Parasitol Res DOI 10.1007/s00436-015-4504-1
ORIGINAL PAPER
Reproduction barrier between two lineages of bed bug (Cimex lectularius) (Heteroptera: Cimicidae) Kamila Wawrocka 1 & Ondřej Balvín 2 & Tomáš Bartonička 1
Received: 24 February 2015 / Accepted: 24 April 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract Populations of bed bugs, Cimex lectularius, have increased in recent years spreading into numerous urban areas across the Western world and making them an increasingly important pest of the twenty-first century. Research into hybridization within and between different lineages of bed bugs can help us to understand processes of micro- and macroevolution in these ectoparasites and may inform the control of this pest species. Hybridization experiments between two host lineages of bed bug (C. lectularius) from Central Europe (Czech Republic), those associated with humans and those with bats, were conducted under laboratory conditions. Number of eggs and early instars were compared between 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 production and later instars, crosses between different host lineages were unsuccessful, although of the mated females possessed sperm in their mesospermaleges and/or seminal conceptacles. These crosses did not even result in egg production. Moreover, in the mixed lineage crosses, mortality rates in
* Tomáš Bartonička [email protected] Kamila Wawrocka [email protected] Ondřej Balvín [email protected] 1
Department of Botany and Zoology, Masaryk University, Kotlářská 2, 611 37 Brno, Czech Republic
2
Department of Ecology, Faculty of Environmental Sciences, Czech University of Life Sciences Prague, Kamýcká 129, 165 21 Prague 6, Czech Republic
adults were higher (51 and 50 % higher in bat and human lineage, respectively) than in those animals from the same lineage. Survival of adults was in pairs from the same locality slightly higher than in pairs from different localities and differed statistically. These results support the existence of postmating barriers and show reproductive isolation between two lineages of C. lectularius. Bat and human host adaptations can promote evolving of such barriers and can be product of alloxenic speciation. Keywords Hybridization . Cimicids . Artificial feeding . Sperm presence
Introduction High genetic diversity in parasites and hosts equip each side of a parasite-host association with evolutionary weapons. In many cases of parasitic insects, speciation events are associated with their hosts (Poulin 2007). If we assume that polymorphism of habitat preference (host body and/or host shelter) exists in the parasite population, there would be a tendency for parasites to survive on the same host species and mate with individuals having similar genotypes. However, gene flow is reduced between parasites of the same species living on two different host species (Brooks and McLennan 1993). When gene flow is dramatically reduced or interrupted, alloxenic speciation may occur and narrow host specificity (Combes and Théron 2000). This specific mode of speciation is driven through a shift of host specificity and subsequent development of reproductive barriers (Euzet and Combes 1980; Mehlhorn 2008). Local adaptation and differentiation can lead to the formation of host races within a species and is considered a major route for sympatric speciation (Coyne and Orr 2004).
Parasitol Res
In this context, two mechanisms may maintain specificity after host speciation and/or host switching (Combes and Théron 2000). In sympatry, according to main biological species concept (Merrell 1981), hybridization should be prevented by (i) recognition concept where individuals may 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 fertilization to occur or produces infertile hybrids (Wheeler and Meier 2000). Consequently, hybridization between lineages or closely related species can increase host range (Detwiler and Criscione 2010), which can be problematic especially in the case of medically important haematophagous insects. One of those is the bed bug (Cimex lectularius), a widely distributed ectoparasite that has enlarged its range in recent years resulting in the increase of cases of infestations in human dwellings (Krueger 2000). Hybridization tests have shown that interspecific mating between C. lectularius and Cimex hemipterus causes high mortality in females and shortens their lifespan, and results in the production of high numbers of infertile eggs (Omori 1939, 1941). There is also an indication that sperm fluids from one species of bed bug appear to be toxic to female of another species. Despite this, it has been shown that under natural conditions, around 69 % of individuals of those two species can successfully mate among each other (Coetzee et al. 1995). Although C. lectularius and Cimex columbarius interbreed freely (Johnson 1939; Titschack 1949), it has been demonstrated that the population of such hybrids will never stabilize in nature and successful mating was connected with the fact that C. columbarius is closely related to C. lectularius (Usinger 1966). Both species are well isolated due to selective mating and high reduction in the number of eggs laid (Ueshima 1964). Members of host races are in general more suited to native hosts than alternative hosts, and offspring they produce with individuals from different native host have reduced fitness (Drès and Mallet 2002); the discontinuity in gene flow caused by physical isolation or assortative mating may lead to incipient speciation (Combes and Théron 2000). Strong genetic (separate evolutionary history based on differences of mtDNA) and morphological divergence (i.e., leg dimensions, body size) between European bed bugs associated with humans and those collected within the roosts of synanthropic bats was proven recently (Balvín et al. 2012). Evidence from host choice, survival rate and development suggests they represent two different lineages—bat and human associated (Wawrocka and Bartonička 2013). These two lineages of bed bug are not geographically isolated, but they parasitize different hosts which in recent decades rarely occupy the same shelter. Therefore, we expect that barriers in their reproduction could also have developed. The main aims of the study were to (1) investigate whether hybrids between
bat- and human-associated bed bugs are produced or not, (2) determine survival and reproduction rates of hybrids, if produced, compared with control samples and finally (3) see how often mating takes place and if the sperm has been transferred if no hybrids are present.
Material and methods Sampling, rearing and feeding of bed bugs Bed bugs (C. lectularius) from different localities in Czech Republic were collected from bats roosts (Hanusovice—A, Prudka—B) and human dwellings (Havirov—C, Znojmo—D). Bed bugs associated with bats were sampled from nursery colonies of the greater mouseeared bat (Myotis myotis) and the Geoffroy’s bat (Myotis emarginatus), roosting in church attics. The bat bugs were collected using soft tweezers or exhaustors and placed in plastic boxes (10×10×5 cm) lined with soft paper. Bed bugs associated with humans were collected in heavily infested apartments directly before pest control operations were carried out. All bugs were separated into adult individuals, early instars (first to third) and late instars (fourth to fifth), and stored in separate tubes (1 cm×7 cm) closed with cotton and filled with a piece of paper (1 cm×4 cm). All samples were kept under stable microclimatic conditions in thermostatically controlled apparatus (ST2, EkoAparatura, Poland) under stable temperature (23±0.1 °C) and humidity (75±10 %) which according to Omori (1941) and Usinger (1966) is most suitable for the development of C. lectularius. Bugs were fed every 5 days with human blood (laboratory commercial blood, Japan Medical Supply). Blood was warmed up in water bath (NE4-14D, Clifton Range) till 37 °C, and bugs were fed by artificial skin (Durex condoms) for ca. 30 min (until ca. 80 % of individuals took blood meal). Experimental setting First crosses were composed of mixed lineages: human×batassociated bug (interspecific mating), and second from bugs from the same lineage (intraspecific). In order to control for the possibility of pre-mating barriers due to geographical separation, crosses were made with animals of the same lineage from (a) the same locality and (b) different localities. All three combinations (interspecific, intraspecific—within and between localities mating) were in both sex directions. In mixed samples, we had 60 males and 60 females (30 females from human lineage, 30 from bat lineage, the same for males). To ensure that offspring only occurred from experimental crosses, virgin females were obtained from collected instars and kept separately from males until experiment. In total, 60 pairs for interspecific mating and 120 pairs (60 within locality
Parasitol Res
In order to determine if sperm has been transferred, mixed, within locality and between localities, pairs were placed on petri dishes and observed after feeding. Directly after, mating was observed and females were checked for sperm presence, first under the microscope and later, if it was necessary, by the removal of the seminal conceptacle and microscopical examination of the contents (Stutt and Siva-Jothy 2001). In total, 15 pairs (15 males and 15 females) for within localities, 15 between localities and 25 pairs for mixed samples were investigated. Seminal conceptacles were removed from 5 females from interspecific matings and 4 from intraspecific (as a control).
than in females from intraspecific crosses (Table 1). Log rank test for groups of females from intra- and interspecific mating showed their survival differed significantly. Females from mixed crosses had lower survival rates compared with females from within a locality (χ2 =58.70, p
ORIGINAL PAPER
Reproduction barrier between two lineages of bed bug (Cimex lectularius) (Heteroptera: Cimicidae) Kamila Wawrocka 1 & Ondřej Balvín 2 & Tomáš Bartonička 1
Received: 24 February 2015 / Accepted: 24 April 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract Populations of bed bugs, Cimex lectularius, have increased in recent years spreading into numerous urban areas across the Western world and making them an increasingly important pest of the twenty-first century. Research into hybridization within and between different lineages of bed bugs can help us to understand processes of micro- and macroevolution in these ectoparasites and may inform the control of this pest species. Hybridization experiments between two host lineages of bed bug (C. lectularius) from Central Europe (Czech Republic), those associated with humans and those with bats, were conducted under laboratory conditions. Number of eggs and early instars were compared between 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 production and later instars, crosses between different host lineages were unsuccessful, although of the mated females possessed sperm in their mesospermaleges and/or seminal conceptacles. These crosses did not even result in egg production. Moreover, in the mixed lineage crosses, mortality rates in
* Tomáš Bartonička [email protected] Kamila Wawrocka [email protected] Ondřej Balvín [email protected] 1
Department of Botany and Zoology, Masaryk University, Kotlářská 2, 611 37 Brno, Czech Republic
2
Department of Ecology, Faculty of Environmental Sciences, Czech University of Life Sciences Prague, Kamýcká 129, 165 21 Prague 6, Czech Republic
adults were higher (51 and 50 % higher in bat and human lineage, respectively) than in those animals from the same lineage. Survival of adults was in pairs from the same locality slightly higher than in pairs from different localities and differed statistically. These results support the existence of postmating barriers and show reproductive isolation between two lineages of C. lectularius. Bat and human host adaptations can promote evolving of such barriers and can be product of alloxenic speciation. Keywords Hybridization . Cimicids . Artificial feeding . Sperm presence
Introduction High genetic diversity in parasites and hosts equip each side of a parasite-host association with evolutionary weapons. In many cases of parasitic insects, speciation events are associated with their hosts (Poulin 2007). If we assume that polymorphism of habitat preference (host body and/or host shelter) exists in the parasite population, there would be a tendency for parasites to survive on the same host species and mate with individuals having similar genotypes. However, gene flow is reduced between parasites of the same species living on two different host species (Brooks and McLennan 1993). When gene flow is dramatically reduced or interrupted, alloxenic speciation may occur and narrow host specificity (Combes and Théron 2000). This specific mode of speciation is driven through a shift of host specificity and subsequent development of reproductive barriers (Euzet and Combes 1980; Mehlhorn 2008). Local adaptation and differentiation can lead to the formation of host races within a species and is considered a major route for sympatric speciation (Coyne and Orr 2004).
Parasitol Res
In this context, two mechanisms may maintain specificity after host speciation and/or host switching (Combes and Théron 2000). In sympatry, according to main biological species concept (Merrell 1981), hybridization should be prevented by (i) recognition concept where individuals may 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 fertilization to occur or produces infertile hybrids (Wheeler and Meier 2000). Consequently, hybridization between lineages or closely related species can increase host range (Detwiler and Criscione 2010), which can be problematic especially in the case of medically important haematophagous insects. One of those is the bed bug (Cimex lectularius), a widely distributed ectoparasite that has enlarged its range in recent years resulting in the increase of cases of infestations in human dwellings (Krueger 2000). Hybridization tests have shown that interspecific mating between C. lectularius and Cimex hemipterus causes high mortality in females and shortens their lifespan, and results in the production of high numbers of infertile eggs (Omori 1939, 1941). There is also an indication that sperm fluids from one species of bed bug appear to be toxic to female of another species. Despite this, it has been shown that under natural conditions, around 69 % of individuals of those two species can successfully mate among each other (Coetzee et al. 1995). Although C. lectularius and Cimex columbarius interbreed freely (Johnson 1939; Titschack 1949), it has been demonstrated that the population of such hybrids will never stabilize in nature and successful mating was connected with the fact that C. columbarius is closely related to C. lectularius (Usinger 1966). Both species are well isolated due to selective mating and high reduction in the number of eggs laid (Ueshima 1964). Members of host races are in general more suited to native hosts than alternative hosts, and offspring they produce with individuals from different native host have reduced fitness (Drès and Mallet 2002); the discontinuity in gene flow caused by physical isolation or assortative mating may lead to incipient speciation (Combes and Théron 2000). Strong genetic (separate evolutionary history based on differences of mtDNA) and morphological divergence (i.e., leg dimensions, body size) between European bed bugs associated with humans and those collected within the roosts of synanthropic bats was proven recently (Balvín et al. 2012). Evidence from host choice, survival rate and development suggests they represent two different lineages—bat and human associated (Wawrocka and Bartonička 2013). These two lineages of bed bug are not geographically isolated, but they parasitize different hosts which in recent decades rarely occupy the same shelter. Therefore, we expect that barriers in their reproduction could also have developed. The main aims of the study were to (1) investigate whether hybrids between
bat- and human-associated bed bugs are produced or not, (2) determine survival and reproduction rates of hybrids, if produced, compared with control samples and finally (3) see how often mating takes place and if the sperm has been transferred if no hybrids are present.
Material and methods Sampling, rearing and feeding of bed bugs Bed bugs (C. lectularius) from different localities in Czech Republic were collected from bats roosts (Hanusovice—A, Prudka—B) and human dwellings (Havirov—C, Znojmo—D). Bed bugs associated with bats were sampled from nursery colonies of the greater mouseeared bat (Myotis myotis) and the Geoffroy’s bat (Myotis emarginatus), roosting in church attics. The bat bugs were collected using soft tweezers or exhaustors and placed in plastic boxes (10×10×5 cm) lined with soft paper. Bed bugs associated with humans were collected in heavily infested apartments directly before pest control operations were carried out. All bugs were separated into adult individuals, early instars (first to third) and late instars (fourth to fifth), and stored in separate tubes (1 cm×7 cm) closed with cotton and filled with a piece of paper (1 cm×4 cm). All samples were kept under stable microclimatic conditions in thermostatically controlled apparatus (ST2, EkoAparatura, Poland) under stable temperature (23±0.1 °C) and humidity (75±10 %) which according to Omori (1941) and Usinger (1966) is most suitable for the development of C. lectularius. Bugs were fed every 5 days with human blood (laboratory commercial blood, Japan Medical Supply). Blood was warmed up in water bath (NE4-14D, Clifton Range) till 37 °C, and bugs were fed by artificial skin (Durex condoms) for ca. 30 min (until ca. 80 % of individuals took blood meal). Experimental setting First crosses were composed of mixed lineages: human×batassociated bug (interspecific mating), and second from bugs from the same lineage (intraspecific). In order to control for the possibility of pre-mating barriers due to geographical separation, crosses were made with animals of the same lineage from (a) the same locality and (b) different localities. All three combinations (interspecific, intraspecific—within and between localities mating) were in both sex directions. In mixed samples, we had 60 males and 60 females (30 females from human lineage, 30 from bat lineage, the same for males). To ensure that offspring only occurred from experimental crosses, virgin females were obtained from collected instars and kept separately from males until experiment. In total, 60 pairs for interspecific mating and 120 pairs (60 within locality
Parasitol Res
In order to determine if sperm has been transferred, mixed, within locality and between localities, pairs were placed on petri dishes and observed after feeding. Directly after, mating was observed and females were checked for sperm presence, first under the microscope and later, if it was necessary, by the removal of the seminal conceptacle and microscopical examination of the contents (Stutt and Siva-Jothy 2001). In total, 15 pairs (15 males and 15 females) for within localities, 15 between localities and 25 pairs for mixed samples were investigated. Seminal conceptacles were removed from 5 females from interspecific matings and 4 from intraspecific (as a control).
than in females from intraspecific crosses (Table 1). Log rank test for groups of females from intra- and interspecific mating showed their survival differed significantly. Females from mixed crosses had lower survival rates compared with females from within a locality (χ2 =58.70, p
