Immunocompetence and Parasitism in Hybrids of Cyprinoid Fish

FACULTY OF SCIENCE

Immunocompetence and parasitism

in hybrids of cyprinoid fish

Ph.D. Thesis

VADYM KRASNOVYD

Supervisor: prof. RNDr. Andrea Vetešníková Šimková, Ph.D.

Department of Botany and Zoology

Brno 2020

Bibliographic Entry

Author: Mgr. Vadym Krasnovyd Faculty of Science, Masaryk University Department of Botany and Zoology

Title of Thesis: Immunocompetence and parasitism in hybrids of cyprinoid fish

Degree Program: PřF D-EKEB Ecological and Evolutionary Biology

Specialization: PARA Parasitology

Supervisor: prof. RNDr. Andrea Vetešníková Šimková, Ph.D. Faculty of Science, Masaryk University Department of Botany and Zoology

Academic Year: 2020/2021

Number of Pages: 148

Keywords: Interspecies hybridization, cyprinoids, immune response,

maternal ancestry, monogeneans, parasite community,

heterosis effect, host-parasite interactions, coadaptation

Bibliografický záznam

Author: Mgr. Vadym Krasnovyd Přírodovědecká fakulta, Masarykova univerzita Ústav botaniky a zoologie

Název práce: Imunokompetence a parazitismus u hybridů kaprovitých ryb

Studijní program: D-EKEB Ekologická a evoluční biologie

Specializace: PARA Parazitologie

Vedoucí práce: prof. RNDr. Andrea Vetešníková Šimková, Ph.D. Přírodovědecká fakulta, Masarykova univerzita Ústav botaniky a zoologie

Akademický rok: 2020/2021

Počet stran: 148

Klíčová slova: Mezidruhová hybridizace, kaprovité ryby, imunitní odpověď, mateřská linie, Monogenea, společenstva parazitů, heterózní efekt, hostitelsko-parazitické interakce, koadaptace

Abstract Fish hybrids and their parasites represent interesting models for evolutionary ecology. The modified immune response, ecological plasticity, maternal ancestry and vigour of hybrid fish are supposed to impact the diversity and distribution of their parasites. In the study A, common bream (Abramis brama), roach (Rutilus rutilus) and their hybrids were investigated. Metazoan parasite abundance and prevalence were higher in parental species when compared to their hybrids. Temporal and spatial effects on the parasite distribution were analysed. The effect of maternal ancestry of hybrids on digenean and crustacean infection level was confirmed. Asymmetrical distribution of parental species-specific parasites in favour of roach-specific parasites in hybrids was discovered. The effect of interspecific hybridization on vigour, physiology and immunity of cyprinoid fish was investigated (study B). Generally, hybrids tended to express intermediate characters of these traits. Role of the heterozygote advantage for F1 hybrids was proposed. Maternal origin of the hybrids was suggested to be involved in the expression of some branches of non-specific immunity. The experimental monogenean infection in pure breeds of silver bream (Blicca bjoerkna) and common bream and F1 cross-breeds under the condition of similar frequencies of pure and hybrid genotypes was investigated (study C). Potential effect of the maternal origin of hybrids on monogenean abundance was tested. Each of pure species harboured specific monogenean fauna. Hybrids harboured all monogenean species specifically infecting one or the other parental species. Monogenean infection level especially that of Dactylogyrus specific to A. brama was low in hybrids. We found no obvious effect of the maternal origin of hybrids on monogenean abundance, which indicates that the mtDNA of hybrids is not an important predictor of host-specific monogenean infection. This may suggest that mitochondrial genes are not strongly involved in the coadaptation between monogeneans and their associated hosts. The asymmetry in the distribution of A. brama specific parasites and B. bjoerkna-specific parasites in hybrids suggests similarity in the molecular components of the immune mechanisms between hybrids and B. bjoerkna. In conclusion, the necessity for genomic studies focused on the genes involved in reciprocal genetic co-adaptations of host-specific parasites and their associated hosts is highlighted by this study. Investigation of inherited protective immunological mechanisms that limit monogenean infection may shed light on the asymmetry in the presence of host-specific parasites in hybrids. Abstrakt Hybridi ryb a jejich paraziti představují zajímavé modely v evoluční ekologii. Modifikovaná imunitní odpověď, ekologická plasticita, mateřský původ a vitalita hybridů ryb mohou ovlivňovat diverzitu a distribuci jejich cizopasníků. Ve studii A byli studováni cejn velký (Abramis brama), plotice obecná (Rutilus rutilus) a hybridi první filiální generace (F1). Abundance a prevalence mnohobuněčných parazitů byla vyšší u rodičovských druhů ve srovnání s jejich hybridy. Analyzovány byly časový a prostorový vliv na distribuci parazitů. Prokázán byl vliv mateřského původu hybridů na míru parazitární infekce korýšů a motolic. Byla zjištěna asymetrická distribuce parazitů specifických pro rodičovské druhy u hybridů ve prospěch parazitů specifických pro plotici obecnou. Byl studován vliv mezidruhové hybridizace na vitalitu, fyziologii a imunitu (studie B). Hybridi měli obecně tendenci vykazovat střední hodnoty většiny měřených parametrů. Podpořena byla hypotéze heterozygotní výhody hybridů F1 generace. Zaznamenán byl efekt mateřského původu hybridů na expresi některých linií nespecifické imunity. Ve studii C byla analyzována experimentální infekce zástupci taxonu Monogenea u linií Blicca bjoerkna (cejnek malý), A. brama a jejich F1 hybridů v podmínkách podobných četností čistých a hybridních genotypů. Testován byl možný vliv mateřského původu hybridů na abundanci monogeneí. Oba druhy, B. bjoerkna i A. brama, byly parazitovány hostitelsky specifickými zástupci taxonu Monogenea, u obou druhů byla zaznamenána podobná míra parazitární infekce. Hybridi byli parazitováni všemi druhy monogeneí hostitelsky specifickými pro B. bjoerkna nebo A. brama. Míra infekce zástupci taxonu Monogenea, zejména infekce druhy rodu Dactylogyrus specifických pro A. brama, byla nízká u hybridů. Nebyl prokázán vliv mateřského původu hybridů na abundanci monogeneí. Výsledky ukazují, že mtDNA hybridů není důležitým indikátorem infekce druhy taxonu Monogenea specifickými pro hostitele, což může naznačovat, že mitochondriální geny nejsou zapojeny do koadaptace specifických monogeneí a jejich asociovaných hostitelů. Asymetrická distribuce parazitů druhově specifických pro A. brama a druhově specifických pro B. bjoerkna u hybridů ukazuje na podobnost mezi molekulárními složkami imunitních mechanismů mezi hybridy a B. bjoerkna. Z práce vyplývá potřeba genomických studií zaměřených na geny podílející se na vzájemných koadaptacích specifických parazitů a jejich hostitelů. Studium dědičnosti obranných imunitních mechanismů limitujících infekci zástupci Monogenea může také osvětlit asymetrii v míre vnímavosti hybridů k hostitelsky specifickým parazitům. Acknowledgement First of all, I would like to express my deepest gratitude to my supervisor prof. RNDr. Andrea Vetešníková Šimková, Ph.D. for her kind help, patience with me, and support. I would like also to express my sincere gratitude to Lukaš Vetešník for his help and contribution during all of these years to this work.

I would like to thank Pavel Jurajda and colleagues from the Institute of Vertebrate Biology, Academy of Sciences of the Czech Republic (and the whole team involved) who kindly helped with fish sampling. Also, I would like to thank my friends, labmates and colleagues from Department of Botany and Zoology, Faculty of Science, Masaryk University for helping with the fish dissections. I am thankful to Markéta Ondračková for the opportunity to be involved in the several dissections and field trips to Germany. I want to thank Pavel Hyršl and Jiří Jarkovský for all of their help. I want to thank Peter Stuart for kind help with the English revision of the thesis.

I am grateful for the opportunity provided to work in the Department of Botany and Zoology, Faculty of Science, Masaryk University.

This thesis was funded by Czech Science Foundation, projects No. P505/12/G112 and No. P505/12/0375.

In the end, I would like to thank my family and friends for keeping me motivated and keeping my “wheels rolling” even when it was extremely hard.

The last, but not least, my sincere gratitude goes to my schoolteacher of biology and chemistry Bondar' Ludmila Yaroslavna, who was kind and patient enough to teach such a wild child as I was in the past.

Original papers and contribution of the author to the papers included in the thesis Thesis includes 2 scientific papers and 1 manuscript under revision.

Paper A Krasnovyd, V., Vetešník, L., Gettová, L., Civáňová, K., & Šimková, A. (2017). Patterns of parasite distribution in the hybrids of non-congeneric cyprinid fish species: is asymmetry in parasite infection the result of limited coadaptation? International Journal for Parasitology, 47(8), 471-483.

Vadym Krasnovyd (VK) processed parasitological material, identified parasite species, performed mitochondrial identification of fish specimens. He was involved in the fish sampling and dissection and parasite collection. He prepared database. VK contributed to manuscript preparation.

Paper B Šimková, A., Janáč, M., Hyršl, P., Krasnovyd, V., & Vetešník, L. Vigour-related traits and immunity in hybrids of evolutionary divergent cyprinid species: advantages of hybrid heterosis? (resubmitted to Journal of Fish Biology)

VK was involved in the fish sampling and processing of material collected from fish specimens. He prepared database and partially participated on manuscript writing.

Paper C Krasnovyd, V., Vetešník, L., & Šimková, A. (2020). Distribution of host-specific parasites in hybrids of phylogenetically related fish: the effects of genotype frequency and maternal ancestry? Parasites & Vectors, 13(1), 1-11.

VK was actively participated on the processing of parasitological material and identified parasite species. He contributed to the writing of the manuscript.

Table of contents INTRODUCTION ...... 10

AIMS OF THE THESIS ...... 11

LITERATURE OVERVIEW ...... 12

Hybridization ...... 12

3.1.1 Natural and anthropogenic factors contributing to hybridization ...... 12

3.1.2 Species invasion and hybrid zones ...... 13

3.1.3 Genetic introgression and mitochondrial origin of hybrids ...... 14

3.1.4 Adaptive introgression and cytonuclear incompatibility ...... 16

3.1.5 Heterosis effect and transgressive segregation ...... 19

3.1.6 Evolutionary role of hybridization ...... 20

Fish hybridization ...... 23

3.2.1 Role of hybridization in evolution of cyprinoids ...... 23

3.2.2 Hybridization success of cyprinoids ...... 23

3.2.1 Intermediacy of traits: plasticity of hybrids ...... 24

Parasite infection in hybrids of cyprinoids ...... 25

3.3.1 Role of parasites in genetic introgression of hosts ...... 25

3.3.2 Distribution of parasites in parental species and their hybrids ...... 27

3.3.3 Host specificity and the system of co-adapted gene complexes ...... 31

3.3.4 Immunocompetence and parasitism in hybrids ...... 32

MATERIALS AND METHODS ...... 34

A. brama × R. rutilus hybridization ...... 36

4.1.1 Fish sampling and parasite collection ...... 36

4.1.2 Molecular identification ...... 37

4.1.3 Physiological measurements and fish condition ...... 38

4.1.4 Immunological and haematological analyses ...... 39

A. brama × B. bjoerkna hybridization ...... 40

4.2.1 Experimental design and cross-breeding ...... 40

RESULTS ...... 42

DISCUSSION ...... 49

CONCLUTIONS AND PERSPECTIVES ...... 55

REFERENCES ...... 57

LIST OF PUBLICATIONS ...... 80

PAPER A ...... 81

PAPER B ...... 95

PAPER C ...... 137

INTRODUCTION

From the point of view of parasitology, hybrid fish represent potential bridges for the parasites between hybridizing pure species. Therefore, host hybridization may influence host- parasite co-evolution by interruption of the co-adapted gene complexes between fish hosts and their especially host-specific parasites and, thus, may alter assymetry in the proportion of parental-specific parasites in hybrids. Sage et al. (1986) and Moulia et al. (1991) suggested that interruption in host-parasite co-adaptations i.e. breakdown in the host-parasite coadaptation system of genes due to hybridization might result in the high susceptibility of hybrid specimens to parasites. The roles of hybrids in the functioning of the host-parasite co-evolution and parasite transmission among the pure species have not been thoroughly investigated yet. Novel characteristics of hybrid genotypes (mitochondrial and nuclear origin) influence the overall fish condition. Heterosis advantage of hybrids is an interesting feature of cross-breeds which may alter parasite load in hybrids via enhancing of overall vigour. Fish hybrids exhibit intermediate traits in morphology and ecology to their parental species. Fish hybridization can lead to the establishment of novel adaptations gained by the transfer from parentals (genetic introgression) or the creation of the favourable mutations affecting parasite distribution in hybrids and limiting affinity of specific parasite to the host. Introgression transmitted elements of parental immunity may be differently pronounced in hybrids and, thus, reflected in the resistance/susceptibility to the parasite infection. Highly host-specific monogeneans and their cyprinoid hosts represent suitable models for the investigation of the effect of hybridization on the host-parasite assosiations evolved during coevolutionary arm races. On one hand, heterosis effect of the F1 hybrids may lead to the resistance to the parasites due to high vigour of hybrids. On the other hand, interuption of the defence mechanisms and cytonuclear breakdown in hybrids may cause their susceptibility to the parasite infection. Therefore, hybridization between cyprinoids represents the unique opportunity to the role of evolutionary divergence of hosts limiting the presence of host-specific parasites.

AIMS OF THE THESIS

• To investigate pattern of metazoan parasite distribution in evolutionarily divergent non-congeneric cyprinoid species – common bream (Abramis brama) and roach (Rutilus rutilus) and their hybrids coexisting in nature.

• To examine the potential physiological and immune aspects of heterosis effect in F1 hybrids of non-congeneric cyprinoids A. brama and R. rutilus

• To analyse the monogenean infection in pure breeds of silver bream (Blicca bjoerkna) and A. brama (two cyprinoid species with low evolutionary divergence) and cross-breeds (F1 generation) under the experimental condition of similar frequencies of pure and hybrid genotypes with specific focus on the potential effect of the maternal origin of hybrids on monogenean infection.

LITERATURE OVERVIEW

Hybridization

Hybridization of the plants and animals became well known after the works of Charles Darwin and Gregor Johann Mendel (Darwin, 1859; Mendel, 1866). Their works took into consideration the importance of hybridization in natural and laboratory conditions with special insight on hybrid superiority. Patterns of heredity with implementations to the plant and animal evolution revealed the importance of hybridization at the level of phylogenetic speciation and diversification (e.g. Barrett, 2010; Manriquez, 2010). Cross-breeding between animal species due to breakdown of the existing reproductive barriers is a frequently occurring phenomenon which has an influence on species evolution (Mayr, 1963; Barton and Hewitt, 1989; Barton, 2001). The appearance of cross-breeding in animals may occur between closely related or distant taxonomic units (Mallet, 2007). The breakdown of the barriers between species has appeared due to natural (Almodóvar et al., 2012) or anthropogenic disturbances (Hasselman et al., 2014) (i.e. habitat transformation, species invasion etc.). Reproductive barriers between species play a role against hybridization (e.g. Barton and Bengtsson, 1986; Goldberg and Lande, 2007; Monteiro et al., 2012; Lafon-Placette and Köhler, 2016). They include prezygotic barriers (i.e. gamete incompatibility, habitat isolation, difference in reproductive period, incompatible courtship behaviour; anatomical or physiological incompatibilities) and postzygotic barriers (i.e. zygote mortality, hybrid inferiority, hybrid sterility). The prezygotic barriers prevent the fertilization (Shen et al., 2015). The postzygotic barriers are targeted on the limitation of the viability (life success) or the fertilization capacity of the hybrids (Russell, 2003).

3.1.1 Natural and anthropogenic factors contributing to hybridization

From the larger spatial scales, natural factors contributing to species hybridization are linked to natural processes in the ecosystem (Cox and Moore, 2016), therefore they are mainly related to landscape dynamics (e.g. glaciation and plate tectonics) and consequent habitat transformation (Lu et al., 2001, Pillon et al., 2007). At smaller spatial scales, spatio-temporal variation affects species and formation of the hybrid zones (Carson et al., 2012). According to Hubbs (1955), hybridization among fishes was

LITERATURE OVERVIEW

documented to be closely-associated with the stability of the biotope (i.e. marine and tropical environments are more stable than temperate and freshwater habitats). Likewise, anthropogenic activity altering the natural habitats can cause hybridization among the indigenous species that do not hybridize under natural conditions (Neuffer et al., 1999; Bleeker and Hurka, 2001). In the last decade, number of studies focusing on indirect human-mediated habitat disturbances has been increased (e.g. climate change, global warming), some of them indicating the impact of such disturbances on hybridization (Garroway et al., 2010; Potts et al., 2014; Miralles et al., 2016). Direct anthropogenic influence on the natural habitats mainly caused by human activities is related to urbanization, infrastructure development and environmental management (e.g. eutrophication, dam construction, forestation, urbanization, water contamination etc.) (Fisher et al., 2006; Heath et al., 2010; Vonlanthen et al., 2012; Hasselman et al., 2014). Anthropogenic disturbances also caused the changes in interspecific interactions among species (Grabenstein and Taylor, 2018). For example, hybridization between divergent lineages that naturally occur in herrings living in sympatry (alewife (Alosa pseudoharengus) and blueback shad (Alosa aestivalis)) caused by anthropogenic habitat disturbance (dam construction on the Roanoke River) in Kerr Reservoir, North Carolina/Virginia (USA) was documented (Hasselman et al., 2014). Another example was showed in mammals, hybridization between sympatric North American flying squirrel species – northern flying squirrel (Glaucomys sabrinus) and southern flying squirrel (Glaucomys volans) induced by climate change was discovered recently (Garroway et al., 2010).

3.1.2 Species invasion and hybrid zones

Accelerated by climate change, expansions of aquatic and terrestrial species implement hybridization by the interaction between invasive and native species associated with the formation of the hybrid zones (Allendorf and Leary, 1998; Ainouche et al., 2009; Ryan et al., 2009; Ouanes et al., 2011; Potts et al., 2013; Muhlfeld et al., 2014). Hybrid zones occur when different taxa meet and mate, resulting in at least some offspring of mixed ancestry (Barton and Hewitt, 1989; Heslop-Harrison, 1990). It was confirmed that hybrid zones move in space and time, with significant consequences for evolutionary and conservation biology of hybridizing species, though such movement is often perceived as rare (Buggs, 2007; Ellstrand, 1992; Pérez-Suárez et al., 1994; Wolf et al., 2001; Liu et al., 2010). 13

LITERATURE OVERVIEW

For example, the introgression between the native Pecos pupfish (Cyprinodon pecosensis) and the introduced, as a baitfish, sheephead minnow (Cyprinodon variegatus) in Pecos river (Texas) generated a hybrid swarm. After 5 years of introduction of the sheephead minnow, it was found that the native Pecos pupfish was excluded from approximately half of its original range by hybrids (Echelle and Connor, 1989). Species invasions caused by the anthropogenic introduction of species to a novel habitat are quite frequent lately. For instance, hybridizations of Nile tilapia (Oreochromis niloticus) × three spotted tilapia (Oreochromis andersonii) and O. niloticus × longfin tilapia (Oreochromis macrochir) were recently discovered in Kafue River, Zambia, after the introduction of O. niloticus outside its native range for the purpose of aquaculture (Deines et al., 2014). Many fish species (e.g. common bream; silver bream; roach; bleak (Alburnus alburnus), asp (Aspius aspius), European bitterling (Rhodeus amarus), topmouth gudgeon (Pseudorasbora parva)) have been documented as invasive species with the consecutive hybridization with indigenous fish species (Gherardi et al., 2008). However, fish hybridization is used as a tool for aquaculture improvement (i.e. to increase growth rate) to transfer advantageous traits between species (e.g. disease resistance, salinity tolerance), to reduce fertility, to manipulate with sexual dimorphism, to increase harvestability or environmental tolerances) (Bartley et al., 2000). Escapes of hybrids from fish farms used for aquaculture purposes might threaten the wild populations (Senanan et al., 2004). These threats could result from competition, predation, and genetic introgression (Campton, 1987; Ferguson et al., 2007; Houde et al., 2010; Jensen et al., 2010). Furthermore, trade of ornamental fish species has been recognised as an important factor contributing to the introduction of invasive species which often results in hybridization of the invasive species with the indigenous one (Mendoza-Alfaro et al., 2012).

3.1.3 Genetic introgression and mitochondrial origin of hybrids

Introgressive hybridization is a cross-breeding with the incorporation of alleles from one species into the gene pool of the second species via backcrossing (Anderson and Hubricht 1938). Rapidly evolving molecular techniques highlighted attention to the importance of this neglected problem, which is not always apparent from morphological studies (Rhymer and Simberloff, 1996). Such an introgression with the specific direction of gene flow (unidirectional introgression) can cause replacement of the alleles in one population with the alleles from another population (Baack and Riesenberg, 2007). Many 14

LITERATURE OVERVIEW

cases of introgressive genetic incorporation caused by hybridization among plants (e.g. oaks, cottonwood trees, sunflowers) and animals (e.g. mice, fruit flies, mosquitoes) have been documented (Martinsen et al., 2001; Payseur et al., 2004; Turner et al., 2005; Macholán et al., 2007; Machado et al., 2007; Yatabe et al., 2007). Concerning fish, Hata et al. (2019) investigated hybridization between native slender bitterling (Tanakia lanceolata) and invasive oily bitterling (Tanakia limbata), which was recently introduced to western Japan. Hybridization with the introduced T. limbata was described as a potential threat to the native population of T. lanceolata caused by genetic introgression and replacement of its niche in streams (Hata et al., 2019). Hybrid zones in cyprinoids, for example, between introduced common nase (Chondrostoma nasus nasus) and native Southwest European nase (C. toxostoma toxostoma) (Costedoat et al., 2005) and between introduced common barbel (Barbus barbus) and Mediterranean barbel (Barbus meridionalis) (Crespin et al., 1999), are well known cases of introgressive hybridization in the rivers in southern France. Hybrids as a pathway for gene flow carry maternal ancestry (mitochondrial DNA - mtDNA) inherited from one of the parental species (Martinsen et al., 2001). Thus, the possibilities of inheritance of different maternal ancestry and reversal dispersion among parentals via backcrossing increase genetic variability in the populations and contribute to the overall genetic diversity (Bazin et al., 2006). Content of the mitochondrial DNA exerts strong selective pressure on mitochondria, hence selection favours mitochondria compatible by the interplay of mitochondrial protein complexes, which encode separately in the nuclear and mitochondrial genomes (Burton et al., 2013; Burgess, 2019). Intergenomic compatibility between nuclear and mitochondrial genomes is critical for both physiological performance and the evolutionary trajectory of the nuclear genome (Harada et al., 2019). Discordance of the nuclear and mitochondrial DNA for different populations of animals and hybridization- mediated mitochondrial introgression were investigated by studies related to the biogeographical historical reconstructions of the populations and areas of species distribution (Pérez-Suárez et al., 1994; Liu et al., 2010). According to Toews and Brelsford (2012), mammals and fish have a higher frequency of discordance between nuclear and mitochondrial DNA compared to other groups. Mitochondrial introgression with the consequent discordance between nuclear and mitochondrial DNA mainly results from sex-bias (Toews and Brelsford, 2012). For example, introgression of mitochondrial DNA could be favoured by female-biased dispersal in stable contact zones as was 15

LITERATURE OVERVIEW

documented for African elephant species (Roca et al., 2005) or by male-biased dispersal in the context of spatial invasion for hare and lemur species (Lawson Handley and Perrin, 2007; Petit and Excoffier, 2009). The direction of the mitochondrial introgression (see Fig. 1) is often unrelated to the direction of the nuclear hybridization (Wielstra and Artzen, 2012; Bonnet et al., 2017). Moreover, the high mitochondrial mutation rate and mitochondrial DNA introgression have impact on the amount and nature of genes exchanged between hybridizing lineages as was observed for sparrow species (Italian sparrow (Passer italiae), the house sparrow (Passer domesticus) and Spanish sparrow (Passer hispaniolensis)) and their hybrids (Trier et al., 2014).

3.1.4 Adaptive introgression and cytonuclear incompatibility

Current genetic variation (i.e. already present genetic diversity), novel mutations and genetic components as a result of introgression (Olson-Manning et al., 2012; Hedrick, 2013; Schmickl et al., 2017) were defined as primary sources of the overall genetic diversity in populations. Genetic introgression between species may result in hybrid incompatibility by the transfer of recessive or lethal alleles (Cattani and Presgraves, 2009). In contrast, gene flow caused by introgressive hybridization could allow the transfer of advantageous alleles between species (Beaumont and Balding, 2004; Gosset and Bierne, 2013). Unidirectional hybridization (i.e. revealed by asymmetric proportion of maternal ancestry in the population), its causes and consequences were investigated by several studies (Roberts et al., 2009; Hartog et al., 2010; Haynes et al., 2011). Unidirectional genetic introgression was suggested to be linked with species ecology (e.g. successful species invasion, mating preferences, spawning behaviour and restrictions linked to the habitat preferences and environmental conditions during reproduction) (Pearson, 2000; While et al., 2015). For example, Korean sand lance (Ammodytes heian) and Japanese sand lance (A. japonicus) from the northwest Pacific Ocean are strongly influenced by both the paleo-climatic change and the contemporary local oceanic current pattern (Kim et al., 2017). This study revealed that the characteristics of local oceanic current resulted in unidirectional gene flow between A. heian and A. japonicus. The difference in the mutation rate and the number of potentially obtained adaptations (e.g. beneficial changes) is higher in a mitochondrial genotype when compared to a nuclear genotype (Xu et al., 2006; Sloan et al., 2012). For instance, 16

LITERATURE OVERVIEW

mitochondrial DNA variations (i.e. nonsynonymous mutations harboured by two lines of Sherpa-specific lineages related to Complex I function) in the highland adaptation to low oxygen environment and its role in coding core subunits of oxidative phosphorylation in mitochondria were highlighted as important adaptation in the high altitude for Sherpa people in Tibetan highlands (Kang et al., 2013). The ability of mtDNA to accumulate the mutations with limited recombination may lead to a wide range of genetic variability which may be recognised as a novel basis for adaptation (Feng et al., 2015). The maternal origin may indirectly influence the effectivity of a defence mechanism (immune response) despite the pattern of compensatory coevolution in which nuclear genotypes have repeatedly evolved in reparation of the mitochondrial function within cytonuclear cooperation (Edmands et al., 2009; Yue et al., 2013; Zhang et al., 2013; Barreto et al., 2018). The mitochondrial genes are involved in cellular energy production (ATP synthesis), thus, they indirectly affect the life dynamics (McBride et al., 2006; Shen et al., 2016). For example, lower levels of the cytochrome c oxidase encoded by components of both nuclear and mitochondrial genes in the population, with mitochondrial introgression were observed in splashpool copepod (Tigriopus californicus) (Edmands and Burton, 1999). Disruption of the mitochondrial gene regulation and overall cytonuclear cooperation resulted in changes of mitochondrial effectivity which was shown to affect the fitness of hybrids (Ellison and Burton, 2006). Study conducted by Innocenti et al. (2011) explored genome-wide variation in nuclear gene expression across strains of fruit fly (Drosophila melanogaster) that differ only in the origin of their mitochondrial genomes. Authors showed that sex-specific selection enables deleterious mutations to accumulate in mitochondrial genomes if these mutations exert male-specific effects. In their study, mitochondrial polymorphism had few effects on nuclear gene expression in females but had major effects in males, modifying nearly 10% of transcripts. Furthermore, Shen et al. (2016) suggested that mutation causing the reduction of mitochondria to 50% can affect the life span of an organism through activation of stress pathways. Thus, disruption of coadaptation between nuclear and mitochondrial genes and accumulated mutations contribute to the phenomenon of hybrid breakdown (Sloan et al., 2012).

LITERATURE OVERVIEW

Fig. 1. A schematic representation of asymmetric mitochondrial DNA introgression via hybridization and species displacement according to Wielstra and Artzen (2012)

LITERATURE OVERVIEW

3.1.5 Heterosis effect and transgressive segregation

Hybrid vigour resulting from the heterosis effect plays an important role in the biology of hybrids (Shull, 1948). Heterosis in fish hybrids is related to the development, growth, resistance to environmental factors, disease and parasite infection (e.g. Hashimoto et al., 2012). Heterosis effect is frequently used in the aquacultural proposes (Einum and Fleming, 1997; Bryden et al., 2004; Hashimoto et al., 2012; Rahman et al., 2013). For example, the hybrids of channel catfish (Ictalurus punctatus) and blue catfish (Ictalurus furcatus) show fast growth, better feed conversion, survival and disease resistance (Bosworth et al., 2004). Another example, black pacu (Colossoma macropomum) × small-scaled pacu (Piaractus mesopotamicus) hybrids exhibit a faster growth rate compared to the parental species and are tolerant to low temperature; C. macropomum × red-bellied pacu (Piaractus brachypomus) hybrids showed better growth and productivity when compared to the parentals (Calcagnotto et al., 1999; Martino, 2002). Hybrids expressed dominant (Davenport, 1908) or over-dominant (Shull, 1908) superiority. The dominance hypothesis for heterosis suggests that favourable dominant alleles mask deleterious recessive alleles in a heterozygote (heterozygote advantage). (Davenport, 1908). Heterozygote advantage contributes most to genetic variation in fitness (Charlesworth and Willis, 2009). In contrast, the overdominance hypothesis suggests that the heterozygote is superior relative to both homozygotes (Shull, 1908). Indeed, hybrids of freshwater serrasalmid fish - P. mesopotamicus and C. macropomum, exhibit favourably interacting gene combinations, making them superior to their parents in growing performance (Mourad et al., 2018; Costa et al., 2020). Heterosis has been documented as dosage-sensitive (Birchler and Veitia, 2010). Explanation of the dosage-sensitivity is based on the expression of responses in hybrids that are not corresponded to gene function predicted by the molecular basis of complementation (Birchler et al., 2010). Therefore, particular slightly deleterious alleles may be localised in quantitative trait loci that are typically dosage-sensitive to some degree. For instance, long-term research on dosage-dependent modifiers of a single phenotype in Drosophila confirmed the contribution of several genes with the molecular basis of transcriptional or signal transduction functions with dosage sensitivity (Birchler et al., 2001). In the transcriptome-based study of Comings and MacMurray (2000) heterosis was explained as the establishment of more favourable gene expression levels

LITERATURE OVERVIEW

in the hybrids compared to the parents. For example, according to Comings and MacMurray (2000) following genes were documented to be associated with molecular heterosis in humans: ADRA2C, C3 complement, DRD1, DRD2, DRD3, DRD4, ESR1, HP, HBB, HLA-DR DQ, HTR2A, properdin B, SLC6A4, PNMT, and secretor. Authors suggested that molecular heterosis could be exhibited on the level of the gene regulation, on the level of the protein subunits or based on the mutant alleles selective advantage. Transgressive segregation is distinct from heterosis because it appears mainly in the F2 generation and might be fixed in the population, thus, it may persist permanently (Guindon et al., 2019). It was stated that transgressive segregation is related to the individuals that exceed parental traits values in either a negative or positive direction (Rieseberg et al., 1999; Bell and Travis, 2005). Rieseberg et al. (1999) considered several possible genetic causes for transgressive segregation in hybrids: 1) high mutation rate in hybrids; 2) reduced developmental stability; 3) nonadditivity of allelic effects between loci, also known as epistatic effect; 4) nonadditivity of allelic effects within a locus or overdominance; 5) the unmasking of rare recessive alleles that are normally heterozygous in the parental taxa; 6) changed chromosome number; 7) the complementation of additive alleles that are inherited from each parental line. Rieseberg et al. (1999) hypothesised that genetic distance between parents is the origin of heterosis and it is positively correlated with the frequency of transgressive segregation.

3.1.6 Evolutionary role of hybridization

According to Cortés-Ortiz et al. (2019), there are several evolutionary outcomes of hybridization: 1) hybrid zone with the exchange of some alleles between parentals (bidirectional introgression); 2) fusion of two divergent species lineages into a one lineage/species resulting from hybridization and backcrossing; 3) introgression between hybridised species in which unidirectional genetic input appears; 4) speciation caused by assortative individual mating among hybrids (i.e., without backcrossing with parental taxa) (see Fig. 2). There is no doubt that hybridization plays an integral role in the diversification process and may significantly enhance differentiation in species evolution (Sætre, 2013). Increase of phenotypical and genetic variability as a result of genetic introgression is one of the most pronounced impacts from the point of view of evolution (Zalapa et al., 2010). Thus, hybridization generates a new combination of alleles, hence enhanced adaptive differentiation (Abbott et al., 2013). 20

LITERATURE OVERVIEW

Hybridization greatly contributes to the shaping and functioning of ecosystems (Brasier, 2001; Lambrinos, 2004; Smith et al., 2013). Hybrids expand in marginal areas of parental species habitats and hybridization appears frequently at boundaries of species ranges, confirming the role of hybridization in range expansion, distribution, and abundance of species (Willis et al., 2006; Morales and Dujon, 2012). There are two mechanisms by which hybridization could increase success of establishment in sexually reproducing species. First, hybridization could result in permanent changes to the genetic composition of the population by increasing genetic variance on which selection could operate or by eliminating deleterious recessive alleles (Lee, 2002). Second, heterosis might increase fitness in the F1 generation (Drake, 2006). For example, evidence that coral hybrids colonize marginal habitats distinct from those of parental species and that hybridization may be more frequent at peripheral boundaries of species was confirmed for several species of Acropora by Willis et al. (2006). Hybridization might accelerate speciation either by transferring genetic material (introgression) or through the establishment of polyploidization which is well- documented, for instance, as polyploidy among amphibians (Chapman and Burke, 2007; Chen, 2010; Schmid et al., 2015) and fish (Leggatt and Iwama, 2003; Liu et al., 2007; Piferrer et al., 2009). Due to the pairing between homoeologous chromosomes established polyploids may have an advantage over fixed heterozygosity of homoeologous alleles (Comai, 2005). Polyploids with their high plasticity of genome structure, as manifested by tolerance to changing chromosome numbers (aneuploidy and polyploidy), genome size, retro/transposable element mobility, insertions, deletions, and epigenome restructuring, contribute to overall genetic diversity and participate in the exploration of the new niches or to outcompete progenitor species (Feldman and Levy, 2005; Leitch and Leitch, 2008; Feldman et al., 2012). Polyploid speciation involves the production of allopolyploids, which by definition contain three or more sets of chromosomes from two different species (Willis et al., 2006). Cherfas et al. (1994) documented that triploid fish could arise from fertilization of diploid eggs in gibel carp (Carassius gibelio) × common carp (Cyprinus carpio) hybrids by the normal spermatozoa. Among fish, interspecies triploids of goldfish (Carassius auratus) female × C. carpio male have been produced for large-scale commercial purpose due to their fast growth and sterility (Liu et al., 2001). Moreover, Liu et al. (2001) highlighted that production of triploids by crossing tetraploids with diploids is preferable due to economic reasons. 21

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Fig. 2. Evolutionary outcomes caused by natural hybridization (according to Cortés-Ortiz et al., 2019). A–D represent different taxa.

LITERATURE OVERVIEW

Fish hybridization

3.2.1 Role of hybridization in evolution of cyprinoids

According to the latest studies the phylogenetic status of Cyprinidae within Cypriniformes still remains under the active discussion (Imoto et al., 2013; Yang et al., 2015; Tan and Armbruster, 2018). After the recent evaluation of the Cyprinidae, the currently revised suborder Cyprinoidei was proposed (more than 1300 species) which consists of twelve families: Acheilognathidae; Sundadanionidae; Danionidae; Gobionidae; Leptobarbidae; Xenocyprididae; Cyprinidae; Paedocypridae; Psilorhynchidae; Tanichthyidae; Tincidae and Leuciscidae (Schönhuth et al., 2018). Represented by six subfamilies, the Leuciscidae is one of the most diverse groups of cyprinoids (contains 672 species in 90 genera according to Eschmeyer’s catalogue of fishes (Fricke et al., 2020)) widely distributed in North America and northern Eurasia (Schönhuth et al., 2018). Probability of the hybridization among fish species is much higher when compared to other animal tax (Turner, 1999). Such rate of hybridization in fish is possible due to external fertilization, weak reproductive isolation mechanisms, unequal abundances of hybridizing species, competition for limited spawning habitats and susceptibility to secondary contacts between recently evolved species (Hubbs, 1955). Hybridization plays an important role in the phylogeny within Cyprinoidei. For example, two independent hybridization events between polyploid ancestors and Cyprinion were suggested for evolution of Torini, Cyprinini, Spinibarbini, and Barbini tribes (Yang et al., 2015).

3.2.2 Hybridization success of cyprinoids

The high frequency of the hybrids for the most abundant fish species within Cyprinoidei was documented (Briolay et al., 1998; Crespin et al., 1999; Elvira et al., 1990; Gozlan and Beyer, 2006; Hopkins and Eisenhour, 2008; Vetešník et al., 2009; Broughton et al., 2011). Hybrid zones for cyprinoids across Europe were documented (Crespin et al., 1999; Costedoat et al., 2005). Hybridization in leuciscids especially including common bream, silver bream and roach is frequent in nature and, currently, is well documented across European waters (Swinney and Coles, 1982; Cowx, 1983; Economidis and Wheeler, 1989; Nzau Matondo et al., 2007; Nzau Matondo et al., 2010a; Hayden et al., 2014; Konopiński and Amirowicz, 2018).

LITERATURE OVERVIEW

Roach, common bream and silver bream live in sympatry in nature and use the same type of spawning habitats under temporal overlapping of the spawning periods (Pitts, 1994; Pitts et al., 1997; Nzau Matondo et al., 2008). The intermediacy of the morphological traits with some parental variants in the interspecific hybrids of these three cyprinoids was documented (Nzau Matondo et al., 2008). Concerning reproductive behaviour of roach and silver bream, both species are polygamous, they exhibit phytophilous spawning with the nonterritorial, nonaggressive, sneaking male behaviour (Nzau Matondo et al., 2008; Nzau Matondo et al., 2010a; Nzau Matondo et al., 2013). Common bream exhibits polygamous and phytophilous spawning also, but with territorial and aggressive male behaviour (Poncin et al., 1996; Nzau Matondo et al., 2009). Hybridization success, morphological traits of F1 hybrids and different behavioural aspects of silver bream, common bream and roach promoting the hybridization was investigated by artificial cross-breeding conducted in several experimental studies (Wood and Jordan, 1987; Nzau Matondo et al., 2008; Nzau Matondo et al., 2009; Nzau Matondo et al., 2010a). A study conducted by Kuparinen et al. (2014) discovered dominance of the hybrids with A. brama maternal origin in Lake Iso Ruuhij ̈arvi in Finland when hybridization between common bream and roach was reported. The authors suggested that the primary mechanism of hybridization between roach and common bream goes mainly via cross- breeding of females of the common bream and males of the roach, even though offspring of these cross-breeding described as viable (Nzau Matondo et al., 2010b) and success of such reproduction is low (Hayden et al., 2010; Toscano et al., 2010). Moreover, Nzau Matondo et al. (2007) pointed out that the advantage in survival for hybrids was strongly influenced by the maternal origin.

3.2.1 Intermediacy of traits: plasticity of hybrids

The phenotypes of fish hybrids may exceed the range of phenotypes in the corresponding parental lineages (Corse et al., 2012). For most studies, a comparison of the morphology revealed the tendency of fish hybrids to express the intermediate or even similar morphology to their parental species (Ferguson and Danzmann, 1987; Wood and Jordan, 1987; Demarais et al., 1992; Legendre et al., 1992; Nzao Matondo et al., 2008). Hybrids of cyprinoids may exhibit wider ecological plasticity in comparison to parentals due to intermediacy of the inherited parental traits and, thus, facilitating wider ecological

LITERATURE OVERVIEW

plasticity in comparison to the pure species range of feeding or habitat preferences (Corse et al., 2009; Hayden et al., 2010; Toscano et al., 2010). However, analyses of experimentally produced cyprinoid hybrids have indicated that phenotypical characters may diverge from intermediacy. For example, two sets of the experimental lines produced by cross-breeding of creek chub (Semotilus atromaculatus) with central stoneroller (Campostoma anomalum), redside dace (Clinostomus elongatus), hornyhead chub (Nocomis biguttatus), and eastern blacknose dace (Rhinichthys atratulus) revealed inconsistency of the results of two repeated experimental cross-breeding with certain degree of the deviation from the intermediacy in the several phenotype parameters of the hybrids (Ross and Cavender, 1981). The study focusing on the effect of hybridization on fish immunity and physiology in cyprinoid fish, common carp, gibel carp and their respective hybrids suggested that the genetic introgression plays an important role for hybrid vigour (Šimková et al., 2015). In their study, the intermediate glucose and cholesterol levels and intermediate intestine– body size index in hybrids were suggested to be an outcome of genetic introgression influencing the range of food utilizable for C. carpio × C. gibelio hybrids likewise their nutritional status, metabolic activity, and energy intake. Leukocyte count and complement activity in hybrids were also intermediate between parental species. Šimková et al. (2015) suggested that intermediate physiological and immune traits in fish hybrids (blood biochemistry, fish physiology and immunity) may represent an advantage for hybrid vigour.

Parasite infection in hybrids of cyprinoids

3.3.1 Role of parasites in genetic introgression of hosts

The hybrid bridge hypothesis is based on prediction that the hybrids may be considered as bridge gaps or intermediate link (”bridge”) for parasite transmission between parental species (Floate and Whitham, 1993). Hybrid intermediates in morphology and genetics were described as gradient continuums (e.g. F1, F2, backcrosses, etc.) which play the role of a bridge for parasite transmission by the host shifting from the one parental species to another (Dupont and Crivelli, 1988; Roderick, 1997). For instance, the role of hybrids as potential bridges between parentals was confirmed in the case of cyprinoids B. meridionalis × B. barbus (Le Brun et al., 1992; Gettová et al., 2016). Gettová et al. (2016) observed the trend towards higher monogenean

LITERATURE OVERVIEW

diversity in hybrids when compared to parental taxa. Authors suggested that hybrids might represent “bridges” for parasite infection between B. barbus and B. meridionalis. Parasitism contributes to the divergence of natural host populations and ecologically mediated host speciation (Karvonen and Seehausen, 2012). Most of the studies dedicated to the ecological speciation have focused on habitat and/or trophic specialization (Hendry et al., 2009); role of resource competition as main triggers of the divergence and reproductive isolation in the populations (Rundle et al., 2005). Parasites have been recognised by many studies as important forces of divergent selection (Price et al., 1986) or mediators of species coexistence (Freeland, 1983). Coevolution in host-parasite interactions may either facilitate hybridization and gene flow or isolation and speciation, depending on the dynamics of coevolution (Summers et al., 2003). Parasites appear to have different influences on the success of adaptive genetic introgression in the animal hybrid zones (Theodosopoulos et al., 2019). Parasites contribute to the maintenance of the species barriers, preventing hybridization by increased vigour costs of hybrids and reducing the gene flow, as it was documented for the pinworms in the hybrids of two house mouse subspecies Mus musculus musculus and M. m. domesticus (Sage et al., 1986; Moulia et al., 1991; Baird et al., 2012). Interestingly, Le Brun et al. (1992) discovered that the parasite intensity of Diplozoon gracile was correlated with genetic introgression and host behaviour in hybrids of B. barbus and B. meridionalis. The transition from a low introgression rate and low parasite prevalence in B. barbus via hybrid zone with intermediate values of introgression and parasite prevalence in hybrids to a high introgression rate and a high prevalence in B. meridionalis was found. Three different types of habits i.e. river zones (from upstream zone via hybrid zone to downstream zone) were associated with three host-hybridization levels and with three parasite prevalence levels (Le Brun et al., 1992). Parasites can cause the opposite effect, thus intensify the gene flow via decreasing the vigour cost for hybrids. It was observed for rosellas hybrids (crimson rosella (Platycercus elegans) and yellow rosella (Platycercus flavelous) that viral infection was lower in comparison to the heterozygotic parental species (Eastwood et al., 2017). Nevertheless, some studies highlighted that parasites do not affect the host species reproductive barriers i.e. no effect on gene flow with equivalent fitness costs for both pure species and hybrids. It was shown that sea lice infection was similar in Arctic char (Salvelinus alpinus), Atlantic salmon (Salmo salar) and their hybrids (Fleming et al., 2014; Frenzl et al., 2014). 26

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3.3.2 Distribution of parasites in parental species and their hybrids

Different aspects of the host biology (i.e. behaviour, physiology, condition status, immunocompetence) and environmental factors (i.e. temperature, seasonality) in relation to parasite load were investigated in order to explain specific trends of parasite infection observed in hybrid systems (Moulia et al., 1991; Stuart-Fox et al., 2009; Schoebel et al., 2011; Dias and Tavares-Dias, 2015). Fritz et al. (1994) suggested four generalised static scenarios of parasite infection for hybrids and their parental species: a) additive (or intermediate); b) dominance; c) hybrid resistance; d) hybrid susceptibility (see Fig. 3). The additive scenario describes hybrid resistance as not different from the average resistance of the parental species. The dominance scenario describes hybrid resistance as similar to either resistant or susceptible parental taxon, thus hybrid resistance might resemble one of the parental species. Hybrid resistance scenario is based on the prediction that hybrids reveal higher resistance than each of parents. Hybrid susceptibility scenario predicts that hybrids express lower resistance to parasite infection than each of parental species. However, the results of several studies revealed the inconsistency of the static scenarios in hybrid systems (Jackson and Tinsley, 2003; Parris, 2004; Wolinska et al., 2007a). Wolinska et al. (2007b) proposed a dynamic scenario for the parasite distribution in hybrids and parental species in the line with prediction of the Red Queen dynamic (see Fig. 4) based on the frequency-dependent selection during host-parasite arms racing. The Red Queen hypothesis originally formulated by Van Valen (1973) describes host-parasite co-evolutionary interactions based on the frequency-dependent selection resulting in reciprocal arms racing between host and parasite. Genetic co-adaptation between host and parasite determines the success of frequency-dependent selection, therefore parasite genotypes associated with the most common host genotypes will be favoured by natural selection (Morran et al., 2011). Dynamic scenario of the parasite distribution was tested in Daphnia hybrid complex (Wolinska et al., 2007a). The dynamic scenario of parasite distribution in hybrids and their parental species includes several stages, each stage representing by different static scenario. Highly susceptible hybrids are overloaded by parasites (stage 1) due to lower fitness; their frequency is high. However, due to the rapid ability of parasites to adapt to the most common host genotype, the parasite load in hybrids declines (stage 2) and the hybrids become released from the parasite infection (stage 3). Release of hybrids from the parasite

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pressure is associated with the increase in frequency of hybrids. Parasites adapt again to the most common i.e. hybrid genotype (stages 4 and 5). Since hybrids that occur frequently become the most common taxa, this phenomenon is repeated continuously.

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Fig. 3. Four static infection scenarios proposed by Fritz et al. (1994), modified by Wolinska et al. (2007b). Parasite infection in parental species (in blue and yellow colours) and hybrids (in green colour) are plotted on the y-axes.

LITERATURE OVERVIEW

Fig. 4 Dynamic scenario of the parasite distribution in hybrids and their parentals proposed by Wolinska et al. (2007b). Hypothetical infection levels of parental species (in blue and yellow colours) and hybrids (green colour) are plotted on the y-axes.

LITERATURE OVERVIEW

3.3.3 Host specificity and the system of co-adapted gene complexes

Host specificity or the affinity of the parasite to the associated host species was reported among different groups of parasites (Poulin, 1992). The majority of monogenean species are highly host-specific (i.e. members of the Dactylogyrus genus are known as parasites with a narrow host specificity to their cyprinoid host species) and well known as a diverse parasite group (Lambert and El Gharbi, 1995; Sasal et al., 1999; Šimková et al., 2001; Šimková et al., 2006a; Desdevises, 2007; Šimková and Morand, 2008). Several levels of host specificity were defined (i.e. basic, structural, phylogenetic or biogeographic specificity) (Desdevises et al., 2002; Poulin et al., 2011; Pojmanska and Niewiadomska, 2012). Various patterns of resistance and susceptibility to parasite infection were observed in hybridizing systems (Sage et al., 1986; Dupont and Crivelli, 1988; Boecklen and Spellenberg 1990; Le Brun et al., 1992). In some studies, hybrids were observed as more susceptible to the parasite infection than both parentals, for example, hybrids of A. alburnus and South European roach (Rutilus rubilio) from Lake Mikri Prespa (Greece) were more susceptible to parasite infection than parental species (Dupont and Crivelli, 1988). This finding is in line with the observation on the mice hybrid zone and susceptibility of the mice hybrids to the parasitic helminths with the first suggestion of interruption of the system of the gene co-adaptation, i.e. co-adapted gene systems regulating parasite infection are broken down in hybrids due to high gene introgression (Sage et al., 1986; Moulia et al., 1991). Indeed, in the study conducted on metazoan parasites in fish - B. barbus, B. meridionalis and their hybrids in three river basins (Loire River, Rhône River and Argens River) in France, authors pointed out that parasite transmission is related to the introgressive hybridization from invasive B. barbus to endemic B. meridionalis (Gettová et al., 2016). The study of metazoan parasite infection in hybrids of two leuciscids - P. toxostoma and C. nasus (Durance and Ardeche Rivers) from Southern France (Šimková et al., 2012) revealed high monogenean abundance in both allopatric and sympatric populations of invasive C. nasus. Trematoda were found as the dominant component of parasite communities of P. toxostoma from the allopatric population likely linked to different feeding or habitat conditions. Introduction of host-specific monogeneans - Dactylogyrus species expanded via C. nasus into introduced areas to P. toxostoma was shown. The intensity of monogenean infection was low in the genotypes of P. toxostoma and

LITERATURE OVERVIEW

recombinant genotypes of hybrids even though they were weakly susceptible to Dactylogyrus transmitted from C. nasus. Host-parasite coevolutionary associations were highlighted as the most important factor contributing to the distribution of the host- specific monogeneans of the genus Dactylogyrus among pure fish species and their hybrids (Šimková et al., 2012). In the study of Šimková et al. (2013), hybrids of two cyprinoids, C. carpio and C. gibelio, were infected by higher number of parasite species but with lower parasite abundance in comparison with parental species. Authors showed that interspecies hybridization affects host specificity of ecto- and endoparasites. Parasite species exhibiting different degrees of host specificity for C. carpio and C. gibelio were found in parasite communities of hybrids. In their study, the immune mechanisms specific to parental species were proposed as potential mechanisms explaining the low abundance of parasites in C. gibelio × C. carpio hybrids. Šimková et al. (2013) also suggested that genetically-based host traits related to susceptibility to the specific parasites might be transferred as a dominant trait via interspecific hybridization in cyprinoids. Overall, the hypothesis of a broken system of gene co-adaptation tested on hybrids of cyprinoids (i.e. the studies of P. toxostoma × C. nasus (Šimková et al., 2012) and C. carpio × C. gibelio (Šimková et al., 2013)) was not confirmed.

3.3.4 Immunocompetence and parasitism in hybrids

The different selection pressures are involved in the host-parasite co-evolution. On one hand, the pressure of the ecological and sexual selection strongly contributes against hybrids (Hatfield and Schluter, 1996; Vamosi et al., 2000). On the other hand, high genetic variation as a result of hybridization shaped by natural selection may result in an adaptive advantage for hybrids. Host resistance loci are predicted to frequently evolve to co-express multiple alleles which were able to recognize a wider diversity of parasite genotypes, while parasite infection loci are supposed to evolve to express only a single allele (Nuismer and Otto, 2005). Nuismer and Otto (2005) predicted that the co-expressed alleles of the parasite should be eliminated by the complex defence of the host, thus, expression of only a single allele would be sufficient to determine the success of the parasite. The genes of major histocompatibility complex (MHC) are one of the most representative and investigated immune genes in hybrids (Clarke and Kirby, 1966; Stet et al., 1998; Šimková et al., 2013b; Šimková, 2017; Ozturk et al., 2019). MHC is a highly polymorphic complex of genes (Graser et al., 1996; Ottová et al., 2005; Seifertová and 32

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Šimková, 2011; Nadachowska-Brzyska et al., 2012) responsible for the coding of the surface glycoproteins and playing an important role in the antigen recognition of the pathogens by T-lymphocytes (adaptive immunity). Parasite-mediated selection (Bernatchez and Landry, 2003); sexual selection based on the mate choice (Reusch et al., 2001; Ottová et al., 2007; Consuegra et al., 2008; Šimková, 2017) and effects of the gene duplication and recombination were identified as the main contributing forces to the overall high MHC diversity (Reusch and Langefors, 2005). Overall genetic variability of the MHC genes in fish indicates that MHC variability may be linked with susceptibility to parasites (e.g. Kurtz et al., 2004; Šimková et al., 2006b; Smith et al., 2011). Indeed, Šimková et al. (2006b) observed a positive association between MHC II DAB genes variability and parasite species diversity for European cyprinoids. Intermediate number of MHC alleles in F1 hybrids of cyprinoid fishes in comparison to pure species was found and it was considered as beneficial for hybrids, hence, parasite load of pure species-specific coevolving parasites is low (Šimková et al., 2013b; Šimková, 2017). Eizaguirre et al. (2012) investigated hybrids of three-spined stickleback (Gasterosteus aculeatus) and showed that evolution of locally adapted MHC allele pool was shaped by parasite-mediated diversifying selection. They hypothesised that species rich parasite communities should drive the evolution of MHC diversity in certain MHC genotypes. Šimková (2017) proposed that parasite-mediated and sexual selections facilitate the genetic introgression of MHC alleles via hybridization in closely-related fish species.

MATERIALS AND METHODS

Two hybridizing systems of cyprinoid species (representatives of Leuciscidae) were studied. First one represents evolutionary divergent and morphological different roach and common bream (Fig. 5A, B, C). The hybrids of F1 generation obtained from nature were used for this study. Second one represents species with low evolutionary divergent and with high morphological similarity (Fig. 6A, B, C, D). In this part, pure breed and cross-breed lines were prepared by artificial breeding.

Fig. 5. Photos of common bream (A) and roach (B). (Photos by Y. Kutsokon).

MATERIALS AND METHODS

Fig. 6. Breed lines of A. brama (A), B. bjoerkna (B), hybrid with maternal A. brama (C) and hybrid with maternal B. bjoerkna (D).

MATERIALS AND METHODS

A. brama × R. rutilus hybridization

4.1.1 Fish sampling and parasite collection Fish species (R. rutilus; A. brama and their respective hybrids) living in nature were collected during the spring and autumn periods of three consecutive years (2011, 2012 and 2013) from the Hamry reservoir (Fig. 7) (49° 44' 00" N, 15° 56' 00" E; the Czech Republic). Moreover, sampling of fish was also performed in the Brno Reservoir (49° 14' 18" N, 16° 30' 29" E; the Czech Republic) during autumn 2013. All fish specimens were transported live to the laboratory and sacrificed by severing the spine. Fish were dissected within 48 h according to classical parasitological dissection procedures (Ergens and Lom, 1970). All metazoan parasites were collected and fixed in 70% ethanol, 4% formaldehyde, or a mixture of glycerine ammonium picrate for further identification using Olympus BX50 light microscope equipped with phase contrast, differential interference contrast (DIC), and Olympus Stream Motion 1.9.2 digital image analysis software (Olympus Optical Co., Japan). Level of the parasite infection was expressed by prevalence, abundance and intensity of infection calculated following Bush et al. (1997)

Fig. 7. Hamry Reservoir, the Czech Republic.

MATERIALS AND METHODS

4.1.2 Molecular identification

Fish specimens (R. rutilus; A. brama and their respective hybrids) were identified using morphological characters (number of gill rakers, number of scales in the lateral line, number of scales in the dorsal fin to lateral line, number of scales in the anal fin to lateral line, and number of branched rays in the anal fin) (Wood and Jordan, 1987) and molecular markers (partial mitochondrial cyt b gene (1120 bp fragment) and 12 microsatellite loci, see details below). Fin clip from each specimen was preserved in 96% ethanol for the further molecular identification. Genomic DNA (gDNA) was extracted using a DNAeasy Blood & Tissue Kit (Qiagen, Germany) according to the manufacturer’s recommendations, with the modification for overnight incubation in lysis buffer containing proteinase K. Isolated gDNA was used as a template for PCRs.

4.1.2.1 Microsatelites

The microsatellite loci were selected following published studies (Mesquita et al., 2003; Barinova et al., 2004; Turner et al., 2004; Muenzel et al., 2007; Vyskočilová et al., 2007; Dubut et al., 2009, 2010). Twelve microsatellite markers (LC52, LCO4, LC32, BL2-114, CtoG-075, LC254, LleA-150, Lsou05, N7G5, Rru4, CtoF-172 and CnaD-112) were used. The descriptions of microsatellite markers and genetic diversity characteristics (i.e. number of alleles per locus, allelic richness, observed and expected heterozygosities, and deviation from the Hardy-Weinberg equilibrium for each locus) for the studied populations were calculated in GenAlEx (Peakall and Smouse, 2006) and FSTAT (Goudet, 1995) software. Multiplex PCR amplification was performed in a total volume of 15 µl of PCR mixture comprised of 1x PPP master mix (TopBio, Czech Republic), an appropriate primer mixture for set A or set B and 50 ng/L of gDNA as a template. The cycling conditions were as follows: initial denaturation at 95°C/3 min; 30 cycles of denaturation at 95°C/30 s, primer annealing for 1.5 min at 53°C for set A and 60°C for set B, and 72°C/1 min extension; final amplification at 72°C/10 min. The analysis of amplified microsatellite fragments was performed on an ABI 3130 Genetic Analyser (Applied Biosystems, USA) by using 1 µl of undiluted PCR product, mixed together with 0.3 µl of LIZ500 (Applied Biosystems), and 12 µl of Hi-Di formamide (Applied Biosystems). Genotypes were scored by GeneMapper v. 3.7 (Applied Biosystems). Additionally, the MICRO-CHECKER program (Van Oosterhout et al., 2004) was used to check for microsatellite null alleles, and the Bayesian clustering method implemented in NewHybrids 1.1 (Andreson and Thompson, 2002) was applied for the identification of common bream, roach, and their hybrids. The NewHybrids program was run with 37

MATERIALS AND METHODS

Jeffreys-type prior, and with 100,000 iterations after a 10,000 burn-in period including no prior population information.

4.1.2.2 Mitochondrial cyt b gene

Cytochrome b gene was used to trace the maternal origin of fish specimens. Cytochrome b gene fragment amplification took place in a final volume of 30 μl comprising 1x PCR buffer, 1.5 mM MgCL2, 0.2 mM dNTP, 0.2 μM of each primer (H16526 and L15267 following Briolay et al. (1998)), 0.5 U Taq DNA polymerase (Invitrogen, USA), and 10 ng/L of template gDNA. PCR was performed following the conditions described in Briolay et al. (1998). PCR products were checked on 1.5% agarose gel and then purified using a High Pure PCR Product Purification Kit (Roche Diagnostics, Germany). Sequencing reactions were carried out using a Big Dye Terminator v 3.1 Cycle Sequencing kit (Applied Biosystems). Amplicons were subsequently purified using a BigDye XTerminator Purification Kit (Applied Biosystems) and analysed on an ABI 3130 Genetic Analyser (Applied Biosystems). All sequences were analysed using Sequencher 2.3.4 (Gene Codes Corp., USA) and aligned using Clustal W multiple alignment (Thompson et al., 1994) implemented in Bioedit 7.2.5.0 (Hall, 1999). The sequences were deposited in GenBank under accession numbers from KX588534 to KX588552.

4.1.3 Physiological measurements and fish condition

4.1.3.1 Physiological indexes and determination of glucose level

Each fish specimen (R. rutilus; A. brama and their respective hybrids) was measured and weighed (e.g. total length (in cm) and total body weight (in grams)). Regarding the internal organs, the gonads, spleen and liver were weighted, and their weights were used to calculate basic physiological indexes. Fulton’s condition factor (CF) was calculated as follows: CF = (total fish weight/length of fish)×100. Investment of the reproduction was expressed by the gonado-somatic index (GSI) and calculated as follows: GSI = (gonad weight/total fish weight)×100. The spleen-somatic index (SSI) was used to evaluate investment to immunity and was expressed as follows: SSI = (spleen weight/total fish weight)×100. Fish condition (energetic status) was expressed by hepato-somatic index (HSI) and calculated as follows: HSI = (liver weight/total fish weight)×100. The level of glucose in plasma was analysed following the instructions provided in the commercial enzyme kit (Glu L 1000, PLIVA-Lachema, Czech Republic). Samples in

MATERIALS AND METHODS

duplicates were analysed with a plate reader (Tecan Sunrise, USA) and a concentration of glucose in samples (mmol/l) against the glucose standard solution was determined.

4.1.4 Immunological and haematological analyses

4.1.4.1 Fish haematology

Only the specimens of R. rutilus; A. brama and their respective hybrids collected in 2013 from Hamry reservoir were used for the analyses of immune and haematological parameters. Blood samples were obtained from each specimen immediately after sampling using caudal vein puncture according to Pravda and Svobodová (2003) and were put in the microtubules with heparin (10µl, 5 000 U/1ml, Zentiva). Immediately after sampling, erythrocyte count (in T.l-1), haematocrit value, haemoglobin content, and leukocyte count (in G.l-1) were determined according to Svobodová et al. (1991). Erythrocyte and leukocyte counts were performed in Bürker's haemocytometer after staining with Natt–Herrick solution. Heparinised microcapillaries (75 mm) were used to measure haematocrit value. Blood samples were centrifuged in microcapillaries using a haematocrit centrifuge at 12,000 g for 3 min. Haematocrit was analysed according to Svobodová et al. (1986). Haemoglobin content was analysed photometrically (540 nm; Helios Unicam, USA) in Kampen–Zijlster transformation medium. White-blood cells count was estimated following Pravda and Svobodová (2003).

4.1.4.2 Respiratory burst activity

The respiratory burst activity of phagocytes was measured as luminol-enhanced chemiluminescence using a luminometer (LM01-T, Immunotech, Czech Republic) and opsonised Zymosan A as an activator (Kubala et al., 1996; Nikoskelainen et al., 2004). The maximal intensity of respiratory burst (peak of expressed chemiluminescence in relative light units, RLU) was used as a measure of respiratory burst activity following Buchtíková et al. (2011) and Šimková et al. (2015).

4.1.4.3 Complement activity

The total bacteriolytic activity of plasma, including all pathways of complement activation, was determined using a bioluminescence-based method according to Buchtíková et al. (2011). Transformed bacteria E. coli K12 with the luxABCDE gene were exposed at laboratory temperature to fish plasma. The light emission of the reaction was positively correlated with the viability of E. coli, which was measured using an LM01-T luminometer (Immunotech, Czech Republic). The relative measure of 39

MATERIALS AND METHODS

complement activity was estimated by computing the difference between the maximum time of measurement (equal to 4 hours) and the time necessary to kill 50% of E. coli by complement (in hours).

4.1.4.4 Lysozyme concentration

The lysozyme concentration in skin mucus was assessed in vitro by radial diffusion in agarose gel mixed with Micrococcus luteus (CCM 169). A volume 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, the mean diffusion zone was measured and the concentration of lysozyme in the sample was converted to mg per ml of mucus according to a calibration curve. For details see Poisot et al. (2009).

4.1.4.5 Determination of total IgM level

The level of total IgM in plasma was analysed by precipitation with zinc sulphate (McEwan et al., 1970). This method is based on the 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 the commercially available kit (Bio-Rad, USA) and a plate reader (Tecan, Sunrise, USA) before and after precipitation. The concentration of IgM in the sample (in g.l-1) was calculated as the difference between the total protein and protein contained in the supernatant after precipitation and centrifugation.

A. brama × B. bjoerkna hybridization

4.2.1 Experimental design and cross-breeding

The sampling of fish specimens (B. bjoerkna and A. brama) was performed by electrofishing from the River Dyje near the city of Břeclav (48° 38′N; 16° 56 E; Morava River basin, Czech Republic). Specimens of each species (3 per species) were randomly selected among sampled fish and were checked for the presence of monogenean parasites. The experiment was conducted in well aerated and filtered tanks. Fish females were stimulated for ovulation by carp pituitary (two doses, 0.3 and 2.7 mg.kg-1 24 h and 12 h before propagation). Males were stimulated for spermiation by carp pituitary (1 mg.kg-1 24 h before propagation). Stimulation was followed by consequent increasing the water temperature to 22°C. Oocytes of ovulating females were obtained by dry method. Sperm

MATERIALS AND METHODS

was sampled according to Linhart et al. (2003). Hatchery water was used for gamete activation. Artificial spawning based on the individual pair mating was performed using the following parental combinations: (1) female and male B. bjoerkna, (2) female and male A. brama, (3) female B. bjoerkna and male A. brama, and (4) female A. brama and male B. bjoerkna. As a result of artificial spawning, four lines of offspring were obtained: B. blicca, A. brama and two lines of F1 hybrids (one with A. brama maternal origin and the next with B. bjoerkna maternal origin). A total of 15 specimens: 8 of A. brama and 7 of B. bjoerkna captured from the nature were placed in the tank with aerated water. In order to initiate the infection, the batches of breed lines of pure fish species (A. brama and B. bjoerkna) and crossbreed lines representing F1 hybrids with the different maternal ancestry were kept with the infected specimens caught in nature. The experiment lasted three weeks, then all fish specimens were sacrificed and dissected following classical parasitological dissection procedures (Ergens and Lom, 1970). All monogenean parasites were collected and fixed in mixture of glycerine ammonium picrate for further identification.

RESULTS

Main results included in two published papers and one manuscript are presented in two sections; the first section summarised the results of the studies of A. brama and R. rutilus hybridization, the second section summarised the results of the study of A. brama and B. bjoerkna hybridization. Full texts of the papers are included in consecutive chapters as Paper A, Paper B and Paper C. The first section summarised the results focused on immunocompetence and parasitism in evolutionary divergent cyprinoid fish species and their hybrids. The second section summarised the results on parasite distribution in phylogenetically closely related cyprinoids and their hybrids.

RESULTS

Section I – Immunocompetence and parasitism in non- congeneric cyprinoid fish species and their hybrids

Paper A Krasnovyd, V., Vetešník, L., Gettová, L., Civáňová, K., & Šimková, A. (2017) Patterns of parasite distribution in the hybrids of non-congeneric cyprinid fish species: Is asymmetry in parasite infection the result of limited coadaptation? International Journal for Parasitology 47(8):471-483. Paper B Šimková, A., Janáč, M., Hyršl, P., Krasnovyd, V., & Vetešník, L. (2020) Vigour- related traits and immunity in hybrids of evolutionary divergent cyprinoid species: advantages of hybrid heterosis? (resubmitted to the Journal of Fish Biology)

The intergeneric hybridization between common bream and roach is probably one of the most investigated known case of hybridization among cyprinoids. Hybridization between common bream and roach represents a unique opportunity to shed light on the cross-breeding of evolutionarily divergent species and limitations associated to host- specific parasites in their hybrids. The potential role of mtDNA in the expression of hybrid vigour (which may be linked to host susceptibility to parasite infection) was poorly investigated in fish hybrids until now. The aim of Paper A is to compare parasite diversity (measured by parasite species richness) and parasite infection level (measured by parasite abundance and prevalence) between F1 hybrids and their parental species. Four static scenarios (Fritz et al., 1994) and one dynamic scenario based on the Red Queen hypothesis (Wolinska et al., 2007b) have been proposed to explain the pattern of parasite distribution in hybridizing species. The maternal effect (i.e. the effect of mtDNA) on the parasite load and diversity was investigated in hybrids. Pattern of parasite distribution among parental taxa and their hybrids for the Hamry Reservoir was in line with the hybrid resistance scenario. Hybrids harboured the majority of parasite species found in parental species. Metazoan parasite abundance and prevalence were higher in parental species when compared with their respective hybrids. Nevertheless, total parasite species richness was higher in hybrids. The presence of the majority of parasites specific to parental species (with the Dactylogyrus genus being dominant in the parasite communities) in hybrids suggests the limitation of genetic coadaptation between a host and its specific parasites. However, the low abundance of

RESULTS

parental-specific parasite species in hybrids indicates that hybrid genomes have limited susceptibility to the host-specific monogeneans. Hybrids harboured all common Dactylogyrus spp. originating from roach, and exhibited high prevalence and relatively high parasite abundance for three common Dactylogyrus spp. (although levels of infection were lower in hybrids than in parental roach), infection by Dactylogyrus spp. originating from common bream was very restricted. This asymmetrical distribution of parasites primarily reflects the different susceptibilities of hybrids to parental gill specialists of the Dactylogyrus genus. This may suggest that hybrid genomes are incompatible or very poorly compatible with common bream-specific parasites and are more compatible with roach-specific parasites. Similar composition of parasite communities was found for R. rutilus, A. brama and their hybrids in two distant localities i.e. Hamry and Brno reservoirs (except for Dactylogyrus falcatus, the dominant species in parasite communities of common bream in the Brno reservoir). In both reservoirs, the total parasite species richness was higher in hybrids when compared with each of the parental species. Concerning Dactylogyrus parasites, the prevalence of species infected both roach and hybrids and the mean abundance of the most common roach-specific parasite Dactylogyrus crucifer were always higher in roach when compared to hybrids. Hybrids exhibiting common bream mtDNA were more parasitised by crustaceans and digeneans than hybrids exhibiting roach mtDNA. Higher infection by digeneans (Diplostomum spp. and Tylodelphis clavata) and crustaceans (Argulus foliaceus and Ergasilus sieboldi) was reported in hybrids when compared with parental R. rutilus and A. brama. Differences in the ecology of hybrids in comparison to their parental species (Hayden et al., 2010; Toscano et al., 2010) were proposed as potential explanation of digenean and crustacean load. The broader trophic spectrum and larger habitat range of hybrids may be associated with high parasite species richness of endoparasites and generalist ectoparasites. The maternal ancestry of hybrids did not affect monogeneans representing the majority of host-specific parasites. Impaired immune mechanisms in hybrids, promoting higher recognition of roach-specific parasites or the ecology of hybrids more similar to roach, resulting in different levels of exposure to the specific parasites of each parental species, may represent possible mechanisms responsible for the asymmetrical distribution of parental species-specific parasites. The aim of Paper B is to examine the potential physiological and immune aspects of hybrid heterosis by comparing the physiological condition, haematological parameters

RESULTS

and selected immune parameters between hybrids and parental fish species, roach and common bream. Regarding the somatic condition of fish expressed by condition factor, no significant differences between fish groups were found in spring. Significant difference in condition factor between roach and hybrids in autumn was documented. In general, hybrids were lowest in the values of condition factor when compared to both parentals. High values of HSI in spring were detected in roach, while common bream reached the highest values of HSI in comparison to roach and hybrids in autumn. Significant differences between HSI in spring were confirmed only between pure species, while HSI in common bream significantly differs from roach and hybrids in autumn. Values of GSI were significantly different only between roach and common bream in autumn, with hybrids tending to reach intermediate values. Nevertheless, significant differences in GSI between hybrids and parentals in spring were not found. Concerning the SSI values, the major effects of sex and season on SSI were revealed. Hybrids and roach reached significantly higher SSI compared to common bream in spring. A significant difference in SSI between common bream and roach was confirmed in autumn, with hybrids expressed intermediate values of SSI. This trend of intermediate immunocompetence revealed by SSI in hybrids supports the hypothesis of heterosis advantage. Effects of the fish group, sex and season were observed in the form of interactions of factors on investigated lysozyme concentration from the fish mucus. Two immune parameters IgM level (specific immunity) and respiratory burst (non-specific immunity) showed very similar trends of distribution among parental species and their hybrids. The IgM level and respiratory burst values in roach males significantly differed from males of common bream and hybrids, with the opposite trends in females (i.e. females of roach achieved lower levels of IgM and respiratory burst when compared to common bream and hybrids). Hybrids tended to express intermediate level of IgM level and respiratory burst when compared to roach and common bream. Comparison of the complement activity showed that roach reached significantly higher values when compared to common bream and hybrids. Contrary, the glucose level was significantly lower in roach compared to common bream and hybrids. In addition, a strong difference between two divergent species, common bream and roach, for each parameter of haematological profile was found. Erythrocyte count and haemoglobin values were significantly higher in roach than compared to hybrids and common bream. Leukocyte count in hybrids tended to reach intermediate values between

RESULTS

common bream and roach in autumn. Significant differences in the leukocyte profile were revealed between common bream and both roach and hybrids. No significant effect of maternal origin of hybrids was found for on vigour-related traits. However, the trend of maternal origin effect on complement activity and respiratory burst was revealed. This study suggests that the high evolutionary divergence between hybridizing species may generate intermediate level of the physiological and immune traits in hybrids.

RESULTS

Section II – Patterns of the parasite distribution in phylogenetically closely-related cyprinoids and their hybrids

Host specificity as a result of coevolution between parasite specialists and their associated hosts is well known. However, novel host genotypes generated by hybridization and interruption of host-parasite co-adaptation in the hybrids are little studied. We hypothesised that host-parasite co-adaptation limits the infection of host- specific parasites in hybrid genotypes. The experimental monogenean infection in pure breeds of Blicca bjoerkna and Abramis brama and cross-breeds (F1 generation) was examined. Moreover, the potential effect of the maternal origin of hybrids (potential coadaptation at the level of mitochondrial genes) on monogenean abundance was also investigated. Pure breeds of two cyprinoids and two cross-breeds (with B. bjoerkna or A. brama in the maternal position) were exposed to infection by monogeneans naturally occurring in silver bream and common bream. The hybrid resistance scenario was confirmed i.e. monogenean infection levels in hybrids was lower in comparison to pure breeds under conditions of similar frequencies of pure and hybrid genotypes. Similar level of total monogenean infection in silver bream and common bream was found. Parasite species richness was higher in hybrids when compared to common bream and silver bream. Each of common bream and silver bream harboured specific monogenean species. Hybrids harboured all monogenean taxa specifically infecting both parental species. Overall, there was no effect of the maternal origin of hybrids on monogenean abundance (only Paradiplozoon bliccae exhibited a significant difference in abundance between hybrids with silver bream and common bream maternal origins). Asymmetry in the distribution of specific parasites in favour of specialists of B. bjoerkna in the monogenean communities of hybrids was found. These results were in line with previous study on common bream and roach demonstrating higher proportion of the monogenean species specific to one of the parental fish species (Paper A). This study indicates that the mtDNA of hybrids is not a limiting factor for host- specific monogenean infection. The asymmetry of species-specific parasites suggests

RESULTS

similarity between the immune mechanisms in hybrids and B. bjoerkna. Our results revealed a different degree of host-parasite coadaptation in specific parasites of A. brama and B. bjoerkna.

DISCUSSION

The hybridization between common bream and roach is most probably the best- documented case of hybridization in cyprinoids. Our study A was focused on the investigation of pattern of parasite distribution in common bream × roach hybrids and their parental species living in the natural habitats. Hybrids harboured more parasite species than each of their parental species. Nevertheless, the abundance of metazoan parasites in hybrids was lower when compared with each of the parental species, which was in line with the hybrid resistance scenario proposed by Fritz et al. (1994) and with the hypothesis of high hybrid vigour for the F1 generation (Ellison and Burton, 2006). The same pattern in the parasite abundance was observed for monogeneans in hybrids of silver bream and common bream in the study C. Majority of the monogeneans reported in A. brama or B. bjoerkna from nature were also found in experimental specimens. We found that total abundance of monogeneans was higher in parental species in comparison to hybrids. Nevertheless, the significant difference in total monogenean abundance was confirmed only between B. bjoerkna and hybrids. Our studies A and C supports the high vigour of F1 hybrids (i.e. heterosis advantage) revealed by lower metazoan parasite abundance in F1 hybrids when compared with each of the parental species. These findings are on the contrary to a previous study performed in fish demonstrating the high hybrid susceptibility to metazoan parasites in the case of the natural hybrids of cyprinoids, A. alburnus and R. rubilio, from Lake Mikri Prespa, Greece (Dupont and Crivelli, 1988). The high metazoan load in the hybrids of A. alburnus and R. rubilio supports hybrid susceptibility scenario which might be related to the specific intrinsic features of their hybrids (resistance to parasites, parasite affinity), spatial and trophic positions of the hybrids of the cyprinoid species involved in the hybridization (Dupont and Crivelli, 1988). The presented study C was focused on the distribution of host-specific parasites in the F1 hybrids of A. brama and B. bjoerkna. This is the first experimental study investigating the distribution of host-specific monogeneans in F1 hybrids of cyprinids under the conditions of similar proportions of parental and hybrid genotypes; and unaffected immunity of breed and crossbreed lines by any previous infection. According to Wolinska et al. (2007b), the dynamic infection scenario predicts that host-parasite coevolution is a force driving infection patterns in hybridizing host systems. Thus, considering the frequencies of parental species and their hybrids are important when interpreting the patterns of parasite infection in hybridizing hosts.

DISCUSSION

In study A, the frequency of common bream × roach hybrids was low in two investigated localities. In contrast, the high frequency of common bream × roach hybrids for Irish and Finnish Lakes was documented (Hayden et al., 2010; Kuparinen et al., 2014). The studies A and C indicate that parasite distribution in cyprinoid hybrids is not driven by the coevolutionary dynamic based on the Red Queen hypothesis, a scenario proposed by Wolinska et al. (2007b). Pattern of parasite distribution in common bream and roach is in line with the static scenario proposed by Fritz et al. (1994) (i.e. hybrid resistance scenario in which the high resistance of hybrids when compared to parental taxa is hypothesised). The asymmetrical distribution of parasites specific to parental fish species was found in common bream × roach hybrids (study A). This asymmetry was shown for parasite species richness and parasite abundance. Similarly, asymmetry in distribution of parasites specific to parental fish species was also observed in common bream × silver bream hybrids in the experimental study (study C). In the case of common bream and roach living in nature, the parasite communities of hybrids were shifted toward a higher proportion of host-specific parasites of R. rutilus, which was the species achieving higher monogenean diversity but lower abundance when compared to A. brama. In the case of experimental infection of common bream and silver bream, the asymmetry in the monogenean communities of hybrids toward a higher abundance of B. bjoerkna-specific parasites was found. Such an unequal representation of parental species – specific parasites in hybrids may suggest the asymmetrical inheritance of protective immunological mechanisms. Alternatively, it may indicate different degrees of coadaptation between different parental species and their specific parasites. Jackson and Tinsley (2003) suggested that reciprocal co-adaptation between hosts and associated specific parasite genotypes was disrupted which may indicate that fully adapted parasites unable for transmission from one parental species to another. In the study of hybridizing house mice subspecies a high level of parasite infection was documented in hybrids (Sage et al., 1986). Authors suggested that broken system of host- parasite genetic coadaptation might be considered as possible explanation of the pattern of parasite distribution in hybrids. Šimková et al. (2012) suggested that host-parasite coevolutionary associations are major factors that limiting the distribution of host-specific parasites in pure and hybrid specimens across hybrid zones. In the experimental study of common bream and silver bream, each parental species harboured unique monogenean fauna (except for G. vimbi). Hybrids harboured all parasites specific to A. brama and B. bjoerkna, mostly at lower intensities of infection when compared to parental species. This 50

DISCUSSION

pattern of distribution of parental species-specific parasite in hybrids likely results from a lack of genetic co-adaptation between the host-specific parasite and associated host genomes which was also suggested by the study of hybridizing system of common bream and roach from nature. The hybrid bridge hypothesis suggests that hybrids act as a bridge between parental species transferring their parasites (Le Brun et al., 1992; Floate and Whitham, 1993). However, to test the hybrid bridge hypothesis, other experiments with hybridizing systems of A. brama and B. bjoerkna (or other fish species with host-specific parasites) will need to be performed in the future, these focusing on the different rate of genetic introgression between two species (the F1 generation, back-crosses, and the F2 generation of hybrids) and the potential to transfer host-specific parasites. Nevertheless, it seems that the hybridization of A. brama and B. bjoerkna does not represent a serious threat to the genetic integrity of these species (due to low frequency of hybrids in nature) and presents only a minimal risk concerning transferring host-specific parasites between two cyprinoid species (due to host-parasite co-adaptation). In the study of common bream and roach living in nature, we investigated associations between maternal ancestry of F1 hybrids and parasite infection including the whole range of metazoan parasites. The broader trophic spectrum and wide range of the occupied habitats of hybrids may also explain their high parasite species richness. The results of study A were in line with Šimková et al. (2013) who also suggested that high parasite species richness of the C. carpio × C. gibelio hybrids seems to be linked with their trophic range. Surprisingly, this study of common bream and roach hybridization suggests that there is a potential maternal effect (i.e. the effect of introgression) determining the infectivity of common generalist parasites to which both parental taxa are susceptible. Higher susceptibility to metazoan parasite infection in common bream × roach hybrids compared to pure species was found for crustacean and digenean parasites. Previously, the potential role of mtDNA in the patterns of parasite infection in fish hybrids was not investigated. Previous studies demonstrated unidirectional introgression of mDNA in fish and suggested that the direction of introgression is favoured by ecological constraints or mating behaviour (Roberts et al., 2009; Haynes et al., 2011; Šimková et al., 2013a). It has been also suggested that biased gene flow could allow advantageous alleles to be transferred between hybridizing cyprinoids (Haynes et al., 2011). Previously published studies documented the high frequencies of roach × common bream hybrids exhibited common bream mtDNA (Hayden et al., 2010, Kuparinen et al., 2014). Nevertheless, 51

DISCUSSION

frequencies of the hybrids with different maternal origins in the present study of common bream and roach were relatively equal. In the study of experimental infection of common bream and silver bream, the potential effect of mtDNA on the presence of host-specific parasites i.e. monogeneans was examined. Cytonuclear incompatibility may cause hybrid breakdown even in the F1 generation of hybrids (Chou and Leu, 2015; Johnson, 2010). Chou and Leu (2015) stated that some diseases related to mitochondrial DNA are pathogenic only if they are associated with specific nuclear DNA of hosts. However, in the study of phylogenetically closely related cyprinoids – common bream and silver bream, no obvious evidence of the mtDNA effect on the level of infection of host-specific monogeneans in hybrids was found. It seems that mitochondrial genes are not primarily involved in the association between fish hosts and their specific parasites. Paradiplozoon bliccae, a strict specialist of B. bjoerkna, was the only parasite species exhibiting a significant difference in abundance between silver bream and hybrids with common bream maternal origin. This result cannot clearly support the effect of mtDNA on the parasite infection due to the low parasite intensity of infection. However, even with no statistical support, our data indicate a higher intensity of infection by B. bjoerkna-specific parasites in hybrid specimens with B. bjoerkna maternal position. Regarding the study focused on physiology and immunocompetence in common bream, roach and their hybrids, GSI was not statistically different between hybrids and each of the pure fish species in spring. However, GSI of hybrids was intermediate between roach and common bream in autumn. No difference in reproductive potential between F1 hybrids with different maternal origin was found in our study. Previously, Šimková et al. (2015) showed that common carp × gibel carp hybrids exhibit a trend of intermediate GSI between parental species in spring. However, Stoumboudi et al., (1993) observed lower GSI in hybrids than those of the parental Mesopotamian barb (Capoeta damascina) and Jordan barbel (Barbus longiceps). Eventrough, condition factor in hybrids tend to attain lower values in comparison to both parentals in autumn; however inter-fish-group comparison did not reveal any significant differences for roach, common bream and hybrids in spring. Differences among fish groups are most likely linked to the trophic preferences and diet availuability by each season. Previously, wide trophic plasticity of roach and common bream hybrids was documented (Hayden et al., 2010; Toscano et al., 2010). Šimková et al. (2015) showed no effects of the sampling period, sex and fish group on index of immunocompetence, SSI. However, the present study on common bream, 52

DISCUSSION

roach and their hybrids revealed inter-fish-group differences affected by sex and season. Values of SSI in hybrids tended to be intermediate between both parents, which is in line with the hypothesis of heterosis advantage. The physiological and immune parameters of the hybrids were investigated by Šimková et al. (2013b; 2015) who found that only white cells count and complement activity tended to reach the values intermediate between the values of parental species. Values of the complement activity of roach was significantly higher in comparison to common bream and hybrids.In the present study, higher complement activity was reported in hybrids with roach maternal origin when compared to hybrids with common bream maternal origin. The majority of immune parameters were primarily affected by season (i.e. reaching higher values in spring than in autumn). These results reflect those of Lamková et al. (2007) and Rohlenová et al. (2011) who suggested that that such a pattern in cyprinoid fish is potentially associated with higher parasite infection. Concerning lysozyme activity (from skin mucus), roach females and hybrids tended to reach higher values in spring. This finding is in the line with our study A when higher parasite species richness for roach and hybrids in comparison to common bream was also documented in spring. In our study focused on parasite distribution on roach, common bream and their hybrids, it was shown that the abundance of some metazoan parasite species differs between hybrids with common bream maternal origin and those with roach maternal origin which may suggest that maternally inherited genotype is playing an important role in the expression of some immune parameters influencing the parasite infection in hybrids. Two other immune parameters analysed in common bream, roach and their hybrids, IgM level (specific immunity) and respiratory burst (non-specific immunity), revealed a similar trend of distribution among parental species and their hybrids. Males of roach reached higher IgM level and respiratory burst when compared to common bream and hybrids. In contrast, females of roach reached lower IgM level and respiratory burst when compared to common bream and hybrids. The trend toward the intermediate immunity found in hybrids of common bream and roach may indicate that some components of immune responses in hybrids are shared with each of the parental species. Cnaani et al. (2004) showed that hybrids of blue tilapia (Oreochromis aureus) and Mozambique tilapia (Oreochromis mossambicus) characterised by improved innate immune response to stress due to heterosis in comparison to pure parental species. The present study on common bream, roach and their 53

DISCUSSION

hybrids supports this hypothesis as it was evidenced that hybrids shared some components of immune responses with each of the parental species. Common bream and roach hybrids expressed the intermediate level of glucose between parental species. Brougher et al. (2005) showed lower level of glucose and higher haemoglobin concentration in hybrids of white bass (Morone chrysops) and striped bass (Morone saxatilis) when compared to M. saxatilis indicating more efficient oxidative metabolism and lower energy-related losses to increased stress in hybrids. Alternatively, intermediate glucose level in common bream x roach hybrids may be linked to difference in the trophic range between hybrids and parental species. These results are in line with the study of Šimková et al. (2015) who also documented the intermediated level of glucose for F1 hybrids of common carp and gibel carp. We evidenced strong difference in haematological profile between common bream and roach. Roach reached the highest values of red cells count, haematocrit and haemoglobin. Haematocrit of male hybrids was more similar to roach. However, red cells count and haemoglobin of hybrids were more similar to common bream. Haematocrit and haemoglobin have been used as the measure of oxidative efficiencies in fish (see Brougher et al., 2005) and haematocrit is also considered as a measure of secondary stress response (see Crespel et al., 2011). Thus, our results might indicate an intermediate level of oxidative efficiency in hybrids. In contrast to the study of Brougher et al. (2005) who showed the elevated level of haemoglobin in white bass × striped bass hybrids when compared with M. saxatilis. Dalziel et al. (2012) identified the genetically based differences in haematocrit by investigating the resident and migratory populations of G. aculeatus and their F1 hybrids. No effect of hybridization on red cells count, haematocrit and haemoglobin were previously recognised for the hybrids of common carp and gibel carp by Šimková et al. (2015). The genetically based differences or evolutionary distances between common bream and roach may also explain the differences in haematological profile in our study.

CONCLUTIONS AND PERSPECTIVES

Host-parasite relationship in hybridizing host systems is one of the most attractive targets for the investigation of the species ecology, biology and evolution. Rapid evolution within the hybrid zones and the various evolutionary pressures give the advantage of using large-scale studies for investigation of deeper host-parasite coevolutionary patterns and parasite infection dynamics in the hybridizing host populations. The objective of this thesis was to investigate the effect of hybridization on the level of parasitism in cyprinoids in natural and experimental conditions as well as on physiology and immunocompetence in cyprinoids from natural habitat. We showed the asymmetrical distribution of parental-species-specific parasites in hybrids. Asymmetry was shown for parasite species richness and parasite abundance. Asymmetry in the proportion of parental-species-specific parasites in hybrids was discovered for the first time. We suggested that impaired immune mechanisms in hybrids are restricted to the recognition of parental-species-specific parasites of one parental species or, alternatively, hybrid genomes may be incompatible with the specific parasites of one parental species. The majority of molecular factors responsible for host specificity remain unidentified. Host-specific factors required by parasites are either not expressed in hybrids in sufficient quantities or factors responsible for parasite resistance may be newly created or inherited from the resistant parent which may alter their immunocompetence (Jackson and Tinsley, 2003). Hosoya et al. (2013) identified quantitative trait loci responsible for the host recognition of oncomiracidia Heterobothrium okamotoi in pufferfish hybrids – tiger pufferfish (Takifugu rubripes) and grass pufferfish (Takifugu niphobles). Quantitative trait loci, including immunity-related genes such as Irak4, Muc2 and Muc5ac, associated with the host specificity of the H. okamotoi was characterised as more important for parasite growth and survival than during initial host recognition at the time of attachment. Currently, localization of the genetically functional parts of the host genome and coadaptation of the parasite genome related to their host-parasite co-evolution (especially in fish and monogeneans) is unknown. With respect to future studies, search of the genetic markers involved in host-parasite coevolution is strongly recommended. This thesis was also focussed to investigate the parasite distribution in parental fishes and their hybrids taking into consideration static and dynamic scenarios of parasite infection. So far, different coevolutionary processes are suggested to be driven by the Red Queen dynamics (Woolhouse et al., 2002). First, the arms race dynamic is characterised by absence of the frequency-dependent selection, in which both host and parasite

CONCLUTIONS AND PERSPECTIVES

continually accumulate adaptive mutations (Gandon et al., 2008). Second, the fluctuating selection dynamic is associated with host-parasite genotype fluctuations in frequencies over time because of negative frequency-dependent selection. Recently, there is a lack of studies conducted on cyprinoid hosts and their co-adapted parasites (especially monogeneans) with evidence for such coevolutionary processes. We hypothetised that maternal origin of the hybrids plays a significant role in the expression of some branches of fish immunity. The inherited gene pool and functional co-operation between nuclear and mitochondrial genetic material of the hybrids represents next poorly investigated topic. Recent work of Chou and Leu (2015) highlighted the cytonuclear co-operation from the point of view of Red Queen dynamics, explaining cyto-nuclear cooperation by the parasitic nature of the mitochondrial origin in which the deleterious effect of mtDNA variations will be revealed when they are introduced into a specific nuclear background. Our results indicated that mitochondrial genes are not involved in the coadaptation between monogeneans and their associated hosts. However, this conclusion needs other verification based on simultaneous transcriptomic analyses of parasite and host. The potential role of hybridization should be applied even for ecological studies when the identification of fish species has to be based on the combination of the suitable molecular and morphological markers. For example, misidentification of Lisbon arched- mouth nase (Iberochondrostoma olisiponensis) and molecular data obtained only for maternally inherited mitochondrial genes (applied for phylogenetic reconstruction) possessed serious questions on the assignment of taxonomical status of this fish (Gante et al. 2007; Sousa-Santos et al., 2014)). Many cases of hybridization are still under active investigation by implementing improved molecular techniques (Twyford and Ennos, 2012). Therefore, additional investigation of hybridization among fish species (phylogenetically distant and closely related) is needed in the future.

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LIST OF PUBLICATIONS

Paper A:

Krasnovyd, V., Vetešník, L., Gettová, L., Civáňová, K., & Šimková, A. (2017) Patterns of parasite distribution in the hybrids of non-congeneric cyprinid fish species: is asymmetry in parasite infection the result of limited coadaptation? International Journal for Parasitology 47 (8): 471-483

Paper B:

Šimková, A., Janáč, M., Hyršl, P., Krasnovyd, V., & Vetešník, L. (2020) Vigour- related traits and immunity in hybrids of evolutionary divergent cyprinid species: advantages of hybrid heterosis? (resubmitted to Journal of Fish Biology)

Paper C:

Krasnovyd, V., Vetešník, L., & Šimková, A. (2020) Distribution of host-specific parasites in hybrids of phylogenetically related fish: the effects of genotype frequency and maternal ancestry? Parasites & Vectors, 13(1), 1-11.

PAPER A

Patterns of parasite distribution in the hybrids of non- congeneric cyprinid fish species: is asymmetry in parasite infection

the result of limited coadaptation?

International Journal for Parasitology 47 (8): 471-483 (2017)

Krasnovyd, V., Vetešnik, L., Gettová, L., Civáňová, K., & Šimková, A.

International Journal for Parasitology 47 (2017) 471–483

Contents lists available at ScienceDirect

International Journal for Parasitology

journal homepage: www.elsevier.com/locate/ijpara

Patterns of parasite distribution in the hybrids of non-congeneric cyprinid ﬁsh species: is asymmetry in parasite infection the result of limited coadaptation? q ⇑ Vadym Krasnovyd a, Lukáš Vetešník b, Lenka Gettová a, Kristína Civánˇová a, Andrea Šimková a, a Department of Botany and Zoology, Faculty of Science, Masaryk University, Kotlárˇská 2, 611 37 Brno, Czech Republic b Institute of Vertebrate Biology, Academy of Sciences of the Czech Republic, v.v.i., Kveˇtná 8, 603 65 Brno, Czech Republic article info abstract

Article history: Hybrids and their parasite diversity represent interesting models for evolutionary ecology. The modified Received 11 October 2016 immune response, shifted ecology, inheritance, and maternal ancestry of hybrid host fish are supposed to Received in revised form 15 January 2017 affect the diversity of their parasite communities. The pattern of metazoan parasite distribution in non- Accepted 18 January 2017 congeneric cyprinids – common bream (Abramis brama) and roach (Rutilus rutilus) (species with different Available online 3 March 2017 morphology and ecology, and harbouring different specific parasites) – and their hybrids was analysed. Four static alternative scenarios based on parasite infection levels in hybrids and parental taxa are Keywords: known. The hybrid resistance scenario predicts that hybrids are more resistant than parental taxa, result- Interspecific hybrids ing in low parasite infection in hybrids. This scenario is principally consistent with hybrid heterosis Cyprinid fish Parasite communities advantage. In accordance with this prediction, metazoan parasite abundance and prevalence were higher Host specificity in parental species when compared with their hybrids. Alternatively, the dynamic Red Queen scenario of Maternal ancestry infection in hybridising systems predicts parasite adaptation to common hosts. Temporal (six sampling events) and spatial (two sampling sites) aspects as possible factors influencing parasite distribution were analysed. We found no support for this hypothesis, i.e. no changes in the frequency of hybrids or their parental species and no changes in parasite infection in parental species or hybrids were found in the different time periods. The effect of maternal ancestry on infection level was evident; hybrids exhibiting common bream mtDNA were more strongly parasitized by digeneans and crustaceans than hybrids exhibiting roach mtDNA. Hybrids harboured a majority of the specific parasites of both parental species; however, the level of infection of common bream-specific parasites (especially monogeneans) in hybrids was low. Such an asymmetrical distribution of parental species-specific parasites in hybrids may suggest the limited inheritance of protective immunological mechanisms from one parental species and reveal stronger coadaptation between common bream and its specific parasites. Ó 2017 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.

1. Introduction zygote mortality, limited hybrid viability and hybrid sterility. How- ever, in spite of these barriers, hybridization in different animal Hybridization is a process with significant consequences in groups has been documented. Concerning fish, cyprinids are one ecology and evolution, either favouring or limiting species diver- of the groups with the highest frequencies of hybridization sity (Moulia, 1999). Generally, animals have a weaker affinity for (Scribner et al., 2001). In fish, the possibility to produce hybrids the production of hybrids due to the existing barriers between spe- exists not only for congeneric species with phylogenetically recent cies (Abbott et al., 2013), e.g. there are forms of prezygotic isolation divergence (Almodóvar et al., 2008) but also for species from dif- including geographical, spatial, behavioural and reproductive (the ferent genera (e.g. Vetešník et al., 2009; Hayden et al., 2010). existence of mechanical barriers, genetic incompatibility and Interspecific fish hybrids (i.e. representatives of the F1 genera- gamete mortality) isolation, and of postzygotic isolation including tion) are often characterised by intermediate morphological phenotypes and intermediate ecology with respect to the two

q parental species (e.g. Hubbs, 1955; Giudice, 1966; Park et al., Note: The mtDNA sequences associated with this article are available in 2006; Toscano et al., 2010; Almodóvar et al., 2012). However, devi- GenBank under accession numbers from KX588534 to KX588552. ⇑ Corresponding author. Fax: +420 549498331. ations from morphological intermediacy in hybrids have been E-mail address: [email protected] (A. Šimková). reported in some studies (Ferguson and Danzmann, 1987 and the http://dx.doi.org/10.1016/j.ijpara.2017.01.003 0020-7519/Ó 2017 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved. 472 V. Krasnovyd et al. / International Journal for Parasitology 47 (2017) 471–483 references included therein), with such deviations resulting from total metazoan parasite abundance was lower when compared genetic dominance (Simon and Noble, 1968), the effect of modifier with each of the parental species (Šimková et al., 2013), supporting genes (Ross and Cavender, 1981), and delays in developmental the hybrid resistance scenario (Fritz et al., 1994). However, until rates (Leary et al., 1983). The processes of hybridization generate now, to our knowledge no study investigating the temporal and new morphological, physiological and genetic features in hybrids, spatial aspects of parasite distribution in fish hybrids and their par- with abilities to occupy non-specific niches when compared with ental species has been performed. Following the dynamic scenario, their parental taxa (Toscano et al., 2010). In this respect, hybridiza- negative frequency-dependent selection may drive the parasite tion may also be viewed as an opportunity to create new habitats infection levels in hybrids and parental species over time, as was for parasites. The modified and often large ecological niches of shown by Wolinska et al. (2007b). Investigating the parasite distri- hybrids may influence parasite species richness and diversity bution in a hybridising fish host system at different time points (Stolzenberg et al., 2009). Four static alternative scenarios explain- may clarify whether the host–parasite interactions in fish hybridising parasite infection levels in hybrids versus those in parental taxa ing systems are driven by coevolutionary dynamics predicted by were proposed by Fritz et al. (1994). They are (i) the additive sce- the Red Queen hypothesis (i.e. the parasite distributions at differ- nario – parental taxa differ in parasite abundance, but hybrid resis- ent time points reflect different static scenarios) or are stable over tance does not differ from the average resistance of parental taxa; time (i.e. the parasite distributions investigated at all time points (ii) the dominance scenario – hybrid resistance is similar to that of reflect one static scenario). either resistant or susceptible parental taxa, i.e. parasite infection The potential role of mtDNA determining hybrid vigour has levels are similar in hybrids and one parental taxa; (iii) the hybrid been little studied even though the direction of genetic introgres- resistance scenario – hybrids are more resistant than parental taxa, sion determining the level of host-specific parasites (such as i.e. the parasite infection level is lower in hybrids when compared monogeneans) was already highlighted by Le Brun et al. (1992). with each of the parental taxa; and (iv) the hybrid susceptibility Rand et al. (2004) suggested that hybrid incompatibilities (derived scenario – hybrids are less resistant than parental taxa, i.e. the par- from deleterious epistatic interactions between divergent parental asite infection level is higher in hybrids when compared with each genotypes, as proposed by Dobzhansky (1937) and Muller (1942)) of the parental taxa; each of these scenarios suggesting a different result from the disruption of nuclear-mitochondrial gene interac- underlying genetic mechanism (Fritz et al., 2003). However, differ- tions, and that the genetic differences between hybrids are ent host–parasite systems may support different static infection restricted to cytoplasmic genetic elements such as mtDNA. scenarios, as suggested by Wolinska et al. (2007b) after summaris- Ellison and Burton (2008) highlighted the role played by mater- ing 86 studies. Wolinska et al. (2007b) highlighted the fact that sta- nally inherited mitochondrial genomes in hybrid breakdown in tic infection scenarios revealed by specific studies were related to the marine copepod Tigriopus californicus, but to our knowledge the two following situations: (i) in the case of field studies, data the potential role of mtDNA in the expression of hybrid vigour from a single season were used, or (ii) in the case of laboratory (which may be linked to parasite infection level) was not investi- studies, parasites were isolated from a single locality or reared gated in fish hybrids. However, Bakke et al. (1999) showed, in on a single host organism. For this reason, they proposed a the case of salmonids, some similarity in Gyrodactylus infection dynamic scenario based on negative frequency-dependent selec- (Monogenea) between pure species and hybrids with the same tion which incorporated the idea of host–parasite coevolution pre- maternal ancestry. dicted by the Red Queen hypothesis into the analysis of parasite Our study focused on intergeneric hybridization between com- distributions in hybrid and parental taxa. More specifically, mon bream (Abramis brama) and roach (Rutilus rutilus), both mem- dynamic infection in a hybridising host system represents the bers of the subfamily Leuciscinae within the Cyprinidae. Roach and coevolutionary cycle and includes the following steps. First, common bream are common species with a wide range of distribu- hybrids that are over-infected by parasites exhibit reduced fitness, tion in many European drainages (Economidis and Wheeler, 1989). which results in a decrease in their frequency. In the next step, par- These fish species have the same spawning period and require- asites search for, and adapt to, a common host genotype (here, par- ments (e.g. Jurajda et al., 2004) and usually co-occur in the same ental taxa) and increase the infection level in this genotype. As a type of river habitat (Nzau Matondo et al., 2008). As a result, nat- consequence, the hybrids are under-infected and may start to ural common bream x roach hybridization has been documented increase their frequency. The parasites adapt again to the more fre- throughout Europe (e.g. Cowx, 1983; Economidis and Wheeler, quent hybrids. These processes are repeated over time. According 1989; Toscano et al., 2010). In some areas, however, these hybrids to Wolinska et al. (2007b), a host–parasite system may appear in occur at an unprecedented frequency, often exceeding that of each one of seven particular static infection scenarios at a given time parental species (Hayden et al., 2010; Kuparinen et al., 2014). point, i.e. in a particular part of the coevolutionary cycle. The These studies documented that the vast majority of common dynamic scenario was applied to explain the coexistence of hybrid bream x roach hybrids represent the F1 generation and suggested and parental Daphnia in a long-term study (Wolinska et al., 2006, that post-F1 hybridization is negligible. The low occurrence of 2007a). backcrosses and F2 hybrids suggests that F1 hybrids experience fit- Several studies focused on the investigation of parasite load in ness disadvantages compared with pure species (Hayden et al., wild living hybrid fish (i.e. Dupont and Crivelli, 1988; Šimková 2010; Toscano et al., 2010). Common bream x roach hybrids et al., 2012, 2013). A higher abundance of metazoan parasites express intermediate morphological phenotypes, display an inter- and higher species richness were reported in intergeneric hybrids mediate niche breadth and trophic position, and use a broader of cyprinid fish from Lake Mikri Prespa (northern Greece) when trophic spectrum compared with parental species (Wood and compared with their parental species Alburnus alburnus and Rutilus Jordan, 1987; Toscano et al., 2010; Hayden et al., 2011). The high rubilio (Dupont and Crivelli, 1988). Considering coadaptation survival of these hybrids at early developmental stages may reflect between host–specific parasites and host genotypes, the high sus- the capacity of hybrids to thrive easily under the same living con- ceptibility of hybrid specimens to specific parasites suggests a ditions as their parental species (Nzau Matondo et al., 2007). breakdown in the host–parasite coadaptation system of genes Common bream and roach, two non-congeneric cyprinid spe- due to hybridization, as originally proposed by Sage et al. (1986) cies with high evolutionary divergence, exhibit different morphol- and Moulia et al. (1991). In contrast, it was shown that F1 hybrids ogy and ecology, and harbour unique metazoan parasite fauna, of phylogenetically closely related cyprinid species (Cyprinus carpio especially concerning their specific monogenean parasites and Carassius gibelio) harboured more parasite species, but their (Moravec, 2001). As mentioned above, metazoan parasite fauna V. Krasnovyd et al. / International Journal for Parasitology 47 (2017) 471–483 473 were previously studied in hybrids of the phylogenetically closely species richness, i.e. the number of parasite species found in an related and morphologically similar cyprinid species C. carpio and individual host, or by the Brillouin diversity index (Magurran, C. gibelio by Šimková et al. (2013), one of the parental species har- 1988)) and (ii) parasite infection (measured by parasite abundance, bouring no strictly specific parasite species (i.e. parasite species i.e. the number of parasite specimens in an individual host). Con- infecting a single host species) and both fish species sharing some cerning parasite abundance, we applied metazoan parasite abun- specific parasite species due to their close phylogenetic relation- dance and the parasite abundance of a given parasitic group. ship. In contrast to C. carpio and C. gibelio, no specific parasite spe- Metazoan parasite abundance refers to the total number of all cies are shared between morphologically and molecularly metazoan parasite specimens found in an individual host. The par- divergent common bream and roach. For this reason, hybridization asite abundance of a given parasitic group refers to the number of between these two cyprinids represents an extraordinary opportu- parasite specimens belonging to a given parasite group (e.g. the nity to investigate the extent to which the crossing of evolutionar- Monogenea or the Digenea). At the level of host group (i.e. common ily distant species limits the presence of host-specific parasites. bream, roach, hybrids with roach maternal origin, and hybrids with The aim of our study was to compare parasite diversity (mea- common bream maternal origin), parasite load was expressed sured by parasite species richness) and parasite infection level using (i) parasite species richness (i.e. the total number of parasite (measured by parasite abundance and prevalence) between F1 species in each host group) and (ii) parasite infection measured by hybrids and their parental species, common bream and roach, in prevalence and mean abundance. Prevalence (the percentage of order to explore which of four static scenarios (Fritz et al., 1994) infected fish in each sample) and mean abundance (the mean num- fits the parasite distribution. We included both the temporal aspect ber of parasite specimens per host taking into account both (six field sampling events within a given locality) and the spatial infected and uninfected hosts) were calculated for each parasite aspect (two different localities, the Hamry and Brno reservoirs, species in each host group following Bush et al. (1997). Host speci- Czech Republic, sampled during the same time period) to investi- ficity for parasite species was evaluated using a checklist of pub- gate whether parasite distribution (and parasite infection level) lished records for parasite species over a period of more than in the fish hybridising system studied follows the prediction of 100 years (Moravec, 2001). Fish were sacrificed by severing the the dynamic scenario based on the Red Queen hypothesis proposed spinal cord in accordance with Law 246/1992 of the Czech Repub- by Wolinska et al. (2007b) or is stable over time (i.e. it follows one lic. The study (whose conditions are defined by document n. static scenario). Finally, we investigated whether there is a mater- 031/2011) was approved by the Animal Care and Use Committee nal effect (i.e. the effect of a particular fish species being in the of the Faculty of Science, Masaryk University in Brno (Czech maternal position during interspecific crossing) on the load of Republic). especially host-specific parasites in hybrids, as previously suggested by Bakke et al. (1999). 2.3. Morphological identification

The morphological identification of all fish specimens investi- 2. Materials and methods gated in this study was performed using five meristic traits (number of gill rakers, number of scales in the lateral line, number of 2.1. Fish sampling and parasite collection scales in the dorsal fin to lateral line, number of scales in the anal fin to lateral line, and number of branched rays in the anal fin; Sup- Common bream, roach, and their respective hybrids were col- plementary Table S1). Standard length and body width were also lected from the Hamry Reservoir (49° 440 0000 N, 15° 560 0000 E; recorded for all specimens. In addition, 16 morphometric traits the Czech Republic) during the spring and autumn periods of three (body depth, head length, head width, head depth, preorbital dis- consecutive years (2011, 2012 and 2013). In addition, to estimate tance, postorbital distance, interorbital distance, diameter of the the spatial effect on the pattern of parasite distribution in hybrids eye, predorsal length, prepectoral length, preventral length, pre- and parental taxa, supplementary sampling of fish was performed anal length, length of the anal fin, depth of the anal fin, length of in the Brno Reservoir (49° 140 1800 N, 16° 300 2900 E; the Czech the dorsal fin, and depth of the dorsal fin) were compared between Republic) during autumn 2013. parental species and their hybrids using a random sample of 10 Concerning the sampling size for the Hamry Reservoir (see specimens of common bream, 10 specimens of roach, and 10 spec- Table 1 for the total sampling size for parental species and their imens of hybrids collected during spring, and 10 specimens of each hybrids), 12–23 common bream specimens, 13–21 roach speci- fish group collected during autumn of 2011 (Supplementary mens, and 7–20 hybrid specimens per sampling event were Table S1). Fish analysed for 16 morphometric traits were not used included in this study. Fish of the following body sizes (mean ± S. for parasite evaluation, as extended manipulation with fish nega- D.) were sampled using gill nets over a period of 3 days during each tively affects the number of ectoparasites (especially the presence field trip: common bream 28.4 ± 3.8 cm, roach 20.8 ± 2.8 cm, and of Gyrodactylus parasites in the fins). hybrids 22.7 ± 6.4 cm. The sampling size for the Brno Reservoir is included in Table 2. All fish specimens were transported live to 2.4. Molecular identification the laboratory and dissected within 48 h following classical parasitological dissection procedures (Ergens and Lom, 1970). All meta- All fish specimens (including both parental species and their zoan parasites were removed and fixed in 70% ethanol, 4% hybrids) were also identified using molecular markers, i.e. a partial formaldehyde, or a glycerin ammonium picrate mixture for further sequence of the cytochrome b gene (1120 bp fragment) and identification to species level using an Olympus BX50 light micro- microsatellite loci. Genomic DNA (gDNA) was extracted from 96% scope equipped with phase contrast, differential interference con- ethanol-preserved fin tissue samples (0.5 0.5 cm) using a trast (DIC), and Olympus Stream Motion 1.9.2 digital image DNAeasy Blood & Tissue Kit (Qiagen, Germany) according to the analysis software (Olympus Optical Co., Japan). manufacturer’s recommendations, with the modification of overnight incubation in lysis buffer containing proteinase K at room 2.2. Parasite load temperature. Isolated gDNA was used as a template for PCRs. Microsatellite loci were used for the identification of common At the level of individual host, parasite load was expressed bream, roach, and their hybrids. The appropriate microsatellite loci using two measures: (i) parasite diversity (expressed by parasite were selected following published studies (Mesquita et al., 2003; 474 V. Krasnovyd et al. / International Journal for Parasitology 47 (2017) 471–483

Table 1 Parasite abundance (A, mean ± S.D.) and prevalence (P, in %) in each group of ﬁsh hosts (Rutilus rutilus (RR), Abramis brama (AB), and hybrids of both maternal origins (RR/roach maternal origin, and AB/common bream maternal origin, are indicated in parentheses)) collected from the Hamry Reservoir, Czech Republic.

Parasite species Rutilus rutilus (n = 107) Abramis brama (n = 111) Hybrids (RR) (n = 46) Hybrids (AB) (n = 47) APAPAPAP MONOGENEA Dactylogyrus crucifer 38.48 ± 36.85a 96b – – 4.15 ± 6.21 76 3.49 ± 4.42 74 Dactylogyrus cabaleroi 3.19 ± 6.72a 45b – – 0.24 ± 0.94 11 0.11 ± 0.37 9 Dactylogyrus nanus 7.91 ± 8.26a 84b – – 2.15 ± 3.49 59 1.81 ± 2.69 57 Dactylogyrus suecicus 2.92 ± 4.51a 69b – – 1.96 ± 2.95 52 2.36 ± 4.87 51 Dactylogyrus fallax 0.02 ± 0.14 3 – – 0.02 ± 0.15 2 – – Dactylogyrus similis 1.23 ± 2.82a 36b – – 0.39 ± 0.94 17 0.19 ± 0.61 11 Dactylogyrus sphyrna 0.24 ± 0.62 17b – – – – 0.17 ± 0.48 13 Dactylogyrus rarrisimus 0.02 ± 0.14 3 – – – – – – Dactylogyrus micracanthus 0.14 ± 0.46 11 – – 0.09 ± 0.28 9 0.11 ± 0.37 9 Dactylogyrus rutili 0.18 ± 0.47 15b – – 0.07 ± 0,25 7 0.04 ± 0.2 4 Dactylogyrus auriculatus – – 53.32 ± 67.95a 68b 0.15 ± 0.42 13 0.13 ± 0.49 9 Dactylogyrus wunderi – – 16.49 ± 14.46a 93b 0.02 ± 0.15 2 – – Dactylogyrus zandti – – 16.68 ± 15.05 93 – – – – Gyrodactylus elegans – – 1.41 ± 10.15 15 0.04 ± 0.2 4 0.15 ± 0.41 13 Gyrodactylus vimbi 0.58 ± 2.42 11 3.27 ± 12.49 13 0.09 ± 0.35 7 0.06 ± 0.24 6 Gyrodactylus carassii 0.87 ± 6.89 11b 0.04 ± 0.3 2 0.02 ± 0.15 2 – – Paradiplozoon bliccae – – – – – – 0.04 ± 0.2 4 Paradiplozoon homoion 0.04 ± 0.23 4 – – 0.02 ± 0.15 2 0.15 ± 0.46 11 Diplozoon paradoxum – – 0.04 ± 0.19 4 – – – – ACANTHOCEPHALA Neoechinorhynchus rutili 0.36 ± 1.61 11b 0.05 ± 0.34 2 – – 0.09 ± 0.45 4 DIGENEA Sphaerostoma bramae – – 0.13 ± 0.76 4 0.13 ± 0.87 2 0.09 ± 0.58 2 Apharyngostrigea cornu – – – – 0.72 ± 2.92 7 0.19 ± 1.3 2 Diplostomum spp. 0.88 ± 1.86 33b 1.08 ± 2.41 35b 0.65 ± 1.03 41 2.02 ± 2.87 49 Tylodelphys clavata 0.3 ± 1.08a 11b 0.68 ± 5.21a 5b 1.74 ± 4.24 26 7.23 ± 15.71 45 CESTODA Caryophyllaeus laticeps 0.03 ± 0.29 2 0.23 ± 1.07 7 – – – – Caryophyllaeides fennica 0.16 ± 0.63 8 0.03 ± 0.28 1 – – – – Caryophyllaeus brachycollis 0.08 ± 0.33 7 0.02 ± 0.19 1 – – – – Caryophyllaeus sp. larva 0.36 ± 1.57 9 0.39 ± 2.1 8 0.11 ± 0.6 4 0.21 ± 0.5 17 Ligula intestinalis – – 0.02 ± 0.19 1 – – – – CRUSTACEA Argulus foliaceus 0.56 ± 1.44a 22b 2.87 ± 6.4 44 0.85 ± 2.01 35 1.38 ± 3.15 43 Ergasilus sieboldi 0.26 ± 0.74a 18b 3.5 ± 7.41 71 3.15 ± 4.06 70 11.51 ± 15.85 66 NEMATODA Contracaecum sp. – – 0.12 ± 0.5 7 - - 0.04 ± 0.2 4

a Signiﬁcant differences between a given parental species and hybrids (taking all hybrids together) as revealed by the Mann–Whitney test. b Signiﬁcant differences between a given parental species and hybrids as revealed by a Fisher exact test.

Barinova et al., 2004; Turner et al., 2004; Muenzel et al., 2007; luted PCR product, mixed together with 0.3 ml of LIZ500 (Applied Vyskocˇilová et al., 2007; Dubut et al., 2009, 2010). Twelve Biosystems), and 12 ml of Hi-Di formamide (Applied Biosystems). microsatellite markers (LC52, LCO4, LC32, BL2-114, CtoG-075, Genotypes were scored by GeneMapper v. 3.7 (Applied Biosys- LC254, LleA-150, Lsou05, N7G5, Rru4, CtoF-172 and CnaD-112) tems). Finally, the MICRO-CHECKER programme (Van Oosterhout pooled into two multiplex sets were used in our study. The et al., 2004) was used to check for microsatellite null alleles, and descriptions of microsatellite markers and genetic diversity char- the Bayesian clustering method implemented in NewHybrids 1.1 acteristics (i.e. number of alleles per locus, allelic richness, (Anderson and Thompson, 2002) was used for the identification observed and expected heterozygosities, and deviation from the of common bream, roach, and their hybrids. The NewHybrids pro- Hardy–Weinberg equilibrium for each locus) for the studied popu- gramme was run with Jeffreys-type prior, and with 100,000 itera- lations calculated in GenAlEx (Peakall and Smouse, 2006) and tions after a 10,000 burn-in period including no prior population FSTAT (Goudet, 1995) software are listed in Supplementary information. Table S2. Multiplex PCR amplification was performed in a total vol- Fish determination based on microsatellite markers was subse- ume of 15 ml of PCR mixture comprised of 1 PPP master mix (Top- quently supported by cytochrome b identification. Cytochrome b Bio, Czech Republic), an appropriate primer mixture for set A or set was also used to trace the maternal origins of hybrids. Cytochrome B (see Supplementary Table S2), and 50 ng/L of gDNA as a template. b gene fragment amplification took place in a final volume of 30 ll

The cycling conditions were as follows: initial denaturation at comprising 1 PCR buffer, 1.5 mM MgCL2, 0.2 mM dNTP, 0.2 lMof 95 °C/3 min; 30 cycles of denaturation at 95 °C/30 s, primer each primer (H16526 and L15267 designed by Briolay et al. annealing for 1.5 min at 53 °C for set A and 60 °C for set B, and (1998)), 0.5 U Taq DNA polymerase (Invitrogen, USA), and 10 ng/ 72 °C/1 min extension; final amplification at 72 °C/10 min. The L of template gDNA. PCR was performed following the conditions analysis of amplified microsatellite fragments was performed on described in Briolay et al. (1998). PCR products were checked on an ABI 3130 Genetic Analyser (Applied Biosystems, USA) by means 1.5% agarose gel and then purified using a High Pure PCR Product of standardised techniques and chemistry, i.e. using 1 ml of undi- Purification Kit (Roche Diagnostics, Germany). Sequencing reac- V. Krasnovyd et al. / International Journal for Parasitology 47 (2017) 471–483 475

Table 2 Parasite abundance (A, mean ± S.D.) and prevalence (P, in %) in each group of ﬁsh host (Rutilus rutilus (RR), Abramis brama (AB) and hybrids of both maternal origins (RR/ roach maternal origin and AB/common bream maternal origin, are indicated in parentheses)) collected from the Brno Reservoir (Czech Republic) in autumn 2013.

Parasite species Rutilus rutilus (n = 15) Abramis brama (n = 15) Hybrids (RR) (n = 7) Hybrids (AB) (n =6) APAPAPAP MONOGENEA Dactylogyrus crucifer 2.59 ± 3.02 79 – – 0.86 ± 0.64 71 0.17 ± 0.37 17 Dactylogyrus nanus 0.44 ± 0.83 71 – – 0.14 ± 0.35 14 0.67 ± 0.75 50 Dactylogyrus suecicus 0.89 ± 1.23 71 – – 0.43 ± 0.49 43 0.67 ± 1.11 33 Dactylogyrus sphyrna – – – – – – 0.17 ± 0.37 17 Dactylogyrus rarrisimus 0.07 ± 0.26 14 – – – – – – Dactylogyrus micracanthus 0.26 ± 0.52 36 – – – – 0.17 ± 0.37 17 Dactylogyrus rutili 0.19 ± 0.47 29 – – 0.43 ± 0.73 29 0.17 ± 0.37 17 Dactylogyrus falcatus – – 9.67 ± 5.41 100 – – – – Dactylogyrus auriculatus – – 0.73 ± 0.68 60 – – – – Dactylogyrus wunderi – – 2.73 ± 2.77 87 – – – – Dactylogyrus zandti – – 11.93 ± 7.08 93 – – – – Paradiplozoon bliccae – – – – – – 0.17 ± 0.37 17 Paradiplozoon homoion – – 0.20 ± 0.54 13 0.43 ± 1.05 14 0.17 ± 0.37 17 Diplozoon paradoxum – – 0.07 ± 0.25 7 – – – – ACANTHOCEPHALA Neoechinorhynchus rutili – – 0.07 ± 0.25 7 – – – – DIGENEA Sphaerostoma bramae – – 1.27 ± 2.41 27 – – – – Diplostomum spp. – – 1.20 ± 1.60 47 2.00 ± 2.20 57 0.67 ± 0.94 33 Tylodelphys clavata 0.04 ± 0.19 7 – – 0.29 ± 0.70 14 – – CRUSTACEA Argulus foliaceus – – 0.13 ± 0.34 13 – – 0.17 ± 0.37 17 Ergasilus sieboldi – – 0.67 ± 1.30 33 0.14 ± 0.35 14 0.67 ± 1.11 33 tions were carried out using a Big Dye Terminator v 3.1 Cycle The effect of the maternal ancestry of hybrids (i.e. the effect of a Sequencing kit (Applied Biosystems). Amplicons were subse- fish species being in the maternal position) on parasite load (mea- quently purified using a BigDye XTerminator Purification Kit sured by parasite species richness, metazoan parasite abundance, (Applied Biosystems) and analysed on an ABI 3130 Genetic Analy- and the parasite abundance of the most numerous parasite group ser (Applied Biosystems). All sequences were analysed using or species) was tested using GLZ analyses with a negative binomial Sequencher 2.3.4 (Gene Codes Corp., USA) and aligned using Clus- distribution. The significance of factor categories was calculated tal W multiple alignment (Thompson et al., 1994) implemented in directly in a GLZ. The effect of the year of investigation and the Bioedit 7.2.5.0 (Hall, 1999). The sequences were deposited in Gen- effect of the period of investigation (spring or autumn) were Bank under accession numbers from KX588534 to KX588552. included in the GLZ as predictors, and standard body size was used as a covariate. Differences in the species richness and abundance of parental 2.5. Statistical analyses host-specific parasites in hybrids (i.e. asymmetrical infection by common bream-specific and roach-specific parasites present in Morphometric traits between hybrids and each of the parental the hybrids) were tested using the Wilcoxon pair test. In this study, species were compared using ANOVA followed by the Tukey post a roach-specific parasite is defined as a parasite species present in hoc test. All morphometric traits were corrected for standard body roach but absent in common bream, and a common bream-specific length. Bonferroni correction was applied for multiple tests. parasite is defined as a parasite present in common bream but Differences in the abundance of parasite species between the absent in roach. Based on cytochrome b sequence analysis, the parental species (roach or common bream) and hybrids were hybrids were separated into two groups: hybrids with roach tested using the Mann–Whitney test. Differences in prevalence maternal origin and hybrids with common bream maternal origin. between parental species and hybrids were tested using the Fisher To test the effect of the maternal origin of hybrids on the asymmet- exact test. rical infection, the difference between roach-specific parasite spe- The effect of fish host (i.e. the effect of the factor termed ‘‘fish cies richness (or abundance) and common bream-specific parasite group” representing the three categories – common bream, roach species richness (or abundance) was calculated and this difference and hybrids) on parasite load (measured by parasite species rich- was used as a dependent variable in the Mann–Whitney test. ness, metazoan parasite abundance, and the abundance of the most All above mentioned statistical analyses were performed for the numerous parasite groups or species) was tested using generalised dataset from the Hamry Reservoir. For the Brno reservoir, due to linear model (GLZ) analyses with negative binomial distribution. the low sampling size (related to permission to perform only one The significance of factor categories was calculated directly in a sampling campaign), non-parametric Kruskal–Wallis (KW) ANOVA GLZ. The effect of different years of investigation and the effect with multiple comparisons was applied to compare parasite abun- of the period of investigation (spring or autumn) were included dance between parental species and hybrids. Statistical analyses in the model as the second and third predictors (i.e. categorical were performed in SPSS Statistics 24. variables), and standard body length was used as a covariate. In addition to GLZ analyses performed directly on sampled data for each fish group within each sampling event, we performed 1000 2.6. Data accessibility random samplings using a subset equivalent in size to the number of hybrids in the group with the minimum sampling size (i.e. seven All supplementary files associated with this article, i.e. Supple- for autumn 2013). A GLZ was then calculated using each random mentary Fig. S1 and Supplementary Tables S1–S4 are available at sampling. Mendeley Data http://dx.doi.org/10.17632/47bj9wbfct.3. 476 V. Krasnovyd et al. / International Journal for Parasitology 47 (2017) 471–483

3. Results Dactylogyrus auriculatus, and Diplozoon paradoxum are strictly specific parasites of common bream or congeneric specialists (par- 3.1. Determination of hybrids asite species restricted to the closely-related cyprinid species of the genus Abramis). In the Hamry Reservoir, the frequency of hybrids was low (rang- In the Hamry Reservoir, at the level of fish group, the hybrid ing from 0.51% to 0.8% of specimens in the randomly collected group was parasitized by 25 parasite species (Table 1). When com- samples of common bream, roach, and hybrids). In the random sea- mon bream, roach and hybrids were compared at the level of fish sonal samples of both species, common bream represented 87% of group in the spring and autumn of each of three consecutive years, 5647 specimens caught in 2011, 91% of 3522 specimens caught in hybrids harboured more parasite species when compared with 2012, and 86% of 3108 specimens caught in 2013, while roach rep- each of the parental species. This was observed in each sampled resented 12% of specimens caught in 2011, 8% of specimens caught period of each year except autumn 2012 (Fig. 1). Hybrids har- in 2012, and 13% of specimens caught in 2013. The total densities boured the majority of parasite species found in roach and com- of both parental fish species decreased over three consecutive mon bream as well as two parasite species which were not years due to programmed biomanipulation (a 1.8-fold reduction identified in parental species (Paradiplozoon bliccae and Apharyn- in their numbers between 2011 and 2013). In the Brno reservoir, gostrigea cornu). In contrast, the mean abundance and prevalence roach represented 32% of the total number of fish specimens sam- of the majority of monogenean species shared between parental pled in our study (the fish community was composed of 15 fish species and hybrids were higher in parental species compared with species with roach recorded as the second most dominant species). hybrids (see the Mann–Whitney test for abundance and the Fisher The population density of roach was higher when compared with exact test for prevalence in Table 1). common bream (roach represented 86% of specimens while com- In the Hamry Reservoir, with respect to parasites found strictly mon bream represented just 13% in random samples of common in roach, only one roach-specific monogenean parasite, Dactylo- bream and roach), and the proportion of hybrids was also very gyrus rarissimus, was completely absent in hybrids (although the low (less than 1%). abundance of this parasite was very low in roach). Concerning par- For analyses, approximately the same numbers of each of the asites found strictly in common bream in high prevalence and parental species and hybrids (if this was possible) were selected abundance (Table 1), D. zandti was absent in hybrids, and only from the randomly caught fish (see Tables 1 and 2 for sampling one specimen of D. wunderi was found in a single hybrid specimen. sizes). With respect to meristic traits (Supplementary Table S1), In addition, the host-specific ectoparasite D. paradoxum and hybrids expressed values intermediate between roach and com- endoparasite Ligula intestinalis, for which common bream is the mon bream. With respect to metric and morphometric traits (Sup- most commonly reported host species (Host–parasite database, plementary Table S1), four of the 18 traits analysed (standard Natural History Museum, http://www.nhm.ac.uk/research-cura- length, body depth, length of anal fin, and depth of anal fin) had tion/scientific-resources/taxonomy-systematics/host-para- significantly higher values in hybrids when compared with com- sites/database/), were also absent in hybrids. No adult stage of mon bream, and significantly lower values in hybrids when com- Caryophyllaeus (Cestoda) was present in hybrids, whilst larval pared with roach (P < 0.05). Other morphometric traits in hybrids Caryophyllaeus was present in both parental species and their (except for the diameter of the eye) tended to be intermediate hybrids (see Table 1). between roach and common bream (even though the differences In the Hamry Reservoir, with respect to parasite infection, four were not statistically significant) or were more similar between Dactylogyrus spp. parasitizing roach and three Dactylogyrus spp. the hybrids and one of the two parental species (Supplementary parasitizing common bream represented species with the highest Table S1). values of prevalence and abundance (Table 1). Three Dactylogyrus Using cytochrome b sequence analysis, all specimens of roach spp. found on roach also exhibited high prevalence in hybrids even exhibited mtDNA of roach and all specimens of common bream though their abundance in hybrids was lower when compared exhibited mtDNA of common bream. Concerning hybrids, 46 of with roach (P < 0.05, see Table 1). the hybrids exhibited mtDNA of roach, and 47 of the hybrids exhib- A similar composition of parasite communities was found for ited mtDNA of common bream. parental species in the Hamry and Brno reservoirs (except for Ten microsatellite markers were successfully amplified in both Dactylogyrus falcatus, the dominant species in parasite communi- parental species and hybrids, while two markers, LC32 and LC52, ties of common bream in the Brno reservoir). In both reservoirs, were excluded from the analyses as they revealed multiallelic the total parasite species richness was higher in hybrids when amplification and were not suitable for the reliable determination compared with each of the parental species. In the Brno reservoir, of parental species or their hybrids. The MICRO-CHECKER pro- concerning Dactylogyrus parasites (the most abundant parasite gramme showed no evidence of null alleles in the studied popula- group), the prevalence of species shared between roach and tions. NewHybrids analysis confirmed the morphological hybrids and the mean abundance of the most common roach par- determination and mitochondrial ancestry of parental species asite Dactylogyrus crucifer were always higher in parental roach and their hybrids, and assigned all hybrid individuals as F1 hybrids (Table 2). Metazoan parasite abundance and monogenean abun- (see Supplementary Fig. S1). dance were higher in common bream when compared with roach and hybrids (KW ANOVA, multiple comparisons, P < 0.001). No common bream-specific Dactylogyrus spp. were found in hybrids. 3.2. Metazoan parasite communities in parental species and their hybrids 3.3. Interspecies hybridization affecting parasite abundance

In the Hamry Reservoir, roach was parasitized by 22 parasite Using data from the Hamry locality, the effect of hybridization species (Table 1). Among those, 10 Dactylogyrus spp. (some of these (represented by the variable termed ‘fish group’ with three cate- Dactylogyrus spp. were roach-specific) and Paradiplozoon homoion gories: common bream, roach, and hybrids) on parasite abundance were present only in roach. Common bream was parasitized by was analysed. Significant effects of fish group, seasonal period, year 19 parasite species (Table 1). Among those, eight species were pre- of sampling, and fish body size on metazoan parasite abundance sent only in common bream. Following Moravec (2001), the mono- were found (Table 3, Supplementary Table S3). Higher metazoan genean parasites Dactylogyrus wunderi, Dactylogyrus zandti, parasite abundance was found in both parental species when com- V. Krasnovyd et al. / International Journal for Parasitology 47 (2017) 471–483 477

Fig. 1. Total parasite species richness per fish group (shown by columns) in each sampling event from the Hamry Reservoir, Czech Republic. High parasite species richness in hybrids (H) when compared with roach (RR) and common bream (AB) is shown in each sampling event (sampling events are separated by vertical lines). Sample size for each fish group within each sampling event is indicated. pared with hybrids (P < 0.001). In addition, higher metazoan para- for monogenean abundance and Dactylogyrus abundance. Fish site abundance was found in common bream when compared with group was significant (P < 0.05) for monogenean as well as for roach (P < 0.001). Fish reached higher metazoan parasite abun- Dactylogyrus abundance across the whole randomly constructed dance in spring than in autumn (P < 0.001) and lower metazoan 1000 sample data set. parasite abundance in 2013 when compared with 2011 and 2012 Significant effects of fish group, seasonal period, year of sam- (P < 0.05). A GLZ calculated on 1000 random samplings confirmed pling, and body size on crustacean abundance, digenean abun- the significant effect of fish group, i.e. fish group was significant dance and cestodean abundance were found (Table 3, (P < 0.05) in the whole randomly constructed 1000 sample data set. Supplementary Table S3, Fig. 2B–D). Using random sampling with Monogenea represented more than 80% of the metazoan para- an equivalent sample size, the significant effect of fish group was site abundance in 86% of roach specimens and in 80% of common confirmed for crustacean abundance (for each of 1000 random bream specimens, but only in 24% of hybrid specimens. Fish group, samplings with P < 0.05) and for digenean abundance (for 997 of seasonal period, and year of sampling were found to have an effect 1000 random samplings with P < 0.05). Concerning cestodean on monogenean abundance (Table 3, Supplementary Table S3, abundance, only 413 of 1000 random samplings revealed the sig- Fig. 2A) and on the abundance of Dactylogyrus, which was the most nificant effect of fish group. Hybrids were more parasitized by numerous parasite genus, especially in parental species (Table 1). digeneans compared with parental fish (P < 0.001). This was pri- The effects of seasonal period and year of sampling on metazoan marily due to parasitization by Diplostomum spp. and Tylodelphys parasite abundance, as described above, were found to be the same clavata (Table 1). Tylodelphys clavata exhibited significantly higher

Table 3 Generalised linear model (GLZ) analyses documenting the effects of ﬁsh host (the effect termed ‘ﬁsh group’) on parasite load (the effects of sampling period, year of collection, and body size were taken into account in the analyses).

Dependent variable Effects Wald statistic P Likelihood ratio Chi-square P Metazoan parasite abundance Fish group 71.40 <0.001 183.44 <0.001 Seasonal period 39.71 <0.001 Year of collection 12.55 0.002 Standard length (cm) 11.50 0.001 Monogenean abundance Fish group 216.43 <0.001 Seasonal period 54.63 <0.001 285.06 <0.001 Year of collection 10.88 0.004 Standard length (cm) 0.08 0.774 Crustacean abundance Fish group 114.10 <0.001 288.07 <0.001 Seasonal period 6.13 0.013 Year of collection 16.65 <0.001 Standard length (cm) 64.08 <0.001 Digenean abundance Fish group 92.15 <0.001 180.25 <0.001 Seasonal period 7.18 0.007 Year of collection 8.24 0.016 Standard length (cm) 22.12 <0.001 Cestodean abundance Fish group 11.45 0.003 128.45 <0.001 Seasonal period 25.73 <0.001 Year of collection 21.99 <0.001 Standard length (cm) 10.18 0.001 478 V. Krasnovyd et al. / International Journal for Parasitology 47 (2017) 471–483

abundance was similar in common bream and hybrids, but significantly lower in roach (P < 0.001). The pattern of seasonal variation in crustacean abundance was found to be similar to that in the abundance of ectoparasitic monogeneans (i.e. higher abundance in spring and lower abundance in autumn, P = 0.013). Ergasilus sieboldi exhibited high prevalence in common bream and hybrids, and was one of the parasite species with the highest abundances in hybrids (Table 1). Hybrids were less parasitized by cestodeans when compared with roach (P = 0.001) and common bream (however P = 0.089). As the presence of cestodeans was almost completely restricted to spring, this effect was demontrated solely for this period (Fig. 2D). No effect of ﬁsh group on parasite diversity (measured by parasite species richness or the Brillouin index of diversity) was found (P > 0.05). The same results were found using 1000 random samplings.

3.4. The effect of the maternal origin of hybrids on their parasite communities

At the level of hybrid group, hybrids of common bream maternal origin harboured 22 parasite species, while hybrids of roach maternal origin harboured 21 parasite species (Table 1). No obvious maternal effect on the presence of common bream- or roach- restricted parasites was found. Metazoan parasite abundance and parasite diversity measured by the Brillouin index of diversity were not affected by the maternal origin of hybrids (GLZ, P = 0.11 and P = 0.49 respectively). GLZs were not signiﬁcant when parasite species richness, monogenean abundance, or Dactylogyrus abundance were considered as measures of parasite load (P > 0.05). In contrast, the maternal origin of hybrids was found to have a signiﬁcant effect on digenean abundance and crustacean abundance (Table 4, Supplementary Table S4). Hybrids exhibiting common bream mtDNA were more parasitized by crustaceans and digeneans than hybrids exhibiting roach mtDNA (P = 0.002 in both cases, Fig. 3).

3.5. Asymmetrical distribution of parental species-speciﬁc parasites in hybrids

Due particularly to the presence of the most diverse Dactylo- gyrus group from roach and the restriction of Dactylogyrus from common bream in common bream roach hybrids, the parasite abundance and species richness of parasites from roach were higher than those of parasites from common bream in the hybrids (Wilcoxon matched pairs test, P < 0.001, Fig. 4). The Mann–Whit- ney test showed no effect of the maternal ancestry of hybrids on the difference in parasite species richness or parasite abundance between roach- and common bream-speciﬁc parasites (P > 0.05).

4. Discussion

The present study showed that hybrids harboured more parasite species than each of their parental species, but that their abundance of metazoan parasites was lower when compared with each of the parental species, which supports the hybrid resistance scenario proposed as one of four static scenarios (additive, dominance, Fig. 2. Box plots of abundances of monogeneans (A), crustaceans (B), digeneans (C) hybrid resistance and hybrid susceptibility) by Fritz et al. (1994). and cestodeans (D) in roach (RR), common bream (AB) and their hybrids (H) in each However, hybrids showed different susceptibilities to different sampling event (spring and autumn of three consecutive years) undertaken in the parasite species (or parasite groups). The rate of hybridization Hamry Reservoir, Czech Republic. Sampling events are separated by vertical lines. between common bream and roach was very low both over time and in different spaces, which may explain the same pattern of par- abundance in hybrids than in each of the parental species (KW asite distribution in hybrids in all sampling events. The absence of ANOVA P < 0.001, multiple comparisons P < 0.01). Crustacean temporal variation in the frequency of parental species and their hybrids, which is likely associated with the absence of temporal V. Krasnovyd et al. / International Journal for Parasitology 47 (2017) 471–483 479

Table 4 Generalised linear model (GLZ) analyses documenting the effects of the maternal origin of hybrids on parasite load (the effects of seasonal period, year of collection, and body size were taken into account in the analyses).

Dependent variable Effects Wald statistic P Likelihood ratio Chi-square P Digenean abundance Seasonal period 2.92 0.087 51.36 <0.001 Maternal origin 9.88 0.002 Year of collection 0.01 0.996 Standard length (cm) 17.46 <0.001 Crustacean abundance Seasonal period 2.72 0.099 64.13 <0.001 Maternal origin 9.19 0.002 Year of collection 7.75 0.021 Standard length (cm) 25.79 <0.001

Fig. 3. Box plots of abundances of digeneans (A) and crustaceans (B) in hybrids with roach maternal origin (H (RR)), and common bream maternal origin (H (AB)). The data on hybrids from the Hamry Reservoir, Czech Republic, are presented.

Fig. 4. Asymmetrical distribution of parental species-specific parasites in hybrids collected from the Hamry Reservoir, Czech Republic. Parasite species richness (A) and parasite abundance (B) of roach–specific and common bream–specific parasites in hybrids. changes in parasite distribution among these groups, indicates that asymmetrically infected more by parasites of roach than by para- the hybridising fish and their parasites in our study were not dri- sites of common bream (especially infection by Dactylogyrus spp. ven by the coevolutionary dynamic scenario based on the Red including a high proportion of species-specific parasites), poten- Queen hypothesis. The maternal ancestry of hybrids affected the tially suggesting different coadaptation between the two parental abundances of endoparasitic digeneans and ectoparasitic crus- species and their specific parasites. taceans, but did not affect ectoparasitic monogeneans which Our study focusing on the hybridization of these two evolution- account for the majority of host-specific parasites. Hybrids were arily highly divergent species investigated hybrid vigour measured 480 V. Krasnovyd et al. / International Journal for Parasitology 47 (2017) 471–483 by the level of parasite infection. The crossing of two differentiated we propose that in the case where the rate of hybridization is very species may have a negative impact on the vigour of their hybrid low, most likely due to some ecological or behavioural constraints offspring (known as hybrid breakdown or outbreeding depression). between two potentially hybridising species (such constraints may F1 hybrids are frequently characterised by a high level of hybrid evidently act over a long time period), parasites may not act as a vigour but the mismatch of mitochondrial and nuclear genomes selective force to induce the Red Queen dynamic. However, we also in the F2 and F3 generations and paternal backcrosses results in point out that the pattern of parasite infection in parental taxa and reduced fitness (Rand et al., 2004; Burton et al., 2013). Our study their hybrids among different cyprinid hybridising systems seems revealed higher total parasite species richness but lower metazoan to be related to the population density of potentially hybridising parasite abundance in F1 hybrids when compared with each of the parental taxa and especially to the rate of hybridization. parental taxa, which may support the high vigour of F1 hybrids (i.e. We showed that hybrids of common bream and roach exhibit heterosis advantage). Parasite communities of hybrids included different susceptibilities to different parasite species. Generally, parasites specific to parental species and widely distributed (even interactions between host genotypes and parasite genotypes if not strictly specific) parasites of parental species, as well as gen- resulting from reciprocal genetic coadaptation are hypothesised eralist parasites (i.e. parasite species shared between common to have an important role in determining host susceptibility to bream and roach). With regard to endoparasites and generalist host-specific parasites. However, the molecular factors responsible ectoparasites (such as crustaceans), the broader trophic spectrum for host specificity remain unidentified. To explain resistance or and larger habitat range of hybrids may explain their high parasite low susceptibility to host-specific monogeneans, Jackson and species richness. However, the majority of parasites specific to par- Tinsley (2003) proposed that either some host-specific factors ental species were ectoparasitic monogeneans. A similar pattern in required by the parasite are not produced by hybrids in sufficient the compositions of parasite communities of hybrids was previ- quantities (this is linked to the inherited co-dominant expression ously reported for the two hybridising phylogenetically closely of resistance in F1 hybrids) or effective anti-parasitic factors may related cyprinids C. carpio and C. gibelio by Šimková et al. (2013). be inherited from the resistant parent or, alternatively, some novel The distribution of metazoan parasite abundance (and especially property may emerge in hybrids that alters their susceptibility. the distribution of Dactylogyrus parasites belonging to the Monoge- Hosoya et al. (2013) used F2 hybrids to determine the genetic nea) in hybrids and parental species in our study follows the regions of pufferfish responsible for the host specificity of the hybrid resistance scenario (Fritz et al., 1994) and is in accordance monogenean parasite Heterobothrium okamotoi. They identified with the hypothesis of high hybrid vigour for the F1 generation some quantitative trait loci (QTL) responsible for the initial host (Ellison and Burton, 2008). However, the high susceptibility (mea- recognition of oncomiracidia and even more important QTL sured by parasite infection) to metazoan parasites (especially of responsible for parasite growth and survival. The phenomenon of the genus Dactylogyrus) of the natural hybrids of two cyprinid spe- different host susceptibilities to parasite species with different life cies, A. alburnus and R. rubilio, from Lake Mikri Prespa, (Dupont and traits was proposed to explain why mouse hybrids (i.e. hybrids of Crivelli, 1988) is in line with the hybrid susceptibility scenario. We Mus musculus musculus and Mus musculus domesticus) are more may question how the different patterns of Dactylogyrus distribu- susceptible to some parasite species but not to others. Derothe tion in different cyprinid models can be explained. According to et al. (2001) proposed that hybrid susceptibility would only apply Wolinska et al. (2007b), the dynamic infection scenario predicts to parasites which exerted enough constraints on their host to that host–parasite coevolution is a force driving infection patterns induce the selection of co-adapted genes of resistance. However, in hybridising host systems. From this point of view, the frequen- this would only be applicable to host-specific parasites. Our study cies of parental taxa and their hybrids are important when investi- revealed different trends of infection when comparing parasites gating the patterns of parasite infection in hybridising with specialist and generalist life strategies. Higher infection by communities. In our study, the frequency of common two digenean parasite genera – Diplostomum spp. and T. clavata – bream roach hybrids was low in both the Hamry and Brno reser- was reported in hybrids when compared with parental taxa. These voirs. In contrast, a very high frequency of common bream x roach two parasite species are able naturally to infect a wide range of hybrids was found in Ireland following the introduction of non- host species, with fish representing intermediate hosts, which are native roach (Hayden et al., 2010), but also reported in native areas actively selected by free-living larval stages. Similarly, two crus- of common bream and roach distribution in the Finnish Lakes tacean species, Argulus foliaceus and E. sieboldi, tended to infect (Kuparinen et al., 2014). When focusing on studies providing infor- hybrids more frequently than parental roach. No genetic coadapta- mation about the frequency of hybrids and parasite infection, the tion can be expected between such generalist parasites and host concept of different frequencies of hybrids being associated with genotypes. Thus, our finding may indicate instead that widely dis- different parasite loads, i.e. a high parasite load in highly frequent tributed parasite species with a wider range of potential host spe- hybrids of A. alburnus and R. rubilio and a low parasite load in gen- cies than host-specific parasites may confer disadvantages to fish erally infrequent hybrids of common bream and roach, is in line hybrids. with the negative frequency-dependent selection suggested by Our study suggests that infection by some parasites is linked to the dynamic infection scenario (based on the Red Queen hypothe- the maternal ancestry of hybrids. Previously published studies sis) proposed by Wolinska et al. (2007b). Even though Wolinska indicated a hybridization bias in favour of unidirectional hybridiza- et al. (2007b) suggested not only that host–parasite coevolution tion concerning maternal ancestry (Roberts et al., 2009; Haynes in hybridising host systems is driven by the Red Queen dynamic et al., 2011; Šimková et al., 2013), which is most likely explained but also that this mechanism acts together with a genetic predis- by ecological constraints or mating behaviour such as male aggres- position to infection and a fluctuating environment affecting para- siveness or different spawning time periods in hybridising species. site infection, our study cannot support the dynamic infection Haynes et al. (2011) suggested that such biased gene flow could scenario. Our results are in line with the hybrid resistance scenario, allow advantageous alleles (e.g. resistance to viruses specifically as we reported the same pattern of parasite distribution among infecting one parental taxa) to move between two phylogenetically parental taxa and their hybrids at six different sampling time closely related cyprinid species, namely C. carpio and C. auratus. points (two seasonal periods in three consecutive years) for the Equal proportions of hybrids exhibiting common bream and roach Hamry Reservoir. Our supplementary study from the Brno Reser- mitochondrial DNA were found at the two sites sampled in our voir seems to support the dominance scenario (although this result study, even though these two sites differed in the frequencies of should be interpreted carefully due to the low sampling size). Thus, parental species. In the Hamry Reservoir, common bream was V. Krasnovyd et al. / International Journal for Parasitology 47 (2017) 471–483 481 the dominant species and roach was the second dominant species ogyrus spp. specific to common bream, was observed in the Brno in fish communities. In the Brno Reservoir, silver bream (Blicca reservoir, i.e. at the site with a low level of total parasite infection bjoekna) was the dominant and roach was the second dominant and with a higher population density for roach. This may suggest species in fish communities. Previously published studies docu- that hybrid genomes are incompatible or very poorly compatible menting the high frequencies of common bream roach hybrids with common bream-specific parasites but are susceptible to infec- showed that the majority of hybrids exhibited common bream tion by roach-specific parasites (even under conditions of low pop- mtDNA (Hayden et al., 2010; Kuparinen et al., 2014). The ulation density for roach). Alternatively, impaired immune hybridization barrier between female roach and male common mechanisms in hybrids restricted to the recognition of parental bream is most likely ecological, i.e. either the territorial behaviour species-specific parasites of just one parent or the ecology of of male common bream or differences in the relative abundances hybrids more similar to one parental species (i.e. roach in this case) of both species (a higher population density for roach) were pro- resulting in different levels of exposure to the specific parasites of posed as possible explanations for this asymmetrical distribution each parental species may represent other possible mechanisms of mtDNA in hybrids (Hayden et al., 2010). Until now, the potential responsible for the asymmetrical distribution of parental species- role of mtDNA in the expression of hybrid vigour (which may be specific parasites. linked to parasite infection level) was not investigated in fish Our study showed that metazoan parasite abundance and hybrids. In our study, we examined whether the maternal ancestry prevalence were higher in parental species – in this case, roach of F1 hybrids had an effect on parasite infection in two cyprinid and common bream – when compared with their hybrids. How- species with relatively high evolutionary divergence. Surprisingly, ever, total parasite species richness was higher in hybrids. The our study seems to suggest that there is a potential maternal effect presence of the majority of parasites specific to parental species determining the infectivity of common generalist parasites to (mainly monogeneans) in hybrids may suggest the interruption which both parental taxa are susceptible. Common bream was of co-adapted genes between a host and its specific parasites. How- more often infected by the generalist ectoparasite group Crustacea ever, the low abundance of parasites specific to parental species in and the most abundant endoparasite group Digenea when com- hybrids may indicate that hybrid genomes limit their susceptibility pared with roach, and hybrids exhibiting common bream mtDNA to these parasites that primarily coevolve with their hosts. Further, were more intensively parasitized than hybrids exhibiting roach the maternal origin of hybrids was shown to have an effect on the mtDNA (which was especially demonstrated for the Hamry local- abundance of digeneans and crustaceans, potentially reflecting ity). This difference between hybrids of common bream and roach some differences in their ecology. Hybrids were asymmetrically maternal origin suggests the potential difference in their ecology infected more by parasites of roach than by parasites of common i.e. hybrids of common bream maternal origin probably have more bream (representatives of the highly diversified Dactylogyrus group similar ecology to common bream while hybrids of roach maternal from roach were frequently reported in hybrids, whilst infection by origin probably have more similar ecology to roach. However, this Dactylogyrus from common bream was very limited). We propose hypothesis needs to be analysed in the future. Nevertheless, no dif- that such an unequal representation of parasites specific to paren- ferences in total parasite species richness or in the presence or tal species in hybrids could suggest the asymmetrical inheritance infection levels of parental-specific gill parasites (i.e. monoge- of protective immunological mechanisms limiting, in particular, neans) were observed between hybrids of different mitochondrial monogenean infection from one parental species (in our study, origin, which is in line with the nuclear-mitochondrial interaction limiting Dactylogyrus infection from common bream). In addition, model proposed by Rand et al. (2004). Thus, following this model, it may indicate different degrees of coadaptation between different F1 hybrids (in contrast to the F2 generation or back-crosses) con- parental species and their specific parasites (i.e. stronger coadapta- tain a full haploid complement of nuclear genes co-adapted to tion between common bream and its specific parasites than mtDNA and suffer no loss of fitness. between roach and its specific parasites) or, alternatively, the role We showed the asymmetrical distribution of specific parasites played by ecology. of parental fish in common bream x roach hybrids. This asymmetry was shown for parasite species richness and parasite abundance. To our knowledge, such asymmetrical parasite distribution in fish hybrids, i.e. the susceptibility to parasite species specific to one Acknowledgements parental species or to parasites widely distributed on this one parental species on the one hand, and the restriction to specific para- This study was funded by The Czech Science Foundation, pro- sites to the other parental species on the other hand, has not, to our ject No. P505/12/0375. We are grateful to Pavel Jurajda and his col- knowledge, been documented before. This asymmetrical distribu- leagues from the Institute of Vertebrate Biology, Academy of tion of parasites primarily reflects the different susceptibilities of Sciences of the Czech Republic for help with fishing; our colleagues hybrids to parental species-specific gill parasites of the Dactylo- and students from the Laboratory of Parasitology, Department of gyrus genus. While hybrids harboured all common Dactylogyrus Botany and Zoology, Faculty of Science, Masaryk University, Czech spp. originating from roach, and exhibited high prevalence and Republic, for help with fish dissection and parasite collection; and moderate-to-high parasite abundance for three common Dactylo- Kristy´ na Koukalová for her assistance in the molecular laboratory. gyrus spp. (although levels of infection were lower in hybrids than We thank the management of the Elbe River Basin Water Author- in parental roach), infection by Dactylogyrus spp. originating from ity, Czech Republic for permission to sample at the Hamry Reser- common bream was very restricted. Dactylogyrus wunderi and D. voir, Czech Republic. We kindly thank Matthew Nicholls for the zandti, two species with very high prevalence and high abundance English revision of the final draft. in common bream, were absent in hybrids (in the Hamry Reservoir, a single hybrid specimen was found to be infected by a single specimen of D. wunderi, most likely by accident); meanwhile, hybrid susceptibility to infection by D. auriculatus (reaching very high Appendix A. Supplementary data abundance in parental common bream) was very limited in hybrids from the Hamry Reservoir, in contrast to the infection pat- Supplementary data associated with this article can be found, in tern observed for D. crucifer, the most abundant parasite in roach. the online version, at http://dx.doi.org/10.1016/j.ijpara.2017.01. Even stronger restriction, i.e. the complete absence of four Dactyl- 003. 482 V. Krasnovyd et al. / International Journal for Parasitology 47 (2017) 471–483

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the Danube River Basin, Czech Republic: Morphological, karyological and Wolinska, J., Keller, B., Manca, M., Spaak, P., 2007a. Parasite survey of a Daphnia molecular characteristics. J. Fish Biol. 74, 1669–1676. hybrid complex: host-specificity and environment determine infection. J. Anim. Vyskocˇilová, M., Šimková, A., Martin, J.F., 2007. Isolation and characterization of Ecol. 76, 191–200. microsatellites in Leuciscus cephalus (Cypriniformes, Cyprinidae) and cross- Wolinska, J., Lively, C.M., Spaak, P., 2007b. Parasites in hybridizing communities: species amplification within the family Cyprinidae. Mol. Ecol. Notes 7, 1150– the Red Queen again? Trends Parasitol. 24, 121–126. 1154. Wood, A.B., Jordan, D.R., 1987. Fertility of roach bream hybrids, Rutilus rutilus Wolinska, J., Bittner, K., Ebert, D., Spaak, P., 2006. The coexistence of hybrid and (L.) Abramis brama (L.), and their identification. J. Fish Biol. 30, 249–261. parental Daphnia: the role of parasites. Proc. Biol. Sci. 273, 1977–1983. PAPER B

PAPER B

Vigour-related traits and immunity in hybrids of evolutionary

divergent cyprinid species: advantages of hybrid heterosis?

resubmitted to Journal of Fish biology

Šimková, A., Janáč, M., Hyršl, P., Krasnovyd, V., & Vetešník, L.

PAPER C

Distribution of host-specific parasites in hybrids of phylogenetically related fish: the effect of genotype frequency and maternal ancestry?

Parasites & Vectors, 13(1), 1-11. (2020)

Krasnovyd, V., Vetešnik, L., & Šimková, A.

137

Krasnovyd et al. Parasites Vectors (2020) 13:402 https://doi.org/10.1186/s13071-020-04271-3 Parasites & Vectors

RESEARCH Open Access Distribution of host‑specifc parasites in hybrids of phylogenetically related fsh: the efects of genotype frequency and maternal ancestry? Vadym Krasnovyd1, Lukáš Vetešník2 and Andrea Šimková1*

Abstract Background: Host specifcity is one of the outputs of the coevolution between parasites and their associated hosts. Several scenarios have been proposed to explain the pattern of parasite distribution in parental and hybrid genotypes ranging from hybrid resistance to hybrid susceptibility. We hypothesized that host-parasite co-adaptation limits the infection of host-specifc parasites in hybrid genotypes even under the condition of the high frequency of hybrids. The experimental monogenean infection in pure breeds of Blicca bjoerkna and Abramis brama and cross-breeds (the F1 generation of hybrids) under the condition of similar frequencies of pure and hybrid genotypes was investigated. We also examined the potential efect of the maternal origin of hybrids (potential co-adaptation at the level of mitochondrial genes) on monogenean abundance. Methods: Pure breeds of two cyprinids and two cross-breeds (one with B. bjoerkna, the next with A. brama in the maternal positions) were exposed to infection by monogeneans naturally occurring in B. bjoerkna and A. brama. The experiment was run under similar frequencies of the four breed lines. Results: We showed similar levels of monogenean infection in B. bjoerkna and A. brama. However, each species harboured specifc monogenean fauna. Hybrids harboured all monogenean species specifcally infecting one or the other species. Monogenean infection levels, especially those of Dactylogyrus specifc to A. brama, were lower in hybrids. For the majority of host-specifc parasites, there was no efect of the maternal origin of hybrids on monogenean abundance. Asymmetry was found in the distribution of specifc parasites in favour of specialists of B. bjoerkna in the monogenean communities of hybrids. Conclusions: Our results indicate that the maternal mtDNA of hybrids is not an important predictor of host-specifc monogenean infection, which may suggest that mitochondrial genes are not strongly involved in the coadaptation between monogeneans and their associated hosts. The asymmetry of species-specifc parasites suggests similarity between the molecular components of the immune mechanisms in hybrids and B. bjoerkna. Our results revealed a diference between the degree of host-parasite coadaptation in specifc parasites of A. brama and the degree of host- parasite coadaptation in specifc parasites of B. bjoerkna and their associated hosts. Keywords: Hybridization, Cyprinid fsh, Monogenean infection, Host specifcity, Coadaptation

*Correspondence: [email protected] 1 Department of Botany and Zoology, Faculty of Science, Masaryk University, Kotlářská 2, 611 37 Brno, Czech Republic Full list of author information is available at the end of the article

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Background the substrate structure of the fsh host (i.e. the diferent Te evolution of host-parasite relationships is associated structure of gill flaments of “wrong” hosts) [27, 28]. with reciprocal genetic co-adaptations between the two Diferences in monogenean species richness and abun- interacting partners [1, 2]. Such host-parasite gene-to- dance between hybrids and their parental species were gene interactions appeared during co-evolution and long- previously shown in fsh [6, 7, 16, 29]. Hybrids of cypri- running arms races between parasite adaptations and nid fsh species are infected by a majority of the parasites host defense mechanisms [3]. Te level of parasite infec- specifc to one or the other parental species in the case tion is regulated by a co-adapted genetic system, which of evolutionarily distant cyprinid species [7], or infected was originally suggested to explain the pattern of parasite by parasites specifc to both parental species in the case distribution in two species of house mice (Mus muscu- of evolutionarily closely-related cyprinid species [6]. lus and M. domesticus) and their natural hybrids [4, 5]. Interestingly, an asymmetrical distribution of parental Host-parasite genetic co-adaptation is more pronounced species-specifc parasites (especially specifc monogene- in host-specifc parasites, thus limiting their infection ans representing gill ectoparasites) was found in hybrids in hybrid genomes [6, 7]. However, several scenarios of of A. brama and R. rutilus. Tis may indicate the limited parasite infection in hybridizing hosts were proposed to inheritance of defense mechanisms from one parental explain the distribution of parasites among hybrids and species and suggests diferent degrees of host-parasite their parental taxa [8, 9]. Wolinska et al. [9], in their coadaptation between diferent host-specifc parasites dynamic scenario, hypothesized that hybrid genomes and their associated hosts [7, 19]. are more parasitized than parental genomes under the In the present study, two widely distributed cyprinid condition of the high frequency of hybrids. In addition, species in central European waters exhibiting low evolu- it seems that the maternal ancestry of hybrids may also tionary divergence and high morphological similarities infuence the infection level of some parasite species, as were studied: silver bream (Blicca bjoerkna) and com- was shown for digenean and crustacean species in F1 mon bream (A. brama). With respect to these species, the hybrids of the evolutionarily divergent, and morphologi- intermediate survival rate of the F1 generation, hybrid cally and ecologically diferent cyprinid species, the com- fertility, and the capacity to produce high quality gam- mon bream (Abramis brama) and roach (Rutilus rutilus) etes have previously been documented [24, 30, 31]. Blicca [7]. bjoerkna and A. brama also exhibit similar ecology. Each Fish hybridization is a frequent event in nature [10, 11]. of these cyprinid species harbours specifc monogenean Such hybridization very often results from the introduc- parasites [32]. tion or invasion of non-native fsh species [12–16] caused Te aim of this study was to use breed lines of B. by direct anthropogenic activities (agriculture, fsh farm- bjoerkna and A. brama and crossbreed lines representing and waterbodies transformation) or human-mediated ing the F1 generation (i.e. using fsh whose immunity disturbances of nature (climate change, interspecies com- was not previously afected by any infection) in order petition or limited living resources). Generally, the pres- to investigate the susceptibility of the F1 hybrids of two ence of F1 hybrids is documented in many wild-living phylogenetically closely-related cyprinid species to the cyprinids even when the frequency of hybrids is rather specifc monogenean parasites of the parental species low [6, 16–22]. Cyprinid hybrids exhibit high larval and to test whether the maternal ancestry of F1 hybrids resistance to environmental disturbances (osmotic and (mtDNA) has an efect on the level of monogenean para- thermal conditions), wider ecological plasticity, greater site infection. fasting abilities in comparison to their parents, and limited susceptibility to parasite infection [7, 23–25]. From the evolutionary point of view, hybridization Methods generates novel morphological and genetic variants of Specimens of B. bjoerkna (3 females and 3 males) organisms. However, novel features of hybrid organisms and A. brama (3 females and 3 males) were caught may afect the compatibilities between hosts and spe- by electrofshing from the River Dyje near the city of cifc parasites. Generally, host-parasite incompatibility Břeclav (48.7566N, 16.8701E; Morava River basin, (predicted by the resistance hypothesis) or, alternatively, Czech Republic) and transported alive to the facil- host-parasite compatibility (revealed as phenotypic ity of the Institute of Vertebrate Biology, Czech Acad- or genotypic compatibility proposed by the matching emy of Sciences. Fish separated according to sex into hypothesis) result from a wide range of molecular deter- two well-aerated tanks were stimulated for ovula- minants [26]. Moreover, the incompatibility between fsh tion/spermiation by carp pituitary (females received 2 and their ectoparasitic monogeneans might arise from doses, 0.3 and 2.7 mg/kg 24 h and 12 h before propagation, respectively; males received 1 mg/kg 24 h before Krasnovyd et al. Parasites Vectors (2020) 13:402 Page 3 of 11

propagation) and by subsequently increasing the water study were classifed as B. bjoerkna-specifc parasites. temperature to 22 °C. Oocytes of ovulating females Te delimitation of host specifcity for monogenean were obtained by the dry method and sperm was sam- species primarily originated from the checklist of fsh- pled according to Linhart et al. [33]. Hatchery water parasite records from the Czech and Slovak Republics was used for gamete activation. Artifcial spawning by Moravec [38]. For some monogenean species, one of based on individual pair mating was performed using the two cyprinid species examined in this study repre- the following parental combinations: (i) female and sents the most common host species in central European male B. bjoerkna; (ii) female and male A. brama; (iii) rivers within a wide range of potential host species; for female B. bjoerkna and male A. brama; and (iv) female example, Dactylogyrus sphyrna is the most common par- A. brama and male B. bjoerkna. As a result of artifcial asite of B. bjoerkna within the range of central European spawning, four lines of ofspring were obtained: pure cyprinids [38–40]. Such a parasite species was also con- breed B. bjoerkna; pure breed A. brama; and two cross- sidered as host specifc in the present study. breed lines, F1 hybrids with A. brama maternal origin Kruskal-Wallis H-test followed by multiple comparison (mtDNA) and F1 hybrids with B. bjoerkna maternal ori- tests was applied to test the diferences in monogenean gin (mtDNA). Four lines of ofspring were reared sub- abundance among and between fsh breeds. As host-spe- sequently up to 1-year-old. cifc parasites were not shared between A. brama and B. Te experimental infection was conducted in a well bjoerkna, the Kruskal-Wallis H-test followed by multiple aerated and fltered tank. A total of 15 specimens: 8 comparisons was performed to compare the abundance of A. brama and 7 of B. bjoerkna captured from nature of specifc parasites (i) between A. brama and the two (the River Dyje near the city of Břeclav) were placed in hybrid lines, i.e. F1 hybrids with A. brama maternal ori- the tank with aerated water. To initiate infection, the gin and F1 hybrids with B. bjoerkna maternal origin, and batches of the A. brama and B. bjoerkna breed lines and (ii) between B. bjoerkna and the two abovementioned the crossbreed lines were kept in the same tank with the hybrid lines. Asymmetrical infection by host-specifc infected specimens of A. brama and B. bjoerkna caught parasites in F1 hybrids with diferent maternal origins in nature. (i.e. diferences in the parasite species richness and abun- Tree weeks after the initiation of the experiment, all dance of A. brama-specifc parasites and B. bjoerkna-spe- fsh specimens were sacrifced by severing the spinal cord cifc parasites between F1 hybrids of diferent maternal in accordance with Law 246/1992 of the Czech Repub- origins) was tested using the Wilcoxon signed-rank test. lic and subsequently dissected following Ergens & Lom Te Mann-Whitney U-test was used to test the efect of [34]. All parasite specimens were collected, fxed in GAP the maternal origin of hybrids on the asymmetrical infec- (glycerine-ammonium picrate) following Malmberg [35], tion by host-specifc parasites in F1 hybrids; i.e. the dif- and identifed on the basis of the species-specifc scle- ference between B. bjoerkna-specifc parasite species rotized structures of the attachment organ (haptor) and richness (or abundance) and A. brama-specifc parasite reproductive organs following Gusev [36] using a light species richness (or abundance) was used as a dependent microscope (Olympus BX 51; Olympus Life and Mate- variable in the Mann-Whitney U-test. Statistical analy- rial Science Europe GMBH, Hamburg, Germany) with ses were performed using Statistica 13.3 for Windows phase-contrast, diferential interference contrast, and a (TIBCO software Inc., Palo Alto, CA, USA). digital image analysis system (Stream motion). Prevalence and intensity of infection were calculated Results for each parasite species in each of the host lines (A. Te examination of wild living fsh specimens revealed brama, B. bjoerkna, F1 hybrids with maternal origin 6 monogenean species in A. brama and 4 monogenean of A. brama and F1 hybrids with maternal origin of B. species in B. bjoerkna, all monogeneans were found bjoerkna). In accordance with Bush et al. [37], prevalence on the gills (Table 1). No diference in monogenean as a percentage of infected fsh of a given host line by a abundance was found between the two cyprinid spe- given parasite species, and mean intensity of infection cies (Mann-Whitney U-test: U(15) = 16.5, Z = − 1.27, (expressed by mean ± standard deviation) as a number of P = 0.203). Using fsh from artifcial breeding, signifcant parasite specimens of a given species per infected host, diferences in total monogenean abundance among A. were calculated. Te range of intensity of infection was brama, B. bjoerkna, and F1 hybrids were found (Kruskal- included for each parasite species in each of host lines. Wallis H-test: H(2, 126) = 6.04, P = 0.049). Even when Te monogenean species present on A. brama and monogenean abundance tended to reach higher values absent on B. bjoerkna in this study were classifed as in common bream and silver bream when compared to A. brama-specifc parasites. Te monogenean species hybrids, multiple comparisons revealed only a signifcant present on B. bjoerkna and absent on A. brama in this diference between F1 hybrids and B. bjoerkna (P = 0.043) Krasnovyd et al. Parasites Vectors (2020) 13:402 Page 4 of 11

(Fig. 1a). Dactylogurus parasites had the dominant posi- least numerous D. auriculatus between A. brama and tion in the monogenean communities of all the investi- hybrids (Kruskal-Wallis H-test: H(2, 87) = 0.51, P = 0.775) gated fsh breed lines (A. brama, B. bjoerkna, F1 hybrids was found. with A. brama maternal origin and F1 hybrids with B. Concerning Dactylogyrus specifc to B. bjoerkna, the bjoerkna maternal origin). Te diferences in Dacty- Kruskal-Wallis H-test followed by multiple comparisons logyrus abundance among A. brama, B. bjoerkna, and revealed a weakly signifcant diference in the abundance hybrids were not signifcant (Kruskal-Wallis H-test: H(2, of only D. sphyrna between B. bjoerkna and hybrids with 126) = 2.90, P = 0.235, Fig. 1b). Dactylogyrus zandti and D. B. bjoerkna maternal origin (Kruskal-Wallis H-test: H(2, falcatus were the parasite species with the highest infec- 106) = 6.99, P = 0.03, multiple comparison: P = 0.046). tion levels in the experimentally infected breed line of A. Concerning P. bliccae, a signifcant diference was found brama. Concerning host-specifc parasites of A. brama between B. bjoerkna and hybrids with A. brama maternal present in hybrids, D. zandti achieved the highest infec- origin (Kruskal-Wallis H-test: H(2, 106) = 21.58, P < 0.001, tion level (Table 2). On B. bjoerkna, D. sphyrna was the multiple comparison: P = 0.009). Te diference between most prevalent parasite, reaching also the highest inten- B. bjoerkna and hybrids with B. bjoerkna maternal origin sity of infection, followed by the intensities of infection was not signifcant (multiple comparison: P = 0.052). of D. distinguendus and Paradiplozoon bliccae (Table 2). Gyrodactylus vimbi was the only monogenean species Dactylogyrus sphyrna was also the monogenean spe- infecting both pure breed lines and both lines of hybrids cies exhibiting the highest infection level (measured by (Table 2). No signifcant diferences in the abundance prevalence and intensity of infection) in the F1 hybrids of G. vimbi were found among the four groups of fsh of both maternal origins. Monogenean communities of (Kruskal-Wallis H-test: H(3, 126) = 4.33, P = 0.228). the A. brama breed line consisted of 5 monogenean spe- Te Wilcoxon signed-rank test revealed asymmetry in cies; 4 of them host-specifc Dactylogyrus species, which the abundance of host-specifc monogeneans in favor of were also identifed in the source specimens of this spe- parasites specifc to B. bjoerkna (Fig. 2a). Te abundance cies (Tables 1, 2). Two species, Gyrodactylus elegans of B. bjoerkna-specifc parasites was higher than the and Diplozoon paradoxum, were found in source speci- abundance of A. brama-specifc parasites in the hybrid mens of A. brama from nature but were not identifed in line with B. bjoerkna maternal origin (Z = 2.99, P = 0.003) experimentally infected specimens of this cyprinid spe- and the hybrid line with A. brama maternal origin cies. Monogenean communities of the B. bjoerkna breed (Z = 2.98, P = 0.003). Te species richness of B. bjoerkna- line consisted of 5 species; 4 of them were found in the specifc parasites was higher than the species richness sample of source specimens of B. bjoerkna collected in of A. brama-specifc parasites in the hybrid line with B. nature (Tables 1, 2). Te monogenean communities of F1 bjoerkna maternal origin (P = 0.049) and the hybrid line hybrids exhibited higher monogenean species richness with A. brama maternal origin (P = 0.003) (Fig. 2b). Te in comparison to those of parental species, i.e. all mono- Mann-Whitney U-test test showed no efect of the mater- genean species found in breed lines of A. brama and B. nal origin of hybrids on the diference in monogenean bjoerkna were also present in F1 hybrids. species richness (U(67) = 462.00, Z = 1.20, P = 0.230) or Concerning Dactylogyrus spp. specifc to A. brama, abundance (U(67) = 552.00, Z = 0.07, P = 0.945) between a signifcant diference (Kruskal-Wallis H-test: H(2, A. brama-specifc and B. bjoerkna-specifc parasites. 87) = 17.09, P = 0.002, multiple comparisons between A. brama and F1 hybrids with A. brama maternal origin: P < 0.001 and between A. brama and F1 hybrids with B. Discussion bjoerkna material origin: P = 0.002) was revealed for total Te presented study focused on the distribution of host- Dactylogyrus abundance. Te same diference was found specifc parasites (gill monogeneans) in the F1 hybrids for the abundance of 3 Dactylogyrus species, i.e. D. fal- of cyprinid species with low evolutionary divergence, A. catus (Kruskal-Wallis H-test: H(2, 87) = 18.82, P < 0.001, brama and B. bjoerkna. Although there is phylogenetic multiple comparisons: P < 0.001 and P = 0.006, respec- proximity between these cyprinid species [41], they har- tively), D. wunderi (Kruskal-Wallis H-test: H(2, 87) = 24.38, bor diferent host-specifc monogenean species, espe- P < 0.001, multiple comparisons: P = 0.005 and P = 0.008, cially representatives of Dactylogyrus. Te majority of respectively), and D. zandti (Kruskal-Wallis H-test: H(2, host-specifc Dactylogyrus in European cyprinid hosts 87) = 9.84, P = 0.007, multiple comparisons: P = 0.033 and evolved by intra-host speciation (speciation within host P = 0.042, respectively). However, no diferences in total species) [39], i.e. D. zandti and D. wunderi specifc to A. Dactylogyrus abundance or the abundance of individ- brama evolved by intra-host speciation within A. brama, ual Dactylogyrus species between the 2 lines of hybrids and D. distinguendus, D. cornu and D. cornoides specifc (P > 0.05) were found. No diference in the abundance of to B. bjoerkna evolved by intra-host speciation within B. Krasnovyd et al. Parasites Vectors (2020) 13:402 Page 5 of 11

Table 1 Intensity of monogenean infection (MI, mean standard deviation), range of intensity of infection (I, minimum-maximum), ± and prevalence (P, in %) for each monogenean species in A. brama and B. bjoerkna captured from nature Parasite species Abramis brama (n 8) Blicca bjoerkna (n 7) = = MI I P MI I P

Dactylogyrus auriculatus 1.67 1.16 1–3 38 – – – ± Dactylogyrus wunderi 4.00 2.65 1–6 38 – – – ± Dactylogyrus zandti 9.80 8.90 2–22 63 – – – ± Dactylogyrus falcatus 1.67 0.58 1–2 38 – – – ± Dactylogyrus sphyrna – – – 2.00 1.41 1–3 29 ± Dactylogyrus cornu – – – 4.75 4.35 1–9 57 ± Dactylogyrus distinguendus – – – 1.75 1.5 1–4 57 ± Gyrodactylus vimbi – – – 1.50 0.71 1–2 29 ± Gyrodactylus elegans 1 – 13 – – – Diplozoon paradoxum 2 2 63 – – –

bjoerkna. Both A. brama and B. bjoerkna are also para- wild fsh specimens and was successfully reproducing in sitized by Dactylogyrus species, representatives of other experimental B. bjoerkna and both lines of hybrids. phylogenetic lineages unrelated to those originating from We found that the total abundance of monogeneans intra-host speciation (more specifcally, D. auriculatus of (7 of 9 monogenean species belonged to Dactylogyrus) A. brama and D. sphyrna of B. bjoerkna likely originate tended to be higher in parental species when compared from host switching). Despite the phylogenetic proximity to hybrids (even if the diference with respect to total of A. brama and B. bjoerkna, their Dactylogyrus species monogenean abundance was signifcant only between B. (or lineages) evolved independently (i.e. Dactylogyrus bjoerkna and hybrids). However, for the majority of host- species host-specifc to A. brama and Dactylogyrus spe- specifc parasites (especially for Dactylogyrus specifc to cies host-specifc to B. bjoerkna are not phylogenetically A. brama), we found higher abundance in a correspond- closely related [39]). ing pure species when compared to parasite abundance Te present work is the frst experimental study inves- in hybrids. Fritz et al. [42] proposed four static scenarios tigating the distribution of host-specifc monogeneans in explaining the pattern of parasite distribution in parental F1 hybrids of cyprinids under the following conditions: species and their hybrids. Te dominance scenario pre- (i) the experiment was run using similar proportions of dicts similar resistance between hybrids and one of the parental and hybrid genotypes; and (ii) the immunity of parental taxa; thus, parasite infection levels in hybrids breed and crossbreed lines was not afected by any pre- and one parental taxon are similar. Te hybrid resistance vious infection prior to experimental infection by mono- scenario predicts the superior resistance of hybrids when geneans (in contrast to the previous studies of Šimková compared to parental taxa. Terefore, the parasite infec- et al. [6] and Krasnovyd et al. [7] investigating natural tion level is lower in hybrids when compared to each of parasite infection of cyprinids). the parentals. Te additive scenario predicts a diference We applied co-habitation design of experiment when in parasite abundance between parental taxa; however, specimens of breed and crossbreed lines were placed hybrid resistance does not difer from the average resist- together with wild A. brama and B. bjoerkna. Almost ance of parental taxa. Te susceptibility scenario is based all monogenean parasites reported in A. brama or B. on the prediction of the lower resistance of hybrid indi- bjoerkna from nature were also found in experimental viduals when compared to parental taxa. In this case, the specimens. Te absence of two monogenean species in parasite infection level is higher in hybrids when com- experimental specimens is a result of low parasite abun- pared to each of the parental taxa. dance in wild fsh specimens (for G. elegans) or failed Wolinska et al. [9] proposed a dynamic scenario parasite reproduction under our experimental conditions based on the frequencies of common and rare geno- (for D. paradoxum). In contrast, P. bliccae, host-specifc types, i.e. frequency-based selection, to explain the parasite of B. bjoerkna, was not identifed in our low sam- temporal dynamics in parasite infection in hybridizing ple size of dissected wild fsh specimens, but this diplo- systems of hosts. Such a dynamic scenario incorporates zoid species was evidently formerly present in cohabited all the phases described as four static scenarios by Fritz et al. [42]. Krasnovyd et al. [7] investigated the pattern Krasnovyd et al. Parasites Vectors (2020) 13:402 Page 6 of 11

a 18

14 e 12 bundanc

a 10

8 ogenean n mo

l 6 ta To 4

0 Mean BB BBAB ABBB AB Mean±SE

b 18

14 e 12

10 s abundanc ru

ogy 8 ctyl

6 Total Da 4

0 Mean BB BBAB ABBBAB Mean±SE Fig. 1 Total monogenean abundance (a) and Dactylogyrus abundance (b) in breed lines of B. bjoerkna, A. brama and hybrids. Abbreviations: AB, Abramis brama; BB, Blicca bjoerkna; ABBB, hybrids with maternal A. brama; BBAB, hybrids with maternal B. bjoerkna Krasnovyd et al. Parasites Vectors (2020) 13:402 Page 7 of 11

Table 2 Intensity of monogenean infection (MI, mean standard deviation), range of intensity of infection (I, minimum-maximum), ± and prevalence (P, in %) for each monogenean species in pure A. brama, pure B. bjoerkna and hybrids Parasite species Abramis brama (n 20) Blicca bjoerkna (n 39) F1 hybrids with maternal F1 hybrids with maternal = = origin of A. brama (n 36) origin of B. bjoerkna (n 31) = = MI I P MI I P MI I P MI I P

Dactylogyrus auriculatus 1 1 10 – – – 1 1 6 1 1 10 Dactylogyrus wunderi 4.00 3.00 1–10 55 – – – 1 1 8 1 1 10 ± Dactylogyrus zandti 7.93 8.93 1–33 70 – – – 7.36 8.24 1–24 31 4.90 5.59 1–19 32 ± ± ± Dactylogyrus falcatus 6.13 5.50 1–18 75 – – – 2.78 2.77 1–8 25 2.30 1.25 1–4 32 ± ± ± Dactylogyrus sphyrna – – – 11.83 9.34 1–31 79 7.08 7.02 1–27 69 9.80 10.38 1–41 48 ± ± ± Dactylogyrus cornu – – – 3.53 2.61 1–9 38 1.73 1.27 1–4 31 2.75 2.49 1–8 26 ± ± ± Dactylogyrus distinguendus – – – 5.77 5.78 1–19 33 3.36 2.95 1–10 25 3.73 4.05 1–15 35 ± ± ± Gyrodactylus vimbi 1 1 15 1 1 8 2 – 3 3.40 4.28 1–11 16 ± Paradiplozoon bliccae – – – 5.50 4.13 1–13 41 1 – 3 1.33 0.58 1–2 10 ± ±

of parasite distribution in two non-congeneric cyprin- species transferring their parasites. However, concern- ids with high evolutionary divergence, A. brama and R. ing strictly specifc parasites, reciprocal co-adaptation rutilus, and their F1 hybrids. Tey showed that hybrids between hosts and associated parasite genotypes is exhibit lower parasite intensities (especially a lower hypothesized [46]. Tis may indicate that fully adapted intensity of infection by host-specifc parasites) than parasites cannot be transmitted through the “hybrid parental species, this pattern of infection was revealed in bridge” from one parental species to another. Sage et al. a diferent time and space. Te same fnding was demon- [4] documented a high level of parasite infection in two strated by Šimková et al. [6] using two phylogenetically house mice species (Mus musculus and M. domesticus) closely-related cyprinids, i.e. Cyprinus carpio and Caras- and their natural hybrids, and suggested that a bro- sius gibelio, that share some Dactylogyrus species exhibit- ken system of host-parasite genetic coadaptation might ing so-called phylogenetic specifcity, i.e. parasite species explain their fnding. In the present study, each parental restricted to phylogenetically closely-related host species species harbored unique monogenean fauna (except for (for details see Šimková et al. [40]). Krasnovyd et al. [7] G. vimbi, which was shared by both species), and inter- suggested that the absence of a pattern proposed by the specifc hybrids resulting from artifcial breeding har- dynamic scenario of Wolinska et al. [9] is related to the bored all parasites specifc to A. brama and B. bjoerkna, constantly low frequency of hybrid genotypes in nature. mostly at lower intensities of infection when compared to In fact, a very low frequency of hybrids was reported in parental species. Tis pattern of distribution of parental both previous studies [6, 7] investigating the parasite dis- species-specifc parasite in hybrids likely results from a tribution in hybridizing cyprinids. In contrast, Dupont lack of genetic co-adaptation between the host-specifc & Crivelli [43] reported a high level of parasite infec- parasite and associated host genome (here the genome of tion in hybridizing Rutilus rubilio and Alburnus albur- A. brama or genome of B. bjoerkna) as suggested by Kras- nus from Lake Mikri Prespa in northern Greece, where novyd et al. [7]. Some monogenean species expressed an extremely high rate of hybridization was reported. In similar infection levels in parental species and F1 hybrids; the present study, we set our experimental conditions these were the three host-specifc Dactylogyrus species: by using similar proportions of A. brama, B. bjoerkna D. distinguendus; D. auriculatus; D. cornu; and the gener- and hybrid genotypes. Our observation of the trend of a alist G. vimbi. However, the last three species were found higher overall abundance of monogeneans and of signif- in very low intensities in parental species and hybrids. cantly higher abundances of the majority of host-specifc To test the hybrid bridge hypothesis, other experiments parasites in corresponding associated host species when with hybridizing systems of A. brama and B. bjoerkna (or compared to hybrids may suggest, in contrast to the other fsh species with host-specifc parasites) will need hypothesis of Wolinska et al. [9], that the frequency of to be performed in the future, these focusing on the dif- host genotypes (pure breeds versus crossbreeds) does not ferent rate of genetic introgression between two species play a signifcant role in host-specifc parasite selection. (the F1 generation, back-crosses, and the F2 generation Te hybrid bridge hypothesis [44, 45] suggests that of hybrids) and the potential to transfer host-specifc hybrid individuals act as a bridge between parental parasites. However, the hybridization of A. brama and Krasnovyd et al. Parasites Vectors (2020) 13:402 Page 8 of 11

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ch 1.1 ri s ie 1.0 ec Sp 0.9

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0.5 Mean AB hybrids (AB) AB hybrids (BB) BB hybrids (AB) BB hybrids (BB) Mean±SE Fig. 2 Asymmetrical distribution of parental-specifc parasites in hybrids with diferent maternal origins. Abundance (a) and species richness (b) of A. brama-specifc parasites (indicated as AB in parentheses on the x-axis) and B. bjoerkna -specifc parasites (indicated as BB in parentheses on the x-axis) in hybrids with A. brama maternal origin (AB hybrids) and hybrids with B. bjoerkna maternal origin (BB hybrids) Krasnovyd et al. Parasites Vectors (2020) 13:402 Page 9 of 11

B. bjoerkna does not represent a serious threat to the A. brama even exhibited slightly higher total monoge- genetic integrity of these species (because of the low fre- nean species richness. quency of hybrids in nature) and presents only a minimal risk with respect to transferring host-specifc parasites Conclusions (because of host-parasite co-adaptation). Our results confrm that the presence of host-specifc In the present study, we also examined the poten- parasites is afected by hybridization. We showed that tial efect of mtDNA on the presence of host-specifc host-specifc monogeneans reached higher levels of parasites. Cyto-nuclear incompatibility, i.e. incompat- infection in pure species when compared to their recip- ibilities between the alleles of mitochondrial and nuclear rocal intergeneric hybrids under the condition of there genomes resulting from hybridization, may cause being similar proportions of pure species genotypes hybrid breakdown even in the F1 generation of hybrids and hybrid genotypes, which again supports the static [47]. Chou & Leu [47] stated that some diseases related scenario of hybrid resistance. Tis was more strongly to mitochondrial DNA are pathogenic only in certain evidenced for the abundant Dactylogyrus specifc to A. nuclear backgrounds of hosts. However, in our study, brama. Our fndings contradict Wollinska et al. [9], who no obvious evidence of an mtDNA efect on the level of hypothesized that host genotypes present in higher fre- infection of host-specifc monogeneans in hybrids was quency are the target of parasite selection, i.e. if hybrids found. It seems that maternal origin, i.e. represented represent the frequent genotype (i.e. common geno- by mitochondrial genes, is not primarily involved in type) they are more susceptible to parasite infection. the association between fsh hosts and their specifc However, we suggest that the low intensity of infec- parasites. Paradiplozoon bliccae, a strict specialist of B. tion by host-specifc monogeneans in cyprinid hybrids bjoerkna, was the only parasite species exhibiting a sig- results from a lack of genetic co-adaptation. Monoge- nifcant diference in abundance between silver bream nean communities exhibited a shift toward a higher and hybrids with common bream maternal origin. Due proportion of host-specifc parasites of B. bjoerkna to the low parasite intensity of infection, this fnding can- potentially resulting from diferent inheritances of not clearly support the efect of mtDNA on the presence immune protective mechanisms from both parental and/or level of parasite infection. However, even with no species. Te maternal origin of hybrids has no principal statistical support, our data indicate a higher intensity of role in determining the presence of host-specifc para- infection by B. bjoerkna-specifc parasites in hybrid spec- sites, which seems to suggest that mitochondrial genes imens with B. bjoerkna maternal position. are not primarily involved in co-evolutionary associa- Šimková et al. [19] suggested host-parasite co-evolu- tions between cyprinid hosts and specifc monogenean tionary associations as a major factor limiting the distri- parasites. We highlight the need for genomic studies bution of host-specifc Dactylogyrus parasites in pure and to identify the genes involved in reciprocal genetic co- hybrid specimens across hybrid zones. In a study focusing adaptations of host-specifc parasites and their associ- on the temporal and spatial variation of parasite infection ated hosts. in a hybridizing system of cyprinid species with high evolutionary divergence, Krasnovyd et al. [7] demonstrated asymmetry in the proportions of parental species-specifc Abbreviations F1 hybrids: frst flial generation of ofspring produced by intercrossing of parasites in the parasite communities of hybrids. Tey diferent parental species; F2 hybrids: second flial generation of ofspring pro- suggested that the diferent inheritances of immune produced by intercrossing of F1 individuals; mtDNA: mitochondrial DNA which is tective mechanisms from two parental species or alter- located in mitochondria within eukaryotic cells. natively the diferent rates of co-adaptation between Acknowledgements specifc parasites and their associated hosts may explain We are grateful to Chahrazed Rahmouni, Imanne Rahmouni, Lenka Gettová, this asymmetry. In their study, the parasite communi- Tomaš Pakosta, Michal Benovics, Kristýna Koukalová, Gabriela Vágnerová, Adam Potrok and Mária Seifertová from the Laboratory of Parasitology, ties of hybrids were shifted toward a higher proportion Department of Botany and Zoology, Faculty of Science, Masaryk University of host-specifc parasites (especially monogeneans) of R. for help with fsh dissection and parasite collection. We kindly thank Matthew rutilus, which was the species achieving higher monoge- Nicholls for English revision of the fnal draft. nean diversity but lower parasite (and also monogenean) Authors’ contributions abundance when compared to A. brama. In our study, we AŠ designed the study, organized and conducted the experiment, analyzed reported asymmetry in the monogenean communities of the data, wrote the manuscript, and obtained funding for the study. VK performed morphological analyses, prepared the source data fles, and partici- hybrids toward a higher abundance of B. bjoerkna-spe- pated in the preparation of the draft. LV conducted the artifcial breeding and cifc parasites. However, source fsh caught in nature as participated in the experiment and the writing of the manuscript. All authors well as breed lines of A. brama and B. bjoerkna exhibited read and approved the fnal manuscript. no diferences in monogenean intensity of infection, and Krasnovyd et al. Parasites Vectors (2020) 13:402 Page 10 of 11

Funding 16. Gettová L, Gilles A, Šimková A. Metazoan parasite communities: support The realization of this study and the persons involved were funded by the for the biological invasion of Barbus barbus and its hybridization with the Czech Science Foundation, Project no. 19-10088S. endemic Barbus meridionalis. Parasites Vectors. 2016;9:588. 17. Vetešník L, Halačka K, Papoušek I, Mendel J, Šimková A. The frst record of Availability of data and materials a natural hybrid of the roach Rutilus rutilus and nase Chondrostoma nasus Data upon which the conclusions are based are provided within the article. in the Danube River Basin, Czech Republic: morphological, karyological and molecular characteristics. J Fish Biol. 2009;74:1669–76. Ethics approval and consent to participate 18. Hayden B, Pulcini D, Kelly-Quinn M, O’Grady M, Cafrey J, McGrath A, The study was approved by the Animal Care and Use Committee of the Fac- Mariani S. Hybridisation between two cyprinid fshes in a novel habitat: ulty of Science, Masaryk University in Brno (Czech Republic). genetics, morphology and life-history traits. BMC Evol Biol. 2010;10:169. 19. Šimková A, Navrátilová P, Dávidová M, Ondračková M, Sinama M, Chappaz Consent for publication R, Gilles A, Costedoat C. Does invasive Chondrostoma nasus shift the Not applicable. parasite community structure of endemic Parachondrostoma toxostoma in sympatric zones? Parasites Vectors. 2012;5:200. Competing interests 20. Šimková A, Vojtek L, Halačka K, Hyršl P, Vetešník L. The efect of hybridi- The authors declare that they have no competing interests. zation on fsh physiology, immunity and blood biochemistry: a case study in hybridizing Cyprinus carpio and Carassius gibelio (Cyprinidae). Author details Aquaculture. 2015;435:381–9. 1 Department of Botany and Zoology, Faculty of Science, Masaryk University, 21. Levin BA, Gandlin AA, Simonov ES, Levina MA, Barmintseva AE, Japosh- Kotlářská 2, 611 37 Brno, Czech Republic. 2 Institute of Vertebrate Biology, vili B. Phylogeny, phylogeography and hybridization of Caucasian bar- Czech Academy of Sciences, Květná 8, 603 65 Brno, Czech Republic. bels of the genus Barbus (Actinopterygii, Cyprinidae). Mol Phylogenet Evol. 2019;135:31–44. Received: 29 March 2020 Accepted: 30 July 2020 22. Ramoejane M, Gouws G, Swartz ER, Sidlauskas BL, Weyl OLF. Molecu- lar and morphological evidence reveals hybridisation between two endemic cyprinid fshes. J Fish Biol. 2020;96:1234–50. 23. Crespin L. Spatially varying natural selection in a fsh hybrid zone. J Fish Biol. 2002;61:696–711. References 24. Nzau Matondo B, Ovidio M, Poncin P, Kakesa TA, Wamuini LS, Philippart 1. Crofton HD. A model of host-parasite relationships. Parasitology. JC. Hybridization success of three common European cyprinid species, 1971;63:343. 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