštině) Kortet R, Taskinen J, Sinisalo T, Jokinen I (2003a) Breeding-related seasonal changes in immunocompetence, health state and condition of the cyprinid fish, Rutilus rutilus, L. Biological Journal of the Linnean Society, 78, 117-127. Kortet R, Vainikka A, Rantala MJ, Jokinen I, Taskinen J (2003b) Sexual ornamentation, androgens and papillomatosis in male roach (Rutilus rutilus). Evolutionary Ecology Research, 5, 411-419. Kortet R, Vainikka A, Rantala MJ, Taskinen J (2004) Sperm quality, secondary sexual characters and parasitism in roach (Rutilus rutilus L.). Biological Journal of the Linnean Society, 81, 111-117. Koskivaara M, Tellervo EV, Prost M (1991a) Seasonal occurrence of gyrodactylid monogeneans on the roach (Rutilus rutilus) and variations between four lakes of differing water quality in Finland. Aqua Fennica, 21, 47-55.

SEZNAM LITERATURY

Koskivaara M, Valtonen ET, Prost M (1991b) Dactylogyrids on the gills of roach in Central Finland: Features of infection and species composition. International Journal for Parasitology, 21, 565-572. Koubková B, Baruš V, Prokeš M, Dyková I (2004) Raphidascaris acus (Bloch, 1779) larvae infections of the stone loach, Barbatula barbatula (L.), from the River Hana, Czech Republic. Journal of Fish Diseases, 27, 65-71. Kouřil J, Hamáčková J, Hulová I, Barthová J (1999) Hormonální indukce ovulace u kapra pomocí čištěného extraktu kapří hypofýzy, Edice metodik VÚRH Vodňany, Vodňany. Kouřil J, Hamáčková J, Svobodová Z (1978) Fluctuation of relative hepatopancreas weight in the course of the year in brood tench (Tinca tinca L.) kept in ponds (in Czech). Bulletin VÚRH Vodňany, 4, 7-11. Kubokawa K, Watanabe T, Yoshioka M, Iwata M (1999) Effects of acute stress on plasma cortisol, sex steroid hormone and glucose levels in male and female sockeye salmon during the breeding season. Aquaculture, 172, 335-349. Kurtz J, Kalbe M, Langefors S, Mayer I, Milinski M, Hasselquist D (2007) An experimental test of the immunocompetence handicap hypothesis in a teleost fish: 11-ketotestosterone suppresses innate immunity in three-spined sticklebacks. American Naturalist, 170, 509-519. Kurtz J, Sauer KP (1999) The immunocompetence handicap hypothesis: testing the genetic predictions. Proceedings of the Royal Society of London Series B-Biological Sciences, 266, 2515-2522. Lamas J, Santos Y, Bruno DW, Toranzo AE, Oanadon R (1994) Nonspecific cellular responses of rainbow trout to Vibrio anguillarum and its extracellular products (ecps). Journal of Fish Biology, 45, 839-854. Lamková K (2006) Vztah imunokompetence jelce tlouště (Leuciscus cephalus) a parazitace mnohobuněčnými cizopasníky. Diplomová práce, Masarykova univerzita, Přírodovědecká fakulta, Brno. Langston AL, Johnstone R, Ellis AE (2001) The kinetics of the hypoferraemic response and changes in levels of alternative complement activity in diploid and triploid Atlantic salmon, following injection of lipopolysaccharide. Fish & Shellfish Immunology, 11, 333-345. Le Morvan C, Troutaud D, Deschaux P (1998) Differential effects of temperature on specific and nonspecific immune defences in fish. Journal of Experimental Biology, 201, 165- 168. Lebedeva DI (2006) Seasonal dynamics of the population structure of Sphaerostomum globiporum (Trematoda: Opecoelidae) maritae in Ladoga Lake. Parazitologiia, 40, 185- 191. Lecklin T, Nikinmaa M (1998) Erythropoiesis in Arctic charr is not stimulated by anaemia. Journal of Fish Biology, 53, 1169-1177. Lefebvre F, Mounaix B, Poizat G, Crivelli AJ (2004) Impacts of the swimbladder nematode Anguillicola crassus on Anguilla anguilla: variations in liver and spleen masses. Journal of Fish Biology, 64, 435-447. Leggatt RA, Iwama GK (2003) Occurrence of polyploidy in the fishes. Reviews in Fish Biology and Fisheries, 13, 237-246. Lehtinen J, Virta M, Lilius EM (2003) Fluoro-luminometric real-time measurement of bacterial viability and killing. Journal of Microbiological Methods, 55, 173-186.

SEZNAM LITERATURY

Linhart O, Kvasnička P, Flajšhans M, Kasal A, Ráb P, Paleček J, Šlechta V, Hamáčkova J, Prokeš M (1995) Genetic studies with tench, Tinca tinca L.: induced meiotic gynogenesis and sex reversal. Aquaculture, 132, 239-251. Linhart O, Rodina M, Kocour M, Gela D (2006) Insemination, fertilization and gamete management in tench, Tinca tinca (L.). Aquaculture International, 14, 61-73. Lipský L (1998) Krajinná ekologie pro studenty geografických oborů, Karolinum, Praha. Lucas A (1996) Physical concepts of bioenergetics. In: Bioenergetics of aquaticanimals (ed by Lucas A). Taylor & Francis, France. Lusková V (1997) Annual cycles and normal values of hematological parameters in fishes, Acta Scientiarum Naturalium, Brno. Lynshiang DS, Gupta BBP (2000) Role of thyroidal and testicular hormones in regulation of tissue respiration in male air-breathing fish, Clarias batrachus (Linn.). Indian Journal of Experimental Biology, 7, 705–712. Malek M (2001) Effects of the digenean parasites Labratrema minimus and Cryptocotyle concavum on the growth parameters of Pomatoschistus microps and P. minutus from Southwest Wales. Parasitology Research, 87, 349-355. Malmberg G (1970) Excretory systems and marginal hooks as a basis for systematics of Gyrodactylus (Trematoda, Monogenea). Arkiv Fur Zoologi, 23, 1-235. Manning MJ (1994) Immunology. A comparative approach. In: Fishes (ed by Turner RJ). Wiley, New York, pp. 69–100. Martínez-Porchas M, Martínez-Córdova LR, Ramos-Enriquez R (2009) Cortisol and Glucose: Reliable indicators of fish stress? Pan-American Journal of Aquatic Sciences, 4, 158- 178. Maule AG, Schrock R, Slater C, Fitzpatrick MS, Schreck CB (1996) Immune and endocrine responses of adult chinook salmon during freshwater immigration and sexual maturation. Fish & Shellfish Immunology, 6, 221-233. Maxime V (2008) The physiology of triploid fish: current knowledge and comparisons with diploid fish. Fish and Fisheries, 9, 67-78. McEwan AD, Fischer EW, Selman IE (1970) Observations on the immune globulin levels of neonatal calves and their relationship to disease. Journal of Comparative Pathology, 80, 259-265. McKnight IM (1966) A hematological study on the mountain whitefish, Prosopium williamsoni. Journal of the Fisheries Research Board of Canada, 23, 45–64. Mehta M, Woo PTK (2002) Acquired cell-mediated protection in rainbow trout, Oncorhynchus mykiss, against the haemoflagellate, Cryptobia salmositica. Parasitology Research, 88, 956-962. Milla S, Depiereux S, Kestemont P (2011) The effects of estrogenic and androgenic endocrine disruptors on the immune system of fish: a review. Ecotoxicology, 20, 305-319. Modrá H, Svobodová Z, Kolařová J (1998) Comparison of differential leukocyte counts in fish of economic and indicator importance. Acta Veterinaria Brno, 67, 215-226. Moens LN, van der Ven K, Van Remortel P, Del-Favero J, De Coen WM (2006) Expression profiling of endocrine-disrupting compounds using a customized Cyprinus carpio cDNA microarray. Toxicological Sciences, 93, 298-310. Moller AP (1997) Immune defence, extra-pair paternity, and sexual selection in birds. Proceedings of the Royal Society of London Series B-Biological Sciences, 264, 561-566.

SEZNAM LITERATURY

Molnár K, Majoros G, Csaba G, Székely C (2003) Pathology of Atractolytocestus huronensis Anthony, 1958 (Cestoda, Caryophyllaeidae) in Hungarian pond-farmed common carp. Acta Parasitologica, 48, 222-228. Morand S, Poulin R (2000) Nematode parasite species richness and the evolution of spleen size in birds. Canadian Journal of Zoology-Revue Canadienne De Zoologie, 78, 1356- 1360. Moravec F (1994) Parasitic nematodes of freshwater fishes of Europe, Academia, Praha. Munro AD, Scott AP, Lam TJ (1990) Reproductive Seasonality in Teleosts: Environmental Influences, CRC Press, Boca Raton, FL. Mylonas CC, Fostier A, Zanuy S (2010) Broodstock management and hormonal manipulations of fish reproduction. General and Comparative Endocrinology, 165, 516-534. Nagy A, Rajki K, Horvath L, Csanyj V (1978) Genetic analysis in carp (Cyprinus carpio) using gynogenesis. Herdity, 43, 35-40. Nakamura S, Imai N, Ohya S, Kobayashi H (1989) Oxygen uptake rate and hematological characteristics in triploid amago salmon, Oncorhynchus masou macrostomus. Memoirs of the Faculty of Agriculture of Kinki University, 22, 31-38. Niewiadomska K (2003) Pasożyty ryb Polski (klucze do oznaczania). Przywry-Digenea, Polskie Towarzystwo Parazytologiczne, Warszawa. (v polštině) Nikoskelainen S, Bylund G, Lilius EM (2004) Effect of environmental temperature on rainbow trout (Oncorhynchus mykiss) innate immunity. Developmental & Comparative Immunology, 28, 581-592. Nikoskelainen S, Lehtinen J, Lilius EM (2002) Bacteriolytic activity of rainbow trout (Oncorhynchus mykiss) complement. Developmental & Comparative Immunology, 26, 797-804. Ohtaka A, Saito T, Kakizaki T, Ogasawara S, Ohtomo C, Nagasawa K (2002) Seasonal and regional occurrence of Acanthocephalus sp. (Acanthocephala: Echinorhynchidae) in fishes and isopods (Asellus hilgendorfi) in a lake system in northern Japan. Limnology, 3, 143-150. Ohtsu J (1993) Size distribution of neutrophils and lymphocytes in triploid masu salmon, Oncorhynchus masou, and changes in their number in circulating blood after administration of liquid paraffin. Bulletin of the Toyama Prefectural Livestock Experiment Station, 4, 1-9. Okada H (1985) Studies on the artificial sex control in rainbow trout, Salmo gairdneri. Scientific Report of Hokkaido Fish Hatchery, 40, 1–49. Ondračková M, Reichard M, Jurajda P, Gelnar M (2004) Seasonal dynamics of Posthodiplostomum cuticola (Digenea, Diplostomatidae) metacercariae and parasite- enhanced growth of juvenile host fish. Parasitology Research, 93, 131-136. Oros M, Hanzelová V, Scholz T (2004) The cestode Atractolytocestus huronensis (Caryophyllidea) continues to spread in Europe: new data on the helminth parasite of the common carp. Diseases of Aquatic Organisms, 62, 115-119. Ottová E, Šimková A, Jurajda P, Dávidová M, Ondračková M, Pečínková M, Gelnar M (2005) Sexual ornamentation and parasite infection in males of common bream (Abramis brama): a reflection of immunocompetence status or simple cost of reproduction? Evolutionary Ecology Research, 7, 581-593. Ozerov MY, Lumme J, Pakk P, Rintamaki P, Zietara MS, Barskaya Y, Lebedeva D, Saadre E, Gross R, Primmer CR, Vasemagi A (2010) High Gyrodactylus salaris infection rate in triploid Atlantic salmon Salmo salar. Diseases of Aquatic Organisms, 91, 129-136.

SEZNAM LITERATURY

Pečínková M, Vollestad LA, Koubková B, Huml J, Jurajda P, Gelnar M (2007) The relationship between developmental instability of gudgeon Gobio gobio and abundance or morphology of its ectoparasite Paradiplozoon homoion (Monogenea). Journal of Fish Biology, 71, 1358-1370. Peter RE, Lin HR, Van der Kraak G, Little M (1993) Releasing hormones, dopamine antagonists and induced spawning. In: Recent Advances in Aquaculture (ed. by Muir JF, Roberts RJ). Blackwell Scientific, Oxford, pp. 25–30. Peter RE, Yu KL (1997) Neuroendocrine regulation of ovulation in fishes: Basic and applied aspects. Reviews in Fish Biology and Fisheries, 7, 173-197. Piersma T, Lindstrom A (1997) Rapid reversible changes in organ size as a component of adaptive behaviour. Trends in Ecology & Evolution, 12, 134-138. Pietrock M, Scholz T (2000) Morphometrics and seasonal occurrence of metacestodes of Neogryporhynchus cheilancristrotus (Cyclophyllidea : Dilepididae) in the blue bream (Abramis ballerus) from the Oder River (Germany/Poland). Folia Parasitologica, 47, 181-185. Pipota J, Linhart O (1986) Gynogensis in carp, Cyprinus carpio L. and tench, Tinca tinca L. induced by 60Co radiation in highly homogeneous radiating field. Radiation Physics and Chemistry, 28, 589-590. Poisot T, Šimková A, Hyršl P, Morand S (2009) Interactions between immunocompetence, somatic condition and parasitism in the chub Leuciscus cephalus in early spring. Journal of Fish Biology, 75, 1667-1682. Pojmańska T (1995) Seasonal dynamics of occurrence and reproduction of some parasites in four cyprinid fish cultured in ponds. Monogenea. Acta Parasitol. Polonica, 40, 79–84. Pottinger TG, Pickering AD (1987) Androgen levels and erythrocytosis in maturing brown trout, Salmo trutta L. Fish Physiology and Biochemistry, 3, 121-126. Poulin R, Morand S (2000) The diversity of parasites. Quarterly Review of Biology, 75, 277- 293. Pravda D, Svobodová Z (2003) Hematologie ryb. Veterinární hematologie (ed. Doubek J). Noviko, Brno, pp. 381-397. Radhakrishnan S, Beevi M, Deepthi GR, Radhakrishnan T (2010) Philometra cephalus (Nematoda) infection in the gonads of the long-arm mullet Valamugil cunnesius : host- parasite relation. Indian Journal of Fisheries, 57, 33-37. Rahkonen R, Pasternack M (1998) Effect of experimental Diphyllobotrium dentriticum infection on the blood leucocytes pattern of brown trout at two temperature levels. Boreal Environment Research, 3, 381-386. Redondo MJ, Palenzuela O, Alvarez-Pellitero P (2004) Studies on transmission and life cycle of Enteromyxum scophthalmi (Myxozoa), an enteric parasite of turbot Scophthalmus maximus. Folia Parasitologica, 51, 188-198. Richards DT, Hoole D, Lewis JW, Ewens E, Arme C (1994) Changes in the cellular composition of the spleen and pronephros of carp Cyprinus carpio infected with the blood fluke Sanguinicola inermis (Trematoda, Sanguinicolidae). Diseases of Aquatic Organisms, 19, 173-179. Roberts ML, Buchanan KL, Evans MR (2004) Testing the immunocompetence handicap hypothesis: a review of the evidence. Animal Behaviour, 68, 227-239. Roed KH, Fjalestad K, Larsen HJ, Midthjel L (1992) The genetic variation in haemolytic activity in Atlantic salmon (Salmo salar L.). Journal of Fish Biology, 40, 739-750. Roff DA (1992) The Evolution of Life Histories, Chapman & Hall, Inc., Routledge.

SEZNAM LITERATURY

Rohde K, Hayward C, Heap M (1995) Aspects of the ecology of metazoan ectoparasites of marine fishes. International Journal for Parasitology, 25, 945-970. Ros AFH, Ferreira C, Serrao Santos R, Oliveira RF (2006) Regulation of immunocompetence by different androgen metabolites in a blenny with alternative reproductive tactics. Journal of Experimental Zoology Part a-Comparative Experimental Biology, 305A, 986- 994. Rotllant J, Tort L (1997) Cortisol and glucose responses after acute stress by net handling in the sparid red porgy previously subjected to crowding stress. Journal of Fish Biology, 51, 21-28. Rubio-Godoy M, Porter R, Tinsley RC (2004) Evidence of complement-mediated killing of Discocotyle sagittata (Platyhelminthes, Monogenea) oncomiracidia. Fish & Shellfish Immunology, 17, 95-103. Saha NR, Usami T, Suzuki Y (2002) Seasonal changes in the immune activities of common carp (Cyprinus carpio). Fish Physiology and Biochemistry, 26, 379-387. Saha NR, Usami T, Suzuki Y (2004) In vitro effects of steroid hormones on IgM-secreting cells and IgM secretion in common carp (Cyprinus carpio). Fish & Shellfish Immunology, 17, 149-158. Sánchez C, Babin M, Tomillo J, Ubeira FM, Domínguez J (1993) Quantification of low levels of rainbow trout immunoglobulin by enzyme immunoassay using two monoclonal antibodies. Veterinary Immunology and Immunopathology, 36, 65-74. Saurabh S, Sahoo PK (2008) Lysozyme: an important defence molecule of fish innate immune system. Aquaculture Research, 39, 223-239. Scott AL, Rogers WA, Klesius PH (1985) Chemiluminescence by peripheral blood phagocytes from channel catfish: function of opsonin and temperature. Developmental and Comparative Immunology, 9, 241-250. Secombes CJ (1996) The nonspecific immune system: Cellular defence. In: The Fish Immune System – Organism, Pathogen,and Environment (ed. by Iwama G, Nakanishi T). Academic Press, San Diego, pp. 63-103. Sharp GJE, Pike AW, Secombes CJ (1992) Sequential development of the immune response in rainbow trout Oncorhynchus mykiss (Walbaum, 1792) to experimental plerocercoid infections of Diphyllobothrium dendriticum (Nitzsch, 1824). Parasitology, 104, 169- 178. Sheldon BC, Verhulst S (1996) Ecological immunology: Costly parasite defences and trade-offs in evolutionary ecology. Trends in Ecology & Evolution, 11, 317-321. Scharsack JP, Kalbe M, Derner R, Kurtz J, Milinski M (2004) Modulation of granulocyte responses in three-spined sticklebacks Gasterosteus aculeatus infected with the tapeworm Schistocephalus solidus. Diseases of Aquatic Organisms, 59, 141-150. Scholz T (1989) Amphilinida and Cestoda, parasites of fish in Czechoslovakia, Acta Scientiarum Naturalium, Brno. Sitja-Bobadilla A (2008) Living off a fish: A trade-off between parasites and the immune system. Fish & Shellfish Immunology, 25, 358-372. Sitja-Bobadilla A, Alvarez-Pellitero P (2009) Experimental transmission of Sparicotyle chrysophrii (Monogenea: Polyopisthocotylea) to gilthead seabream (Sparus aurata) and histopathology of the infection. Folia Parasitologica, 56, 143-151. Sitja-Bobadilla A, Redondo MJ, Bermudez R, Palenzuela O, Ferreiro I, Riaza A, Quiroga I, Nieto JM, Alvarez-Pellitero P (2006) Innate and adaptive immune responses of turbot,

SEZNAM LITERATURY

Scophthalmus maximus (L.), following experimental infection with Enteromyxum scophthalmi (Myxosporea : Myxozoa). Fish & Shellfish Immunology, 21, 485-500. Skarstein F, Folstad I (1996) Sexual dichromatism and the immunocompetence handicap: An observational approach using Arctic charr. Oikos, 76, 359-367. Skarstein F, Folstad I, Liljedal S (2001) Whether to reproduce or not: immune suppression and costs of parasites during reproduction in the Arctic charr. Canadian Journal of Zoology- Revue Canadienne De Zoologie, 79, 271-278. Slater CH, Schreck CB (1997) Physiological levels of testosterone kill salmonid leukocytes in vitro. General and Comparative Endocrinology, 106, 113-119. Smith RL, Paul AJ, Paul JM (1990) Seasonal changes in energy and energy cost of spawning in Gulf of Alaska Pacific cod. Journal of Fish Biology, 36, 307-316. Stillwell EJ, Benfey TJ (1996) Hemoglobin level, metabolic rate, opercular abduction rate and swimming efficiency in female triploid brook trout (Salvelinus fontinalis). Fish Physiology and Biochemistry, 15, 377-383. Sumantadinata K, Taniguchi N, Sugiarto (1990) Increased variance of quantitative characters in the 2 types of gynogenetic diploids of indonesian common carp. Nippon Suisan Gakkaishi, 56, 1979-1986. Suzuki R, Nakanishi T, Oshiro T (1985) Survival, growth and sterility of induced triploids in the cyprinid loach Misgurnus anguillicaudatus. Bulletin of the Japanese Society of Scientific Fisheries, 51, 889-894. Suzuki Y, Orito M, Iigo M, Kezuka H, Kobayashi M, Aida K (1996) Seasonal changes in blood IgM levels in goldfish, with special reference to water temperature and gonadal maturation. Fisheries Science, 62, 754-759. Suzuki Y, Otaka T, Sato S, Hou YY, Aida K (1997) Reproduction related immunoglobulin changes in rainbow trout. Fish Physiology and Biochemistry, 17, 415-421. Svobodová Z, Flajšhans M, Kolářová J, Modrá H, Svoboda M, Vajcová V (2001) Leukocyte profiles of diploid and triploid tench, Tinca tinca L. Aquaculture, 198, 159-168. Svobodová Z, Kolářová J (2004) A review of the diseases and contaminant related mortalities of tench (Tinca tinca L.). Veterinary Medicine, 49, 19-34. Svobodová Z, Kolářová J, Flajšhans M (1998) The first findings of the differences in complete blood count between diploid and triploid tench, Tinca tinca L. Acta Veterinaria Brno, 67, 243-248. Svobodová Z, Pravda D, Paláčková J (1986) Jednotné hematologické metody hematologického vyšetřování ryb, Edice Metodik, Vodňany. Šimková A, Jarkovský J, Koubková B, Baruš V, Prokeš M (2005) Associations between fish reproductive cycle and the dynamics of metazoan parasite infection. Parasitology Research, 95, 65-72. Šimková A, Lafond T, Ondračková M, Jurajda P, Ottová E, Morand S (2008) Parasitism, life history traits and immune defence in cyprinid fish from Central Europe. Bmc Evolutionary Biology, 8:29, doi:10.1186/1471-2148-8-29. Tanriverdi F, Silveira LFG, MacColl GS, Bouloux PMG (2003) The hypothalamic-pituitary- gonadal axis: immune function and autoimmunity. Journal of Endocrinology, 176, 293- 304. Taskinen J, Kortet R (2002) Dead and alive parasites: sexual ornaments signal resistance in the male fish, Rutilus rutilus. Evolutionary Ecology Research, 4, 919-929.

SEZNAM LITERATURY

Taylor M, Hoole D (1989) Ligula intestinalis (L.) (Cestoda, Pseudophyllidea): plerocercoid induced changes in the spleen and pronephros of roach, Rutilus rutilus (L.), and gudgeon, Gobio gobio (L.). Journal of Fish Biology, 34, 583-596. Thilagam H, Gopalakrishnan S, Bo J, Wang KJ (2009) Effect of 17beta-estradiol on the immunocompetence of Japanese sea bass (Lateolabrax japonicus). Environmental Toxicology and Chemistry, 28, 1722-1731. Thomas MB, Thomas W, Hornstein TM, Hedman SC (1999) Seasonal leukocyte and erythrocyte counts in fathead minnows. Journal of Fish Biology, 54, 1116-1118. Thompson RM, Mouritsen KN, Poulin R (2005) Importance of parasites and their life cycle characteristics in determining the structure of a large marine food web. Journal of Animal Ecology, 74, 77-85. Thorgaard GH (1983) Chromosome set manipulation and sex control in fish. In: Fish Physiology (ed. by Hoar WS, Randall DJ, Donaldson EM). Academic Press, New York, pp. 405-434. Tiwary BK, Kirubagaran R, Ray AK (2004) The biology of triploid fish. Reviews in Fish Biology and Fisheries, 14, 391-402. Vainikka A, Jokinen EI, Kortet R, Paukku S, Pirhonen J, Rantala MJ, Taskinen J (2005) Effects of testosterone and beta-glucan on immune functions in tench. Journal of Fish Biology, 66, 348-361. Vainikka A, Taskinen J, Loytynoja K, Jokinen E, Kortet R (2009) Measured immunocompetence relates to the proportion of dead parasites in a wild roach population. Functional Ecology, 187-195. Van den Heuvel MR, Landman MJ, Finley MA, West DW (2008) Altered physiology of rainbow trout in response to modified energy intake combined with pulp and paper effluent exposure. Ecotoxicology and Environmental Safety, 69, 187-198. Van Kampen EJ, Zijlstra WG (1961) Standardization of hemoglobimetry: the hemiglobincyanide method. Clinica Chimica Acta, 6, 538-544. Vanvuren JHJ, Hattingh J (1978) Seasonal study of hematology of wild freshwater fish. Journal of Fish Biology, 13, 305-313. Vijayan MM, Pereira C, Grau EG, Iwama GK (1997) Metabolic responses associated with confinement stress in tilapia: The role of cortisol. Comparative Biochemistry and Physiology C-Pharmacology Toxicology & Endocrinology, 116, 89-95. Virta M, Karp M, Rönnemaa S, Lilius EM (1997) Kinetic measurement of the membranolytic activity of serum complement using bioluminescent bacteria. Journal of Immunological Methods, 201, 215-221. Vladimirov VL (1960) Morphology and biology of Posthodiplostomum cuticola cercaria (Nodrmann, 1832)—the agent of black spot disease. Doklady biological sciences USSR, 135, 1009–1011. (v ruštině) Volf P, Horák P, kol. a (2007) Paraziti a jejich biologie. Triton, Praha, pp. 318. Wakelin D (1996) Immunity to parasites: how parasitic infections are controlled. University Press, Cambridge. Watanuki H, Yamaguchi T, Sakai M (2002) Suppression in function of phagocytic cells in common carp Cyprinus carpio L. injected with estradiol, progesterone or 11- ketotestosterone. Comparative Biochemistry and Physiology C-Toxicology & Pharmacology, 132, 407-413. Wedekind C (1992) Detailed information about parasites revealed by sexual ornamentation. Proceedings of the Royal Society of London B, 247, 169-174.

SEZNAM LITERATURY

Wedekind C, Folstad I (1994) Adaptive or nonadaptive immunosupression by sex hormones? American Naturalist, 143, 936-938. Williams TD, Diab AM, George SG, Sabine V, Chipman JK (2007) Gene expression responses of European flounder (Platichthys flesus) to 17-beta estradiol. Toxicology Letters, 168, 236-248. Yamaguchi T, Watanuki H, Sakai M (2001) Effects of estradiol, progesterone and testosterone on the function of carp, Cyprinus carpio, phagocytes in vitro. Comparative Biochemistry and Physiology C-Toxicology & Pharmacology, 129, 49-55. Yamamoto A, Iida T (1994) Hematological characteristics of triploid Rainbow trout. Fish Pathology, 29, 239-243. Yamamoto A, Iida T (1995a) Nonspecific defense activities of triploid Rainbow trout. Fish Pathology, 30, 107-110. Yamamoto A, Iida T (1995b) Susceptibility of triploid rainbow trout to IHN, furunculosis and vibriosis. Fish Pathology, 30, 69-70. Yamamoto E (1999) Studies on sex-manipulation and production of cloned populations in hirame, Paralichthys olivaceus (Temminck et Schlegel). Aquaculture, 173, 235-246. Yaron Z (1995) Endocrine control of gametogenesis and spawning induction in the carp. Aquaculture, 129, 49-73. Zahavi A (1975) Mate selection - a selection for a handicap. Journal of Theoretical Biology, 53, 205-214. Zahavi A (1977) The cost of honesty (further remarks on the handicap principle). Journal of Theoretical Biology, 67, 603-605. Zapata AG, Varas A, Torroba M (1992) Seasonal variations in the immune system of lower vertebrates. Immunology Today, 13, 142-147. Zohar Y, Goren A, Tosky M, Pagelson G, Leibovitz D, Koch Y (1989) The bioactivity of gonadotropin-releasing hormones and its regulation in the gilthead seabream, Sparus aurata: in vivo and in vitro studies. Fish Physiology and Biochemistry, 7, 59–67. Zohar Y, Mylonas CC (2001) Endocrine manipulations of spawning in cultured fish: from hormones to genes. Aquaculture, 197, 99-136.

internetové zdroje:

URL1: http://cs.wikipedia.org/wiki/Hematokrit

URL2: http://www.imunol-usti.cz/?stranka=bunecnai-imunita

PUBLIKACE TVOŘÍCÍ DIZERTAČNÍ PRÁCI

8 PUBLIKACE TVOŘÍCÍ DIZERTAČNÍ PRÁCI

Článek A:

LAMKOVÁ K, ŠIMKOVÁ A, PALÍKOVÁ M, JURAJDA P, LOJEK A (2007): Seasonal changes of immunocompetence and parasitism in chub (Leuciscus cephalus), a freshwater cyprinid fish. Parasitology Research 101: 775-789.

Článek B:

ROHLENOVÁ K, ŠIMKOVÁ A (2010): Are the immunocompetence and the presence of metazoan parasites in cyprinid fish affected by reproductive efforts of cyprinid fish? Journal of Biomedicine and Biotechnology. ID 418382.

Článek C:

ROHLENOVÁ K, MORAND S, HYRŠL P, TOLÁROVÁ S, FLAJŠHANS M, ŠIMKOVÁ A (2011): Are fish immune systems really affected by parasites? An immunoecological study of common carp (Cyprinus carpio). Parasites and Vectors 4:120.

Článek D:

ROHLENOVÁ K, DÁVIDOVÁ M., HYRŠL P., TOLÁROVÁ S., FLAJŠHANS M., ŠIMKOVÁ A: The physiological and immunological status of tench, Tinca tinca L.: the effects of hormonal stimulation and ploidy character determined by chromosomal manipulation (připravováno pro Aquaculture)

Článek A

Seasonal changes of immunocompetence and parasitism in chub (Leuciscus cephalus), a freshwater cyprinid fish

Parasitology Research 101: 775-789 (2007)

LAMKOVÁ K, ŠIMKOVÁ A, PALÍKOVÁ M, JURAJDA P, LOJEK A

Parasitol Res DOI 10.1007/s00436-007-0546-3

ORIGINAL PAPER

Seasonal changes of immunocompetence and parasitism in chub (Leuciscus cephalus), a freshwater cyprinid fish

Karolína Lamková & Andrea Šimková & Miroslava Palíková & Pavel Jurajda & Antonín Lojek

Received: 2 February 2007 /Accepted: 16 April 2007 # Springer-Verlag 2007

Abstract Seasonal variation of water characteristics, pre- indicating higher investment in spleen size in April (after dominantly temperature, is considered to strongly affect winterizing) and August (post-breeding with low gonado- fish physiology and immunology. In nature, this variation somatic index [GSI]). The significant seasonal differences directly influences the life cycle especially in fish parasites, in erythrocyte and leukocyte cell counts, as well as in but the infection of parasites is also altered by the host phagocyte count affecting respiratory burst, were recorded. immune response. This study is aimed to analyze the The general trend of leukocyte composition was similar in seasonal changes in selected physiological and immuno- all seasons investigated; however, the changes in proportion logical parameters, the latter a potential measure of fish of different neutrophilic cells were reported. The parasite immunocompetence. Moreover, the seasonal changes in diversity and the infection parameters in parasite commu- metazoan parasite infection were investigated, and the nities were highest in spring and early summer. When potential associations between fish physiology, immuno- comparing parasite abundance in infracommunities among competence, and parasitism were estimated. No differences seasons, the statistically highest values were observed in in gender were recognized for immunological parameters. April and June for Monogenea, in April and November for The significant differences in the spleen–somatic index Acanthocephala, and in April for Cestoda. The positive were found among fish samples of different seasons correlations between GSI and the parasite abundance of groups with higher infection parameters were found in males. Moreover, the positive association between Mono- K. Lamková : A. Šimková (*) Department of Botany and Zoology, Faculty of Science, genea as the dominant parasite group and respiratory burst Masaryk University, was observed. The higher investment in condition and the Kotlářská 2, seasonal variation in GSI were associated with a decrease 611 37 Brno, Czech Republic immune function measured by spleen size or leukocyte cell e-mail: [email protected] count especially for males suggesting the seasonal energy M. Palíková allocation between immune function and somatic or University of Veterinary and Pharmaceutical Sciences, reproductive investment. Palackého 1/3, 612 42 Brno, Czech Republic

P. Jurajda Introduction Institute of Vertebrate Biology, Academy of Science of the Czech Republic, The close relationship that exists between teleost fish and their Květná 8, 603 65 Brno, Czech Republic environment is the basis of a wide variety of studies. Fish have a body temperature that is essentially influenced by the temper- A. Lojek ature of the surrounding water, and therefore their physiological Institute of Biophysics, functions and also immune system are affected by the changes Academy of Science of the Czech Republic, Královopolská 135, of environmental temperature (Cuchens and Clem 1977;Clem 612 65 Brno, Czech Republic et al. 1984;Stolenetal.1984;Rowleyetal.1988). Parasitol Res

However, the studies investigating seasonal changes in that acquired immunity, rather than innate immunity immune defenses in fish are scarce (for instance Hutchinson (phagocytosis), is more sensitive to low temperatures and and Manning 1996; Le Morvan et al. 1998; Scapigliati et al. induces immunosuppression. Phagocytosis may also play an 1999; Bowden et al. 2004; Kumari et al. 2006), and the important role in maintaining the good condition of a fish at majority of studies investigating the differential effects of low environmental temperatures (Ainsworth et al. 1991). temperature on innate or acquired immune defenses are However, the analyses of phagocytic activity in in vitro provided in experimental conditions. Nevertheless, the assays demonstrated higher activity at room temperature in information concerning the seasonal influence on the im- spring samples when comparing with winter samples, but a mune function could be helpful for understanding the greater activity was observed in the winter sample of tench energetic trade-off between different functions in fish, as (Tinca tinca), a cyprinid fish at a seasonal temperature the investment in many functions can vary in time because (Collazos et al. 1995a). Chemiluminescence (CL) of head of seasonal effects. kidney phagocytes as a potential measure of immunocom- Fish blood is a suitable hematological and biochemical petence in fish has been also applied in recent published parameter strongly influenced by endogenic and exogenic studies (Kortet et al. 2003a;Vainikkaetal.2004). factors: environmental influences (including seasonal vari- Recently, the measuring of immunological parameters ation of water characters such as temperature or oxygen plays a central role for the investigation of the potential level), stress effect of anthropogenic origin, biological associations of immunocompetence (defined as the ability aspects (sex, age, etc.), and condition and health (e.g., to produce a normal immune response) and parasitism pathogenic and parasitic diseases) of those aquatic verte- (Manning 1994; Owens and Wilson 1999). The mecha- brates (Heath 1990). Seasonality is chiefly due to variation nisms that enable host species to resist bacterial, viral, or in water temperature, which has an immediate effect on fish parasitic infection are most probably a complex and metabolism (Martínez et al. 1994). The differences because multifactorial combination of specific and nonspecific and of seasonal changes are observed in hematocrit and humoral and cellular parameters (Secombes and Olivier erythrocyte counts (McKnight 1966; Collazos et al. 1998). 1996). When investigating the associations between host Moreover, the differential leukocyte count, as an important immunity and parasitism in fish living in natural conditions, characteristic of the fish health state and useful for the first point of interest should be to obtain the knowledge measuring the immune function, is considered being whether the considered parameters connected with fish dependent on environmental effect as other hematological physiology and immunology are affected by seasonal characteristics (Wester et al. 1994). changes and how is the presence and abundance of par- Spleen plays a highly important role in hemopoiesis and asites subjected to those changes. immune reactivity of teleost fish producing antibodies and Concerning metazoan parasites of fish, there are two participating in clearance of pathogens and foreign particles primary factors affecting parasite abundance: the host itself from the blood stream (Dalmo et al. 1997). This secondary and environmental factors of the host habitat (Rohde 1984; lymphatic organ is widely used as a simply measurable Esch et al. 1990; Rohde et al. 1995). Water temperature is immunological variable with a potential role in immune major abiotic factor influencing the duration of the life response against parasites in fish (Manning 1994;Kortetetal. cycle of ectoparasitic Monogenea (Chubb 1977; Hanek and 2003a;Ottováetal.2005, 2007). Measuring of the relative Fernando 1978; Koskivaara et al. 1991). However, the spleen size is the simplest method relying on estimating the diverse genera of this parasite group respond differently to absolute or relative abundance of immunologically active water temperature changes. Generally, there is a high cells (Owens and Wilson 1999). Several studies have abundance of dactylogyrids reported during summer and illustrated that size of lymphoid organs depends on fish an increase in abundance of gyrodactylids in autumn condition (Piersma and Lindström 1997). (Koskivaara 1992). The occurrence of endoparasitic species Killing ability of phagocytes (including monocyte– that develop by a complex life cycle is, moreover, macrophage cell lineage and the neutrophils) is one of the connected with the presence of an intermediate host whose basic immune mechanisms that fish use to protect them- life cycle can also be affected by seasonal changes (for selves against a pathogen attack (Finco-Kent and Thune instance, the arthropods for Acanthocephala as shown by 1987). The first encounter of the host with a foreign Crompton 1985). organism guides to mobilization of phagocytic cells Therefore, the present study is aimed to analyze the (Bellanti and Kadlec 1985). Similarly to other physiolog- seasonal changes in selected physiological and immunological and immunological parameters, an effect of the ical parameters as the potential measures of fish health status environmental temperature on fish phagocytosis is also or immunocompetence. Moreover, the seasonal changes in known (Cuchens and Clem 1977; Clem et al. 1984; Stolen the infection of all metazoan parasites and the changes in et al. 1984). A study by Ainsworth et al. (1991) denoted parasite diversity as well as potential associations between Parasitol Res fish physiology, immunocompetence, and parasitism were ), and

analyzed. K

Materials and methods GSI

Fish sampling

A total of 83 chub (Leuciscus cephalus, Cyprinidae), 62 males and 21 females, were collected during four seasons in 2004 (i.e., April as spring, June as early summer, August as K late summer, and November as autumn) from one locality situated on the confluence of the Svitava and Svratka rivers, belonging to the Morava river basin on the periphery of Brno (Czech Republic). The fish were caught by electrofishing and immediately after catching, a blood sample (340 μl) was collected from each fish by the heart puncture method (Modrá et al. 1998) using heparinized syringes in heparinized microtubes and mixed with the heparin diluted 10× with 0.7% NaCl to retain the number of heparin units in the measured blood according to the methodology written by Kubala et al. (1996). The fishes were placed in a tank containing water from the same location, oxygenated and transported to the laboratory. During storage in the laboratory tanks, the original water was maintained, and a standard aquarium filter was used for water filtration. All fish individuals were killed within 24 h. Each individual males), total length, body weight, weight of gonads, weight of spleen, condition factor ( M was measured (total and standard lengths in millimeters), weighed (in grams), and a complete dissection of the fish according to Ergens and Lom (1970) was performed. females, F The physiological parameters including gonad weight (in grams), hematocrit and erythrocyte count, and the selected immunological parameters including spleen weight (in grams), leukocyte count, differential leukocyte cell counts, phagocyte count, and respiratory burst (RB) activity were measured.

We also calculated the relative body weight (condition Total lengthMean ±SD Body weight Mean Gonad weight ±SD Mean Spleen weight ±SD Mean ±SD Mean ±SD Mean ±SD factor, K) using the equation: K = constant × somatic weight 3 (g)/(standard length [cm]) and relative size of gonads n (gonado-somatic index, GSI): GSI = gonad weight (g)/body weight (g) × 100. The spleen–somatic index (SSI) was FF 3F 243.33 11 ±35.47 289.55F 3 135.03 ±19.44 303.33 4 ±69.69 254.44 ±4.16 298.00 18.70 ±65.49 299.25 ±27.95 ±3.54 12.65 ±39.04 347.68 ±10.61 0.24 6.37 ±181.9 0.31 ±0.01 16.55 ±0.61 ±0.09 2.76 ±9.82 0.36 1.67 ±2.43 0.41 ±0.12 ±0.23 13.54 ±0.17 1.79 4.80 ±5.23 1.99 ±0.15 ±3.17 ±0.98 2.14 4.38 ±0.16 ±1.15 calculated as spleen weight (g)/body weight (g) × 100. represents the number of fish collected (

According to Bolger and Connolly (1989), the assumption n of the condition factor is that the heavier the fish is in relation to its length, the better is its general condition. The means and standard deviations (± SD) of the selected parameters are shown separately for males and females in Table 1.

Parasite collection and determination

Fish were investigated for all metazoan parasites. A light Data on the fish investigated: microscope (Olympus BX 50) with phase-contrast, differ- gonado-somatic index (GSI): mean ± SD are shown Sampling date Water temperature °C Sex AprilJuneAugust 10.5November 15.4 16.6 10.9 M 16 M M 239.13 9 M ±24.01 19 248.89 18 120.78 238.63 ±42.52 277.72 ±48.92 ±34.79 178.28 ±31.01 13.66 140.21 ±85.82 235.94 ±8.09 ±53.22 7.65 ±92.13 2.72 0.17 8.13 ±5.39 ±1.38 ±0.05 ±3.75 0.24 1.47 0.22 0.29 ±0.08 ±0.47 ±0.09 1.84 ±0.14 10.04 1.63 1.77 ±0.16 ±4.62 ±0.30 3.96 ±0.21 1.89 3.41 ±1.76 ±0.74 ±0.80 ential interference contrast (DIC according to Nomarski), Table 1 Parasitol Res and digital image analysis (Micro Image 4.0 for Windows) species according to Bush et al. (1997). Parasite infra- was used for parasite determination and measurements. communities and component communities were defined Ectoparasites (Monogenea, Hirudinea, and Mollusca) and following Margolis et al. (1982) and Bush et al. (1997). endoparasites (Digenea, Cestoda, Acanthocephala, and For classification of the diversity in parasite communities, Nematoda) were determined using recent keys and meth- we used Shannon’s diversity index, Shannon’s evenness odology (Ergens and Lom 1970; Gusev 1985; Khotenovsky index, Simpson’s index (for component communities), and 1985; Scholz 1989; Moravec 1994). All recorded speci- Brillouin’s index diversity for infracommunities (the mens of metazoan parasites, Monogenea in glycerin– calculations were performed following Magurran 1983). amonium picrate, Nematoda in glycerin–ethanol, and other The data of parasite abundance and several immunological metazoan parasites (Digenea, Cestoda, Acanthocephala and and physiological variables (e.g., erythrocyte count, lym- Mollusca) in 4% formalin, were fixed. Digenea and phocyte count) did not fit the normal distribution; Cestoda were subsequently stained using IAC carmine therefore, we used a nonparametric analysis of variance (Georgiev et al. 1986). (Kruskal–Wallis test, KW) for multiple comparisons and the Mann–Whitney (MW) test for pair group comparison Blood analyses for all investigated variables. Spearman’s rank correlation coefficient was used to investigate the associations be- Differential cell counts were estimated in whole-blood tween parasite abundance and physiological or immuno- smears according to standard Pappenheim using May– logical variables. As the spleen weight and gonad weight Grünwald and Giemsa–Romanovsky stains (Svobodová et were correlated with total body weight, we calculated the al. 1991). This method allows differentiating a variety of GSI or SSI before performing correlation analyses. All white blood cells and also the developmental stages of statistical analyses were executed using Statistica 7.1. these cells. Erythrocyte and leukocyte counts were executed in Bürker’s hemocytometer after staining with Natt–Herick solution (Svobodová et al. 1986; Lusková 1997). The Results heparinized microcapillaries (75 mm) were used to measure hematocrit. The blood samples were centrifuged in micro- Gender differences capillaries using a hematocrit centrifuge at 12,000g for 3 min (Svobodová et al. 1986). The gender differences in immunological and physiological variables were tested using the MW test for the total sample size Respiratory burst activity (i.e., including four seasons) and for the early summer sample (June) with enough individuals of females and males. A Two types of CL signals were measured for each sample: significant difference in total weight (p=0.0001), total body (1) activated CL with opsonised zymosan and (2) sponta- length (p=0.003), erythrocyte count (p=0.0267), and partial neous CL (without an activator). hematocrit (p=0.0450) were found between males and The CL emission was measured in 5-min intervals during females in the total sample size. The gender difference had 100 min (i.e., a total of 20 measurements were recorded) to no significant effect on K factor,SSI,GSI,gonadweight,RB, obtain the kinetic curves for each sample. The peak value of leukocyte count, and parasite abundance (p<0.05). Therefore, the kinetic curve corresponding to the maximal value of CL we executed the analyses using the compiled data on females signal was used as the CL value for each sample. The peak of and males. However, we also performed the supplementary the CL signal, i.e., the maximal value of RB, was used for analyses of associations for males from all seasonal samples statistical comparison. For a graphical interpretation of the and analysis only for females samples collected in the early CL signal, the conversion of maximal values of RB to 1,000 summer (i.e., June), when the equivalent proportion of males phagocytes was applied. CL measurements were carried out and females were sampled. with a Luminometer Junior (Checklight; 380–630 nm) according to methodology of Kubala et al. (1996). Analysis of physiological and immunological parameters

Data analysis During the present study, differences among investigated periods for SSI, GSI, erythrocyte, and leukocyte count were Epidemiological characteristics as a prevalence (percentage found. Using the KW test, we observed a statistical of infected host individuals in each sample), intensity of significant effect of the season on GSI (N=76; p<0.0001), infection (number of parasites per infected host), and SSI (N=81, p=0.0165), erythrocyte count (N=82, p=0.002), abundance (mean number of parasites per host individual and leukocyte count (N=82, p=0.0001). Using the MW test in each seasonal sample) were calculated for each parasite for comparison between seasons, we recorded the statistically Parasitol Res higher values of SSI in August, than in June and November The seasonal differences in phagocyte count and RB (p<0.05), and also in April when comparing with were analyzed. The KW test revealed the significant November (Fig. 1). The maximal value of GSI was found difference among seasons for RB adjusted for 1,000 in April (p<0.001). The highest value of erythrocyte count phagocytes (p<0.05). The tendency for seasonal differences wasrecordedinAprilandNovember(p<0.05) and leukocyte was found in the phagocyte cell count (although p=0.076). count in June and August (p<0.05). Dynamics of erythrocyte The MW test indicated a significantly higher phagocyte and leukocyte counts in four periods are shown in Fig. 2. count in April (p<0.05 when comparing April with June The dynamics of the CL signal (representing the RB) was and November) and the lowest values of RB (i.e., the also analyzed in each sample. The correlation between kinetics lowest values for the recorded peak of CL) in November of activated and spontaneous CL signals using Spearman’s (p<0.05 for the values of maximum CL adjusted for 1,000 correlation was recorded for the majority of individuals in three phagocytes and p<0.0001 for the nonadjusted values). periods of investigation (Table 2). In August, the significant The seasonal changes in CL adjusted and not adjusted for correlation between activated and spontaneous oxidative burst the phagocyte count are showed in Fig. 4. was recorded only for half of the samples. The percentage distribution of individual types of Seasonal changes in parasite infection leukocytes in four seasons is shown in Fig. 3. By studying differential leukocyte counts, only small variances in the Metazoan parasite species belonging to seven groups were composition of white blood cell components were revealed. found: Monogenea, Digenea, Cestoda, Nematoda, Acan- The lymphocytes were the most dominant group of white thocephala, Hirudinea, and Mollusca. Monogenea was the cells; their number reached approximately constant values species richest group (including Dactylogyrus vistulae, D. (from 94.7 to 96.9%) in all seasons investigated. The fallax, D. folkmanovae, Paradiplozoon megan, Gyrodacty- highest percentage of monocytes was recorded in August lus vimbi, G. gracilihamatus, G. lomi and G. prostae). The and November (about 1.9%). The total count of neutrophils presence of all species observed during the whole study is was the highest in April (3.8%). However, the more obvious shown in Table 3. From a total of 24 parasite species found, differences were observed in proportion to the developmental the presence of nine of them was not affected by seasonal stages of neutrophilic granulocytes (including myelocytes, changes. The presence of ten species was connected with metamyelocytes, bands, and segments), indicating the highest spring and/or early summer (including two species of proportion of neutrophilic bands in April and November, the Monogenea, two species of Digenea, three species of increasing proportion of myelocytes in June, and the Cestoda, one species of Nematoda, and larval Mollusca). increasing number of metamyelocytes in August. On the other hand, the presence of Philometra abdominalis

Fig. 1 Seasonal changes in SSI 0.32 (spleen–somatic index) 0.30 0.28 0.26 0.24 0.22 0.20 0.18 0.16

SSI (%) 0.14 0.12 0.10 0.08 0.06 0.04 0.02 Mean 0.00 Mean±SE April June August November Mean±2*SD Sampling dates Parasitol Res

Fig. 2 Seasonal changes in 2.6 erythrocyte and leukocyte counts 2.4

2.2

2.0

1.8 ) -1 1.6

1.4

1.2 Erythrocytes (T.l 1.0

0.8

0.6

0.4 Mean 0.2 Mean±SE April June August November Min-Max Sampling dates

260

240

220

200

180

) 160 -1

140

120

Leukocytes (G.l 100

20 Mean 0 Mean±SE April June August November Min-Max Sampling dates

(Nematoda) with the autumn sample and the presence of were calculated (Table 4). The KW and MW tests revealed Gyrodactylus prostae with the early spring and autumn the seasonal differences for the following parasite groups: samples were associated. Monogenea reached the highest abundance in April and The epidemiological characteristics were influenced by June (KW test, p<0.0001; MW test between each two the seasonal changes. When comparing the four periods seasons p<0.01), Acanthocephala in April and November investigated, we detected some variations in those parame- (KW test, p=0.0035; MW test, p<0.05), and we further ters calculated for parasite groups or parasite species. observed a tendency of increased abundance of Cestoda in The abundance (mean ± SD), intensity of infection April (KW test, p=0.0254 but p>0.05 for between season (minimum–maximum), and prevalence of parasite groups comparison by MW test). When comparing parasite species Parasitol Res

Table 2 Spearman’s correlation between activated and spontaneous Acanthocephalus anguillae was recorded in April (KW test, respiratory burst: the numbers of significant correlations at p<0.05 are p=0.0049); the abundance values were significantly shown, n.s. indicates an insignificant correlation higher in April comparing to August (MW test, p<0.05). Activated versus spontaneous Abundance of Pomporhynchus laevis was significantly higher in April and November when comparing with Sampling date p<0.05 n.s. August (KW test, p=0.029, MW test, p<0.05). April 19 1 The species diversity estimated by all index’s used had a June 20 3 tendency to decrease from April to November (see Table 5). August 10 11 In samples collected at similar temperatures in summer, i.e., November 20 2 June and August, the identical values of species evenness and dominance were found; even the Shannon’s index diversity was higher in June than August. The KW test abundance among seasons, we found the highest abundance showed the among-samples differences for the diversity of in April and the lowest abundance in November for infracommunities calculated using Brillouin’sdiversity Dactylogyrus spp. (KW test, p<0.0001; MW test p<0.01). index (N=83, p<0.0001). The diversity of infracommuni- The abundance of Gyrodactylus spp. was also higher in ties, similarly to the diversity in component communities, April when compared to other samples (KW test, p<0.0001; reached the highest value in April and decreased in MW test, p<0.01). The trend of higher abundance of November.

April 2004 August 2004

0.95% 0.95% 0.33% 1.90% 0.58% 0.32% 0.56% 0.84% 1.53% 3.80% 1.89% 94.67% 95.65% 0.50% 1.00% 0.22%

Lymfocytes Monocytes Blasts Lymfocytes Monocytes Blasts Neutrophilic metamyelocytes Neutrophilic myelocytes Neutrophilic metamyelocytes Neutrophilic bands Neutrophilic myelocytes Neutrophilic bands Neutrophilic segments Neutrophilic segments

June 2004 November 2004

1.10% 1.86% 0.27% 0.10% 0.23%

1.15% 0.18%

96.95% 1.85% 0.99% 96.92% 0.36% 0.30%

0.30% 0.18% 0.10%

Lymfocytes Monocytes Blasts Lymfocytes Monocytes Blasts Neutrophilic myelocytes Neutrophilic metamyelocytes Neutrophilic bands Neutrophilic myelocytes Neutrophilic metamyelocytes Neutrophilic bands Neutrophilic segments Neutrophilic segments

Fig. 3 Seasonal changes in leukocyte differential count Parasitol Res

Fig. 4 The peak of kinetics of respiratory burst in all seasons investigated. The data are log transformed

The seasonal differences in the associations ship with the lymphocyte count in the total sample size (R= among immunity, physiological parameters −0.3212) and April sample. Similarly, the positive correla- and parasite infection tion between RB and the phagocyte count (their activity is connected with RB) in the total sample size (R=0.3607) as The final analysis of the experiment was conducted to well as in April and November samples (R=0.7107 and investigate the potential correlations between immunocom- 0.4839, respectively) and a negative correlation between the petence, physiological parameters, and parasitism that phagocyte count and lymphocyte count in the total sample could be subjected to seasonal changes (Fig. 5). size (R=−0.9846) and each seasonal sample were found Two kinds of analyses were performed using: (1) all (the last association reflects a proportional increase in sampled from all four sampling periods to show a general phagocyte count connected with an decrease in lymphocyte trend of associations and (2) each seasonal sample to test count calculated per 100 leukocytes using differential whether seasonal changes of immunological and physio- leukocyte count). The leukocyte count was negatively logical parameters reflect the changes in parasite abundance. correlated with GSI in the total sample size (R=−0.3557). We found three types of statistically significant relation- Further, we observed the significant positive correlation ships observed for: (a) the total sample size but not significant between erythrocyte count and hematocrit (not shown in for any seasonal sample, (b) several seasonal samples (but not Fig. 5, R=0.3760). significant for the total sample size), and (c) the total sample The general trend was that the abundance of two parasite size as well as several seasonal samples. groups (Monogenea and Acanthocephala) increased with When comparing Spearman’s rank correlation among the the GSI (R=0.2458 and 0.3122, respectively). We found a four time periods investigated, several associations between significantly positive relationship between the monogenean the parameters connected with physiological status, immu- abundance and RB (R=0.3484) in the total sample size. We nity, and parasite abundance exposed the seasonal variation. also found significant relationships between the abundance A significant negative relationship was found between the of parasite groups (Monogenea, Digenea, and Cestoda) in spleen weight and condition factor (R=−0.3801) after the total sample size and in two seasonal samples with the correcting the spleen weight for the fish body weight (using highest total parasite abundance (see Table 4 and Fig. 5). No SSI) in the total sample size. When analyzing the seasonal significant relationship was found between immunological samples, this correlation was significant in August (R= or physiological parameters and total metazoan parasite −0.5720) and November (R=−0.4331). The RB revealed a abundance (p>0.05). No significant relationships were significant positive association with the leukocyte count in found between physiological or immunological variables the total sample size (R=0.2493) and a negative relation- and parasite abundance of Mollusca or Nematoda, as well as Parasitol Res

Table 3 Presence (+) and absence (−) of parasite species in individual sampling dates

Parasite group Parasite species Sampling date

April June August November

Monogenea Dactylogyrus vistulae +++ + Dactylogyrus fallax ++−− Dactylogyrus folkmanovae +++ + Paradiplozoon megan +++ + Gyrodactylus vimbi +++ + Gyrodactylus gracilihamatus +++ + Gyrodactylus lomi − + −− Gyrodactylus prostae + −− + Digenea Sphaerostomum bramae +++ + Diplostomum larv. sp. + − ++ Apharyngostrigea cornu − + −− Metagonimus yokogawai − + −− Metorchis intermedius +++ + Strigeidae sp. −−++ Cestoda Caryophyllaeus laticeps + −− − Caryophyllaeus brachycollis ++−− Caryophyllaeus fennica ++− + Proteocephalus torulosus − + −− Nematoda Pseudocapillaria tomentosa − + −− Philometra abdominalis −−− + Acanthocephala Pomporhynchus laevis +++ + Acanthocephalus anguillae ++−− Hirudinea Piscicola geometra +++ + Mollusca Glochidium spp. (larval stage) + −− −

no association was found between their abundance and other Discussion parasites groups; therefore, they are not included in Fig. 5. The analyses including only males in total sample size Immunity and physiological parameters affected indicated that the GSI is correlated with the following by seasonal changes parameters: negatively with SSI (R=−0.3371) and leukocyte count (R=−0.4267); the last association was also significant in Our analyses focused on the complex study of physiological and April (R=−0.7538) and positively with erythrocyte count (R= immunological parameters. Even the seasonal dynamics of 0.2789) and Acanthocephala abundance (R=0.3865). The those parameters have been already studied in different fish positive associations found between GSI and several variables models; the analyses were mostly orientated to follow the are as follows: Cestoda abundance in June (R=0.7723), dynamic of one or two selected immunological parameters (for phagocyte count in August (R=0.5456), and hematocrit in instance Collazos et al. 1994, 1995a, b, 1998; Hutchinson and November (R=0.4840). When separately analyzing the SSI Manning 1996). As was demonstrated by our study, several for females and males, negative correlations were found immunological and physiological parameters measured in between SSI and the following variables: Acanthocephala natural fish populations are subjected to seasonal changes. abundance for males in the total sample size and June (R= Generally, it is considered that many immunological variables −0.3119 and −0.8051, respectively) and condition factor in are predominantly influenced by the seasonal changes affecting total and August samples (R=−0.4286 and −0.5561). A fish metabolism or in connection to the fish reproductive cycle positive correlation was found between SSI and leukocyte (Zapata et al. 1992; Scapigliati et al. 1999). In our studies, we count (R=0.2891) in the total sample size. Concerning the only analyzed changes in the GSI among four seasons analyses for females in June, when the equivalent proportion investigated as a measure of reproductive investment. Howev- of both genders were recorded, significant negative correla- er, we can predict that the seasonal gonad development is tions between (1) erythrocyte count and Digenea abundance connected with an elevated level of steroid hormones that (R=0.6778) and (2) hematocrit and both SSI and GSI were could suppress the immune function and increase the risk of foundinfemales(R=−0.4669 and −0.7973, respectively). the host being attacked by pathogens and/or parasites. The Parasitol Res

Table 4 Basic epidemiological characteristics: abundance (mean ± SD), intensity of infection and prevalence (percentage of infected hosts) of all metazoan parasite groups

Parasite group Abundance ±SD Intensity of infection Prevalence Abundance ±SD Intensity of Prevalence (Min–Max) (in %) infection (in %) (Min–Max)

April June Monogenea 49.79 ±26.96 16–119 100 44.40 ±51.53 2–213 100 Hirudinea 0.21 ±0.63 0–2 11 0.35 ±0.99 0–415 Mollusca 2.32 ±3.62 0–13 58 0 – 00 Acanthocephala 4.89 ±3.96 0–15 89 3.65 ±4.69 0–17 75 Digenea 22.68 ±43.6 0–189 58 12.50 ±20.51 0–71 70 Cestoda 19.95 ±69.88 0–305 26 1.05 ±2.65 0–920 Nematoda 0 – 0 0 0.05 ±0.22 0–15 August November Monogenea 25.86 ±17.41 3–73 100 9.23 ±10.87 0–50 91 Hirudinea 0.05 ±0.21 0–1 5 0.09 ±0.29 0–19 Mollusca 0 – 000– 00 Acanthocephala 1.95 ±3.5 0–12 36 4.09 ±3.79 0–16 95 Digenea 9.64 ±12.96 0–54 77 23.91 ±37.45 0–136 68 Cestoda 0 – 0 0 0.05 ±0.21 0–15 Nematoda 0 – 0 0 0.27 ±1.28 0–65

associations between steroid hormones and the presence of et al. 1998). In the presented analyses conducted in chub, parasites were previously demonstrated in roach, Rutilus rutilus the highest values were reported from summer samples (Kortetetal.2003b; Vainikka et al. 2004). However, the (June and August) compared to the periods with low water immunomodulatory effects of steroid hormones in cyprinid temperature (April and November), corresponding poten- fish are still in question (Vainikka et al. 2004). tially to the decline in lymphocyte function, i.e., the The seasonal variations, observed for the parameters immunosuppression in winter and immunostimulation in investigated in our study, were similar to the previous summer (Collazos et al. 1994, 1998; Hardie et al. 1994). studies performed in cyprinid fish. We recorded the differences Until now, the number of studies about differential white in erythrocyte count and hematocrit values between females and blood cell counts in fish has been limited (for instance Modrá males in chub. A small gender difference in those hematological et al. 1998; Vainikka et al. 2004). Vainikka et al. (2004) parameters were previously documented in tench, with a similar investigated the developmental stages of neutrophilic granu- tendency in both genders to decrease hematocrit in autumn and locytes and demonstrated that the maximal values of winter in comparison to spring and summer (Collazos et al. neutrophilic granulocytes occurred in spring samples in roach 1998). In our analyses of hematological parameters in chub, (R. rutilus) populations. This finding was also supported by the highest values of erythrocyte count was observed in April our data recorded for chub. However, we also observed the and November, i.e., in the periods of low water temperature. changes in the proportion to different development stages of These findings demonstrate that the levels of blood cells are neutrophilic cells, which could suggest either that this affected by seasonal changes and can potentially reflect an composition is directly under seasonal influence or indirectly adaptation to water temperature connected with the declining affected by an established parasite or pathogen infection level of oxygen dissolved in the water in the summer periods. because of changes in water temperature (through the The highest values of white bloods cells in tench were influence on parasite life cycle). reported in summer and autumn samples, and a decrease in Generally, the immunosuppressive effect of water tem- winter for both females and males was observed (Collazos perature on the innate immune response is reported in fish. However, phagocytosis in fish was demonstrated as being Table 5 The indexes of parasite diversity and dominance in sampling resistant to low temperatures (Collazos et al. 1998). No dates clear relationship was observed between RB and water April June August November temperature in our study; nevertheless, some trends were recognized from our analyses. We recorded the lowest ’ Shannon s index 2.44 1.94 1.85 1.78 values of RB, phagocytes, and also leukocyte count in ’ Simpson s index 0.11 0.21 0.21 0.26 November in comparison to other periods investigated. This Shannon’s evenness 0.79 0.63 0.7 0.63 result indicates that the decreasing reactivity of the immune Parasitol Res

Fig. 5 The associations between physiological and immunological parameters and the abundance of parasite groups in four periods investigated using Spearman’s rank correlation coefficient. The positive (+) and negative (−) correlations (at p<0.05) recorded for individual sampling dates are shown. The correlations found only in the pooled data set are indicated by dashed windows (positive correlations) or grating windows (negative correlations). SSI Spleen–somatic index, K condition factor, GSI gonado-somatic index, RB respiratory burst, LYM lymphocyte count, PH phagocyte count, ERY erythrocyte count, LEU leukocyte count, MON Monogenea, HIR Hirudinea, ACA Acanthocephala, CES Cestoda Parasitol Res system (innate branch of immunity) in this period may be The presence of endoparasites of the groups Cestoda and due to slumping exposure of parasites in autumn (see Nematoda, several species of Digenea, as well as ectoparasitic below). Other observation resulting from our study was the larval stages of Mollusca (glochidium) was seasonally deter- higher intrasample variance in the values of CL signal in mined in the present study. The seasonal dynamic of glochidium April and August periods even if the variance in phagocyte was studied for three species of freshwater fish, where higher numbers was approximately similar (not shown in abundance was connected predominantly with spring (Blažek “Results”), demonstrating the differences in innate immu- and Gelnar 2006). For endoparasites, the efficiency of nity among fish individuals. This could be potentially intermediate hosts and the successful transfer to definitive connectedwithhighparasiteorpathogeninfectionin hosts are crucial for their seasonal dynamics. Our research individual hosts as parasites are known to be aggregated in showed similar results to previous studies. For instance, the hosts (the parasite abundance fitted to binomial distribution). presence of crustacean intermediate hosts for Proteocephalus Finally, a strong correlation between spontaneous and sagittus (Hanzelová and Gerdeaux 2003) or seasonal compo- activated RB was found with the exception of August. This sition of fish food for nematode Raphidascaris acus (Šimková fact can by explained by the different developmental stages et al. 2005) are the factors facilitating the success of the of neutrophilic granulocytes recorded in the different complex life cycle. These features could also explain the seasonal periods, as the distribution of those development seasonal repartition of the endoparasite species in chub. stages is an important factor for its ability to release the free oxygen radicals after stimulation. Associations between physiological parameters, However, our results contradict to that of Kortet et al. immunity and parasitism (2003a) in roach, when the maximum phagocytic activity of head kidney granulocytes was recorded in October. O’Neill The general prediction from the life history theory is that an (1985) found that the phagocytic capacity of brown trout organism has a limited account of energy, which can be neutrophils declined with falling assay temperature, but distributed between different functions. In this study, we Ainsworth et al. (1991) in channel catfish (Ictalurus hypothesized that if the investment in one function increases punctatus) observed a similar phagocytic index in low and in a given season, than the investment in another function high temperature, but the differences in the percentage should decrease. However, the methods used for analyzing phagocytosis was increased at high temperature in compar- must be suitable to detect the investment in a function. The ison to low temperature. However, those studies did not hypothesis of trade-off could be applied to explain the take into account the seasonal effect. associations in the seasonal investment between spleen size and gonad mass (especially for males). A seasonal decrease Seasonal changes in parasite community composition in gonad mass was connected with increasing spleen size in and parasite infection our study; a similar pattern was previously found in roach (Kortet et al. 2003a). Moreover, the negative correlation Our comparison of four seasonal samples demonstrated the between GSI and both immunological measurements SSI highest parasite diversity in chub in spring and decreased and leukocyte count (another potential measure of immu- toward autumn. However, species dominance and evenness nocompetence) was reported for males of chub. was comparable within two summer and autumn samples. The negative correlation between the condition factor and The composition of parasite communities was the result of SSI was found in the total sample size as well as in two both seasonal influence and water temperature. The presence sampling periods (August and November). This relationship of several parasites as well as their intensity of infection is was previously found in common bream (Abramis brama) associated with low water temperature. This is generally collected during breeding season and interpreted as a trade- observed for viviparous Monogenea belonging to the genus off between investment in different life functions, i.e., Gyrodactylus (Koskivaara 1992; Šimková et al. 2005). A between somatic condition and immune defense (Ottová significantly higher abundance was recorded for this genus et al. 2005). In our study, the period of breeding was not in spring and autumn samples; however, the occurrence of included. However, the correlation observed between several Gyrodactylus species was retained in all periods spleen size and condition factor in chub was significant in investigated. In concern with the oviparous Dactylogyrus two periods with different water temperature. Therefore, we species, their presence as well as the high parameters of suggest that the seasonal changes did not strongly affect infection is connected with an increase in water temperature this association, and we hypothesize that the investment in (Koskivaara et al. 1991; Koskivaara 1992; Šimková et al. somatic condition and immune function could be different 2001) even if several Dactylogyrus species are present in when comparing the spring and early summer periods with spring or autumn. In the present study, D. fallax was found August (the period not affected by reproduction) and as a parasite restricted to spring and early summer. November (the period near winterizing when the feeding Parasitol Res resources for fish are already limited, although the fish should In our study, positive correlations were demonstrated be in a good condition). Recently, the spleen size was widely between GSI and parasite abundance of Monogenea and applied as a measure of immunocompetence in intraspecific Acanthocephala in the total sample size. The positive studies using fish (Skarstein et al. 2001;Kortetetal.2003a; associations between GSI and parasites abundance were Ottová et al. 2005); this measure reflects overall immuno- previously documented, for instance, P. sagittus (Cestoda) competence related to innate and acquired mechanisms. The increases in abundance in both genders in spring, and it was condition factor represents a measure of relative body weight hypothesized that parasites life cycle could be synchronized and could reflect host vigor. The previous study in common with the beginning of host reproduction, probably induced bream illustrated the positive relationship between condition by an increasing level of steroid hormones in fish (Šimková factor and the diversity in major histocompatibility genes et al. 2005). The same association was documented in our (MHC) by Ottová et al. (2007). Considering the hypotheses study, i.e., a positive correlation between GSI and Cestode that higher diversity in immune genes like MHC represents abundance in males in June, i.e., a few weeks after spawning. the ability to present a wider range of antigen peptides to Moreover, we recognized a positive correlation between pathogens and parasites, this suggests that the individuals abundance of Acanthocephala and GSI in the total sample size. better, equipped by their genetically based immune predispo- Similarly, Heins et al. (1999) showed the seasonal pattern of sition, reach a good condition status. cestode infection compatible with host reproduction but without Recently, several studies were conducted to investigate a host immune response suggesting evolutionary host–parasite the wide range of immunological and physiological variables interactions. The same study showed a positive correlations to try to estimate their potential associations with metazoan between gonad mass and abundance of Gyrodactylus species parasites (for instance Kortet et al. 2003b; Vainikka et al. (Monogenea) and R. acus (Nematoda) suggesting either the 2004). Our analyses suggest that the associations between immunosuppressive roles of steroid hormones or the reproduc- immunological or physiological variables and metazoan tive energetic costs. However, such a hypothesis needs further parasites are scarce. We observed a positive association investigation into chub, including the comparison of spawning between RB (the peak of CL signal) and abundance of with pre-spawning and post-spawning periods. Monogenea, a dominant metazoan parasite group in freshwater fish. The phagocyte counts as well as the values of RB Acknowledgments This study was supported by the Grant Agency converted to phagocyte counts follow the similar trend as of the Czech Republic, Project no. 524/04/1128. The MS preparation monogenean abundance, i.e., the decrease in November. A was funded by the Grant Agency of the Czech Republic, Project no. 524/07/0188. The field study and hematological analyses were similar tendency was recorded for parasite species diversity partially funded by a Research Project from Masaryk University, in both infracommunity (i.e., individual fish) and component Brno, Project no. MSM 0021622416. KL and AŠ were supported by community level (i.e., fish population). the Ministry of Education, Youth and Sports of the Czech Republic Even the comparison of immunological parameters such as (Project no. LC522, Ichthyoparasitology Research Centre). We thank Jana Benešová from the Institute of Experimental Biology, Faculty of SSI, leukocyte counts, and RB as well as parasite abundance Science, Masaryk University, Brno, for valuable help with physiolog- showed no significant results between genders as was also ical analyses; Martina Pečínková, Martina Dávidová, Radim Blažek, demonstrated by several studies (for instance Vainikka et al. Radim Sonnek, and Eva Řehulková from the Laboratory of Parasitol- 2004); we also executed the analyses considering females ogy, Institute of Botany and Zoology, Faculty of Science, Masaryk University, Brno, for kindly helping with parasite dissection. We thank and males separately in June (and insuring a comparable Jiří Huml, Michal Janáč, and Matej Polačik for their valuable sample size for both genders). These analyses demonstrated assistance in field sampling. The authors thank the Moravian Anglers a negative correlation between spleen size and the Acantho- Union for supporting the research in their entire district and for cephala abundance in males and a positive correlation purchasing the experimental fish. We are very grateful to Maria Vass for the correction of English. between erythrocyte count and Digenea as well as hematocrit being important physiological parameter in females, which suggest that small differences in immune investment between genders could occur. References The investment in gonad size is predicted to be different in females and males; however, we did not confirm a Ainsworth AJ, Dexiang C, Waterstrat PR, Greenway T (1991) Effect significant difference between genders. This may be due to of temperature on the immune system of channel catfish (Ictalurus punctatus). I. Leucocyte distribution and phagocyte a low proportion of females in three of the four periods function in the anterior kidney at 10°C. Comp Biochem Physiol investigated or the differences in gonad size are not so A 100:907–912 obvious between genders because of chubs collected in Bellanti JA, Kadlec JV (1985) General immunology. In: Bellanti JA – periods excluding the breeding period, where the effect of (ed) Immunology III. Saunders, Philadelphia, PA, pp 16 53 Blažek R, Gelnar M (2006) Temporal and spatial distribution of season is more important. In the future, further investigation glochidial larval stages of European unionid mussels (Mollusca: including a higher female sample could clarify this finding. Unionidae) on host fishes. Folia Parasitol 53(2):98–106 Parasitol Res

Bolger T, Connolly PL (1989) The selection of suitable indices for the (Limanda limanda L) sampled from Lyme Bay, UK. Fish measurement and analysis of fish condition. J Fish Biol 34:171–182 Shellfish Immunol 6:473–482 Bowden TJ, Butler R, Bricknell IR (2004) Seasonal variation of serum Khotenovsky IA (1985) Fauna SSSR, Monogenea. Nauka, Leningrad lysozyme levels in Atlantic halibut (Hippoglossus hippoglossus (In Russian) L.). Fish Shellfish Immunol 17:129–135 Kortet R, Taskinen J, Sinisalo T, Jokinen I (2003a) Breeding-related Bush AO, Lafferty KD, Lotz JM, Shostak AW (1997) Parasitol- seasonal changes in immunocompetence, health state and ogy meets ecology on its own terms: Margolis et al revisited. condition of the cyprinid fish, Rutilus rutilus, L. Biol J Linn J Parasitol 83:575–583 Soc 78:117–127 Chubb JC (1977) Seasonal occurrence of helminths in freshwater Kortet R, Vainikka A, Rantala MJ, Jokinen EI, Taskinen J (2003b) fishes. Part I. Monogenea. Adv Parasitol 15:133–192 Sexual ornamentation, androgens and papillomatosis in male Clem LW, Faulmann E, Miller NW, Ellsaesser CF, Lobb CJ, Cuchens roach (Rutilus rutilus). Evol Ecol Res 5:411–419 MA (1984) Temperature-mediated processes in teleost immunity: Koskivaara M (1992) Environmental factors affecting monogeneans differential effect of in vitro and in vivo temperatures on parasitic on freshwater fishes. Parasitol Today 8:339–342 mitogenic responses of channel catfish lymfocytes. Dev Comp Koskivaara M, Valtonen ET, Prost M (1991) Dactylogyrids on the Immunol 8:313–322 gills of roach in Central Finland: features of infection and species Collazos ME, Ortega E, Barriga C (1994) Effect of temperature on the composition. Int J Parasitol 21:565–572 immune system of a cyprinid fish (Tinca tinca, L.). Blood Kubala L, Lojek A, Číž M, Vondráček J, Dušková M, Slavíková H phagocyte function at low temperature. Fish Shellfish Immunol (1996) Determination of phagocyte activity in whole blood of 4:231–238 carp (Cyprinus carpio) by luminol-enhanced chemiluminiscence. Collazos ME, Barriga C, Ortega E (1995a) Seasonal variations in the Vet Med 41:323–327 immune system of cyprinid Tinca tinca—phagocytic function. Kumari J, Sahoo PK, Swain T, Sahoo SK, Sahu AK, Mohanty BR Comp Immunol Microbiol Infect Dis 18:105–113 (2006) Seasonal variation in the innate immune parameters of the Collazos ME, Barriga C, Ortega E (1995b) Seasonal-changes in Asian catfish Clarias batrachus. Aquaculture 252:121–127 phagocytic capacity and superoxide anion production of blood Le Morvan C, Troutaud D, Deschaux P (1998) Differential effects of phagocytes from tench (Tinca tinca, L). J Comp Physiol B temperature on specific and non-specific immune defences in 165:71–76 fish. J Exp Biol 201:165–168 Collazos ME, Ortega E, Barriga C, Rodrìguez AB (1998) Seasonal Lusková V (1997) Annual cycles and normal values of hematological variation in haematological parameters in male and female Tinca parameters in fishes. Acta Sci Nat Brno 31:70 tinca. Mol Cell Biochem 183:165–168 Magurran A (1983) Ecological diversity and its measurement. Croom Crompton DWT (1985) Reproduction. In: Crompton DWT, Nickol Helm, London BB (eds) Biology of the Acanthocephala. Cambridge Univ. Press, Manning MJ (1994) Fishes. In: Turner RJ (ed) Immunology. A Cambridge, pp 213–271 comparative approach. Wiley, New York, pp 69–100 Cuchens MA, Clem LW (1977) Phylogeny of lymphocyte hetero- Margolis L, Esch GW, Holmes JC, Kuris AM, Schod GA (1982) The geneity. II. Differential effects of temperature on fish T-like use of ecological terms in parasitology (report of an ad hoc and B-like cells. Cell Immunol 34:219–230 committee of the American Society of Parasitologists). J Parasitol Dalmo RA, Ingebritsen K, Bøgwald J (1997) Nonspecific defence 68:131–133 mechanism in fish, with particular reference to the reticuloendo- Martínez FJ, García-Riera MP, Canteras M, De Costa J, Zamora S thelial system (RES). J Fish Dis 20:241–273 (1994) Blood parameters in rainbow trout (Oncorhynchus Ergens R, Lom J (1970) Causative agents of parasitic fish diseases. mykiss): Simultaneous influence of various factors. Comp Academia, Prague (in Czech) Biochem Physiol 107A:95–100 Esch GW, Bush AO, Aho JM (1990) Parasite communities: patterns McKnight IM (1966) A hematological study on the mountain and processes. Chapman & Hall, London, UK whitefish, Prosopium williamsoni. J Fish Res Board Can Finco-Kent D, Thune RL (1987) Phagocytosis by catfish neutrophils. 23:45–64 J Fish Biol 31A:41–49 Modrá H, Svobodová Z, Kolářová J (1998) Comparison of differential Georgiev B, Bisekov V, Genov T (1986) The staining method for leukocyte counts in fish of economic and indicator importance. cestodes with iron acetocarmine. Helminthologia 23:279–281 Acta Vet Brno 67:215–226 Gusev AV (1985) In: Bauer ON (ed) Metazoan parasites. Part I. Moravec F (1994) Parasitic nematodes of freshwater fishes of Europe. Identification key to parasites of freshwater fish. vol. 2. Nauka, Academia, Praha Leningrad (in Russian) O’Neill JG (1985) An in vitro study of polymorphonuclear phagocy- Hanek C, Fernando CH (1978) The role of season, habitat, host age tosis and the effect of temperature. In: Manning MJ, Tatner MF and sex on gill parasitism of Lepomis gibbosus (L.). Can J Zool (eds) Fish immunology. Academic, New York, pp 47–55 56:1247–1250 Ottová E, Šimková A, Jurajda P, Dávidová M, Ondráčková M, Hanzelová V, Gerdeaux D (2003) Seasonal occurrence of the Pečínková M, Gelnar M (2005) Sexual ornamentation and tapeworm Proteocephalus longicollis and its transmission from parasite infection in males of common bream (Abramis brama): copepod intermediate host to fish. Parasitol Res 91:130–136 a reflection of immunocompetence status or simple cost of Hardie IJ, Fletcher TC, Secombes CJ (1994) Effect of temperature on reproduction? Evol Ecol Res 7:581–593 macrophage activation and the production of macrophage Ottová E, Šimková A, Morand S (2007) The role of major histocompat- activating factor by rainbow trout (Oncorhynchus mykiss) ibility complex diversity in vigour of fish males (Abramis brama L.) leucocytes. Dev Comp Immunol 18:57–66 and parasite selection. Biol J Linn Soc 90 (in press) Heath AG (1990) Summary and perspective. Am Fish Soc Symp Owens IPF, Wilson K (1999) Immunocompetence: a neglected life history 8:183–191 trait or conspicuous red herring? Trends Ecol Evol 14:170–172 Heins DC, Singer SS, Baker JA (1999) Virulence of the cestode Piersma T, Lindström Å (1997) Rapid reversible changes in organ size Schistocephalus solidus and reproduction in infected threespine as a component of adaptive behaviour. Trends Ecol Evol 12:134– stickleback, Gasterosteus aculeatus. Can J Zool 77:1967–1974 138 Hutchinson TH, Manning MJ (1996) Seasonal trends in serum Rohde K (1984) Ecology of marine parasites. Helgol Meeresunters lysozyme activity and total protein concentration in dab 37:5–33 Parasitol Res

Rohde K, Hayward C, Heap M (1995) Aspects of the ecology of Svobodová Z, Pravda D, Paláčková J (1986) Universal methods of metazoan ectoparasites of marine fishes. Int J Parasitol 25:945–970 hematological investigations in fish. Edice Metodik, no. 22. Rowley AF, Hunt TC, Page M, Mainwaring G (1988) Fish. In: VÚRH Vodňany, Czech Republic (In Czech) Rowley AF, Ratcliffe NA (eds) Vertebrate blood cells. Cam- Svobodová Z, Pravda D, Paláčková J (1991) Unified methods of bridge Univ. Press, Cambridge, pp 19–128 haematological examination of fish. Manuals of research institute Scapigliati G, Scalia D, Marras A, Meloni S, Mazzini M (1999) of fish culture and hydrobiology, vol 22. University of South Immunoglobulin levels in the teleost sea bass Dicentrarchus Bohemia, Vodňany, p 331 labrax (L.) in relation to age, season, and water oxygenation. Šimková A, Sasal P, Kadlec D, Gelnar M (2001) Water tem- Aquaculture 174:207–212 perature influencing dactylogyrid species communities in Scholz T (1989) Amphilinida and Cestoda, parasites of fish in roach, Rutilus rutilus, in the Czech Republic. J Helminthol 75: Czechoslovakia. Acta Sci Nat Brno 23:1–56 373–383 Secombes CJ, Olivier G (1996) Host–pathogen interactions in Šimková A, Jarkovský J, Koubková B, Baruš V, Prokeš M (2005) salmonids. In: Bernoth EM, Ellis AE, Midtlyng P, Olivier G, Associations between fish reproductive cycle and the dynamics Smith P (eds) Furunculosis—Multidisciplinary Fish Disease of metazoan parasite infection. Parasitol Res 95:65–72 Research. Academic, London, pp 269–296 Vainikka A, Jokinen EI, Kortet R, Taskinen J (2004) Gender- and Skarstein F, Folstad I, Liljedal S (2001) Whether to reprodukce or not: season dependent relationships between testosterone, oestradiol immune suppression and cista of parasites during reproduction in and immune functions in wild roach. J Fish Biol 64:227–240 the Arctic charr. Can J Zool 79:271–278 Wester PW, Vethaak AD, van Muiswinkel WB (1994) Fish biomarkers Stolen JS, Ganh T, Kasper V, Nagle JJ (1984) The effect of in immunotoxicology. Toxicology 86:213–232 environmental temperature on the immune response of a marine Zapata AG, Varas A, Torrba M (1992) Seasonal variations in the immune teleost (Paralichthys dentatus). Dev Comp Immunol 8:89–98 system of lower vertebrates. Immunology Today 13:142–147

Článek B

Are the immunocompetence and the presence of metazoan parasites in cyprinid fish affected by reproductive efforts of cyprinid fish?

Journal of Biomedicine and Biotechnology. ID 418382 (2010)

ROHLENOVÁ K, ŠIMKOVÁ A

Hindawi Publishing Corporation Journal of Biomedicine and Biotechnology Volume 2010, Article ID 418382, 14 pages doi:10.1155/2010/418382

Research Article Are the Immunocompetence and the Presence of Metazoan Parasites in Cyprinid Fish Affected by Reproductive Efforts of Cyprinid Fish?

Karolına´ RohlenovaandAndrea´ Simkovˇ a´

Department of Botany and Zoology, Faculty of Science, Masaryk University, Kotla´ˇrska´ 2, 611 37 Brno, Czech Republic

Correspondence should be addressed to Andrea Simkovˇ a,´ [email protected]

Received 31 July 2009; Accepted 23 October 2009

Academic Editor: Jorge Morales-Montor

Copyright © 2010 K. RohlenovaandA.´ Simkovˇ a.´ This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Each organism has the limited resources of energy that is distributed among important life traits. A trade-off between immune response and other physiological demands of organism especially costly reproduction is expected. Leuciscus cephalus, the cyprinid fish, was investigated during three periods varying in reproductive investment, that is, before-breeding, breeding, and after- breeding periods. We tested whether a potentially limited investment in immunity during the breeding is associated with higher susceptibility to the metazoan parasites. Following the immunocompetence handicap and sperm protection hypotheses, males expressing more elaborated sexual ornamentation should produce better quality sperm and be more parasitized. We found that reproductive investments in fish play an important role for energy allocation into somatic condition, immunity, and reproduction. The immune parameters including respiratory burst and leukocyte count were higher in breeding; however, parasite species richness and abundance appeared low. Males investing more in spawning tubercles reached high spermatocrite and were more parasitized by digeneans.

1. Introduction Immune system of each organism could be a subject of rapid temporal changes due to limited resources of energy, Life-history theory predicts that each organism has a limited especially in reproductive period, such as breeding in fish. amount of energy, which is allocated among the traits The changes should be achieved either by trade-off between connected with maintenance, reproduction, and growth [1, immunity and reproduction or by adaptive or nonadaptive 2]. One of the most important life traits involving the immunomodulative action of sex hormones [8]. The costs potential life trade-offs is the ability of immune system to paid for reproduction due to immunosuppression by steroid defend hosts against parasites. Thus recently, the investment hormones were suggested for salmonid Artic charr (Salveli- in immune defence is a matter of great concern in the nus alpinus), following the observation that spawning males immunoecological or evolutionary immunology studies [3]. were more susceptible to parasite infection and had smaller There are several studies investigating the importance of spleen size in comparison with the resting males [9]. A trade- immunological competence as a potential determinant of off in energy allocation could also explain a negative rela- reproductive success and fitness predominantly using birds tionship between immunocompetence (measured by spleen as a model (e.g., [4–7]). The main presumption of those size) and condition factor (measurement of fish vigour and studies is that the host immune system is exposed to the health) found in fish males, previously observed in common variety of parasite species; therefore, hosts have to develop bream (Abramis brama) during breeding period [10]. The adequately strong immune response to reduce the fitness cost sexual ornamentation in fish males during spawning seems due to parasitism. to reflect the potential costs of reproduction caused by 2 Journal of Biomedicine and Biotechnology higher parasite infection although there was no effect on hypotheses, we tested whether males expressing more elab- immunocompetence measured by spleen size. orated sexual ornamentation produce better quality sperm Following the hypotheses of evolution of sexual selection, (measured by sperm density) and are parasitized by high the expressed sexual traits like sexual ornamentation could parasite species richness due to potential immunosuppresion be considered as a male handicap playing a key role by steroid hormones. in mate choice. The more intensive expression of sexual trait indicates the presence and effectiveness of genes for resistance (so-called “good genes”) against pathogens and 2. Materials and Methods parasites [11]. Many studies using different models have been 2.1. Fish Sampling. A total of 90 males of chub (Leuciscus conducted to investigate the associations between parasite cephalus, Cyprinidae) were collected in 2005 during three loads and intensity of sexual ornamentation but their results different periods in relation to reproduction investment. are various (see [12–14]). Studies based on measurements Those periods included before breeding in early May, breed- of immune function provided stronger evidence for the ing in late May, and after breeding in late June. The fish was Hamilton and Zuk hypothesis than those based on mea- collected from the same locality situated on the confluence sures of parasite load [13]. Immunocompetence handicap of the Svitava and Svratka rivers, belonging to the Morava hypothesis introduced by Folstad and Karter [15]isbased river basin on the periphery of Brno (Czech Republic). on presumption of dualistic role of steroid hormones. On The fish individuals were captured using electrofishing the one hand, those hormones stimulate the increase in the and immediately a blood sample (340 μL) was taken from expression of sexual ornamentation, but on the other hand, individually electronarcotized fish. Cardiac puncture using they decrease the resistance and immune defence of individ- heparinized syringes was used as an optimal sampling uals by immunosupression. The role of steroid hormones, method in electronarcosis to collect fish blood [25]. Blood mainly in testosterone-mediated immunosuppression, was samples were collected in heparinized microtubes and mixed also highlighted by sperm protection hypothesis [16, 17]. with heparin diluted 10x with 0.7% NaCl to retain the Males able to tolerate the negative effect of testosterone number of heparin units in the measured blood according produce more developed sexual traits and also the sperm to the methodology written by Kubala et al. [26]. After of better quality. Therefore, they are supposed to be more blood sampling, the fish were placed in a tank containing susceptible to parasites because their immune response is water collected from the same location and then transported suppressed by testosterone. This hypothesis was partially to the laboratory. During the storage in the laboratory supported in roach (Rutilus rutilus), a freshwater cyprinid tanks, the original water temperature was maintained and a fish [18]. standard aquarium filter was used for water filtration. All fish Leuciscus cephalus is the cyprinid fish spawning usually to individuals were killed within 24 hours. Each individual was sand or gravel bottom from May to June [19]. The spawning measured (total and standard lengths in mm) and weighed period is determined by water temperature. During the (in g), and a complete parasitological dissection of the fish spawning period, males produce keratin-based epidermal according to Ergens and Lom [27]wasperformed. nodules—breeding tubercles—which are invoked by several The physiological parameters, including gonad weight pituitary and sex hormones [20]. The breeding tubercles and spermatocrit, hematocrit, and erythrocyte counts, and may have a role as a status badge or as a sign of quality the selected immune parameters, including spleen weight, [21], or they are important in mate choice indicating male’s leukocrit and leukocyte counts, differential leukocyte cell parasite load to females [22] and resistance to parasites counts, phagocyte count, and respiratory burst activity, were or pathogens [23]. Taskinen and Kortet [23]founda measured. We calculated relative body weight (condition positive relationship between proportions of dead Rhipido- factor, K) using the equation: K = constant × somatic cotyle campanula (Digenea) and sexual ornamentation in weight (g)/(standard length (cm))3. According to Bolger and three populations of roach in accordance with Hamilton- Connolly [28], the assumption of the condition factor is that Zuk hypothesis. Skarstein and Folstad [24]documenteda the heavier the fish is in relation to its length. Therefore, it negative association between lymphocyte density and sexual is better to use the general condition rather than the weight ornamentation (red spawning coloration) in Artic charr and length particularly. The relative size of gonads, that is, which may indicate the increased investment in sexual gonado-somatic index, GSI, was calculated as follows: GSI ornamentation at the costs of immunity. = gonad weight (g)/body weight (g) × 100. Spleen-somatic The aim of the present study was to investigate the index, SSI, was calculated as spleen weight (g)/body weight changes in immune defence, condition status, reproduc- (g) × 100. tion parameters, and parasitism in selected cyprinid fish species in the periods of different reproductive investment, following the hypothesis of potential trade-offsinenergy 2.2. Parasite Collection and Determination. The complete allocation. We supposed and tested whether a potentially dissection of fish was performed using the method of Ergens limited investment in immunity is associated with higher and Lom [27]. Fish individuals were investigated for all meta- susceptibility to the metazoan parasites (including especially zoan parasites. Ectoparasites (Monogenea, Hirudinea, and helminths) during the period of high reproductive invest- Mollusca) and endoparasite helminths (Digenea, Cestoda, ment (i.e., spawning period). Finally, following the predic- Acanthocephala, and Nematoda) were determined using tions of immunocompetence handicap and sperm protection recent keys and methodology [27, 29–32]. All recorded Journal of Biomedicine and Biotechnology 3 specimens of metazoan parasites, that is, Monogenea in (percentage of infected host individuals in each period) glycerin-amonium picrate, Nematoda in glycerin-ethanol and intensity of infection (number of parasites per an and other metazoan parasites (Digenea, Cestoda, Acantho- infected host) according to Bush et al. [38]. Moreover, cephala, and Mollusca) in 4% formalin were fixed. Digenea abundance as a number of parasites in all investigated and Cestoda were subsequently stained using IAC carmine hosts in each period was calculated. Only parasite groups [33]. A light microscope (Olympus BX 50) with phase- with high epidemiological values, including Monogenea, contrast, differential interference contrast (DIC according Digenea, Cestoda, and Acanthocephala, were used for sta- to Nomarski), and Digital Image Analysis (Micro Image tistical analyses. The differences in physiological, immune 4.0 for Windows) was used for parasite determination and parameters, and parasitism among 3 investigated periods measurements. were tested using ANOVA. Tukey HSD post hoc test was applied for multiple pairwise comparisons. The potential 2.3. Blood Analyses. Differential cell counts were estimated relationships among the studied variables were analyzed in whole blood smears stained panoptically by the Pappen- using a Principal Component Analysis (PCA) and Pearson’s ffi heim technique (May-Grunwald¨ and Giemsa-Romanovsky) correlation coe cient. Bonferroni correction for multiple [34]. We considered 200 leukocytes and classified them tests was applied. Most of variables analyzed did not using morphology into following categories: lymphocytes, fit a normal distribution, and therefore, they required a monocytes, blasts, and neutrophiles (including the differ- transformation using logarithm (for GSI, SSI, respiratory ent neutrophiles stages, i.e., myelocytes, metamyelocytes, burst, count of erythrocytes, and number of head tubercles), bands and segments) according to Svobodova´ et al. [34]. arcsine square root (for spermatocrit), and square root (for Erythrocyte (in T.l−1) and leukocyte (in G.l−1)countswere parasite abundance and total parasite species richness). No executed in Burker’s¨ hemocytometer. Heparinized blood was transformation was necessary for condition factor and count diluted with Natt-Herick solution at 1 : 200 ratio in special of phagocytes. GSI and SSI instead of gonad or spleen weights − ff 25 mL flask [35, 36]. Hematocrit and leukocrit (in l.l 1)were were used in PCA to eliminate the e ect of body weight on measured using heparinized microcapillaries 75 mm long gonad or spleen weights. All statistical analyses were executed and 60 μm in inner volume. Centrifugation was carried out using Statistica 8.0. with a hematocrit centrifuge at 12000 g for 3 minutes [35]. Differential leukocyte count and total count of leukocytes were used to calculate the relative count of phagocytes 3. Results (G.l−1). According to Sterzlˇ [37] concerning neutrophiles, only metamyelocytes or older stages than metamyelocytes are 3.1. Physiological and Immunological Parameters. As can ff considered to have phagocytic ability. Therefore, the relative be concluded from the results, the significant di erences count of phagocytes in our study included monocytes, were found in the measured physiological and immune neutrophilic metamyelocytes, bands, and segments. parameters when comparing the three periods which varied in the investment in reproductive activity (i.e., before- breeding, breeding, and after-breeding). The mean and 2.4. Respiratory Burst Activity. Chemiluminescence (CL) standard deviations (± SD) of total body length, body activated with opsonised zymosan was measured in each fish weight, gonad weight, spleen weight, and condition factor of sample. The chemiluminescence emission (in relative light fish within each period investigated are shown in Table 1. units, RLU) was measured in five-minute intervals during We tested the differences in physiological and immune 100minutes(i.e.,atotalof20measurementsweretaken)to parameters among 3 investigated periods using ANOVA. obtain the kinetic curves for each sample. The peak of the CL The statistically significant differences were found for GSI signal, that is, the maximal value of a respiratory burst (RB), (F = 106.250, P < .0001), condition factor (F = 7.150, was used for each individual. CL measurements were carried 2,82 2,87 P = .0013), respiratory burst (F = 7.113, P = .0014), out with a Luminometer Junior (Checklight; 380–630 nm) 2,84 erythrocyte (F = 3.704, P = .0286) and leukocyte (F according to methodology of Kubala et al. [26]. 2,87 2,87 = 6.151, P = .0032) counts, hematocrit (F2,85 = 3.491, P = .0349), and leukocrit (F2,85 = 3.331, P = .0405). The 2.5. Sperm Quality and Sexual Ornamentation. The selected highest values of GSI, condition factor, and hematocrit parameters potentially reflecting the quality of male individ- were recorded in before-breeding period while erythrocyte uals were measured only during the spawning period. Sper- count revealed the highest values in after-breeding period. −1 matocrit (in l.l ), a measurement of sperm concentration The maximal values of immune parameters, including expressing the percentage of a given volume of milt occupied respiratory burst, leukocyte count, and leukocrit, were found by sperm cells was analyzed according to Kortet et al. [18]. in breeding period. However, no significant difference was The microcapillaries with milt samples were centrifuged for shown for SSI and phagocyte count among three periods of 5 minutes at 12000 g. We calculated all breeding tubercles, different reproductive investments (ANOVA, P > .05). Using as secondary sexual ornaments in fish, on the head and Tukey HSD post hoc test we compared differences between operculum of each individual. all pairs of periods. First, we detected significant differences between all periods for GSI (P < .05). Consequently, we 2.6. Data Analysis. Each parasite group was characterized found the significant differences of before-breeding period in by the following epidemiological parameters: prevalence comparison with both breeding and after-breeding periods 4 Journal of Biomedicine and Biotechnology

Table 1: Data on the ﬁsh investigated: n presents the number of males collected, total body length, body weight, gonad weight, spleen weight, and condition factor (K): means ± SD are shown.

Total length (mm) Body weight (g) Gonad weight (g) Spleen weight (g) K Sampling period n Mean ±SD Mean ±SD Mean ±SD Mean ±SD Mean ±SD Before-breeding 29 271.59 ±3.25 245.37 ±106.88 21.88 ±10.98 0.27 ±0.13 1.92 ±0.22 Breeding 31 270.39 ±26.93 215.97 ±61.39 10.30 ±3.57 0.25 ±0.09 1.76 ±0.17 After-breeding 30 260.52 ±71.48 215.49 ±63.92 10.51 ±3.79 0.25 ±0.08 1.60 ±0.43

Before-breeding for condition factor and leukocyte count. Further, the significant differences (P < .05) were also found between before-breeding and breeding periods for respiratory burst 1.38% 0.59% and hematocrit and between before breeding and after- 0.45% 0.34% breeding for erythrocyte count and leukocrit. As revealed by comparison of differential leukocyte count 95.38% 2.79% 1.34% among the three periods studied, only small variability in the composition of white blood cell components was found. Blood of chub showed lymphocytic character, that 0.52% is, lymphocytes prevailed in the white blood cell. The proportion of lymphocytes reached the similar values in (a) all three periods. Only a small difference was found in the proportions of monocytes, total neutrophilic granulocytes, and blasts in relation to three periods investigated (Figure 1). Breeding Nevertheless, the variations were found in proportions of the developmental stages of neutrophilic granulocytes including early ontogenetic stages, that is, myelocytes and 1.61% metamyelocytes as well as late ontogenetic stages, that 0.35% is, bands and segments. We recorded the low proportion 1.26% of neutrophilic myelocytes and metamyelocytes and the 95.48% 2.55% high proportion of bands and segments in before-breeding 0.39% period. On the other hand, in breeding and after-breeding 1.68% periods, we found higher proportions of early ontogenetic 0.23% stages (myelocytes and metamyelocytes) compared with other development stages of neutrophiles. The percentage distribution of individual types of leukocytes during three (b) periods investigated is shown in Figure 1.

After-breeding 3.2. Parasite Infection. Metazoan parasite species belonging to seven groups including ectoparasitic Monogenea, Hirudinea, Mollusca, and endoparasitic Digenea, Cestoda, 0.7% Acanthocephala, and Nematoda were found. Monogenea 0.07% 1.5% was the species richest group, including eight species. The presence of all parasite species in three periods investigated 95.9% 3.33% is shown in Table 2. From a total 22 parasite species, 12 of 0.9% them occurred in all three periods studied, 7 species were 0.73% present only in before-breeding and after-breeding periods 0.2% (i.e., those parasites were absent in breeding period), and 2 parasite species were found solely in breeding period. When comparing the three periods, we detected a variation in epidemiological characteristics including abundance, Lymphocytes N. myelocytes Monoctyes N. metamyelocytes intensity of infection, and prevalence. Those characteristics Blasts N. bands for each parasite group are shown in Table 3.ANOVAtest Neutrophilic granulocytes N. segments revealed statistically significant differences in abundance among three periods investigated for three parasite groups: (c) Monogenea (F2,87 = 10.887, P < .0001), Digenea (F2,87 = Figure 1: Changes in leukocyte differential count in three periods 10.257, P = .0001), and Cestoda (F2,87 = 4.745, P = .0111). investigated. The significant differences for these parasite groups were Journal of Biomedicine and Biotechnology 5

Table 2: Presence (+) and absence (−) of parasite species in each sampling period. Hatching areas indicate the presence of a given species in all three periods investigated.

Samplingperiod Parasite group Parasite species Before-breeding Breeding After-breeding Monogenea Dactylogyrus vistulae +++ Dactylogyrus fallax +++ Dactylogyrus folkmanovae +++ Paradiplozoon megan +++ Gyrodactylus vimbi +++ Gyrodactylus lomi +++ Gyrodactylus prostae − + − Gyrodactylus gasterostei + − + Digenea Sphaerostomum bramae +++ Diplostomum sp.larv. +++ Metorchis xanthosomus +++ Asymphylodora imitans + − + Strigeidae gen. sp. + − + Cestoda Caryophyllaeus fimbriceps + − + Caryophyllaeus brachycollis + − + Proteocephalus torulosus +++ Nematoda Philometra ovata − + − Rhabdochona denudata + −− Raphidascaris acus + − + Acanthocephala Pomporhynchus laevis +++ Hirudinea Piscicola geometra +++ Mollusca Glochidium spp. + − + also found after using Bonferonni correction (P < .05). The and two numerous parasite species of Digenea, that is, highest abundance of Monogenea was recorded in after- Sphaerostomum bramae and Metorchis xanthosomus,were breeding period. Digenea and Cestoda reached the maximal treated separately because of different location (intestine or values of abundance in before-breeding period. Using post skin) and strategy (adult or larvae stages) in hosts. The hoc test for pairwise comparison, we found statistically graphic outputs of PCA analyses using parasite abundance significant differences between before-breeding and after- as a measurement of parasite load in Figures 2–4(a), (b) breeding as well as breeding and after-breeding periods and total parasite species richness in Figures 2–4(c), (d) are for abundance of Monogenea and Digenea (both P < .05). shown. The first three axes explain the highest proportion Further, the significant differences for Cestoda were seen of total variability and therefore they are retained for graphic between before- and breeding periods (P < .05). interpretation (see Table 4 for the variability explained by the first three axes). In before-breeding period, the analyses showed that fish 3.3. Associations between Immunity, Physiological Parameters, condition in before-breeding period was positively corre- and Parasite Infection. The potential correlations between lated to abundance of Metorchis xanthosomus (R = 0.4129, immunocompetence, physiological parameters and para- P < .0001) and negatively to Sphaerostomum bramae (R = sitism were investigated in three periods varying in the −0.6582, P = .040) (see Figures 2(a) and 2(b)). Following reproduction investment. After transforming data to achieve PCA results, GSI is negatively associated to Monogenea the normal distribution, the following parameters were used abundance (Figure 2(b)). Moreover, GSI is associated to SSI for the analyses: (1) immunological data (SSI, respiratory in before-breeding period (Figures 2(c) and 2(d)). The total burst, and count of phagocytes), (2) physiological data (GSI, parasite species richness increased with higher SSI and lower condition factor, count of erythrocytes, spermatocrit, and GSI (Figure 2(c)). Nevertheless, the correlations between number of head tubercles), and (3) parasitism. For each those variables were not significant (P > .05). period two measurements of parasitism were applied, that In breeding period, we found a positive correlation is, total parasite species richness and parasite abundance. between the number of head tubercles and spermatocrit (R = The second one includes abundances of the most numerous 0.4009, P = .038) (see Figures 3(a), 3(b),and3(c)). Following parasite groups, that is, Monogenea (all of them gill and PCA results, GSI tends to be positively associated with both skin parasites), Pomporhynchus laevis (Acanthocephala), spermatocrit and number of head tubercles (Figures 3(a) and Cestoda (including three intestinal parasite species) and 3(c)). However, the correlations were not significant 6 Journal of Biomedicine and Biotechnology

Table 3: Basic epidemiological characteristics: abundance (mean ± SD), intensity of infection (min-max), and prevalence (percentage of infected hosts) of all metazoan parasite groups.

Parasite group Abundance ±SD Intensity of infection Prevalence (in %) Before-breeding Monogenea 49.76 ±27.97 8–116 100.00 Hirudinea 0.10 ±0.41 0–2 6.90 Mollusca 0.03 ±0.19 0–1 3.45 Acanthocephala 3.97 ±5.34 0–21 75.86 Digenea 71.83 ±76.79 0–378 96.55 Cestoda 2.03 ±6.74 0–36 27.59 Nematoda 0.10 ±0.31 0–1 10.34 Breeding Monogenea 42.61 ±48.46 0–186 90.32 Hirudinea 0.03 ±0.18 0–1 3.23 Mollusca — — — — Acanthocephala 3.52 ±6.52 0–34 67.74 Digenea 58.13 ±114.3 0–474 87.10 Cestoda 0.03 ±0.18 0–1 3.23 Nematoda 0.10 ±0.3 0–1 9.68 After-breeding Monogenea 92.73 ±59.11 1–234 100.00 Hirudinea 0.30 ±0.7 0–3 20.00 Mollusca 0.27 ±1.46 0–8 3.33 Acanthocephala 3.60 ±3.8 0–14 76.67 Digenea 14.17 ±34.04 0–180 56.67 Cestoda 0.20 ±0.55 0–2 13.33 Nematoda 0.03 ±0.18 0–1 3.33

(P > .05). Even if there was no correlation between GSI 4. Discussion and head tubercles, a positive correlation between gonads and head tubercles was found using gonad weight (R = 4.1. Differences in Immunity and Physiology. In this study, 0.3943, P = .042). Moreover, spleen weight was positively the differences in fish condition and immunity were hypoth- correlated to spermatocrit (R = 0.4652, P = .014) (not esized in relation to reproductive effort of fish. Therefore, shown in the figure). Further, count of phagocytes was we analyzed several immune and physiological parameters of positively correlated to total parasite species richness (R chub, selected cyprinid fish species in three periods which = 0.4958, P = .009) (Figures 3(c) and 3(d))aswellas varied in reproductive investment, that is, before-breeding, abundance of Pomporhynchus laevis (Acanthocephala) was breeding, and after-breeding periods. The selected immune positively associated to SSI (R = 0.4434, P = .021) (Figure parameters are widely applied in immunoecological studies 3(a)) in breeding. Moreover, a positive correlation between of fish. When comparing the immune and physiological abundance of Metorchis xanthosomus and spermatocrite (R variables measurements, we found significant changes in all = 0.4421, P = .021) (Figures 3(a) and 3(b))andanegative parameters except relative weight of spleen and phagocyte correlation between total parasite species richness and count count. In spite of no significant change in relative phagocyte of erythrocytes (R = −0.3822, P = .049) (Figures 3(c) and count, the activity of phagocytes measured by respiratory 3(d)) were found. burst showed significant changes among three periods Using PCA and Pearson’s correlation for the data from investigated. The differences in proportion of developmental after-breeding period, abundance of Cestoda was negatively stages of neutrophiles were also found among the periods of correlated to fish condition (R = −0.3768, P = .048) and different reproductive investments. It may suggest that the phagocyte count (R =−0.4355, P = .021) (Figures 4(a) total phagocyte activity is potentially affected by neutrophile and 4(b)). A negative correlation was also found between profile rather than by total number of phagocytes. Moreover, phagocyte count and abundance of Sphaerostomum bramae the individual activity level of neutrophile cells is probably (R = −0.4268, P = .024) (Figures 4(a) and 4(b)). Finally, involved in the total signal of respiratory burst. Further, negative correlation between total parasite species richness in spite of the fact that the changes in the differential and both phagocyte count (R = −0.3871, P = .042) and SSI leukocyte count are considered to be one of the most sensitive (R = −0.4059, P = .032) were found (see Figures 4(c) and indicators of acute stress in fish [39], we did not record any 4(d)). noticeable change in percentage of lymphocytes and total Journal of Biomedicine and Biotechnology 7

1 1

Ph RB GSI 0.5 0.5 SSI PoLa Cest SpBr Cest 62% 19% . . RB K Ery K 0 0 SpBr PoLa MeXa Ph

Factor 2: 14 GSI Factor 3: 13

−0.5 −0.5 MeXa SSI Mono Mono Ery

−1 −1 −1 −0.50 0.51 −1 −0.50 0.51 Factor 1: 21.12% Factor 1: 21.12% (a) (b)

1 1 Ph GSI RB

0.5 0.5 ToTa K SSI Ery 54% 62% Ph Ery . . RB ToTa K 0 0

Factor 2: 20 GSI Factor 3: 16 SSI −0.5 −0.5

−1 −1 −1 −0.50 0.51 −1 −0.50 0.51 Factor 1: 24.77% Factor 1: 24.77% (c) (d)

Figure 2: Principal component analysis (PCA) to evaluate the associations within physiology, immunity, and parasitism in before-breeding period. K: condition factor; GSI: gonado-somatic index; SSI: spleen-somatic index; RB: respiratory burst; Ph: phagocyte count; Ery: erythrocyte count; Mono: Monogenea; Cest: Cestoda; MeXa: Metorchis xanthosomus;SpBr:Sphaerostomum bramae;PoLa:Pomporhynchus laevis; ToTa: total parasite species richness. neutrophiles among the periods investigated. Generally, the condition factor among the periods of different reproductive acute stress (mainly related to the changes of glucocorticoid effort. Fish were in the best condition in before-breeding level) induces both neutrophilia and lymphopenia in fish period and subsequently condition factor decreased in [40], although rarely only lymphopenia is reported [41]. spawning and after breeding. Spawning is considered to In the before-breeding period, we supposed that fish be physically demanding and stressful period [28, 42, 43]; invest more energy into the gonad development. Fish males therefore, we suggest that the fish accumulate its energy reached the highest values of GSI before breeding and reserves before breeding to obtain the sufficient resources for GSI sharply decreased in spawning. The similar changes spawning. as observed for gonad weight measured by gonadosomatic Moreover, in spawning period we revealed the highest index were observed for relative body weight measured by values of measured immune variables including respiratory 8 Journal of Biomedicine and Biotechnology

1 1

Ph Mono RB 0.5 Cest 0.5 PoLa

SSI MeXa SpBr Cest 25% 84% . Mono . MeXa 0 0 Ph RB Sperm Head tub GSI Factor 2: 15 Factor 3: 14 K SpBr Sperm K Ery − SSI Ery − 0.5 Head tub GSI 0.5 PoLa

−1 −1 −1 −0.50 0.51 −1 −0.50 0.51 Factor 1: 21.34% Factor 1: 21.34% (a) (b)

1 1

Sperm SSI 0.5 SSI 0.5 Head tub Ph GSI Ery Ery Ph 7% . 33% ToTa . 0 0 RB K ToTa K Sperm Factor 3: 15 Factor 2: 18 GSI −0 5 −0 5 . . RB Head tub

−1 −1 −1 −0.50 0.51 −1 −0.50 0.51 Factor 1: 22.35% Factor 1: 22.35% (c) (d)

Figure 3: Principal component analysis (PCA) to evaluate the associations within physiology, immunity, and parasitism in breeding period. For abbreviations see legend of Figure 2. burst, leukocyte count, and leukocrit. An increased number steroid hormones is considered to play a key role especially of leukocytes is considered to be common consequence in spawning, fish physiology in a given place and time is of infection (i.e., [44]). Surprisingly, we found that total influenced also by water temperature, quality of water and parasite diversity is lower in spawning in comparison with other abiotic factors as well as biotic interactions which may before-breeding and after-breeding periods. However, the influence a current physiological status of individuals. high values of immune parameters may also reflect the extensive stress due to spawning or alternatively those values may be induced by pathogens, such as protozoa or viruses 4.2. Differences in Parasite Infection and a Link with Immunity. which were not investigated in the present study. The parasite life cycle and real infection parameters of However, we should take into consideration that fish parasites within a given host individual or population are physiology and immunity is also affected by abiotic as well influenced by host physiology and immunity (e.g., [45– as biotic factors in each sampled period. Even if the level of 51]). In our study, we tried to evaluate the differences in Journal of Biomedicine and Biotechnology 9

1 1

GSI

MeXa 0.5 0.5 Cest PoLa Mono

43% SpBr 80% . . Ph Mono PoLa 0 Ph 0 GSI K RB SSI RB Factor 2: 15 Factor 3: 14 Cest Ery MeXa −0.5 −0.5 SpBr K SSI Ery

−1 −1 −1 −0.50 0.51 −1 −0.50 0.51 Factor 1: 19.97% Factor 1: 19.97% (a) (b)

1 1

K Ery GSI

0.5 0.5

ToTa RB K

72% 92% ToTa . . SSI RB Ery 0 0 Ph SSI Factor 2: 21 Factor 3: 15

−0.5 −0.5 GSI Ph

−1 −1 −1 −0.50 0.51 −1 −0.50 0.51 Factor 1: 26.36% Factor 1: 26.36% (c) (d)

Figure 4: Principal component analysis (PCA) to evaluate the associations within physiology, immunity and parasitism in after-breeding period. For abbreviations see legend of Figure 2. metazoan parasite infection in three periods which varied couldreflectthechangesinmonogeneaninfectionwhen in the investment in fish reproduction and likewise we comparing periods varied in reproductive investment. For studied the potential associations between selected immune example, the production of specific immunoglobulin against parameters and parasite load. gill monogeneans in different fish species (see [49, 52, Abundance of Monogenea (a group of gill and skin 53]) or the role of complement for parasite infection in parasites) reached relatively high values in before-breeding salmonid fishes was shown in experimental studies (e.g., period, subsequently decreased in breeding, and finally [54–56]). The association between infection by the gill or switched over to maximal values in after-breeding period. skin monogeneans and mucus lysozyme activity was also Although we found no correlation between measured found [52, 57, 58]. Further, parasites dispose of various immune variables and infection of Monogenea, we suggest mechanisms whereby evade or cope with fish immune that it is possible that other measurements of immunity response. For instance, several species of monogeneans look (which are not involved in recent immnoecological studies) for host sites where the immune response is not strong 10 Journal of Biomedicine and Biotechnology

Table 4: The axes of PCA explaining the total variability in the date immune defence mechanisms than the ones investigated in set. our study. In the reviews by Secombes and Chappell [46] and Alvarez-Pellitero [71], the various immune mechanisms Period Parasitism measure axis 1 axis 2 axis 3 against helminths have been summarized (incl. Cestoda), Parasite abundance 21.12 14.19 13.62 Before-breeding that is, antibody, inflammation, or complement response, Species richness 24.77 20.62 16.54 which may be caused by parasite infection. Finally, as already Parasite abundance 21.34 15.25 14.84 mentioned, no relationship between infections of Cestoda Breeding Species richness 22.35 18.33 15.78 and measured immune variables may be related to parasite invasion mechanisms, especially “antigen-based strategies.” Parasite abundance 19.97 15.43 14.80 After-breeding For instance, Bothriocephalus scorpii have the ability to bind Species richness 26.36 21.72 15.92 C-reactive protein to avoid the recognition by host’s immune system [72]. Total abundance of Digenea reached the maximal values enough to kill them. Other parasites seem to have evolved in before-breeding period and decreased toward after- and adapted their life cycles with host age or season in breeding period. Two numerous digenean parasite species, which the host immune system is weaker (see [59]) or Sphaerostomum bramae and Metorchis xanthosomus,were the monogeneans incorporate the host molecules into their analyzed separately regarding the differences within host surface to evade host immune system [49]. location and life strategy. Chub investigated in our study As mentioned above, the immune parameters are also was parasitized by Sphaerostomum bramae adults, located in influenced by water temperature and other environmental intestine, and larval stages of Metorchis xanthosomus, located factors (e.g., [60–66]). For instance, Poisot et al. [67] in skin. Concerning Sphaerostomum bramae, we found the investigated the effect of several immune parameters on significantly highest abundance in before-breeding period, metazoan parasite abundance in chub after winterizing decreased values in breeding, and subsequently marked period associated with a rapid temperature increase. They decrease after breeding (not shown in results). Evans [73] did not find any correlation between mucus activity and showed the high infection level of immature Sphaerostomum monogeneans, the most numerous metazoan parasite group, bramae during autumn and winter, subsequently rapid whose infection is expected to be associated with mucous maturation of these parasites in spring, which was accom- activity. However, they found a positive correlation between panied by decrease in infection level. Further, abundance respiratory burst and parasitism. A positive association of Metorchis xanthosomus also significantly changed among between respiratory burst and monogenean abundance three periods investigated in our study reaching the high was also found in seasonal study of chub [68], which values in before-breeding period, maximal values in breeding showed a decrease for both immune and parasite variables period, and sharp decrease in after-breeding period. The in November and increase toward spring and summer. larval stages of Diplostomum sp. represent other digenean Although the highest values of respiratory burst in our study species infecting chub. For this parasite species we observed were observed in breeding period, surprisingly the level of no significant difference when comparing three periods infection by monogeneans was low. We could hypothesize of different reproductive investments. Diplostomum species that spawning behaviour in chub prevents transmission of belongs to the group of fish parasites that develop in gill and skin parasites with direct life cycle. However, such immunoprivileged host tissues, that is, eye in this case, where hypothesis should be tested in future. High increase of host barriers prevent or limit the immune response [74]. This monogenean abundance in after-breeding period suggests may explain why no associations with measured immune that fish weakened by spawning become more susceptible to parameters were found in our study of chub. monogenean infection. The measured immune parameters Abundance of Pomporhynchus laevis, intestinal “spiny- in after-breeding period reached the lower values in compar- headed” worms, was positively correlated to SSI as well ison with breeding period. Therefore, we suppose that the as to leukocyte count (not seen in results). Although a values of measured immune parameters during the breeding little information is available on immune reactions to reflect the stress caused by spawning rather than current acanthocephalan parasites of fishes, helminth infections may parasite infection. significantly alter the number of leucocytes in the circulation Concerning Cestoda, a group of intestinal parasites, the as well as in lymphoid organs, such as the spleen and kidney decrease in abundance was revealed in breeding period [45, 75, 76]. when comparing with before-breeding period. Those results could indicate the temporal presence of intermediate hosts (i.e., [69]) or reflect the changes associated with parasite 4.3. Trade-Off between Immunity and Reproduction and the life cycle. For instance, Scholz and Moravec [70]recorded Role of Parasites. The central hypothesis of our study is a reaching maximum values of prevalence and mean intensity trade-off between immune response and other physiological of Proteocephalus torulosus in March whilst no parasites demands because of limited energy resources for each occurred in fish during summer and autumn. However, our organism. Therefore, we estimated the costs of reproduction study showed that this cestode species is present in all periods paid by weakened immune response. If the immunity varying in reproductive investment in fish. It is possible and reproduction are traded off, the potential associations that current infection by Cestodes is reflected in other between those traits are expected. Moreover, we could Journal of Biomedicine and Biotechnology 11 suppose that an individual, which immunity is weakened, digenean parasites Sphaerostomum bramae was found. Even should be more susceptible to the infection by pathogens and if no relationship was found between head tubercles and par- parasites. asite species richness or abundance, the significant positive Following the prediction of energy allocation, we correlation between the immune measures and parasitism expected the trade-off between immunity and reproduction was found in spawning. during the before-breeding period, considering the high In after-breeding period we expected and confirmed that investment in gonad development. Even if the correlation condition status of fish decreased when comparing with between GSI and relative spleen size was not significant, breeding period. Fish are weakened after spawning and this we found a trend for negative association between those may explain the increase of monogenean abundance due to variables. On the other hand, no trend of association relatively rapid direct life cycle. The investment in somatic between GSI and SSI was found in breeding and after- condition seems to play an important role for parasite breeding periods. Using PCA, the total parasite species establishing. This may explain the fact that fish in worse richness was positively associated with SSI, which suggests condition are more parasitized by Cestoda. The immunity that hosts parasitized by wider spectrum of parasite species is no more suppressed by steroid hormones in this period. dispose a large spleen because they invested more energy in We found a negative correlation between phagocyte count immune defence. The positive correlation between spleen and both parasite abundance (Cestoda and Sphaerostomum size and species richness of nematode parasites was found bramae) and total parasite species richness. Generally, the in interspecies study of males birds, using comparative high immune response (i.e., high number of phagocytes analysis, suggesting a causative role for parasitic nematodes in this case) leads to the decrease of parasite infection. in the evolution of avian spleen size [77]. Concerning fish, However, Viney et al. [80] highlighted the fact that due to the positive correlation between spleen size and parasite the autoimmunity, “immunologically more” may not mean abundance was found in interspecific comparative study necessarily “better.” Further, it was proposed that rather of cyprinid fish for females although the correlation was than the intensity of immune responses, it is recommended lacking for males [78]. Surprisingly, using PCA and Pearson to determine the optimal immune response, that is, the correlation we found the reverse relationships between fish effective protection of individuals against infection as well as condition and two most abundant digenean parasites in measurement of individuals’ fitness. before-breeding period. Condition factor was negatively correlated with abundance of Sphaerostomum bramae and positively with Metorchis xanthosomus.Asmentionedabove, 5. Conclusion ff these two parasite species di er in strategy and location We conclude that the reproductive investments in fish within fish host. Sphaerostomum bramae is an adult living in related to breeding play an important role for energy intestine of definitive host. On the other hand, metacercariae allocation among condition, immunity, and reproduction. of Metorchis xanthosomus parasitizing in fish skin are long- The investments in those traits and parasitism differ among life, resting, and intermediate stages of the parasite. Fish before-breeding, breeding, and after-breeding periods. The as intermediate host infected by metacercariae of Metorchis high values of immune parameters in breeding did not reflect species are ingested by the definitive host (i.e., carnivores ff a current infection by metazoan parasites but probably reflect feeding in aquatic habitats). Thus, the di erent relationship the stress due to spawning or may be caused by protozoan between the abundance of those digenean parasite species ff ff or viral infection. A high reproductive e ort associated and condition factor could be explained by a di erent with spawning leads to higher digenean infection in more parasite life strategy. ornamented individuals and/or males possessing the sperm In spawning period, following the sperm protection of better quality. hypothesis [16, 17], we expected and confirmed that fish males highly investing into the spawning ornamentation, that is, breeding tubercles, possess the sperm of a better Acknowledgments quality (measured by spermatocrite) than less ornamented males. Such males should be more susceptible to the diseases This study was supported by the Grant Agency of the regarding weakened immunity. In accordance with this Czech Republic, Project no. 524/07/0188. The field study prediction, we found that males with high spermatocrite and haematological analyses were partially funded by a values dispose of large spleen. Moreover, fish males with Research Project from Masaryk University, Brno, Project no. high spermatocrite value were parasitized by higher number MSM 0021622416. K. RohlenovawasfundedbyRector’s´ of digenean species Metorchis xanthosomus parasitizing fish Programme in Support of MU Students’ Creative Activities. skin. Due to potential immunosuprresion by testosterone A. Simkovˇ a´ was supported by the Ministry of Education, as highlighted by immunocompetence handicap hypothesis Youth and Sports of the Czech Republic (Project no. [15, 79], immunity of males should be weakened when LC522, Ichthyoparasitology Research Centre). The authors investing more extensively in spawning ornamentation, thank Martina Pecˇ´ınkova,´ Martina Davidov´ a,´ Radim Blazek,ˇ and then, those males should be more parasitized. Our Radim Sonnek, and Eva Rehulkovˇ a´ from the Laboratory of results partially confirmed the immunocompetence hand- Parasitology, Institute of Botany and Zoology, Faculty of icap hypothesis. A significant positive correlation between Science, Masaryk University, Brno for kindly helping with number of head breeding tubercles and abundance of parasite dissection. They thank Miroslava Pal´ıkovafrom´ 12 Journal of Biomedicine and Biotechnology the University of Veterinary and Pharmaceutical Sciences, [16] I. Folstad and F. Skarstein, “Is male germ line control creating Brno, and Anton´ın Lojek from the Institute of Biophysics, avenues for female choice?” Behavioral Ecology, vol. 8, no. 1, Academy of Science of the Czech Republic, for their help pp. 109–112, 1997. with immunology and haematology methodology and Pavel [17] S. Liljedal, I. Folstad, and F. Skarstein, “Secondary sex traits, Jurajda, Jirˇ´ı Huml, Michal Janać,ˇ and Matej Polacikˇ from parasites, immunity and ejaculate quality in the Arctic charr,” the Institute of Vertebrate Biology, Academy of Science of Proceedings of the Royal Society B, vol. 266, no. 1431, pp. 1893– the Czech Republic, for their valuable assistance in field 1898, 1999. sampling. They thank the Moravian Anglers Union for [18] R. Kortet, A. Vainikka, M. J. Rantala, and J. Taskinen, “Sperm quality, secondary sexual characters and parasitism in roach supporting the research in their entire district and for (Rutilus rutilus L.),” Biological Journal of the Linnean Society, purchasing the experimental fish. vol. 81, no. 1, pp. 111–117, 2004. [19] V. BarusandO.Oliva,ˇ Lampreys (Petromyzones) and Fishes References (Osteichthyes). Fauna of the Czech and Slovak Republics, Academia, Praha, Czech Republic, 1995. [1] D. A. Roff, The Evolution of Life Histories: Theory and Analysis, [20] M. L. Wiley and B. B. Collette, “Breeding tubercles and contact Chapman & Hall, New York, NY, USA, 1992. organs in fishes: their occurrence, structure and significance,” [2] S. C. Stearns, The Evolution of Life Histories, Oxford University Bulletin of the American Museum of Natural History, vol. 143, Press, Oxford, UK, 1992. no. 3, pp. 143–216, 1970. [21] R. Kortet, J. Taskinen, A. Vainikka, and H. Ylonen,¨ “Breeding [3] B. C. Sheldon and S. Verhulst, “Ecological immunology: costly tubercles, papillomatosis and dominance behaviour of male parasite defences and trade-offs in evolutionary ecology,” roach (Rutilus rutilus) during the spawning period,” Ethology, Trends in Ecology & Evolution, vol. 11, no. 8, pp. 317–321, vol. 110, no. 8, pp. 591–601, 2004. 1996. [22] C. Wedekind, “Detailed information about parasites revealed [4] A. P. Møller, “Immune defence, extra-pair paternity, and by sexual ornamentation,” Proceedings of the Royal Society B, sexual selection in birds,” Proceedings of the Royal Society B, vol. 247, no. 1320, pp. 169–174, 1992. vol. 264, no. 1381, pp. 561–566, 1997. [23] J. Taskinen and R. Kortet, “Dead and alive parasites: sexual [5] T. Szep´ and A. P. Møller, “Cost of parasitism and host immune ornaments signal resistance in the male fish, Rutilus rutilus,” defence in the sand martin Riparia riparia: a role for parent- Evolutionary Ecology Research, vol. 4, no. 6, pp. 919–929, 2002. ff o spring conflict?” Oecologia, vol. 119, no. 1, pp. 9–15, 1999. [24] F. Skarstein and I. Folstad, “Sexual dichromatism and the [6] D. Hasselquist, M. F. Wasson, and D. W. Winkler, “Humoral immunocompetence handicap: an observational approach immunocompetence correlates with date of egg-laying and using Arctic charr,” Oikos, vol. 76, no. 2, pp. 359–367, 1996. reflects work load in female tree swallows,” Behavioral Ecology, [25] H. Modra,´ Z. Svobodova,´ and J. Kola´rovˇ a,´ “Comparison of vol. 12, no. 1, pp. 93–97, 2001. differential leukocyte counts in fish of economic and indicator [7] A. P. Møller and N. Saino, “Immune response and survival,” importance,” Acta Veterinaria Brno, vol. 67, no. 4, pp. 215–226, Oikos, vol. 104, no. 2, pp. 299–304, 2004. 1998. [8] N. Hillgarth, M. Ramenofsky, and J. Wingfield, “Testosterone [26] L. Kubala, A. Lojek, M. Cˇ ´ız,ˇ J. Vondraćek,ˇ M. Duskovˇ a,´ and sexual selection,” Behavioral Ecology, vol. 8, no. 1, pp. 108– and H. Slav´ıkova,´ “Determination of phagocyte activity in 112, 1997. whole blood of carp (Cyprinus carpio) by luminol-enhanced [9] F. Skarstein, I. Folstad, and S. Liljedal, “Whether to reproduce chemiluminescence,” Veterinarni Medicina, vol. 41, no. 10, pp. or not: Immune suppression and costs of parasites during 323–327, 1996. reproduction in the Arctic charr,” Canadian Journal of Zoology, [27] R. Ergens and J. Lom, Causative Agents of Parasitic Fish vol. 79, no. 2, pp. 271–278, 2001. Diseases, Academia, Prague, Czech Republic, 1970. [10] E. Ottova,´ A. Simkovˇ a,´ P. Jurajda, et al., “Sexual ornamen- [28] T. Bolger and P. L. Connolly, “The selection of suitable indices tation and parasite infection in males of common bream for the measurement and analysis of fish condition,” Journal of (Abramis brama): a reflection of immunocompetence status or Fish Biology, vol. 34, no. 2, pp. 171–182, 1989. simple cost of reproduction?” Evolutionary Ecology Research, [29] A. V. Gusev, “Metazoan parasites—part I,” in Identification Key vol. 7, no. 4, pp. 581–593, 2005. to Parasites of Freshwater Fish, O. N. Bauer, Ed., vol. 2, p. 424, Nauka, Leningrad, Russia, 1985. [11] W. D. Hamilton and M. Zuk, “Heritable true fitness and bright [30] I. A. Khotenovsky, Fauna of the SSSR, Monogenea,Nauka, birds: a role for parasites?” Science, vol. 218, no. 4570, pp. 384– Leningrad, Russia, 1985. 387, 1982. [31] T. Scholz, “Amphilinida and Cestoda, parasites of fish in [12] W. J. Hamilton and R. Poulin, “The Hamilton and Zuk Czechoslovakia,” Acta Scientiarum Naturalium Academiae hypothesis revisited: a meta-analytical approach,” Behaviour, Scientiarum Bohemicae Brno, vol. 23, no. 4, pp. 1–56, 1989. vol. 134, no. 4-5, pp. 299–320, 1997. [32] F. Moravec, Parasitic Nematodes of Freshwater Fishes of Europe, [13] A. P. Møller, P. Christe, and E. Lux, “Parasitism, host immune Academia and Kluwer Academic Publishers, Praha, Czech function, and sexual selection,” Quarterly Review of Biology, Republic, 1994. vol. 74, no. 1, pp. 3–20, 1999. [33] B. Georgiev, V. Bisekov, and T. Genov, “The staining method [14] M. L. Roberts, K. L. Buchanan, and M. R. Evans, “Testing for cestodes with iron acetocarmine,” Helminthologia, vol. 23, the immunocompetence handicap hypothesis: a review of the pp. 279–281, 1986. evidence,” Animal Behaviour, vol. 68, no. 2, pp. 227–239, 2004. [34] Z. Svobodova,´ D. Pravda, and J. Palaćkovˇ a,´ Unified Methods [15] I. Folstad and A. J. Karter, “Parasites, bright males, and the of Haematological Examination of Fish, vol. 22, Manuals immunocompetence handicap,” American Naturalist, vol. 139, of Research Institute of Fish Culture and Hydrobiology, no. 3, pp. 603–622, 1992. University of South Bohemia, Vodnany,ˇ Czech Republic, 1991. Journal of Biomedicine and Biotechnology 13

[35] Z. Svobodova,´ D. Pravda, and J. Palaćkovˇ a,´ Universal Methods [52] V. L. Vladimirov, “The immunity of fishes in the case of Haematological Investigations in Fish, Edice Metodik, no. 22, of dactylogyrosis,” Parasitologiya, vol. 5, pp. 51–58, 1971 VURH,´ Vodnany,ˇ Czech Republic, 1986. (Russian). [36] V.Luskova,´ “Annual cycles and normal values of hematological [53] K. Buchmann, “A note on the humoral immune response parameters in fishes,” Acta Scientiarum Naturalium Academiae of infected Anguilla anguilla against the gill monogenean Scientiarum Bohemicae Brno, vol. 31, no. 5, p. 70, 1997. Pseudodactylogyrus bini,” Fish & Shellfish Immunology, vol. 3, [37] J. Sterzl,ˇ The Immune System and Its Physiological Functions, no. 5, pp. 397–399, 1993. Czech Society for Immunology, Prague, Czech Republic, 1993. [54] K. Buchmann, “Binding and lethal effect of complement [38]A.O.Bush,K.D.Lafferty,J.M.Lotz,andA.W.Shostak, from Oncorhynchus mykiss on Gyrodactylus derjavini (Platy- “Parasitology meets ecology on its own terms: margolis et al. helminthes: Monogenea),” Diseases of Aquatic Organisms, vol. revisited,” Journal of Parasitology, vol. 83, no. 4, pp. 575–583, 32, no. 3, pp. 195–200, 1998. 1997. [55] P. D. Harris, A. Soleng, and T. A. Bakke, “Killing of Gyrodacty- [39] G. A. Wedemeyer, B. A. Barton, and D. J. McLeay, “Stress and lus salaris (Platyhelminthes, Monogenea) mediated by host acclimation,” in Methods for Fish Biology,C.B.Schreckand complement,” Parasitology, vol. 117, no. 2, pp. 137–143, 1998. P. B. Moyle, Eds., pp. 451–489, American Fisheries Society, [56] M. Rubio-Godoy, R. Porter, and R. C. Tinsley, “Evidence of Bethesda, Md, USA, 1990. complement-mediated killing of Discocotyle sagittata (Platy- [40] A. L. Pulsford, S. Lemaire-Gony, M. Tomlinson, N. Colling- helminthes, Monogenea) oncomiracidia,” Fish & Shellfish wood, and P. J. Glynn, “Effects of acute stress on the Immunology, vol. 17, no. 2, pp. 95–103, 2004. immune system of the dab, Limanda limanda,” Comparative [57] K. Buchmann and A. Uldal, “Gyrodactylus derjavini infections Biochemistry and Physiology C, vol. 109, no. 2, pp. 129–139, in four salmonids: comparative host susceptibility and site 1994. selection of parasites,” Disease of Aquatic Organisms, vol. 28, [41] A. Larsson, K. J. Lehtinen, and C. Haux, “Biochemical no. 3, pp. 201–209, 1997. and hematological effects of a titanium dioxide industrial [58] K. Buchmann and J. Bresciani, “Microenvironment of Gyro- effluent on fish,” Bulletin of Environmental Contamination and dactylus derjavini on rainbow trout Oncorhynchus mykiss: Toxicology, vol. 25, no. 3, pp. 427–435, 1980. association between mucous cell density in skin and site [42] K. R. Munkittrick and J. F. Leatherland, “Haematocrit values selection,” Parasitology Research, vol. 84, no. 1, pp. 17–24, in feral goldfish, Carassius auratus L., as indicators of the 1997. health of the population,” Journal of Fish Biology, vol. 23, pp. [59] A. Sitja-Bobadilla, “Living off afish:atrade-off between par- 153–161, 1982. asites and the immune system,” Fish & Shellfish Immunology, [43] P. W. Wester, A. D. Vethaak, and W. B. van Muiswinkel, “Fish vol. 25, no. 4, pp. 358–372, 2008. as biomarkers in immunotoxicology,” Toxicology, vol. 86, no. [60] J. E. Bly and L. W. Clem, “Temperature and teleost immune 3, pp. 213–232, 1994. functions,” Fish & Shellfish Immunology, vol. 2, no. 3, pp. 159– [44] A. E. Ellis, “The function of teleost fish lymphocytes in relation 171, 1992. to inflammation,” International Journal of Tissue Reactions, vol. [61] M. E. Collazos, E. Ortega, and C. Barriga, “Effect of tempera- 8, no. 4, pp. 263–270, 1986. ture on the immune system of a cyprinid fish (Tinca tinca,L). [45]D.T.Richards,D.Hoole,J.W.Lewis,E.Evans,andC. Blood phagocyte function at low temperature,” Fish & Shellfish Arme, “Changes in the cellular composition of the spleen and Immunology, vol. 4, no. 3, pp. 231–238, 1994. pronephros of carp Cyprinus carpio infected with the blood [62] T. H. Hutchinson and M. J. Manning, “Seasonal trends in fluke Sanguinicola inermis (Trematoda: Sanguinicolidae),” serum lysozyme activity and total protein concentration in Diseases of Aquatic Organisms, vol. 19, no. 3, pp. 173–179, dab (Limanda limanda L.) sampled from Lyme Bay, U.K,” Fish 1994. & Shellfish Immunology, vol. 6, no. 7, pp. 473–482, 1996. [46] C. J. Secombes and L. H. Chappell, “Fish immune responses to [63] G. Scapigliati, D. Scalia, A. Marras, S. Meloni, and M. Mazzini, experimental and natural infection with helminth parasites,” “Immunoglobulin levels in the teleost sea bass Dicentrarchus Annual Review of Fish Diseases, vol. 6, pp. 167–177, 1996. labrax (L.) in relation to age, season, and water oxygenation,” [47] K. Buchmann, “Immune mechanisms in fish skin against Aquaculture, vol. 174, no. 3-4, pp. 207–212, 1999. monogeneans—a model,” Folia Parasitologica, vol. 46, no. 1, [64] A. L. Langston, R. Hoare, M. Stefansson, R. Fitzgerald, H. pp. 1–9, 1999. Wergeland, and M. Mulcahy, “The effect of temperature on [48] S. R. M. Jones, “The occurrence and mechanisms of innate non-specific defence parameters of three strains of juvenile immunity against parasites in fish,” Developmental and Com- Atlantic halibut (Hippoglossus hippoglossus L.),” Fish & Shell- parative Immunology, vol. 25, no. 8-9, pp. 841–852, 2001. fish Immunology, vol. 12, no. 1, pp. 61–76, 2002. [49] K. Buchmann and T. Lindenstrøm, “Interactions between [65] A. Hernandez´ and L. Tort, “Annual variation of complement, monogenean parasites and their fish hosts,” International lysozyme and haemagglutinin levels in serum of the gilthead Journal for Parasitology, vol. 32, no. 3, pp. 309–319, 2002. sea bream Sparus aurata,” Fish & Shellfish Immunology, vol. [50] T. K. Hatice, Z. Erdogan, and R. Coz-Rakovac, “The occur- 15, no. 5, pp. 479–481, 2003. rence of Ligula intestinalis (L.) observed in chub (Leuciscus [66] T. J. Bowden, R. Butler, and I. R. Bricknell, “Seasonal variation cephalus L.) from Caparlipatlak Dam lake, Ivrindi-Balikesir, ofserumlysozymelevelsinAtlantichalibut(Hippoglossus Turkey,” Periodicum Biologorum, vol. 108, no. 2, pp. 183–187, hippoglossus L.),” Fish & Shellfish Immunology, vol. 17, no. 2, 2006. pp. 129–135, 2004. [51] G. Munoz,A.S.Grutter,andT.H.Cribb,“Structureof˜ [67] T. Poisot, A. Simkovˇ a,´ P. Hyrsl,ˇ and S. Morand, “Interactions the parasite communities of a coral reef fish assemblage between immunocompetence, somatic condition and para- (Labridae): testing ecological and phylogenetic host factors,” sitism in the chub Leuciscus cephalus in early spring,” Journal Journal of Parasitology, vol. 93, no. 1, pp. 17–30, 2007. of Fish Biology, vol. 75, no. 7, pp. 1667–1682, 2009. 14 Journal of Biomedicine and Biotechnology

[68] K. Lamkova,´ A. Simkovˇ a,´ M. Pal´ıkova,´ P.Jurajda, and A. Lojek, “Seasonal changes of immunocompetence and parasitism in chub (Leuciscus cephalus), a freshwater cyprinid fish,” Parasitology Research, vol. 101, no. 3, pp. 775–789, 2007. [69] V. Hanzelova´ and D. Gerdeaux, “Seasonal occurrence of the tapeworm Proteocephalus longicollis and its transmission from copepod intermediate host to fish,” Parasitology Research, vol. 91, no. 2, pp. 130–136, 2003. [70] T. Scholz and F. Moravec, “Seasonal dynamics of Pro- teocephalus torulosus (Cestoda: Proteocephalidae) in barbel (Barbus barbus) from the Jihlava River, Czech Republic,” Folia Parasitologica, vol. 41, no. 4, pp. 253–257, 1994. [71] P. Alvarez-Pellitero, “Fish immunity and parasite infections: from innate immunity to immunoprophylactic prospects,” Veterinary Immunology and Immunopathology, vol. 126, no. 3- 4, pp. 171–198, 2008. [72] T. C. Fletcher, A. White, and B. A. Baldo, “Isolation of a phosphorylcholine-containing component from the turbot tapeworm, Bothriocephalus scorpii (Muller),¨ and its reaction with C-reactive protein,” Parasite Immunology, vol. 2, no. 4, pp. 237–248, 1980. [73] N. A. Evans, “The occurrence of Sphaerostoma bramae (Digenea: Allocreadiidae) in the roach from the Worcester- Birmingham canal,” Journal of Helminthology, vol. 51, no. 3, pp. 189–196, 1977. [74] M. Kalbe and J. Kurtz, “Local differences in immunocompetence reflect resistance of sticklebacks against the eye fluke Diplostomum pseudospathaceum,” Parasitology, vol. 132, no. 1, pp. 105–116, 2006. [75] A. Murad and S. Mustafa, “Blood parameters of catfish, Heteropneustes fossilis (Bloch), parasitized by metacercariae of Diplostomum sp,” Journal of Fish Diseases, vol. 11, no. 4, pp. 365–368, 1988. [76] M. Taylor and D. Hoole, “Ligula intestinalis (L.) (Cestoda: Pseudophyllidea): plerocercoid-induced changes in the spleen and pronephros of roach, Rutilus rutilus (L.), and gudgeon, Gobio gobio (L.),” Journal of Fish Biology, vol. 34, no. 4, pp. 583–596, 1989. [77] S. Morand and R. Poulin, “Nematode parasite species richness and the evolution of spleen size in birds,” Canadian Journal of Zoology, vol. 78, no. 8, pp. 1356–1360, 2000. [78] A. Simkovˇ a,´ T. Lafond, M. Ondrackovˇ a,´ P. Jurajda, E. Ottova,´ and S. Morand, “Parasitism, life history traits and immune defence in cyprinid fish from Central Europe,” BMC Evolu- tionary Biology, vol. 8, no. 29, pp. 1–11, 2008. [79] C. Wedekind and I. Folstad, “Adaptive or nonadaptive immunosuppression by sex hormones?” The American Natu- ralist, vol. 143, no. 5, pp. 936–938, 1994. [80] M. E. Viney, E. M. Riley, and K. L. Buchanan, “Optimal immune responses: immunocompetence revisited,” Trends in Ecology & Evolution, vol. 20, no. 12, pp. 665–669, 2005.

Článek C

Are fish immune systems really affected by parasites? An immunoecological study of common carp (Cyprinus carpio)

Parasites and Vectors 4: 120 (2011)

ROHLENOVÁ K, MORAND S, HYRŠL P, TOLÁROVÁ S, FLAJŠHANS M, ŠIMKOVÁ A

Rohlenová et al. Parasites & Vectors 2011, 4:120 http://www.parasitesandvectors.com/content/4/1/120

RESEARCH Open Access Are fish immune systems really affected by parasites? an immunoecological study of common carp (Cyprinus carpio) Karolína Rohlenová1, Serge Morand2, Pavel Hyršl3,Soňa Tolarová3, Martin Flajšhans4 and Andrea Šimková1*

Abstract Background: The basic function of the immune system is to protect an organism against infection in order to minimize the fitness costs of being infected. According to life-history theory, energy resources are in a trade-off between the costly demands of immunity and other physiological demands. Concerning fish, both physiology and immunity are influenced by seasonal changes (i.e. temporal variation) associated to the changes of abiotic factors (such as primarily water temperature) and interactions with pathogens and parasites. In this study, we investigated the potential associations between the physiology and immunocompetence of common carp (Cyprinus carpio) collected during five different periods of a given year. Our sampling included the periods with temporal variability and thus, it presented a different level in exposure to parasites. We analyzed which of two factors, seasonality or parasitism, had the strongest impact on changes in fish physiology and immunity. Results: We found that seasonal changes play a key role in affecting the analyzed measurements of physiology, immunity and parasitism. The correlation analysis revealed the relationships between the measures of overall host physiology, immunity and parasite load when temporal variability effect was removed. When analyzing separately parasite groups with different life-strategies, we found that fish with a worse condition status were infected more by monogeneans, representing the most abundant parasite group. The high infection by cestodes seems to activate the phagocytes. A weak relationship was found between spleen size and abundance of trematodes when taking into account seasonal changes. Conclusions: Even if no direct trade-off between the measures of host immunity and physiology was confirmed when taking into account the seasonality, it seems that seasonal variability affects host immunity and physiology through energy allocation in a trade-off between life important functions, especially reproduction and fish condition. Host immunity measures were not found to be in a trade-off with the investigated physiological traits or functions, but we confirmed the immunosuppressive role of 11-ketotestosterone on fish immunity measured by complement activity. We suggest that the different parasite life-strategies influence different aspects of host physiology and activate the different immunity pathways.

Background also strongly influenced by water temperature changes Physiology and immunity in fish, a group of poikilother- [1,2]. mic vertebrates, are strongly influenced by both abiotic To determine whether the observed status of fish phy- and biotic factors. Water temperature is generally con- siology results from abiotic changes or reflects the level sidered as the strongest abiotic factor which affects fish of parasite infestation is very difficult in natural condi- physiology including immune functions. However, the tions because of the confounding effects of several abio- infection dynamics of fish parasites and pathogens is tic and biotic factors including parasitism, often varying in space and time. Recently, many studies have focused * Correspondence: [email protected] on the abiotic effects, especially of water temperature, 1Department of Botany and Zoology, Faculty of Science, Masaryk University, on physiological and immunological mechanisms in poi- ř Kotlá ská 2, 611 37 Brno, Czech Republic kilothermic organisms, like fish. The majority of Full list of author information is available at the end of the article

© 2011 Rohlenová et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Rohlenová et al. Parasites & Vectors 2011, 4:120 Page 2 of 18 http://www.parasitesandvectors.com/content/4/1/120

immunological studies have suggested an immune-sup- Furthermore Folstad and Karter [28] introduced the pression effect associated with a decrease in water tem- immunocompetence handicap hypothesis, which pre- perature [3-7]. Moreover, the immunosuppressive effects dicts that the steroid hormones responsible for the pro- of polychlorinated biphenyls are known in fish species duction of sexual signals in males may cause [e.g. [8,9]]. Teleost fish possess similar immune system immunosuppression especially during the reproductive mechanisms to mammals - both non-specific (innate or period. Many recent studies have been conducted to test natural) and specific (acquired or adaptive) [10]. How- whether the expression of sexual ornamentation is asso- ever, substantial differences exist between the immune ciated to immunosuppression in fish [e.g. [29,30]]. systems of poikilo- and homoiothermic organisms. The aims of the present study were to analyze the According to Ainsworth et al. [11], the specific branch effects of seasonal changes (i.e. temporal variability) on of immunity is more sensitive than the non-specific (a) selected physiological and immune parameters and defence at lower water temperature, and was assumed on (b) the infection by metazoan parasites. We exam- to be more important for poikilothermic than for homo- ined the potential associations between immunity, phy- iothermic vertebrates [12]. Moreover, Le Morvan et al. siology and parasite infection following the assumption [5] suggested that, at low water temperature, the non- of trade-offs in energy allocation using the total invest- specific defence of fish immune system tends to offset ment in immunity, physiology and total measure of specific immune suppression until the specific immune parasitism as well as analyzing each measure of immu- system adapts. nity and physiology separately. We tried to estimate Several studies have reported that the decrease in whether the seasonal changes of abiotic environment water temperature may cause the suppression of are the main driver of immune activation in order to acquired immunity, with the components of innate face risks of being parasitized or whether the observed immunity being relatively independent of water tem- associations between immunity and physiology are the perature [13]. Other seasonally-dependent events like results of trade-off without being affected by seasonal spawning in fish could more strongly influence immu- changes. Finally, we tested the hypothesis of immuno- nity than water temperature [14]. However, many stu- suppression by 11-ketotestosteron in fish males. dies have also shown how water temperature drives the seasonal changes in parasite infection, mainly because Methods parasite reproduction and survival of free-living infective Fish sampling stages of parasites are dependent on a specific range of A total number of 160 three-to-four-year-old individuals temperature [15]. of common carp (Cyprinus carpio Linnaeus 1758, Cypri- Close interactions occur likewise between fish host nidae), including 87 males and 73 females were collected and parasites. The interactions between fish physiology from a pond-farmed population (Vodňany, South Bohe- (associated with host size, age, sex etc.) and the level of mia, Czech Republic) in five selected months in 2007 parasite infection have been relatively well documented and 2008. Each sample represented a different season [16,17]. However, there have been few studies investigat- and diverse water temperature, i.e. June 2007 - early ing the effects of seasonal changes on selected measures summer (16°C), August 2007 - late summer (18.4°C), of host physiology in relation to parasite load [e.g. [18]], November 2007 - autumn (4.9°C), February 2008 - win- and fish immune response has mostly been studied ter (2.8°C) and April 2008 - spring (7.5°C). The tem- solely in relation to parasite infection [19,20]. perature on the day of collection was measured. The The contribution of immunoecological studies has fish were sampled using seine netting and then sepa- gained a place of central importance [21,22]. Life-history rated according to their sex. Samples of blood were theory is at the core of immunoecological studies. The taken immediately from each specimen from the caudal principle assumption is that each organism has a limited vein using heparinized syringes following Pravda and amount of energy which is allocated to different funda- Svobodová [31]. Blood was preserved in microtubes with mental functions (i.e. maintenance including immune heparin (50 U/ml, Zentiva). Blood samples used for defence, reproduction and growth) in accordance with measuring oxidative burst activity and haematology (dif- current needs [23]. The activation of the immune sys- ferential leukocyte counts and total leukocyte counts) tem is energetically costly [24,25]. Therefore, trade-offs were processed immediately after blood collection. are expected to occur as hosts infected by parasites Blood samples used for other immunological analyses should invest energy into immune responses, at the (complement activity, IgM) and 11-ketotestosterone expense of other physiological tasks [26]. However, opti- concentration were deep-frozen at -80°C. mum host defence is governed by a parasite-mediated Each fish individual was intramuscularly tagged for allocation trade-off between growth and immune func- later identification using a P.I.T. tag (134.2 kHz, AEG tion (see Tschirren and Richner [27]). ID-162, AEG Co., Ulm, Germany) in the left side of the Rohlenová et al. Parasites & Vectors 2011, 4:120 Page 3 of 18 http://www.parasitesandvectors.com/content/4/1/120

dorsal part close to the first hard dorsal fin ray. Fish of measurement (equal to 4 h) and the time necessary were transported to the laboratory in tanks containing for killing 50% E. coli by complement (in h). For the original water from the pond. Fish were killed by sever- details see Buchtíková et al. [42]. ing the spine. Each individual was measured (total length in centimetres) and weighed (in grams). A com- The determination of total IgM level plete parasitological dissection for all metazoan parasites The major component of fish specific humoral defence was immediately performed for 145 individuals (includ- is immunoglobulin M (IgM), although IgD [43] and ing 80 males and 65 females) according to Ergens and even IgZ and IgT [44,45] have also been recently Lom [32]. described. Clear seasonal changes of plasma IgM levels were found to be related to water temperature and/or Respiratory burst activity gonad maturation [46]. The production of specific Phagocytes (granulocytes and monocytes/macrophages) immunoglobulin against gill monogeneans [16] or other areconsideredtobethefirstlineofimmunedefence helminths [47] was observed. Specific antibodies play an against pathogens overcoming the natural barriers. essential role in cytolytic or cytotoxic mechanisms, such These cells have the ability to engulf and kill pathogens as in the activation of the complement system (classical during the so-called “oxidative burst” leading to produc- pathway) or helping leukocytes to adhere to the parasite tion of reactive oxygen species [33]. Seasonal changes in surface, presumably through Fc-like receptors [48]. phagocytic activity have been studied in different fish The total IgM level was determined using precipita- species [34,11]. tion with zinc sulphate (0.7 mM ZnSO4.7H2O, pH = Respiratory burst activity was measured as luminol- 5.8) [49]. The concentration of IgM in the sample (in g/ enhanced chemiluminescence using a luminometer l) was calculated as the difference between total proteins (LM01-T, Immunotech, Czech Republic) and opsonised (commercial kit, Bio-Rad, USA) and proteins in the Zymosan A as activator [35,36]. The maximal intensity supernatant after precipitation and centrifugation. of respiratory burst (peak in relative light units - RLU) was evaluated in this study. 11-ketotestosterone level The immune system is affected by the level of steroid The measurement of complement activity in plasma hormones. The 11-ketotesterone (11-KT) is a major Among other non-specific humoral factors (i.e. non-cel- androgen in the majority of teleost fish, responsible for lular defence mechanisms) complement system plays an sexual behaviour and spermatogenesis, found in higher important role in natural defence against pathogens. levels in the blood plasma or serum in males than in Complement contains a series of serum proteins that females [50]. As already mentioned, these hormones are activated using either a classical (antibody depen- have an immunosuppressive effect. The level of 11-keto- dent) or alternative pathway. Complement participates testosterone in male plasma was analyzed following the in lytic, pro-inflammatory, chemotactic and opsonic protocol provided in the commercial competitive activities, thus it forms the connection between non- enzyme immunoassay kits (Cayman Chemical, Estonia). specific humoral and cellular mechanisms (i.e. phagocyte For the details see Buchtíková et al. [42]. responses) [37]. One of the most important and well known complement functions is the capacity to create Haematological analyses pores in the membrane of the pathogens’ surface and The determinants of white-blood cell count (including thereby kill them. Hernández et al. [7] reported a close leukocyte and lymphocyte counts, and differential leuko- relationship between water temperature and the level of cyte counts) are considered to be an important para- complement activity. The role of complement in mono- meter of fish health status. Like other haematological genean infection was demonstrated in salmonid fish parameters, white-blood cell counts depend on various [38,39]. abiotic and biotic factors such as water temperature, Complement activity was measured according to Virta environmental stress, fish sex and age [51,52]. According et al. [40] and Nikoskelainen et al. [41] with modifica- to Ruane et al. [53], fish infected by parasites signifi- tions. The total complement activity (including all acti- cantly changed their haematological parameters. Leuko- vation pathways) of plasma was determined using a cyte counts can be applied as a measure of general bioluminescent strain of Escherichia coli (K12luxAmp, immune response. The increased leukocyte counts and kindly provided by University of Turku, Finland). The shift values towards the myeloid line (especially a high light emission measured by LM01-T luminometer was number of myelocytes and metamyelocytes) reflect the positively correlated with the viability of E. coli.The current infection or inflammation [54]. relative measure of complement activity was estimated Leukocyte counts (in g/l) were determined according by computing the difference between the maximal time to the methodology of Svobodová et al. [55] and Rohlenová et al. Parasites & Vectors 2011, 4:120 Page 4 of 18 http://www.parasitesandvectors.com/content/4/1/120

Lusková [56] using Natt-Herrick solution. A differential Statistical analyses leukocyte profile was assessed following Lamková et al. Most of the measured parameters did not fit a normal [18]. We estimated the percentage distribution of all distribution and required transformation logarithm, types of white blood cells. However, we used only the hyperbolic arcsine, hyperbolic arcsine square root or leukocyte count and the relative count of lymphocytes square root transformations. First, preliminary analyses and phagocytes (in g/l) for statistical analyses (see Roh- testing the effects of season and sex on parasitism, phy- lenová et al. [30]). Finally, haemoglobin content (Hb) siology and immunity were performed using one-way was analyzed photometrically (540 nm; Helios Unicam, ANOVAs. USA) in Kampen-Zijlster transformation medium. Following Poisot et al. [71], we used an indicator of parasite community structure based on principal com- Spleen size ponent analysis (PCA) that takes into account all para- The spleen, the thymus, but also the head and trunk of site species and the number of individuals for each the kidney belong to the principal lymphomyeloid tis- parasite species within host individuals. In the same sues of teleosts. The spleen as a secondary lymphatic manner, we used an indicator of immunity based on and scavenging organ plays an important role in haema- PCA that takes into account all immune variables topoiesis, antigen degradation and antibody production (spleen-somatic index, leukocyte, lymphocyte and pha- processing [57]. This organ is also known to act as an gocyte counts, IgM level, respiratory burst and comple- erythrocyte reservoir [58]. The spleen size of fish is ment activity). Finally, an indicator of physiology was widely used as a simple measurable immune parameter also based on PCA that takes into account parameters with a potential role in immune response against para- linked to physiology (condition factor, gonado-somatic site infection [59-61]. A link between spleen size and index, hepato-somatic index and haemoglobin). fish condition was also previously documented [62]. In We extracted values of the first three principal com- this study, we measured spleen weight (in grams at ponents (PCs) of each of these PCAs (i.e. principal com- accuracy 0.001 g). The spleen-somatic index (SSI) was ponents having eigenvalues over one were considered calculated as spleen weight (g)/body weight (g) × 100. following Vainikka et al. [72]). These PCs represent a measure of parasitism (PCs of parasites), a measure of Other physiological parameters immunity (PCs of immune variables) or a measure of We measured liver and gonad weight (in grams). We physiology (PCs of physiological variables) respectively. calculated the relative body weight (i.e. condition fac- Then, we performed Pearson correlation between sam- tor, K) using the equation: K = constant × somatic pling period and all three PCs for each parasitism, weight (g)/(standard length [cm])3 according to Bolger immunity and physiology. Further, partial correlations and Connolly [63]. The relative size of gonad (i.e. controlling for sampling period were performed to ana- gonado-somatic index, GSI) was calculated as follows: lyze the potential relationships between parasitism, GSI = gonad weight (g)/body weight (g) × 100, and the immunity and physiology. Next, GLM analyses were relative size of liver (i.e. hepato-somatic index, HSI) performed to investigate the potential associations was calculated as follows: HSI = liver weight (g)/body between parasite abundance and all immune and phy- weight (g) × 100. siological variables taking into account the sampling period. Last, GLM analyses were conducted to analyze Parasite collection and determination the associations between immune and physiological vari- Fish hosts were investigated for all metazoan parasites ables taking into account the effect of sampling period (Table 1). Collected parasites were determined using or alternatively the effects of both sampling period and recent keys [64-68]. Due to the very high parasite sex following the preliminary analyses. As the spleen, abundance on fish gills and because Dactylogyrus (see gonad and liver weights were correlated with total body Table 1), in particular, die quickly after the killing of weight; we used SSI, GSI or HSI. All statistical analyses the fish, we collected these parasites only from the were executed using Statistica 9.0 for Windows. right side of the gills following Kadlec et al. [69]. For the details on parasite fixation and identification see Results Lamková et al. [18]. Epidemiological characteristics Seasonal changes of parasite infection and gender such as prevalence (percentage of infected host indivi- differences duals in each sample), intensity of infection (number The basic characteristics of parasite infection were esti- of parasites per infected host), and abundance (mean mated for each parasite species within each sampling number of parasites per host individual in each seaso- period (Table 1). The metazoan parasites belonging to nal sample) were calculated for each parasite species six parasitic groups were found on common carp according to Bush et al. [70]. including ectoparasitic Monogenea, Crustacea, Mollusca http://www.parasitesandvectors.com/content/4/1/120 Table 1 Parasite abundance, intensity of infection and prevalence Rohlenová Abundance Intensity of infection Prevalence Abundance Intensity of infection Prevalence Abundance Intensity of infection Prevalence ±SD (min-max) (%) ±SD (min-max) (%) ±SD (min-max) (%)

Parasites Parasite species Early summer Late summer Autumn al et .

Monog D. molnari Ergens & Dulma, 79.04 ± 6-204 100 1065.63 ± 267-2450 100 1816.62 ± 159-6879 100 Vectors & Parasites 1969 54.61 592.69 1623.86 D. extensus Mueller & Van 16.33 ± 9.55 1-40 100 179.39 ± 4-611 100 99.95 ± 0-1212 97 Cleave, 1932 132.67 222.96 D. falciformis Achmerow, 1952 7.16 ± 8.77 0-32 84 63.9 ± 46.05 3-167 100 207.54 ± 30-528 100 156.32 D. achmerowi Gussev, 1955 3.55 ± 5.08 0-20 72 36.21 ± 0-81 97 46.16 ± 0-133 97 21.65 35.16 2011, D. anchoratus (Dujardin, 1845) 0.08 ± 0.28 0-1 8 2.3 ± 3.82 0-12 30 1 ± 3.93 0-19 7 4 Gyrodactylus spp. 0.88 ± 1.9 0-8 28 - - - 0.8 ± 1.32 0-4 33 :120 Eudiplozoon nipponicum 8.44 ± 4.08 2-20 100 22.77 ± 0-53 97 1.1 ± 1.63 0-7 47 (Goto, 1891) 17.45 Crusta Argulus foliaceus (Linnaeus, 13.4 ± 11.22 3-47 100 9.27 ± 7.17 0-27 87 8.57 ± 5.79 0-25 97 1758) Ergasilus sieboldi Nordmann, 0.04 ± 0.2 0-1 4 - - - 0.07 ± 0.25 0-1 7 1832 Cesto Antractolytocestus huronensis 6.24 ± 15.30 0-56 28 472.83 ± 0-5014 83 19.73 ± 0-119 43 Anthony 1958 964.82 36.26 Khawia sinensis Hsü, 1935 0.16 ± 0.55 0-2 8 - - - 0.5 ± 1.43 0-6 17 Valipora campylancristrota - - - 0.17 ± 0.65 0-3 7 - - - (Wedl, 1855) Dige Diplostomum larv sp. 5.28 ± 6.94 0-31 68 1.97 ± 3.50 0-12 33 6.13 ± 7.74 0-31 73 Moll Glochidium spp. - - - 0.07 ± 0.37 0-2 3 - - - Hirud Piscicola geometra (Linnaeus, 0.04 ± 0.2 0-1 4 - - - 0.13 ± 0.43 0-2 10 1761) Winter Spring Monog D. molnari Ergens & Dulma, 81.72 ± 0-825 97 229.56 ± 23-3653 100 1969 144.99 662.57 D. extensus Mueller & Van 1.25 ± 2.55 0-9 33 2.59 ± 7.08 0-38 59 Cleave, 1932 D. falciformis Achmerow, 1952 3.99 ± 12.15 0-67 67 30.58 ± 0-803 86 148.64 D. achmerowi Gussev, 1955 12.69 ± 0-161 90 12.92 ± 0-172 97 30.36 31.04 D. anchoratus (Dujardin, 1845) ------Gyrodactylus spp. 303.83 ± 0-5664 70 2.14 ± 2.29 0-8 66 1094.16 18 of 5 Page Eudiplozoon nipponicum 0.43 ± 0.86 0-3 27 0.3 ± 0.53 0-2 33 (Goto, 1891) http://www.parasitesandvectors.com/content/4/1/120 Rohlenová tal et . aaie Vectors & Parasites 2011, 4 Table 1 Parasite abundance, intensity of infection and prevalence (Continued) :120 Crusta Argulus foliaceus (Linnaeus, - - - 0.03 ± 0.19 0-1 3 1758) Ergasilus sieboldi Nordmann, 0.03 ± 0.18 0-1 3 - - - 1832 Cesto Antractolytocestus huronensis ------Anthony 1958 Khawia sinensis Hsü, 1935 1.37 ± 3.52 0-15 20 0.62 ± 1.40 0-6 24 Valipora campylancristrota 0.03 ± 0.18 0-1 3 - - - (Wedl, 1855) Dige Diplostomum larv sp. 3.7 ± 4.02 0-16 80 4.05 ± 3.43 0-14 86 Moll Glochidium spp. ------Hirud Piscicola geometra (Linnaeus, 0.03 ± 0.18 0-1 3 - - - 1761)

Parasite abundance (mean with standard deviation), intensity of infection (minimum and maximum values) and prevalence for each metazoan parasite species found on common carp collected within each sampled period. Monogenea (Monog), Crustacea (Crusta), Cestoda (Cesto), Digenea (Dige), Mollusca (Moll), and Hirudinea (Hirud). ae6o 18 of 6 Page Rohlenová et al. Parasites & Vectors 2011, 4:120 Page 7 of 18 http://www.parasitesandvectors.com/content/4/1/120

and Hirudinea, and endoparasitic Cestoda and Digenea. decreasedfromearlysummertospring(Figure1B) No Nematoda or Acanthocephala were observed. Mono- resulting from the seasonal variation of Argulus foliaceus genea was the species’ richest and most numerous (see Table 1). Cestoda reached highest abundance in late group (almost 90% of total parasite abundance) and summer (Figure 1C) resulting from the seasonal varia- included Eudiplozoon nipponicum, five Dactylogyrus spe- tion of Antractolytocestus huronensis (see Table 1). Only cies (four of them present in all sampling periods) and a slight seasonal variation in abundance of larval viviparous Gyrodactylus species. In addition, three spe- Digenea was found (Figure 1D). ANOVA showed a cies of Cestoda, two species of Crustacea, one species of weak significant effect of host sex only on the abun- HirudineaandonespeciesofDigeneawerefound.The dance of Cestoda (F1, 142 = 4.1041, p = 0.045) with larval stages of Mollusca were undetermined. Following higher abundance values recorded in females. the variability in abundance among the various metazoan parasites (see Table 1), only the parasite Seasonal effect on host physiology and immunity and groups with high abundance were included in statistical gender differences analyses i.e. Monogenea as the most abundant group, All measured variables were influenced by the sampling Crustacea and Cestoda characterized by the presence of period: condition factor (F4, 139 = 11.619, p < 0.0001), one dominant species and one or two rare species, and GSI (F4, 137 = 19.927, p < 0.0001), HSI (F4, 139 = 42.483, Digenea represented only by larval stages of Diplosto- p < 0.0001), haemoglobin concentration (F4, 150 = mum species. Due to low abundance Hirudinea and 30.747, p < 0.0001), SSI (F4, 139 = 8.630, p < 0.0001), Mollusca were not included into statistical analyses. leukocyte count (F4, 150 = 26.741, p < 0.0001), lympho- Using one-way ANOVA, significant effects of sam- cyte count (F4, 149 = 7.484, p < 0.0001), phagocyte count pling period were observed on the abundance of Mono- (F4, 150 = 17.871, p < 0.0001), respiratory burst (F4, 150 = genea (F4,139 = 66.951, p < 0.0001), Cestoda (F4, 139 = 14.131, p < 0.0001), IgM concentration (F4, 149 = 7.484, 44.108, p < 0.0001) and Crustacea (F4, 139 = 25.707, p < p < 0.0001), activity of complement (F4, 133 = 29.293, p 0.0001). A marginal but significant effect of sampling < 0.0001) and concentration of 11-ketotestosterone mea- period on the abundance of Digenea was found (F4, 132 sured in males (F4,84 = 4.541, p < 0.01). = 2.488, p = 0.046). Clear patterns emerge for Monoge- The highest values of condition factor and HSI were nea with the highest values of abundance observed in detected in winter and spring (Figure 2A, C), whereas GSI late summer and autumn (Figure 1A) due to peak infec- reached the highest values in summer and autumn (Figure tion of Dactylogyrus. D. molnari, in particular, reached 2B). The highest values of haemoglobin concentration extremely high abundance. Viviparous Gyrodactylus spe- (Figure 2D) and SSI (Figure 3A) were found in early sum- cies were present only in winter (in very high abun- mer. Lymphocyte counts (Figure 3B) and total leukocyte dance) and spring (Table 1). Abundances in Crustacea counts (not shown) showed similar seasonal variations with high values in autumn and spring and low values in winter. On the other hand, the phagocyte count increased 3.6 18 A Monogenea 16 B Crustacea 3.4 from summer to winter and reached maximum values in 14 3.2 12 3.0 spring (Figure 3C). Values of respiratory burst were low in 10 2.8 8 late summer and significantly increased in autumn and the 2.6 6 2.4 4 following periods (Figure 3D). The highest IgM concentra- 2 2.2 0 2.0 tion level was recorded in early summer and autumn (Fig- -2 1.8 -4 ure 3E). The concentration level of 11-ketotestosterone in 1.6 -6 4 9 males increased in spring (not shown), whereas comple- Digenea C Cestoda 8 D

3 7 ment activity (considered for both males and females) 6 achieved its lowest values in spring (Figure 3F). 2 5 4 The effect of sex on each immune and physiological 1 3 2 variable was tested using ANOVA. Significant differences l a rv 1 e 0 int ce

n were revealed only for IgM concentration (F1,152 = 34.265, de ﬁ

0 n co

95 . -1 -1 0 p < 0.0001) with significant higher values in females, and early late autumn winter spring early late autumn winter spring summer summer summer summer for haemoglobin concentration (F = 24.588, p < Sampling period Sampling period 1, 153 Figure 1 The changes in parasite abundance in relation to 0.0001) with significant higher values in males. sampling period. Detailed legend: The changes in abundance of (A) Monogenea, (B) Crustacea, (C) Cestoda and (D) Digenea in Parasitism versus immunity and physiology: the effect of relation to sampling period. Log transformation for abundance of sampling period Monogenea and hyperbolic arcsine square root transformation for PCA performed on parasites showed that the first three Cestoda were applied. axes accounted for most of the total variability in the Rohlenová et al. Parasites & Vectors 2011, 4:120 Page 8 of 18 http://www.parasitesandvectors.com/content/4/1/120

0.54 data set. The first axis explained 42.0%, the second

0.52 A explained 26.3% and the third explained 21.1% of the

0.50 total variability in the parasitological data set (see Figure

0.48 4A for the representation using first and second axes).

0.46 The values of the first three PCs were extracted as a

on factor measure of parasitism (PCs 1, 2 and 3 for parasitism).

ti 0.44

0.42 PC1 for parasitism was negatively related to abundance Condi 0.40 of Monogenea, Cestoda and Crustacea. PC2 for parasit-

0.38 ism was positively related to abundance of Digenea and

0.36 finally, PC3 for parasitism was positively related to

0.34 abundance of Crustacea and negatively to abundance of Monogenea(Table2).ThePCAonphysiologyshowed 3.4 that the first three axes accounted for most of the total 3.2 B

3.0 variability in the physiological data set. The first axis

2.8 explained 52.9%, the second explained 20.5% and the c index

ti 2.6 third axis explained 17.2% of the variability in the data

2.4 set (see Figure 4B for the representation using first and

2.2 second axes). The values of the first three PCs were Gonado-soma 2.0 extracted as a measure of physiology (PCs 1, 2 and 3 for

1.8 physiology). PC1 for physiology was negatively related

1.6 most clearly to LSI and condition and positively to GSI

1.4 and haemoglobin concentration. PC2 for physiology was negatively related to haemoglobin concentration and 0.8 C condition. PC3 for physiology was positively related to 0.7 GSI (Table 2). Finally, we performed similar analyses for the immune variables. PCA showed that the first three 0.6 axes accounted for most of the total variability in the c index ti data set, with the first axis explaining 33.1%, the second 0.5 explaining 22.0% and the third explaining 16.4% of the 0.4

Hepato-soma variability in the data set (see Figure 4C for the representation using first and second axes). The values of the 0.3 first three PCs were extracted as a measure of immunity

0.2 (PCs 1, 2 and 3 for immunity). PC1 for immunity was most clearly related to leukocyte, lymphocyte and pha- 2.20 D gocyte counts, and respiratory burst. PC2 for immunity 2.15 was positively related to phagocyte count, respiratory 2.10 on

ti burst and SSI, but negatively related to lymphocyte 2.05 count. Finally, PC3 for immunity was indicative of the 2.00 complement system activity and, IgM level (Table 2). 1.95 Sampling period was significantly correlated with PC1

1.90 l and PC3 for parasitism, PC1 for physiology and PC1 a rv Haemoglobin concentra Haemoglobin te

1.85 in and PC2 for immunity (p < 0.001). The seasonal varia- ce n de

ﬁ tion of parasitism, immunity and physiology using PC1

1.80 n co is shown in Figure 4D-F). After correcting for sampling 1.75 0.95 early late autumn winter spring period (Table 3), the significant negative correlation summer summer between PC1 for parasitism and two PCs for physiology Sampling period as well as between PC3 for parasitism and PC2 for phy- Figure 2 The changes in physiological variables. Detailed legend: The changes in the following physiological variables (A) siology were found. Moreover, PC1 for parasitism with condition factor, (B) gonado-somatic index, (C) hepato-somatic PC2 for immunity and PC2 for parasitism with PC1 for index and (D) haemoglobin concentration in relation to sampling immunity were significantly positively correlated. Finally, period. Log transformation for condition factor, hepato-somatic PC2 for immunity was significantly negatively correlated index and haemoglobin concentration; and hyperbolic arcsine with PC1 and PC2 for physiology, but PC3 for immunity square root transformation for gonado-somatic index were applied. was significantly positively correlated with PC2 and PC3 for physiology. Rohlenová et al. Parasites & Vectors 2011, 4:120 Page 9 of 18 http://www.parasitesandvectors.com/content/4/1/120

-0.40 2.6 B A 2.5 -0.45 2.4 -0.50 2.3

c index c -0.55 2.2 ti

-0.60 2.1 Lymphocytes 2.0

Spleen-soma -0.65 1.9 -0.70 1.8

-0.75 1.7 2.0 14 1.9 C 13 D 1.8 12 1.7 11 10 1.6 9 1.5 8

Phagocytes 1.4 7

1.3 burst Respiratory 6 1.2 5 1.1 4 1.0 3 0.9 2

24 1.4 E F 22 1.3

20 1.2

18 1.1

16 1.0

IgM Complement TP Complement 14 0.9 l a rv 12 0.8 te in ce n de

0.7 ﬁ 10 n co

95 . 8 0.6 0 early late autumn winter spring early late autumn winter spring summer summer summer summer Sampling period Sampling period Figure 3 The changes in immune variables. Detailed legend: The changes in the following immune variables (A) spleen-somatic index, (B) lymphocyte count, (C) phagocyte count, (D) respiratory burst activity, (E) IgM level and (F) complement activity in relation to sampling period. Log transformation for spleen-somatic index; hyperbolic arcsine square root transformation for phagocyte count, lymphocyte count and complement activity; and square root transformation for respiratory burst were applied.

The different parasite life strategies: a link with host performed (Table 4). Except for the case of Digenea, the physiology and immunology abundance of all other analyzed parasite groups (i.e. GLM analyses on the abundance of different parasite Monogenea, Cestoda and Crustacea) was found to be groups (representing parasites with different life strate- significantly dependent on sampling period. Moreover, gies) as a function of immune and physiological vari- significant relationships between the abundance of ables and taking into account sampling periods were Monogenea and two physiological variables, i.e. Rohlenová et al. Parasites & Vectors 2011, 4:120 Page 10 of 18 http://www.parasitesandvectors.com/content/4/1/120

1.0 1.5 A DIGE D 1.0

0.5 0.5 CRUSTA sm

MONOG ti 0.0 0.0 CESTO -0.5 PC 2: 26.34 % 2: 26.34 PC PC 1 for Parasi PC 1 for -0.5 -1.0

-1.5 -1.0 -2.0 -1.0 -0.5 0.0 0.5 1.0 PC 1: 41.99 %

1.5 1.0 B E 1.0

0.5 0.5

0.0 GSI 0.0 HSI 2: 20.53% -0.5 PC PC 1 for Physiology PC 1 for -0.5 K -1.0 Hb -1.5 -1.0 -2.0 -1.0 -0.5 0.0 0.5 1.0 PC 1: 52.92 %

2.5 1.0 C F 2.0 SSI RB 0.5 Phag 1.5

% 1.0 Comp 0.0 0.5 PC 2: 22.00 2: 22.00 PC

Leu Immunity PC 1 for 0.0

-0.5 IgM Lym -0.5 dence interval -1.0 ﬁ -1.0 -1.5 0.95 con -1.0 -0.5 0.0 0.5 1.0 early late autumn winter spring PC 1: 33.11 % summer summer Sampling period Figure 4 PCA on parasitism, physiology and immunity in common carp. Detailed legend: Principal component analyses on (A) parasitism including abundance of Monogenea (MONOG), Crustacea (CRUSTA), Cestoda (CESTO), and Digenea (DIGE), (B) physiological variables including condition factor (K), gonado-somatic index (GSI), hepato-somatic index (HSI), haemoglobin concentration (Hb), and (C) immune variables including spleen-somatic index (SSI), leukocyte (Leu), lymphocyte (Lym) and phagocyte count (Phag), respiratory burst (RB), IgM concentration (IgM) and complement activity (Comp). The changes of (D) index of parasitism (using PC1 of parasitism), (E) index of physiology (using PC1 of physiology) and (F) index of immunity (using PC1 of immunity) in relation to sampling periods are shown. Rohlenová et al. Parasites & Vectors 2011, 4:120 Page 11 of 18 http://www.parasitesandvectors.com/content/4/1/120

Table 2 Component scores of the three principal (measured by GSI) and linked to host immunity mea- components of host physiology, host immunity and sured by respiratory burst and complement activity, parasitism which suggests a potential trade-off between immunity Physiology PC1 PC2 PC3 and reproduction. The reversed patterns of seasonal K -0.67 -0.55 0.46 changes for 11-KT (not shown) and complement activity GSI 0.75 0.00 0.60 (Figure 3F) levels were revealed. Phagocyte counts were LSI -0.87 -0.07 -0.07 related only to respiratory burst (a measure of phagocyte Haemoglobin 0.60 -0.72 -0.34 activity). However, no relationships among the different Cumulative R2 52.92 73.45 90.62 measures of immunity or between immunity and phy- Immunity PC1 PC2 PC3 siology were found when taking into account the sam- SSI 0.10 0.56 -0.11 pling period (p > 0.05). Leukocyte count 0.94 -0.25 -0.04 The last analysis was restricted to the spring season Respiratory burst 0.54 0.57 0.41 and to males as the level of 11-ketotestosterone was Complement -0.30 0.01 0.76 only significantly higher in this period (following the IgM concentration 0.18 -0.43 0.61 results of ANOVA). A significant negative relationship Lymphocyte count 0.77 -0.56 -0.10 between 11-ketotestosterone and the level of total com- Phagocyte count 0.65 0.59 0.03 plement pathway was found (N = 12, b = -0.78, p > Cumulative R2 33.11 55.11 71.52 0.01) suggesting the potential immunosuppression by Parasitism PC1 PC2 PC3 steroid hormones (Figure 5). There were no significant relationships for any other immune variables in the Monogenea -0.80 0.16 -0.40 spring period (p > 0.05). Crustacea -0.56 0.20 0.80 Cestoda -0.85 -0.21 -0.16 Discussion Digenea 0.07 0.97 -0.13 The relationship between abiotic environment and Cumulative R2 41.99 68.33 89.45 parasite infection The most important parameters contributing to the principal components are shown in bold. Changes in parasite abundance in relation to their life- cycle have been generally considered to be influenced by both host environment and host physiology [2,73]. Dif- condition factor and haemoglobin concentration, were ferences in the seasonal dynamic of abundance changes found. The abundance of Cestoda was significantly among different parasite groups are then predetermined related to phagocyte count and respiratory burst when by parasite life-strategies. Moreover, both the presence taking into account the effects of sampling period and and efficiency of intermediate hosts play an important sex (both effects were included in GLM following the role in the transmission of endoparasites (e.g. [74]). In results of one-way ANOVA). GLM analyses showed a our study, we confirmed that seasonality influence the significant partial relationship between the abundance of abundance of Monogenea, Crustacea and Cestoda. Digenea and SSI, although the model was not significant Change in water temperature (one of the principal cues (see Table 4). of seasonality) is commonly regarded as one of the most important factors determining the presence and abun- Associations between host immunity and physiology dance of Monogenea [1]. We observed a different seaso- Using GLM analyses, associations between host immu- nal pattern of the abundance changes in oviparous gill nity (i.e. SSI, phagocyte count, respiratory burst, comple- parasites of Dactylogyrus and Eudiplozoon (with maxi- ment activity and IgM concentration) and physiology mum abundance observed in summer) compared to (condition factor, GSI, HSI and haemoglobin) taking viviparous Gyrodactylus species (with maximum abun- into account the effect of sampling period or, alterna- dance in winter). Dactylogyrus species were the most tively, the effects of both sampling period and sex, were abundant parasites. The general trend associated with analyzed. All variables of immunity and physiology, their life-cycle (direct-transmitted ectoparasites) is that except 11-ketotestosterone concentration, appeared sta- any increase in temperature leads to an increase of their tistically dependent on sampling period (Table 5). Sig- population densities [75]. Our results demonstrating the nificant relationships between condition factor, GSI and high abundance of all Dactylogyrus species of common HSI were found when taking into account the sampling carp in summer confirmed that water temperature is the period. Haemoglobin concentration was related to GSI main factor determining the high abundance of all Dac- and affected by both sampling period and sex. In addi- tylogyrus species of common carp. tion, 11-ketotestosterone concentration measured in Adult stages of Atractolytocestus huronensis repre- males was significantly related to gonad weight sented the dominant cestode species of common carp in Rohlenová et al. Parasites & Vectors 2011, 4:120 Page 12 of 18 http://www.parasitesandvectors.com/content/4/1/120

Table 3 Partial correlations controlling for sampling period PC1 for PC2 for PC3 for PC1 for PC2 for PC3 for PC1 for PC2 for Parasitism Parasitism Parasitism Physiology Physiology Physiology Immunity Immunity PC1 for 1 Parasitism PC2 for 0.039 1 Parasitism PC3 for 0.221 -0.070 1 Parasitism PC1 for -0.300 0.058 -0.138 1 Physiology PC2 for -0.464 0.145 -0.333 0.121 1 Physiology PC3 for -0.108 -0.043 -0.026 -0.078 -0.011 1 Physiology PC1 for -0.045 0.187 0.067 0.065 -0.177 0.075 1 Immunity PC2 for 0.485 0.014 0.152 -0.283 -0.194 -0.101 -0.137 1 Immunity PC3 for -0.105 0.095 -0.060 -0.099 0.235 0.385 0.148 0.026 Immunity Statistical significant correlations (p < 0.05) are shown in bold. this study. The abundance of cestode infection can be blindness [78]. A marginal effect of seasonality on the connected with the temporal presence of intermediate abundance of this species was observed. The highest hosts. The highest cestode abundance was recorded in abundance values were found in early summer and late summer. Similar seasonal changes in the abundance autumn. Similar findings have been documented in sev- of A. huronensis were previously reported for German eral studies (e.g. [79,80]), and Burrough [81] suggested pond-farmed carp [76]. Moreover, this cestode species that the first peak of infection (early summer) may was the only parasite affected weakly by host sex. Reim- probably come from the snails that survived through the chen and Nosil [77], who monitored the level of parasit- winter. A second peak of infection may occur in autumn ism in a population of threespined stickleback from snails that hatched during the spring period. (Gasterosteus aculeatus), showed that females were Crustacea were represented by an abundant species, more likely to be parasitized by the cestode Schistoce- Argulus foliaceus, which is considered to be an obligate phalus solidus (Cestoda) and suggested that this sex- branchiuran ectoparasite infecting many freshwater fish biased infection could be connected with a dietary niche species. Some Argulus species are able to tolerate a wide variation, which may result in differential exposure to range of water temperatures (e.g. [82]). Argulus folia- infected intermediate hosts. ceus, a common species in Europe, is known to reach Digeneans parasitizing common carp were represented high abundance on their hosts during late summer and mainly by the larval stage (metacercaria) of Diplosto- early autumn (e.g. [83,84]), which was also found here. mum species, which live in the eyes of this second inter- However, no infection was observed in winter. Hakalahti mediate fish host. This ubiquitous parasite causes andValtonen[85]showedalowabundanceofArgulus cataracts, reduces fish vision, and may even induce total coregoni in fish during winter suggesting that this

Table 4 GLM analyses on the relationship between parasite abundance, immunity and physiology Dependent variable Independent variables SS Df F p Total F (p) Monogenea Condition factor 1.653 1 11.144 0.001 Haemoglobin 0.588 1 3.966 0.049 Sampling 13.619 4 22.958 0.000 22.209 (< 0.0001) Crustacea Sampling 1248.215 4 7.114 0.000 5.826 (< 0.0001) Cestoda Respiratory burst 5.357 1 5.639 0.019 Phagocytes 7.865 1 8.278 0.005 Sampling 56.846 4 14.958 0.000 8.841 (< 0.0001) Digenea SSI 174.815 1 5.648 0.019 1.683 (0.074) GLM analyses on the relationship between parasite abundance and immune or physiological variables, taking into account sampling period. Rohlenová et al. Parasites & Vectors 2011, 4:120 Page 13 of 18 http://www.parasitesandvectors.com/content/4/1/120

Table 5 GLM analyses on the relationship between host immunity and physiology Dependent variable Independent variables SS Df F p Total F (p) Condition factor GSI 0.018 1 4.628 0.034 HSI 0.059 1 15.24 0.000 Haemoglobin 0.019 1 5.039 0.027 Sampling 0.131 4 8.505 0.000 6.629 (< 0.0001) GSI Condition factor 1.275 1 4.628 0.034 HSI 3.948 1 14.329 0.000 Haemoglobin 2.321 1 8.423 0.004 IgM 4.237 1 15.378 0.000 Sampling 5.853 4 5.311 0.001 11.34 (< 0.0001) HSI Condition factor 0.152 1 15.24 0.000 GSI 0.143 1 14.329 0.000 Sampling 0.514 4 12.916 0.000 20.054 (< 0.001) Haemoglobin GSI 0.043 1 7.681 0.007 Sampling 0.351 4 15.763 0.000 Sex 0.129 1 23.191 0.000 11.384 (< 0.001) SSI Sampling 0.44 4 6.446 0.000 3.691 (0.0001) Phagocytes Respiratory burst 2.924 1 35.009 0.000 Sampling 1.871 4 5.602 0.000 10.771 (< 0.0001) Respiratory burst Phagocytes 402.309 1 35.009 0.000 Sampling 242.144 4 5.268 0.001 9.922 (< 0.0001) IgM concentration Sampling 378.458 4 4.372 0.003 Sex 504.633 1 23.316 0.000 Sampling_Sex 597.857 4 6.906 0.000 7.773 (< 0.0001) Complement Sampling 1.972 4 19.414 0.000 9.189 (< 0.0001) 11-ketotestosterone GSI 6586.91 1 25.556 0.000 Respiratory burst 1791.337 1 6.95 0.011 Complement 3611.305 1 14.011 0.000 6.524 (< 0.0001) GLM analyses on the relationship between immune and physiological parameters, taking into account sampling period and sex effect. species can survive winter due to overwintering egg store energy in muscle tissues or in the liver (glycogen) stages laid in autumn. during periods of high food and energy intake [86]. Therefore, both condition factor and the relative size of The link between host immunity and physiology liver (HSI) are recommended as an indirect indicator of We hypothesized that fish investing more in immunity energy status [86]. The gonado-somatic index (GSI) should show less investment in other physiological func- represents an accurate assessment of reproductive tions. Moreover, we also hypothesized that seasonality maturity. Negative relationships were found between acts as an important factor determining the levels of fish fish energy status (measured either by the condition fac- physiology and immunological activity. Using PCA, we tor or HSI) and GSI, suggesting the existence of a trade- revealed that one PC for physiology and two PCs for off, with a decreasing fish condition in the period of immunity (from three PCs analyzed) were correlated gonad formation and reproduction. A significant rela- with seasonality suggesting that the majority of mea- tionship was also observed between haemoglobin con- sured variables are under seasonal changes. The differ- centration and GSI. The seasonal variation in ent PCs for immunity and physiology were correlated haemoglobin concentration is potentially related to var- with different variables (mainly when comparing PC1 iation in water temperature and variation in water oxy- and 2 to PC3). This fact together with the significant gen concentration. Fish adapt via increases in total correlations between PCs of immunity and physiology haemoglobin concentration or by other mechanisms may suggest the potential relationships between different such as changes in red cell nucleoside triphosphate con- immunity and physiology variables. centration [87,88]. The spawning process may affect We conducted analyses among selected measures of haematological parameters [89], which may explain the host status related to condition and reproduction and of observed relationship between haemoglobin concentra- immunity taking into account the sampling period. Fish tion and GSI. Moreover, the concentration of Rohlenová et al. Parasites & Vectors 2011, 4:120 Page 14 of 18 http://www.parasitesandvectors.com/content/4/1/120

1.4 synthesis. Suzuki et al. [95] followed the annual changes of IgM in three strains of rainbow trout under constant 1.3 water temperature and natural day length, and showed 1.2 that the IgM concentration level varied due to the 1.1 immunosuppression effect induced by sex hormones. 1.0 Here, we did not find any clear association between sea- vity ti 0.9 sonal changes (potentially related to water temperature) 0.8 and IgM concentration level. The lowest water tempera-

0.7 ture was recorded in autumn and winter whilst the low- Complement ac

0.6 est concentration in IgM level was observed in winter and spring. Moreover, IgM concentration was the only 0.5 immune variable affected by host sex, with a signifi- 0.4 cantly lower IgM concentration in males. Although, the 0.3 -20 0 20 40 60 80 100 120 140 160 immunosuppressive effect of testosterone and/or gonad 11-Ketotestosterone concentration maturation on IgM has already been demonstrated in Figure 5 The negative relationship between the level of 11- rainbow trout [96], this effect has not been investigated ketotestosterone and the activity of complement in spring. in common carp before our study. Other hormones like cortisol may also reduce the IgM secretion, as shown in salmonid fish [97]. haemoglobin was the single physiological parameter that The annual fluctuation in complement activity was differs between females and males, which has already investigated in gilthead sea bream, Sparus aurata by been observed in other fish species [51]. Hernández et al. [7]. These authors showed increasing Although significant relationships were found between complement activity in warm months probably in rela- PCs of physiology and immunity suggesting the poten- tion to higher metabolic activity at higher temperatures tial trade-off associations between physiology and pha- in poikilothermic organisms. In this study, the total gocyte activity or SSI (using PC2 for immunity and PCs complement activity decreased in spring but not in win- 1 and 2 for physiology) and between physiology and ter at the coldest temperature. Seasonal changes (poten- complement activity or IgM (using PC3 for immunity tially related to water temperature) does not seem to and PCs 2 and 3 for physiology), no associations affect the total complement activity and its decrease in between individual immune variables and physiological spring could be related to reproduction (see below its variables were found using GLM. However, host immu- relationship with 11-ketotestosterone). nity was strongly dependent on the sampling period, Phagocyte counts and phagocyte activity (measured by which confirms that seasonality is the driving force of the respiratory burst) were both affected by seasonality, immune variation in fish. although these variables were highly correlated. Seasonal We hypothesized that higher investments in somatic variability in phagocyte activity has been investigated in condition could be associated with lower investments in several fish species with inconsistent conclusions. An immune defence. Although, our analyses revealed no immunosuppressive effect of water temperature on the significant relationship between condition factor and innate immune response in catfish Ictalurus punctatus spleen size, both these variables showed similar seasonal [11] and in tench, Tinca tinca [98,99] has been dynamics. The highest values of SSI were found in early observed. In accordance with these studies, we recorded summer followed by decreasing values in late summer. low values of respiratory burst in late summer, where Our findings are not in agreement with previous studies water temperature is higher comparing to other period in roach Rutilus rutilus [90] or in Arctic charr, Salveli- investigated. High values of respiratory burst were nus alpinus [91], where spleen size was shown to recorded from autumn to spring. Phagocyte activity decrease in the breeding season. seems not to be suppressed by water temperature in Concentration levels of IgM were also dependent on several fish species e.g. rainbow trout under laboratory the sampling period with low IgM values recorded in conditions [35]. winter and spring. Previous studies on IgM concentra- 11-ketotestosterone is considered to be a major andro- tion showed a decrease in IgM level during the winter gen hormone in teleost fish (see review by Borg [50]). period, probably related to water temperature, in rain- This hormone influences spermatogenesis and effects bow trout Oncorhynchus mykiss [46] and goldfish Caras- the expression of secondary sexual characters and repro- sius auratus [92]. According to Avtalion [93] and Stolen ductive behavior. It also suppresses several immune et al. [94], low water temperature causes a selective sup- functions. Steroid hormones have dualistic functions: pression of in vitro T cell responses and antibody they increase the expression of elaborated sexual Rohlenová et al. Parasites & Vectors 2011, 4:120 Page 15 of 18 http://www.parasitesandvectors.com/content/4/1/120

ornamentation but decrease the immunocompetence of may contribute to the decrease in haemoglobin concen- an individual. This observed mechanism is at the basis tration. Sitja-Bobadilla and Alvarez-Pellitero [101] sug- of the immune-handicap hypothesis [28]. We found a gested that even a low intensity of infection by a positive association between gonad size (measured by monogenean species (Sparicotyle chrysophrii)may GSI) and 11-ketotestosterone concentration. Moreover, induce fish anaemia, and activation of fish haematopo- we found support for the immunosuppressive role of esis leading to an increase in immature erythrocytes 11-ketotestosterone, with negative relationships observed with lower amounts of haemoglobin. between the concentration of 11-ketotestosterone and We found a significant relationship between the abun- two immune parameters - total complement activity and dance of Cestoda and counts of peripheral blood phago- respiratory burst (taking into account the sampling per- cytes. Moreover, the immunological activity of iod). Using linear regression (not included in the phagocytes measured by the respiratory burst was also results), only complement activity was negatively corre- found to be associated with cestode infection (taking lated with concentration of 11-ketotestosterone. A sig- into account the sampling period). The abundance of nificant immunosuppressive effect of 11- cestodes (A. huronensis was the dominant species) was ketotestosterone on complement activity was also found negatively related to both these measures of immunity when using only spring data, i.e. where concentrations using linear regression (notshownintheresultssec- of 11-ketotestosterone were the highest and correlated tion). We could hypothesize that, during a period of low with gonad development. parasite infection, phagocytes are present in peripheral blood ready to react quickly to an entering antigen The role of the season: host immunity and physiology derived from parasites. However, during high parasite versus total parasite load infection, the phagocytes and other blood cells colonize One aim of our study was to investigate the potential the tissues surrounding the attachment organs of A. link between parasitism and host immunity or physiol- huronensis [102]. A low activity of phagocytes, measured ogy on two different scales. First, using the computed by respiratory burst, was also demonstrated in fish PCs we showed the significant role of seasonality on experimentally infected by the cestode Schistocephalus each of parasitism, immunity and physiology. solidus [103] or in gynogenetic form of triploid Caras- After correcting for sampling period, the negative rela- sius auratus infected by metacercaria of Metagonnimus tionships between parasitism and physiology were found, sp. [104]. Other examples demonstrating the depression which suggests that a “good” physiological status reflects of oxidative burst and/or the impairment of phagocyte a host’s ability to escape from parasitism (especially con- activity induced by parasites are given by Alvarez-Pelli- cerning Monogenea, Crustacea and Cestoda). Moreover, tero [19]. In addition, some parasites are able to exploit parasitism was positively related to immunity, which the host immune reaction in order to improve their could indicate that despite of strong effect of seasonality attachment to the host tissue. Alvarez-Pellitero [19] on fish immunity, this system (or at least some immune documented that attachment of the cestode Cyathoce- pathways) is activated by increasing level of parasite phalus truncatus in the fish pyloric caeca was facilitated infection. by an inflammatory reaction. However, Vainikka et al. [72] observed the positive correlation between parasite Parasitism versus host immunity and physiology: effect of loads and the relative proportion of phagocytes in blood season or a real association? of roach (Rutilus rutilus). They also showed that func- Effects of seasonal variability on the parasite abundance tional characteristics of these cells were positively of several parasite groups that differ according to life related to the proportion of dead Rhipidocotyle campa- strategy were observed. A significant relationship nula (Digenea) which may indicate that the chemilumi- between monogeneans, the most abundant ectoparasite nescence method is a suitable measure to estimate group, and the condition of fish was found, taking into functional immunocompetence in fish. account the sampling period. The opposite seasonal pat- A significant relationship was observed between the terns in fish condition and in the abundance of monoge- abundance of digeneans and SSI. However, using simple neans (Figures 1A and 2A) suggest that high infection linear regression (not shown in the results section), a by these parasites should be detrimental to fish. positive relationship was found between larval digeneans A negative relationship between haemoglobin concen- of Diplostomum species and relative spleen size. Skar- tration and the same parasites was also observed. The stein et al. [90] suggested that large spleen in fish may presence of Eudiplozoon nipponicum, a haematophagous reflect the ability to respond to parasite infection or monogenean species with intracellular blood digestion may indicate high immunological activity against already [100] and a body size 25-60 times higher than the aver- established infection. The associations between spleen agebodysizeofDactylogyrus and Gyrodactylus species, size and parasitism by metazoan parasites have been Rohlenová et al. Parasites & Vectors 2011, 4:120 Page 16 of 18 http://www.parasitesandvectors.com/content/4/1/120

tested in many intraspecific studies (e.g. [29,59]), but a Kotlářská 2, 611 37, Brno, Czech Republic. 4University of South Bohemia České Budějovice, Research Institute of Fish Culture and Hydrobiology in significant relationship is rarely reported [e.g. [30,61]]. Vodňany, Zátiší 728/II, 389 25 Czech Republic. Vainikka et al. [72] did not find any associations between spleen size and parasite counts in roach sug- Authors’ contributions AŠ designed this study. KR and AŠ drafted the manuscript. SM significantly gesting that spleen size might not represent the measure contributed in drafting the statistical part of manuscript. SM, MF and PH of immunocompetence in roachandthusthisvariable involved in revising for important content and discussing the results. All should be interpret with caution in immunoecological authors contributed to acquisition of data, data analysis or data interpretation. All authors read and approved the final version of the studies. Moreover, they suggest that it is difficult to manuscript. interpret causal relationships when using only correlation study to analyze the associations between immune Competing interests variables and parasite load and therefore, they propose The authors declare that they have no competing interests. that the experimental studies are needed. Received: 11 March 2011 Accepted: 27 June 2011 Published: 27 June 2011 Conclusions References Our study showed that host immunity and physiology, as 1. Koskivaara M, Tellervo EV, Prost M: Seasonal occurrence of gyrodactylid well as parasite infection, are highly dependent on seaso- monogeneans on the roach (Rutilus rutilus) and variations between four nal variability (i.e. temporal variation) potentially related lakes of differing water quality in Finland. Aqua Fenn 1991, 21:47-55. 2. Rohde K, Hayward C, Heap M: Aspects of the ecology of metazoan to the changes of water temperature (one of the principal ectoparasites of marine fishes. Int J Parasitol 1995, 25:945-970. cues of seasonality), although several other abiotic char- 3. Bly JE, Clem LW: Temperature and teleost immune functions. Fish Shellfish acteristics of the water environment may play a part. Immun 1992, 2:159-171. 4. Hutchinson TH, Manning MJ: Seasonal trends in serum lysozyme activity Nevertheless, we confirmed the associations between and total protein concentration in dab (Limanda limanda L.) sampled parasitism and both host physiology and immunity after from Lyme Bay, UK. Fish Shellfish Immun 1996, 6:473-482. correction for temporal variability. When considering 5. Le Morvan C, Troutaud D, Deschaux P: Differential effects of temperature on specific and nonspecific immune defences in fish. J Exp Biol 1998, parasites with different life strategies, and taking into 201:165-168. account the effects of seasonality, fish in a worse physio- 6. Langston AL, Hoare R, Stefansson M, Fitzgerald R, Wergeland H, Mulcahy M: logical condition suffer from a higher level of infection by The effect of temperature on non-specific defence parameters of three strains of juvenile Atlantic halibut (Hippoglossus hippoglossus L.). Fish the abundant ectoparasitic monogeneans. The infection Shellfish Immun 2002, 12:61-76. by cestodes seems to activate several mechanisms of the 7. Hernández A, Tort L: Annual variation of complement, lysozyme and immune system and particularly phagocyte activity. Sea- haemagglutinin levels in serum of the gilthead sea bream Sparus aurata. Fish Shellfish Immun 2003, 15:479-481. sonal variability affects host immunity and physiology 8. Duffy JE, Carlson E, Li Y, Prophete C, Zelikoff JT: Impact of polychlorinated through energy allocation in a trade-off between impor- biphenyls (PCBs) on the immune function of fish: age as a variable in tant functions, i.e. reproduction and fish condition. How- determining adverse outcome. Mar Environ Res 2002, 54:559-563. 9. Carlson E, Zelikoff J: The immune system of fish: a target organ of toxicity ever, the measures of host immunity were not found to Washington DC: Taylor and Francis; 2008. be in a direct trade-off with the investigated physiological 10. Du Pasquier L: Evolution of the Immune System New York: Raven Press; 1993. traits or functions, but the immunosuppressive role of 11. Ainsworth AJ, Dexiang C, Waterstrat PR, Greenway T: Effect of temperature on the immune system of channel catfish (Ictalurus punctatus). I. 11-ketotestosterone was observed. Leucocyte distribution and phagocyte function in the anterior kidney at 10°C. Comp Biochem Phys A 1991, 100:907-912. Acknowledgements and Funding 12. Ellis AE: Innate host defense mechanisms of fish against viruses and bacteria. Dev Comp Immunol 2001, 25:827-839. This study was funded by the Grant Agency of the 13. Magnadóttir B, Jónsdóttir H, Helgason S, Björnsson B, Jørgensen TO, Czech Republic, project No. 524/07/0188. MF was also Pilström L: Humoral immune parameters in Atlantic cod (Gadus morhua supported by the Ministry of Education project L.) - II. The effects of size and gender under different environmental conditions. Comp Biochem Phys B 1999, 122:181-188. MSM6007665809. KR was funded by the Ichthyoparasi- 14. Saha NR, Usami T, Suzuki Y: Seasonal changes in the immune activities of tology Research Centre of the Ministry of Education, common carp (Cyprinus carpio). Fish Physiol Biochem 2002, 26:379-387. Youth and Sports of the Czech Republic LC 522 and 15. Aydogdu A, Altunel FN: Helminth parasites (Plathelminthes) of common ’ carp (Cyprinus carpio L.) in Iznik Lake. B Eur Assoc Fish Pat 2002, partially by the Rector s Programme in Support of MU 22:343-348. Students’ Creative Activities. AŠ was supported by the 16. Buchmann K, Lindenstrøm T: Interactions between monogenean parasites Research Project of Masaryk University (No. and their fish hosts. Int J Parasitol 2002, 32:309-319. 17. Muñoz G, Grutter AS, Cribb TH: Structure of the parasite communities of MSM0021622416) a coral reef fish assemblage (Labridae): Testing ecological and phylogenetic host factors. J Parasitol 2007, 93:17-30. 18. Lamková K, Šimková A, Palíková M, Jurajda P, Lojek A: Seasonal changes of Author details immunocompetence and parasitism in chub (Leuciscus cephalus), a 1 Department of Botany and Zoology, Faculty of Science, Masaryk University, freshwater cyprinid fish. Parasitol Res 2007, 101:775-789. 2 Kotlářská 2, 611 37 Brno, Czech Republic. University of Montpellier 2, CNRS- 19. Alvarez-Pellitero P: Fish immunity and parasite infections: from innate IRD, Institute of Evolutionary Sciences, CC065, 34095, Montpellier, France. immunity to immunoprophylactic prospects. Vet Immunol Immunop 2008, 3 Institute of Experimental Biology, Faculty of Science, Masaryk University, 126:171-198. Rohlenová et al. Parasites & Vectors 2011, 4:120 Page 17 of 18 http://www.parasitesandvectors.com/content/4/1/120

20. Sitja-Bobadilla A: Living off a fish: A trade-off between parasites and the 46. Sánchez C, Babin M, Tomillo J, Ubeira FM, Domínguez J: Quantification of immune system. Fish Shellfish Immun 2008, 25:358-372. low levels of rainbow trout immunoglobulin by enzyme immunoassay 21. Sorci G, Bouliner T, Gauthier-Clerc M, Faivre B: Écologie évolutive de la using two monoclonal antibodies. Vet Immunol Immunopathol 1993, Réponse Immunitaire (in French) Bruxelles: De Boeck & Larcier; 2007. 36:65-74. 22. Šimková A, Lafond T, Ondračková M, Jurajda P, Ottová E, Morand S: 47. Secombes CJ, Chappell LH: Fish immune responses to experimental and Parasitism, life history traits and immune defence in cyprinid fish from natural infection with helminth parasites. Annu Rev Fish Dis 1996, Central Europe. BMC Evol Biol 2008, 8:29. 6:167-177. 23. Roff DA: The Evolution of Life Histories Routledge: Chapman & Hall, Inc.; 48. Griffin BR: Opsonic effect of rainbow trout (Salmo gairdneri) antibody on 1992. phagocytosis of Yersinia ruckeri by trout leukocytes. Dev Comp Immunol 24. Keymer AE, Read AF: Behavioural Ecology: the Impact of Parasitism in 1983, 7:253-259. Parasite-Host Associations: Coexistence or Conflict? Oxford: University Press; 49. McEwan AD, Fischer EW, Selman IE: Observations on the immune globulin 1991. levels of neonatal calves and their relationship to disease. J Comp Pathol 25. Zuk M, Stoehr AM: Immune defense and host life history. Am Nat 2002, 1970, 80:259-265. 160:S9-S22. 50. Borg B: Androgens in teleost fishes. Comp Biochem Phys C 1994, 26. Owens IPF, Wilson K: Immunocompetence: a neglected life history trait or 109:219-245. conspicuous red herring? Trends Ecol Evol 1999, 14:170-172. 51. Lusková V: Annual Cycles and Normal Values of Hematological Parameters in 27. Tschirren B, Richner H: Parasites shape the optimal investment in Fishes Brno: Acta Scientiarum Naturalium; 1997. immunity. Proc Biol Sci 2006, 273:1773-1777. 52. Modrá H, Svobodová Z, Kolářová J: Comparison of differential leukocyte 28. Folstad I, Karter AJ: Parasites, bright males, and the immunocompetence counts in fish of economic and indicator importance. Acta Vet Brno 1998, handicap. Am Nat 1992, 139:603-622. 67:215-226. 29. Ottová E, Šimková A, Jurajda P, Dávidová M, Ondračková M, Pečínková M, 53. Ruane NM, Nolan DT, Rotllant J, Costelloe J, Bonga SEW: Experimental Gelnar M: Sexual ornamentation and parasite infection in males of exposure of rainbow trout Oncorhynchus mykiss (Walbaum) to the common bream (Abramis brama): a reflection of immunocompetence infective stages of the sea louse Lepeophtheirus salmonis (Kroyer) status or simple cost of reproduction? Evol Ecol Res 2005, 7:581-593. influences the physiological response to an acute stressor. Fish Shellfish 30. Rohlenová K, Šimková A: Are the immunocompetence and the presence Immun 2000, 10:451-463. of metazoan parasites in cyprinid fish affected by reproductive efforts of 54. Doubek J: Veterinary Haematology (in Czech) Brno: Noviko; 2003. cyprinid fish? J Biomed Biotechnol 2010, Article Number:418382. 55. Svobodová Z, Pravda D, Paláčková J: Universal methods of hematological 31. Pravda D, Svobodová Z: Haematology of Fishes (in Czech) Brno: Noviko; investigations in fish (in Czech) Vodňany: Edice Metodik; 1986. 2003. 56. Lusková V: Annual cycles and normal values of hematological parameters in 32. Ergens R, Lom J: Causative Agents of Parasitic Fish Diseases (in Czech) Prague: fishes Brno: Acta Scientiarum Naturalium; 1997. Academia; 1970. 57. Manning MJ: Fishes. In Immunology, A comparative approach. Edited by: 33. Secombes CJ: The nonspecific immune system: Cellular defence. In The Turner RJ. New York: Wiley; 1994:69-100. Fish Immune System - Organism, Pathogen and Environment. Edited by: 58. Dalmo RA, Ingebrigtsen K, Bogwald J: Non-specific defence mechanisms Iwama G, Nakanishi T. San Diego: Academic Press; 1996:63-103. in fish, with particular reference to the reticuloendothelial system (RES). 34. Scott AL, Rogers WA, Klesius PH: Chemiluminescence by peripheral blood J Fish Dis 1997, 20:241-273. phagocytes from channel catfish: function of opsonin and temperature. 59. Taskinen J, Kortet R: Dead and alive parasites: sexual ornaments signal Dev Comp Immunol 1985, 9:241-250. resistance in the male fish, Rutilus rutilus. Evol Ecol Res 2002, 4:919-929. 35. Nikoskelainen S, Bylund G, Lilius EM: Effect of environmental temperature 60. Kortet R, Taskinen J: Parasitism, condition and number of front head on rainbow trout (Oncorhynchus mykiss) innate immunity. Dev Comp breeding tubercles in roach (Rutilus rutilus L.). Ecol Freshw Fish 2004, Immunol 2004, 28:581-592. 13:119-124. 36. Kubala L, Lojek A, Číž M, Vondráček J, Dušková M, Slavíková H: 61. Lefebvre F, Mounaix B, Poizat G, Crivelli AJ: Impacts of the swimbladder Determination of phagocyte activity in whole blood of carp (Cyprinus nematode Anguillicola crassus on Anguilla anguilla: variations in liver and carpio) by luminol-enhanced chemiluminescence. Vet Med (in Czech) spleen masses. J Fish Biol 2004, 64:435-447. 1996, 41:323-327. 62. Piersma T, Lindström Å: Rapid reversible changes in organ size as a 37. Ellis AE: Immunity to bacteria in fish. Fish Shellfish Immun 1999, 9:291-308. component of adaptive behaviour. Trends Ecol Evol 1997, 12:134-138. 38. Buchmann K: Binding and lethal effect of complement from 63. Bolger T, Connolly PL: The selection of suitable indexes for the Oncorhynchus mykiss on Gyrodactylus derjavini (Platyhelminthes: measurement and analysis of fish condition. J Fish Biol 1989, 34:171-182. Monogenea). Dis Aquat Organ 1998, 32:195-200. 64. Malmberg G: Excretory systems and marginal hooks as a basis for 39. Harris PD, Soleng A, Bakke TA: Killing of Gyrodactylus salaris systematics of Gyrodactylus (Trematoda, Monogenea). Ark Zool 1970, (Platyhelminthes, Monogenea) mediated by host complement. 23:1-235. Parasitology 1998, 117:137-143. 65. Georgiev B, Biserkov V, Genov T: In toto staining method for cestodes 40. Virta M, Karp M, Rönnemaa S, Lilius EM: Kinetic measurement of the with iron acetocarmine. Helminthologia 1986, 23:279-291. membranolytic activity of serum complement using bioluminescent 66. Gusev AV: Part I. Identification Key to Parasites of Freshwater Fish (in Russian) bacteria. J Immunol Methods 1997, 201:215-221. Leningrad: Nauka; 1985. 41. Nikoskelainen S, Lehtinen J, Lilius EM: Bacteriolytic activity of rainbow 67. Khotenovsky IA: Monogenea (in Russian) Leningrad: Nauka; 1985. trout (Oncorhynchus mykiss) complement. Dev Comp Immunol 2002, 68. Scholz T: Amphilinida and Cestoda, Parasites of Fish in Czechoslovakia Brno: 26:797-804. Acta Scientiarum Naturalium; 1989. 42. Buchtíková S, Vetešníková Šimková A, Rohlenová K, Flajšhans M, Lojek A, 69. Kadlec D, Šimková A, Gelnar M: The microhabitat distribution of two Esa-Matti Lilius, HyršlP:The seasonal changes in innate immunity of the Dactylogyrus species parasitizing the gills of the barbel, Barbus barbus. J common carp (Cyprinus carpio). Aquaculture 2011, 318:169-175. Helminthol 2003, 77:317-325. 43. Harding FA, Amemiya CT, Litman RT, Cohen N, Litman GW: Two distinct 70. Bush AO, Lafferty KD, Lotz JM, Shostak AW: Parasitology meets ecology on immunoglobulin heavy chain isotypes in a primitive, cartilaginous fish, its own terms: Margolis et al revisited. J Parasitol 1997, 83:575-583. Raja erinacea. Nucleic Acids Res 1990, 18:6369-6376. 71. Poisot T, Šimková A, Hyršl P, Morand S: Interactions between 44. Danilová N, Bussmann J, Jekosch K, Steiner LA: The immunoglobulin heavy- immunocompetence, somatic condition and parasitism in the chub chain locus in zebrafish: identification and expression of a previously Leuciscus cephalus in early spring. J Fish Biol 2009, 75:1667-1682. unknown isotype, immunoglobulin Z. Nat Immunol 2005, 6:295-302. 72. Vainikka A, Taskinen J, Loytynoja K, Jokinen E, Kortet R: Measured 45. Hansen JD, Landis ED, Phillips RB: Discovery of a unique Ig heavy-chain immunocompetence relates to the proportion of dead parasites in a isotype (IgT) in rainbow trout: Implications for a distinctive B cell wild roach population. Funct Ecol 2009, 23:187-195. developmental pathway in teleost fish. P Natl Acad Sci USA 2005, 73. Esch GW, Bush AO, Aho JM: Parasite Communities: Patterns and Progresses 102:6919-6924. London: Chapman and Hall; 1990. Rohlenová et al. Parasites & Vectors 2011, 4:120 Page 18 of 18 http://www.parasitesandvectors.com/content/4/1/120

74. Hanzelová V, Gerdeaux D: Seasonal occurrence of the tapeworm 98. Collazos ME, Barriga C, Ortega E: Seasonal changes in phagocytic capacity Proteocephalus longicollis and its transmission from copepod and superoxide anion production of blood phagocytes from tench intermediate host to fish. Parasitol Res 2003, 91:130-136. (Tinca tinca, L.). J Comp Physiol B 1995, 165:71-76. 75. Chubb JC: Seasonal ocurrence of helminths in freshwater fishes Part. I. 99. Collazos ME, Ortega E, Barriga C: Effect of temperature on the immune Monogenea. Adv Parasit 1977, 15:133-139. system of a cyprinid fish (Tinca tinca, L). Blood phagocyte function at 76. Kappe A, Seifert T, El-Nobi G, Brauer G: Occurrence of Atractolytocestus low temperature. Fish Shellfish Immun 1994, 4:231-238. huronensis (Cestoda, Caryophyllaeidae) in German pond-farmed 100. Smyth JD, Halton DW: The Physiology of Trematodes Cambridge: Cambridge common carp Cyprinus carpio. Dis Aquat Organ 2006, 70:255-259. University Press; 1983. 77. Reimchen TE, Nosil P: Ecological causes of sex-biased parasitism in 101. Sitja-Bobadilla A, Alvarez-Pellitero P: Experimental transmission of threespine stickleback. Biol J Linn Soc 2001, 73:51-63. Sparicotyle chrysophrii (Monogenea: Polyopisthocotylea) to gilthead 78. Ersdal C, Midtlyng PJ, Jarp J: An epidemiological study of cataracts in seabream (Sparus aurata) and histopathology of the infection. Folia seawater farmed Atlantic salmon (Salmo salar). Dis Aquat Organ 2001, Parasit 2009, 56:143-151. 45:229-236. 102. Molnár K, Majoros G, Csaba G, Székely C: Pathology of Atractolytocestus 79. Pennycuick L: Seasonal variations in the parasite infections in a huronensis Anthony, 1958 (Cestoda, Caryophyllaeidae) in Hungarian population of three-spinned sticklebacks, Gasterosteus aculeatus L. pond-farmed common carp. Acta Parasitol 2003, 48:222-228. Parasitology 1971, 63:378-388. 103. Scharsack JP, Kalbe M, Derner R, Kurtz J, Milinski M: Modulation of 80. McKeown CA, Irwin SWB: Accumulation of Diplostomum spp. (Digenea: granulocyte responses in three-spined sticklebacks Gasterosteus Diplostomatidae) Metacercariae in the eyes of 0+ and 1+ roach (Rutilus aculeatus infected with the tapeworm Schistocephalus solidus. Dis Aquat rutilus). Int J Parasitol 1997, 27:377-380. Organ 2004, 59:141-150. 81. Burrough RJ: The population biology of two species of eyefluke, 104. Hakoyama H, Nishimura T, Matsubara N, Iguchi K: Difference in parasite Diplostomum spathaceum and Tylodelphys clavata, in roach and rudd. J load and nonspecific immune reaction between sexual and gynogenetic Fish Biol 1978, 13:19-32. forms of Carassius auratus. Biol J Linn Soc 2001, 72:401-407. 82. Walker PD, Russon IJ, Duijf R, Bonga SEW: The effect of temperature on the biology, survival and viability of the fish parasite, Argulus japonicus doi:10.1186/1756-3305-4-120 Thiele. Comp Biochem Phys A 2005, 141:S90-S90. Cite this article as: Rohlenová et al.: Are fish immune systems really 83. Hakalahti T, Pasternak AF, Valtonen ET: Seasonal dynamics of egg laying affected by parasites? an immunoecological study of common carp and egg-laying strategy of the ectoparasite Argulus coregoni (Crustacea: (Cyprinus carpio). Parasites & Vectors 2011 4:120. Branchiura). Parasitology 2004, 128:655-660. 84. Harrison AJ, Gault NFS, Dick JTA: Seasonal and vertical patterns of egg- laying by the freshwater fish louse Argulus foliaceus (Crustacea: Branchiura). Dis Aquat Organ 2006, 68:167-173. 85. Hakalahti T, Valtonen ET: Population structure and recruitment of the ectoparasite Argulus coregoni Thorell (Crustacea: Branchiura) on a fish farm. Parasitology 2003, 127:79-85. 86. Busacker GP, Adelman IR, Goolish EM: Methods for Fish Biology Maryland: American Fisheries Society; 1990. 87. Johansen K, Weber RE: On the adaptability of haemoglobin function to environmental conditions. In Perspectives in Experimental Biology. Edited by: Davies PS. Oxford: Pergamon; 1976:219-234. 88. Weber RE: Functional significance and structural basis of multiple hemoglobins with special reference to ectothermic vertebrates. In Animal Nutrition and Transport Processes, 2 - Transport, Respiration and Excretion: Comparative and Environmental Aspects. Edited by: Truchot JP, Lahlou B. Basel: Karger; 1990:58-75. 89. Lenhardt M: Seasonal changes in some blood chemistry parameters and in relative liver and gonad weights of pike (Esox lucius L.) from the river danube. J Fish Biol 1992, 40:709-718. 90. Kortet R, Taskinen J, Sinisalo T, Jokinen I: Breeding-related seasonal changes in immunocompetence, health state and condition of the cyprinid fish, Rutilus rutilus,L.Biol J Linn Soc 2003, 78:117-127. 91. Skarstein F, Folstad I, Liljedal S: Whether to reproduce or not: immune suppression and costs of parasites during reproduction in the Arctic charr. Can J Zoolog 2001, 79:271-278. 92. Suzuki Y, Orito M, Iigo M, Kezuka H, Kobayashi M, Aida K: Seasonal changes in blood IgM levels in goldfish, with special reference to water temperature and gonadal maturation. Fisheries Sci 1996, 62:754-759. 93. Avtalion RR: Temperature effect on antibody production and immunological memory, in carp (Cyprinus carpio) immunized against bovine serum albumin (BSA). Immunology 1969, 17:927-931. Submit your next manuscript to BioMed Central 94. Stolen JS, Gahn T, Kasper V, Nagle JJ: The effect of environmental and take full advantage of: temperature on the immune response of a marine teleost (Paralichthys dentatus). Dev Comp Immunol 1984, 8:89-98. 95. Suzuki Y, Otaka T, Sato S, Hou YY, Aida K: Reproduction related • Convenient online submission immunoglobulin changes in rainbow trout. Fish Physiol Biochem 1997, • Thorough peer review 17:415-421. • No space constraints or color ﬁgure charges 96. Hou Y, Suzuki Y, Aida K: Changes in immunoglobulin producing cells in response to gonadal maturation in rainbow trout. Fisheries Sci 1999, • Immediate publication on acceptance 65:844-849. • Inclusion in PubMed, CAS, Scopus and Google Scholar 97. Hou Y, Suzuki Y, Aida K: Effects of steroids on the antibody producing • Research which is freely available for redistribution activity of lymphocytes in rainbow trout. Fisheries Sci 1999, 65:850-855.

Submit your manuscript at www.biomedcentral.com/submit

Článek D

The physiological and immunological status of tench, Tinca tinca L.: the effects of hormonal stimulation and ploidy character determined by chromosomal manipulation

připravováno pro Aquaculture

ROHLENOVÁ K, DÁVIDOVÁ M., HYRŠL P., TOLÁROVÁ S., FLAJŠHANS M., ŠIMKOVÁ A

The physiological and immunological status of tench, Tinca tinca L.: the effects of hormonal stimulation and ploidy character determined by chromosomal manipulation

Rohlenová K.1, Dávidová M. 1, Hyršl P. 2, Tolarová S.2, Flajšhans M.3 & Šimková A. 1

1Department of Botany and Zoology, Faculty of Science, Masaryk University, Kotlářská 2,

611 37 Brno, Czech Republic

3Institute of Experimental Biology, Faculty of Science, Masaryk University, Kotlářská 2, 611

37, Brno, Czech Republic

3 University of South Bohemia in Česke Budějovice, Faculty of Fisheries and Protection of

Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses,

Vodňany, Zátiší 728/II, 389 25 Czech Republic

*Corresponding author: Andrea Šimková, Department of Botany and Zoology, Faculty of

Science, Masaryk University, Kotlářská 2, 611 37 Brno, Czech Republic, Phone: +420-

549497363; Fax: +420-541211214; E-mail: [email protected]

1 Abstract

The methods of genetic modifications have an important position in commercial aquaculture.

The fish, artificially obtained by induced polyploidy or gynogenesis, possess many different life traits that render them interesting for basic and applied researches. Therefore, the aim of this study was to compare the important components of physiology and immunology among amphimictic diploid, induced triploid and meiotic gynogenetic tench (Tinca tinca), and to test the effect of hormonal stimulation on physiology and immunity. The ploidy level affected fish condition and hepato-somatic index. The significant lower condition was detected in triploid females when compared to diploid females. Significant differences among populations were detected for blood cell counts (erythrocytes, leukocytes and phagocytes) in both genders. The highest cell counts were found in diploids. Moreover, the non-specific immunity (phagocyte activity measured by respiratory burst) differed between diploid and triploid tench. No significant effect of population on other parameters of non-specific immunity (complement activity and lysozyme activity) and specific immunity (IgM concentration) was found. Our results suggest that induced triploid tench kept in aquaculture under the same conditions as diploids dispose lower condition and their immunocompetence is reduced.

The hormonal treatment is commonly used in aquaculture to induce the spermiation, ovulation and spawning. The hormonal stimulation of tench significantly influenced physiological

(haemoglobin and glucose) and immunological (respiratory burst activity and lysozyme concentration) parameters in both males and females. In all tench populations, the level of glucose decreased after stimulation. The obtained results are probably connected with repeated stressors such is fish handling which can cause desensitization and attenuation of the neuroendocrine and metabolic responses. The increasing values of haemoglobin, lysozyme concentration and respiratory burst were found after hormonal stimulation. The changes in immunological parameters could be connected with secondary stress responses. The same

2 effect of hormonal stimulation on 11-ketotestosterone concentration and both lysozyme concentration and respiratory burst suggests a potential link between this steroid hormone and non-specific immunity.

Introduction

In connection with increasing fish production, a hormonal treatment has an important position in commercial aquaculture. It is commonly applied to stimulate the feed consumption, to increase body growth efficiency (Hunt et al., 2000; Silverstein et al., 2000; Leedom et al.,

2002), to modify the immunological function (Sakai et al., 1996; Narnaware et al., 1998) and to induce the reproduction in many fish species (Mylonas & Zohar, 2001; Adebayo &

Fagbenro, 2004; Fagbenro, 2004; Asturiano et al., 2005; Mylonas et al., 2010). Hormonal stimulation of fish reproduction is applied primarily in fish held in captivity, which often exhibit several forms of reproductive dysfunctions. In females, there are problems with final oocyte maturation, ovulation or spawning, while in the case of males, the milt production is reduced, or reaches a lower quality (Yaron, 1995; Mylonas & Zohar, 2001; Zohar & Mylonas,

2001; Viveiros et al., 2002). Artificial reproduction is does not depend on the seasonally determined spawning in natural conditions, which may represent the numerous advantages in pisciculture. The first hormones, extracts of fish pituitary glands, inducing the reproduction of fish, have been utilized since 1930s (Billard, 1989). Over time, many studies have used the hormonal approaches including synthetic hormones to induce spawning in fish living in captivity (Peter et al., 1988; Halder et al., 1991; Tucker, 1994; Mylonas & Zohar, 2001; Zohar

& Mylonas, 2001; Adebayo & Fagbenro, 2004; Zakes & Szczepkowski, 2004; Asturiano et al., 2005; Schiavone et al., 2006; Wojtczak et al., 2007). Recently, the most common technique to induce spawning in cultured fish has been the application either of gonadotropin- releasing hormone (GnRH) or its synthetic analogue version (GnRHa). These hormones have been successfully used to induce the spermiation, ovulation and spawning in various groups

3 of fish including mainly cyprinids and salmonids (Mikolajczyk et al., 2003; Arabaci & Sari,

2004; Rutaisire & Booth, 2004; Wojtczak et al., 2007; Mikolajczyk et al., 2008; Podhorec &

Kouřil, 2009) .

Reproduction is an important life trait; the investment in reproduction affects each fish organism. In the case of artificial reproduction, which is induced by rapid hormonal change unlike natural spawning, fish could be stressed by hormonal application, handling, and changes of water temperature. Prior to hormonal manipulations, genetic modifications have been often applied in fish commercial aquaculture (Flajšhans et al., 1993; Flajšhans et al.,

1995; Arai, 1997; Benfey, 1999; Gomelsky, 2003). In general, the chromosomal manipulations have been applied to induce the polyploidy and changes in mode of reproduction such as gynogenesis, androgenesis and other sex manipulations. Polyploidy, the multiplication of entire sets of chromosomes beyond the normal set of two, is phenomenon also known in teleosts. Artificially induced polyploidy has been used in aquaculture to produce sterility or improve production (Donaldson & Devlin, 1996). Triploid fish are interesting models for physiological research due to their specific characteristics – e.g. their sterility, faster growth, longer lifespan and better quality of muscle tissue when compared with diploids (Ihssen et al., 1990). Such differences are associated with biological cost connected with reproduction. Diploid individuals invest source of energy into the development of gametes, whereas genetically manipulated fish like induced triploids invest into their body growth (Beaumont et al., 2003). However, a few studies showed the contradictory results, e.g. according to Benfey (2001) triploid salmonids in commercial aquaculture tend to have reduced growth and survival with evidence of chronic stress.

In some cases, the chromosomal manipulation is connected with the modification of reproduction mode. In fish, gynogenesis is the asexual reproduction leading to all-female population (e.g. Yamamoto, 1999) so that the resulting fish possess many traits that render

4 them interesting for basic research. In gynogenesis, although the spermatozoa do not participate in creation of new genotype in offspring, females require the presence of sperm to stimulate their eggs. The mitotic diploid or meiotic diploid gynogenetic populations can be produced following the suppression of first mitotic or second meiotic divisions after fertilization using inactivated sperm (Purdom, 1983; Ihssen et al., 1990). Gynogenetic diploids have been induced in many fish species (Ihssen et al., 1990; Linhart et al., 1995a; Na-

Nakorn, 1995; Arai, 1997). The life traits, e.g. growth, survival or morphology, were compared between gynogenetic diploids and sexually-reproducing diploid females in some fish species. In Misgurnus anguillicaudatus (Suzuki et al., 1985) and Salmo salar (Johnstone

& Stet, 1995), gynogenetic fish was growing faster than sexually-reproducing diploids. In contrast with these studies, gynogenetic individuals of Clarias macrocephalus (Na-Nakorn,

1995) and Gnathopogon caerulescens (Fujioka, 1998) revealed similar growth rates to sexually-reproducing diploids.

In aquaculture, the different resistance to diseases between triploid and diploid fish could be one of important aspects of production triploid fish commercially. Generally, triploid salmonids appear to be less resistant to various pathogens compared with diploid fish (e.g.

Dunham, 2004; Ozerov et al., 2010). However, some other studies showed the similar resistance to infection in diploid and triploid fish (e.g. Inada et al., 1990; Yamamoto & Iida,

1995b; Piačková & Flajšhans, 2006). Further, fish resistance may be also connected with experimental design and might differ among fish species (Budino et al., 2006).

The tench (Tinca tinca L.) is a popular cyprinid fish, cultivated extensively in European ponds since the middle ages. The Czech Republic belongs to one of the major producers of marketable tench in Europe, although its production depends either on natural spawning of selected fish under controlled conditions, or completely on artificial reproduction (Flajšhans,

1997). Carp pituitary extract and synthetic analogues of gonadotropin-releasing hormone

5 (GnRHa) are very effective for induction of tench reproduction (Kouřil et al., 1986; Linhart et al., 1995a). Hormonal treatment is frequently applied especially in tench males, when this stimulation has a positive effect on the volume and quality of the sperm (Caille et al., 2006).

Moreover, tench is an object of chromosomal manipulation associated with modified reproduction (such is gynogenesis) in order to increase fish production.

The aim of the present work was to evaluate the effect of hormonal stimulation and population effect (i.e. amphimictic diploid, induced triploid and meiotic gynogenic populations) on the selected physiological and immunological parameters of tench. The character of population was either non-influenced by chromosomal manipulation (i.e. amphimictic diploids) or determined by chromosomal manipulation associated with modified mode of reproduction (i.e. induced triploids and gynogenetic diploids).

6 Material and methods

Fish sampling

A total of 93 five-year-old individuals of tench (Tinca tinca, L.) were collected from pond- farmed Vodňany population (University of South Bohemia in České Budějovice, Faculty of

Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and

Biodiversity of Hydrocenoses, České Budějovice, Czech Republic) during one week in June

2008. Sampled fish included amphimictic diploid (2n) – 22 females and 16 males, induced triploid (3n) – 15 females and 20 males and 20 meiotic gynogenetic (MeiG) tench. Eggs for amphimictic diploid (2n) individuals were incubated intact, triploidy was reached by exposing the eggs to a cold shock (0-2°C, 35 min) starting 2-5 min after gamete activation following

Flajšhans et al. (1993) and Flajšhans & Linhart (2000), and finally, diploid gynogenic population was induced by meiotic gynogenesis. For induction of gynogenetic development, carp sperm genome was inactivated by gamma irradiation (Co-60; 1400 GY dose) and after gamete activation, cold shock (0-2°C, 30 min) was applied following Linhart et al. (1995b).

Level of ploidity in larval stage was assessed as relative DNA content by flow cytometry according Lecommandeur et al. (1994).

In the day of sampling, all fish were caught using a seine net. Individuals of each population

(i.e. amphimictic diploid, induced triploid and meiotic gynogenetic populations) were selected into two groups, i.e. non-stimulated and stimulated. The group of stimulated fish was separated in tanks at 20–23°C and hormonal treatment to induce production of gametes according to methodology by Rodina (2004) and Linhart (2006) was applied. Carp pituitary suspension for the stimulation of males and GnRH analog [D-Ala6, GnRH ProNHEt,

Kobarelin] for the stimulation of females were used. Both stimulants were injected intramuscularly into the dorsal part of the fish body.

7 The group of non-stimulated fish was analyzed immediately after fish sampling. The group of stimulated fish was analyzed two days after hormonal treatment. Blood samples and mucus were obtained from each individual. Blood samples were taken from caudal vein using methodology according to Pravda & Svobodová (2003) and were put in the microtubules with heparin (10µl, 5 000 U/1ml, Zentiva). Blood for analysis of respiratory burst and haematology

(differential leukocyte count, total leukocyte and erythrocyte count, hematocrit and haemoglobin concentration) was processed immediately after collection. Blood plasma samples for other analyses (IgM, 11-ketotestosterone concentration, glucose concentration and complement activity) were deep-frozen (-80°C). The samples of mucus were used for analysis of lysozyme activity performed immediately after mucus collection.

After sampling, each individual was tagged with a P.I.T. tag for later identification (134.2 kHz, AEG ID-162, AEG Co., Ulm, Germany) intramuscularly into the left side of the dorsal part close to the first hard dorsal fin ray. Fish in the original water from the pond were transported to the laboratory and humanely killed by severing the spine. Subsequently, each individual was measured and weighed, e.g. total length (in mm) and total body weight (in grams). Fulton’s condition index was calculated following the equation: KF = [body weight

(in g)/ (total length (in cm))3 ] × 100000 (Anderson & Neumann, 1996). Moreover, the weights of visceral organs, e.g. gonads, spleen and liver (in grams) were measured, and relative size of each organ, i.e. spleen-somatic index (SSI), gonado-somatic index (GSI) and hepato-somatic index (HSI) was calculated. For the details see (Rohlenová et al., 2011).

Haematological analyses

Leukocyte and erythrocyte counts, haematocrit, haemoglobin concentration and differential leukocyte count have been considered as important physiological or immunological parameters of fish health state. The analysis of total leukocyte (in G.l-1) and erythrocyte count

8 (in T.l-1) were performed according to methodology of Svobodová et al. (1986) and Lusková

(1997). Haemoglobin content (Hb) was analyzed photometrically (540 nm; Helios Unicam,

USA) in Kampen–Zijlster transformation medium. Differential leukocyte profile was assessed from blood smears stained with Hemacolor set (Merck Co., Darmstadt, Germany). Further, we estimated the percentage distribution of all types of white blood cells (Pravda &

Svobodová, 2003). Moreover, the relative counts of lymphocytes and phagocytes (in g/l) were calculated prior to performing statistical analyses (see Rohlenová & Šimková, 2010).

Haematocrit was analyzed according to Svobodová et al. (1986).

Imunological and physiological parameters

Respiratory burst activity

Respiratory burst activity was measured as luminol enhanced chemiluminescence using a luminometer (LM01-T, Immunotech, Czech Republic) and opsonised using Zymosan A as activator (Kubala et al. 1996, Nikoskelainen et al. 2004). In the present study, the maximal intensity of respiratory burst (peak in relative light units i.e. RLU) was evaluated. For the details see (Buchtíková et al., 2011).

Complement activity

The total bacteriolytic activity (TA) including all pathways was determined using a bioluminescence-based method. Transformed bacteria E. coli K12 with luxABCDE gene were exposed at laboratory temperature to plasma obtained from T. tinca. The light emission of the reaction is positively correlated with the viability of E. coli which was measured using LM01-

T luminometer (Immunotech, Czech Republic). The relative complement activity was calculated prior to performing statistical analyses. For the details see (Buchtíková et al.,

2011).

9 Lysozyme concentration

The lysozyme concentration was assessed in vitro by radial diffusion in agarose gel mixed with Micrococcus luteus (CCM 169). Samples of 15 μl of mucus from individual fish were applied into the well cut in the agarose gel placed on glass plates, and incubated at room temperature (20°C). After twenty four hours, mean diffusion zone was measured and the concentration of lysozyme in the sample was converted to mg per ml of mucus according to calibration curve. For details see Poisot et al. (2009).

Determination of total IgM level

The level of total IgM in plasma was analyzed by precipitation with zinc sulphate. This method is based on specific dehydration of immunoglobulins by 0.7 mM ZnSO4.7H2O (pH =

5.8). The immunoglobulins originated from the solution and were removed by centrifugation.

The following quantification of IgM was based on the total level of proteins in the sample using commercially available kit (Bio-Rad, USA) and plate reader (Tecan, Sunrise, USA) before and after precipitation. The concentration of IgM in the sample (in g/l) was calculated as the difference between the total protein and protein contained in the supernatant after precipitation and centrifugation.

Determination of total glucose level

The level of glucose in plasma was analyzed following the instructions provided in the commercial enzyme kit (Glu L 1000, PLIVA-Lachema, Czech Republic). Samples in duplicates were analyzed with a plate reader (Tecan Sunrise, USA) and a concentration of glucose in samples (mmol/l) against the glucose standard solution was determined.

10 11-Ketotestosterone determination

The level of 11-ketotestosterone in male plasma was analyzed following the instructions provided in the commercial competitive enzyme immunoassay kits (Cayman Chemical,

Estonia). The plates with samples were analyzed with a plate reader (Tecan Sunrise, USA) and a concentration of 11-KT (in pg.ml−1) was calculated according to manufacturer's instructions. For details see (Buchtíková et al., 2011).

Statistical analyses

The following physiological parameters were analyzed: total body length, KF, GSI, HSI, erythrocyte count, haematocrit, haemoglobin, and glucose concentration. In males, an 11- ketotestosterone concentration was also included in the analyses. The analyzed immunological parameters included: SSI, leukocyte count, relative lymphocyte and phagocyte counts, respiratory burst activity, relative activity of complement, IgM concentration and lysozyme concentration. The data on total fish body length and haematological parameters

(i.e. erythrocyte count, haematocrit and haemoglobin concentration) fitted a normal distribution. Other data required transformation prior to parametric tests. The following transformations were applied: log-transformation for KF, GSI, SSI, LSI, complement activity, phagocyte and lymphocyte counts and glucose concentration; square root transformation for leukocyte count, IgM concentration and 11-ketotestosterone concentration, and finally hyperbolic arcsine square root transformation for respiratory burst and lysozyme concentration. First, using non-stimulated fish, general linear models (GLM), taking into account total body length, were applied to test the gender effect on immunological and physiological parameters in amphimictic diploid and induced triploid populations. One-way

ANOVA was used to test the effect of population on total body length of tench. Following the results of first GLM analysis and considering the fact that the different hormonal stimulation

11 was applied in males and females, the next GLM analyses followed by Tukey HSD post-hoc tests were performed separately for males and females. The effects of hormonal stimulation

(i.e. stimulated and non-stimulated) and population (amphimictic diploid, induced triploid and gynogenetic diploid) on immunological and physiological parameters were tested. As the spleen, gonad and liver weights were correlated with total body weight, we used SSI, GSI and

HSI in statistical analyses to eliminate the effect of body size. All statistical analyses were executed using Statistica 9.0 for Windows.

RESULTS

Gender effect on immunology and physiology of non-stimulated tench

First, using GLM analysis, the gender effect on measured immunological and physiological parameters taking into account a total body length was tested in non-stimulated tench (Table

1). Gender effect was analyzed separately for amphimictic diploids and induced triploids.

The significant effect of sex on GSI in amphimictic diploid fish was found. Higher values of

GSI were found in females. Gender effect was also found for complement activity (Table 1).

A weak gender effect (but total model was not significant p = 0.058) was found for phagocyte count. Higher values of both immunological parameters were found in males. Further, effect of sex on immunological and physiological parameters was tested in induced triploid tench.

Using GLM analysis, the statistical significant effect of sex on GSI and respiratory burst activity was found. Higher values of GSI were found in females whereas respiratory burst activity was higher in males.

The effects of population and hormonal stimulation in females

First, using One-way ANOVA, the effect of population on total body length of tench was tested. The effect of population on total body length was not significant (p > 0.05). Next, the effect of hormonal stimulation on a female total body length was tested using GLM analysis.

12 No difference in total body length between non-stimulated and stimulated females was found

(p > 0.05).

Consequently, using GLM, the effects of population and hormonal stimulation on each of measured immunological and physiological variables taking into account the total body length were tested for females (Table 2). In females, the total body length significantly influenced only one of the measured immunological parameter, i.e. respiratory burst activity.

Using the general linear models with the effects of population and hormonal stimulation, we found that the character of population related to ploidy level and mode of reproduction had significant effect on many physiological and immunological parameters in females, i.e. spleen

(SSI), blood cell counts (erythrocytes, leukocytes, lymphocytes), and respiratory burst (Table

2). A weak population effect on condition factor and phagocyte count was found (however, the total model was not significant). Using the GLM including only population effect and taking into account the total body length, the significant effect of population was also found for HSI (F for total model = 3.676, p = 0.019, F for population effect = 4.673, p = 0.014) and confirmed for condition factor (F for total model = 3.742, p = 0.017, F for population effect =

4.97, p = 0.011) and phagocyte count (F for total model = 3.829, p = 0.015, F for population effect = 5.655, p = 0.006). All these variables except for SSI and HSI showed the same trend, i.e. higher values in amphimictic diploid females and meiotic gynogenic females compared with induced triploids (see Fig. 1A-C for condition factor, erythrocytes, leukocytes and Fig.

2A for respiratory burst). Using Tukey HSD post hoc test, the significant differences (p <

0.05) for all above listed parameters between induced triploids and amphimictic diploids were found. Moreover, the significant differences (p < 0.05) for all of these parameters except respiratory burst and phagocyte count were also found between induced triploids and meiotic gynogenic females. The lowest values of SSI and HSI in amphimictic diploid females and the

13 highest values of these indices in meiotic gynogenic females were found (Tukey HSD post hoc test, p <0.05).

The significant effect of hormonal stimulation on two physiological and two immunological parameters was found i.e. haemoglobin, glucose concentration, lysozyme concentration, and respiratory burst. Using GLM including the population effect and hormonal stimulation effect taking into account the total body length, the significant effect of hormonal stimulation on lysozyme, and glucose concentration was found (Table 2). Using the general linear models including only hormonal stimulation and taking into account the total body length, the significant effect of hormonal stimulation on haemoglobin was also found (F for total model =

4.198, p = 0.020, F for hormonal stimulation effect = 7.712, p = 0.008). Respiratory burst was the only immunological parameter in females affected by both population and hormonal stimulation (Figure 2A) and moreover, the interaction between both effects was significant

(Table 2). The significant interaction between ploidy and stimulation was also found for SSI.

Concerning hormonal stimulation, the higher values of haemoglobin, respiratory burst and lysozyme concentration were found in stimulated females (Fig. 2A-B). On the other hand, glucose concentration decreased after stimulation of females (Fig. 2C).

The effects of population and hormonal stimulation in males

First, the effect of hormonal stimulation on male total body length was tested using GLM analysis. No difference in total body length between non-stimulated and stimulated males was found (p > 0.05).

Subsequently, using GLM, the effects of population and hormonal stimulation on each of measured immunological and physiological variables taking into account a total body length were tested in males (Table 3). In males, the total body length significantly influenced two immunological parameter i.e. respiratory burst activity and leukocyte count.

14 Erythrocyte count (Figure 1B) and respiratory burst (Figure 2A) were affected by both population and hormonal stimulation effects. The effects of both factors were also found for

11-ketotestosteron concentration, however, the total model was not significant (Table 3). A significant effect of population on phagocyte count and a weak population effect on leukocyte count (Figure 1C) in males were also found. The higher values of all above listed parameters were found in amphimictic diploid males when compared with induced triploids. Hormonal stimulation had moreover a significant effect on other three parameters i.e. haemoglobin, lysozyme concentration, and glucose concentration in males (Figure 2B-C). Using GLM including the population effect and hormonal stimulation effect and taking into account the total body length, the significant effect of hormonal stimulation on lysozyme and glucose concentration was found (Table 3). Using the general linear models including only hormonal stimulation and taking into account the total body length, the significant effect of hormonal stimulation on haemoglobin was also found (F for total model = 3.783, p = 0.033, F for hormonal stimulation effect = 7.333, p = 0.011). Whereas the hormonal stimulation led to decreasing of the glucose concentration, the values of other immunological and physiological parameters significantly affected by stimulation were higher in stimulated fish when compared to non-stimulated fish.

Differential leukocyte count in tench populations

The differential leukocyte count was analyzed for meiotic gynogenetic females and separately for males and females in amphimictic diploid and induced triploid populations. The proportion of all white blood cell types (i.e. lymphocytes, monocytes and neutrophiles including myelocytes, metamyelocytes, bands and segments) in different fish groups (i.e. non- stimulated and stimulated 2n males, 2n females, 3n males, 3n females and meiotic gynogenetic females) is shown in Fig. 3. The blood was clearly of lymphocytary character,

15 i.e. lymphocytes dominated in the leukocyte counts. In non-stimulated fish, the proportion of lymphocytes varied from 88.3% for amphimictic diploid males to 96.6% for non-stimulated meiotic gynogenetic females. A weak increase of lymphocytes after hormonal stimulation was observed only in males (both amphimictic diploid and induced triploid). In non-stimulated fish, the proportion of monocytes varied from 0% (induced triploid females) to 4.56%

(induced triploid males). Low proportion of monocytes (less than 1%) was found in all female groups after hormonal stimulation. The decrease of monocytes due to hormonal stimulation was found in induced triploid males. In non-stimulated fish, neutrohiles represented from

3.1% (for meiotic gynogenetic females) to 10.4% (for amphimictid diploid males) of total blood cells when the neutrophile proportion in diploid males was at least twice higher than the neutrophile proportion in other fish groups. Approximately, the same proportion of neutrophiles was found between non-stimulated and stimulated induced triploid males, and amphimictic diploid females and meiotic gynogenetic females. However, a decrease of neutrophile proportion after hormonal stimulation was found in induced triploid females and was especially obvious in amphimictic diploid males. Concerning the proportions of different neutrophile cell types, metamyelocytes were the most frequently observed cells of the granulocyte series. In non-stimulated fish, the proportion of metamyelocytes varied from

2.2% to 6.9%. Hormonal stimulation led to similar changes of metamyelocyte proportion in induced triploid females and amphimictic diploid males as described for total neutrophile count. Other types of neutrophilic granulocytes were rare with the proportion less than 2% of total leukocyte count (Fig. 3).

Discussion

The present study was conducted on the analyses of selected measures of fish condition, reproduction and immunity in amphimictic diploid, induced triploid and meiotic gynogenetic

16 populations of tench living in aquaculture. Moreover, the effect of hormonal stimulation on all measured physiological and immunological parameters was studied.

Effect of ploidy level on physiology and immunity of fish

Generally, triploid fish are sterile, but yet show different degrees of gonad development and dispose of larger but fewer cells in most tissues and organs compared to diploids (Benfey,

1999). Therefore, a better growth which may be observed in triploids probably resulted from their sterility and different energy consumption when compared with diploids. According to

Beaumont et al. (2003), diploid individuals invest their sources of energy into the reproduction connected with gamete development whereas triploids invest energy into body growth. However, Flajšhans & Linhart (2000) reviewed the studies that aimed to test the hypothesis of higher growth rate of triploids compared to diploids and evidenced many studies not supporting this hypothesis (e.g. Gervai et al., 1980; Cherfas et al., 1994; Bonnet et al., 1999). Benfey (2001) showed that growth and survival of triploid salmonids is reduced when compared with diploids and evidenced that triploids fish tend to be chronically stressed.

Fish store their energy in muscles and liver (in form of glycogen). Therefore, condition factor and hepato-somatic index (HSI) may be used as an indirect indicator of energy status

(Busacker et al., 1990). The indicator of reproductive investment in fish is the gonado-somatic index (GSI), which represents an accurate assessment of reproductive maturity. In the present study, we found that condition factor of induced triploids females was lower than condition factor of amphimictic diploid females and meiotic gynogenetic females. The same trend was observed for amphimictic diploid males and induced triploid males, although this difference was not significant (p > 0.05). These results did not confirm the hypothesis of positive effect of sterility on somatic condition of triploids. Any growth advantage of triploids, even when the diploid controls were fully mature, was not observed for example in the sea bass,

Dicentrarchus labrax (Felip et al., 2001). However, fish grow performances are closely

17 dependent on conditions in which fish are reared (Cassani & Caton, 1986; Galbreath et al.,

1994; McCarthy et al., 1996). When the conditions of communal rearing are used, the triploids may exhibit lower grow performances than diploids. This may not necessarily mean that triploid grow worse, but the observations may be connected with their low aggressiveness and ability to compete for food (Maxime, 2008). The information on growth and maturation in gynogenetic fish are still limited. Our study showed the similar condition factor in amphimictic diploid females and meiotic gynogenetic females of tench which was also previously shown in the sea bass, Dicentrarchus labrax (Peruzzi et al., 2004).

The gonad weight and derived index of reproduction (GSI) differ among diploids, triploids and meiotic gynogens (Flajšhans et al., 1993; Peruzzi et al., 2004). In the present study, the difference in GSI for both genders in amphimictic diploids of tench when compared with induced triploids was not found. Felip et al. (2001) showed that prior to sexual maturation of the sea bass, Dicentrarchus labrax, GSI of triploid males was similar to the one observed in diploid males. The increasing differences in the GSI between diploid and triploid males just before fish spawning are connected with gonad maturation. When the gonads of diploid and triploid tench were macroscopically examined, the differences in histological composition between diploid and triploid individuals were found (see Flajšhans, 1997). 11- ketotestosterone is a major androgen in majority teleosts, responsible for sexual behavior and spermatogenesis (Borg, 1994). In this study, the character of population linked to ploidy status determined by chromosomal manipulation seems to partially affect the level of this steroid hormone. The higher values of 11-ketotestosterone were found in diploid males when compared with triploids although this difference was not statistically significant, suggesting that in the present study level of steroid hormones was not associated with development of gonads.

18 In our study, HSI, indicator of energy status, and SSI, a measure of immunocompetence, were analyzed in amphimictic diploid, induced triploid and meiotic gynogenetic populations.

Spleen is one of the principal lymphomyeloid tissues of teleosts. In fish, spleen size is used as a simple measurable immunological parameter with potential role in immune response against parasite infection (e.g. Kortet & Taskinen, 2004; Taskinen & Kortet, 2002). We found significant differences in HSI and SSI among female groups, the lowest values were found in amphimictic diploid females, the higher values were found in induced triploid, and they increased again in gynogenetic females. The similar trend of lower values of HSI and SSI was also found in amphimictic diploid males when compared with induced triploid males although the differences were not statistically significant (not shown in results). Hepato-somatic index was previously analyzed in several fish species with different ploidy level (e.g. Johnson et al.,

1986; Felip et al., 2001; Buchtová et al., 2003) but little is still known about spleen size in fish species when the populations of different ploidy level occur naturally or are the result of chromosomal manipulation in artificial conditions. Buchtová et al. (2003) studied amphimictic diploids and induced triploids of tench and suggested that the effect of ploidy level was reflected in significantly higher weight of hepatopancreas in induced triploid females at T3 and T3+ age and higher weight of spleen in triploid females and males at T3+ age when compared with diploids of the same age. However, when the correction for body weight was applied, i.e. HSI and relative spleen size were used, HSI was lower in triploid than in diploid females. The significant reduction of hepatopancreas weight expressed by HSI in triploids when compared with diploids was also observed in Oncorhynchus kisutch (Johnson et al., 1986) and Dicentrarchus labrax (Felip et al., 2001). These results cannot be explicitly interpreted because the weight of internal organs of fish can be affected by various internal

(age, sex, muscular activity, health) and external (water temperature, oxygen content in water, food supply) factors (Kouřil et al., 1978). Moreover, the weight of hepatopancreas correlates

19 with glycogen deposited in, and with its ability to water retention (Svobodová & Kocová,

1977), thus the differences in hepatopancreas between diploids and triploids could reflect the different glycogen reserve.

The haematological parameters in diploid and triploid fish were previously extensively studied (e.g. Benfey et al., 1984; Suzuki et al., 1985; Biron & Benfey, 1994; Nakamura et al.,

1989; Svobodová et al., 2001). As already mentioned above, triploids dispose higher cell count than diploid individuals, but also the different immunological activity and subsequently lower resistance against pathogen infection in triploids were observed (Dunham, 2004;

Ozerov et al., 2010). Many studies investigating blood cell profiles in diploid and triploid fish have concentrated on erythrocytes and oxygenation capacity of blood (e.g. Biron & Benfey,

1994; Yamamoto & Iida, 1994; Ranzani-Paiva et al., 1998; Benfey & Biron, 2000).

Comparison of leukocyte count and the analysis of leukocyte profiles in diploid and triploid fish are rather rare (Ranzani-Paiva et al., 1998; Svobodová et al., 1998; Svobodová et al.,

2001). In the present study, the significant differences in erythrocyte, leukocyte and phagocyte counts for both females and males among amphimictic diploid, induced triploid and meiotic gynogenetic populations, were found i.e. the higher counts of these blood cells were found in amphimictic diploids or meiotic gynogens of tench when compared with induced triploids. Our findings support other studies performed in different fish species which showed increasing ploidy level associated with decreasing erythrocyte count (e.g. Svobodová et al., 1998; Peruzzi et al., 2005; Vetešník et al., 2006; Gao et al., 2007). Moreover, the ploidy level is associated with erythrocyte size and haemoglobin content (Benfey, 1999). However, in the present study, no significant difference was observed for haemoglobin concentration.

In our study of tench, the similar trend as observed for erythrocytes was revealed for leukocytes and also when analyzing phagocytes separately, i.e. lower counts of leukocytes and phagocytes in triploids than in diploids was found. However, Svobodová et al. (1998)

20 found that the leukocyte count of triploid tench was insignificantly lesser compared to diploids. Other analyses showed no significant difference in total leukocyte count between diploid and triploid 3-year-old tench (Svobodová et al., 2001).

Moreover, in the present study, the different immune response using the parameter of specific immunity (IgM) and parameters of non-specific immunity (lysozyme concentration, phagocyte count, respiratory burst activity and complement activity) were analyzed in amphimictic diploids, induced triploids and meiotic gynogens of tench. However, only non- specific immunity, phagocyte count and phagocyte activity measured by respiratory burst differed among populations in both genders. Both parameters of non-specific immunity analyzed in our study were lower in induced triploids when compared to amphimictic diploids.

According to Budino et al. (2006) large phagocytic cells in triploids have the high surface membrane and cell volume, and therefore the higher capacity of individual phagocytes is supposed in triploids when compared to diploids. However, when taking into account the number of phagocytes converted to one microliter of blood, significant difference in total phagocytic activity was similar between diploids and triploids of turbot (Psetta maxima)

(Budino et al., 2006). The similar results were also shown for rainbow trout (Oncorhynchus mykiss) (Yamamoto & Iida, 1995a). In our study, functional activity of phagocytes using respiratory burst measured in the same blood volume was analyzed. In both genders, the higher values of phagocyte count in diploids than in triploids connected with higher respiratory burst were found. It could support the hypothesis that triploid fish investing more intensively and rapidly in their growth have the reduced energy for immune protection.

Moreover, the difference in phagocyte activity between diploid and induced triploid females could suggest the different susceptibility to pathogen infection. In the study of Carassius auratus, a free living cyprinid fish species with diploid sexual form and triploid gynogenetic

21 form, the nitroblue tetrazolium test to measure the level of immune activity of the phagocytes was applied (Hakoyama et al., 2001). Phagocyte activity (i.e. immune activity of neutrophils and monocytes) of sexual diploid females was significantly higher than that of gynogenetic triploid females. This difference in non-specific reaction was suggested to explain the high parasite load in triploids of C. auratus.

Concerning other components of non-specific immunity measured in our study, i.e. complement activity and lysozyme concentration, no significant effect of populations on complement activity and lysozyme activity was observed. No significant difference in complement activity were previously found between diploid and triploid individuals of rainbow trout (Oncorhynchus mykiss) (Yamamoto & Iida, 1995a) and turbot (Psetta maxima)

(Budino et al., 2006). Moreover, in turbot (Psetta maxima), the lysozyme concentration in serum was similar in both diploids and triploids. All these findings suggested the similarity in immunological activity of the non-specific cellular defense in diploids and triploids.

The weak effect of population (p = 0.048) on the component of specific humoral system measured by IgM concentration was found when comparing females of amphimictic diploid with induced triploid and meiotic gynogenetic populations of tench (not shown in results).

However, the total general linear model including the population effect and hormonal stimulation effect was not significant (p = 0.220). General linear model including only population effect revealed a statistically non significant trend of population effect (p = 0.064) on IgM concentration The effect of population found for lymphocyte count was the same as observed trend of population effect on IgM concentration among female groups. The increasing count of lymphocytes in diploid females was followed by increasing production of

IgM antibodies. On the other hand, the decreasing lymphocyte count in triploid females was accompanied by lower IgM production. This suggests the link between number of lymphocytes and IgM production in tench. To our knowledge, there is no study comparing

22 IgM concentration between diploid and triploid fish. Recently, the effects of polyploidy on specific gene expression was investigated in diploids and triploids of chinook salmon

(Oncorhynchus tshawytscha) (Ching et al., 2010). It was shown, that after an immune challenge with the pathogen Vibrio anguillarum, the gene expression for IgM of triploids was reduced when compared to diploids. This finding supported the prediction that under stress triploids displayed reduced performance.

Effect of hormonal stimulation on physiology and immunity of fish

Our study showed that the hormonal stimulation significantly influenced several physiological and immunological parameters in both males and females. Concerning tench physiology, haemoglobin (using GLM including the effect of hormonal stimulation) and glucose concentration (using GLM including population effect and hormonal stimulation effect) were significantly influenced by hormonal stimulation. Among the measured immunological parameters, respiratory burst activity and lysozyme concentration, both representing the components of innate immunity, were affected by hormonal stimulation. Whereas hormonal stimulation led to an increase of haemoglobin and lysozyme and a decrease of glucose concentration in all tench populations and both genders, respiratory burst of meiotic gynogenetic females was not affected by hormonal stimulation. Moreover, the stimulation significantly influenced erythrocyte count and partially influenced 11-ketotestosterone concentration in males.

After hormonal treatment the initiation of reproduction connected with increasing values of

11-ketotestosterone was hypothesized. Following immunocompetence handicap hypothesis

(Folstad & Karter, 1992), immunosupression effect of 11-ketotestosterone is predicted. In the present study, the 11-ketotestosterone concentration increased after stimulation and at the same time, the values of two immunological parameters i.e. lysozym concentration and

23 respiratory burst increased in both amphimictic diploid and induced triploid males. Although the immunosupressive affect of 11-ketotestosterone on lysozyme activity has not been documented in fish yet, the negative effect of androgens including 11-ketotestosterone on activity of phagocytes has been already reported (Watanuki et al., 2002; Yamaguchi et al.,

2001). On the other hand, the immunosupressive role of testosterone was not unambiguously supported in meta –analysis performed across other vertebrates (see Roberts et al., 2004).

The level of glucose in blood serum of fish may be used as an indicator of stress (Busacker et al., 1990). Fish under a stress condition use energy reserves such as glycogen in muscle and liver and subsequently increase the level of glucose in serum (Hoar et al., 1992). The glucose level in blood of fish is a subject of seasonal changes especially those connected with spawning (Hoar et al., 1992). In the present study, we applied the hormonal stimulation, which initiates fish reproduction, and we observed that the level of blood glucose decreased after stimulation in both genders and in all types of populations (i.e. amphimictic diploid, induced triploid and meiotic gynogenetic). In our study, all fish were collected the same day and subsequently glucose level was analyzed in the groups of non-stimulated fish whereas other fish designed to stimulate were transferred to the hatchery and subsequently stimulated.

The investigation of these fish was performed two days after stimulation. Therefore, a decrease of glucose level observed in stimulated fish more likely results from the exposure to stressors such is fish handling. Repeated stress can cause a fish desensitization and attenuation of the neuroendocrine and metabolic responses to subsequent exposure to stressors (Barton et al., 1987); thus, the matching response as a significant decline of glucose level can be observed (Reid et al., 1998).

Primary and secondary physiological responses of fish to environmental stressors were defined by Barton (2002). The secondary responses include glucose level, the changes in haematological (hematocrit, leucokrit and haemoglobin) and immunological (such is

24 lysozyme and antibody production) parameters (see review by Barton, 2002). Therefore, the higher values of haemoglobin, lysozyme concentration and respiratory burst found in males and females and the higher erythrocyte count in males, could be also linked to secondary stress responses.

Differential leukocyte count in tench populations

In all analyzed populations (amphimictic diploid, induced triploid and meiotic gynogenetic) and both genders, blood of lymphocytary character was observed as previously shown for example in diploids and triploids of tench (Tinca tinca) (Svobodová et al., 2001) or rainbow trout (Oncorhynchus mykiss) (Ranzani-Paiva et al., 1998). In 5- year-old tench analyzed in this study, no strong difference in white blood cell profile was found when comparing different populations, likewise no strong effect of hormonal stimulation on differential leukocyte profiles was revealed. Similar results were previously published for 3-year-old and

4-year-old diploid and triplod tench (Svobodová et al., 2001). According to Svobodová et al.

(2001), healthy tench based on clinical and morphological examination have lymphocyte frequency over 90%, monocyte frequency below 1%, and granulocytes are mostly represented by myelocytes and metamyelocytes. In the present study, all fish groups showed approximately normal range of healthy tench except for non-stimulated diploid and triploid males, where the proportion of phagocytes was higher than in other tench groups. Generally, lymphocytopenia connected with neutropenia and/or monocytosis is related to several causes, e.g. parasite infection, spawning, water temperature, etc. (e.g. Nair & Nair, 1983; Pickering,

1986). The most presumptive reason explaining different white blood cell profiles observed in this study is either male-biased gender susceptibility in parasitism or disease (e.g. Reimchen

& Nosil, 2001) or the different resistance of diploid and triploid males (Dunham, 2004;

Ozerov et al., 2010).

25 Acknowledgements

This study (all immunological and a large part of haematological analyses) was funded by the

Grant Agency of the Czech Republic, project No. 524/07/0188. KR was funded by the

Ichthyoparasitology Research Centre of the Ministry of Education, Youth and Sports of the

Czech Republic LC 522 and partially by the Rector’s Programme in Support of MU Students’

Creative Activities. AŠ was supported by the Research Project of Masaryk University (No.

MSM0021622416). This study was partly supported (i.e. fish manipulation and heamatological analyses) by projects CENAKVA CZ.1.05/2.1.00/01.0024 and GAJU

047/2010/Z. We thank Marie Pečená from South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Vodňany for analyzing blood cell profiles and Naďa

Musilová, Iva Přikrylová, Kateřina Francová and Jaroslav Piňos from Department of Botany and Zoology for their help with fish manipulation.

26 References

Adebayo OT, Fagbenro OA (2004) Induced ovulation and spawning of pond raised African giant catfish, Heterobranchus bidorsalis by exogenous hormones. Aquaculture, 242, 229-236. Anderson RO, Neumann RM (1996) Length, weight, and associated structural indices. In: Fisheries Techniques (ed. by Murphy BR, Willis DW). American Fisheries Society, Bethesda, pp. 447–482. Arabaci M, Sari M (2004) Induction of ovulation in endemic pearl mullet (Chalcalburnus tarichi), living in the highly alkaline Lake Van, using GnRHa( D-Ser(tBu)(6), Pro(9)- Net -GnRH) combined with haloperidol. Aquaculture, 238, 529-535. Arai K (1997) Chromosome manipulation. In: Fish DNA (ed. by Aoki T, Takashima F, Hirano T). Koseisha-Koseikaku, Tokyo, pp. 32-62 (In Japanese). Asturiano JF, Perez L, Garzon DL, Penaranda DS, Marco-Jimenez F, Martinez-Llorens S, Tomas A, Jover M (2005) Effect of different methods for the induction of spermiation on semen quality in European eel. Aquaculture Research, 36, 1480-1487. Barton BA (2002) Stress in fishes: A diversity of responses with particular reference to changes in circulating corticosteroids. Integrative and Comparative Biology, 42, 517- 525. Barton BA, Schreck CB, Barton LD (1987) Effects of chronic cortisol administration and daily acute stress on growth, physiological conditions, and stress responses in juvenile rainbow trout. Diseases of Aquatic Organisms, 2, 173-185. Beaumont A, Boudry P, Hoare K (2003) Biotechnology and Genetics in Fisheries and Aquaculture, Blackwell Publishers. Benfey TJ (1999) The physiology and behavior of triploid fishes. Reviews in Fisheries Sciences, 7, 39–67. Benfey TJ (2001) Use of sterile triploid Atlantic salmon (Salmo salar L.) for aquaculture in New Brunswick, Canada. ICES Journal of Marine Science, 58, 525-529. Benfey TJ, Biron M (2000) Acute stress response in triploid rainbow trout (Oncorhynchus mykiss) and brook trout (Salvelinus fontinalis). Aquaculture, 184, 167-176. Benfey TJ, Sutterlin AM, Thompson RJ (1984) Use of erythrocyte measurements to identify triploid salmonids. Canadian Journal of Fisheries and Aquatic Sciences, 41, 980-984. Billard R (1989) Endocrinology and fish culture. Fish Physiology and Biochemistry, 7, 49-58. Biron M, Benfey TJ (1994) Cortisol, glucose and hematocrit changes during acute stress, cohort sampling, and the diel cycle in diploid and triploid brook trout (Salvelinus fontinalis Mitchill). Fish Physiology and Biochemistry, 13, 153-160. Bonnet S, Haffray P, Blanc JM, Vallee F, Vauchez C, Faure A, Fauconneau B (1999) Genetic variation in growth parameters until commercial size in diploid and triploid freshwaterrainbow trout (Oncorhynchus mykiss) and seawater brown trout (Salmo trutta). Aquaculture, 173, 359-375. Borg B (1994) Androgens in teleost fishes. Comparative Biochemistry and Physiology C- Pharmacology Toxicology & Endocrinology, 109, 219-245. Buchtová H, Svobodová Z, Flajšhans M, Vorlová L (2003) Analysis of slaughtering value of diploid and triploid population of tench (Tinca tinca, Linnaeus 1758). Czech Journal of Animal Science, 48, 285-294. Buchtíková S, Vetešníková Šimková A, Rohlenová K, Flajšhans M, Lojek A, Lilius E-M, Hyršl P (2011) The seasonal changes in innate immunity of the common carp (Cyprinus carpio). Aquaculture, 318, 169-175.

27 Budino B, Cal RM, Piazzon MC, Lamas J (2006) The activity of several components of the innate immune system in diploid and triploid turbot. Comparative Biochemistry and Physiology a-Molecular & Integrative Physiology, 145, 108-113. Busacker GP, Adelman IR, Goolish EM (1990) Methods for fish biology, American Fisheries Society, Maryland. Caille N, Rodina M, Kocour M, Gela D, Flajshans M, Linhart O (2006) Quantity, motility and fertility of tench Tinca tinca (L.) sperm in relation to LHRH analogue and carp pituitary treatments. Aquaculture International, 14, 75-87. Cassani JR, Caton WE (1986) Growth comparisons of diploid and triploid grass carp under varying conditions. Progressive Fish-Culturist, 48, 184-187. Cherfas NB, Gomelsky B, Bendom N, Peretz Y, Hulata G (1994) Assessment of triploid common carp (Cyprinus carpio L) for culture. Aquaculture, 127, 11-18. Ching B, Jamieson S, Heath JW, Heath DD, Hubberstey A (2010) Transcriptional differences between triploid and diploid Chinook salmon (Oncorhynchus tshawytscha) during live Vibrio anguillarum challenge. Heredity, 104, 224-234. Donaldson EM, Devlin RH (1996) Uses of biotechnologyto enhance production. In: Principles of Salmonid Culture (ed. by Pennell W, Barton BA). Developments in Aquaculture and Fisheries Science, pp. 969-1020. Dunham R (2004) Aquaculture and fisheries biotechnology: Genetic approaches, CAB International, Cambridge. Fagbenro OA (2004) Soybean meal replacement by roquette (Eruca sativa Miller) seed meal as protein feedstuff in diets for African Catfish, Clarias gariepinus (Burchell 1822), fingerlings. Aquaculture Research, 35, 917-923. Felip A, Piferrer F, Carrillo M, Zanuy S (2001) Comparison of the gonadal development and plasma levels of sex steroid hormones in diploid and triploid sea bass, Dicentrarchus labrax L. Journal of Experimental Zoology, 290, 384-395. Flajšhans M (1997) Reproduction sterility caused by spontaneous triploidy in tench (Tinca tinca). Polish Archives of Hydrobiology, 44, 39-45. Flajšhans M, Linhart O (2000) Production of triploid tench. Manuals of Research Institute of Fish Culture and Hydrobiology, University of South Bohemia, Vodňany (in Czech). Flajšhans M, Linhart O, Kvasnička P (1995) Tench, Tinca tinca (Linnaeus, 1758): a model for chromosomal manipulation studies. Polish Archives of Hydrobiology, 42, 123–131. Flajšhans M, Linhart O, Kvasnička P (1993) Genetic studies of tench (Tinca tinca L) - induced triploidy and tetraploidy and 1st performance data. Aquaculture, 113, 301-312. Folstad I, Karter AJ (1992) Parasites, bright males, and the immunocompetence handicap. American Naturalist, 139, 603-622. Fujioka Y (1998) Survival, growth and sex ratios of gynogenetic diploid honmoroko. Journal of Fish Biology, 52, 430-442. Galbreath PF, Stjean W, Anderson V, Thorgaard GH (1994) Freshwater performance of all female diploid and triploid Atlantic salmon. Aquaculture, 128, 41-49. Gao Z, Wang W, Abbas K, Zhou X, Yang Y, Diana JS, Wang H, Wang H, Li Y, Sun Y (2007) Haematological characterization of loach Misgurnus anguillicaudatus: Comparison among diploid, triploid and tetraploid specimens. Comparative Biochemistry and Physiology a-Molecular & Integrative Physiology, 147, 1001-1008. Gervai J, Peter S, Nagy A, Horvath L, Csanyi V (1980) Induced triploidy in carp, Cyprinus carpio L. Journal of Fish Biology, 17, 667-671. Gomelsky B (2003) Chromosome set manipulation and sex control in common carp: a review. Aquatic Living Resources, 16, 408-415.

28 Hakoyama H, Nishimura T, Matsubara N, Iguchi K (2001) Difference in parasite load and nonspecific immune reaction between sexual and gynogenetic forms of Carassius auratus. Biological Journal of the Linnean Society, 72, 401-407. Halder S, Sen S, Bhattacharya S, Ray AK, Ghosh A, Jhingran AG (1991) Induced spawning of Indian major carps and maturation of a perch and a catfish by murrel gonadotropin releasing hormone, pimozide and calcium. Aquaculture, 97, 373-382. Hoar WS, Randall DJ, Conte PF (1992) Fish Physiology: The Cardiovascular System, Academic Press, London. Hunt AS, Soares JH, Byatt JC, Dahl GE (2000) The effects of exogenous bovine growth hormone and placental lactogen on juvenile striped bass Morone saxatilis feed and growth efficiency. Journal of the World Aquaculture Society, 31, 14-21. Ihssen PE, McKay LR, McMillan I, Phillips RB (1990) Ploidy manipulation and gynogenesis in fishes: Cytogenetic and fisheries applications. Transactions of the American Fisheries Society, 119, 698-717. Inada Y, Yoshimura K, Taniguchi N (1990) Study on the resistance to vibriosis in induced triploid ayu Plecoglossus altivelis. Nippon Suisan Gakkaishi, 56, 1587-1591. Johnson OW, Dickhoff WW, Utter FM (1986) Comparative growth and development of diploid and triploid coho salmon, Oncorhynchus kisutch. Aquaculture, 57, 329-336. Johnstone R, Stet RJM (1995) The prodution of gynogenetic Atlantic salmon, Salmo salar L. Theoretical and Applied Genetics, 90, 819-826. Kortet R, Taskinen J (2004) Parasitism, condition and number of front head breeding tubercles in roach (Rutilus rutilus L.). Ecology of Freshwater Fish, 13, 119-124. Kouřil J, Barth T, Hamáčková J, Flegel M (1986) Induced ovulation in tench (Tinca-Tinca L) by various LH-RH synthetic analogs - effect of site of administration and temperature. Aquaculture, 54, 37-44. Kouřil J, Hamáčková J, Svobodová Z (1978) Fluctuation of relative hepatopancreas weight in the course of the year in brood tench (Tinca tinca L.) kept in ponds. Bulletin VÚRH Vodňany, 4, 7-11 (in Czech). Lecommandeur D, Haffray P, Philippe L (1994) Rapid flow cytometry method for ploidy determination in salmonid eggs. Aquaculture and Fisheries Management, 25, 345– 350. Leedom TA, Uchida K, Yada T, Richman NH, Byatt JC, Collier RJ, Hirano T, Grau EG (2002) Recombinant bovine growth hormone treatment of tilapia: growth response, metabolic clearance, receptor binding and immunoglobulin production. Aquaculture, 207, 359-380. Linhart O, Flajšhans M, Kvasnička P (1995a) Gynogenesis of tench (Tinca tinca L.) after short-term storage of eggs. Aquaculture, 129, 135-135. Linhart O, Kvasnička P, Flajšhans M, Kasal A, Ráb P, Paleček J, Šlechta V, Hamáčková J, Prokes M (1995b) Genetic studies with tench, Tinca tinca L.: induced meiotic gynogenesis and sex reversal. Aquaculture, 132, 239-251. Linhart O, Rodina M, Flajšhans M, Mavrodiev N, Nebesarová J, Gela D, Kocour M (2006) Studies on sperm of diploid and triploid tench, Tinca tinca (L.). Aquaculture International, 14, 9-25. Lusková V (1997) Annual cycles and normal values of hematological parameters in fishes, Acta Scientiarum Naturalium, Brno. Maxime V (2008) The physiology of triploid fish: current knowledge and comparisons with diploid fish. Fish and Fisheries, 9, 67-78. McCarthy ID, Carter CG, Houlihan DF, Johnstone R, Mitchell AI (1996) The performance of all-female diploid and triploid Atlantic salmon smolts on transfer together to sea water. Journal of Fish Biology, 48, 545-548.

29 Mikolajczyk T, Chyb J, Sokolowska-Mikolajczyk M, Enright WJ, Epler P, Filipiak M, Breton B (2003) Attempts to induce an LH surge and ovulation in common carp (Cyprinus carpio L.) by differential application of a potent GnRH analogue, azagly-nafarelin, under laboratory, commercial hatchery, and natural conditions. Aquaculture, 223, 141- 157. Mikolajczyk T, Sokolowska-Mikolajczyk M, Szczerbik P, Duc M, Goryuko K, Dobosz S, Glogowski J, Epler P, Enright WJ (2008) The effects of the GnRH agonist, azagly- nafarelin (Gonazon (TM)), on ovulation and egg viability in the European grayling (Thymallus thymallus L.). Aquaculture, 281, 126-130. Mylonas CC, Fostier A, Zanuy S (2010) Broodstock management and hormonal manipulations of fish reproduction. Generaland Comparative Endocrinology, 165, 516-534. Mylonas CC, Zohar Y (2001) Use of GnRHa-delivery systems for the control of reproduction in fish. Reviews in Fish Biology and Fisheries, 10, 463-491. Na-Nakorn U (1995) Comparison of cold and heat shocks to induce diploid gynogenesis in thai walking catfish (Clarias macrocephalus) and performances of gynogens. Aquatic Living Resources, 8, 333–341. Nair GA, Nair NB (1983) Effect of infestation with the isopod, Alitropus typus M. Edwards (Crustacea: Flabellifera: Aegidae) on the haematological parameters of the host fish, Channa striatus (Bloch). Aquaculture, 30, 11-19. Nakamura S, Imai N, Ohya S, Kobayashi H (1989) Oxygen uptake rate and hematological characteristics in triploid amago salmon, Oncorhynchus masou macrostomus. Memoirs of the Faculty of Agriculture of Kinki University, 22, 31-38. Narnaware YK, Kelly SP, Woo NYS (1998) Stimulation of macrophage phagocytosis and lymphocyte count by exogenous prolactin administration in silver sea bream (Sparus sarba) adapted to hyper- and hypo-osmotic salinities. Veterinary Immunology Immunopathology, 61, 387-391. Ozerov MY, Lumme J, Pakk P, Rintamaki P, Zietara MS, Barskaya Y, Lebedeva D, Saadre E, Gross R, Primmer CR, Vasemagi A (2010) High Gyrodactylus salaris infection rate in triploid Atlantic salmon Salmo salar. Diseases of Aquatic Organisms, 91, 129-136. Peruzzi S, Chatain B, Saillant E, Haffray P, Menu B, Falguiere JC (2004) Production of meiotic gynogenetic and triploid sea bass, Dicentrarchus labrax L. 1. Performances, maturation and carcass quality. Aquaculture, 230, 41-64. Peruzzi S, Varsamos S, Chatain B, Fauvel C, Menu B, Falguiere JC, Severe A, Flik G (2005) Haematological and physiological characteristics of diploid and triploid sea bass, Dicentrarchus labrax L. Aquaculture, 244, 359-367. Peter RE, Lin HR, Vanderkraak G (1988) Induced ovulation and spawning of cultured freshwater fish in China: Advances in application of GnRH analogues and dopamine antagonists. Aquaculture, 74, 1-10. Piačková V, Flajšhans M (2006) Long-term examination of health conditions in monoculture of communally tested amphimictic diploid, diploid gynogenic and triploid tench, Tinca tinca (L.). Aquaculture International, 14, 43-59. Pickering AD (1986) Changes in blood cell composition of the brown trout, Salmo trutta L., during the spawning season. Journal of Fish Biology, 29, 335-347. Podhorec P, Kouřil J (2009) Induction of final oocyte maturation in Cyprinidae fish by hypothalamic factors: a review. Veterinary Medicine, 54, 97-110. Poisot T, Šimková A, Hyršl P, Morand S (2009) Interactions between immunocompetence, somatic condition and parasitism in the chub Leuciscus cephalus in early spring. Journal of Fish Biology, 75, 1667-1682. Pravda D, Svobodová Z (2003) Haematology of fishes, Noviko, Brno (in Czech).

30 Purdom CE (1983) Genetic engineering by the manipulation of chromosomes. Aquaculture, 33, 287-300. Ranzani-Paiva MJT, Tabata YA, Das-Eiras AC (1998) Comparated hematology between diploids and triploids of rainbow trout, Oncorhynchus mykiss Walbaum (Pisces Salmonidae). Revista Brasileira de Zoologia, 15, 1093–1102. Reid SG, Bernier NJ, Perry SF (1998) The adrenergic stress response in fish: control of catecholamine storage and release. Comparative Biochemistry and Physiology C- Pharmacology Toxicology & Endocrinology, 120, 1-27. Reimchen TE, Nosil P (2001) Ecological causes of sex-biased parasitism in threespine stickleback. Biological Journal of the Linnean Society, 73, 51-63. Roberts ML, Buchanan KL, Evans MR (2004) Testing the immunocompetence handicap hypothesis: a review of the evidence. Animal Behaviour, 68, 227-239. Rodina M, Cosson J, Gela D, Linhart O (2004) Kurokura solution as immobilizing medium for spermatozoa of tench (Tinca tinca L.). Aquaculture International, 12, 119-131. Rohlenová K, Morand S, Hyršl P, Tolarová S, Flajšhans M, Šimková A (2011) Are fish immune systems really affected by parasites? A immunoecological study of common carp (Cyprinus carpio). Parasites & Vectors, 4. Rohlenová K, Šimková A (2010) Are the immunocompetence and the presence of metazoan parasites in cyprinid fish affected by reproductive efforts of cyprinid fish? Journal of Biomedicine and Biotechnology. Rutaisire J, Booth AJ (2004) Induced ovulation, spawning, egg incubation, and hatching of the cyprinid fish Labeo victorianus in captivity. Journal of the World Aquaculture Society, 35, 383-391. Sakai M, Kobayashi M, Kawauchi H (1996) In vitro activation of fish phagocytic cells by GH, prolactin and somatolactin. The Journal of Endocrinology, 151, 113-118. Schiavone R, Zilli L, Vilella S, Fauvel C (2006) Human chorionic gonadotropin induces spermatogenesis and spermiation in 1-year-old European sea bass (Dicentrarchus labrax): Assessment of sperm quality. Aquaculture, 255, 522-531. Silverstein JT, Wolters WR, Shimizu M, Dickhoff WW (2000) Bovine growth hormone treatment of channel catfish: strain and temperature effects on growth, plasma IGF-I levels, feed intake and efficiency and body composition. Aquaculture, 190, 77-88. Suzuki R, Oshiro T, Nakanishi T (1985) Survival, growth and fertility of gynogenetic diploids induced in the cyprinid loach, Misgurnus anguillicaudatus. Aquaculture, 48, 45-55. Svobodová Z, Flajšhans M, Kolárová J, Modrá H, Svoboda M, Vajcová V (2001) Leukocyte profiles of diploid and triploid tench, Tinca tinca L. Aquaculture, 198, 159-168. Svobodová Z, Kocová A (1977) The hepatopancreas of the carp: the relation ofweight to glycogen content. Buletin VÚRH Vodňany, 2, 22-24(in Czech). Svobodová Z, Kolářová J, Flajšhans M (1998) The first findings of the differences in complete blood count between diploid and triploid tench, Tinca tinca L. Acta Veterinaria Brno, 67, 243-248. Svobodová Z, Pravda D, Paláčková J (1986) Universal methods of hematological investigations in fish, Edice Metodik, Vodňany. Taskinen J, Kortet R (2002) Dead and alive parasites: sexual ornaments signal resistance in the male fish, Rutilus rutilus. Evolutionary Ecology Research, 4, 919-929. Tucker JW (1994) Spawning by Captive Serranid Fishes: A Review. Journal of The World Aquaculture Society, 25, 345-359. Vetešník L, Halačka K, Lusková V, Lusk S (2006) Erythrocyte profile of diploid and triploid silver crucian carp (Carassius auratus). Acta Veterinaria Brno, 75, 203-207.

31 Viveiros ATM, Fessehaye Y, ter Veld M, Schulz RW, Komen J (2002) Hand-stripping of semen and semen quality after maturational hormone treatments, in African catfish Clarias gariepinus. Aquaculture, 213, 373-386. Watanuki H, Yamaguchi T, Sakai M (2002) Suppression in function of phagocytic cells in common carp Cyprinus carpio L. injected with estradiol, progesterone or 11- ketotestosterone. Comparative Biochemistry and Physiology C-Toxicology & Pharmacology, 132, 407-413. Wojtczak M, Kuzminiski H, Dobosz S, Mikotajczyk T, Dietrich GJ, Kowalski R, Kottowska M, Enright WJ, Ciereszko A (2007) Milt characteristics in European whitefish (Coregonus lavaretus) in relation to season and hormonal stimulation with a gonadotropin-releasing hormone analogue, azagly-nafarelin. In: Biology and Management of Coregonid Fishes - 2005 (ed. by Jankun M, Brzuzan P, Hliwa P, Luczynski M), pp. 171-185. Yamaguchi T, Watanuki H, Sakai M (2001) Effects of estradiol, progesterone and testosterone on the function of carp, Cyprinus carpio, phagocytes in vitro. Comparative Biochemistry and Physiology C-Toxicology & Pharmacology, 129, 49- 55. Yamamoto A, Iida T (1994) Hematological characteristics of triploid Rainbow trout. Fish Pathology, 29, 239-243. Yamamoto A, Iida T (1995a) Nonspecific defense activities of triploid Rainbow trout. Fish Pathology, 30, 107-110. Yamamoto A, Iida T (1995b) Susceptibility of triploid rainbow trout to IHN, furunculosis and vibriosis. Fish Pathology, 30, 69-70. Yamamoto E (1999) Studies on sex-manipulation and production of cloned populations in hirame, Paralichthys olivaceus (Temminck et Schlegel). Aquaculture, 173, 235-246. Yaron Z (1995) Endocrine control of gametogenesis and spawning induction in the carp. Aquaculture, 129, 49-73. Zakes Z, Szczepkowski M (2004) Induction of out-of-season spawning of pikeperch, Sander lucioperca (L.). Aquaculture International, 12, 11-18. Zohar Y, Mylonas CC (2001) Endocrine manipulations of spawning in cultured fish: from hormones to genes. Aquaculture, 197, 99-136.

32 TABLES

Table 1. GLM analyses of the effect of sex, taking into account total body length, on physiological and immunological parameters in non-stimulated amphimictic diploid and induced triploid tench population

Fish group Dependent variable SS df MS F p total F (p) amphimictic diploids GSI 0.962 1 0.962 29.458 < 0.001 46.835 (<0.001) Complement 0.155 1 0.155 18.343 0.001 11.223 (0.002) induced triploids GSI 1.822 1 1.822 13.283 0.003 7.293 (0.008) Respiratory burst 3.562 1 3.562 10.437 0.007 5.547 (0.020)

33 Table 2. GLM analyses of the effect of population (amphimictic diploid, induced triploid and gynogenetic diploid) and hormonal stimulation, taking into account total body length, on physiological and immunological parameters in females

Dependent variable Independent variable SS df MS F p total F (p) KF population 0.018 2 0.009 4.867 0.012 2.284 (0.053) Erythrocyte count population 4.051 2 2.026 27.603 < 0.001 9.978 (<0.001) Glucose concentration stimulation 0.268 1 0.268 16.496 < 0.001 3.975 (0.003} SSI population 0.169 2 0.084 4.793 0.013 3.363 (0.008) population_stimulation 0.167 2 0.083 4.731 0.014 Leukocyte count population 60.061 2 30.031 17.243 < 0.001 6.148 (<0.001) Lymphocyte count population 1.503 2 0.752 18.969 < 0.001 6.672 (<0.001) Phagocyte count population 0.448 2 0.224 4.873 0.012 2.124 (0.067) Respiratory burst total body length 0.799 1 0.799 4.067 0.049 11.162 (<0.001) population 5.275 2 2.637 13.42 < 0.001 stimulation 4.845 1 4.845 24.656 < 0.001 population_stimulation 3.469 2 1.734 8.825 < 0.001 Lysozyme concentration stimulation 5.92 1 5.92 49.593 < 0.001 8.495 (<0.001)

Table 3. GLM analyses of the effect of population (amphimictic diploid, induced triploid and gynogenetic diploid) and hormonal stimulation, taking into account total body length, on physiological and immunological parameters in males

Dependent variable Independent variable SS df MS F p total F (p) Erythrocyte count population 3.371 1 3.371 93.07 < 0.001 30.617 (<0.001) stimulation 0.182 1 0.182 5.023 0.032 Glucose concentration stimulation 0.228 1 0.228 10.301 < 0.01 Leukocyte count total body length 8.886 1 8.886 7.492 0.01 2.586 (0.056) population 4.958 1 4.958 4.181 0.049 Phagocyte count population 0.483 1 0.483 10.578 0.003 3.673 (0.015) Respiratory burst total body length 0.531 1 0.531 4.263 0.048 7.545 (<0.001) population 2.133 1 2.133 17.119 < 0.001 stimulation 1.319 1 1.319 10.589 < 0.001 Lysozyme concentration stimulation 2.497 1 2.497 31.306 < 0.001 10.928 (<0.001) 11-Ketotestosterone concentration population 21908.225 1 21908.225 4.763 0.037 2.317 (0.079} stimulation 20584.286 1 20584.286 4.475 0.043

34 FIGURE LEGENDS

Figure 1. The effect of population on condition factor (A), erythrocyte count (B) and leukocyte count (C).

2n – amphimictic diploids, 3n – induced triploids, G – meiotic gynogenetic females, M – males, F – females, N – stimulated fish, S- non-stimulated fish.

Figure 2. The effects of hormonal stimulation and population on respiratory burst (A), the effect of hormonal stimulation on lysozyme concentration (B) and glucose concentration (C).

2n – amphimictic diploids, 3n – induced triploids, G – meiotic gynogenetic females, M – males, F – females, N – stimulated fish, S- non-stimulated fish.

Figure 3. Differential leukocyte profile. 2n – amphimictic diploids, 3n – induced triploids, G – meiotic gynogenetic females, M – males, F – females, N – stimulated fish, S- non-stimulated fish.

35 Fig. 1 3.06

3.04 A

3.02

3.00

2.98

2.96

2.94 Condition factor

2.92

2.90

2.88

2.86 2n F N 3n F N G F N 2n F S 3n F S G F S 2n M N 3n M N 2n M S 3n M S

2.2 B 2.0

1.8

1.6

1.4

Erythrocyte count 1.2

1.0

0.8

0.6 2n F N 3n F N G F N 2n F S 3n F S G F S 2n M N 3n M N 2n M S 3n M S

8.5

8.0 C

7.5

7.0

6.5

6.0

5.5

5.0 Leukocyte count

4.5

4.0

3.5

3.0

2.5 0.95 conﬁdence interval 2n F N 3n F N G F N 2n F S 3n F S G F S 2n M N 3n M N 2n M S 3n M S group Fig. 2 4.5 A 4.0

3.5

3.0

2.5 Respiratory burst

2.0

1.5

1.0 2n F N 2n F S 2n M N 2n M S 3n F N 3n F S 3n M N 3n M S G F N G F S

2.5 B 2.0

1.5

1.0

0.5

0.0 Lysozyme concentration Lysozyme

-0.5

-1.0

-1.5 2n F N 2n F S 2n M N 2n M S 3n F N 3n F S 3n M N 3n M S G F N G F S

1.2 C

1.1

1.0

0.9

0.8 Glucose concentration

0.7

0.6 0.95 conﬁdence interval 2n F N 2n F S 2n M N 2n M S 3n F N 3n F S 3n M N 3n M S G F N G F S group Fig. 3 2n M N 2n M S

0,71% 1,22% 1,89% 1,29%

3,71% 10,44% 6,89% 6,14% 88,33% 92,57%

1,14% 1,44% 0,22% 0,57%