Bats, Bacteria, and Bat Smell V.2.0: Repeatable Sex-Specific Differences in Scent Organ Microbiota.

Reproducibility is a fundamental principle in science, ensuring reliable and valid findings. However, replication studies are scarce, particularly in ecology, due to the emphasis on novelty for publication. We explored the possibility of replicating original findings in the field of microbial and chemical ecology by conducting a conceptual replication of a previous study analysing the sex-specific differences in the microbial communities inhabiting the wing sacs, a scent organ with crucial functions in olfactory communication, of greater sac-winged bat (Saccopteryx bilineata). In the original study, the skin swabs from the antebrachial wing sacs of the males and wing sac rudiments of the females were analysed using culture-dependent methods to test sex-specific differences. The authors demonstrated that males have lower microbial richness and different microbial composition than females. We attempted to reproduce these findings using 16S rRNA sequencing, which offers improved accuracy in pinpointing microbial members than culture-dependent methods because of advanced statistical methods. Our study validated the original study's findings: Males had a lower microbial richness, and the community composition differed between the sexes. Furthermore, in the current study, males had an increased abundance of bacteria that might potentially be involved in odour production and degradation of malodorous substances and antimicrobial production. Our conceptual replication study corroborated that microbes can play a role in shaping their host's olfactory phenotype and consequently influence sexual selection. Furthermore, the current study emphasises the importance of replication efforts and hopefully encourages a culture that values replication studies in scientific practice.

The smell of hunger: Norway rats provision social partners based on odour cues of need.

When individuals exchange helpful acts reciprocally, increasing the benefit of the receiver can enhance its propensity to return a favour, as pay-offs are typically correlated in iterated interactions. Therefore, reciprocally cooperating animals should consider the relative benefit for the receiver when deciding to help a conspecific. Norway rats (Rattus norvegicus) exchange food reciprocally and thereby take into account both the cost of helping and the potential benefit to the receiver. By using a variant of the sequential iterated prisoner's dilemma paradigm, we show that rats may determine the need of another individual by olfactory cues alone. In an experimental food-exchange task, test subjects were provided with odour cues from hungry or satiated conspecifics located in a different room. Our results show that wild-type Norway rats provide help to a stooge quicker when they receive odour cues from a hungry rather than from a satiated conspecific. Using chemical analysis by gas chromatography-mass spectrometry (GC-MS), we identify seven volatile organic compounds that differ in their abundance between hungry and satiated rats. Combined, this "smell of hunger" can apparently serve as a reliable cue of need in reciprocal cooperation, which supports the hypothesis of honest signalling.

Immune profile predicts survival and reflects senescence in a small, long-lived mammal, the greater sac-winged bat (Saccopteryx bilineata).

The immune system imposes costs that may have to be traded against investment of resources in other costly life-history traits. Yet, it is unknown if a trade-off between immunity and longevity occurs in free-ranging mammals. Here, we tested if age and survival, two aspects associated with longevity, are linked to immune parameters in an 8 g bat species. Using a combination of cross-sectional and longitudinal data, we assessed whether total white blood cell (WBC) counts, bacterial killing ability of the plasma (BKA) and immunoglobulin G (IgG) concentration change with age. Furthermore, we asked if these immune parameters impose costs resulting in decreased survival probabilities. We found that WBC counts decreased with age both within and among individuals. IgG concentrations were higher in older individuals, but did not change with age within individuals. Furthermore, individuals with above average WBC counts or IgG concentration had lower probabilities to survive the next six months. High WBC counts and IgG concentrations may reflect infections with parasites and pathogens, however, individuals that were infected with trypanosomes or nematodes showed neither higher WBC counts or IgG concentrations, nor was infection connected with survival rates. BKA was higher in infected compared with uninfected bats, but not related to age or survival. In conclusion, cellular (WBC) and humoral (IgG) parts of the immune system were both connected to age and survival, but not to parasite infections, which supports the hypothesis that energetically costly immunological defences are traded against other costly life-history traits, leading to a reduced lifespan in this free-ranging mammal.

Frugivory is associated with low measures of plasma oxidative stress and high antioxidant concentration in free-ranging bats.

Oxidative stress--an imbalance between reactive pro- and neutralising antioxidants--damages cell structures and impairs fitness-relevant traits such as longevity and reproduction. Theory predicts that feeding on diets with high antioxidant content such as fruits should reduce oxidative stress; however, there is no support of this idea in free-ranging mammals. Bats cover a large variety of ecological niches, and therefore, we asked if measures of oxidative stress are lower in species with fruit diets. We measured reactive oxygen metabolites (ROM) representing total pro-oxidants produced and antioxidants in the plasma of 33 Neotropical bat species. Species with a fruit diet showed the lowest level of ROM and the highest concentration of antioxidants, followed by omnivorous and animalivorous species. Potentially, frugivorous species ingest more antioxidants with food and thus are able to neutralise more pro-oxidants than species not feeding on fruits, resulting in an overall lower level of oxidative stress. We therefore showed for the first time that measures of oxidative stress vary according to diets in free-ranging mammals.

Inflammatory challenge increases measures of oxidative stress in a free-ranging, long-lived mammal.

Oxidative stress - the imbalance between reactive oxygen species (ROS) and neutralising antioxidants - has been under debate as the main cause of ageing in aerobial organisms. The level of ROS should increase during infection as part of the activation of an immune response, leading to oxidative damage to proteins, lipids and DNA. Yet, it is unknown how long-lived organisms, especially mammals, cope with oxidative stress. Bats are known to carry a variety of zoonotic pathogens and at the same time are, despite their high mass-specific basal metabolic rate, unusually long lived, which may be partly the result of low oxidative damage of organs. Here, we asked whether an immune challenge causes oxidative stress in free-ranging bats, measuring two oxidative stress markers. We injected 20 short-tailed fruit bats (Carollia perspicillata) with bacterially derived lipopolysaccharide (LPS) and 20 individuals with phosphate-buffered saline solution (PBS) as a control. Individuals injected with LPS showed an immune reaction by increased white blood cell count after 24 h, whereas there was no significant change in leukocyte count in control animals. The biological antioxidant potential (BAP) remained the same in both groups, but reactive oxygen metabolites (ROMs) increased after treatment with LPS, indicating a significant increase in oxidative stress in animals when mounting an immune reaction toward the inflammatory challenge. Control individuals did not show a change in oxidative stress markers. We conclude that in a long-lived mammal, even high concentrations of antioxidants do not immediately neutralise free radicals produced during a cellular immune response. Thus, fighting an infection may lead to oxidative stress in bats.

Ecological and dietary correlates of stable hydrogen isotope ratios in fur and body water of syntopic tropical bats.

Hydrogen stable isotope ratios of keratin (delta2H(K)) are increasingly used as endogenous markers to study animal movements, yet it is unclear what factors might influence delta2H(K) in free-ranging animals. We studied hydrogen stable isotope ratios of body water (delta2H(BW)) and fur keratin in 36 bat species (> 400 individuals) from a tropical forest assemblage to evaluate if delta2H(BW) and delta2H(K) are related to body size, trophic position, and movement ecology. We found a relatively large range of delta2H(BW) values (approximately 65 per thousand) across bat species. Our phylogenetically controlled analysis showed that delta2H(BW) was not related to body size, trophic position, or movement ecology of species. The analysis also indicated that delta2H(BW) of primary consumers (i.e., fruit-eating bats) was similar to that of fruit juice, and delta2H(BW) of secondary consumers (i.e., animalivorous bats) was similar to that of surface water. Across bat species, delta2H(K) tended to decrease with increasing delta2H(BW), suggesting that delta2H(K) is not directly linked to delta2H(BW). We further tested whether delta2H(K) varied with a species' trophic position (measured as delta15N) and movement ecology (local or regional species). In syntopic bats, delta2H(K) ranged over 73 per thousand, yet delta2H(K) was related neither to delta15N nor to the movement ecology of species. Fur keratin of secondary consumers was more enriched in 2H by 44 per thousand and in 15N by 3.7 per thousand compared with fur keratin of primary consumers. In an intraspecific analysis, we found that delta2H(K) of an insectivorous bat varied by 20 per thousand between colonies at 4 km distance. Within this species, deltaH(K) was not related to individual delta15N and body mass. Our data suggest that variation in delta2H(K) can be large in bats of tropical assemblages, both across species (range approximately 70 per thousand) and even within sedentary species (range approximately 20 per thousand), and that delta2H(K) is largely affected by the dietary habits of species.

Measures of the constitutive immune system are linked to diet and roosting habits of neotropical bats.

Ecological and social factors are central in the emergence and transmission of infectious diseases, thus bearing the potential for shaping a species' immune functions. Although previous studies demonstrated a link between social factors and the cellular immune system for captive mammals, it is yet poorly understood how ecological factors are connected with the different branches of the immune system in wild populations. Here, we tested how variation in aspects of the constitutive cellular and humoral immune system of free ranging bats is associated with two ecological factors that likely influence the putative risk of species to become infected by parasites and pathogens: diet and shelter. We found that white blood cell counts of 24 syntopic Neotropical bat species varied with the species' diet and body mass. Bats that included at least partially vertebrates in their diet exhibited the highest white blood cell counts, followed by phytophagous and insectivorous species, which is in agreement with the assumption that the immune system varies with the pathogen transmission risk of a trophic level. The soluble part of the constitutive immune response, assessed by an in vitro bacterial killing assay, decreased with increasing roost permanence. Our results suggest that the ecology is an important factor in the evolution of the immune system in bats and probably also other mammals.

Reciprocal cooperation between unrelated rats depends on cost to donor and benefit to recipient.

BACKGROUND: Although evolutionary models of cooperation build on the intuition that costs of the donor and benefits to the receiver are the most general fundamental parameters, it is largely unknown how they affect the decision of animals to cooperate with an unrelated social partner. Here we test experimentally whether costs to the donor and need of the receiver decide about the amount of help provided by unrelated rats in an iterated prisoner's dilemma game. RESULTS: Fourteen unrelated Norway rats were alternately presented to a cooperative or defective partner for whom they could provide food via a mechanical apparatus. Direct costs for this task and the need of the receiver were manipulated in two separate experiments. Rats provided more food to cooperative partners than to defectors (direct reciprocity). The propensity to discriminate between helpful and non-helpful social partners was contingent on costs: An experimentally increased resistance in one Newton steps to pull food for the social partner reduced the help provided to defectors more strongly than the help returned to cooperators. Furthermore, test rats provided more help to hungry receivers that were light or in poor condition, which might suggest empathy, whereas this relationship was inverse when experimental partners were satiated. CONCLUSIONS: In a prisoner's dilemma situation rats seem to take effect of own costs and potential benefits to a receiver when deciding about helping a social partner, which confirms the predictions of reciprocal cooperation. Thus, factors that had been believed to be largely confined to human social behaviour apparently influence the behaviour of other social animals as well, despite widespread scepticism. Therefore our results shed new light on the biological basis of reciprocity.

Rain increases the energy cost of bat flight.

Similar to insects, birds and pterosaurs, bats have evolved powered flight. But in contrast to other flying taxa, only bats are furry. Here, we asked whether flight is impaired when bat pelage and wing membranes get wet. We studied the metabolism of short flights in Carollia sowelli, a bat that is exposed to heavy and frequent rainfall in neotropical rainforests. We expected bats to encounter higher thermoregulatory costs, or to suffer from lowered aerodynamic properties when pelage and wing membranes catch moisture. Therefore, we predicted that wet bats face higher flight costs than dry ones. We quantified the flight metabolism in three treatments: dry bats, wet bats and no rain, wet bats and rain. Dry bats showed metabolic rates predicted by allometry. However, flight metabolism increased twofold when bats were wet, or when they were additionally exposed to rain. We conclude that bats may not avoid rain only because of sensory constraints imposed by raindrops on echolocation, but also because of energetic constraints.