Ph.D. Dissertation

MASARYK UNIVERSITY FACULTY O F S CIENCE DEPARTMENT OF BOTANY AND ZOOLOGY

Ph.D. Dissertation

BRNO 2017 Msc. Willems Kamila

MASARYK UNIVERSITY FACULTY O F S CIENCE DEPARTMENT OF BOTANY AND ZOOLOGY

Intraspecific variability and dispersal propensity of the palaearctic bugs of the genus Cimex (Heteroptera: Cimicidae)

M.Sc. Willems Kamila

Supervisor : doc. Mgr. Tomáš Bartoni čka, Ph.D

Consultant : Mgr. Ond řej Balvín, Ph.D

Bibliografický záznam

Autor: Mgr. Kamila Willems, Přírodov ědecká fakulta, Masarykova Univerzita, Ústav botaniky a zoologie

Vnitrodruhová variabilita a schopnost ší ření u palearktických št ěnic rodu Cimex Název práce: (Heteropetra: Cimicidae)

Studijní program: Biologie Studijní obor: Zoologie

Vedoucí práce: doc. Mgr. Tomáš Bartoni čka, Ph.D

Akademický rok: 2017/2018

Po čet stran: 124

Klí čová slova: Ektoparazit, št ěnice, Cimex, k oevoluce

Bibliographic Entry

Author: M.Sc. Kamila Willems, Faculty of Science, Masaryk University, Department of Botany and Zoology

Title of Thesis: Intraspecific variability and dispersal propensity of the palaearctic bugs of the genus Cimex (Heteroptera: Cimicidae)

Field of Study Zoology

Supervisor: doc. Mgr. Tomáš Bartoni čka, Ph.D

Academic Year: 2017/2018

Number of Pages: 124 Ectoparasites, bed bugs, bat bugs, Cimex , Keywords: coevolution ABSTRACT

Main research was focus on two haematophagous ectoparasitic species from family Cimicidae. First directly related to human - bed bug Cimex lectularius which expands widely last years and is more and more often found in human dwellings. As ectoparasite that can leave freely and use host only during taking a blood meal, and can starve for a very long period of the time, it is hard to exterminate. Knowledge about its biology, ecology and morphology can be very useful in solving many problems connected with its infestation. Especially that it was found to create associations both with humans and other crucial host, namely bats. Regarding those facts our research focused on investigation of sucking abilities of both bat and human associated bugs to estimate level of association with both hosts. We have found that both human and bat associated bed bugs can suck on non-specific for them host. Further observations of successful survival, reproduction and development abilities shown us clearly that in cross-feeding experiment type of the host, specific/non-specific, seems to have an impact on survival, moulting and development rate in both cross-feedings conducted in vitro. Because bed bugs were never found on some bat species, we investigated blood composition with focus on RBC (red blood cells) size and density. We did not find significant differenced in RBC measurements between specific and non- specific bat host species. Following the fact that both human and bat associated bugs can suck on non- specific host, it was important to see if both lineages in case of meeting are being able to mate, reproduce and produce successfully progeny. Further hybridization experiments between two lineages human and bat associated bed bug ( Cimex lectularius ) from Central Europe (Czech Republic) were conducted under laboratory conditions. Couples mix of both lineages (interspecific mating) were compared with couples from same lineage, same locality and same lineage, different localities (intraspecific mating). In case of mixed couples from different lineages creating hybrids was unsuccessful, no eggs no progeny was produced. Second species, indirectly connected with human and closely associated with bat hosts - C. pipistrelli was investigated in terms of transmission by its host. We do not posses much information about bat bugs sucking on humans, nevertheless in many cases bats and humans share same shelter which increases risk of infestation. Transmission experiments were conducted on 2 most common bat bugs vectors – noctule bat ( Nuctalus noctula ) and greater mouse – eared bat ( Myotis myotis ) as well on non common bat species – daubenton´s bat (Myotis daubentonii ). Results clearly confirmed that bat bugs prefer transmission on bigger bat species such as noctule or greater mouse-eared bat than daubenton´s bat. What is more from both bigger bats they shows clear preferences towards noctule bat which in fact is mostly mist-netted bat which carries bugs on his body. Research proved also that bugs that mainly departed from bat box on bats body were mated females not virgins, as a second one has no possibility to start new infestation. Following bat bug transmission possibilities, we conducted genetic research at local and regional scale in Central Europe. Developed polymorphic loci has shown homogenous character in populations of bat bugs from localities across Czech Republic, Slovakia and Ukraine.

Acknowledgements

I would like to express my special appreciation and thanks to my advisor and supervisor, doc. Mgr. Tomáš Bartoni čka, Ph.D who has a tremendous mentor and help for me. I would like to thank him for encouraging me in my research and for allowing me to grow as a research scientist. I would also like to thank also my consultant Mgr. Ond řej Balvín, Ph.D, doc. Mgr. et Mgr. Josef Bryja, Ph.D and Mgr. Adam Kone čný, Ph.D. for all advices and sharing with me their experience and knowledge. And to all the other people that made my PhD time at Masaryk University unforgettable experience.

A special thanks to my family. Words cannot express how grateful I am to my mother and sister for all of the sacrifices that you’ve made on my behalf. Your words of support, encouraging and your patience. I would also like to thank all of my friends all over the Europe, who supported me in writing, and incented me to strive towards my goal. At the end I would like to express appreciation to my beloved husband Wout Willems who spent sleepless nights with me and was always my support in the hardest moments.

Prohlášení

Prohlašuji, že jsem svoji diserta ční práci vypracovala samostatn ě s využitím informa čních zdroj ů, které jsou v práci citovány.

Brno ……………………………… Kamila Willems

1. Introduction ...... 2 1.1 Host specificity ...... 2 1.2 Reproductive isolation in insects – inter- and intraspecific matings...... 5 1.3 Host transmission and genetic structure of bat ectoparasites...... 11 1.4 Traumatic insemination as mating strategy ...... 14 2. Model organisms...... 16 2.1 Bed bug – Cimex lectularius ...... 16 2.2 Bat bug – Cimex pipistrelli...... 17 2.3 Human and bats as bed bug hosts...... 18 2.4 Bat hosts...... 20 2.5 Some hematological aspects of hosts – specific versus non-specific bat host...... 22 3. Main aims of the thesis...... 26 4. Summary of main results ...... 28 4.1 Two different lineages of bed bug (Cimex lectularius) reflected in host specificity...... 29 4.2 Hybridization experiments on bed bugs from two lineages (bat and human associated)...... 31 4.3 Erythrocyte size as potential explanation of host specificity in bed bugs...... 33 4.4 Development of multiplex panels of polymorphic microsatellite loci for bat bug (Cimex pipistrelli): a pilot methodological study...... 34 4.4.1 Abstract...... 34 4.4.2 Microsatelites as genetic tools in insects genetic ...... 35 4.4.3 Co-evolution of host and ectoparasite genetic variability ...... 37 4.4.4 Material and methods...... 40 4.4.5 Results...... 42 4.4.6 Discussion...... 44 4.5 Bat bugs (Cimex pipistrelli) transmission propensity in three bat species...... 50 5. Conclusions and future perspectives ...... 51 6. References ...... 53 7. Manuscripts published and submitted ...... 88 7.1 Bat bugs (Cimex pipistrelli) transmission propensity in three bat species...... 89

1. Introduction

1.1 Host specificity

Host-parasite association is result of long time interactions (Janzen 1980). More than 14 000 insects feed on blood of vertebrates (Adams 1999). They belong to Diptera, Hemiptera, Phthiroptera and Siphonoptera. Host-parazite coevolution might be responsible for specialization in parasitic insects. It will be different in ecto- and endoparasites. Most ectoparasites is more flexible in their environment (host) choice and in a case of unfavorable conditions can easily change host. Host determines survival, reproduction and development success of the parasites and depends on many aspects such as: i) environment, ii) fitness, iii) life style, iv) diversity or even v) age of the host. On the other hand parasites are never neutral for their hosts. They can influence many aspects of host life such as community structure, size of host population, they may decrease fitness, mating and fecundity (e.g. Clayton 1990). Poulin (2007) described two filters that determine host choice by parasite: i) encounter filter, when parasite excludes the host which can not be colonize and used as blood meal source because of behavioural or ecological reasons and ii) compatibility filter which excluds all host individuals on which parasite can not feed because of morphological, physiological and immunological reasons. Next to both of those filters Dick and Patterson (2006) suggested existence of reproductive filter that excludes hosts that may decrease reproduction, fitness or simply when finding mates is impossible, even when both encounter and compatibility filters were overcomed.

In host-parasite association we can distinguish principal and alternative host. In case of principal host species, it is occupied by majority of individuals from parasite population of certain species, while in alternative host we find just a few parasite individuals of certain species, that uses other host by coincidence or as principal host replacement (Poulin and Mouillot 2004). Changes in parasites density (parasite density hypothesis; Combes 2001) such as increase in a number of parasite, rise probability of feeding on accidental alternative or encounter host (Poulin 1998) by overcrowding and higher competition (limited resources; Emelianov 2007). On the other hand principal host hypothesis states that also decrease in host density causes searching for alternative host in purpose to reduce extinction risk (Bush and Kennedy 1994). Alternative host choice can be determined by different factors such as i) host ecological similarity - in the need of choice of alternative host, the one that is closer ecologically to principal host will be selected (Timms and Read 1999), or ii) host phylogenetic similarity, when immunological and physiological characteristic of novel host determines host choice (Combes 2001) and iii) alternative host availability, based on host density, the more abundant is the host,the more stable food source it will be (Grenfell and Harwood 1997). Parasites developed mechanisms to feed on their host and hosts developed mechanisms to avoid the parasites. Some of them developed even immunocompatibility with some taxa (Moller et al. 2004). For parasite tt is important to chose suitable host because his survival, development and reproductive

2 success depends on it (Krasnov et al. 2002). Especially that blood properties vary between different hosts and what comes with it also nutritional value (Harrington et al. 2001). Costs of feeding play also very important role such as digestion cost, locating host, piercing and feeding costs (Poulin 2007) as well as immunological response of the host (Krasnov 2008). If principal host is absent, it might be sometimes beneficial to feed on genetically distant host as it probably posses very weak immune response (Krasnov et al. 2006), contrary to common host which during coevolution processes developed strong immunological response against its parasites (Khokhlova et al. 2004).

According to the niche in which we can find parasites they belong to i) field ectoparasites; which are free living parasites that stay on the body of its host just during feeding time, or to ii) nest ectoparasites - depending on the hosts roost, if roost is not suitable, whole parasite population can extinct, even if host is available and iii) host depending ectoparasites - parasites permanently staying on host body. Also depending on association with their host we can distinguish specialists and generalists parasites. While specialists can survive, develop and reproduce on one host species mainly, generalists can infest many different hosts (flea Ceratophyllus gallinae – ca 80 hosts; Tripet and Richner 1997). Host specificity plays important role in biological control and is described as dependence of parasites life cycle, survival and reproduction success on certain host species. Dick (2007) described specificity as a degree to which parasite species occurs in association with host species, if 5% or more host individuals are infested we can call them specific hosts (Tripet et al. 2002). Such parasites posse usually very narrow amount of host species (Poulin 1992) and evolution in such cases leads to specialization towards those hosts (Combes 1991).

According to Ward (1992) host specificity evolved due to i) host characteristics (ecology, physiology or/and biology) demanded from parasites very specific adaptations, ii) specialization on enemy free - space (predation or competition) and iii) availability of mating partner (Rohde´s hypothesis). Combes (1995) describes many costs connected with being specialists such as a) risk of extinction or impoverishing of host species, b) poorer niche space or c) elimination of parasite by immune system of host (Red Queen hypothesis ) and what’s is more d) risk connected with coincidental feeding on non - specific host to which parasite is totally not adapted. According to Sasal et al. (1999) specialists host seems to be bigger in size than in case of generalist hosts. In principe bigger host has usually more stable life style and ecology and provides better condition for development and reproduction of parasites. Holmes (1973) and others claim that host - parasite evolution promoted specialization to particular host species which can lead to becoming better competitor. However such evolution track has many disadvantages. Mayr (1963) and Poulin (2007) have noted that specialized parasite posses usually lower fitness on non-specific host. What is more such evolutionary pattern claims to be a “dead end”. Specialization develops to overcome host responses but it can be irreversible (Thompson 1994; Poulin et al. 2006). Nevertheless most parasites are specialists (Agosta et al. 2010). In many cases specialists can become generalists while switching

3 host which could be both with a reason or can be totally coincidental. Once new conditions appear, specialists may survive such a switch or not. If specialists survive, we are dealing with ecological fitting, thanks to parasite adaptation capabilities (Agosta and Klemens 2008), shared characteristics with host that is already known for that parasite (Futuyma et al. 1995) or host was already know to parasite ancestors (Futuyma and Mitter 1996). Within specialists and generalists we can also find “faux specialist” and “faux generalists” (Brooks and McLenan 2002), where ecological factors restrict them to a few or a single host: local microclimate, non-concordant distribution or competition, both of them can switch host when needed (in case of extinction, lack of mating partner etc.).

In parasites both genetic and geographic pattern differ in generalists and specialists. Specialists with lower host range are more likely exposed on extinction as they are usually locally adapted and mostly also to a single host species, that’s why they often try to re-colonize. Events such as genetic drift in specialist parasites decrease within population genetic diversity. Parasites are able to survive an absence of their host and posses usually high within-population genetic variation which was reported in ca. 90% of parasites populations.

Bats as a hosts differ in their according to different species in behaviour, roosting places, hunting strategies, morphology and physiology. Especially in a case of ectoparasites, roosting strategy play very important role in ectoparasites loads and accuracy. Some bat species live in more stable roosts both natural and artificial e.g. caves – R.hipposideros , hollow trees or bat boxes - N.noctula and M.bechsteinii or attics – M.myotis ) or temporary such as foliage or barks (e.g. Lasiurus cinereus ). Some bats stay in one roost for most of the summer (maternity colonies of urban bats e.g. M. myotis, Pipistrellus pipstrellus ), some switch summer roosts few times during season (e.g. solitary males - M. daubentonii or forest-dwelling bats as N. noctula ). Social organization of bat species has an impact on occurrence of ectoparasites (Kunz and Fenton 2005). Wingless parasitic insects posses limited migration abilities contrary to actively locomotive species which transport does not depend on movements of its host (e.g. Gandon et al. 1996). Specificity index (SI) was developed to describe host-parasite association and portrays the proportion of each individual parasite on particular host. Wenzel et al (1966) divided parasites on mono- and polyxenous. In principle bat parasites are considered to be host specific (e.g. Maa 1965). Exclusively on bats were found Nycteribidae, Streblidae, Polyctenidae, Ischnopsylidae and Acarina (Maa 1965; Usinger 1966).

Phenomenon such as allopatric host distribution (isolation of the host) takes place when parasites can have access to any other host, but developed during coevolution morphological, physiological and behavioural adaptations makes it later impossible to occupy any other hosts (Brooks and McLennan 1993). For example most bat flies like nycteribiids and streblids are highly bat specific (Scott 1925; Maa 1965). In those cases ecological isolation of host increases specificity level of parasite (e.g. Krasnov et al. 2007).

1.2 Reproductive isolation in insects – inter- and intraspecific matings

Driven by evolution pressure males of all species seek to spreading their genes and being sure that their sperm will be use to produce offsprings. We can divide mating on intraspecific, when mating between individuals of the same species takes place and interspecific while individuals from two different species/subspecies/ or ecotypes mate. It is clear that exists some physiological or/and behavioural interactions between females and males from different lineages especially in case of intraspecific matings e.g. mating with heterospecific males can be costly for females. According to Muller (1939) reproductive isolation between two subspecies is a result of genetic discrepancy. Separated subspecies or lineages developed adaptations to particular environment or host and become divergent, sometimes also at reproductive level. Natural mechanisms were created to avoid hybrids that can be less flexible in their adaptation abilities or unfertile in order to avoid waste of reproductive material and food. Reproductive isolation can be as a result of many factors such as different morphology, isolation by distance or different evolution history. Eberhard (1985) claimed that male genitals were evolved under the pressure of adaptation to females and were created in coevolutionary male - female genitalia processes. Morphological evolution of genitals shown that female genitalia remains relatively invariant (Eberhard 1985). Many authors describe male - female coevolution but also arms – race, as females have more disadvantages, and covers more costs connected with mating and fertilization, maybe therefore males have much higher genitalia divergence. Three main hypothesis describes genitalia evolution : i) pleiotropy hypothesis describing genital evolution as a result of evolution of genetically related traits (Mayr 1963), ii) sexual selection, claiming that variations in males genitalia were leaded by sexual selection (Eberhard 1985) and iii) lock and key hypothesis reffering to mechanical remedies which leaded to pre-insemination isolation (Dufour 1844). In most cases females have choice and can control usage of the sperm (Eberhard 1985). Succesful copulation with female doesn’t mean that particular male will become a father. Choice of partner is driven by sperm competition and recognition, in purpose to save gamets organisms avoid heterospecific mating.

On Earth many evolutionary processes takes place in maintaining biodiversity. One of them is speciation which leads to isolation of the populations. Different barriers can cause split of genetic pool in populations. Between closely related taxa many barriers exist, both prezygotic (pre-and post copulatory) and postzygotic which are mostly followed by geographic isolation. Prezygotic barriers are claimed to be stronger and having bigger imact on taxa isolation, it takes place before zygotes are created and reduces gene flow (Ramsey et al. 2003). Prezygotic mechanisms prevents mating to occur or even prevents meeting of two organisms. Within this prezygotic mechanisms we can mention: 1) habitat isolation – organisms are living in totally different, usually distanced niches, they do not meet in natural environment so are reproductive isolated, 2) behavioural barrier – organisms ignore each

5 other due to different behaviour e.g. mating rituals, signals, outer look, different pheromones produced etc., 3) temporal isolation, while organisms posses different mating times, seasons that do not overlap, 4) mechanical isolation e.g. genitals are incompatible or 5) gametic, where no fertilization takes place even if mating occurred.

(1) Many laboratory experiments were successful to mate two species in which mating was not possible under natural conditions (in their natural habitat), which leaded to the hypothesis that lack of mating in nature was result of ecological or behavioural differences between two species. Sailer (1954) shown before in case of interspecific matings within Cimicidae family (stinkbugs), that while giving bugs a free choice, they will mate 17 times more readily with females of their own species. According to Dobzhansky (1940) evolution starts at the level of divergent population which evolves later to independent races and in the end leads to development of new species. When individuals mate only within their selected habitat and is isolated from other populations, it cause barriers in gene flow and can lead to mating incompatibility. As it was shown before bat and human associated bed bugs differ on their evolutionary history. Research by Balvín et al. (2012a) based on morphometry and COI has shown that on 21 different haplotypes both lineages were sharing just 1 haplotype (H2) which proven that they create two different clades. What’s more detailed research based on mtDNA and microsatellites has confirmed that they are two different host races (Booth et al. 2015). Both lineages were never found together in one roost (Balvín et al. 2012b). Reduced mating ability between individuals from different populations can be result of habitats isolation (Bush 1969, Feder et al. 1994). Also lack of immigrants from different populations can create barriers between them including mating barriers (Nosil 2007).

(2) There is many aspects of behavioural barriers causing no mating. Best known are mating signals e.g. birds (Baker 1991), frogs (Gerhardt 1994), but one of the most diverse in insects e.g. Drosophila (Tomaru and Oguma 1994), grasshoppers (Ingrisch 1995), crickets (Otte 1994) etc. In case of territorial signals only small differences can occur between advertising and aggressive signals (e.g. territorial cricket Boake 1983). Other quite well know reason can be also difference in pheromones secretion which can be not attractive for opposite sex of the other lineage. Most of pheromones seems to be host specific and creates barriers for interspecific matings. Additionally, olfactory experiments on lycosid spiders have shown that substances secreted by one the species excites just males of the same species (Hegdekar and Dondale 1969). It is well known that bed bugs posses different chemoreceptors and pheromones to communicate. They share informations about their mating status - mated/unmated in a case of females (Siva-Jothy and Stutt 2003), produce aggregation pheromones (Levinson and Ilan Bar 1971), alarm pheromones (Marx 1955, Levinson and Ilan Bar 1971 ), nymphal pheromones to avoid traumatic insemination by immature individuals (Harraca et al. 2010). Nymphs of the common bed bug ( Cimex lectularius ) produce anti-aphrodisiac defence against conspecific males. Important role are playing here as well pheromones that stimulate

6 sexual behaviour by attracting opposite sex for mating. Pheromone secretion and response can differ between isolated populations as it is in e.g. Coleoptera where both sexes has shown higher response on pheromones produced by individuals from their own locality or even discriminated individuals of the same sex geographically distinct (Lanier 1966). Discrimination is even stronger where male adaptative hybridization is likely to occur e.g. Drosophila pseudoobscura and Drosophila persimilisi. Such discriminatory aspects can be physiology or morphology of other sex like wings pattern in Calopteryx maculate and X. aequabilis . In many other experiments e.g. on moths ( Helicoverpa armigera ) post-mating barriers were found – despite mating no offsprings were produced or produced hybrids were unfertile.

(3) Temporal reproductive isolation was recorded both in fauna and flora, best example is flowering time in plants (Levin 1978), amphibians breeding seasons (Blair 1941) or breeding in insects (e.g. Smith 1953). Breeding time can differ depending on many mechanisms. It was observed before that reproduction of the bed bugs is correlated with reproduction of their host (bats case). Obviously such pattern will differ between bat host who breeds and creates colony once a year contrary and humans which as a host are contaniousely available. Differences in reproductive timing among populations, or “host races”, were reported before in populations that are isolated over geological time scales (e.g. Clausen et al. 1940). Research on crickets ( Acheta veletis ) has shown that populations of those insects in Northeastern US are seasonally isolated (reproduction timing and overwintering stages; Alexander and Bigelow 1960) and even laboratory breeding was unsuccessful, despite identical communicative sounds. Similarly populations of moth ( Inurois punctigera ) in colder climates are divided for early or late flight periods (Yamamoto and Sota 2009). Next to breeding time other aspects such as postdiapause eclosion time can play important role in population isolation and divergence in insect interactions, even if they belong to the same species (Smith 1988).

(4) Genitalia incompatibility can reduce insemination and gametic incompatibility (Palumbi and Metz 1991) can cause a problem for sperm to fertilize eggs (Lepidoptera, Dopman et al. 2009). In Heteroptera some mechanisms can change shape and/or anatomy of mating organs e.g. asymmetry occurs in males (Ludwig 1937) but more often in females, that determines mating ability and shapes mating preferences. It was proven that shape, parameters of copulatory organs can be an issue in case of interspecific mating (Orius spp., Shapiro et al. 2010). Other reason can be hybrids inaccessibility by for eaxmple early mortality, which reduces existence and survival of hybrids in natural environment. Local males often adapt to local females (Baker and Shore, 1995). Even before Eady (2001) in case of between populations matings has shown higher females preferences of sperm of males adapted to the local females. Two investigated lineages of bed bug are geographically isolated, what is more they were never found in one shelter (Balvín et al. 2012a). Costs of inbreeding or local incompatibility can be the reasons of such response of bed bug females on mating.

(5) Despite mating and fertilization process, sperm still may fail to fertile ovaries. Moreover Mashiko (1992) has proven that even in ovaries sperm selection takes place and that may affects fertilization process. Carayon (1966) did find duplication system in Leptocimex duplicatus , Cragg (1912) did find also double spermaleges in Cimex lectularius (D-females), also D-females were found in cases of Cimex hempiterus (Ludwig 1937) and other species ( Stricticimex brevispinosus ) has shown another abbreviation – non-hemocyclic insemination system (Carayon 1959, 1966). In case of some insects sperm from heterospecific mating is not effectively transported to the storage organs or is transported but is not used and in the end gives non fertilized eggs or no eggs at all (Katakura and Sobu 1986). Wade et al. (1994) has published another prezygotic (postcopulatory) isolation in two beetle species ( Tribolium castaneum and T. freemani ) in which offsprings come from conspecific males. Postcopulatory, prezygotic isolation also favouring conspecific males was found in ground cricets ( Allonemobius fasciatus and A. socius )(Gregory and Howard 1993) or grasshoppers ( Podisma pedestris ) (Hewitt et al. 1989) and Chorthippus parallelus (Bella et al. 1992). Tyler et al. (2013) has discovered that while field cricket was mated with both conspecific and interspecific partners, produced offsprings were from conspecific males. In many cases females preferred conspecific males (Shapiro 2001). Because fertilization takes place in females body, she is able to regulate fertilization process and usage of sperm (Knowlton and Greenwell 1984). Eberhard (1996) has pointed at many possible ways that females may use to get rid of sperm of certain male, such as sperm digestion - females can use sperm as nutrition source, rejection of sperm or control of ovulation. Additionally females evolved many post-copulatory mechanisms to avoid being fertilized by genetically incompatible sperm. Such process was called sperm choice (Zeh and Zeh 1997) or cryptic choice (Thornhill 1983). One of the mechanisms of cryptic sperm choice by females can be muscles control of undertaken sperm (Hall et al. 2001). In process of sperm choice female can even discriminate sperm of certain male (Birkhead 1998) e.g. grasshoppers (Bella et al. 1992). Studies done on insects did proof that female genotype affects fertilization. According to that males are able to successfully fertilize just females with certain genotype (Wilson et al. 1997). Incompatibility in female reproductive system can inhibit sperm transfer to ovaries.

If in some way organisms could overcome prezygotic barriers second defence line appears - postzygotic barriers such as hybrids viability, fertility or breakdown when fertile hybrids are created but with each next generation individuals are weaker and their fertility decreasres.

Nevertheless if hybridization occurs it can lead to creating new species (e.g. in plants ca. 22%) (Rieseberg 1997), but still some conditions must be met as haploid genomes from both parents must be able to produce a new individual. Offspring organism must produce viable gamets and reproductive isolation must exist between hybrids from their parental individuals. Hybrid must be capable to mate with other organism, if successful and new, stable population could be created than creation of new species can take place. In case of ground cricets both inter - and intraspecific males fertilize egg but conspecific male fertilize much more eggs (Larson et al. 2012). Many papers before shown favour of

8 sperm of own species not only in insects but also other invertebrate species (Howard et al. 2009). Post-mating isolation in Bombus bumblebee with very low amount of hatched eggs in hybrids from Bombus hypocrita sapporoensis and B. hypocrite hypocrite . Interspecific matings are costly for females, it cause ca. 50% offsprings reduction, females fecundity can be restored. Research on crossing between even closely related species gives in the end no eggs or very few eggs - reduced oviposition as it was shown in flour beetles (Wade et al. 1994) or in ground crickets (Gregory and Howard 1993).

Mechanism of postzygotic isolation was found in Argentinean populations of A. cajennens e, expressed by the small number of hatched larvae as a result of cross matings between females and males from different ecological regions (Mastropaolo et al. 2011). Differences in sperm morphology can occur even within same species (such as mobility, flagellum length etc.). In case of sperm selection it occurs right after insemination takes place. Negative correlation between sperm components or sperm phenotype may influence fertilization probability (Parker and Begon 1993). Research made on sperm variation (molecular and genetic analysis) has shown that it vary for e.g. in protein components between different populations of the same species. Also other ejaculatory components can induce different mechanisms such as oviposition (Eberhard 1996).

Cimicids such as C. lectularius and C. pipistrelli group belongs to ectoparasites of higher importance and were before tested on possibility of successful mating between species. Cimicids females were shown to has high diversity in the paragenital organs (Carayon 1966). Seems that structure of mating organs is very variable and it is hard to regenerate coevolution of mating organs. Eberhard (1996) has hown that in case of sperm competition variation of female reproductive system influences the success of fertilization by certain male i.e. bruchid beetle (Wilson et al. 1997), marine ascidion (Bishop 1996). In many cases we can observe sterility in hybrids (Graham et al. 1978). In a case of Lygaeidae (Heteroptera) hybridization experiments were not successful or gave very low percentage of hybrids, unfertile eggs, high mortality and unfertile hybrids (Eyles and Blackith 1965). Possible that both divergent lineages - human and bat associated developed different mating systems, diffrent sperm morphology or even whole mating organs and that is the reason of unsuccessful crossing trial. Especially that it was confirmed that they differ at morphological level (Balvín et al. 2012a) which can include also mating organs.

Other possible explanations of reproductive isolation in cimicids could be endosymbionts, which can influence both pre- and postzygotic mechanisms, which was first confirmed in bed bugs already long time ago (Arkwright et al. 1921, Usinger 1966). One of them is Wolbachia, common invertebrate’s symbiont (Hypsa and Aksoy 1997, Rasgon and Scott 2004). It infects around 70% of all insects (Jeyaprakash and Hoy 2000). It was stated that it may play crucial role in bed bugs biology and ecology, for example influencing obtaining of the vitamins (B) that they cannot get from blood meal (De Meillon and Golberg 1947). Wolbachia is also responsible for many reproductive modifications in insects and at different levels causes cytoplasmic incompatibilities, between sperm

9 and egg. One of the example is post-mating incompatibility which was stimulated by Wolbachia in parasitic wasps in crossing between Nasonia giraulti and N. longicornis (Seth et al. 2000). It was shown in Hemiptera ( Orius strigicollis ) that it stimulates infertility between infected males and not infected females and also between individuals infected by different strains of this bacteria (Watanabe et al. 2011). According to Fleur and Wedell (2006) it decreases sperm production and reduces sperm competition in non-virgin males. This symbiont seems to has a big impact on mating abilities. Some researchers did claim that it can be even used to control population densities of medically important insects such as bed bugs. It was already proven before that presence of Wolbachia has impact on crossing between Cimex lectularius and C. columbarius (Jenys) even when antibiotics and treatment with heat were used (Chang 1974).

Both prezygotic and postzygotic mechanisms ensure that only the best adaptations and characteristics must survive and develop further, working against spreading “bad genes” that makes populations/species weak by for e.g. reduction of hybrids survival if they are not able to survive in parental environment (Egan and Funk 2009).

1.3 Host transmission and genetic structure of bat ectoparasites

Ectoparasites association can be result of natural or artificial events. In first case it occurs in natural way while ectoparasite itself colonizes the host or it is transferred by one of the host to another. Second is caused usually by artificial colonization - contamination by humans e.g. in lab, on clothes etc. and is not initiated by ectoparasite itself. Ectoparasites in comparison with endoparasites possesses much higher host switching ability. Host switching is an important aspect of ecology, evolution, epidemiology. Knowledge about this phenomenon is a key to understand many complicated processes occurring in ectoparasites ecosystems. It can be interspecific, when ectoparasite is transmitted between different species or intraspecific when transmission takes place among hosts of the same species. In maternity colonies vertical (and intraspecific) transmission is observed between parents and their offsprings in nests or roosting places, but also horizontal (intra and/or interspecific) between adult individuals (Dubinin 1947, Eichler 1963). Interspecific, between species transmission can influences virology and transmission of some ectoparasites as different bat species often share roosts and maternity colonies. In both cases knowledge of host ecology and behavior is needed to estimate transmission level. The higher dispersion in ectoparasites, the higher within-population genetic diversity (Barrett et al. 2008). Ectoparasites transmission depends on many factors but one of the most important is host mobility and contact with other potential hosts. Contact between potential hosts is usually variable and depends on population or individual behavior (Anderson and May 1991). Nevertheless contact not always means transmission, especially when we talk about species specific ectoparasites. Sociality in host can mean also opposite, ectoparasite number may decrease by intense grooming behavior. In some social groups we have both inter and intraspecific contact e.g. one nest, one niche, roost may be visited or occupied by different individuals of same or other species. What’s more social system differ even among same species depending on individual age, position in hierarchy, space use, time of the year. Isolation of host groups shows in many cases low gene flow between host races (e.g. Ixodes uriae , McCoy et al. 2001). Nevertheless depending just on observations and already gained knowledge is not enough to confirm or decline transmission event. Useful tools in estimation of level of ectoparasites transmission are genetic analysis, which helps to discover dynamic both in ectoparasites and their hosts. Low or lack of genetic differentiation is the best proof that transmission of ectoparasites takes place. Gene flow between host populations is possible thanks to hosts movements and if host of different species, especially in case of specialized ectoparasites never or rarely meet, it leads to genetic diversification. Host- ectoparasite life history has an impact on such behavior (Nadler 1995).

There are many examples of genetic research made on flying animals that can carry ectoparasites on far distances. Research covers both vertical and horizontal transmission.

During research on a bat from genus Pteropus and it’s bat flies – Cyclopodia horsfieldi , where 8 species from different colonies in Malaysia, Vietnam, Cambodia were investigated. Molecular variance did show low genetic variation between species and area, witch proves interspecific transmission (due to sharing roosts even distant one) (Olival et al. 2013). Other research made in Central Europe on Myotis myotis and its ectoparasite Spinturnix myoti and M. bechsteinii and its ectoparasite – Spinturnix bechsteini has shown that first of them has high genetic diversity – no differences among bat populations, while second ectoparasite possesses low genetic diversity with high differentiation among bat populations (van Schaik et al. 2014). Those differences were explained by host ecology and behavior, particularly social system – within and among colonies exchange in M. myotis during roosting, in maternity colonies and during mating.

Other example of both vertical and horizontal transmission are long-tailed jaegers, arctic jaeger ( Stercorarius parasiticus ) and long-tailed jaeger ( S. longicaudus ) breeding in the High Arctic archipelago (Svalbard) and their feather mites from genus Alloptes and Zachvatkinia . Mites from genus Alloptes had morphological similarities on both bird species but high genetic differentiation (new criptic species from genus described). Reasons for such pattern was easy to predict as both jaegers species contact with each other, and Zachvatkinia sp . is much more transmitted between individuals because it occurs on more external regions of the feathers and are easier to be exchanged (Dabert et al. 2015) between contacting birds.

Other case - lices are mostly transmitted from parents to offspring (vertical transmission) than to other individuals, especially from other species. Genetic research conducted by Levin and Parker (2013) revealed that frigate birds ( Fregata minor ) and Nazca boobies ( Sula granti ) living on islands of Galapagos Archipelago were differentiated genetically but their flies Olfersia spinifera and Olfersia aenescens did not (common haplotype). In such situation non-breeding movements hypothesis was proposed, or alternative host is responsible for spreading ectoparasites across islands and birds colonies. They went also into conclusion that adult birds are philopatric but juveniles that could also carry flies were observed to visit often other sites. Similar research on Galapagos on hawk ( Buteo galapagoensis ) and its lice Degaeriella regalis was carried out by Koop et al. (2014). Hawks did have high genetic differentiation among birds populations from different islands, and lices as well. Koop et al. (2014) explained it as typical vertical transmission, as genetic differences occurred also between individuals. Author summarized birds as parasites islands.

Parazitation changes seasonally when breeding, mating and hibernating. Côté and Poulin (1995) stated that physical contact or close spatial association are required for ectoparasites transmission. Bat ectoparasites differ at mobility level, most common in maternity colonies are e.g. fleas, mites (Pearce and O’Shea 2007). Christe et al. (2000) noted vertical mite transmission mother- offspring, whereas fleas mostly undergo their cycle on substrate and both of those ectoparasites are transported to hibernation sites (Czenze and Broders 2011). Bats maternity colonies deliver favorable, stable microclimate conditions (Willis and Brigham 2007) and both mothers and juveniles suffer highest parasitation (e.g. Czenze and Broders 2011, Christe et al. 2007, Christe et al. 2000, McLean and Speakman 1997). Juveniles are always much more parazitated due to weak immunological system and lower grooming abilities (Christe et al. 2000). Males that live usually solitary posses very reduced ectoparasites number as they roost mostly far from maternity colonies (Christe et al. 2003, 2007; Kunz and Lumsden 2003). Swarming behaviour in bats which combines mating and acquainting juveniles with hibernating sites (Fenton 1969) gives bats opportunity to contact with conspecifics. During that behhaviour also ectoparasites exchange can take place. In mites Spinturnix bechsteini , no genetic structure was found which means they are exchanged freely between different bats colonies (Bruyndonckx et al. 2009).

Genus Cimex is often transported on a large distances between bat roosts (Balvín et al. 2012b). They found no genetic differences among bat species based on COI (18s, 28s ribosomal genes). Research on genetic structure and gene flow within and between populations based on mtDNA, microsatellite loci and knock-down resistance gene variants shown lack of gene flow between host associated populations, within human associated populations higher inbreeding and lower genetic variability was observed (Booth et al. 2015). Many research on C. lectularius proven also low genetic variability based on microsatellites (e.g. Fountain et al. 2014, Akhoundi et al. 2015). Morphometrical analysis of bat and human associated C. lectularius combined with genetic analysis of COI and 16S genes, despite morphological adaptations toward bat and human host indicated lack of genetic variability and ended up with host races theory (Balvín et al. 2012a). Morphological research on C . pipistrelli and genetic analysis (COI, nuclear loci, 18S and 28s ribosomal genes) shown 27 mitochondrial haplotypes in 2 distinct haplogroups but still low genetic variability between different bat species (Balvín et al. 2013).

Presence of morphological adaptations towards different hosts as humans and bats combined with low genetic variation between those two host associated bed bug races is best proof that they originated from common ancestor and are result of host switch in far past. Both bed and bat bugs posses limited mobility abilities and their transport depends on their hosts. Seems that despite wide range of occurrence, wide range of occupied bat host species and in many cases isolation by distance of different bat bugs population, we have found that C . pipistrelli shows low genetic variability, meaning somehow gene exchange between different populations and host species takes place.

1.4 Traumatic insemination as mating strategy

Darwin (1871) and the others have described that selection in organisms is driven by sexual selection and sexual conflict. External environment, diseases and opportunistic microbes (Endler 1986; Reinhardt et al. 2005) by injuries during mating can infect female or even cause sperm damage (Otti et al. 2013). Traumatic insemination (TI) is quite unique method of mating known in some organisms. It involves piercing of female abdomen with males hypodermic genitalia and injecting sperm directly to female’s body cavity (Carayon 1966).

Genitalia can create different forms from needle-like to sclerotized sinus or tube. This method of mating is most common in Hemiptera (Arnqvist and Rowe 2005), where at least 7 families mates that way. Despite the fact that this type of mating was considered as costly for females it is quite well known phenomenon which is discovered lately in many other groups of organisms (e.g. Strepsiptera, Beani et al. 2005), spiders Harpactea sadistica (Rezac 2009) and fruit flies ( Drosophila spp., Kamimura 2007). In Drosophila bipectinata males organ is reduced aedeagus and in a case of Harpactea sadistica it is embolus, but all of them cause wound in female abdomen during copulation. Such mating takes place also during homosexual copulation i.e. Xylocoris maculipennis from genus Afrocimex where both females and males posses ectospermalege.

Dominant group in which TI evolved three times independent is intraorder Comicomorpha. At least once it evolved in the superfamily Cimicoidea. In Anthocoridae spermalege shows variation in morphology in different taxa. Polyctenidae shows primitive female organs with lack of spermalege. In Lyctocoridae male mating organ is even more primitive and true ectospermalege in females doesn’t exist. Independent evolution of TI is suspected in Plokiophilidae where females are equipped with paired copulatory tubes and owns non-functional vestigal spermatheca. Nevertheless the first time TI was described and observed in species from Cimicidae , namely in Cimex lectularius (Fig.1.). In this species male pierces females abdomen by needle-like copulating organ and inject sperm to mesospermaleges. Mesospermaleges activate sperm by fluids present in sperm and ca 4 h after mating sperm leave mesospermalege and via hemolymph travels to sperm storage organs - seminal conceptacles. Later threw the canals in the oviduct walls sperms reaches ovaries and fertilization takes place. Bed bugs were used since long as a model to study TI. Structure of mating organs in cimicids is very variable. It is well known that genitalia incompatibility can reduce insemination and cause gametic incompatibility (Palumbi and Metz 1991), and even create the problem for sperm to fertilize eggs (Lepidoptera; Dopman et al. 2009), especially sperm is injected to the wrong place. In case of some insects sperm from heterospecific mating is not effectively transported to the storage organs or is transported but without effects (Katakura and Sobu 1986). In bed bugs sperm must get to mesospermalege as it is responsible for activation of fertilization process (Davis 1965), sperm injected in any other place after getting to haemocel cause high mortality after such copulation and of course fertilization failure. Moreover in females body there are two main

14 types of haemocytes, one type absorb seminal fluids, second is responsible for digesting spermatozoa. Presence of seminal fluid, phagocytes and spermatozoa in mesospermalege were suggested by Berlese (1898) to be used for sperm digestion. Absorption of sperm would deliver female some nutritions (Morrow and Arnqvist 2003). It was proven first time by Harrison and Mellanby (1931) that mating by TI decreases female lifespan due to wounds and pathogens introduction. Nevertheless it was also proven that spermalege minimalize costs of mating, ectospermalege restricts damage and mesospermalege reduce infections. It was already investigated that mostly in case of female in TI copulation increase risk of infection (Kamimura 2007). Otti et al. (2013) reported that in case of bed bug females in copulatory organ and haemolymph is present at least 19 species of bacterias that may hamper sperm viability, while in case of males 3 fungal and 6 bacterial species were described (Reinhardt et al. 2005).

Within organisms mated by TI there is many deviations from typical copulatory organs e.g. in Afrocimex constrictus females can has male form of genitals, also intrasexual mating take place. For solving such situations some sympatric sister species evolved genital incompatibility to avoid such matings (e.g. Coridromius tabitiensis and C. taravao , Tatarnic and Cassis 2013).

2. Model organisms

2.1 Bed bug – Cimex lectularius

Cimex lectularius belongs to Cimicidae family with ca 110 speciesand 24 genera (Henry 2009). All are haematophagous insects (Reinhardt and Siva-Jothy 2007). Bed bugs are directly and indirectly connected to public health. They show worldwide resurgence, keep expanding and are closely associated with human and their shelters. Size of adult individual range between 6 – 7 mm body length and has characteristic odor (Harlan 2006). Body is dorso-flattened with 4 segments on antennas and 3 segments on labium (Usinger 1966). They are thigmotactic, avoiding light insects. Female body is more round while males body is more pointy, they are wingless but posses wing pads. People usually confuse them with beetles or cockroaches, thinking they are just harmless insects. Bed bugs are hemimetabolous insects (Elzinga 1987) with 7 life stages: egg, 5 nymphs and adult. They are really resistant to different temperatures (can survive -15°C), but some temperatures for longer period of the time can be for bed bugs deadly (e.g. 43°C Usinger 1966). Rivnay (1932) estimated that the best humidity vary between 10 – 70% RH but for successful eggs hatching humidity should be definitely higher, the best relative himidity (RH) was reported between 75 – 90%. Environmental conditions regulate behaviour and development of bed bugs , temperature of 25.5° C is best for colonies, optimal temperature is between 28 – 29°C for bed bugs. Threshold for adults and hatching eggs is 13 – 15°C (e.g. Hase 1930). Bigger females seems to take bigger blood meal and lay more eggs with average of 6 – 10 eggs, 5 – 6 days after taking blood meal. Without blood meal females are not intended to copulate (lack of blood meal for approximately 2 weeks, Mellanby 1935).

Front legs of bed bugs are used to grab skin of the host, their amputation cause no feeding (Usinger 1966). Feeding takes an average 10 – 15min for adult bug to suck fully and after taking meal bug comes back to its shelter. They seems not to have much natural enemies, nevertheless some of them leaded to destruction of whole bug colonies. Some insects hunt on them: Reduvius personatus (Quarles 2007), some ants such as Ploiaria domestica (Johnson 1952), Monomorium pharaonis (Howard and Marlatt 1896), Solenopsis geminara rufa Jerolon (India; Negi 1933), Chelifer cancroides (L.) (Kemper 1936). Some bed bugs predators like spider Steabola bipunctata (L.) which was found by Povolný (1957) can kill whole C. lectularius colony (Austerlitz).

In 20´s century bed bugs had the biggest resurgence ever in history and become most common ectoparasite. To be able to ectoparasite on humans or other organism evolution equipped them in many morphological and physiological abilities. Bed bugs shown aggregation behaviour despite that they do not belong to social insects (Wertheim et al. 2004). Such behaviour gives them higher chance for survival, protection, higher chance for mating, faster development or better microclimatic conditions (Dambach and Goehlen 1999). Bed bugs stay closely to their sleeping host (Gangloff-

Kaufmann et al. 2006) and can easily detect it, using host seeking behaviour (Lehane 2005)- appetitive, activation and attraction phase. Bed bugs can find their host thanks to chemoreceptors and ability to detect CO 2, heat or visual characteristics.

2.2 Bat bug – Cimex pipistrelli

Cimex pipistrelli can be differentiated from bed bug C. lectularius just under the microscope by length of hair on pronotum (Fig.1.). Without microscope they all looks like common bed bug, but they have longer bristles, their eyes are less protruding than from bed bug, hind femur is wider, no differences in internal organs or muscles were found (Goddard et al. 2009). Cimex pipistrelli Jenyns, 1839 group is solely associated with bats and from the other taxa, it is delimited by narrow lateral lobes of the pronotum and cleft but also naked paragenital sinus. The group consists of 10 described Palaearctic species (Usinger 1966; Ueshima 1968; Bhat 1974a,b). From Western Palaearctic region three main taxa were described ( C. pipistrelli, C. dissimilis Horvath 1910 and C. stadleri Horvath 1935) and one with hazy taxonomic status ( C. pipistrelli form Singeri, China, 1938). Interpretation of taxa appurtenance is since long a matter of dispute, some claims described taxa are subspecific forms (Stichel 1938, 1959; Wendt 1941; Lansbury 1961), and others treat them as three distinct species (Hedicke 1935; Usinger 1966; Wagner 1967). For example Stichel (1959) or Péricart (1972, 1996) treated C. stadleri as synonymous species to C. dissimilis .

Laboratory crossings between Cimex pipistrelli group from the British Isles and those of Czechoslovakia proven existence of reproduction barrier and supported the existence of at least two separate species in Europe (Usinger 1966). Analysis of samples from C. pipistrelli group from Central Europe (Czech Republic, Slovakia, n=77) based on maximum parasimony (COI) emerged two haplotypes described as A and B (Balvín at al. 2008). What is more each of the species from Cimex pipistrelli group was found to posses different host ranges (e.g. Usinger 1966; Péricart 1972). Populations of Cimex pipistrelli were found to be morphologically differentiated among different bat host species (Balvín et al. 2013). Smaller and less hairy individuals were found from Pipistrellus spp. Nevertheless mtDNA (COI) and four nuclear loci did not shown structuring or reflection of morphological adaptations according to different host species. Cimex pipistrelli group is only one species with high phenotype plasticity. Nyctalus and Pipistrellus spp. are common host of this species (Povolný 1957).

Fig.1. Comparison of pronotum hair between bed bug (A - C. lectularius ) and bat bug (B- C. pipistrelli ). Hair on pronotum of bat bug are longer than width of eye. (picture O. Balvín)

2.3 Human and bats as bed bug hosts

C. lectularius is a pest of global importance (Eddy and Jones 2011). Bed bugs, humans and bats share life end evolution history for a long time (Usinger 1966). According to Sailer (1952) and others, bats were primary host of bed bugs and switch to humans took place at the prehistoric times when both hosts were sharing their roosts, mainly caves. Ryckman et al. (1981) noted that even in far human history people had to deal with bed bugs e.g. Aristotles or Aristophanes. Later, development of humans 8000 – 5000 B.C.: agriculture, villages and other human aggregations caused dynamic bed bugs resurgence. According to Usinger (1966) first record of bed bug comes from England from 1583, since people started to take a closer look at those ectoparasites. In history, there is a lot of examples of coexistence of bed bugs and humans. Not always bed bug was seen as parasitic insect. There are many references about usage of bed bugs in medicine e.g. snakebite, leeches (burning bugs) in Greece or Rome or using mixture of bed bugs with wine, eggs etc. in diseases treatment (Busvine 1976) or even as remedy for malaria (Riley and Johanssen 1938). Bed bugs were always more rife in poor people and servants. In 1800´s they were easily spread and occurred on ships and were often carried among people, finding shelters in their wooden beds with many crevices. In 1900´s discovery of warming houses changed bed bugs life seasonal pattern. It is said that ca. 30% of houses in Europe was infested by this ectoparasite, mostly due to lack of hygiene and overcrowded villages and cities (Potter 2011). In 20´s century expansion of bed bug thanks to communication development was really intense and they started to occur everywhere, not only in human dwellings but also in shops, theatres and restaurants. In 80´s and 90´s people were trying to use different methods to get rid of bed bugs, starting from contact sprays containing arsenic or mercury which were also toxic for the people, threw safer pyrethrum, fumigants, DDT, traps or high temperature (Potter 2008).

Nowedays bed bugs still shows cosmopolitan distribution. In science it is used as a model arthropod to test drugs and chemicals. Despite years of experiments and far-reaching knowledge it is still impossible to create pesticides that will sufficiently help to get rid of those pests. Bed bug is

18 evolving and adapting very fast to new conditions, it spreads for close and far distances by “local dispersion” or “passive dispersal”, it can easily develop huge colony starting from a single, mated female. It harbour relapsing fever, Q fever, tularemie and hepatitis B (El-Masry and Kotkat 1990) and in bats it is a vector of trypanosomes (Gardner and Molyneux 1988). Recent research has shown existence of host races – bat and human associated bed bugs feeding on those two different hosts, they developed morphological differentiation. Humans and bats occupy common shelters (houses, flats etc.). Bats were suspected to be reservoir for bed bugs population recovery (Szalanski et al. 2008).

Humans and bats are hosts of bed bugs nowadays despite that they differ in fact at all levels. What is more humans still seems to be easier and more beneficial host. People live in bigger societies, are practically hairless, living in more enclosed spaces, constitute bigger blood meal reservoir, their blood has stable temperature, they sleep at predictable time and place.

Bed bugs resurgence was promoted by humans behaviour: creating second hand shops, decline in awareness, overcrowding, increased migration connected with tourism, business, improvement of living conditions as central heating , double glazing and global warming (Boase 2008), pest control issues as changing of insecticide products or resistance on them. We can meet them now everywhere: single, multi family houses, flats, schools, hospitals, hotels, theatres, cinemas, public transport (Cleary and Buchanan 2004). Doggett et al. (2012) noticed that more vulnerable individuals suffer from mental problems as insomnia, loss of appetite or social isolation or even depression. There are even cases of post traumatic stress disorder (Goddard and de Shazo 2012). For bed bugs prevention it is recommended to remove cracks from sealing and walls, furnitures isolation from the walls, using metal beds above wooden one, clutter removal and intense vacuuming. Identification of bed bugs infestation can be made by using dish with oil and tape, moat interceptors (e.g. Anderson et al. 2009), or more specialistic canine detection by specially trained dogs (Pfiester et al. 2008). Best known bed bugs infestation treatments are: disposal of infested items, encasements, vacuuming (Frishman 2000), using heat (48°C) (Benoit et al. 2009a), steam, freezing (dry ice) (Benoit 2011)(-17°C), laundering (over 60°)(Naylor and Boase 2010), pyrethroids – syntetic pesticide (Fletcher and Axtell 1993), diatomaceous earth (Benoit et al. 2009b), dichlorvos – organophosphate insecticide (Fletcher and Axtell 1993), chlorfenapyr – pyrrole insecticide (Moore and Miller 2006), fumigation – sulfuryl fluoride gas (Miller and Fisher 2008). Nevertheless the easiest and the cheapest method is personal observation of adults, larva’s and eggs, molted skin or fecal sand characteristic smell.

Bats (with more than 1250 species) are special hosts due to morphological and physiological characteristics as active flight, torpor or hibernation (Altringham 1999). Bats were found between most infested with parasites animals (Moura et al. 2003). As a jost they posses variety of behaviour, morphological and physiological differences, different roosts preferences, might live solitary or create colonies of all sizes, they differe in roost fidelity, they posses fur, they live in enclosed space but only for certain period. Ca. 687 bats ectoparasites are described (Dick et al. 2003) – 4 orders are included:

Dermaptera, Hemiptera, Diptera and Siphonoptera, from those, 6 families are strictly associated with bats. Bats from the ecological and biological point of view constitute perfect ectoparasites hosts. Their roosting microclimat delivers stable and long-lived shelters for many ectoparasites. Roosting place even within single bat species can decide about parazitation loads e.g. warm and cosy attics versus cold and humid caves or mines, it may reduce or increase ectoparasites number, it mating or survival (Marshall 1982; Bartoni čka and Gaisler 2007). In most cases cavity - roosting bats shows higher infestation by bat flies and mites (ter Hofstede and Fenton 2005). M. myotis breeds in caves (southern Europe) and attics (Central Europe) so they are living in different microclimate conditions e.g. higher infection level of Spinturnicidae in M. myotis from caves contrary to M.myotis from attics due to fitness differences in hosts (Uhrin et al. 2010). There is many ectoparasites that are strictly connected with certain bat species because of their roosting place. For example Nycterophilia streblid associated with “hot roost caves” where temperature and humidity is unique. Next to type of the roost and ecology also roost structure, composition of colony (single versus mixed species, one or mixed sexes colonies) or inter-bat spacing and occupancy. That all has impact on ectoparasites composition in bat species. Ectoparasites quantity and quality will be different in bats roosting in trees, foliage, mines (Kunz and Lumsden 2003) and bats roosting in caves and rock crevices with many bat generations using same shelters (Kunz 1982). Roost fidelity promotes parasitic loads and depends on microclimatic structure, safety, exposure and external conditions. Bats posses diverse social system occurring from a single individuals up to groups counted even in millions (Hill and Smith 1984). In many described cases, females have higher infestation than solitary males – e.g. Rhinolophus mehelyi (Sharifi et al. 2003). Grooming and group size has an impact on ectoparasites loads e.g. M. lucifigus and M. septentrionalis (Czenze and Broders 2011).

2.4 Bat hosts

Model bats species used in experiments vary at different ecological aspects and that may play crucial role in bed bugs parasitation, both its quality and quantity. Roosting strategy, foraging behaviour and reproduction can run coevolution bat – bed/bat bug and create one or the other species specific, non-specific or co-incidential host. Bat uses day roosts, night roosts and resting places which can be used few times per night also during unfavourable weather conditions (Kunz 1982). Such roost switching might drive ectoparasites transmission between bats shelters. Bats behaviour or even body size can be connected with spreading bed or bat bugs even on bigger distances. Larger range and body size is usually correlated with more parasites.

During planning our experiments we wanted to chose specific and non-specific bat and bed bugs host species and species that differ both at ecological and physiological level. For genetic research we have chosen greater mouse-eared bat Myotis myotis (big size bat up to 45g), which is the main bed bug host. This bat forages in the forest with sparse understory with preferences towards

20 deciduous forests i.e. beech forests. Colonies are pretty big and occupies mostly attics of the buildings (mainly churches). Males stay separately at the attics, bridges, bat boxes but also hollow trees, bunkers and caves. In middle Europe they create 50 – 3000 individuals per group, more than 90% of females comes back to the same roost in which they were born. Roost fidelity is highest in maternity period (Tuttle 1976) and it increases parasitation level (Kunz 1982). Those big maternity colonies are sometimes mixed with other bat species, mainly M. emarginatus , M. cappacini or M. blythii . Those bats rarely visit another colonies and if yes than just for a short time. This species belongs to regional and migrating bats travelling between 50 and 100km between maternity colony and hibernating place (Dietz et al. 2011). For comparison we have used Myotis daubentonii (medium size bat 6 – 8g) which is specialized in catching aquatic insects from Diptera above the water surface like may flies, caddis flies or Chironomidae (Flavin et al. 2001, Taake 1992). It has flexible foraging behaviour and may hunt as well in a forest as in a parks or gardens but prefers deciduous and mixed forest (Kalko and Schnitzler 1989, Siemers et al. 2001). Opposite to M. myotis it is found in tree natural cavities, bat boxes, bridges etc. It is migratory species that travel less than 150km between summer and winter roost. Females can change their roosts every 2 – 3 days. Materinty colony counts up to 200 individuals (20 - 50 in average)(Greenaway and Hutson 1990). Colonies are found usually in hollow trees, sometimes even bat boxes but also old bridges etc. Males also create small groups. They usually change place every 2 till 5 days but those in the buildings last for longer periods. During the year this species is noted to change colony places even 40 times and hunt in diameter of 6 to 10 km from it (Geiger and Rudolph 2004, Arnold et al. 1998). Males prepare special mating roosts where they mate with females during swarming period (Bogdanowicz 1994). Copulation may take place 15 – 30 min (Bogdanowicz 1994), while exchange of parasites can take place. They were found to create mixed colonies with other bat species e.g. M. nattereri (Bogdanowicz 1983). Nursing females and juveniles posse higher number of parasites what may indicate low or even no grooming behaviour comparing to non-nursing individuals (Bogdanowicz 1994).

Into our rsearch we included also samples from quite big bat - Nyctalus noctula (21 – 40g). This species is suspected to carry bed bugs as most cases where bat bugs were found on bat body comes from this species. Is found mostly in hollow trees but also in bat boxes or in a buildings. Hollow tree roosts are often changed in diameter of 12km. Maternity colonies are usually composed of 20 – 60 females. Males can create groups that occupy hollow trees, ceilings, caves and buildings (up to 20 individuals). Sometimes they share maternity roost with other bats like M. daubentonii or Pipistrellus nathusii . They can hunt even 26km from their roost. Noctule belongs to migratory species that can travel up to 1000km.

In blood feeding experiments both specific and non-specific bat host were used. Bed bugs were fed on bats from species Vespertilio murinus – middle size bat (48 – 64mm) that creates colonies and roosts in tambour cabinets, attics, crevices and buildings, hunts usually at open space, above water

21 ponds, trees, parks. Maternity colonies are composed of 20 – 60 females. Males can create colonies up to 300 individuals. They usually stay up to 7 different roosts. Some populations migrate, longest known distance is 1440km. As well mentioned before greater mouse-eared bat as most common bed bug host and also the smalles bat species used in experimental part - Pipistrellus kuhlii (5 - 8g), synantropic species living in anthropocentric landscape. Hunts in proximity of their roosts, often next to street lights, gardens, parks or water ponds. Colonies occupy buildings – panels, roofs, windows, cracks in concrete, sometimes also bat boxes or hollow trees. Maternities are usually created of 20 – 100 females. They create mixed colonies with Pipistrellus pipistrellus or Hypsugo savii .

In most cases maternity colonies become main parasites reservoir. Knowledge about sucking blood possibilities of bed bugs, their reproduction success and development abilities on non-specific host plays important role in their prevalence. Many bats occupy human shelters or live with them in close proximity. What is more most bats mentioned above and used in our experiments creates mixed colonies both females and also solitary males can meet with other individual of same or other species, they meet during swarming and on hibernation sites in all cases exchange or spread of parasites can take place. It is not coincidence that bigger bat as N. noctula who occupy mostly narrow spaces was found very often to carry cimicids (big body surface and limited grooming abilities). Same as not surprising that M. myotis who creates big maternity colonies is reported to be most infected bat species (good microclimate, weaker immune response during maternity period, high roost fidelity). Smaller bats as pipistrells were found to carry bugs very rarely (small body surface, less stable microclimatic conditions in roosts).

2.5 Some hematological aspects of hosts – specific versus non-specific bat host

Haematophagy evolved some 145 – 65 milion years ago and at least 6 times among arthropods. Blood is very nutritional food that helps to produce eggs and provide longer life expectancy mainly thanks to hemoglobine (Hb). First fossils of parasitic insects comes from half of 18 th century (Bloch 1776) and belongs to Aedes ciliaris L. (Quaternary), Cenozoic lice (Dubinin, 1948, Syberia). Some claims that primary host of blood sucking Diptera belonged to reptiles (Downes 1978). Ectoparasites that feeds on blood originated in Early Cretaceous while mammals emerged in Late Triassic (Carol 1993). In Early Cretaceous vertebrates such as birds or mammals become more common and diverse and so available for parasites. Waage (1979) has desribed that insects initially were plant-sucking and by coincidental bite a vertebrate and developed towards feeding on them. In hemipterans parasitism occur in families Cimicidae, Polyctenidae, Reduviidae and also Lygaeidae. Others suggest that they were originally predators (Cobben 1978). Some insectivorous or phytophagous Hemiptera still bite humans (Myers 1929, Usinger 1934). Haematophagous arthropods posseses number of adaptation for sucking blood. They suck blood

22 directly from the vessel or from haematoma. Blood - feeding insect has piercing and sucking apparatus and within them we can distinguish: lice (Anoplura), true flies (Diptera), fleas (Siphonoptera) and true bugs (Heteroptera). In other groups such apparatus occurs sporadically e.g. moths ( Calpe eustrigata ) (Bänziger 1968). Salvia of parasites has potent immunogens adapting immune response of the host and avoid haemostatic defenses (Ribeiro 1995), what’s more salvia contains components promoting vasodilatation and in C. lectularius it contains additionally nitric oxide (NO). Immune system of the host response on ectoparasite by activating antigen - presenting cells, lymphocytes B and T, complements, antibodies, mast cells, granulocytes and cytokines (Jones et al. 1996). Cimicids midgut contains special digestive cells responsible for absorption and secretion of enzyme, and endocrine cells are playing endocrine function, also regenerative cells are responsible for replacement of ephitelium (e.g. Chapman 1985). Blood insects were divided into different clues depending on blood components they feed on: clues associate the cellular fraction – most blood sucking insects, they suck whole blood but prefer plasma e.g. Tse-tse flies, black flies (Smith and Friend 1982) or mosquitoes (Friend 1978), clues present in blood plasma - cell free plasma is a main parasites meal e.g. Anopheline mosquitos (Aropheles freeborni ) and intermediate clues feeding on both cellular fraction and plasma – e.g. flea Xenopsylla cheopis.

Blood profile reflects condition of the individual (Bjarghov et al. 1976, Nieminen et al. 1979), it can change with season, day time, quality and availability of food, age of animal, sex etc. Lower temperature for example increase Hb and hematocrit level (Horton 1981, Lochmiller et al. 1985). Even stress can change blood profile (Widmaier and Kunz 1993). Decisions about host choice is based on costs/benefits evaluation including costs connected with processing food, its digestion (Kersten and Visser 1996). Digestion abilities on different hosts were reported in many haematophagous arthropods such as ticks (Coons et al. 1986), fleas (Vatschenok et al. 1976) or mosquitoes (Briegel 2003). With digestion of blood many costs are connected such as metabolismus of blood cells, enzymes secretion or excretion by-products (Clements 1992). In principle parasites prefer host which allows them to digest blood with lower energy loss. According to Marshall (1981) parasite must adapt to both chemical and physiological blood properties. Krasnov et al. (2005) has shown that costs of digestion of blood meal in fleas ( Parapulex chephrensis ) on specific host were lower than when fed on non- specific host, what is more digestion took less time in case of specific host blood meal. In the evolutionary track parasites become species - specific feeders adapted to specific blood parameters of their hosts such as proteins, viscosity or lipids components. Some choses their hosts based on certain blood parameters such as amino acid composition in case of mosquitoes which prefers human as their main host (Harrington et al. 2001). Host diet manipulation has impact on ectoparasites such as C. lectularius and Ornithodoros moubata – while fed on rats deficient in thiamine and folic acid caused in C. lectularius inhibited reproduction and in O. moubata slowered development and caused smaller body size, definitely diet composition changes blood components. What is more abnormal

23 development in 5 th instar of C. lectularius was observed while fed on rats protein - deficient (de Meillon et al. 1948). Other experiment with C. lectularius has demonstrated that higher calcium (Ca) level is for them toxic. Krynski et al. (1952) and Nelson (unpubl) have provided informations that some hosts blood might be toxic by creating pyramidal crystals or agglutination within the midgut and death after feeding (e.g. guinea pig or dog and Melophagus ovinus and Linognathus vituli ). Also hormonal level have big impact on feeding, especially testosterone, hydrocortisone, corticosterone (Rothschild and Ford 1964). Variations in blood components can change blood prosperities e.g. decrease in haematocrit cause lower blood viscosity.

All haematophagus insects uses pumps in head capsule to pump actively blood meal. Next to costs and host choice reasons mentioned above quite crucial seems to be also feeding canal radius that determine possibility and time of taking blood meal. Evolution leads to maximalize the size of food canal (diameter of 8 – 10µm), and makes it bit bigger than cells size. In Cimex food canal has 8µm as most of its hosts posses blood capsules which are less than 10qm. Most important blood components are red cells, being main proteins source. In case of erythrocytes of chicken which are about 3 to 5 µm longer in diameter than human host RBC, human blood seem so be better choice as it is more suitable for the narrow food canal of C. lectularius . In Hemiptera there is no digestion in midgut and size of the blood meal depends on sex, age, mating status or feeding history. In some cases specific blood composition of host caused fleas adaptation and made it incompatible with another species (Krasnov et al. 2002).

Any haematological data or database can be used as reference but also help to explain some adaptations of certain species, habitat choice, parasite infestation, activity level and environmental conditions (Wojtaszek et al. 1997). RBC size reflects evolutionary level of species: bigger cells with nucleus or smaller without. There is still little know about bat blood. Bats posses smaller in size but bigger in number RBC comparing to humans (Neuweiler 2000). Such solution helps to increase oxygenation surface, Hb level is so high that allows high oxygen capacity and active, intense flight. Lower level of Hb was found in N. noctula (Schreber 1774), M. myotis (Borkhausen 1797), M. nattereri (Kuhl 1817) and Plecotus auritus (Linnaeus 1758). Bat blood contains the biggest amount of blood cells (26mln/ml), their red cells are smaller than in other mammals but there is much more of them which creates bigger oxygenation surface (Neuweiler 2000). Blood samples from E. serotinus (n=36) were found to have high hemoglobine level and number of erythrocytes which increases their flight efficiency (Wołk and Ruprecht 1988). In most insectivorous bats RBC and WBC parameters are alike (Lidicker and Davis 1955). It was suggested before that bugs has preferences towards some bat species which was suspected to be connected with level of hematocrit (% of erythrocytes) – some bats has “thinner” blood and it makes it easier to feed on them. Human blood was shown easier to drink than for example birds blood which can be the one of the reasons why some ectoparasites switched to humans (Hase 1926, Tawfik 1968). The highest Hb was found in pipistrelle bat – 24g/100ml (Neuweiler 2000).

Species Hematocrit RBC (10^6/µl) Hb conc (g/100ml) Miniopterus schreibersi 57 15.4 17 Pipistrellus pipistrellus 65 14.5 20 Plecotus austriacus 48 12.1 15 Myotis myotis 43 9.3 16 M. nattereri 60 12.6 20 M. daubentoni (hibernation) 49 11.3 16 average for bats 59 12.5 17 small mammals 45 7-8 human 42 4.5 – 6.00 16 pigeon 51 - 17 chicken 28 - 9

Tab1.1. Comparison of some blood properties between chosen bat species, humans, birds and small mammals. (Data from Maina (1989); Thomas and Suthers (1972); Arevalo et al. (1987);Wightman et al. (1987); Wołk and Bogdanowicz (1987); Maina and King (1984); Bassett and Wiederhelm (1984); van der Westhuyzen (1988))

Also number and volume of RBC was different in hibertanting bats of M. daubentonii with bigger number and smaller red cells in males, juveniles of 6 months old had higher number of lymphocytes than adult bats (Wołk and Bogdanowicz 1987). Research on blood serum proteins evaluated no differences between sex in M.myotis – alike in M.myotis and M.blythi (SDS – PAGE method) (Baydemir et al. 2009).

Human blood in comparison contains erythrocytes which has no nuclei and creates 40 – 50% of blood volume, responsible for transport of O 2 and CO 2. RBC are produced in bone narrow stem cells. Diameter of typical RBC in humans vary between 6.2 – 8.2 µm, in females 4 – 5x10 6/µl and in males 5 – 6x10 6/µl. Hb is in 95% composed of red blood cells and is rich in Fe. Plasma is 92% of water, sugar, fat, protein and salt solution 55% of blood volume, responsible for removal of metabolic waste, hormones, lipids, vitamins, hormones or enzymes.

3. Main aims of the thesis

The main aim of the research was to illustrate basic micro-evolutionary mechanisms occurring in two important ectoparasite species from Cimicidae family paraziting two different hosts – human and bats.

1) Balvín et al. (2012) showed deep genetic and morphological divergence between European bed bugs associated with humans and those collected within the roosts of synanthropic bats. Moreover both bug lineages were never found in one roost together. Tthe main aim of that part is to provide further insight into the relationship of C. lectularius with their hosts – bats and human and to give evidence of far-back events, and specialization to particular hosts. We need to designed experiment during which bed bugs collected from bat roosts will be fed on humans (its non-specific host), while bed bugs collected from humans would be fed on bats (as non-specific host). Succesful results would help us to find out presence/existance of host specificity within bed bug and furher to compare survival, reproduction and development rates of adults and possibly their instars on their specific and non-specific hosts.

2) Geographically distant, conspecific populations can posses some incompatibilities (Coyone and Orr 2004). Strong genetic (differences in mtDNA) and morphological divergence (i.e. leg dimensions, hair density) between European bed bugs associated with humans and those collected within the roosts of synanthropic bats was proven recently (Balvin et al. 2012). Following that together with the fact that bed and bat bugs were never found together in one roost and many researches proved that habitat/geographical isolation can most probably be followed by reproductive isolation, the aim of that part is to check how far distinction between bat and human associated bed bugs went. During experiments we investigate whether hybrids between bat and human associated bugs can be created and if hybrids are produced to determine their survival and reproduction rate comparing with control samples and finally if no hybrids present to check how often mating takes place and if sperm is transported.

3) Cimicids have never been found in certain bat species i.e. Plecotus auritus. P. austriacus , Barbastella barbastellus or Rhinolophus hipposideros and R. ferrumequinum . There can be many reasons starting from differences in their biology or ecology or immune response, hormonal status (Jones 1996), roosting strategy and also blood components. All above influences not only the sucking ability but also digestion, survival and development rate of blood sucking parasites (Krasnov 2008). Nevertheless the main reason mentioned by many

researchers in case of haematophagous insect is blood meal itself – its components and chemistry. For example chicken erythrocytes are 11.2 µm in diameter, while human erythrocytes only 6-8 µm (Reinhardt and Siva-Jothy 2007, Benoit 2011), which makes humans an easier host than a chicken for Cimex lectularius (diameter of proboscis canal is 8-12 µm; Hase 1926, Tawfik 1968). Therefore size parameters of erythrocytes may determine the success of feeding. To check that hypothesis some compounds of blood, with focus on RBC (red blood cells) will be measured and compared between different specific bat host species and compared with non-specific host and human blood.

4) It was claimed that bats are the one of the main reservoirs of global recuperation and massive expansion of bed and bat bugs (Szalanski et al. 2008). Synanthropic bats are forced to use alternative roosts, closer to human dwellings. Noctule bat ( Nyctalus noctula ) is the most commonly reported species within Central European cities after 1990 (Hanák et al. 2009) and most of all, mist netted bats of this species are carrying cimicids (Heise 1988, Rupp et al. 2004). Aim of that part will be to check the importance of that bat species for bat bugs ( C. pipistrelli ) migratory events in comparison with two other bats species. Due to the fact that mainly female bugs were found on bats body, it is suggested that they leave the roost in purpose of starting new infestation, second part of the research will be focused on comparison of transmission even between mated females and virgins of bat bugs.

5) Balvín (2008) research on mtDNA has proven that in Czech Republic and Slovakia two different haplotypes of bat bugs are present. More recent research based on morphometry and mtDNA has shown that bat bugs differ morphologically but not genetically between different bat hosts species (Balvín et al. 2013). To obtain more information about fragmentation of the ectoparasite populations, genetic variation and gene flow in bat bug, C. pipistrelli , at local (close populations) and regional (distant populations) scale, we have planned to develop polymorphic microsatellites. Developed microsatellites will be use to investigate if bat bugs genetic variation is reflected in isolation of bats populations or/and bat species. Populations remarkably isolated were expect to show genetic diversity across different localities because despite that bats are highly mobile, bat bugs are rarely found on mist-netted bats outside of the roosts (Balvín et al 2012b).

4. Summary of main results

4.1 Two different lineages of bed bug (Cimex lectularius) reflected in host specificity.

MS I: “Two different lineages of bed bug (Cimex lectularius) reflected in host specificity.”

Wawrocka K., Bartoni čka T.

Published in : Parasitology Research, Germany: Springer, 2013, vol. 112, No 11, p. 3897-3904. ISSN 0932-0113. doi:10.1007/s00436-013-3579-9

Thesis author contribution: Full contribution in laboratory experiments, designing methodology and writing first draft of manuscript

During this part of experiments we have conducted two stages of lab work. First was feeding bat and human associated bed bugs on bat blood using living bats from 2 different species common for bed bugs (Fig.2.). Second stage based on feeding bed bugs on human blood using specially constructed rubber feeders and water bath. Statistical analysis included Kaplan-Meier survival analysis for survival on specific and non-specific host, Log rank test for comparison between lineages and Cox hazard test. During first part of experiment while feeding bugs on bat blood no statistically significant differences were found in any of the lineages, Log rang test comparing survival of instars between bat and human associated groups shown differences in survival at the level of late stages (Log rank test, 4th instars χ² = 9.93, p < 0.05 and 5th instars χ² = 11.33, p < 0.05), while Cox hazard shown existence of differences in survival between two ecotypes ( χ² = 22.51, df = 9, p = 0.007). Second part of experiment while using human blood shown no differences among instars at survival, Log rank tests did not shown significant differences between lineages nor Cox hazard.

Molting among instars did not differ while feeding with bat blood, while comparing lineages differences were visible just in 4 th instar (Log rank test.; 4th instars χ² = 5.91, p < 0.05), Cox hazard did not shown statistically significant differences. Feeding with human blood shown statistically differences just in bat associated group ( χ² = 11.66, df = 4, p = 0.02), no differences between lineages (Log rank and Cox hazard).

Difference between molting probability in case of feeding with bat blood was statistically significant while comparing both lineages ( χ² = 75.18, df = 9 p < 0.001). What is more bat lineage shown low molting probability in early stages and higher in late stages while human lineage opposite. Also period between ecdyses in bed bugs fed on both types of blood differ statistically in bat and human associated bugs (U = 5.0, n.s, U = 11.5, n.s).

Fig .2. Fed and unfed bed bugs – adult males, females and instars.

4.2 Hybridization experiments on bed bugs from two lineages (bat and human associated).

MS III : Reproduction barrier between two lineages of bed bug (Cimex lectularius) (Heteroptera:

Cimicidae)

Published in: Parasitology Research, Springer-Verlag Berlin Heidelberg 2015, 2015, vol. 2015. doi:10.1007/s00436-015-4504-1

Wawrocka K, Bartoni čka T, Balvín O

Thesis author contribution : Full contribution in laboratory experiments, designing methodology and writing manuscript

No progeny or even eggs were produced in interspecific crosses (human with bat associated bugs). Intraspecific crosses from different localities produced both offspring and fertile eggs. Mortality rate in inter- and intraspecific crossing was checked for both sexes. In bed bugs breeding colony in all cases males showed a higher mortality than females. The largest difference in male mortality was observed in mixed samples of both human and bat associated bugs, where it was equal for both lineages at the level of 84%, which is an average 37% higher than in males from intraspecific crosses. Females shown similar pattern with mortality 73% in bat associated and 69% in human associated bed bug from interspecific samples. Statistic analysis - Log rank test for groups of females from intra-and interspecific mating shown their survival differed significantly. Females from mixed crosses had lower survival rates compared with females from within a locality ( χ²= 58.70, p< 0.05) as well with females from between localities ( χ²=27.03, p< 0.05). Survival rates of males were also statistically significant different between males from mixed crosses compared with males from within a locality ( χ² =87.21, p< 0.05) and between localities ( χ² =54.45, p<0.05). Chi-square test was used for comparing survival in males and females within each group and statistically significant differences were observed in all cases except in the comparison between localities (interspecific mating : χ²= 13.506 p < 0.05; intraspecific mating within localities: χ²= 4.046 p < 0.05). Sperm presence was also checked to be sure that mating occurred. Observations of animals on petri dish, showed that mating occurred in 80% of cases within populations (n=12), between populations 67% of mated females was observed (n=10) and in interspecific sets 52% (n=13)(Fig.4.). Within 30 minutes after mating had occurred females were checked for presence of sperm. In 4 of the 5 females from mixed crosses sperm was found in the seminal conceptacle and in all 4 females checked from intraspecific mating sperm was present. In case of interspecific samples mating took a shorter amount of time and varied between

20 - 130 sec (±30) than in case of intraspecific matings (112 - 190 sec (±23)). Differences between mating time were statistically significant ( χ²=39, p< 0.05).

Fig.4. Traumatic insemination in bed bugs on Petri dish.

4.3 Erythrocyte size as potential explanation of host specificity in bed bugs.

MS II: “Erythrocyte size in bats - factor determining host choice in Cimicids (Heteroptera:

Cimicidae)”

Published in: Vespertilio, Skupina pre ochranu netopierov, 2014, vol. 17, No 1, p. 215-220. ISSN 1213-6123

Thesis author contribution: Full contribution in laboratory experiments, designing methodology and writing manuscript

Blood from 12 bat species from seven different genera was described in a paper. Females and males, adults and juveniles of these species were caught : Blood samples were taken and analysed (Fig.3.). The highest hematocrit as well as RBC number was found in Pipistrellus pygmaeus (14.0/10 6/µl). Kluskal – Wallis test did not show statistically significant differences between particular bat species in RBC size. Comparison between sexes within same species did not shown any significant differences either, between adults and juveniles within species no statistically significant differences (U test, p>0.05, n1=4,n2=4) reported. In all checked bats, both specific and non-specific, size of erythrocytes was between 4.5 - 6.9 µm. Five from 12 species shown significantly bigger RBC size in comparison with average RBC size for all checked bat species (5.7 µm) i.e. Eptesicus nilssonii (10% higher), Barbastella barbastellus (5%), Myotis daubentonii (18%), Myotis bechsteinii (15%) and Myotis nattereri (10%). From those species just one ( Barbastella barbastellus ) was found to be not infested by bed bug, nevertheless other species seems to have bigger erythrocytes diameter. Size of RBC in human blood (B+) was estimated on 6.5 µm, which is bigger that in most bat species.

A B

Fig.3. Picture from QuickPHOTO MICRO 2.3, red blood cells of specific host Myotis myotis of bed bug (A) and non-specific bat Plecotus auritus (B).

4.4 Development of multiplex panels of polymorphic microsatellite loci for bat bug (Cimex pipistrelli): a pilot methodological study.

4.4.1 Abstract

Family Cimicidae is composed of 24 genera (Henry 2009) including haematophagous, well know group of blood sucking ectoparasites found to feed on many vertebrates such as mammals, birds, rodents, humans and bats (Usinger 1966). Bats are considered their ancestral hosts from whom they switched later to other hosts such as birds and humans (Usinger 1966; Balvín et al. 2012). Best proof of such consideration is simple fact the majority of cimicid bugs are bat-associated (Balvín et al. 2013). There are many studies conducted on one of the most problematic species, bed bug ( Cimex lectularius ) that experience an extraordinary global resurgence in recent years (Booth 2012). But it was still not enough known about other species from this family which is indirectly connected to humans (and their shelters) namely bat bugs ( Cimex pipistrelli ). Bat bugs as parasites with limited migration abilities was suspected to create subdivided populations with its own organization system, connectivity and dynamic. We used microsatellites to investigate bat bugs population structure. As shown before cimicids posses spatial genetic structure due to dispersal abilities. Microsatellite markers have been developed and used to examine the genetic structure of bat bug (Cimex pipistrelli) at Central European scale (Czech Republic, Slovakia and Ukraine) by utilizing samples from geographically distant populations. Three main question was: how much genetic variation can be expected within a population/bat colony and between bugs populations geographically distant based on microsatellites markers and if genetic variation exist at all. Such questions are relevant to understand dynamics in bat bugs namely regional transmission, evolution and pathogeny. To fill this gap we investigated genetic structure at local scale – within populations and regional scale – between populations across three Central European countries. Bat bugs were collected from three most common host species (Myotis myotis, Nyctalus noctula and Pipistrellus pygmaeus) to determine if any level of host specialization at genetic level occurs and if exist genetic diversity between different populations. Five polymorphic microsatellites were developed for 164 individuals from 18 locations. AMOVA shown greater molecular variance among individuals than among populations. Also in host species comparison higher variance occurred within one host population. Nevertheless populations in close proximity were genetically more similar than distanced populations. The most clustered genetic structure was observed in four localities on border Czech Republic – Slovakia from M. myotis samples . Such results may indicate that bats are able to spread bat bugs on bigger distances and even between different bat species, though such etological observations are missing.

4.4.2 Microsatelites as genetic tools in insects genetic

Development of genetic techniques allow us to understand origin, evolution history, gene flow, genetic diversity of whole species, individuals and whole populations. Thanks to genetic and genomic approach we can follow evolutionary mechanisms hosts and their parasites. In many cases karyology, physiology, behaviour or life history analyses can help with proper taxonomical treatment. In insects world that is still not fully described and discovered many mechanisms cause changes and fade a bit knowledge that we already have about most of them also due to environmental and climate changes. Sultan (1995) suggested two hypotheses the organisms manage different environmental changes in many ways. First is a phenotypic plasticity showing phenotypic differences at the level of populations occupying different environmental niche, despite shared genotype. Second is ecotype hypothesis suggesting that phenotypic variation among populations has its base at genetic level and local adaptations (local adapted ecotypes). First - phenotypic plasticity is an important process of adaptive evolution. West – Eberhard (2003) described this phenomenon as ability to create different phenotypes according to different environmental conditions. Mechanism such as phenotypic plasticity makes possible to single genotype to create more morphological, physiological or/and behavioural forms depending on different environmental conditions (West-Eberhard 1989). Environmental changes even if just temporal may lead to decrease of abundance, reproduction and development. Meyers and Bull (2002) mention two main adaptive variations that organisms established: within and between generations. If environmental fluctuation takes less time than one generation phenotypic plasticity is best solution, if takes longer we observe genetic polymorphisms and the best adapted progeny will survive (Bradshow 1965). Ecotypes variability between generations was described first time by Turessona (1922) as genotypical response of an ecospecies to a particular habitat. Population that is genetically unique and has certain biological characteristics can survive in local environment. Ecotypes create specific ecology, behaviour e.g. body size differences in fishes occupying different biogeographical areas (Jennings et al. 1999). Blood sucking insects to obtain blood meal must face many trade-offs e.g. balancing the risk of feeding on preferable host or/and selection. Selection for host preferences may lead to genetic changes in population same as environment may induce phenotypic plasticity. Sometimes morphological evolution cause small genetical variation that have a obvious impact on phenotype. Investigation of parasites population and genetic dynamic is main way to understand parasite– host interactions. One of the most important tools in this case are molecular markers such as microsatellites which at the beginning were used to research of neurological diseases in humans (Litt and Lutty 1989). Wang et al. (2009a) shown that microsatelites can be used both within and between populations determination of genetic structure. Considering that populations are in equilibrium we can estimate migration rates. Genetic variation exists both within and between populations (Hutchison and Templeton 1999). Population can be genetically similar or divergent, two main mechanisms are responsible for that - genetic drift and gene flow.

Mitochondrial DNA (mtDNA) used in genetic population research weakened by presence of pseudogenes in the nuclear genome (Zhang and Hewitt 1996a). Mechanisms of reducement of interference of mtDNA are not aways successful. What is more mtDNA represents single locus (“looking threw single window of evolution”), effective population size is only 25% of nuclear autosomal sequences which is connected with high alels extinction. Using it as marker in population genetic can cause many problems such as (i) underestimating genetic variability, (ii) genealogical analysis migh by incomplete by missing linkage, (iii) oversimplifying evolutionary reatioship or (iv) uncorect or lack of remote populations processes. In case of Amplified Fragment Length Polymorphisms (AFLPs) are also not best as they are dominant, can be genotyped, are technically demending, deliver less information per loci than microsatellites, can be difficult to ampyfying from older DNA. Nuclear DNA (nDNA) on the other hand migh cause problems as every stage of the research because of recombination in nuclear genomes – information about evolution wil be destroyed by recombination, selection, insertion/deletion – non coding regions, low divergence and poytomy – low divergence or heterozygosity. Microsatellites (msats) play crucial role in research as with its high variability it covers wide range of evolution and genetic processes. Mitochondrial DNA on the other hand is used in phylogeographic and phylogenetic study as very variable, where recombination doesn’t take place (e.g. Balvín et al. 2012). Msats made it possible to detect both hetero- and homozygotic genotypes which can not be done by for example AFLP or RAPD (Random Amplification of Polymorphic DNA). They become most popular in biochemistry, molecular biology, ecology or biodiversity conservation (Web of Science e.g. between 2010 and 2015 930 records). It shows wide usage in many organisms due to many reasons, first of all it is co – dominant, amplified by PCR, has wide distribution in a genome, high polymorphic, transferred between species (Barbara et al. 2007). Next to many advantages microsatellites has also some weak points such as errors being results of for e.g. (i) linkage disequilibrium – outcome of substructuring and inbreed in population causing deviations in alleles associations (Weising et al., 2005); (ii) homoplasy – underestimation of divergence between populations, length can be same but they can differ in sequences, or both length and sequences can be same in both populations but the evolutionary history of one of them is different (Anmarkrud et al., 2008); (iii) null alleles – errors in estimation in alleles frequency (deletions or mutations). Microsatellites due to development of new techniques and overcoming most of difficulties connected with lab equipment and costs of genetic research, become most informative genetic markers detecatable using PCR thanks to easy access to genetic library.

4.4.3 Co-evolution of host and ectoparasite genetic variability

Hosts subdivision can cause reduction of gene flow in parasite what can impact outcome of co-evolutionary interactions – local adaptations, parasite virulence of resistence of the host (Antonovies et al. 1997). Coevolutionary change is driven by antagonistic nature of host-parasite association. These interactions take part in maintainance of genetic diversity, speciation, diversification and reproductive isolation (Schmidt-Hempel 2011). Dynamic of evolution parasite – host was described by different processes such as selective sweeps, NFDS (negative frequency dependent selection) and heterozygote advantage. Theoreticians created two main models for coevolution host – parasite : a) MA – matching alleles (Grosberg and Hort 2000), successful infection is possible only if parasite – host matchies, parasite seaks most commont host and host avoids most common parasites, second b) GFG – gene for gene (Flor 1956), it takes into consideration variable genotypes of host and parasite, selection between broad range generalist and narrow range specialists. There are three crucial factors that determines parasites diversity structure : (i) host dispersal abilities (McCoy et al. 2003), (ii) demographic history (Stefka et al. 2011) and (iii) spectrum of parasitized hosts (Barrett et al. 2008). Differences in gene flow patterns between host and ectoparasite may occur. For example forested areas were found in brown bat´s ( Myotis ucifugus ) parasite C. adjunctus barrier in genetic flow which can be caused due to less possibility of meeting other bats, no opportunities to start colony or high bugs mortality in dense forested area (Talbot et al. 2016). Weak generalist on highly mobile host would always show lower genetic variability than strong generalist on host that shows low movements. Also not every host departure and migration will be connected with carrying bugs, not every bug will move it to new roost (can fell off or being eaten by the host) and not every, even fertilized female will be able to start new colony. Other than main host can be used to migrate e.g. birds or other mammals in case of bat parasites, which can be connected with the level of association of the parasite with host – strong generalist (e.g. C. lectularius ) are connected with phylogenetically different host species contrary to weak generalist (like C. pipistrelli or C. adjunctus ) connected with species that are related with each other as different bat species (Mazé – Guilmo et al. 2016). As mentioned before if ectoparasite is in certain roost doesn’t meant that it comes together with bat species that in that roost occurs. For instance Talbot et al. (2016) shown very low genetic variability at mtDNA in bat bugs samples from different host species in C. adjunctus which is clear proof of systematic change of the host. Seven genetic clusters in bat bugs were found with non- significant isolation by distance while in case of stronger generalist ( C. lectularius ) 11 genetic clusters were found with statistically significant isolation by distance, creating non-equilibrum metapopulation (Benoit et al. 2016). Isolation of hosts is reflected in isolation of their parasites (Thompson et al. 1994). Criscione (2008) showed that insect ectoparasites with limited migration abilities genetic structure is dependent on host distribution and social system e.g. ticks (Ixodes uriae ) on cliff-nesting seabirds ( Rissa tridactyla and Fratercula arctica ) shown different structure dependent on birds movement and their

37 spatial composition (McCoy et al. 2003, 2005). Genetic diversification of populations was also explained by distance between populations. Genetic research in C. adjunctus shown segregation by distance which explained variation in its genetic structure. C. adjunctus shown 10 different clusters while only two of them are linked with bat hosts ( Myotis lucifugus ) (Nadin Davis et al. 2010, Van Hof and Muller 2015). Clearly shows that parasite is more subdivided than its host, which shows that movements of host do not reflect fully genetic structure of its ectoparasite. Higher genetic variability was higher in Cimex adjunctus than in its host Eptesicus fuscus and Myotis lucifugus . Mazé – Guilmo et al. (2016) found that movements of host doesn’t cover movements of bugs. Gene flow can be also sex related both in case of host as well as in parasite. Male bats are migrating more between roosts and colonies than females. Especially during maternity period while females stay in same roosts and must come back to feed their juveniles. Females and juveniles live in a summer in bigger aggregations and posse weaker immunological response (Christe et al. 2000). Males on the other hand living solitary but they may during summer visit bachelor aggregation or even maternity colony (Safi and Kerth 2007). Therefore females posses always higher parasites level (Reickardt and Kerth 2009). During swarming periods males can play role of ectoparasites and viruses vectors between females (van Schaik et al. 2014). Transmission of ectoparasites takes part mainly in autumn, less during hibernation in the winter. Bat males in spring and early summer show low parasites, their number increases during swarming season (Weber 2015a). Ectoparasites from Cimex genus are shown to use bats for migration e.g. C. adjunctus (Talbot et al., 2016). But parasite sex also determines dispersion. Both C. pipistrelli and C. adjunctus did not show genetic association among different bat species. Also dispersal abilities of the host determine horizontal transmission of ectoparasites. Genetic structure of parasites whom dispersal depends on its host movements may vary even across different host populations e.g. if populations are isolated and rarely or never meet. The closer association of parasite with its host the bigger dependence of movements and the higher genetic divergence (e.g. Apodemus sylvaticus and its parasite Heligmosonides polygurys (Nieberding et al. 2004)). Isolation of hosts is reflected in isolation of its ectoparasites (Thompson et al. 1994). Usinger (1966) stated that Cimex spp. rarely disperse on their own despite that they are not fully dependent on their hosts. Cimex pipistrelli and C. adjunctus stay at body of their host only during feeding period and it was shown that in case decline of numbers of host may lead to departure of bugs from colony. What is more Cimex adjunctus was found to switch between different bat host species while at certain moment of the season they shared same roost and parasites exchange took place (Adam et al. 2000). Bats switch their roosts due to different reasons. Fission – fusion and in most cases not all bats move at once so exchange of parasites between different bat species can occur (e.g. Nyctalus noctula , Fortuna et al. 2009;, Myotis bechsteinii, Kert and Konig 1999; Eptesicus fuscus , Willis and Brigham 2004. Another aspect of the host is also behaviour of individual and its personality, more curious and social bats will

38 have more parasites as same as more choosy and less social females mate with less males and so they keep more parasites on their body (Barber and Dingemanse 2010; Webber et al. 2015b). Movements of the parasite depends on social system of the hosts. They occur in groups and interact differently, i.e. 1) genetically connected or not individuals groups with some non random association and close contact (colony) or 2) individuals assemblages mostly occupying same shalter in the same time by coincidence, no social bounds occur (aggregation). Both aggregations and colonies play crucial role in parasite occurrence and dynamic of its population (Weber et al. 2016). Assumpsion that roosting group of bat is is a colony may be misleading than. Stanko et al. (2002) shown that more interactions between hosts occur and probability of parasites transmission/exchange increase in bigger host population contrary to smaller associations or solitary individuals. Bats are highly social animals. They evolved towards social behaviour as it is very beneficial for them in all aspects of their life. Some bat species seems to be preferred above other in term of parasitism, in many cases greater mouse-eared bat ( Myotis myotis ) seems to be favoured. Group size, density and population dynamic seems to be most crucial and this bat species meets all those criteria. What is more research on Cimex transmission shown that this bat is most probable responsible for its movements between colonies and roosting places of that bat (Willems, K řemenová and Bartoni čka in prep.). Allopatric populations can show sequences variability and can share barcode sequences, what can lead to wrong interpretation of species affiliation (cf. Sepsis pyrrhosoma Denise et al. 2009). Such situation was found in cimicids where large morphological variability leaded in evolutionary history in more similar species and taxonomical inconsistency. Mainly three species (C. pipistrelli , C. dissimilis Horváth 1910 and C. stadleri Horváth, 1935) were considered by different researchers due to morphological differences as three totally separate species (Usinger 1966), subspecies (Lansbury 1961) or synonymic species ( C. stadleri and C. dissimilis )( Péricart 1972). From genetic point of view Warren et al. (2015) has shown high genetic divergence of two C. lectularius host races – bat and human associated. Gene flow within and among populations both human and bats associated bugs (mtDNA, microsatellites and knock-down resistance gene variants) shown in bat associated bugs within roosts higher genetic diversity. But there is still little known about second important species - bat bug ( Cimex pipistrelli ). This blood sucking insect associated with bats was also found to feed on human as alternative host (Whyte et al. 2001). Bats are nevertheless main and original host of this species (Horvath 1913). In bigger amount, bat bugs were found in Myotis myotis, Myotis emarginatus, Nyctalus noctula, Pipistrellus spp. roosts. Balvín (2008) research on mtDNA (COI) shown that in Czech Republic and Slovakia exist two different haplotypes of bat bugs – A and B. Type B was found to be homogenous in Czech Republic and Slovakia, one separate sample differed from A and B and it comes from Switzerland, marked as haplotype C. Halpotypes in this case did not reflect bats populations relationships. What is more recent research based on morphometry and mtDNA shown that bat bugs differ morphologically but not genetically between different bat hosts species (Balvín et al. 2013). Balvín et al. (2013) found 27 different mitochondrial haplotypes (based

39 on mtDNA) in West-Palearctic within 69 localities and 12 bat species. Problem with mitochondrial DNA is that it is maternity inherited and will show only its history. In such case more than one genetic marker is needed. In case of male – biased dispersal there will be higher genetic variation in mitochondrial than nuclear DNA while female biased dispersal in which mitochondrial variation will be lower than in nuclear DNA. Females of bed bug ( C. lectularius ) were shown to disperse often than males (Pfiester et al. 2009). The main aim of our research was to check based on developed microsatellite markers if bat bugs genetic variation is reflected in isolation of bats populations. We used microsatellite markers to analyze bug population structure in the area of central and Eastern Europe. First, we specifically analyzed the distribution of genetic variation within and among localities. Second, since the ecological and behavioral variations are often the most visible features of roosting strategies of bat hosts, we therefore focused on the comparison of the population genetic diversity between different host species. In case of host populations (roosts) remarkably isolated geographically we would expect genetic diversity across different localities because despite that bats are highly mobile, bat bugs are rarely found on mist-netted bats outside of their roosts (Balvín et al 2012b).

4.4.4 Material and methods

Data sampling Between 2005 and 2014 bat bugs from 18 (4 localities were excluded due to low DNA level) different localities from Central Europe were collected (Czech Republic -16 localities, Slovakia -5, Ukraine-1). Bat bugs were picked up manually with soft tweezers and exhaustors from bat maternity colonies (attics of churches, castles, hollows in trees and bat boxes) and also during mist-netting bats emerging directly from the roost (Ukraine sample nr 195). In total 164 bugs were collected from 4 different bat species – Myotis myotis (13 localities) Nyctalus noctula (2 localities), Pipistrellus pygmaeus (2 localities) and M. daubentonii (1 locality). Number of host individuals from which bat bugs were collected varied between 10 till 1300. The distance between localities was 18 to 1264km (Jílové u D ěč ína – Verkhivnya, Zhytomyrs'ka region). After collecting, all bugs were stored in 96% ethanol in freezer for DNA extraction.

DNA extraction DNA was extracted from randomly chosen bat bugs, legs were removed using soft tweezers (8 legs per each locality). Qiagen DNassay tissue extraction kit and protocol for tissues was used with 24h tissue digestion in AE Buffer with proteinasis K under 56°C in thermostat (ROTH-CC59.1, Memmert). For each locality DNA samples were prepared (depending on amount of material between 5 and 14 samples - males and females legs). For primers development we obtained three different bat bugs samples represented three different haplotypes - two from Slovakia and one from Czech

Republic. After DNA extraction samples were situated in Qubit fluorometer following Quibt protocol for quantifying DNA, in the end additionally electophoresis was conducted.

Primers design After screening (Genoscreen) we obtained 646 sequences with di-, tri-tetra-nucleotide repeats. We have chosen 15 of them which suited the best, based on motives, Ta, % of G/C etc. (manually and using Primer3Plus calculator) excluding homopolymers and tested them on 8 different localities. Mixture of multi-mix, destilated water, primers and DNA was created. Samples were placed in a cycler (95°-15min, 94°- 60s (denaturation), 55° - 90s (annealing), 72°-60s (extension)- 35cycles and 72°-15min (final extension). Results from PCR shown that 11 from 15 primers works for our samples. For those 11 loci we ordered fluorescent labelled forward primer (PET, VIC, FAM, NED). Again mixture of multi-mix, fluorescent primers, destilated water and DNA was created and after vortexing placed in the cycler for the same procedure as above for non-labelled primers. For fragment analysis mixture of Liz-500 (0.4µl of Gene Scan Size Standard, Applied Biosystems) and formaldehyde (12 µl) with 0.8 µl of PCR product and putted into cycler for denaturation in 95° for 5min. Results from electrophoresis were further analysed in Gene Mapper v3.7 (Applied Biosystems). After creating two multiplexes according to the length of the product and dye, we excluded 6 from 11 markers and obtained in the end 5 polymorphic markers (Tab. 1). During analysis in Gene Mapper v3.7 additionally one more primer appeared – Bmut. AMOVA, HWE and HFL analysis were conducted in GenAlEx 6.501. for each sample number of allels Ne, I, He and Ho was calculated (Tab. 2).

Tab.1. Characteristics of 5 designed microsatellites (Ho – observed heterozygosity, He-Nei´s unbiased estimator of expected heterozygosity, A- number of alleles).

Population No of individuals No of allels Ne I He Ho Pop1 9 1,571 1,171 0,206 0,089 0,098 Pop2 9 1,286 1,127 0,232 0,206 0,156 Pop3 9 2,143 1,647 0,487 0,125 0,284 Pop4 11 1,857 1,488 0,405 0,250 0,256 Pop5 9 2,286 1,531 0,468 0,163 0,262 Pop6 9 2,571 2,160 0,623 0,159 0,340 Pop7 10 1,000 0,989 0,096 0,029 0,069 Pop8 10 1,714 1,420 0,332 0,167 0,20 Pop9 8 1,571 1,405 0,324 0,185 0,223 Pop10 14 1,286 1,169 0,140 0,077 0,093 Pop11 8 1,714 1,288 0,215 0,059 0,110 Pop12 6 1,286 1,199 0,168 0,057 0,114 Pop13 10 2,143 1,577 0,410 0,104 0,227 Pop14 10 2,429 1,772 0,499 0,107 0,266 Pop15 6 1,714 1,629 0,380 0,143 0,238 Pop16 10 1,857 1,656 0,492 0,257 0,324 Pop17 5 2,286 1,722 0,518 0,200 0,297 Pop18 11 1,857 1,571 0,448 0,179 0,291

Tab.2. Calculated values for each from 18 populations: number of alleles, Ne effective population size- , I - Shannon's Information Index, He – expected heterozygosity

4.4.5 Results

During analysis we had to exclude two samples from Slovakia and three samples from Czech Republic as primers did not work for them or worked just for too few samples ( <5 ). Each locality was treated as a separate population for genetic analysis. Total of 164 bat bugs at 5 microsatellites were analyzed. Across all populations we observed 1.222 – 3.333 alleles per locus (with mean =1.473). The mean expected and observed heterozygosity for all loci was 0.214 and 0.142 respectively. Percentage of polymorphic loci varied depending on locality/population between 14 – 85% (with average=52%). In seven from 18 populations we found private allels (unique to a single population) with alleles numbers vary between 0.143 – 0.286. Estimated variance among populations, individuals and within individuals did not differ significantly (0.416, 0.592 and 0.470 respectively) with percentages of molecular variance 32% within individuals, 28% among populations and 40% among individuals. For comparison between different host species percentage of molecular variance (AMOVA) among populations was 29% and 71% within populations. Percentage of polymorphic loci was highest in N. noctula and P. pygmaeus (42-71% and 57-72% respectively), while in M. myotis the percentage of variance was lower (28-57%). Null allele frequencies for each locus and population were analysed following the Expectation Maximization (EM) algorithm of Dempster in FreeNa. Analysis has shown high amount of null alleles from all expected alleles in case of 2 microsatelites number 1 and 7 (mean

NA1 =0,13; mean NA7 =0,21) in such case statistic analysis couldn’t be conduced (biased by the huge null allele proportion). What is more in case of one of those loci lack of genotypes at all for some populatons made it uninformative and gave risk of causing biases in subsequent analyses. Both loci were excluded from further analysis. A Bayesian clustering procedure was implemented in STRUCTURE 2.2 (Pritchard, Stephens and Donnelly, 2000). The Bayesian model assumes two K (unknown) populations, K2 to K5. For K=2 we observed clustered population differentiated on border Czech Republic – Slovakia (Fig.1.). Strong structure was observed using cluster membership K=5 (at which curve reaches the plateau). No visible genetic structure across different host species was observed neither across all populations as genetic variability in both cases was higher within than between populations. Nevertheless stronger genetic similarity we observed in case of closer localities than those at longer distances (Fig.2).

Fig.1. Genetic structure of Cimex pipistrelli obtained using STRUCTURE. Divided on host species, marked on the right site: M. myotis (13), N. noctula (2), P. pygmaeus (2) and M. daubentonii (1). The figures shown for a giving K (K 2 – K5) based on the highest probability run at that K.

Fig.1. Geographic distribution of bat bugs across Czech Republic, Slovakia and Ukraine (STRUCTURE analysis for K=3 for populations/localities; each pie represent separate locality, red circle around the pie – samples from N. noctula , brown circle – M. daubentonii , black circle – P. pygmaeus ).

4.4.6 Discussion

Based on our research we can not draw clear conclusions concerning genetic variability in bat bug ( C. pipistrelli ) due to low number of microsatellites (from 11 developed only 5 appeared to be polymorphic). No clear genetic structure across geographic populations, neither local nor regional scale in C. pipistrelli gives us an idea how homogenous genetic population of that species possible is. Nevertheless in some cases populations that are closer to each other shows more genetic similarity that divergent populations. It definietely puts more pressure for further investigation and optimalization of more microsatellites based on existing already msat library which we developed. Cimex pipistrelli was found before on Nyctalus spp. ( N. leisleri (Kuhl, 1817), N. lasiopterus (Schreber, 1780)) and also Pipistrellus pygmaeus (Leach, 1825), E. serotinus (Southwood and Leston 1959 ), M. bechsteinii (Morkel, 1999) etc. but most numerously on M. myotis (Lederer,1950). We expected different genetic diversity according to host species, as shown before genetic analysis proven existence of two different lineages of this bugs – haplotype A (16 samples) and haplotype B (26 samples) (COI analysis) and also one individual with difference in one base from Switzerland, marked as haplotype C. Moreover, Usinger (1966) suggested the existence of a reproductive barrier between the populations of bat bugs of the Cimex pipistrelli group from the British Isles and those of Czechoslovakia, supporting the existence of at least two separate species in Europe. Moreover mitochondrial (cytochrome oxidase subunit I, COI) and four nuclear loci (internal transcribed spacer

2, 18S and 28s ribosomal genes and elongation factor 1 subunit α) were used showing 27 mitochondrial haplotypes belonging to two distinct haplogroups (on nuclear loci of C. pipistrelli showed almost no variation), but not reflected in bat species contrary to morphological differences found between bat bugs from different bat species (body size and amount of hair, Balvín et al. 2013). On the other hand similar research done on C. adjunctus shown genetic variance among samples associated with different bat species. Additionally there was evident difference between mitochondrial DNA and microsatellite markers used with mitochondrial data showing much less variation among populations associated with different bat species than those from microsatellites (Talbot et al. 2016). As mitochondrial DNA is maternally inherited it will show variation in dispersal and history of the maternal lineage only. Many ectoparasites is highly host-specific which is connected with host ecology and its geographic isolation (Wenzel and Tipton 1966). C. pipistrelli was considered as specialist as it occurs mostly on bats (on human it can not undergo full life cycle)(Southwood 1953). C. adjunctus which belongs with C. pipistrelli to Cimex bat bugs shown 7 genetic clusters among sites with non- significant isolation by distance contrary to bed bug (C. lectularius ) belonging to generalist showing stronger genetic structuring – 11 genetic clusters and significant isolation by distance (Talbot et al. 2016). Bugs vector from that research ( M. lucifugus ) shown long distances migration abilities (up to 647km; Norquai et al. 2013), our investigated bat host – M. myotis migrates on smaller distances but still impressive 200 – 355km (Gaisler t al. 2003). Wenzel et al. (1966) shown that in case of parasites migrating on its hosts random distribution can be shown in case of colonial bats and clumped distribution in solitary bats. However in our results within population genetic differentiation was stronger than between different populations. That together with lack of genetic differentiation between different bat host species shows that bats are good vectors for bat bugs and let them to move between different roosts not only the same but also different bat hosts. Such model may match to genetic structure of other wingless bat ectoparasites, mites. Research on Spinturnix bechsteini between different bat populations shown no genetic structure, mites could freely move among and between bats colonies (Bruyndonekx et al. 2009). It was shown before that those mites can move quickly from one individual bat to another (Christe et al. 2003). Research proven that there is no dispersal barriers, no differentiation between mites paraziting different bat species in one colony. Research by Olival et al. (2013) on Nycteribidae ( Cyclopodia horsfieldi ) also wingless dependent on host mobility parasite of Pteropus species parasite had low population genetic structure even between localities which were 1000 km far from each other. It is serious proof of bats contacting with each other and gene flow between their parasites. Females of M. bechsteinii were found to change roost every 2 – 3 days and research on their parasite Basilia nana did not show either temporal as spatial genetic structure between 13 different colonies between years (Deukeukleire 2012).

In STRUCTURE output we can see that bat bug dispersal abilities of closer populations are greater than further populations. Mainly movement of host at small scale – between closer roosts (18 - 20km distanced) - local homogeneous groups within geographical areas are visible. It could mean that migration is not equal – differentiation between close populations and lack of differentiation between distanced populations. As shown in ticks ( Rissa tridactyla ), their genetic structure was dependent on dynamic of local population of their hosts Fratercula arctica (McCoy et al. 2003). Closed spatial system of M. bechsteinii leaded to higher genetic variability in its mite and creating subpopulations, while in case of M. myotis with totally different population structure and social dynamic genetic structure between its mites was not found ( van Schaik et al. 2014). Therefore some populations can be less genetically different from each other even if they seems to be in close proximity (McCoy et al. 2003). Few previous papers suggested that bugs undergo migration in purpose and that is not coincidental event (e.g. Heise 1988). If wingless ectoparasite such as bat bug want to disperse it is mainly depend on host movements. Dispersing individual might meet a costs due to either decreased fitness, increased mortality or costs connected with adaptation to new environment. Local extinction of population or population collapse may force parasite to migrate to survive and reproduce (Olivieri et al. 1995) for example when maternity colony fall apart or bats move out due to many reseans such as atticks renovations, destroyed roost or changed mlicroclimat. Parasite dispersion depends also on adaptation abilities of certain species (Mopper 1996). Parasites that possess higher mobility are more likely to adapt to new environment. Also one, important reason for dispersal might be inbreeding depression which has an impact on fitness and survival (Charlesworth and Charlesworth 1987; Keller and Waller 2002). Mating between relatives leads to the “dominance” effect (Charlesworth and Willis 2009) an increase expression of deleterious recessives or to the “overdominance” effect (Charlesworth and Willis 2009) – reduction in fitness caused by an increase of homozygosity at loci with heterozygote advantage. Both inbreeding and reduced fitness raise extinction risk of the population (Saccheri et al. 1998). Bats as main hosts of bat bugs are one of the most variety of parasites than any other mammals (Hudson 1972). Nevertheless bats differ in their ecology, roosting behaviour, foraging strategies, habitat preferences. In case of bats some species occur rarely together in one roost, Pipistrellus spp. and Nyctalus spp. are found mostly in crevices as well natural (caves, rocks) as artificial (e.g. buildings) but also in bat boxes and tree holes (e.g. Celuch and Kanuch 2005). There we can find numerous numbers of cimicids (e.g. Bartonička and Gaisler 2007). Despite high differences in ecology, roosting and foraging strategy of bug hosts we did not find clear evidence or reflection at genetic level according to different host species. Despite the fact that bat bugs were rarely found on mist-netted M. myotis their genetic structure did not differ from bugs from other much more evident bugs vectors as Noctule or Pipistrell´s. In a case of bat host same roost was occupied by different species during consecutive years (Lu čan et al. 2009). Most samples we had come from M. myotis

46 species, which reoccupies every year same maternity roost. It was proven before that bugs can be exchanged between close M. myotis roosts (Horá ček 1985). Exchange of ectoparasite between bat hosts can take place in different situations, mainly : 1) during swarming, 2) in maternity colonies, or 3) sharing daily roost. Social system of the host affect reproduction success of its parasite. During swarming males and females meet (Kerth et al. 2003b). Dekeukeleire et al. (2011) shown that females from one colony visit different swarming sites and in total five different colonies may take part in swarming. In M. myotis during swarming it was observed that few females copulate on average four days with one male. Male has few roost that he visits sporadically and few that are his stable roosts (Zahn and Dippel 2009). Christe et al. (2000) proven that maternity colonies creates perfect conditions for parasites development and reproduction. Pregnant/lactating females and juveniles are perfect host for parasites as they posses low immune defence (Reckardt and Kerth 2009). Males rarely roost together with females (Kerth and Morf 2004). Ectoparasites transmission will be different in maternity colonies and solitary individuals. In maternity colony females stay close to each other and that can lead to bugs transmission between individuals. Myotis myotis which is most common bugs host is species that prefers forested habitat. During summer females form bigger colonies (between few till hundreds of individuals) where exchange of ectoparasites can take place, especially that some females can take over some solitary roosts which are closer to foraging sites (e.g. Kerth and Morf 2004). Females can switch between roosts especially after the fledging of the juveniles. Colonies that are on attics change their location according to temperature changes. Males of this species living solitary and are isolated from large females colonies (Zahn and Rainho 2003).Transmission of cimicid Oeciacus vicarious between populations of swallows had its peak in nesting season (Brown and Brown 2005). According to them migration depends on colony size and season. They observed constant transmission of bugs between nests. They mostly migrated during summer – higher chance to find host, most of them were adult as instars doesn’t have a reason to risk dispersion and die due to lack of food. Dick et al. (2003) found that bats has the highest horizontal parasites transmission while being close to each other. Males that live solitary are always very low or not infested. In case of this and few other bats species establishment of new colonies was observed, former maternity colonies were occupied by solitary bats and some solitary roosts become maternity colonies (Spitzenberger and Weiss 2012). Also many aspects of ecological and natural history may influence the population genetic structure of animal ectoparasites. The main factors reducing genetic structure : (a) Highly eagile hosts (definitive, intermediate, paratenic) or vectors, (b) persistent (long-lived) life cycle stages in environment or definitive host, (c) low definitive host specificity, many reservoir hosts or (d) underdispersed distribution of parasites among hosts, (e) life history with frequent meta-population extinction followed definitive hosts required for transmission. In our case definietely highly eagile host plays crucial role in reducing genetic structure and making it more homogenous.

Research made on population genetic structure of the two mite species ( Spinturnix myoti and Spinturnix bechsteini ) in two different bat species from genus Myotis differed strongly as a result of the social system of their hosts (van Schaik et al. 2014). Mouse – eared bat seems to be a bat species with quite homogenous structure of ectoparasites not only in our case but also in case of mites (Spinturnix myoti (van Schaik et al. 2014)). Between different colonies of this bat species very low or lack of genetic differentiation was found and no substructuring. Same as in our case mites shown genetic diversity mainly within population which would suggest easy ectoparasites exchange between different bat hosts belonging to different colonies outside maternity period. Contrary to our results and those from for example S. myoti , population genetic structure of S. bechsteini showed higher nuclear genetic diversity with significant differentiation between populations, creating clearly subpopulations (van Schaik et al. 2014). Such difference was explained by ecology and behaviour characteristic for host species namely M. bechsteinii creating smaller colonies and their tendency to keep less body contact with conspecifics. Moreover it was shown that this host intensively avoid parasites by often roost switching (Reckart and Kerth 2007). In M. myotis there is not many records neither evidence for anti-parasite behaviour shown in M. bechsteinii . Our samples origin mainly from greater mouse- eared bat creating big maternity colonies (lack of smaller sub-divided colonies like in other bat species) with high social interactions as well during maternity, mating as hibernation period that allows to exchange ectoparasites between individuals of different sex and age. Roosts switching is quite well known phenomenon in bats. Bats can switch their roost to be closer to foraging areas (Fleming 1988), to have better microclimate, avoid overcrowded place or to avoid parasites (Bartoni čka and Ruži čková 2012; Reckhard and Kerth 2007). Roosts choice is usually species specific but in many cases we can find few bat species in one roost. In the context of monitoring bat populations in the Czech Republic is annually revised ca 140 shelters summer colonies of M. myotis and more than 20 of them are occupied at the same time by species, which belongs to the non-specific hosts (as Plecotus or R. hipposideros ) (Czech bat conservation trust database, unpubl.). To parasite exchange can take place during sharing tree hollows or bat boxes by different bat species (e.g. Siemers and Swift 2006). In case of tree-dwelling bats in one roost we can find different bats species with different foraging strategies (e.g. N. noctula and N. lesileri ; Bogdanowicz and Ruczynski 2005). Sometimes they share also bat boxes like N.noctula and M. daubentonii (Cerveny and Burger 1987; Walk and Rudolph 2005), or M. bechsteini , P. pipistrellus and N. noctula (pers. observation). Lack of geographic population genetic structure both at local and regional scale in C. pipistrelli could be due to insufficient variance or invariability of the molecular markers used in our research. Nevertheless the most probably explanation taking into consideration long term association bat bug – bat which started long time ago and suffered many co-evolutionary processes genetic exchange followed by high gene flow across bat hosts seems at that moment most logic explanation. Due to the fact that pure samples are coming mostly from one bat species and that we were successful to develop only five fully working polymorphic microsatellites still more research is

48 needed. Taking into account development of more microsatellites based on existing library and investigating more local scale associations including more bat host species and different colonies. Data collected were not suffiecient to respect microstatellites development regarding same localisations because amount of DNA available was limited especially for distant samples from Slovakia or Ukraine, therefore more samples from same and more locations need to be collected in order to create more microsatellies and obtain higher DNA concentration. Behaviour shown by bat bugs seems to be the part of host-parasite paradox of co-evolution. As far as there is opportunity, parasites will migrate. Nevertheless free-living bat bugs who posses legs and are highly mobile, not fully dependen on host for survival and reproduction should naturally show lower genetic variation than ectoparasites fully dependent on their host at all levels of their life cycle (Olival et al. 2003). Bats phylogeny reflects their ecology and it decrease problems connected with adapting to new ecology, behaviour or even immune response. It is easier for them to switch host if they are closer related. Our results based on microsatellites clearly shows that host switching and migration of bat bugs occur and surprisingly is quite common.

4.5 Bat bugs (Cimex pipistrelli) transmission propensity in three bat species

MS V: Bat bugs ( Cimex pipistrelli ) transmission propensity in three bat species

Willems K, Křemenová J, Bartoni čka T

Thesis author contribution : Full contribution in laboratory experiments, designing methodology

Transmission by different bat species

During 30 volary sessions conducted with three bat species we found significant differences between numbers of bat bugs found on the bat bodies (ANOVA, F 2,27 =44.43, p<0.001) and in the box

(F 2,27 =11.98, p<0.001). Only few bat bugs were observed on M. daubentonii (1.6±0.69 bugs per one bat), while pooled fed and unfed numbers of bat bugs were similar on N. noctula body (7.5±1.87 bug) and M. myotis (4.9±2.06 bug) (t-test, t=-1.31, p=0.073).

Futher we found differences in all variables, i.e. numbers of fed bugs (F 2,27 =10.28, p<0.001) and unfed bugs (F2,27 =58.56, p<0.001) on bat body, numbers of fed bugs (F2,27 =23.36, p<0.001) and unfed bugs

(F2,27 =95.77, p<0.001) in bat box. Thorough N. noctula had on its body more unfed bugs (5.7±0.67) comparing with M. myotis (2.2±1.47) (t=-6.82, p<0.001) whilst number of fed bugs on the body of M. myotis (2.1±0.99) and on N. noctula (1.8±0.79) does not differ (t=0.74, p=0.464) (Fig. 2). Bat bugs sucked faster and/or more effective on M. myotis , that on N. noctule , what is obvious from the highest numbers of fed bat bugs found in the box after session. Although the period during that bats have been emerging the box was similar among species similar

(ANOVA, F 2,27 =0.4725, p=0.634), N. noctula left bat box earlier than other species (F2,27 =26.25, p<0.001) (Tab. 1). Morever N. noctula started be active as the leatest from studies bat species

(F2,27 =12.05, p=0.011).

Mated and virgin females transmission Almost 4.5 more females found on N. noctula body were mated females (31% mated females and 7% virgins) (t-test, t=4.35, p<0.01), contrary in a box were found 28% more virgins than mated females (64% mated, 92% virgins) (t=3.12, p=0.014). (Fig. 3). Also 23% less virgin females was found to be fed comparing to mated females, but numbers of fed mated and fed virgin female did not differ significantly (t=1.19, p>0.05).

5. Conclusions and future perspectives

Despite many research that has been done till now on bed bug ( C. lectularius ), still many information is missing. This haematohpagous ectoparasites shows incredible high adaptations capacity. Fact that bat associated bugs can, not only succesully feed on humans but also reproduce and develop on humans blood, can be disturbing and may have a negative impact on humans attitude towards bats and their presence in humans dwellings. In purpose to investigate if offsprings produced from bugs fed on non-specifi host blood stay fertile and are able to develop succesful, fully functional population, more research dealing with that topic would be recommended. Another question is, if possibility of creating hybrids between bat and host associated bugs fed on non- specific host would be possible. It would bringtotally new light on problem of bed bugs infestations.

Many aspects connected with host choice by bed bugs stays yet unclear. There is no exact explanation why some bat species are common hosts of bed bugs and some not. RBC size mentioned by other authors as possible explanation seems not to play crucial role in that case. More detailed research on bat host blood is needed concerning both specific and non-specific bat species, especially regarding nutritional value of blood, it’s temperature or density, as some bats were claimed to have “thinner” blood are being easier to feed on.

Spatial host separation of two lineages of bed bugs – human and bat associated went so far that despite observed mating between lineages, no progeny or even unfertile eggs were produced. Such situation can exclude pre-mating barriers but still pre-zygotic or post-zygotic barriers need to be investigated. Further e.g. comparison of hormones produced by both lineages, detailed observation of sperm after fertilization is highly recommended.

Noctule bat ( N. noctula ) was reported as a main vector of bugs transmission, comparing to other bat species from genus Myotis ( M. Daubentonii or M. myotis ). This fact become confirmed during the lab experiments, comparing Noctule with dubenton´s bat and greater mouse-eared bat, where N. noctula carried most of the bugs on his body. Bugs were found to chose to migrate on bigger bat species. Still it is not fully explained why this situation takes place, body size as possible explanation could be excluded due to the fact that both N. Noctula and M. myotis doesnt differ significantly in body size or body mass. Further comparison concerning thermoregulation differences between those bat species, their torpor behaviour is needed. Genetic structure within and between populations of bat bug ( C. pipistrelli ) based on microsatellites did not show direct diversification between bat bugs populations in Czech Republic, Slovakia or even Ukraine. That proves that bed bugs despite their transmission limits can freely discover, visit and colonize new places, being transmitted by their bat host, creating homogenous genetic population in Central Europe. Due to small number of microsatliites that could be used in our analysis, it is higly recommended to develop more polymorphic loci based on already existing library. Most of our samples comes from one bat species ( M. myotis ). More samples, also from other

51 bat species would be recommended, especially those that are rarely or never found to carry bugs. As Balvin (2008) have found before that bat bugs creats two main haplotypes A and B, would be interesting to see if that pattern is also reflected in microsatellites. More localities included to research could help to have better overwiev on that matter.

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7. Manuscripts published and submitted

7.1 Bat bugs (Cimex pipistrelli) transmission propensity in three bat species

Kamila Willems, Jana K řemenová, Tomáš Bartoni čka*

Department of Botany and Zoology, Masaryk University, Kotlá řská 2, 611 37 Brno, Czech Republic,

[email protected], [email protected], [email protected]

Corresponding author: Tomas Bartonicka, Department of Botany and Zoology, Masaryk University,

Kotlá řská 2, 611 37 Brno, Czech Republic, [email protected], tel: +420 549493095

Informative title: Bat bug transmission

Abstract

Bats as the only mammals possessing flight abilities gives us a wide opportunity to study transmission capability of their ectopatasites. Transmission occurs in purpose and is rarely co incidential and if successful leads to creation of new infestation. As bat bugs ( Cimex pipistrelli ) are not so often found on mist netted bats it was doubtful if they can be transmitted by bats especially on long distances. Previous studies found Nyctalus noctula the most often bat species carrying bat bugs on its body when mist-netted outside of its roosts. Therefore, we tested if bat species differ in numbers and status of bat bugs transmitted on their bodies. It was also shown before that mated females from cimicids, namely C. lectularius , attempt mostly to leave colony to start new infestation, we assumpted it will be also the case in bat bugs. To check the hypotheses we used three different bat species and mated or virgin females to see the numbers of bat bugs on bats emerging from bat box during volary sessions. The lowest numbers of bat bugs were found on M. daubentonii , whilst number of bugs on the body of M. myotis and N. noctula were much higher. Thorough N. noctula had on its body more unfed bugs comparing with M. myotis . On the body of N. noctula mated and virgin females were found approximately in the ratio 4:1. Our experiments clearly show that bat bugs prefer to disperse on bigger bats species such and further bat bugs suck longer on N. noctula comparing M. myotis , which make N. noctula more appropriate as bug vector.

Introduction Limited migratory dispersal behaviour of ectoparasites plays important role in their movements. Dispersal of ectoparasites that stay in host roost and feed occasionally when hosts are in their shelter, i.e. bed bugs ( Cimex lectularius ) and bat bugs ( Cimex pipistrelli ) are driven mainly by a transmission by humans or bats. However bugs can move from place to place even actively, but only within one buiding, i.e. in large attics inhabited by maternity colony (Bartoni čka and R ůži čková 2012) or among

89 different floors in building (Usinger 1966). Genetic research (based on both mitochondrial and nuclear markers) shown that C. pipistrelli create quite homogenous and stable population across Central Europe which indicates they posses high migratory level (Balvín et al. 2008, Balvín et al. 2013,Willems et al. in prep). Different reasons for bugs to disperse were described, with most important searching for new host due to lack of food, to avoid competition or to restart new colony (Pinto et al. 2007). In cimicids feeding efficiency, survival and development depends mainly on their hosts. However bats as host vary in ecology, biology and physiology, roost choice, what may affect their parasites transmission abilities. Roost choice in bats especially play important role, some type of roosts seems to be even species specific (Kunz and Fenton 2003). Roost choice differ also between seasons, between males and females or is releted to reproduction status (e.g. Borkin and Parson 2011). Balvín et al. (2013) found considerable morphological differentiation among batbugs from different bat species, although individuals representing particular mitochondrial haplogroups often live sympatrically and on the same host species. It seems that batbugs are morphologically adapted to a particular bat host despite the low genetic structuring among individuals parasitizing different species of bats. Morphological adaptations depending on host were found also in C. lectularius , when bugs from the lineage feeding on bats has shorter and more hairy legs than those feeding on humans (Balvín et al. 2012a). It was suspected that bats are the one of the main reservoirs of global recuperation and massive expansion of bedbugs (Szalanski et al. 2008). Despite of the fact that there is no evidence that bats could be responsible for bugs expansion (Balvín et al. 2012), synanthropic bats are forced to use alternative roosts near human more pressing than ever. Noctule ( Nyctalus noctula ) is the most commonly reported species within Central European city’s boundaries after 1990 (Hanák et al. 2009). At the same time, it is a species that logically most often gets into conflict with human. Moreover, the refusal of close co-existence with this species is strengthened due to it is believed that this species is a main responsible for carrying cimicids, what was explained that N. noctula most of all mist netted bat species is carring cimicids (Heise 1988, Rupp et al. 2004). Balvín et al. (2012b) found that 75% caught bats with bugs were noctules, what was explained by many aspects. Nyctalus noctula belongs to bigger size bat (up to 30g). It is found mostly in small, closed tree cavities with stable microclimate but also in bat boxes or in crevice-like human shelters (Gebhard and Bogdanowicz 2004). Limited grooming abilities in less spacious shelters together with bigger body surface and the fact that noctule switchs roosts quite often and is far migrating bat (more than 1000 km, Petit and Mayer 2000) can give opportunities for bugs to travel on (Balvín et al. 2012b). Sometimes noctules were found in one roost with other bats like M. daubentonii or Pipistrellus nathusii . Which can be important information concerning carrying cimicids between different roost, when shared with other bat species they can become infested by bugs or carry them further to another roost (Balvín et al. 2012b). However, it is not known whether there are differences in the length of the blood sucking or in time spent on the

90 body of different bat hosts. Differences in such behavior could affect the likelihood of bug transmisson to other shelters by different bat species. In most cases bugs transmitted on mist-netted bats adults with far outweigh amount of adults (Balvín et al. 2012b). When the most abundant early instars were found on bats only sporadically, adult females were the most often. However, to establish a new colony transported females must already be fertilized. Considering all mentioned above in our research we wanted to examine two main hypotheses i) N. noctula bats will carry more bugs on his body than other, same sized Myotis myotis and smaller but also dwelling bat M. daubentonii and ii) mated females of bat bugs ( Cimex pipistrelli ) will be often found on bat body than virgin females, as they can increase their ability to start new colony elsewhere.

Material and methods

Host specificity Twenty five adult female bugs of the Cimex pipistrelli were used in each session. Until the beginning of sessions bat bugs were kept in 20°C, relative humidity 70%, in dark, for 10 days without feeding. Five males of Nyctalus noctula , Myotis daubentonii and Myotis myotis fed each day after a session and had access to water enriched by vitamins ad libitum. All bats were returned to their original colony after the sessions. During captivity, the light regime was natural and air conditions stable. All sessions were held in the outside volary (3 × 3 × 2.5 m) equipped with wooden bat box. In the mornings bugs were placed in bat box occupied by bats, on the small shelf (Fig. 1). Bat box was equipped with a camera (SONY DCR SR 52E) to monitor bat behaviour before emerging. Evening after bats come out from torpor and their body temperature raised, bat bugs started to be attracted by them. First movement in the bat box before emerging, time of the first bat leaving and period of emerging all bats from the box were recorded. After bats left the box, they were caught and amount of bugs on bat body, in box and status of bugs – fed/unfed was recorded. Experiment was repeated for 30 days, i.e. 10 days for N.noctula , 10 days for M.daubentonii and 10 days for M. myotis. All bats were adult males in similar weight range ( M. daubentonii – 8.3±1.4 g, M. myotis – 23.8±2.1 g, N. noctula – 22.7±2.6 g)

Transmission and fertility of bugs Experimental setting was same as in host specifity experiment. However bat box was not divided into two parts and occupied only by five males of N. noctula . We used mated females of bat bugs – one that were day or two after mated with males and virgin females, i.e. females that were freshly molted from 5 th instar and kept separately from males. Virgin females were marked white on abdomen. Similarly to previous experiment 15 mated and 15 virgin bugs were added morning in a box and after bats left bat box, amount of mated females present on bat body and stayed in box was noted as same

91 for virgins and bug status fed/unfed. Experiment was also repeated for 10 days. All bats were captured, handled and temporarily kept in captivity under the licence South Moravian Regional Authority Permit JMK 24451/2013 and 63761/2017. In addition Tomáš Bartoni čka is authorised to handle free-living bats under Certificate of Competency No. CZ01297 (§17, law No 246/1992), No. 922/93-OOP/2884/93 and 137/06/38/MK/E/07 of the Ministry of Environment of the Czech Republic.

Statistical analysis All variables showed a normal distribution. Statistica 12.0 for Windows and SPSS (IMB Statistic 21.0) were used for data analyses. We used one-sample ANOVA to compare three bat species as groups to test numbers and status of bat bugs, i.e. unfed and fed bugs in a box, unfed and fed bugs on a bat and bat emerging behaviour. The significance of numbers of bugs in each status (fed/unfed and mated/virgin) was further analysed using a two-sample t-test and Bonferroni correction when independent tests were being performed simultaneously.

Results

Transmission by different bat species

During 30 volary sessions conducted with three bat species we found significant differences between

numbers of bat bugs found on the bat bodies (ANOVA, F 2,27 =44.43, p<0.001) and in the box

(ANOVA, F 2,27 =0.4725, p=0.634), N. noctula left bat box earlier than other species (F2,27 =26.25, p<0.001) (Tab. 1). Morever N. noctula started be active as the leatest from studies bat species

(F2,27 =12.05, p=0.011)(Tab.1).

Discussion

Both hypotheses that we wanted to examine were confirmed. Our results indicate that the N. noctula transported more bugs from the box than Myotis myotis and M. daubentonii , despite of the fact that Myotis myotis was confirmed as one of the most often host along with M. emarginatus and Pipistrellus spp. (Balvin et al. 2012). Nyctalus noctula seems to be regular vector of bugs (Balvín et al. 2012b). Rupp et al. (2004) found cimicids on 15% of 221 individuals of mist netted N. noctula , while no single bug was found on 793 individuals of the other 17 bat species. Seems that as in nature mechanisms of choice of bat host, which can be used for movement to another bat roost/colony shows the same pattern in laboratory. Lack of presence of bat bugs on other bigger sized mist-netted bats, which are highly infested with those ectoparasites was explained by roosting conditions i.e. M. myotis or M. emarginatus roost in bigger places and thanks to that can easier groom and get rid of bugs than N. noctula that occur in tight crevices (Balvín et al. 2012b). Maternity colonies of this two species create stable blood source due to high roost fidelity and it is not surprising that lactating females of M. myotis or M. emarginatus , were found rarely to carry bugs (Balvín et al. 2012b). Contrary to N. noctula , females and juveniles of M. daubentonii and M. myotis were found to be faithful to some roosting and foraging sites (Kapfer 2008, Zahn 1999), which may create more suitable conditions for bugs to stay in this bat roost rather than to be transmitted to other place. Despite of the similar period during that bats have been emerging the box among species, N. noctula left bat box much earlier than other studied bats and aroused from daily torpor as the leatest species. During day time bat can use torpor to reduce daily energy expenditure (DEE) which can be connected with lower food availability, temperature or both those factors (Coburn and Geisler 1998; Hosken 1997; Kurta 1990). Few studies shown differences in torpor between males and females. Females of Myotis daubentonii fall into torpor rarely during maternity period, while males stay in torpor quite regularly even few hours per day with lowest skin temperature of 16.8°C (Dietz and Kalko 2005). Similarly in other bat species Eptesicus fuscus both females and males felt into torpor but in case of males it was much deeper torpor comparing to reproductive females (Grinevitch et al. 1995; Hamilton and Barclay 1994). What is more, pregnant and non-pregnant females went into torpor much often than lactating females of this species (Audet and Fenton 1988; Chruszcz and Barclay 2002). Lactating females of bats as M. myotis or M. emarginatus also rarely use torpor and stay active for most of the time so they attract bugs most of the day, so the possibility of the

93 coincidental bug transport is less probable comparing to males for example who often use torpor and after being awake attract ectoparasites, that come to the host to feed and can be carried by coincidence on their body (Balvin et al. 2012). If differences between sexes of the same species exist also differences between two species such as N. noctula and M. daubentonii in torpor behaviour and mechanism can be present. N. noctula and M. daubentonii are both dwelling bats, nevertheless their roost sites in trees may vary considerably regarding for example thermal microclimate (Humphrey et al. 1977). Research on roosts choice between N. noctula and M. daubentonii shown similar pattern in choice, nevertheless some differences were found. Boonman (2000) observed that N. noctula were using woodpecker cavities more than M. daubentonii , preferring them above natural cavities, while M. daubentonii preferred oaks over beeches. Both were found to roost at the forest edge (Boonman 2000). Also, bat such as M. daubentonii was noted to use more spacious roosts but still compact, that together with situation of sharing roost with other bat species (e.g. Nyctalus noctula , N. leisleri or M. myotis ) which is often observed (Natuschke 1960, Swift and Racey 1983 etc.) or when bigger amount of individuals of same species occupy one shelter (up to 144 individuals for M. daubentonii ; Encarnação et al. 2005) reduces grooming ability. N. noctula during foraging can change their roost and can be found even 50 km far from their roost (Popa – Lisseanu et al. 2004). That gives bugs opportunity to enlarge its scope and find better places. noctules were found often in human shelters (houses, flats roofs etc.), which they use as roosts, colonies or even hibernating sites. While carrying mated females, they can infest humans with both bat and bed bugs (Balvín et al 2012a). Such pattern is continuously observed and doesn’t have good impact on humans good attitude towards bats, even if C. pipistrelli was found to suck on them but not able to develop (Southwood 1953). Also body size can play crucial role, N. noctula has bigger body surface than M. daubentonii , also wing membrane is bigger (161cm 2 in N. noctula and 98cm 2 in M. daubentonii , Norberg and Rayner 1987, Webb et al. 1992) (Tab. 2). Body mass of Noctule and greater moue-eared bat is very alike so bugs will find both species more attractive than daubenton´s bat. It was shown that bugs

seeking host behaviour depends on concentration of the ambient CO 2. Comparing noctules and mouse

– eared bat with daubenton´s it is quite logic that bigger bat will produce more CO 2 and will attract bugs more towards them. Same in case of response on caradian rhythm which was suggested to attrack bugs towards host (Romero et al. 2010). In M.myotis very low genetic variation was found among bat bugs from different maternity colonies of that bat species (Willems and Bartoni čka, in prep.) which would indicate high level of transmission between different roosts, however no records during field experiments proving such situation, in contrast to N. noctula . On the body of N. noctula we found a higher number of unfed bugs compared to M. daubentonii and M. myotis . It was observed by many chiropterologists that wing membrane of N.

94 noctula is being stronger than in other bats from genus Myotis , even same body size M. myotis (Kunz & Fenton 2003), where mostly bugs are found not only to fed but also being transported (Heise 1988). On the other hand wing membrane of M. myotis posses bigger surface (Tab. 2). Same for mated females who were found much more often on bat body than virgins. Such behaviour was already before confirmed as not coincidental (Heise 1988). Experiments on both adults and nymphs shown that nymphal stages in composition and density experiments stayed closely to adult bugs, preferring staying in groups. Similarly males preferred to stay in groups and were found alone just in rare situation. Opposite, females were often found solitary, separated from other bug groups, meaning they do not produce at all or just in smaller amount aggregation pheromone (Siljander et al. 2007), which allows them to leave group without being followed by other bugs. Pfiester et al. (2008) suggested that this fact help females to move to other niche, develop new hosts and even establish new bugs colony and that females are responsible for bugs movements and dispersal. That would confirm earlier observation of females bed bugs found far from aggregations (Siljander et al. 2008). Also what is interesting most bugs found on mist netted bats were adults from which greater number females (Usinger 1966; Balvín et al. 2012b). In all cases bugs found on bat body were adults (Balvín et al. 2012b), no instars were found, despite that they are also present in bat roosts and colonies. All above highly suggest that female bugs (often mated) stay on host body in purpose of migration and re-colonization of new places. Our experiment clearly shows that bugs prefer to migrate on bigger bats species such as noctule or greater mouse – eared bat than on smaller host. It is not fully clear if body size is the only reason as both bigger species used in our experiment weight alike (ca 40g). Nevertheless comparing both M. myotis is the one that is more preferred above Noctule bat to migrate. What is more we have shown also that migration is more common in mated females of bat bugs. Comparison of day profile of few crucial hosts that are used by bugs to migrate would be recommended. Some bats wake up earlier from torpor than the another which is connected with raising body temperature and having impact on bugs feeding on them – as mentioned before body temperature and emission of CO 2 attracks bugs. Bugs are also learning during their life time and there can be difference between adult, experienced females and freshly developed female virgins in exploration of hosts body, sucking time, used body parts and migration willingness. Because all bats differ in their anatomy would be practical to examine and compare how bugs attack noctules vs other bat species, which body parts play crucial role, which are used to suck and which to migrate on. What is more could be practical to gain knowledge about sucking time and time that bugs spent on different bat host species, because that can also determine not only feeding efficiency but also migration occurrence.

Fig .1. Bat box interior designed for volary experiments on one bat species, on scheme visible landing wooden shelf (black) that allow bugs to freely move in the box.

Fig. 2. Percentage of bugs found during experiments in three bat species. A, b, c show significant differences among numbers of bugs of different variables in one species (p<0.05).

9 1,8

1,6 8

1,4 7

1,2

6 1,0

0,8 5

0,6 number of bat bugs 4

0,4

3 0,2

0,0 2 mated virgin mated virgin A B

Fig. 3. Numbers of mated and virgin females found on N. noctula in one session (A) and number of all fed mated and virgin females during all sessions found on bat bodies (B).

Species/variables Time (min) Period (min) First move (min) M. daubentonii 44.6±8.51 21.0±5.89 146.8±39.09 M. myotis 24.8±5.30 24.8±5.78 224.5±53.42 N. noctula 0.6±14.57 20.5±7.79 62.4±25.61

Tab. 1. Emerging behaviour in studies species. Time – time after sunset, when first bat emerged from the box (mean±SD), period – length of emerging period (mean±SD), first move – first movement of bats in the box before emerging (mean±SD).

Species M (g) B (cm) S (cm 2) ar N/m 2 TI Myotis myotis 26 38 233 6.3 11.2 1.22 Myotis daubentonii 7 25 98 6.3 7 1.22 Nyctalus noctula 26 34 161 7.4 16.1 1.43

Tab. 2. Wings measurements comparison. M (g) – body mass, B (cm) – wing span, S (cm2) – wing area, ar – aspect ratio (B2 S), N/m2 – wing loading, TI – tip length ratio. (From Norberg and Rayner 1987 and Webb et al. 1992).

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ARTICLE I

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Parasitol Res (2013) 112:3897 –3904 DOI 10.1007/s00436-013-3579-9

ORIGINAL PAPER

Two different lineages of bedbug (Cimex lectularius ) reflected in host specificity

Kamila Wawrocka & Tomá š Bartoni čka

Received: 7 June 2013 / Accepted: 12 August 2013 / Published online: 28 August 2013 # Springer-Verlag Berlin Heidelberg 2013

Abstract Co-speciation between host –parasite species is generally Introduction thought to result in mirror-image congruent phylog-enies. For the last several centuries, many bat species have been turning Host specificity and anatomical and morphological adapta-tions synanthropic, especially those that are hosted by bedbugs in are essential for understanding the variability of life strategies Europe. There is evidence of only limited gene flow from the and the evolution of parasitic species. There is a wide list of population of people to the population of bats. This study was parasites that are connected with a host via their life cycle and focused on comparison of survival, develop-ment, and the that fact, next to dispersal limitations, decrease potential host reproduction rate based on cross-feeding exper-iments. In our switching and limit host ’s range. Most parasites occur on a research, we used two bedbugs groups of Cimex lectularius —bat- restricted number of hosts and show some evidence of specificity. and human-associated and respectively as specific/non-specific Specialization for particular hosts may result by fidelity to host bat and commercial human blood. Both lineages show different hosts and sympatric occurrence at the same time, host- different behavior according to their host preferences. During the associated genetic differentiation, and/or restricted but bat blood experiment, we found sig-nificant differences between appreciable mutual gene flow (Dres and Mallet 2002 ). 2 both human- and bat-associated bedbugs (Log rank test fourth χ Under such conditions, closely related parasites may be spe- 2 =9.93, p >0.05; fifth χ = 11.33, p <0.05), while no differences cialized in a particular host. Poulin ( 2007 ) described two host occurred with the human blood experiment between the survival choices by parasite: (1) encounter filter, when parasite ex-cludes levels. In molting, differences between both groups were the host which he can not colonize and feed on because of significant particularly in the case of the bat blood experiment behavioral or ecological reasons and (2) compatibility filter, 2 (fourth χ =5.91, p < 0.05). In the case of the bat blood excluding all host individuals on which parasite can not feed experiment, we found a higher probability of molting in bat- because of morphological, physiological, and immunological associated groups than in human-associated groups. In the case of reasons. Host specificity was defined by Dick and Patterson the human blood experiment, molting probability was stable in (2007 ) as a degree to which a parasite species occurs in both specific and non-specific, showing similar pattern in both association with host species. Host-specific parasites general- cases for all stages. Our results indicate an occurrence of two ly have a major-primary host (5 % or more host individuals ecotypes within the one species C. lectularius. These findings are infested) and a few less frequently used hosts (Tripet et al. support earlier data about morphological and mitochondrial DNA 2002 ). Even generalists show a preference for some species differences. The differentiation of both lineages fits the con-cept above others (Tripet and Richner 1997 ; Johnson et al. 2002 ). of specific host choice. The bedbug, Cimex lectularius Linnaeus, 1758, is likely the most widely known cimicid species. Fast ontogenesis,

high reproductive potential, mobility, and at the same time, a : hidden way of life make bugs (Cimicidae) important ectopar- K. Wawrocka ( *) T. Bartoni čka asites. Despite the fact that bedbugs were found on 8 species of birds, 10 rodents and mustellids, 10 bat species, and Department of Botany and Zoology, Masaryk University, Kotlá řská 2, 611 37 Brno, Czech Republic humans, we recognized only two primary hosts —human and e-mail: [email protected] the greater mouse-eared bat (Myotis myotis) (Povolný 1957 ). T. Bartoni čka Since other hosts have different in ecology and behavior, their e-mail: [email protected] infestation is probably relatively recent (Povolný and Usinger

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1966 ; Usinger 1966 ); the bedbug represents an optimal and lengths of rostral segments and dimension of antennal model for a study on initial stages of host specialization. segments or eyes. If these differences occur, we can expect Since the last decade, many studies of re-emerging differences as well on an ecological and developmental bedbugs in USA and Europe were published (Romero et al. level (Usinger 1966 ). 2007 ; Reinhardt et al. 2008 ). To find out the existence of host specificity within bedbug In some cases, individuals from the Cimicidae family, C. lectularius, we carried out an experiment which is sup- during the absence of the natural host, are able to switch on posed to show not only the ability to suck on different hosts non-specific host (Cimex pipistrelli, Whyte et al. 2001 ; but also to illustrate different survival between two lineages of Cimex dissimilis , Smaha 1976 ). But to make the substitute bedbugs (bat and human associated) under specific and non- host proper for parasites, it has to fulfill certain conditions, specific host feeding. The aim of the study was to check what espe-cially morphological, physiological and behavioral. and if there are some reasons for host choice as specific ones As Reinhardt and Siva-Jothy ( 2007 ) proven, such and how the host choice influence on bugs life expectancy, limitation could be in some cases the size of the red cells. speed of molting, or mortality. Erythrocytes diameter can influence sucking ability (e.g., chicken erythro-cytes are almost twice as large as those in humans, 11 to 6 µm). Moreover, as was shown before, also Material and methods sex, age, and reproductive status has an influence on the reproduction of parasites (Lourenço and Palmeirim 2008 ). Sampling of bedbugs Recent studies suggest that Central European populations of some bat species have shifted their roosting strategy in the last decade, Bedbugs (C . lectularius) associated with bats were sampled with prefab houses becoming places of their most frequent from a nursery colony of greater mouse-eared bats (M . occurrence. Large-scale renovations of those objects, currently myotis ) roosted under roofs of the church in Hanu šovice performed in Central Europe, pose a serious threat to populations (north Moravia, Czech Republic). Other samples of bedbugs of several bat species. In cases when the bats switch their roosts, associated with human host were sampled from hostels in the or they are to dislodge from shelters by renovations of houses, cities of Ostrava and Bohumín (north Moravia, Czech people complain repeatedly about the presence of cimicids in Republic). Bugs were collected with soft forceps and ex- their flats. These bugs intensively feed on people (T. Bartoni čka, hausters into small plastic boxes (10×10×5 cm) lined with soft P. Schnitzerová personal observa-tion). These records not only paper. Together, we had 27 samples (one female and a few come from C. pipistrelli, which is more often found on bat hosts, males) for each host lineage i.e., human- and bat-associated but also from C. lectularius. The ability of C. pipistrelli in bedbugs in each experiment. In each tube appeared different attacking people and sucking on them was confirmed, but they stages (from egg –adult). are not able to develop and undergo a full cycle. Adults bugs that were fed on human blood and laid eggs were successful but Equipment and experimental settings hatched larvas did not want to suck human blood (Southwood 1953 ). According to previous papers, it seems that blood meal During segregation and forming groups, we avoided infesta- temperature, not its specificity, play a crucial role in blood tion of samples with eggs or instars from delivered tubes and feeding experiments (Moloo 1971 ). Balvín et al. ( 2012 ) suggests chose only adult individuals. To sort the samples, individual that switches between the bedbugs were immobilized by sudden “freezing ” at 0 °C for human- and bat-associated groups of C. lectularius are only 10 min. Experimental groups (one female and one or more occasional since their split and that the bedbugs mostly switched males) were stored in separate plastic pellucid tubes (6×1 cm). from humans to bats. Beside the host choice or less fitness of Each tube was equipped with a piece of paper 4×1 cm to let hybrids, this could be also a reason for the degree and shape of the bedbugs move, defecate, and lay eggs on it. All tubes were the mutual gene flow; nevertheless, the limited evidence of the numbered and tightly closed with a piece of cotton. contact of the two populations (bat- and human-associated bugs), Afterwards, they were positioned in a thermostatically con- shows that bats could serve as reservoirs, covers the temporary trolled apparatus (ST2, POL-EKO Aparatura, Poland) under absence of the primary host, and can contribute to the current stable temperature (27±0.1 °C) and humidity (75±10 %) dramatic spread of the bedbug among humans. which according to Omori ( 1941 ) and Usinger ( 1966 ) was Moreover, some previous studies (e.g., Usinger 1966 ; best for the development of C . lectularius . Humidity was Balvín et al. 2012 ) show that bedbugs are also adapted to their controlled using water in a Petri dish on the bottom of the host morphologically. Balvín et al. ( 2012 ) shows differences glass jar with samples and damp cotton. Bugs were fed every in relative leg lengths, i.e., longer legs in human-associated 4 days. To facilitate observations during everyday checks and bedbugs and shorter, stronger legs and more hair in bat- feeding, up-to-date dead individuals and rest from after molting were taken out and stored separately according to associated bedbugs. It also shows differences within widths

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particular instars. All experiments were carried out for all statistic analysis. A mortality (molting) of 50 % in a between April and October 2012. sample was considered significant to finish the experiment and such a session was marked as complete in the database. Other Bat blood feeding groups were evaluated as censored. The differences in survival rate and molting speed among age groups were tested using the Special cylinder tube with one bat and a sample of the bedbugs Kaplan –Meier survival functions. For the Cox proportional were placed together in a dark thermostatically controlled unit hazards model, the chi-square value was estimated as a function (30 °C) for 15 min (cf. Usinger 1966 or Giorgi et al. 2004 ). To of the log-likelihood for the model with all covariates. It was reduce stress for animal and to avoid antiparasitic behavior suspected that the effect of the treatment (exposure to different including eating bugs, the bat was covered with dressing gauze host) on the underlying hazard was not constant; that is, that the and situated in a feeding tube. Before putting bugs into the proportionality assumption may be violated. To check the feeding tube, they were counted to be sure that afterwards, all of differences in molting and survival rate among age groups them were collected back. After feeding period, the bat was then models, we used the Log rank test. We also used the Wilcoxon taken out from the tube, carefully revised, especially wing test to compare survival distribution among groups. Weibull membrane, ears, and uropatagium, and bugs that still were regression was used to estimate molting probability for certain attached into the bat ’s body were delicately removed with instars in both human and bat-associated bedbugs. Differences entomological soft tweezers to counteract crushing, especially between specific and non-specific hosts in amount of the days very soft instars. The number of bugs and their feeding status after which they are able to undergo molting were tested using (fed, unfed) were determined. In total, two non-reproducing Mann –Whitney U test. females of Vespertilio murinus and two males of M. myotis were used and the bats were changed after feeding each two bug groups. Bats were fed ad libitum with a mixed diet consisting of Results crickets (Acheta sp.) and mealworms (Tenebrio molitor) and after experiments, returned back to the colony. The bats were captured, Survival at different host-associated bedbugs handled, and temporarily kept in captivity under the license no. 922/93-OOP/2884/93 and 137/ 06/38/MK/E/07 of the Ministry of During analysis, we excluded individuals who undergo Environment of the Czech Republic. The author has been molting or were alive. No different changes were found in the authorized to operate with free-living bats according to the survival rate within instars in the case of bat blood in bat- certificate of competency no. 104/ 2002-V4 (§ 17 of the law No. associated bedbugs, specific host (Kaplan –Meier survival test, 2 246/1992). χ =7.56, df =4, n.s.) neither in the case of human-associated, 2 non-specific host ( χ =4.61, df =4, n.s.) (Fig. 1). While we Human blood feeding compared both lineages fed on bat blood, we found statistically significant differences in the fourth and fifth instars at 2 Human blood for feeding (laboratory commercial blood, survival rate (Log rank test, fourth instars χ =9.93, p <0.05 2 Japan Medical Supply, B+) was stored in a fridge. Durex and fifth instars χ =11.33, p <0.05). Cox proportional haz- condoms were tightly fitted on the plastic tube, which perfect- ards model for bat blood experiment showed that the differ- ly fit over bug-breeding tubes. During artificial feeding, blood ences between bat and human-associated bugs are 2 was warmed up to 45 °C in the cylinder and 5 ml of blood significant ( χ =22.51, df =9, p =0.007). was added to each roller. It was crucial to give bugs access to Same tests were made also for human blood experiment, the blood meal, thus, the situate roller was low enough and where no statistical differences were found among instars in covered all surface of the condom membrane with blood (cf. human-associated bedbugs and specific host (Kaplan –Meier 2 Montes et al. 2002 ). Feeding tubes were closed and thermo- survival test, χ =7.56, df =4, n.s.), neither in bat-associated 2 statically controlled under stable temperature 27 °C. The bedbugs and non-specific host ( χ =2.08, df =4 n.s.). While most suitable blood temperature was experimentally comparing both associated groups fed on human blood, no established on 37 –38 °C. Too high or too low (<35 and statistically significant differences between instars were >38 °C) didn ’t attract bugs (K. Wawrocka, personal found. Cox model for human blood experiment compared observation). Feeding was con-tinued till the moment bat- and human-associated bugs and did not show 2 when ca 80 % of individuals had taken the blood. statistically significant differences ( χ =15.28, df =9, n.s.).

Statistical analysis Different molting in host lineages

All variables showed a normal distribution after log transfor- Evaluating the different molting rate among instars and host mation. SPSS for Windows 7.0 (IBM Statistics 19) was used lineages we conducted by Kaplan –Meier survival test where

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Cumulative number of survivors

days

Fig. 1 Cumulative survival function for first –fifth instars, showing the functions of instars in case of human blood in human-associated (c) fraction surviving according to different blood meal (specific —a, c/non- and bat-associated bedbugs (d). “Complete ” means at least 50 % specific —b, d). Kaplan –Meier survival functions of instars in case of bat mortality in particular instars blood in bat-associated bedbugs (a); in human-associated (b); survival

we tested the level of molting. We excluded dead individuals statistically significant difference exists in the molting of 2 and those who didn ’t undergo molting, but survived. fourth instars (Log rank test, fourth instars χ =5.91, p < In the case of bat blood experiment, we found no statisti- 0.05). Nevertheless, the Cox model did not show any 2 cally significant differences among all instars of bat and statisti-cally significant differences ( χ =14.61, df =9, n.s.). 2 human-associated bugs (Kaplan –Meier survival test, χ = We tested in the same way human blood experiments and 2 5.023, df =4, n.s.; χ =5.242, df =4, n.s.). When we compared found no significant differences among instars of human- 2 both host lineages between each other, we found that a associated bedbugs ( χ = 1.16, df =4, n.s.) but we found

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2 differences in the case of bat-associated bugs ( χ =11.66, df = 4, probability was stable in both specific and non-specific show- p =0.02). Comparisons of both human- and bat-associated bug ing similar pattern in both cases for all instars (Fig. 2). lineages showed that there is no significant difference between them. Cox model did not show significant differences between Time period between ecdyses 2 molting in both lineages ( χ =10.59, df =9, n.s.). We found the differences between specific and non-specific hosts Probability of molting in the amount of days after which they are able to undergo molting. Analyzed data starts from the first instar till becoming Different probabilities of undergoing molting in time (days) adults. Mann –Whitney U test showed in both cases that ecdyses for certain instars is described by Weibull regression. We time differs significantly only in bat blood experiment (U =5.0, found significant differences between bat- and human- n.s.) as in human blood experiment (U =11.5, n.s.) (Table 1). 2 associated bedbugs in the case of bat blood experiment ( χ =75.18, df =9, p <0.001) as well in the case of human blood 2 experiment ( χ = 54.04, df = 4, p < 0.001). Bat-associated Discussion bug shows low molting in earlier instars (first – third) and higher in later instars (fourth, fifth) and in same case, human- Although in the course of research in the last decades there associated bugs had the highest molting proba-bility of early have been found numerous occasional hosts of bedbugs instars (second) and the lowest in later instars (fourth). In the (Cimex lectularius ), the primary hosts are only bats and case of human blood experiment, molting humans (Usinger 1966 ). Previous studies have suggested that

Probability of metamorphosis Probability of

days

Fig. 2 Probability of molting. Weibull regression for first –fifth instar bat blood in bat-associated bedbugs (a), human-associated bedbugs showing probability of metamorphosis according to different blood meal (b), regression model for human blood experiment in human- (specific a, c/non-specific b, d). Molting probability of instars in case of associated bed-bugs (c), and bat-associated bedbugs (d)

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Table 1 Ecdyses between undergoing molting in certain stages (first — adult) for specific and non-specific host in bat and human blood experiment (days ± SD)

Experiment/age 1st (Specific/ 2nd (Specific/ 3rd (Specific/ 4th (Specific/ 5th (Specific/ Adults (both sexes) stages non-specific) non-specific) non-specific) non-specific) non-specific) (specific/non-specific)

Bat blood 5±0.81/6±0.74 4±0.89/5±0.74 3±0.64/6±1.12 4±0.67/5±0.48 4±0.90/6±0.95 7±0.32/9±0.46 Human blood 4±0.96/6±0.87 3±0.68/5±0.79 7±0.87/10±1.09 8±0.46/15±1.47 4±0.64/2±0.44 9±0.93/7±1.10

bedbugs ’ primary host were bats (Povolný and Usinger myotis and human very often inhabit buildings, populations of 1966 ) and from them, they were transferred on humans. bat and human-associated bedbugs show a minimum ex- Recent studies (Balvín et al. 2012 a) showed an interesting change of individuals between them (Balvín et al. 2012 ). host-associated differentiation of the population of the Balvín et al. ( 2012 ) found that nursery colonies of greater bedbug on both morphological and molecular data. mouse-eared bat are more often parasite by C. pipistrelli than We are seeing probably a gradual ecological niche diversi- C. lectularius and moreover, both bug species have never been fication and perhaps the origin of reproductive isolating bar- found together in one bat roost. However, on human host, C. riers. From this perspective, the study of the host in C . pipistrelli is found very rare always in anecdotic observations, lectularius specificity seems to be a good model for the study especially if an infested apartment is near to temporary of microevolutionary mechanisms connected with speciation abandoned bat roost and hungry bugs are looking for food. It and adaptive radiation within the group (Fig. 3). has long been known, that C . pipistrelli can feed on human In our study, we used two host lineages of C . lectularius — host, but it has a significantly lower survival rate than on a bat- and human-associated bedbugs. In cross-feeding experi- specific host bat (Usinger 1966 ), and at the same time, there is ment type of the host, specific/non-specific, seems to have an a notable reproductive barrier between C . lectularius and C . impact on survival, molting, and development rate in both pipistrelli. This isolation of particular bug populations shows cross-feedings conducted in vitro. The most common bat host a very limited capacity to transport and dispersion to another of the bedbug, M. myotis, is originally the cave-roosting bat. or even new host (cf. Giorgi et al. 2004 ). It is perhaps a coincidence that this species began to inhabit Host switching is usually complicated, especially when the buildings all over Europe only several centuries ago. primary host has a very different life strategy than the potential Moreover C. lectularius is not found in their cave roosts in new host. Bat-associated bugs usually inhabit only these parts of Europe (Simov et al. 2006 ), likely because of the climatic buildings attics which are suitable for bats, but from there, they conditions in caves. The only exception in central Europe is have only a limited ability to move (Bartoni čka and R ůžičková the Hranická abyss, which roost a large nursery colony of M. 2012 ). The frequency of movements between bugs and man is myotis, but the bedbugs were never found on emerging bats hard to monitor and on this issue, we have only limited infor- despite repeated nettings (Z. Řehák in litt.). So, while M . mation (Balvín et al. 2012 ). Adult bedbugs can travel at a speed of 126 cm per minute (Hase 1917 ) and therefore, they should be able to reach large distances very quickly. When bugs can follow

fecal spots, food prints, or pheromone trails by dragging the engorged bugs, (Hase 1917 ; Aldana et al. 2008 ) they can successfully travel to very distant hosts (Kemper 1936 ). In cases when the host is available, bat-associated bugs have no reason to

find a new one. The probability of sucking on humans is therefore not reduced by the distance of infested attics from the people occupied parts of buildings, but also by human effort to eliminate new bug population in the case of successful trans-mission.

Moreover, no cases of movement of C. lectularius from bat host to people in the same building have yet been published. Bugs are adapted to long starving periods. It might confirm Romero et al. ’s (2007 ) experiment, where individuals starved for a shorter time

showed much higher activity and movement level than those who were starving for a few weeks. This might help them to limit Fig. 3 Bed bug (Cimex lectularius) bat-associated. Visible main characteristic differing them from human-associated bugs: stronger and shorter energy loss connected with movements and decrease metabolism. legs, more hair, difference in antennal segments dimension and eye which It appears, therefore, that the bugs are adapted to wait rather than is connected with adaptations to certain host (by O. Balvín) to actively search the

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host. Such strategy could lead between the two host- taxonomical status of both bedbug lineages could be to verify associated bug groups to higher degree of mutual isolation. whether between the two ecotypes, are there any reproductive Blood sucking parasites may favor and choose higher mechanisms limiting the free hybridization of bedbugs. quality hosts who can give them a better blood meal. On the other hand, such host might be hard to feed on (Møller 2000 ; Acknowledgments We are very grateful to J. Gaisler for his valuable Khokhlova et al. 2007 ). Our experiments clearly show the comments on the manuscript and Martin Toman for help with fieldwork and sampling bedbugs associated with human host. This importance of the specific host. However, it can not be as- study was supported by grants from the Ministry of the Environment sumed that the quality of the blood of a bat and human should and the Ministry of Education, Youth and Sports of the Czech be fundamentally different, because other studies showing the Republic No. MSM0021622416,grant No. MUNI/A/0976/2009. gradual isolation of both bedbug populations associated with different host lineages (Povolný and Usinger 1966 ). As suggested before (Hase 1926 ; Tawfik 1968 ), we did not find any significant differences between bat and human red cells diam- References eter (Wawrocka in prep.). The cause of low survival rate on non-specific host might be in immunocompetence (T cell Aldana E, Abramson CI, Lizano E, Vegas R, Sulbaran-Romero E (2008) response) and the condition of the host, as evidenced by the Learning and orientation to odor in the bug Rhodnius prolixus numerous proofs has been "well-fed host strategy" (Christe Stal 1859 under laboratory conditions. Parasitol Res 103:587 – 594. doi: 10.1007/s00436-008-1014-4 et al. 2000 ). Previous research showed already differences at Balvín O, Munclinger P, Kratochvíl L, Vilímová J (2012) Mitochondrial morphological level in case of bedbug according to the host DNA and morphology show independent evolutionary histories of bedbug type —human or bat (Balvín et al. 2012 ). Host specificity has Cimex lectularius (Heteroptera: Cimicidae) on bats and impact not only on the parasite himself but also on his host, humans. Parasitol Res 111:457 –469 including the human. Preferences to some hosts can, on one Bartoni čka T, Gaisler J (2007) Seasonal dynamics in the numbers of hand, decrease feeding efficiency but on the other hand, make parasitic bugs (Heteroptera, Cimicidae): a possible cause of roost switching in bats (Chiroptera, Vespertilionidae). Parasitol Res it easier for parasite to feed on host, whose immune system 100:1323 –1330 and ecology is already well known to him. As Dick et al. Bartoni čka T, R ůžičková LL (2012) Bat bugs (Cimex pipistrelli) and their (2009 ) suggested that lack of some barriers that occur in impact on non-dwelling bats. Parasitol Res 111:1233–1238 Christe P, Arlettaz R, Vogel P (2000) Variation in intensity of a parasitic nature can break down host specificity. Bats, as partly synanthropic species, adapted to new con- mite (Spinturnix myotis) in relation to the reproductive cycle and immunocompetence of its bat host (Myotis myotis ). Ecol Lett ditions and also treat connections with human population 3:207 –212 expansion that impact their natural landscape, food decrease Dick CW, Patterson BD (2007) Against all odds: explaining high host by habitat adaptations, and degradation, forcing bats to use specificity in dispersal-prone parasites. Int J Parasitol 37:871 –876 alternative roosts and foraging sites and move to cities. They Dick CW, Esberard CEL, Graciolli G, Bergallo HG, Gettinger D (2009) start to share shelters with humans (attics, churches, base- Assessing host specificity of obligate ectoparasites in the absence of dispersal barriers. Parasitol Res 105:1345 –1349 ments, vacant buildings, etc.), what provided them cozy, Dres M, Mallet J (2002) Host races in plant-feeding insects and their warm, and safe places that they would not find in the wild. importance in sympatric speciation. Philos Trans R Soc B-Biol Bat –human conflict is a well-known phenomenon. Bat noises, Sci 357:471 –492 droppings, and presence itself is a big problem for bat conser- Giorgi MS, Arlettaz R, Guillaume F, Nusslé S, Ossola C, Vogel P, Christe P (2004) Casual mechanisms underlying host specificity in bat vation. What more is that together with bats appear also their ectoparasites. Oecologia 138:648 –654 parasites as cimicids. It was suspected that bats are the one of Hase A (1917) Die Bettwanze (Cimex lectularius L.), ihr Leben und ihre the main reservoirs of global recuperation of bedbug popula- Bekampfung. Z Angew Entomol 4, 144 pp tions (Szalanski et al. 2008 ) thus they do not possess other Hase A (1926) Über verfahlen zur Untersuchung von Quaddeln und than roosts switching (Bartoni čka and Gaisler 2007 ) the abil- anderen Hauterscheinungen nach Insektenstichen. Z Angew ity to reduce infestation. That fact could unfortunately deepen Entomol 1926:243 –249 Johnson KP, Williams BL, Drown DM, Adams RJ, Clayton DH (2002) antipathy to bats. Nevertheless, there is no evidence at the The population genetics of host specificity: genetic differentiation in genetical level that bats could be responsible for their expan- dove lice (Insecta: Phthiraptera). Mol Ecol 11:25 –38 sion (Balvín et al. 2012 ) which is much more wider in the Kemper H (1936) Die Bettwanze und ihre Bekämpfung. Z Kleintierk case of, for example, poultry (Szalanski et al. 2008 ). Pelztierk 12:1 –107 Khokhlova IS, Fielden LJ, Degen AA, Krasnov BR (2007) Feeding Spreading this information and knowledge among society is performance of fleas on different host species : is phylogenetic crucial in bat conservation. distance between hosts important? Parasitology 139:60 –68 This study showed the significant limitations in the ability Lourenço S, Palmeirim JM (2008) Which factors regulate the reproduc- of survival and molting in confusion of the two primary bug tion of ectoparasites of temperate-zone cave-dwelling bats? Parasitol hosts and it is likely to fit the concept of host races according Res 104:127 –134 Montes C, Cuadrillero C, Villela D (2002) Maintenance of a laboratory to Dres and Mallet ( 2002 ). The next step to elucidate the colony of Cimex lectularius (Hemiptera: Cimicidae) using an artificial feeding technique. J Med Entomol 39:675 –679

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Møller AP (2000) Survival and reproductive rate of mites in relation to Simov N, Ivanova T, Schunger I (2006) Bat-parasitic Cimex species resistance of their barn swallow hosts. Oecologia 124:351 –357 (Hemiptera: Cimicidae) on the Balkan Peninsula, with zoogeograph- Moloo SK (1971) An artificial feeding technique for Glossina . ical remarks on Cimex lectularius, Linnaeus. Zootaxa 1190:59 –68 Parasitology 63:507 –512 Smaha J (1976) Die Fledermauswanze, Cimex dissimilis (Horvath) Omori N (1941) Comparative studies on the ecology and physiology of (Heteroptera: Cimicidae), als Lastling in Paneeltafelhausern. common and tropical bed bugs, with special references to the Anz Schädlingsk, Pflanzen, Umweltschutz 49:139 –141 reactions to temperature and moisture. J Med Assoc Formosa Southwood TRE (1953) The production of fertile eggs by Cimex 60:555 –729 pipistrelli Jenyns (Hem., Cimicidae) when fed on human blood. Poulin R (2007) Evolutionary ecology of parasites, 2nd edn. Princeton Entomol mon mag XC:35 University Press, Princeton Szalanski AL, Austin JW, McKern JA, Steelman CD, Gold RE (2008) Povolný D (1957) Kritická studie o štěnicovitých (Het. Cimicidae) v Mitochondrial and ribosomal internal transcribed spacer 1 diversity of Cimex lectularius (Hemiptera: Cimicidae). J Med Entomol Československu. [Review study on cimicids (Het. Cimicidae) in 45:229 –236 Czechoslovakia] (in Czech). Folia Zool 6:59 –80 Povolný D, Usinger RL (1966) The discovery of a possibly Tawfik MS (1968) Feeding mechanisms and the forces involved in aboriginal population of the bed bug (Cimex lectularius some blood-sucking insects. Quaest Entomol 4:92 –111 Linnaeus, 1958). Acta Mus Moraviae, Sci biol 51:237–242 Tripet F, Richner H (1997) The coevolutionary potential of a ‘generalist ’ Reinhardt K, Siva-Jothy MT (2007) Biology of the bed bugs (Cimicidae). parasite, the hen flea Ceratophyllus gallinae. Parasitology 115:419 –427 Tripet F, Jacot A, Richner H (2002) Larval competition affects the life Ann Rev Entomol 52:351 –374 Reinhardt K, Harder A, Holland S, Hooper J, Leake-Lyall C (2008) Who histories and dispersal behavior of an avian ectoparasite. knows the bed bug? Knowledge of adult bed bug appearance Ecology 83:935 –945 increases with people age in three counties of Great Britain. J Med Usinger RL (1966) Monograph of Cimicidae (Hemiptera – Heteroptera). Entomological Society of America. Thomas Say Entomol 45:956 –958 Romero A, Potter MF, Haynes KF (2007) Insecticide resistance in the Foundation, New York bedbugs: a factor in the pest sudden resurgence? J Med Entomol Whyte AS, Garnett PA, Whittington AE (2001) Bats in the belfry, bugs in bed? Lancet 357:604 44:175 –178

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ARTICLE II

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Parasitol Res DOI 10.1007/s00436-015-4504-1

ORIGINAL PAPER

Reproduction barrier between two lineages of bed bug (Cimex lectularius) (Heteroptera: Cimicidae)

1 2 1 Kamila Wawrocka & Ond řej Balvín & Tomá š Bartoni čka

Received: 24 February 2015 / Accepted: 24 April 2015 # Springer-Verlag Berlin Heidelberg 2015

Abstract Populations of bed bugs, Cimex lectularius, have adults were higher (51 and 50 % higher in bat and human increased in recent years spreading into numerous urban areas lineage, respectively) than in those animals from the same across the Western world and making them an increasingly lineage. Survival of adults was in pairs from the same locality important pest of the twenty-first century. Research into hy- slightly higher than in pairs from different localities and dif- bridization within and between different lineages of bed bugs fered statistically. These results support the existence of post- can help us to understand processes of micro- and macro- mating barriers and show reproductive isolation between two evolution in these ectoparasites and may inform the control of lineages of C. lectularius. Bat and human host adaptations can this pest species. Hybridization experiments between two host promote evolving of such barriers and can be product of lineages of bed bug (C. lectularius) from Central Europe alloxenic speciation. (Czech Republic), those associated with humans and those with bats, were conducted under laboratory conditions. . . . Number of eggs and early instars were compared between Keywords Hybridization Cimicids Artificial feeding Sperm presence crosses of mixed host lineages (interspecific mating) with pairs from the same host lineage, those from the same locality and same lineage from different localities (intraspecific mating). While crosses within host lineages resulted in egg pro- Introduction duction and later instars, crosses between different host lineages were unsuccessful, although of the mated females pos- High genetic diversity in parasites and hosts equip each side of sessed sperm in their mesospermaleges and/or seminal con- a parasite-host association with evolutionary weapons. In many ceptacles. These crosses did not even result in egg production. cases of parasitic insects, speciation events are associ-ated with Moreover, in the mixed lineage crosses, mortality rates in their hosts (Poulin 2007 ). If we assume that poly-morphism of habitat preference (host body and/or host shelter) exists in the parasite population, there would be a tendency for parasites to * Tomá š Bartoni čka survive on the same host species and mate with individuals [email protected] having similar genotypes. However, gene flow is reduced

Kamila Wawrocka between parasites of the same species living on two different host [email protected] species (Brooks and McLennan 1993 ). When gene flow is

Ond řej Balvín dramatically reduced or interrupted, alloxenic speciation may o.Balví[email protected] occur and narrow host specificity (Combes and Théron 2000 ). z This specific mode of speciation is driven through a shift of host specificity and subsequent development of reproductive barriers 1 (Euzet and Combes 1980 ; Mehlhorn 2008 ). Local adaptation and Department of Botany and Zoology, Masaryk University, Kotlá řská 2, 611 37 Brno, Czech Republic differentiation can lead to the formation of host races within a 2 species and is considered a major route for sympatric speciation Department of Ecology, Faculty of Environmental Sciences, (Coyne and Orr 2004 ). Czech University of Life Sciences Prague, Kamýcká 129, 165 21 Prague 6, Czech Republic

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In this context, two mechanisms may maintain specificity bat- and human-associated bed bugs are produced or not, after host speciation and/or host switching (Combes and (2) determine survival and reproduction rates of hybrids, if Théron 2000 ). In sympatry, according to main biological spe- pro-duced, compared with control samples and finally (3) cies concept (Merrell 1981 ), hybridization should be see how often mating takes place and if the sperm has been prevented by (i) recognition concept where individuals may transferred if no hybrids are present. not recognize another as a potential mating partner or (ii) the isolation concept which states that post-mating mechanisms (working according to natural selection) do not allow fertili- Material and methods zation to occur or produces infertile hybrids (Wheeler and Meier 2000 ). Consequently, hybridization between lineages or Sampling, rearing and feeding of bed bugs closely related species can increase host range (Detwiler and Criscione 2010 ), which can be problematic especially in Bed bugs (C. lectularius) from different localities in Czec h R the case of medically important haematophagous insects. epu blic wer e co lle cted from b ats ro osts (Hanusovice —A, One of those is the bed bug (Cimex lectularius), a widely Prudka —B) and human dwellings (Havirov —C, Znojmo — distrib-uted ectoparasite that has enlarged its range in D). Bed bugs associated with bats were sampled from nursery recent years resulting in the increase of cases of colonies of the greater mouse-eared bat (Myotis myotis) and infestations in human dwellings (Krueger 2000 ). the Geoffroy ’s bat (Myotis emarginatus), roosting in church Hybridization tests have shown that interspecific mating attics. The bat bugs were collected using soft tweezers or between C. lectularius and Cimex hemipterus causes high exhaustors and placed in plas-tic boxes (10×10×5 cm) lined mortality in females and shortens their lifespan, and results with soft paper. Bed bugs as-sociated with humans were in the production of high numbers of infertile eggs (Omori collected in heavily infested apart-ments directly before pest 1939 , 1941 ). There is also an indication that sperm fluids from control operations were carried out. one species of bed bug appear to be toxic to female of another All bugs were separated into adult individuals, early instars species. Despite this, it has been shown that under natural (first to third) and late instars (fourth to fifth), and stored in conditions, around 69 % of individuals of those two species can separate tubes (1 cm×7 cm) closed with cotton and filled with successfully mate among each other (Coetzee et al. 1995 ). a piece of paper (1 cm×4 cm). All samples were kept under Although C. lectularius and Cimex columbarius interbreed freely stable microclimatic conditions in thermostatically controlled (Johnson 1939 ; Titschack 1949 ), it has been demon- apparatus (ST2, EkoAparatura, Poland) under stable temper- strated that the population of such hybrids will never ature (23±0.1 °C) and humidity (75±10 %) which according to stabilize in nature and successful mating was connected Omori ( 1941 ) and Usinger ( 1966 ) is most suitable for the with the fact that C. columbarius is closely related to C. development of C. lectularius. Bugs were fed every 5 days lectularius (Usinger 1966 ). Both species are well isolated with human blood (laboratory commercial blood, Japan due to selective mating and high reduction in the number Medical Supply). Blood was warmed up in water bath (NE4- 14D, Clifton Range) till 37 °C, and bugs were fed by artificial of eggs laid (Ueshima 1964 ). Members of host races are in general more suited to native skin (Durex condoms) for ca. 30 min (until ca. 80 % of hosts than alternative hosts, and offspring they produce with individuals took blood meal). individuals from different native host have reduced fitness (Drès and Mallet 2002 ); the discontinuity in gene flow caused Experimental setting by physical isolation or assortative mating may lead to incip- First crosses were composed of mixed lineages: human×bat- ient speciation (Combes and Théron 2000 ). Strong genetic (separate evolutionary history based on dif- associated bug (interspecific mating), and second from bugs ferences of mtDNA) and morphological divergence (i.e., leg from the same lineage (intraspecific). In order to control for dimensions, body size) between European bed bugs associat- the possibility of pre-mating barriers due to geographical sep- ed with humans and those collected within the roosts of aration, crosses were made with animals of the same lineage synanthropic bats was proven recently (Balvín et al. 2012 ). from (a) the same locality and (b) different localities. All three

Evidence from host choice, survival rate and development combinations (interspecific, intraspecific —within and be- suggests they represent two different lineages —bat and hu- tween localities mating) were in both sex directions. In mixed samples, we had 60 males and 60 females (30 females from man associated (Wawrocka and Bartoni čka 2013 ). These two lineages of bed bug are not geographically isolated, but human lineage, 30 from bat lineage, the same for males). To they parasitize different hosts which in recent decades rarely ensure that offspring only occurred from experimental occupy the same shelter. Therefore, we expect that barriers in crosses, virgin females were obtained from collected instars their reproduction could also have developed. The main aims and kept separately from males until experiment. In total, 60 of the study were to (1) investigate whether hybrids between pairs for interspecific mating and 120 pairs (60 within locality

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Parasitol Res and 60 between localities) for intraspecific mating were than in females from intraspecific crosses (Table 1). Log rank pre-pared. Each pair was reared in separate plastic tube and test for groups of females from intra- and interspecific mating checked every day for the presence of eggs/instars and the showed their survival differed significantly. Females from number of dead individuals. Each experiment was mixed crosses had lower survival rates compared with females 2 conducted for 60 days (12 feeding sessions). from within a locality ( χ =58.70, p<0.05) as well with fe- 2 males from between localities ( χ =27.03, p<0.05). Similarly, Presence of sperm survival rates of males were also significantly different between males from mixed crosses compared with males from 2 In order to determine if sperm has been transferred, mixed, within within a locality ( χ =87.21, p<0.05) and between localities locality and between localities, pairs were placed on petri dishes 2 (χ =54.45, p<0.05). For the comparison of survival in males and observed after feeding. Directly after, mating was observed and females within each group chi-square test was used, and and females were checked for sperm presence, first under the statistically significant differences were observed in all cases microscope and later, if it was necessary, by the removal of the except in the comparison between localities (Table 2). seminal conceptacle and microscopical exam-ination of the Weibull model showed highest probability of death in time contents (Stutt and Siva-Jothy 2001 ). In total, 15 period (feeding sessions) in the case of females from mixed pairs (15 males and 15 females) for within localities, 15 pairs. Males from different localities and same localities be-tween localities and 25 pairs for mixed samples were together with females from different localities have shown a investi-gated. Seminal conceptacles were removed from 5 similar failure probability pattern, while males from mixed females from interspecific matings and 4 from intraspecific pairs in contrast to females from this group have shown (as a control). lowest failure probability in time (Fig. 1).

Statistical analysis Sperm presence

All variables showed a normal distribution. Statistica 12.0 for In 12 pairs originating from the same population, 80 % mated; Windows and SPSS (IMB Statistic 21.0) were used for data in 10 pairs from different populations, 67 % mated; and in 13 analyses. Mortality in samples was marked as complete in the interspecific sets, 52 % mated. During observations of female database (0), the rest as censored (1). Weibull regression was abdomens under the microscope, the movement of sperm used to compare the probability of death between different from ectospermalege to mesospermalege was observed, and sample sets and sexes. A log rank test was used to determine later (within 4 h after mating), the movement deeper into the differences in survival rate of males, females and number of body cavity and seminal conceptacles. Females checked di- eggs among groups. Sex differences between interspecific and rectly after mating (within 30 min) contained sperm. In 4 of intraspecific matings in survival were tested using chi-square the 5 females from mixed crosses, sperm was found in the test. The significance of mating time differences between the seminal conceptacle, and in all 4 females checked from intra- two groups (inter- vs. intraspecific) was further analyzed specific mating, sperm was present. In case of interspecific using a t test. samples, mating took a shorter amount of time and varied between 20 and 130 s (mean 52±30) than in the case of intraspecific matings (112 –190 s, mean 137±23). Differences be- Results tween mating time were statistically significant (t = 39, p <0.05). Inter- and intraspecific mating

Crosses between human- and bat-associated bed bugs failed to Discussion produce eggs (even unfertile ones) or progeny. Crosses within lineages but from different localities produced both fertile Bed bugs are cosmopolitan species with Palearctic origin eggs and offsprings. In all cases, males showed a higher mor- (Usinger 1966 ); their main hosts were bats, and later, humans tality than females. The largest difference in male mortality who shared with them in the same caves and, as a result, rate was observed in mixed samples of both human- and bat- became secondary hosts. With time, humans moved out of associated bugs, where it was equal for both lineages at the caves and created alternative dwellings, probably taking bed level of 84 %, which is an average 37 % higher than in males bugs with them. Presumably, that is when divergence of both from intraspecific crosses (Table 1). Females have shown a lineages occurred and specialization started. Bat- and human- similar pattern with mortality at the level of 73 % in bat- associated bed bugs are quite isolated and differ at the mor- associated and 69 % in human-associated bed bug from inter- phological level showing adaptation to their host (bat or hu- specific samples, which was 31 and 24 %, respectively, higher man) (Balvín et al. 2012 ). Such host speciation, which

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Table 1 Percentage of survivors in adult bed bugs and eggs produced in different time intervals (15, 30, 45 and 60 days) in mixed pairs (bat- human-associated bugs), within locality and between localities

Time 15 days (survivals/new egg) 30 days (survivals/new egg) 45 days (survivals/new egg) 60 days (survivals/new egg)

Gender/eggs M F E M F E M F E M F E Mixed pairs 91 91 0 62 74 0 71 56 0 16 29 0 Within locality 97 98 192 91 92 46 76 85 22 41 58 20 Between localities 94 97 174 86 89 36 71 79 17 35 40 5

M male, F female, E eggs

includes physiological and morphological adaptations, is correlated with a higher possibility for successful cross mating reflected in their phenotype. In this case, ancestral (Arnold 1992 ). Yet, previous research on crossings between even closely related species can result in very few or no eggs —reduced oviposition, i.e., flour beetles (Wade et al. population had been split together with host division into 1994 ) or in ground crickets (Gregory and Howard 1993). In probable that they are mechanically incompatible. Bed bugs mate using traumatic insemination (Arnqvist and Rowe 2005 ), when males inject their sperm to the female two lineages. Research by Balvín et al. (2012 ) and Booth mesospermaleges. We observed successful sperm injection in half of interspecific pairs. Therefore, more probably is that reproductive incompat-ibility is created further on the transportation of sperm as cimicid females possess very complex copulatory organs in-cluding a diversified paragenital system (Carayon 1966 ). In case of some insects, sperm et al. ( 2015 ) has shown that bat- and human-associated from heterospecific mating is not effectively transported to storage organs or is transported, but is not compatible and results in either no eggs or eggs that are not fertile (Katakura and Sobu 1986 ). Eberhard ( 1996 ) has bugs create indeed two different clades. other studies, i.e., on moths (Helicoverpa armigera), post- In our experiment on interspecific mating between bat- mating barriers were found —they could mate, but no off- and human-associated bugs, neither eggs nor progeny was springs or infertile hybrids were produced. The existence obtain-ed. Our study has thus suggested a reproductive of reproductive isolation can be a result of many factors incompatibil-ity between two lineages of bed bug (C. such as different morphologies, isolation by distance or lectularius). It sup-ports our assumptions that along with different evo-lutionary histories. Reproductive isolation morphological differ-ences, genetic divergence and host between two sub-species is a result of genetic discrepancy specificity bed bugs asso-ciated with humans and bats are (Muller 1942 ). Separated subspecies or lineages develop also probably reproductively isolated. adaptations to par-ticular environments or host and they Reproductive isolation can develop on a number of differ- become divergent, sometimes also on mating level. ent levels (i) pre-mating level when we can observe pre- In case of Cimex, we observed mainly post-mating incom- zygotic incompatibility, connected mainly with allochronic patibility which caused the death of females after insemination isolation, habitat isolation, behavioural or sexual isolation; and also decrease the production of fertile eggs both in closer and (ii) post-mating level with pre-zygotic isolation connected related species (C. lectularius×C. columbarius) and wider related with mechanic and gametic incompatibility and post-zygotic (C. lectularius×C. hemipterus). Mating between bugs of both that cause death of zygotes, sterility of hybrids, decrease of lineages occurred, but no progeny was produced, clearly eggs amount or/and hybrids unviability (Nosil et al. 2005 ). indicating the occurrence of post-mating barriers (pre- or/and Generally, the lower genetic distance between species is post-zygotic). Pre-mating barriers cannot be ex-cluded because of a low percentage of interspecific pairs that copulated. Such post- mating but pre-zygotic barriers can have two forms: (1) Table 2 Percentage of death in intra- and interspecific mating crosses mechanic —reduced sperm transmission or (2) gametic — for both sexes incompatibility does not allow fertilization to occur (Nosil et al. Cross mating Death (%) Survival between sexes 2005 ). Although the structure of mating organs in 2 (χ test) ♂ ♀

Interspecific mating Mh×Fb 84 73 χ2 =13.506, p<0.05 Mb×Fh 84 69 Intraspecific crossing bl bl 2 Mh ×Fh 63 57 χ =2.128, p>0.05 bl bl Mb ×Fb 66 62 wl wl 2 Mh ×Fh 60 46 χ =4.046, p<0.05 wl wl Mb ×Fb 57 48

Statistic comparison of survival between females and males in each 2 group ( χ test for two independent groups) Mh male human lineage, Fh female human lineage, Mb male bat lineage, Fb female bat lineage; indexes: bl between localities, wl within localities

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Fig. 1 Weibull regression showing probability of death in all three samples sets (mixed, between and within localities) for females and males (Mm Males, mixed pairs; Mb males between population; Mw males within population; Fm females, mixed pairs; Fb females between population, Fw females within population)

shown that in the case of sperm competition, variation of during sperm competition when the female desires to get rid of female reproductive system influences success of fertilization sperm after copulating with a few different males and chooses the by certain male. Such a situation is found in, e.g., bruchid sperm of the best one. Additionally, females evolved many post- beetle by Wilson et al. ( 1997 ) or marine ascidian by Bishop copulatory mechanisms to avoid being fertilized by ge-netically ( 1996 ). In bed bugs, sperm must get to the mesospermalege as incompatible sperm acceptance (Zeh and Zeh 1997 ). it is here that fertilization occurs (Davis 1965 ), sperm injected One of the mechanisms of sperm choice by females can be in any other place may enter the haemocel causing high mor- female muscle control of sperm (Eberhard 1985 ). tality after such copulation and of course fertilization failure. Studies done on insects have shown that female genotype In our experimental matings, we observed sperm injection affects fertilization. Males are able to successfully fertilize into mesospermalege regions in all mated females (inter- and only females of a certain genotype (Wilson et al. 1997 ). in-traspecific). In female spermalege, there are two main types Incompatibility in female reproductive track can inhibit sperm of haemocytes —one type is able to absorb seminal fluids and transfer to the ovaries. Evolution controls evolving of differ- second is responsible for digesting spermatozoa. The appear- ences in sperm morphology that can occur even within species ance of sperm which is non-specific and unknown for female (e.g., its mobility, flagellum length). Sperm varies in proteins can result in an immune attack response and result in fertili- between different populations of the same species (Eberhard zation failure, especially if that sperm is injected to 1996 ). haemolymph where it is treated as foreign matter. Moreover, A further reason for physiological incompatibility can some accessory gland products injected to female body can be endosymbionts which first were discovered in bed bugs have negative effects (Davis 1965 ). Mashiko ( 1992 ) has (Arkwright et al. 1921 ; Usinger 1966 ). One of these is shown that sperm selection may occur in the ovaries and Wolbachia, a common invertebrate symbiont (Hypsa and may affect fertilization (Macrobrachium nipponense). Aksoy 1997 ; Rasgon and Scott 2004 ; Baldo et al. 2006 ). It Mating with heterospecific males can be costly for females infects around 70 % of all insects (Jeyaprakash and Hoy (Howard et al. 2009 ). Tyler et al. ( 2013 ) discovered that while 2000 ). It plays a crucial role in vitamin absorption and is also field cricket mated with both conspecific and interspecific responsible for reproductive modifications in insects and at partners, offspring were only from conspecific. In many cases, different levels causing cytoplasmic incompatibilities, between sperm and egg. It can also cause a decrease in sperm females preferred conspecific males (Shapiro 2001 ), when local males often adapt to the local females (Holman and Kokko production and reduces sperm competition in non-virgin 2014 ). Even in the case of between population mating, it has been males Fleur and Wedell ( 2006 ). What is more, Wolbachia shown that there is a higher preferences of sperm of males occurrence was confirmed in crossing between C. lectularius adapted to the local female reproduction environment (Eady and C. columbarius (Ueshima 1964 ). Siva-Jothy ( 2006 ) sug- 2001 ). Females are able to regulate fertilization and usage of gested that sexually transmitted bacteria can cause reproductive isolation between populations. Therefore, more experi- sperm (Knowlton and Greenwell 1984 ). Eberhard (1996 ) pointed many possible ways that females may get rid ments are needed to test whether sperm is able to access the of the sperm of certain males. Haemocytes may attack and ovaries and fertilize eggs after interspecific mating has consume sperm (Carayon 1966 ), which is a useful mechanism occurred.

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Another possible reason of lineage incompatibility could Booth W, Balvín O, Vargo EL, Vilímová J, Schal C (2015) Host associ- be pheromone secretion that may not attract the opposite sex ation drives genetic divergence in the bed bug, Cimex lectularius. Mol Ecol. doi: 10.1111/mec.13086 of other lineages. Most of the pheromones seem to be host Brooks DR, McLennan DA (1993) Parascript: parasites and the language specific and create barriers for interspecific matings. Bed bugs of evolution. Smithsonian Institution Press, Washington, DC possess different chemoreceptors and emit diverse phero- Carayon J (1966) Paragenital system. In: Usinger RL (ed) Monograph of mones to communicate; they send information about their Cimicidae (Hemiptera –Heteroptera). College Park, MD: Thomas Say Foundation, Entomol Soc of Amer, pp 81 –166 mating status —mated or unmated in a case of female (Siva- Coetzee M, Hunt RH, Walpole DE (1995) Interpretation of mating Jothy and Stutt 2003 ), aggregation pheromones (Levinson and be-tween two bedbug taxa in a zone of sympatry in KwaZulu, Ilan Bar 1971 ), alarm pheromones (Marx 1955 ; Levinson and South Africa. In: Lambert DH, Spenzer HG (eds) Speciation and Ilan Bar 1971 ) and nymphal pheromones against traumatic the rec-ognition concept: theory and application. Johns Hopkins insemination to avoid mating (Harraca et al. 2010 ). Many University Press, Baltimore, pp 175 –190 Combes C, Théron A (2000) Metazoan parasites and resource heteroge- previous studies have proven that pheromone secretion and neity: constraints and benefits. Int J Parasitol 30:299 –304 response can differ between isolated populations where both Coyne JA, Orr HA (2004) Speciation. Sinauer, Sunderland sexes have shown a higher response on pheromones produced Davis NT (1965) Studies of the reproductive physiology of by individuals from their own locality or even discriminated Cimicidae (Hemiptera). II. Artificial insemination and the function of seminal fluid. J Insect Physiol 11:355 –366 geographically distinct individuals of the same gender Detwiler JT, Criscione CD (2010) An infectious topic in reticulate evo- (Coleoptera: Scotylidae in Lanier et al. 1972 ). That could ex- lution: introgression and hybridization in animal parasites. Genes 1, plain why only half of observed interspecific pairs mated. Our doi: 102 –12310.3390/genes1010102 study has shown a reproductive isolation between two Drès M, Mallet J (2002) Host races in plant-feeding insects and their importance in sympatric speciation. Philos Trans R Soc Lond B lineages of C. lectularius. Divergent host adaptations can pro- 357:471 –492 mote evolving of such barriers (Nosil 2007 ) and can be a Eady PE (2001) Postcopulatory, prezygotic reproductive isolation. J Zool product of alloxenic speciation (Muller 1942 ; Mayr 1963 ; 253:47 –52 Mehlhorn 2008 ). However, to understand the base of such Eberhard WG (1996) Female control: sexual selection by cryptic female choice. Princeton Univ Press, Princeton divergence at reproductive level, it is necessary to conduct Eberhard WG (1985) Sexual selection and animal genitalia. Harvard further research on pre- (ex. behavioural, ecological) and Univ Press, Cambridge post-mating (ex. mechanical, gametic) isolation barriers Euzet L, Combes C (1980) Les problèmes de l ’espèce chez les be-tween those two lineages. animaux parasites. Bull Soc Zool France 40:239 –285 Fleur ECC, Wedell N (2006) Wolbachia infection reduces sperm

compet-itive ability in an insect. Proc R Soc B 273:1455 –1458 Acknowledgments We are very grateful to John Haddow and Henry Gregory PG, Howard DJ (1993) Laboratory hybridization studies of Schofield for their valuable comments on the manuscript and Martin Allonemobius fasciatus and A. socius (Orthoptera: Gryllidae). Ann Toman and Zden ěk Bu řič for their help with fieldwork and sampling Entomol Soc Am 86:694 –701 bed bugs. Harraca V, Ryne C, Ignell R (2010) Nymphs of the common bed bug

(Cimex lectularius) produce anti-aphrodisiac defence against conspecific males. BMC Biol 8:121. doi: 10.1186/1741-7007-8-121 References Holman L, Kokko H (2014) Local adaptation and the evolution of female choice. In: Hunt J, Hosken D (eds) Genotype-by-Environment Interactions and Sexual Selection. Wiley - Blackwell, Hoboken, Arkwright JA, Atkin EE, Bacot A (1921) An autoradiographic NJ, pp 41 –62 investiga-tion of sperm movements in the female reproductive Howard DJ, Palumbi SR, Birge LM, Manier MK (2009) Sperm and tract. Proc R Soc Lond B Biol Sci 263:369 –376 speciation. In: Birkhead TR, Hosken DJ, Pitnick S (eds) Sperm Arnold ML (1992) Natural hybridization as an evolutionary process. biology: an evolutionary perspective. Academic, London, pp Annu Rev Ecol Syst 23:237 –261 368 – 403 Arnqvist G, Rowe L (2005) Sexual conflict. Princeton Univ Press, Hypsa V, Aksoy S (1997) Phylogenetic characterization of two Princeton transovarially transmitted endosymbionts of the bed bug Cimex Baker AM, Shore JS (2005) Pollen competition in Turnera ulmifolia lectularius (Heteroptera: Cimicidae). Insect Mol Biol 6:301 –304 (Turneraceae). Am J Bot 82:171 –725 Jeyaprakash A, Hoy MA (2000) Long PCR improves Wolbachia DNA Baldo L, Hotopp JCD, Jolley KA, Bordenstein SR, Biber SA, amplification: wsp sequences found in 76 % of sixty-three arthropod Chounhurry RR, Hayashi C, Maiden MCJ, Tettelin H, Werren JH species. Insect Mol Biol 9:393 –405 Johnson CG (1939) Taxonomic characters, variability and relative growth (2006) Multilocus sequence typing system for the endosymbiont Wolbachia pipientis. Appl Environ Microbiol 72:7098–7110 Balvín in Cimex lectularius L. and C. columbarius Jenyns (Heteropt. O, Munclinger P, Kratochvíl L, Vilímová J (2012) Mitochondrial Cimicidae). Trans R Entomol Soc Lond 89:543 –568 DNA and morphology show independent evolutionary histories of Katakura H, Sobu H (1986) Cause of low hatchability by the interspecific bed bug Cimex lectularius (Heteroptera: Cimicidae) on bats and mating in a pair of sympatric ladybirds (Insecta, Coleoptera, Coccinellidae): incapacitation of alien sperm and death of hybrid humans. Parasitol Res 111:457 –469 Bishop JDD (1996) Female control of paternity in the internally fertilizing embryos. Zool Sci 3:315 –322 compound ascidian Diplosoma listerianum. I. Autoradiographic in- Knowlton N, Greenwell SR (1984) Male sperm competition avoidance vestigation of sperm movements in the female reproductive tract. mechanisms: the influence of female interests. In: Smith RL (ed) Sperm competition and the evolution of animal mating system. Proc R Soc Lond B Biol Sci 263:369 –376 Academic, London, pp 61 –84

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Krueger L (2000) Don ’t get bitten by the resurgence of bed bugs. Pest Rasgon JL, Scott TW (2004) Phylogenetic characterization of Control 68:58 –64 Wolbachia symbionts infecting Cimex lectularius L. and Lanier GN, Birch MC, Schmitz RF, Furniss MM (1972) Pheromones of Oeciacus vicarius Horvath (Hemiptera: Cimicidae). J Med Ips pini (Coleoptera: Scolytidae): variation in response among three Entomol 41:1175 –1178 populations. Can Entomol 104:1917 –1923 Shapiro LH (2001) Asymmetric assortative mating between two hybrid- Levinson HZ, Ilan Bar AR (1971) Assembling and alerting scents izing Orchelium katydids (Orthoptera: Tettigoniidae). Am Midl Nat pro-duced by the bed bug, Cimex lectularius L. Experientia 27:102 – 145:423 –427 103 Marx R (1955) Über die Wirtsfindung und die Bedeutung des Siva-Jothy MT (2006) Trauma, disease and collateral damage: conflict in artspezifischen Duftstoffes bei Cimex lectularius Linné. Parasitol cimicids. Philos Trans R Soc B 361:269 –275 Res 17:41 –72 Siva-Jothy MT, Stutt AD (2003) A matter of taste: direct detection of Mashiko K (1992) Genetic egg and clutch size variations in mating status in the bed bug. Proc R Soc B 270:649 –652 freshwater prawn populations. Oikos 63:454 –458 Stutt AD, Siva-Jothy MT (2001) Traumatic insemination and sexual con- Mayr E (1963) Animal species and evolution. Harvard Univ Press, flict in the bed bug Cimex lectularius. Proc Natl Acad Sci U S A 98: Cambridge 5683 –5687 Mehlhorn H (2008) Encyclopedia of parasitology, 3rd edn. Springer, Titschack E (1949) Die bettwanze und die taubenwanze. Verh dt Ges Berlin angew Entomol 11:71 –77 Merrell DJ (1981) Ecological genetics. Univ of Minnesota Press, Tyler F, Harrison XA, Bretman A, Veen T, Rodríguez-Muñoz R, Minneapolis Tregenza T (2013) Multiple post-mating barriers to hybridization Muller HJ (1942) Isolating mechanisms, evolution and temperature. in field crickets. Mol Ecol 22:1640 –1649 Biopolym Symp 6:71–125 Ueshima N (1964) Experiments on reproductive isolation in Cimex Nosil P (2007) Divergent host plant adaptation and reproductive lectularius and Cimex columbarius. Pan Pac Entomol 40:47 –53 Usinger isolation between ecotypes of Timema cristinae walking sticks. RL (1966) Monograph of Cimicidae: (Hemiptera-Heteroptera). Thomas Am Nat 169: 151 –162 Say Foundation, Entomol Soc of Amer, New York Nosil P, Vines TH, Funk DJ (2005) Perspective: reproductive Wade MJ, Patterson H, Chang NW, Johnson NA (1994) Postcopulatory, isolation caused by natural selection against immigrants from prezygotic isolation in flour beetles. Heredity 72:163 –167 divergent hab-itats. Evolution 59:705 –719 Wawrocka K, Bartoni čka T (2013) Two different lineages of bed bug Omori N (1939) Experimental studies on the cohabitation and crossing of (Cimex lectularius) reflected in host specificity. Parasitol Res 112: two species of bed-bugs (Cimex lectularius L. and C. hemipterus F.) 3897 –3904 and on the effects of interchanging of males of one species for the Wheeler QD, Meier R (2000) Species concept and phylogenetic other, every alternate days upon the fecundity and longevity of fe- theory: a debate. Columbia Univ Press, New York males of each species. Act J Med Trop 1:127 –154 Wilson N, Tubman SC, Eady PE, Robertson GW (1997) Female Omori N (1941) Comparative studies on the ecology and physiology of geno-type affects male success in sperm competition. Proc Biol common and tropical bed bugs, with special reference to the reac- Sci 264: 1491 –1495 tions to temperature and moisture. J Med Assoc Taiwan 60:555 –729 Zeh JA, Zeh DW (1997) The evolution of polyandry II: post-copulatory Poulin R (2007) Evolutionary ecology of parasites, 2nd edn. Princeton defences against genetic incompatibility. Proc R Soc Lond B Biol Univ Press, New Jersey Sci 264:69 –75

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Vespert ilio 17 : 215–220, 2014 ISSN 1213-6123

Erythrocyte size as one of potential causes of host preferences in cimicids (Heteroptera: Cimicidae: Cimex )

Kamila W awrocka & Tomáš B artoni čka

Department of Botany and Zoology , Faculty of Science , Masaryk University , Kotlá řská 2 , CZ–611 37 Brno , Czech Republic ; kamila .freeme@gmail .com ; bartonic@sci .muni .cz

Abstract . Cimicids are haematophagous insects whose life cycle, reproduction and survival rate depends on the blood of its hosts. Blood ingredients play a crucial role there. Two lineages have been identified in Cimex lectularius – bat- and human-associated bed bugs. Also bat bugs C. pipistrelli differ in particular bat hosts. We found some differences between the two lineages of bed bugs in the speed of moulting, length of life and reproduction success in cross-host experiments. It was considered that the bug proboscis could be very narrow and that red blood cells might not be able to pass through it. Therefore the first aim of this study was to find out whether the red blood cell (RBC) size has an impact on the occurrence of cimicids in bat and human host. Except one observation on Plecotus auritus , bat bugs never occurred in certain bat species i.e. Barbastella barbastellus , Rhinolophus hipposideros and Plecotus austriacus . We classified them as non-specific bug hosts, while the other bat species as specific hosts. The second aim of this study was to compare RBC size in specific and non-specific bat hosts. We collected blood samples from seven bat genera represented by 12 vespertilionid species and one rhinolophid species. Diameters of red cells were measured. We found some differences between the bat species, however, there was no clear correlation in erythrocyte size between specific and non-specific bat hosts and humans. Therefore RBC size is probably not the reason why some bat species are not parasitized by cimicids. Bat and bed bugs, RBC, hematocrit, bats, human

Introduction

Two Cimex species connected with bats, viz. Cimex lectularius Linnaeus, 1758 (bed bug) and Cimex pipistrelli Jenyns, 1839 (bat bug), posses numerous adaptations to ecology and anatomy of their hosts. Nevertheless they have never been found together in the same roost (Balvín et al. in prep.). Moreover, two different host lineages – bat- and human–associated – seem to exist within Cimex lectularius (Wawrocka & Bartoni čka 2013) which, according to Balvín et al. (2012a), never met even in evolutionary line. They differ at the genetic and morphological scale, which is a clear adaptation to a specific host. Similarly, existence of many ecotypes associated with different bat species was shown in Cimex pipistrelli (Balvín et al. 2013). Cimicids have never been found in certain bat species i.e. Plecotus auritus , P. austriacus , Barbastella barbastellus or Rhinolophus hipposideros and R . ferrumequinum , except one newly described finding from Ukraine (Balvín et al. 2012b). One of the explanations may be their, a bit different than in the other species, roosting strategy that plays an important role and affects bat behaviour, occurrence or diversity (Kunz 1982, Findley 1993). Immune response, hormonal status (Jones 1996), roosting strategy and also blood components influence not only the sucking ability but also digestion, survival and development rate of blood sucking parasites (Krasnov

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2008). Zedníková (2010) showed that C. pipistrelli is able to feed on non-specific species under laboratory conditions. However we do not know how sucking on non–specific (non-hosted in natural habitats) species affects ontogeny, survival in F1 and F2 generations, fertility or oviposition. RBCs, as the main blood components, seem to be the hardest items to digest due to their high protein amount (Gooding 1972). Therefore size parameters of erythrocytes may determine the success of sucking. For example chicken erythrocytes are 11.2 µm in diameter, while human erythrocytes only 6–8 µm (Reinhardt & Siva-Jothy 2007, Benoit 2011). This fact makes humans a better host than chicken for Cimex lectularius whose proboscis canal is 8–12 µm in diameter (Hase 1926, Tawfik 1968). The aim of this study was to find out whether the size of RBC could be a reason why some bat species are commonly parasitized by cimicids while others not. Therefore we measured RBC size, hematocrit and also the amount of red cells in specific and non-specific bat hosts of bed and bat bugs. We did the same for human commercial blood.

Material and Methods

Blood samples were taken from the median vein of bat wings with scalpel and bleeding was stopped immediately by antiseptic liquid (Betadine). A small drop of blood was situated on a glass slide and coloured with May-Grundwald and Giemsa colours. The smallest (width) and largest (length) dimensions of red blood cells were measured using program QuickPHOTO MICRO 2.3. We measured 20 red cells for each microscope slide at 100× magnification using immerse oil (Fig. 1). With syringe (Hamilton, Chromoservis) we took 4 µl of blood and diluted it 200 times in physiological salt. Bürker glass was used to count and compare RBC in specific and non-specific hosts. The total RBC number was counted manually; mean from 10 visual fields (1 mm 2) was taken. Counting was carried out at 200× magnification. This sample was compared with a slide made in the same way from human commercial blood (laboratory commercial blood, Japan Medical Supply, B+). RBC size was counted for each species, sex (male/female) and status (adult/juvenile). To compare red cell size among species, the Kruskal – Wallis test was conducted. Statistical comparison of parameters in sex and age was made using the Mann-Whitney U test where possible. Blood from 12 bat species from seven different genera. Therefore we caught females and males, adults and juveniles of these species: Rhinolophus hipposideros (Borkhausen, 1797) (n=1), Myotis myotis (Borkhausen, 1797) (n=2), M. bechsteinii (Kuhl, 1817) (n=1), M . nattereri (n=1), M . mystacinus (Kuhl, 1817) (n=1), M . brandtii (Eversmann, 1845) (n=1), M. daubentonii (Kuhl, 1817) (n=1), Vespertilio murinus Linnaeus, 1758 (n=1), Eptesicus nilssonii (Keyserling et Blasius, 1839) (n=2), Pipistrellus pygmaeus (Leach, 1825) (n=2), Barbastella barbastellus (Schreber, 1774) (n=1), and Plecotus auritus (Linnaeus, 1758) (n=5).

Fig. 1. Red blood cells of Myotis myotis , a specific host of the bed bug (left) and Plecotus auritus , a non-specific host (right), as viewed in the QuickPHOTO MICRO 2.3.

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Table 1. Size of red blood cells in selected bat species and man (min size = the smallest dimension, width; max size = the largest dimension, length) species mean min size±SD (µm) mean max size±SD (µm)

Rhinolophus hipposideros 4.3±0.42 4.6±0.43 Myotis myotis 5.6±0.55 5.9±0.45 Myotis daubentonii 6.6±0.41 6.9±0.51 Myotis brandtii 4.5±0.82 4.8±0.86 Myotis bechsteinii 6.5±0.51 6.6±0.49 Myotis nattereri 6.2±0.38 6.4±0.40 Myotis mystacinus 5.3±0.26 5.7±0.32 Pipistrellus pygmaeus 5.2±0.39 5.6±0.40 Eptesicus nilssonii 6.1±0.26 6.4±0.37 Vespertilio murinus 5.6±0.39 5.8±0.46 Barbastella barbastellus 5.8±0.74 6.2±0.61 Plecotus auritus 5.4±0.66 5.7±0.66 human 6.4±0.31 6.6±0.43

Results

Comparison between sexes within the same species did not show any significant differences in RBC size (U test, p>0.05, n 1=3, n 2=3) and neither did the comparison between adults and juveniles within the species (U test, p>0.05, n 1=4, n 2=4). The Kruskal-Wallis test did not reveal statistically significant differences between particular bat species (H=15, df=11, p=0.18). In all tested bats, both specific and non-specific hosts, the size of erythrocytes ranged between 4.5–6.9 µm. Nevertheless, five of 12 species showed a significantly larger RBC size (Table 1) in comparison with the mean RBC size for all studied bat species (5.7 µm), i.e. Eptesicus nilssonii (10% higher), Barbastella barbastellus (5%), Myotis daubentonii (18%), M . bechsteinii (15%), and M . nattereri (10%). Out of these species, only Barbastella barbastellus was not infested by cimicids, however, the other species have larger erythrocytes. The size of RBC in human blood (B+) was estimated at 6.5 µm, which is a higher value than in most bat species. Finally, we compared the number of RBCs in bats and humans. Hematocrit level as well as the number of RBCs is higher in bats than in humans (156% higher in Myotis nattereri , 141% in M. daubentonii , 192% in Pipistrellus pygmaeus , and 79% in Vespertilio murinus ) (Table 2). The highest hematocrit level as well as RBC number was found in Pipistrellus pygmaeus .

Table 2. Comparison between human and bat blood structure. Hematocrit (percentage of volume of red blood cells per unit blood) and amount of RBC in µl. species hematocrit (%) RBC (10 6/µl)

Myotis myotis 41 9.1 Myotis nattereri 58 12.3 Myotis daubentonii 52 11.6 Pipistrellus pygmaeus 61 14.0 Plecotus auritus 49 12.3 Vespertilio murinus 46 8.6 human 42 4.8

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Discussion

Specific and non-specific bat hosts Feeding success of blood-sucking insects depends on physiological and nutritional conditions of their host. Differences in blood chemistry and physiology may be one of the mechanisms determining host preferences in bats. Poulin (2007) described two main filters that determine host choice by parasites, one of them being a compatibility filter that excludes all host individuals on which parasites cannot feed for morphological, physiological and immunological reasons. That is why haematological data on bats could be important to understand host choice in cimicids and their species specificity. Previous research has shown that many components and aspects of host blood (e.g. T-lymphocytes, antibodies, mast cells or granulocytes) influence not only the sucking ability but also digestion, survival and development rate of parasites (Krasnov 2008). Size of red cells is definitely one of the important factors (cf. Reinhadt & Siva-Jothy 2007). Specific host seems to be a good reservoir of blood meal and bugs, in order to avoid risk, prefer to feed less often but more intensively. In other hosts (non-specific), after taking blood meal, bugs did not continue till repletion (Barbarin et al. 2013, personal observation). We did not find significant differences in the amount of red cells between the studied bat species (specific: Myotis myotis , M. nattereri , M . daubentonii , Pipistrellus pipistrellus , and non-specific: Plecotus auritus ), which means that we cannot find explanation for host preferences in RBC density (Table 1). Neverthe-less, according to a blood analysis carried out in the Egyptian fruit bat ( Rousettus aegyptiacus ) (Korine et al. 1999), blood profile changes between seasons and also depends on activity of the animal and reflects animal fitness. It has been shown that in the wild, Cimex pipistrelli does not occur or only very rarely in certain bat hosts ( Barbastella barbastellus , Plecotus auritus , P. austriacus , and Rhinolophus hipposideros ), but it seems to be able to feed on them under laboratory conditions (Zednikova 2010). The easiest explanation for the absence of bugs in these bats would be that specific bug hosts do not meet non-specific hosts in the roosts, where the transmission of ectoparasites is most likely. However, in the case of roosts in attics, there are quite often more species of bats roosting together. During monitoring of bat populations in the Czech Republic, about 140 roosts of nursery colonies of Myotis myotis are checked annually and over 20 of them are shared with some of the species ranking among non-specific hosts (such as Plecotus spp. or Rhinolophus hipposideros ) (Czech Bat Conservation Trust database, unpubl.). Despite that, it seems that the transfer to a new host is very rare. B. barbastellus prefers to roost in crevices of dead beech trees, shows frequent roost switching behaviour and forms quite small colonies (Russo et al. 2004), which makes it not suit-able for cimicids. Moreover, this species never shares its roosts with other bat species, especially the bug specific hosts. Rhinolophus bats, originally cave-dwelling species (Jepsen 1970), shows night roosting activity with frequent switches and their day roosts are found in barns, stables, garages as well as in caves or underground tunnels and cellars (Knight & Jones 2009). Caves, which usually offer a wide thermal range (Tuttle & Stevenson 1978), are very cold for bugs in the temperate zone (Balvín et al. 2012b). Nevertheless, many studies indicate that blood composition and chemical components are an important issue in the case of bed bugs. In Cimex lectularius , the proboscis canal is a simple tube and blood is stored in midgut. Erythrocyte diameter can influence sucking ability of the bugs, especially when it is larger than the internal diameter of the proboscis tube. Feeding experiments using rabbit, chicken, cavia and human blood showed that human blood was most convenient for the bugs and kept their survival at the highest level (Barbarin et al. 2013). These results sug-gest that technical and chemical blood components which differ in the above mentioned hosts

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122 may affect blood intake in the bed bug, however, the authors did not measure RBC diameter. In our study we did not find significant differences between humans and bats in RBC size (range 4.5–6.9 µm in bats, 6.2–6.8 µm in human), though the size of human RBC is at the upper limit of the bat range.

Bat and human lineage of Cimex lectularius Bats are quite atypical hosts in terms of the short time bugs have to feed on them, and that is probably why evolution favoured maximization of the diameter of the proboscis canal. Percen- tage of hematocrit in the host blood has an impact on host choice and feeding time. Decrease of hematocrit would shorten the time needed to take full meal (Daniel & Kingsolver 1983). Myotis myotis has the lowest level of hematoctrit (41%) of all tested bat species. This fact might explain why it is the most common host of Cimex lectularius . Similarly as Neuweiler (2000), we confir-med that bats possess a higher number of erythrocytes than humans, with the highest number in Pipistrellus pipistrellus (14.0). High concentration of hemoglobine in bat RBC gives them quite high oxygen capacity at the level of 30% which is almost twice more than in the case of ground dwelling mammals (Neuweiler 2000). We also found that red cell size is smaller in bats than in humans and this together with their higher numbers helps bats to manage oxygen exchange during such energy consuming activity as flight is. All blood-sucking insects had to develop special mechanisms for blood intake. Occurrence of bugs in certain host species is determined by many factors such as temperature, host availability but also morphology of mouthparts and saliva components (Guarneri et al. 2000, Sant’Anna et al. 2001). Marcus & Safier (1993) proved that bed bug saliva contains special molecules that neutralize haemostatic answers of host body. This is mostly apyrase, an inhibitor of Factor Xa, responsible for the coagulation cascade (Valenzuela et al. 1996), and nitrophorin that enables nitric oxide (NO) transports (Valenzuela et al. 1995). These molecules thus halt immune system response and help to avoid blood coagulation. One of the reasons why some bat species are not infested by cimicids in the wild may be a stronger immune defence and the higher number of white blood cells (eozynofiles or neutrofiles). Therefore more studies on bat blood components are needed to find out which factors determine certain species as specific or non-specific hosts.

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GoodinG p. H., 1972: Digest ive pr ocesses of haemat ophagous insect s. I. A l it er at ur e r eview. Quaestiones Entomologicae , 8: 5–60. Guarneri a. a., p ereira M. H. & d ioTaiuTi O., 2000: Influence of the blood meal source on the development of Triatoma infestans , Triatoma brasiliensis , Triatoma sordida , and Triatoma pseudomaculata (Heteroptera, Reduviidae). Journal of Medical Entomology , 37 : 373–379. Hase a., 1926: Über ver fahl en zur Unt er suchung von Quaddel n und ander en Haut er scheinungen nach Insektenstichen. Zeitschrift für Angewandte Entomologie , 1926 : 243–249 Jepsen G. l., 1970: Bat or igins and evol ut ion. Pp.: 1–64. In: W iMsaTT W.A. (ed.): Biology of Bats . Academic Press, New York, 406 pp. Jones c. J., 1996: The immunol ogy of host -ect opar asit ic art hr opod r el at ionships. Pp.: 150–174. In: WiKel S. K. (ed.): The Immunology of Host-Ectoparasitic Arthropod Relationships . CAB International. Wall-ingford, Oxon, UK, 331 pp. KniGHT T. & J ones G., 2009: Importance of night roost s for bat conservat ion: roost ing behaviour of t he l esser horseshoe bat Rhinolophus hipposideros . Endangered Species Research , 8: 79–86. Korine c., Z inder o. & a rad Z., 1999: Diurnal and seasonal changes in bl ood composit ion of t he free-l iving Egyptian fruit bat ( Rousettus aegyptiacus ). Journal of Comparative Physiology B , 169 : 280–286. Krasnov b. r., 2008: Functional and Evolutionary Ecology of Fleas: A Model for Ecological Parasitology . Cambridge University Press, Cambridge, UK, 593 pp. KunZ T. H., 1982: Roost ing ecol ogy. Pp.: 1–55. In: K unZ T. H. (ed.): Ecology of Bats . Plenum Press, New York, xviii+425 pp. Marcus a. J. & s aFier l. b., 1993: Thromboregul at ion: Mult icel l ul ar modul at ion of pl at el et react ivit y in hemostasis and thrombosis. The Federation of American Societies for Evolutionary Biology Journal , 7: 515–522. neuWeiler G., 2000: The Biology of Bats . Oxford Univeristy Press, New York, 310 pp. poulin r., 2007: Evolutionary Ecology of Parasites . Second Edition . Princeton University Press, Princeton, 342 pp. reinHarT K. & s iva -JoTHy M. T., 2007: Biol ogy of t he Bed Bugs (Cimicidae). Annual Review of Entomo- logy , 52 : 351–374. russo d., G isTrone l., J ones G. & M aZZoleni S., 2004: Roost selection by barbastelle bats ( Barbastella barbastellus , Chiroptera: Vespertilionidae) in beech woodlands of central Italy: consequences for conservation. Biological Conservation , 117 : 73–81. sanT ’ a nna M. r. v., d ioTaiuTi l., G onTiJo a. F. & p ereira M. H., 2001: Feeding behaviour of mor phol ogi- cally similar Rhodnius species: influence of mechanical characteristics and salivary function. Annales de la Societe Belge de Medecine Tropicale , 65 : 149–164. TaWF iK M. s., 1968: Feeding mechanisms and t he for ces involved in some bl ood-sucking insect s. Quaes- tiones Entomologicae , 4: 92–111. TuTTle M. d. & s Tevenson d. e., 1978: Var iat ion in t he cave envir onment and it s biol ogical impl icat ions. Pp.: 108–121. In: Z uber R., c HesTer J., G ilberT S. & r Hodes D. (eds.): Proceedings of the National Cave Management Sympozium . Adobe Press, Albuquerque, 140 pp. valenZuela J. G., W alKer F. a. & r ibeiro J. M. C., 1995: A sal ivary nit hr opor in (nit r ic-oxide-car rying hemoprotein) in the bed bug Cimex lectularius . Journal of Experimental Biology , 7: 1519–1526. valenZuela J. G., G uiMares J. a. & r ibeiro J. M. C., 1996: A novel inhibit or of Fact or X act ivat ion fr om the salivary glands of the bed bug Cimex lectularius . Expert Parasitology , 83 : 184–190. WaWrocKa K. & b artoni čka T., 2013: Two differ ent l ineages of bed bug ( Cimex lectularius ) reflected in host specificity. Parasitology Research , 112 : 3897–3904. Zedníková K., 2010: Přežívání št ěnic ( Cimex pipistrelli ) v r ůzných mikroklimatických podmínkách [ Survival of Bat Bugs (Cimex pipistrelli ) under Different Microclimatic Conditions ]. Unpubl. MSc Thesis. Palacký University, Olomouc, 52 pp (in Czech). received on 17 May 2014

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List of publications:

WAWROCKA K.J.,BARTONI ČKA, T.,REITER 2013 Pipistrellus kuhlii, a bat species breeding and hibernating in the Czech Republic. Vespertilio, Praha, Revúca: ČESON Praha, SON Revúca, 2013, vol. 16, No 1, p. 351-356. ISSN 1213-6123.

WAWROCKA K.J.,BARTONI ČKA, T. 2013 Two different lineages of bedbug (Cimex lectularius) reflected in host specificity. Parasitology Research, Germany: Springer, 2013, vol. 112, No 11, p. 3897-3904. ISSN 0932-0113. doi:10.1007/s00436- 013-3579-9

WAWROCKA KJ,BARTONI ČKA, T. 2014. Erythrocyte size as one of potential causes of host preferences in cimicids (Cimex spp., Heteroptera: Cimicidae). Vespertilio, Skupina pre ochranu netopierov, 2014, vol. 17, No 1, p. 215-220. ISSN 1213-6123.

WAWROCKA K.J.,BARTONI ČKA, T 2015 Reproduction barrier between two lineages of bed bug (Cimex lectularius)(Heteroptera: Cimicidae). Parasitology Research, Springer-Verlag Berlin Heidelberg 2015, 2015, vol. 2015. doi:10.1007/s00436-015-4504-1

WILLEMS K.,KREMENOVA J,BARTONI ČKA T. 2017. Bat bugs (Cimex pipistrelli) transmission propensity in three bat species. (submitted).

WILLEMS K.,WILLEMS W. 2016. Bats in bunkers and other fortifications in Świnouj ście and surroundings ( West Pomeranian ) (in prep.).

Conferences:

WAWROCKA K., MONIUSZKO H., KOWALSKA – GORALSKA M. 2011. Impact of cleaning chemicals on survival of Lemna minor. National Conference of Biology Students 2011, Pozna ń (oral presentation)

WAWROCKA K., KOKUREWICZ T. 2011. Comparison of foraging activity time of females mouse-eared bat (Myotis myotis) during two summer seasons in Natura 2000 site “Nietoperek” “. International conference of students societies, Lublin 2011. (3rd place in Environmental section) (poster)

WAWROCKA K., KOKUREWICZ T., BOERS K.,KAAL R., WILLEMS W., BORGENS F. 2011. Influence of temperature on site selection by hibernating bats. European Bat Research Sympozium, 2011 Lithuania (poster)

WAWROCKA K.J. 2011. Foraging preferences of lactating females of greater- mouse eared bat (Myotis myotis) in "Nietoperek" Natura 2000 site. In 5th Conference Bats of Sudets, Abstracts of the 5th Conference Bats of Sudets, Bartosova Pec. 2011 (oral presentation)

WAWROCKA K.,BARTONI ČKA T. 2012. Host specificity in bed bug (Cimex lectularius): a cross-feeding experiment. In From Biotechnology to Environmental Protection. The interdisciplinary meeting of young naturalists.The VIIth International Conference of Young Naturalists

WAWROCKA K, BARTONI ČKA T. 2013. Host specificity of bedbugs (Cimex lectularius) In Zoologické dny 7.-8.2. 2013. 2013. ISBN 978-80-87189-14-6 (oral presentation)

WAWROCKA K.,WILLEMS W. 2013. Habitat use of Myotis emarginatus in a small-scale cultural landscape. In 3nd International Berlin Bat Meeting: Bats in the Antropocene 1.-3.03.2013. 2013. ISBN 978-3-9815637-1-9 (poster)

KOKUREWICZ T.,BONGERS F.,CIECHANOWSKI M.,DUVERGE L.,GLOVER A.,HADDOW J.,RACHWALD A.,RUSINSKI M.,SCHMIDT C.,SCHOFIELD H.,WAWROCKA K.J.,WILLEMS W.,ZAPART A. 2014 Bat research and conservation in "Nietoperek" bat reserve (Western Poland). In XIIIth European Bat Research Sympozium. 2014. ISBN 978-953-6904-30-3 (oral presentation)

WAWROCKA K.J.,WILLEMS W. 2014 Creating a bat inventarisation in Flemish Brabant by use of complementary research methods . In VIIIth European Bat Research Symposium. 2014. ISBN 978-953-6904-30-3 (poster)

WAWROCKA K.J.,BARTONI ČKA, T. 2014 Erythrocyte size in bats - factor determining host choice in Cimicids (Heteroptera: Cimicidae). In VIIIth European Bat Research Symposium. 2014. ISBN 978-953-6904-30-3 (poster)

WAWROCKA K.J. 2014 Host specificity in bed bugs and its implication for bat conservation. In VIIIth European Bat Research Symposium. 2014. ISBN 978-953- 6904-30-3 (oral presentation)

WAWROCKA K.J.,BARTONI ČKA, T. 2014 Multiplex panels of polymorphic microsatellite loci for bat bug (Cimex pipistrelli) in Central Europe. In Xth European Congress of Entomology, University of York, UK. 2014 (poster)

WAWROCKA K.J.,BARTONI ČKA, T 2014 Štenice a netopý ři v praktické ochran ě (Bed bugs and bats in practical conservation). In Chiropterologický seminár, ChiSe 2014. 2014 (oral presentation)

Projects and internships:

1. Monitoring the numbers of bats in the Nature 2000 area “Nietoperek” – “Winter censuses 2010 – 2016

2. Swarming research in fort Walem (Mechelen, Belgium). Bat working group Flanders, 2011.

3. Swarming research in fort Oelegem (Ranst, Belgium). Bat working group Flanders, 2011.

4. Bats in forests and parks in the provincie Flemish Brabant. Natuurpunt and province Flemish Brabant, 2011-2012.

5. Greater Mouse-eared bat and Bechstein’s Bat in Flemish Brabant. Natuurpunt and province Flemish Brabant, 2012-2013.

6. WNS – white nose syndrome project (GCAR), Masaryk University (2012)

7. Nature connections for bats in South Limburg. Natuurpunt and province Limburg, 2013-2014.

8. WNS – shite nose syndrome project (GCAR), Masaryk University (2013)

9. Bat monitoring in Natura2000-areas in the Brussels Capital Region. Natuurpunt, Natagora and BIM, 2013-2017.

10. Development of a research method for the swarming behavior of bats in the Antwerp fort belts with the aim of obtaining a good state of conservation. Natuurpunt and ANB Antwerpen, 2013-2016

11. Specialist at Masaryk University, Department of Botany and Zoology, Vertebratology Group

12. Bats on (church) attics in Flemish Brabant. Natuurpunt and province Flemish Brabant, 2014

13. Ecologic monitoring of core areas Greenplan city of Antwerp. Bat research. Natuurpunt and City of Antwerpen (ongoing)

14. Internship in Natuurpunt vzw, Mechelen, Belgium (2011 – 2016)

CURRICULUM VITAE

Name: Willems Kamila

Address: Ertbrandstraat 154 2950 Kapellen

Tel: 0032 48 512 6970

Email: [email protected]

Date of birth: 03/07/1987

Qualifications:

2011 - onward PhD in Biology, Faculty of Science, Departament of Botany and Zoology Masaryk University, Brno, Czech Republic

2009 – 2011 Wrocław University of Environmental and Life Sciences, Biology, Insitute of Biology, Departement of Vertebrate Ecology and Paleontology,specialization : Environmental biology, Msc

2006 – 2009 Wrocław University of Environmental and Life Sciences, Applied biology, Insitute of Biology, Bsc

Languages : Polish – native English – B2 Dutch – B1 Czech – A2