Changes in body size and fertility due to artificial and natural feeding of laboratory common bed bugs (Heteroptera: Cimicidae).

Rearing common bed bugs (Cimex lectularius L.) and other hematophagous insects is essential for basic, medical, and pest-control research. Logistically, acquiring fresh blood can be a challenge, while biologically, the eventual effects of different rearing and blood preparation protocols on bed bug genotype and phenotype pose a risk of biased research results. Using bed bug populations that are either bat- (BL) or human-related (HL), we tested the short- and long-term effects of rearing bugs on live bats or human volunteers, or artificially on CPDA (citrate phosphate dextrose, adenine)-treated blood, measuring meal size, body size, and fertility. We found that artificial feeding did not affect meal size compared with feeding on natural hosts. Long-term rearing across many generations of HL on CPDA-preserved blood led to reduced body size and fertility compared with populations reared on human volunteers. Blood preservatives increased the proportion of sterile eggs even after a single feed. Finally, our results indicated that laboratory reared bed bugs were smaller, regardless of the blood source, than wild bugs. Similar effects of artificial feeding or laboratory rearing alone should be considered in future studies using bed bug cultures to choose an appropriate rearing protocol. With regard to switching between bat and human hosts, HL took smaller meals and BL had lower fertility when fed on bats than when fed on humans. We attribute these results to methodological constrains, specifically the inconsistency of bat feeding, rather than to host specialization. Nevertheless, BL can be easily reared using human blood and artificial feeding systems.

Despite genetic isolation in sympatry, post-copulatory reproductive barriers have not evolved between bat- and human-associated common bedbugs (Cimex lectularius L.).

BACKGROUND: The common bedbug Cimex lectularius is a widespread ectoparasite on humans and bats. Two genetically isolated lineages, parasitizing either human (HL) or bat (BL) hosts, have been suggested to differentiate because of their distinct ecology. The distribution range of BL is within that of HL and bedbugs live mostly on synanthropic bat hosts. This sympatric co-occurrence predicts strong reproductive isolation at the post-copulatory level. RESULTS: We tested the post-copulatory barrier in three BL and three HL populations in reciprocal crosses, using a common-garden blood diet that was novel to both lineages. We excluded pre-copulation isolation mechanisms and studied egg-laying rates after a single mating until the depletion of sperm, and the fitness of the resulting offspring. We found a higher sperm storage capability in BL, likely reflecting the different seasonal availability of HL and BL hosts. We also observed a notable variation in sperm function at the population level within lineages and significant differences in fecundity and offspring fitness between lineages. However, no difference in egg numbers or offspring fitness was observed between within- and between-lineage crosses. CONCLUSIONS: Differences in sperm storage or egg-laying rates between HL and BL that we found did not affect reproductive isolation. Neither did the population-specific variation in sperm function. Overall, our results show no post-copulatory reproductive isolation between the lineages. How genetic differentiation in sympatry is maintained in the absence of a post-copulatory barrier between BL and HL remains to be investigated.

Age-related mating rates among ecologically distinct lineages of bedbugs, Cimex lectularius.

Understanding how many mates an animal has in its lifetime is a critical factor in sexual selection. At the same time, differences in an organism's ecology, such as the quantity and quality of food, could be reflected in different mating rates. Mating rate had a significant effect on female net fitness (i.e., lifetime offspring production), however, laboratory measurements cannot well mirror the situation in wild. The common bedbug (Cimex lectularius) is a well-established model for studying traumatic insemination and sexual conflict. The species comprises two host lineages that feed on bats (BL) or humans (HL). HL can constantly feed on human hosts throughout the year, while BLs feed only during summer months when their bat hosts occupy the roosts. Because mating in female bedbugs is closely linked to foraging, this system provides a valuable model to study mating variation in the field. We established a new method for estimating age-dependent mating rates of females in the wild by relating the fluorescent pigment accumulation in the eyes of females to the number of mating scars that manifest as melanized spots caused by the injection of sperm through the wall of the female abdomen by the male into the spermalege. In addition, using laboratory bedbugs we found that three and a half observed matings on average lead to one observed melanized mating scar. Although young BL and HL females (with low pteridine concentrations) did not differ in the number of matings, the mating rate increased with age only in HL but not in BL females. We sampled on average older BL than HL females. The lack of access to food (bat blood) during winter could explain the lack of increase in the number of scars with age in BL. In species where mating leaves visible marks, using fluorescent pigments to determine female age (applicable to most arthropods) could be an important tool to study sexual selection and mating rate in the wild. The method can help formulate sustainable and biologically lucid approaches for their control.

Effects of diesel exhaust particles on the health and survival of the buff-tailed bumblebee Bombus terrestris after acute and chronic oral exposure.

The diesel-powered transportation sector is a major producer of environmental pollution in the form of micro- and nanoscale diesel exhaust particles (DEP). Pollinators, such as wild bees, may inhale DEP or ingest it orally through plant nectar. However, if these insects are adversely affected by DEP is largely unknown. To investigate potential health threats of DEP to pollinators, we exposed individuals of Bombus terrestris to different concentrations of DEP. We analysed the polycyclic aromatic hydrocarbons (PAH) content of DEP since these are known to elicit adverse effects on invertebrates. We investigated the dose-dependent effects of those well-characterized DEP on survival and fat body content, as a proxy for the insects' health condition, in acute and chronic oral exposure experiments. Acute oral exposure to DEP showed no dose-dependent effects on survival or fat body content of B. terrestris. However, we could show dose-dependent effects after chronic oral exposure with high doses of DEP where significantly increased mortality was observed. Further, there was no dose-dependent effect of DEP on the fat body content after exposure. Our results give insights into how the accumulation of high concentrations of DEP e.g., near heavily trafficked sites, can influence insect pollinators' health and survival.

Diesel exhaust particles alter gut microbiome and gene expression in the bumblebee Bombus terrestris.

Insect decline is a major threat to ecosystems around the world as they provide many important functions, such as pollination or pest control. Pollution is one of the main reasons for the decline, alongside changes in land use, global warming, and invasive species. While negative impacts of pesticides are well-studied, there is still a lack of knowledge about the effects of other anthropogenic pollutants, such as airborne particulate matter, on insects. To address this, we exposed workers of the bumblebee Bombus terrestris to sublethal doses of diesel exhaust particles (DEPs) and brake dust, orally or via air. After 7 days, we looked at the composition of the gut microbiome and tracked changes in gene expression. While there were no changes in the other treatments, oral DEP exposure significantly altered the structure of the gut microbiome. In particular, the core bacterium Snodgrassella had a decreased abundance in the DEP treatment. Similarly, transcriptome analysis revealed changes in gene expression after oral DEP exposure, but not in the other treatments. The changes are related to metabolism and signal transduction, which indicates a general stress response. Taken together, our results suggest potential health effects of DEP exposure on insects, here shown in bumblebees, as gut dysbiosis may increase the susceptibility of bumblebees to pathogens, while a general stress response may lower available energy resources. Those effects may exacerbate under natural conditions where insects face a multiple-stressor environment.

Comparison of fitness effects in the earthworm Eisenia fetida after exposure to single or multiple anthropogenic pollutants.

Terrestrial ecosystems are exposed to many anthropogenic pollutants. Non-target effects of pesticides and fertilizers have put agricultural intensification in the focus as a driver for biodiversity loss. However, other pollutants, such as heavy metals, particulate matter, or microplastic also enter the environment, e.g. via traffic and industrial activities in urban areas. As soil acts as a potential sink for such pollutants, soil invertebrates like earthworms may be particularly affected by them. Under natural conditions soil invertebrates will likely be exposed to combinations of pollutants simultaneously, which may result in stronger negative effects if pollutants act synergistically. Within this work we study how multiple pollutants affect the soil-dwelling, substrate feeding earthworm Eisenia fetida. We compared the effects of the single stressors, polystyrene microplastic fragments, polystyrene fibers, brake dust and carbon black, with the combined effect of these pollutants when applied as a mixture. Endpoints measured were survival, increase in body weight, reproductive fitness, and changes in three oxidative stress markers (glutathione S-transferase, catalase and malondialdehyde). We found that among single pollutant treatments, brake dust imposed the strongest negative effects on earthworms in all measured endpoints including increased mortality rates. Sub-lethal effects were found for all pollutants. Exposing earthworms to all four pollutants simultaneously led to effects on mortality and oxidative stress markers that were smaller than expected by the respective null models. These antagonistic effects are likely a result of the adsorption of toxic substances found in brake dust to the other pollutants. With this study we show that effects of combinations of pollutants cannot necessarily be predicted from their individual effects and that combined effects will likely depend on identity and concentration of the pollutants.

Seminal fluid and sperm diluent affect sperm metabolism in an insect: Evidence from NAD(P)H and flavin adenine dinucleotide autofluorescence lifetime imaging.

Sperm metabolism is fundamental to sperm motility and male fertility. Its measurement is still in its infancy, and recommendations do not exist as to whether or how to standardize laboratory procedures. Here, using the sperm of an insect, the common bedbug, Cimex lectularius, we demonstrate that standardization of sperm metabolism is required with respect to the artificial sperm storage medium and a natural medium, the seminal fluid. We used fluorescence lifetime imaging microscopy (FLIM) in combination with time-correlated single-photon counting (TCSPC) to quantify sperm metabolism based on the fluorescent properties of autofluorescent coenzymes, NAD(P)H and flavin adenine dinucleotide. Autofluorescence lifetimes (decay times) differ for the free and protein-bound state of the co-enzymes, and their relative contributions to the lifetime signal serve to characterize the metabolic state of cells. We found that artificial storage medium and seminal fluid separately, and additively, affected sperm metabolism. In a medium containing sugars and amino acids (Grace's Insect medium), sperm showed increased glycolysis compared with a commonly used storage medium, phosphate-buffered saline (PBS). Adding seminal fluid to the sperm additionally increased oxidative phosphorylation, likely reflecting increased energy production of sperm during activation. Our study provides a protocol to measure sperm metabolism independently from motility, stresses that protocol standardizations for sperm measurements should be implemented and, for the first time, demonstrates that seminal fluid alters sperm metabolism. Equivalent protocol standardizations should be imposed on metabolic investigations of human sperm samples.

Male diet affects female fitness and sperm competition in human- and bat-associated lineages of the common bedbug, Cimex lectularius.

Sperm performance can vary in ecologically divergent populations, but it is often not clear whether the environment per se or genomic differences arising from divergent selection cause the difference. One powerful and easily manipulated environmental effect is diet. Populations of bedbugs (Cimex lectularius) naturally feed either on bat or human blood. These are diverging genetically into a bat-associated and a human-associated lineage. To measure how male diet affects sperm performance, we kept males of two HL and BL populations each on either their own or the foreign diet. Then we investigated male reproductive success in a single mating and sperm competition context. We found that male diet affected female fecundity and changed the outcome of sperm competition, at least in the human lineage. However, this influence of diet on sperm performance was moulded by an interaction. Bat blood generally had a beneficial effect on sperm competitiveness and seemed to be a better food source in both lineages. Few studies have examined the effects of male diet on sperm performance generally, and sperm competition specifically. Our results reinforce the importance to consider the environment in which sperm are produced. In the absence of gene flow, such differences may increase reproductive isolation. In the presence of gene flow, however, the generally better sperm performance after consuming bat blood suggests that the diet is likely to homogenise rather than isolate populations.

Individual vs. Combined Short-Term Effects of Soil Pollutants on Colony Founding in a Common Ant Species.

Insects are integral to terrestrial life and provide essential ecosystem functions such as pollination and nutrient cycling. Due to massive declines in insect biomass, abundance, or species richness in recent years, the focus has turned to find their causes. Anthropogenic pollution is among the main drivers of insect declines. Research addressing the effects of pollutants concentrates on aquatic insects and pollinators, despite the apparent risk of contaminated soils. Pollutants accumulating in the soil might pose a significant threat because concentrations tend to be high and different pollutants are present simultaneously. Here, we exposed queens of the black garden ant Lasius niger at the colony founding stage to different concentrations and combinations of pollutants (brake dust, soot, microplastic particles and fibers, manure) to determine dose-dependent effects and interactions between stressors. As proxies for colony founding success, we measured queen survival, the development time of the different life stages, the brood weight, and the number of offspring. Over the course of the experiment queen mortality was very low and similar across treatments. Only high manure concentrations affected the colony founding success. Eggs from queens exposed to high manure concentrations took longer to hatch, which resulted in a delayed emergence of workers. Also, fewer pupae and workers were raised by those queens. Brake dust, soot and plastic particles did not visibly affect colony founding success, neither as single nor as multiple stressors. The application of manure, however, affected colony founding in L. niger negatively underlining the issue of excessive manure application to our environment. Even though anthropogenic soil pollutants seem to have little short-term effects on ant colony founding, studies will have to elucidate potential long-term effects as a colony grows.

Identification and age-dependence of pteridines in bed bugs (Cimex lectularius) and bat bugs (C. pipistrelli) using liquid chromatography-tandem mass spectrometry.

Determining the age of free-living insects, particularly of blood-sucking species, is important for human health because such knowledge critically influences the estimates of biting frequency and vectoring ability. Genetic age determination is currently not available. Pteridines gradually accumulate in the eyes of insects and their concentrations is the prevailing method. Despite of their stability, published extractions differ considerably, including for standards, for mixtures of pteridines and even for light conditions. This methodological inconsistency among studies is likely to influence age estimates severely and to hamper their comparability. Therefore we reviewed methodological steps across 106 studies to identify methodological denominators and results across studies. Second, we experimentally test how different pteridines vary in their age calibration curves in, common bed (Cimex lectularius) and bat bugs (C. pipistrelli). Here we show that the accumulation of particular pteridines varied between a) different populations and b) rearing temperatures but not c) with the impact of light conditions during extraction or d) the type of blood consumed by the bugs. To optimize the extraction of pteridines and measuring concentrations, we recommend the simultaneous measurement of more than one standard and subsequently to select those that show consistent changes over time to differentiate among age cohorts.

Mating changes the genital microbiome in both sexes of the common bedbug Cimex lectularius across populations.

Many bacteria live on host surfaces, in cells and in specific organ systems. In comparison with gut microbiomes, the bacterial communities of reproductive organs (genital microbiomes) have received little attention. During mating, male and female genitalia interact and copulatory wounds occur, providing an entrance for sexually transmitted microbes. Besides being potentially harmful to the host, invading microbes might interact with resident genital microbes and affect immunity. Apart from the investigation of sexually transmitted symbionts, few studies have addressed how mating changes genital microbiomes. We dissected reproductive organs from virgin and mated common bedbugs, Cimex lectularius L., and sequenced their microbiomes to investigate composition and mating-induced changes. We show that mating changes the genital microbiomes, suggesting bacteria are sexually transmitted. Also, genital microbiomes varied between populations and the sexes. This provides evidence for local and sex-specific adaptation of bacteria and hosts, suggesting bacteria might play an important role in shaping the evolution of reproductive traits. Coadaptation of genital microbiomes and reproductive traits might further lead to reproductive isolation between populations, giving reproductive ecology an important role in speciation. Future studies should investigate the transmission dynamics between the sexes and populations to uncover potential reproductive barriers.

Female immunity in response to sexually transmitted opportunistic bacteria in the common bedbug Cimex lectularius.

Besides typical sexually transmitted microbes, even environmental, opportunistic microbes have been found in copulatory organs of insects and even humans. To date, only one study has experimentally investigated the sexual transmission of opportunistic microbes from male to female insects, whereas nothing is known about the transmission from females to males. Even if opportunistic microbes do not cause infection upon transmission, they might eventually become harmful if they multiply inside the female. While the immune system of females is often assumed to target sexually transmitted microbes, most studies ignore the role of mating-associated opportunistic microbes. Variation in immunity between populations has been linked to parasite or bacteria prevalence but no study has ever addressed between-population differences in immune responses to sexually transmitted opportunistic microbes. We here show that bacteria applied to the copulatory organs of common bedbugs, Cimex lectularius, are sexually transmitted to the opposite sex at a high rate, including the transmission from female to male. Bacterial growth in the female sperm-receiving organ was inhibited over the first hours after introduction, but after this initial inhibition bacterial numbers increased, suggesting a shift of investment from immune defence towards reproduction. However, 24 h after the injection of bacteria, male components, or saline as a control, the sperm-receiving organ showed lysozyme-like activity and inhibited the growth of Gram-negative and Gram-positive bacteria in vitro, potentially to mop up the remaining bacteria. Contrasting our prediction, neither bacterial growth nor immune responses differed between populations. Future studies should link transmission dynamics, immune responses and fitness effects in both sexes. Experimental manipulation of environmental bacteria could be used to investigate how transmission frequency and toxicity of sexually transmitted opportunistic microbes shapes bacteria clearance and immune responses across populations.

Pollutants and Their Interaction with Diseases of Social Hymenoptera.

Many insect species, including social insects, are currently declining in abundance and diversity. Pollutants such as pesticides, heavy metals, or airborne fine particulate matter from agricultural and industrial sources are among the factors driving this decline. While these pollutants can have direct detrimental effects, they can also result in negative interactive effects when social insects are simultaneously exposed to multiple stressors. For example, sublethal effects of pollutants can increase the disease susceptibility of social insects, and thereby jeopardize their survival. Here we review how pesticides, heavy metals, or airborne fine particulate matter interact with social insect physiology and especially the insects' immune system. We then give an overview of the current knowledge of the interactive effects of these pollutants with pathogens or parasites. While the effects of pesticide exposure on social insects and their interactions with pathogens have been relatively well studied, the effects of other pollutants, such as heavy metals in soil or fine particulate matter from combustion, vehicular transport, agriculture, and coal mining are still largely unknown. We therefore provide an overview of urgently needed knowledge in order to mitigate the decline of social insects.

Transmission, Tropism, and Biological Impacts of Torix Rickettsia in the Common Bed Bug Cimex lectularius (Hemiptera: Cimicidae).

The torix group of Rickettsia have been recorded from a wide assemblage of invertebrates, but details of transmission and biological impacts on the host have rarely been established. The common bed bug (Cimex lectularius) is a hemipteran insect which lives as an obligatory hematophagous pest of humans and is host to a primary Wolbachia symbiont and two facultative symbionts, a BEV-like symbiont, and a torix group Rickettsia. In this study, we first note the presence of a single Rickettsia strain in multiple laboratory bed bug isolates derived from Europe and Africa. Importantly, we discovered that the Rickettsia has segregated in two laboratory strains, providing infected and uninfected isogenic lines for study. Crosses with these lines established transmission was purely maternal. Fluorescence in-situ hybridization analysis indicates Rickettsia infection in oocytes, bacteriomes, and other somatic tissues. We found no evidence that Rickettsia infection was associated with sex ratio distortion activity, but Rickettsia infected individuals developed from first instar to adult more slowly. The impact of Rickettsia on fecundity and fertility resulted in infected females producing fewer fertile eggs. However, we could not find any evidence for cytoplasmic incompatibility associated with Rickettsia presence. These data imply the existence of an unknown benefit to C. lectularius carrying Rickettsia that awaits further research.

Regular Wounding in a Natural System: Bacteria Associated With Reproductive Organs of Bedbugs and Their Quorum Sensing Abilities.

During wounding, tissues are disrupted so that bacteria can easily enter the host and trigger a host response. Both the host response and bacterial communication can occur through quorum sensing (QS) and quorum sensing inhibition (QSI). Here, we characterize the effect of wounding on the host-associated bacterial community of the bed bug. This is a model system where the male is wounding the female during every mating. Whereas several aspects of the microbial involvement during wounding have been previously examined, it is not clear to what extent QS and QSI play a role. We find that the microbiome differs depending on mating and feeding status of female bedbugs and is specific to the location of isolation. Most organs of bedbugs harbor bacteria, which are capable of both QS and QSI signaling. By focusing on the prokaryotic quorum communication system, we provide a baseline for future research in this unique system. We advocate the bedbug system as suitable for studying the effects of bacteria on reproduction and for addressing prokaryote and eukaryote communication during wounding.

Condition-dependent ejaculate production affects male mating behavior in the common bedbug Cimex lectularius.

Food availability in the environment is often low and variable, constraining organisms in their resource allocation to different life-history traits. For example, variation in food availability is likely to induce condition-dependent investment in reproduction. Further, diet has been shown to affect ejaculate size, composition and quality. How these effects translate into male reproductive success or change male mating behavior is still largely unknown. Here, we concentrated on the effect of meal size on ejaculate production, male reproductive success and mating behavior in the common bedbug Cimex lectularius. We analyzed the production of sperm and seminal fluid within three different feeding regimes in six different populations. Males receiving large meals produced significantly more sperm and seminal fluid than males receiving small meals or no meals at all. While such condition-dependent ejaculate production did not affect the number of offspring produced after a single mating, food-restricted males could perform significantly fewer matings than fully fed males. Therefore, in a multiple mating context food-restricted males paid a fitness cost and might have to adjust their mating strategy according to the ejaculate available to them. Our results indicate that meal size has no direct effect on ejaculate quality, but food availability forces a condition-dependent mating rate on males. Environmental variation translating into variation in male reproductive traits reveals that natural selection can interact with sexual selection and shape reproductive traits. As males can modulate their ejaculate size depending on the mating situation, future studies are needed to elucidate whether environmental variation affecting the amount of ejaculate available might induce different mating strategies.

Do microsporidia function as "biological weapon" for Harmonia axyridis under natural conditions?

Invasive alien species, such as the multicoloured Asian ladybird Harmonia axyridis, are often regarded as major drivers of biodiversity loss. Therefore understanding which characteristics or mechanisms contribute to their invasive success is important. Here the role of symbiotic microsporidia in the hemolymph of H. axyridis was investigated in the context of intraguild predation between wild-caught H. axyridis and the native ladybird species Coccinella septempunctata. The microsporidia were recently discussed to contribute to the unpalatability of Harmonia for other coccinellids during intraguild predation and to function as "biological weapons". In the present study, visual detection of microsporidia in hemolymph samples revealed that 73.5% of H. axyridis were infected. Intraguild predation experiments between larvae of the two species showed a significant competitive advantage for H. axyridis, even against larger larvae of C. septempunctata. Adult C. septempunctata always killed and fed on H. axyridis larvae. However only 11.4% (4 of 47) of C. septempunctata that fed on infected H. axyridis died within 4 months. In contrast to previous studies this suggests that microsporidia or harmonine, the chemical defense compound of H. axyridis, do not lead to death of C. septempunctata preying on larvae of H. axyridis. Instead our results support the idea that competitive advantage during intraguild predation greatly facilitates the success of H. axyridis and that this may help this highly invasive species to outcompete native species. The impact of microsporidia on Harmonia itself as well as on interspecific interactions require further studies.

Outbreeding effects in an inbreeding insect, Cimex lectularius.

In some species, populations with few founding individuals can be resilient to extreme inbreeding. Inbreeding seems to be the norm in the common bed bug, Cimex lectularius, a flightless insect that, nevertheless, can reach large deme sizes and persist successfully. However, bed bugs can also be dispersed passively by humans, exposing inbred populations to gene flow from genetically distant populations. The introduction of genetic variation through this outbreeding could lead to increased fitness (heterosis) or be costly by causing a loss of local adaptation or exposing genetic incompatibility between populations (outbreeding depression). Here, we addressed how inbreeding within demes and outbreeding between distant populations impact fitness over two generations in this re-emerging public health pest. We compared fitness traits of families that were inbred (mimicking reproduction following a founder event) or outbred (mimicking reproduction following a gene flow event). We found that outbreeding led to increased starvation resistance compared to inbred families, but this benefit was lost after two generations of outbreeding. No other fitness benefits of outbreeding were observed in either generation, including no differences in fecundity between the two treatments. Resilience to inbreeding is likely to result from the history of small founder events in the bed bug. Outbreeding benefits may only be detectable under stress and when heterozygosity is maximized without disruption of coadaptation. We discuss the consequences of these results both in terms of inbreeding and outbreeding in populations with genetic and spatial structuring, as well as for the recent resurgence of bed bug populations.

Genitalia-associated microbes in insects.

In sexual reproduction different types of symbiotic relationships between insects and microbes have become established. For example, some bacteria have evolved almost exclusive vertical transmission and even define the compatibility of insect mating partners. Many strictly sexually transmitted diseases have also been described in insects. Apart from such rather specific relationships the role of opportunistic infections in the reproductive process has been widely neglected. Opportunistic microbes transmitted passively during mating might impose an energetic cost, as the immune system will need to be alert and will use resources to fight potential intruders. Through mating wounds and contaminated reproductive organs opportunistic microbes might be transferred to mating partners and even enter the body cavity. Females as the "receiving" sex are particularly likely to have evolved adaptations to avoid or reduce opportunistic infections. Males of several species show highly complex seminal fluids, which as well as containing components that influence a males' fertilization success, also possess antimicrobial substances. The role of antimicrobials in the reproductive process is not well understood. Some evidence hints at the protection of sperm against microbes, indicating a role for natural selection in shaping the evolution of reproductive traits. By highlighting the potential importance of microbes in sexual selection and their role in reproduction in general I will make a case for studies in sexual selection, especially the ones investigating postcopulatory processes, that should incorporate environmental, as well as genotypic variation, in reproductive traits.

Unifying external and internal immune defences.

External immune defence, such as antimicrobial secretions, is not generally viewed as part of the immune system. Nevertheless, it constitutes a first barrier to pathogens and manipulates the microbial environment. Hygienic measures from the protection of oneself or conspecifics, of the nesting site, or of stored food might be more efficient with secreted antimicrobials. Here, we argue that antimicrobial secretions represent an extended arm of the immune system, forming an underappreciated selective force in the evolution of immune systems. Integrating external immunity into the immune system and general host physiology provides an amenable concept for the understanding of immune system variation and life-history trade-offs. Future research should evaluate complementary or additive roles of antimicrobial secretions in relation to internal immunity.