bioRxiv preprint doi: https://doi.org/10.1101/819300; this version posted October 28, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Research article 2 3 4 Mating changes the genital microbiome in both sexes of the common bedbug 5 Cimex lectularius across populations 6 7 8 Running title: Mating-induced changes in bedbug genital microbiomes 9 10 11 Sara Bellinvia1, Paul R. Johnston2, Susan Mbedi3,4, Oliver Otti1 12 13 14 1 Animal Population Ecology, Animal Ecology I, University of Bayreuth, Universitätsstraße 30, 95440 15 Bayreuth, Germany 16 2 Institute for Biology, Free University Berlin, Königin-Luise-Straße 1-3, 14195 Berlin, Germany. 17 3 Museum für Naturkunde - Leibniz-Institute for Evolution and Biodiversity Research, Invalidenstraße 18 43, 10115 Berlin. 19 4 Berlin Center for Genomics in Biodiversity Research (BeGenDiv), Königin-Luise-Straße 1-3, 14195 20 Berlin, Germany. 21 22 23 Corresponding author: Sara Bellinvia 24 Phone: +49921552646, e-mail: [email protected] 1 bioRxiv preprint doi: https://doi.org/10.1101/819300; this version posted October 28, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 25 ABSTRACT 26 Many bacteria live on host surfaces, in cells, and specific organ systems. Although gut microbiomes of 27 many organisms are well-documented, the bacterial communities of reproductive organs, i.e. genital 28 microbiomes, have received little attention. During mating, male and female genitalia interact and 29 copulatory wounds can occur, providing an entrance for sexually transmitted microbes. Besides being 30 potentially harmful to the host, invading microbes might interact with resident genital microbes and 31 affect immunity. While sexual transmission of individual symbiont species is relatively well- 32 understood, few studies have addressed how mating changes genital microbiomes. Here, we 33 characterize male and female genital microbiomes in four different populations of the common 34 bedbug Cimex lectularius and investigate mating-induced changes. We dissected reproductive organs 35 from virgin and mated individuals to sequence their genital microbiomes. We show that mating 36 changes genital microbiomes, suggesting bacteria are sexually transmitted. This raises the question 37 how the host’s physiological and immunological mechanisms control mating-induced changes in their 38 genital microbiomes. Also, genital microbiomes varied between populations and the sexes. This 39 provides evidence for local and sex-specific adaptation of bacteria and hosts, again suggesting that 40 bacteria might play an important role in shaping the evolution of reproductive traits. Coadaptation of 41 genital microbiomes and reproductive traits might further lead to reproductive isolation between 42 populations, giving reproductive ecology an important role in speciation. Future studies should 43 investigate the transmission dynamics between the sexes and populations to uncover potential 44 reproductive barriers. 2 bioRxiv preprint doi: https://doi.org/10.1101/819300; this version posted October 28, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 45 INTRODUCTION 46 Animals have intimate associations with bacteria. These bacteria are found on host surfaces, within 47 and between host cells, or are associated with specific organ systems, such as the gut1–3. Also, 48 reproductive organs of a variety of animals often harbor several different bacterial species. Microbes 49 have been found on male reproductive organs and in semen of vertebrates4–9, but also in female 50 reproductive organs of birds and mammals, both including humans5,10–13. In insects, besides 51 intracellular reproductive manipulators (reviewed in14), extracellular microbes have been found both 52 on the male copulatory organ and within the female reproductive organs (reviewed in15, also see16,17). 53 Interestingly, the microbiomes of whole body homogenates or the gut are sex-specific in a variety of 54 species18–20. Possible reasons could be differences in behaviors or feeding strategies, different 55 functions of the two sexes in the ecosystem, or different roles in reproduction. Supporting the latter, 56 pronounced differences exist between the genital microbiomes of female and male red-winged 57 blackbirds5 and bedbugs16,17, respectively. However, the role of genital microbes in reproduction and 58 the exact composition of these genital microbiomes is still unknown for most insect species. 59 Microbiomes are dynamic and react to environmental change. Diet21,22, age23, climate24, and even time 60 of day25 alter the composition of the gut microbiome of humans, mice and lizards. Some life-history 61 events affect multiple microbiomes at the same time, for instance pregnancy changes the vaginal, gut, 62 and oral microbiomes26. Genital microbiomes might be subject to similar changes. For instance, 63 women have a distinct vaginal microbiome before and after the menopause27. An obvious pattern that 64 has the potential to affect genital microbiomes is mating as the microbiomes of both sexes encounter 65 each other and potentially interact. Therefore, physiological changes in anticipation to the production 66 of offspring might occur. To date, little is known about the potential effects of mating on the 67 composition of genital microbiomes, especially in species other than humans. 68 Microbes are sexually transmitted in a large range of species (e.g.28–30). Not only typical sexually 69 transmitted microbes (STM) are transferred during mating, also opportunistic microbes (OM) 70 inhabiting the genitalia17,31,32 might be transferred. Possible entry ports are genital openings and 71 copulatory wounds, which frequently occur for example in insects, spiders, flatworms and 3 bioRxiv preprint doi: https://doi.org/10.1101/819300; this version posted October 28, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 72 gastropods33. These OM might have a more direct effect compared to STM: Once transferred into the 73 female genital tract, sperm will be exposed to a rich microbial flora7,10. Several bacterial species, such 74 as Escherichia coli34–36, Pseudomonas aeruginosa37, and Staphylococcus aureus38 can decrease sperm 75 motility and agglutinate spermatozoa in humans. In insects, bacteria from the environment have the 76 potential to increase sperm mortality39. Therefore, male reproductive success might depend on the 77 microbe communities within the female, and males would be selected to protect their sperm. Females 78 could be invaded by STM or sexually transmitted OM, disturbing the present genital microbiome. The 79 intestinal microbiota of mice, for instance, was shown to be altered by Salmonella typhimurium40 and 80 Citrobacter rodentium41. Copulatory wounding during mating further increases the risk of 81 opportunistic infection not only in invertebrate species42,43, but also in humans44. Both males and 82 females might be subject to a considerable cost of reproduction, even if OM do not always become 83 pathogenic. Hosts should invest in reducing or regulating the number of STMs or sexually transmitted 84 OM to prevent uncontrolled growth, most likely by using their immune system. However, sexually 85 transmitted bacteria disturbing the composition of the genital microbiome might select not only for a 86 host immune response, but also for defensive responses in the resident microbiota16. Therefore, the 87 host and its endosymbionts have a mutual interest in keeping invading bacteria in check. In some 88 organisms, the resident microbiota is part of the interaction with invading microbes45–48. 89 If genital microbiomes are subject to OM, an adaptation to environmental microbes seems 90 conceivable. Indeed, the human vaginal microbiome seems to depend on ancestry: American women 91 of African, Asian, European, and Hispanic ancestry harbored distinct vaginal microbiomes11 and African 92 ancestry was related to larger alpha diversity of the vaginal microbiome compared to European 93 ancestry49, raising the question whether there are conserved differences in the community 94 composition between populations. Despite the potential relevance for speciation, not much is known 95 about the adaptation of insect genital microbiomes to different host populations which might lead to 96 reproductive isolation. Another unresolved question is how genital microbiomes are affected by 97 mating and whether induced changes differ between populations. Unlike in humans such mating- 98 induced changes can be experimentally investigated in insects with relatively little effort17. 4 bioRxiv preprint doi: https://doi.org/10.1101/819300; this version posted October 28, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 99 Bedbugs are an interesting system to study mating-induced changes of insect genital microbiomes 100 because several organs are involved in reproduction. During mating, these organs might be invaded by 101 microbes that use the routes of ejaculate transfer as entrance to the reproductive