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

doi:10.21203/rs.3.rs-3551111/v1

Download PDF

Research Article

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

https://doi.org/10.21203/rs.3.rs-3551111/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 25 Mar, 2024

Read the published version in Microbial Ecology →

Version 1

posted

You are reading this latest preprint version

Background: 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 collected and 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. Recent molecular techniques offer improved accuracy in pinpointing microbial members than culture-dependent methods because only a limited proportion of the symbiotic microbiota is cultivable. For this reason, we attempted to reproduce these findings using 16s rRNA sequencing and advanced statistical methods.

Results: Our study corroborated 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.

Conclusions: Our conceptual replication study on the wing-sac microbiota of S. bilineata validated the original findings, corroborating 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.

Conceptual replication

Greater sac-winged bats

microbiome

scent gland microbiota

olfactory communication

chemical signalling

sex-specific

Reproducibility is a fundamental principle of scientific practice, ensuring the reliability, objectivity and validity of the findings. Replication studies are the cornerstones of reproducibility in terms of testing robustness and should be considered as the safeguard against errors, biases, and even scientific misconduct [1]. Conceptual replication, where the researchers repeat the original study by making deliberate modifications in the methodology to reproduce the findings of the original study [2, 3], has a particular epistemological function: they allow the progression of science in increments, using a more advanced method. Nevertheless, the novelty of the findings has become a prerequisite for publication, making such studies extremely rare, also in ecology [4, 5]. In other words, the scientific community trades off reproducibility with novelty, deepening one of the most prominent problems, the replication crisis [5]. Today, in several fields, including ecology, most of the knowledge on several hypotheses comes solely from first-of-its-kind studies.

One hypothesis which can benefit from validation by conceptual replication is the fermentation hypothesis for chemical recognition, an important concept in olfactory social communication. This theory postulates that odorant molecules produced by symbiotic microorganisms residing in mammalian scent organs as metabolic by-products can contribute to individuals' scent gland secretions and thereby are consequently involved in chemical signalling [6–8]. Accordingly, mammalian scent organs, being moist, warm and nutrient-rich, offer a matchless environment for microbial growth [6, 9]. As the composition and structure of these microbial communities are, at least to some extent, determined by host factors such as taxonomy, life-history traits, genetics and social interactions, microbially produced odours can broadcast complementary information on these underlying host factors [7, 9–12]. Empirical studies have demonstrated that microbially-produced odours might encode cues on taxonomic identity [13], sex [14, 15], age [15, 16], group membership [13–15, 17], reproductive cycle [18], and social status [14] of different mammalian hosts.

Scent gland microbiota was also proposed to play a role in the mate choice decisions of a Neotropical bat species, the greater sac-winged bat (Saccopteryx bilineata; [19]. S. bilineata is an insect-feeding bat species with a harem polygynous mating system[20–22], where a single male defends its harem consisting of up to eight females the whole year [20]. Colonies comprise several harem groups and peripheral males that roost close to the harem territories [20]. The mating season is restricted to a few weeks per year, and females give birth to a single offspring[23]. Although harem males sire more offspring than peripheral males, they do not have exclusive access to the females in their harem and only sire approximately 30% of the young within their territory [24]. The high frequency of extra-harem paternity can be explained by the larger size of the females, which gives them an advantage during agonistic encounters. Consequently, female choice is an essential component of the reproductive ecology of this species, and male fitness depends on advertising their quality [21, 25].

Chemical cues play an important role in the mate choice decisions of this species [22, 26, 27]. Males have pouch-like scent organs in the antebrachial wing (Fig. 1A), which are used to store odoriferous secretions, while females only have the rudiments of these sacs (Fig. 1B) [20]. During courtship, males exhibit hovering flights and fan the odiferous substances from their wing sacs towards females [22, 26]. Wing sac odours carry information on species [28] and individual identity [26], sexual maturity[29] and the geographic distance between colonies [27, 30]. The wing sacs lack glandular tissue and consequently do not produce any secretions [31]. Males clean up and refill these organs every day via a two-step ritual [24, 28, 32, 33]. In the first step, they take up some urine into their mouths and then lick their wing sacs [28, 32–34]. In the second step, they fill the wing sacs with liquids from the genital and gular regions [28, 32–34]. This stereotypic perfume blending behaviour can take up to an hour. It was proposed that males perform this energetically costly and time-consuming behaviour to control microbial growth in the wing sac to minimise microbial fermentation and to generate individual-specific olfactory profiles [19] Indeed, Voigt and colleagues [19] found that samples originating from the wing sacs of males had lower microbial richness than the samples collected from the wing sac rudiments of females. Undoubtedly providing pioneering insides into the sex-specific alterations in the microbiota of scent organs of S. bilineata, the study was conducted using culture-dependent methods (i.e. by growing the bacteria in culture media). Today we know that only a small proportion of the symbiotic microbiota can be cultured, and consequently, relatively recent molecular techniques can provide better resolution in identifying microbial members [35].

Here, we performed a conceptual replication study to characterise wing-sac microbiota collected from two Costa Rican populations of the S. bilineata, using a culture-independent molecular method, 16s ribosomal RNA sequencing, and novel statistical techniques. We evaluated the feasibility of reproducing the findings by Voigt and colleagues [19]. We also tested whether broader identification coverage provided through 16s RNA sequencing can provide a deeper understanding of the sex-specific regulation of the wing sac microbiota.

We sequenced the hypervariable V3–V4 region of the 16S rRNA gene from wing sac swabs originating from 56 bats (41 males, 15 females) from two different Costa Rican populations (Supplementary Table 1). After the exclusion of two samples due to unsuccessful amplification, our dataset consisted of 54 samples (39 males, 15 females) and 277 different operational taxonomic units (OTU), with a total read count of 4089801.16. We identified seven microbial phyla, with the domination of Proteobacteria (78.23%), Firmicutes (15.63%), and Bacteroidota (6.11%). At a finer taxonomic scale, identified taxa corresponded to 74 microbial families (Fig. 2).

Sex-specific differences in microbial richness, diversity and composition

The number of females and males, retained in our final dataset, was 15 and 39, respectively. Samples collected from females contained, on average, 43.2 individual OTUs (minimum: 19; maximum: 71; SD:14.21) with an average read count of 107896.2 (SD: 103380.93). Samples collected from males contained, on average, 33.5 individual OTUs (minimum: 14; maximum: 75; SD:11.8) with an average read count of 63368.15 (SD: 93468.92).

We found sex-specific differences in the community richness as measured by two different metrics: Males have a lower observed number of OTUs (LM Sex [M], β= -0.76 ± 0.32, 95% CI [-1.40 - -0.11], p = 0.022) (Fig. 3A) and Chao 1 (LM Sex [M], β= -0.85 ± 0.33, 95% CI [-1.50 - -0.19], p = 0.012) (Fig. 3B). However, we did not find any significant differences in the Shannon diversity index, which measures the diversity and evenness of the microbial communities, between males and females (LM Sex [M], β= -0.19 ± 0.24, 95% CI [-0.66-0.29], p = 0.435) (Fig. 3C).

Microbial community composition also differed between males and females. Although the most abundant microbial family was Moraxellaceae in both males (44.70%) and females (38.46%), the most abundant families exhibited prominent differences between sexes (Fig. 2). In females, the most abundant microbial families were Pseudomonadaceae (7.69%), Enterobacteriaceae (7.24%), Weeksellaceae (7.17%), Aeromonadaceae (6.24%) and Yersiniaceae (6.05%) (Fig. 2A). In males, the communities dominated by Erwiniaceae (12.36%), Staphylococcaceae (12.00%), Enterobacteriaceae (7.87%), Xanthomonadaceae (7.045%), Enterococcaceae (5.54%) (Fig. 2B). When we statistically tested the observed compositional differences between the sexes using PERMANOVA, we found significant differences in the models based on Jaccard (R² = 0.027; p = 0.042) and Bray-Curtis (R² = 0.041; p = 0.01) resemblance matrices. Minimal compositional overlap between males and females was also visually supported by PCoA plots generated based on these two resemblance matrices (Fig. 4). Nevertheless, the sex-specific differences were not evident in the PERMANOVA models based on unweighted (R² = 0.023; p = 0.233) and weighted UniFrac resemblance (R² = 0.028; p = 0.162). PERMDISP analyses did not reveal any statistical difference in the homogeneity of group dispersion between males and females (all p values obtained from permutes were larger than 0.05), indicating that the significant PERMANOVA results were not caused by differences in dispersion among the groups.

We also identified the differentially abundant OTUs between samples collected from males and females using beta-binomial regression models and controlling for differential variability across sexes. Overall, we found 13 differentially abundant OTUs (Fig. 5, see Supplementary Table S2 for finer taxonomic assignments). Of these, six were significantly more abundant in female hosts. Notably, although males have overall lower microbial richness, we seven OTUs were significantly more abundant in this group. Two OTUs showing a higher abundance in males belong to the microbial families containing lactic acid bacteria responsible for fermentation, Aerococcaceae and Carnobacteriaceae [36]. Furthermore, one of the OTUs with increased abundance in males belongs to the Enterobacterales order. Some of the species in this taxon are known to produce volatile organic compounds that contribute to the smell of cheese [37]. One of the OTUs belongs to the Bacillus genus of Bacillaceae family (Supplementary Table S2). Some Bacillus species are known to break down malodorous volatile organic compounds [38, 39]. One of the OTUs exhibiting an increased abundance in males belongs to the Micrococcales order, which contains some antimicrobial-producing bacteria [40].

Microbial communities did not differ between the populations

The linear models did not reveal any significant differences in any of the alpha diversity measures between the two populations (Population [Palo Verde]; observed number of OTUs: β = -0.06 ± 0.29, 95% CI [-0.63–0.52], p = 0.848); Chao 1: β = 0.02 ± 0.29, 95% CI [-0.57–0.61], p = 0.934); Shannon's diversity index: β = 0.34 ± 0.21, 95% CI [-0.09-0.77], p = 0.114). PERMANOVA models did not show significant differences between the two colonies (all p values were larger than 0.05). Furthermore, we also tested whether samples collected from spatially closer locations have more similar microbial communities than geographically distant ones by testing the correlations between the distance matrix of the geographical coordinates of the sampling based on the Haversine distances and microbial resemblance matrices. Based on the Mantel test, there was no correlation between spatial proximity and microbial composition (all p values were larger than 0.05).

In our conceptual replication study, we repeated the study by Voigt and colleagues [19], which demonstrated sex-specific differences in the microbiota in the sexually selected scent organ of S. bilineata by adopting some methodological incremental improvements. Complementary to the original study, which relied on culture-dependent identification techniques, we employed a culture-independent molecular method, 16s rRNA gene sequencing, and advanced statistical method to characterise wing-sac microbiota of S. bilineata. Unsurprisingly, our study identified more microbial taxa (277 versus 40) than the one of Voigt and colleagues [19], corroborating that culture-dependent methods can identify only a small proportion of the symbiotic bacteria that can be identified by 16s rRNA gene sequencing. Nevertheless, our findings are consistent with the original study: wing-sac microbiota of S. bilineata exhibit sex-specific differences.

In line with the original study, microbial richness is lower in wing sacs of males than wing sac rudiments of females. In contrast to these findings, in wild spotted hyenas (Crocuta Crocuta; [41] and white-tailed deer (Odocoileus virginianus; [42], males have richer scent gland microbiota than females. Considering that the wing sacs of the males are filled with potentially nutrient-rich excretions and are more humid than female rudiments, one can expect a higher microbial richness. Therefore, the observed sex-specific patterns in greater sac-winged bats cannot be attributable to morphological differences in the antebrachial wing membrane alone. Voigt and colleagues [19] proposed the male-specific behaviour of wing sac cleaning and refilling as one of the potential explanations for controlling microbial growth. During the first phase of this time-consuming and energetically costly ritual, males take up some urine into their mouths and then lick their wing sacs [28, 32–34]. Saliva is known to contain antimicrobial peptides [43] and can potentially inhibit microbial growth. Furthermore, urine is proposed to posses antimicrobial properties due to its hypertonic nature with a low pH and high concentrations of urea, which collectively deter the growth of most bacteria [44, 45].

Furthermore, we observed an increased abundance of operational taxonomic units (OTUs) belonging to the Micrococcales order in males. Some species in this taxon can produce antimicrobial substances inhibiting the growth of numerous bacteria [40], suggesting that regulation of this complex ecosystem might involve microbe-microbe interactions. Rojas-Gätjens and colleagues [40] suggested that Micrococcales species residing in the sloth fur could control hair microbiota in two sloth species.

Consistent with the original study, we also found sex-specific compositional differences in the scent organ microbiota. This finding is also in line with the previous studies showing sex-specific compositional differences in scent gland microbiota of wild spotted hyenas [41], meerkats (Suricata suricatta; [15], and owl monkeys (Aotus nancymaae; [46]. Among the OTUs exhibiting higher abundance in males, some belong to taxa containing potential odour producers, suggesting a potential role of microbially produced volatiles in the male scent profile [19]. One of the OTUs that are more abundant in males belongs to the Bacillus genus. Strikingly, some members of this taxon are known to break down malodorous volatile organic compounds [38, 39]. Nevertheless, it is important to note that the production of odours by bacteria is a complex process influenced by various factors, including the specific strain, environmental conditions, and the presence of substrates or nutrients.

The scarcity of replication studies, particularly in certain fields like ecology, hinders incremental progress and challenges scientific practice integrity. In this respect, our conceptual replication study investigating sex-specific differences in the wing-sac microbiota of S. bilineata fills an important gap. Employing modern molecular techniques and advanced statistical methods, we validated the original study's findings. Our results add to the existing knowledge on the potential impact of microbially produced volatiles in shaping the scent profiles of male wing-sac bats, underlining the importance of replication efforts in corroborating scientific findings. Furthermore, our research opens new windows to study potential mechanisms behind the sex-specific regulation of this complex ecosystem and the role of microbial symbionts on the sexual selection of their hosts. We hope our study also encourages the scientific community to promote a culture that values replication studies as integral to the scientific process.

Sample collection

Samples used in this study were derived from 56 individuals from two populations (Supplementary Table S1), Palo Verde National Park (10.378884°N; 85.285158°E) and Golfito (9.588213°N; 83.916920°E) in Costa Rica separated by a distance of approximately 300 km (Fig. 1C), during November and December 2018. The capturing protocol was described by Schneeberger and colleagues [27]. Microbial samples were collected from the area around the wing sacs of males and wing sac rudiments of females using a sterile nylon flocked swab (ESwab, Copan Italia, Italy). Swabs were immediately transferred to liquid Amies medium and stored at − 20°C. The samples were shipped to Germany on dry ice.

DNA extraction and library preparation

Microbial DNA was extracted using BiOstic Bacteremia DNA Isolation Kit (MOBIO Laboratories, Carlsbad, CA, USA) following the manufacturer's protocol. Subsequently, we amplified the hypervariable V3–V4 region of the 16S ribosomal RNA (rRNA) gene, following the Illumina 16S Metagenomic Library Preparation Guide. The details of the library preparation steps were described by Maraci and colleagues [47]. The two samples were excluded from the analyses due to the unsuccessful amplification after three attempts. The final amplicon pool contained the libraries of 54 biological samples and three blank controls for DNA extraction and amplification and was sequenced on the Illumina MiSeq system (Illumina, Inc., San Diego, CA, USA).

Data Analyses

The details of the bioinformatic processing were described in detail [47]. In short, MiSeq PE reads were assembled in an iterative manner using Flash v1.2.11 [48]. For the adapter clipping step, we used cutadapt v1.18 [49]. De-replication, alignment, filtering and de-noising were performed with mothur v1.41.3 [50]. Chimaera checking and operational taxonomic unit (OTU) clustering were performed using USEARCH v8.0.1477 [51]. Taxonomic classification was performed based on the full SILVA database v132 [52].

All consecutive statistical analyses were performed in R version 4.0.0 [53]. We discarded all OTUs classified as archaea, mitochondria or chloroplasts as an initial quality filtering step. For the alpha diversity analyses, we estimated alpha diversity based on the observed number of OTUs, Chao 1 as the measure of the microbial richness [54], and Shannon's diversity index, which accounts for both abundance and evenness of the taxa present [55]. We tested whether the alpha diversity metrics for normality and applied necessary transformation in case of non-normal distribution. Subsequently, we tested whether these metrics differ between sexes and areas using the linear model, using the lm function of R package stats. The residuals of the models were inspected visually.

The compositional differences between sexes were visualised based on the microbial family level taxonomy by stacked bar plots produced by ggplot2 version 3.3.2 [56]. Before beta diversity analyses, we implemented (log10(x + 1)) transformation to deal with unequal sequence coverage. Then, we generated the dissimilarity matrices based on Jaccard [57], Bray–Curtis [58], unweighted UniFrac [59] and weighted UniFrac [60] resemblances. The beta group dissimilarities between sexes were visualised using Principal Coordinate Analysis (PCoA) Plot, implemented using the Vegan package version 2.5-6 [61]. We also statistically tested the differences between samples collected from males and females and different areas by permutational multivariate analyses of variance (PERMANOVA) [62] with 9999 permutations. The homogeneity of group dispersions was also tested using PERMDISP. as implemented by the betadisper function in the Vegan package [61]. We also investigated the spatial structuring of the microbial communities by test to test the correlations between the distance matrix of the geographical coordinates of the sampling based on the Haversine distances and microbial resemblance matrices using a mantel test.

The differentially abundant OTUs between sexes were identified using the Corncob package [63], which estimates taxa-specific differential abundances by building beta-binomial regression models, controlling for differential variability across the covariate of interest. We set the significance threshold for p values to 0.05 after Benjamini and Hochberg FDR correction [64].

Bray–Curtis

LMM

Linear mixed models

OTU

Operational taxonomic unit

PERMANOVA

permutational multivariate analysis of variance

rRNA

Ribosomal RNA

Weighted UniFrac

Ethics approval and consent to participate

The sample collection procedures were approved by National System of Conservation Areas, Regional Directorate of Cordillera Volcanica Central Biosphere Reserve (Nº SINAC-ACC-PI-R-113-2018530.421630–1,18.4.2002).

Consent for publication

Not applicable

Availability of data and material

The datasets generated during the current study can be found in the European Nucleotide Archive repository, Project ID: PRJEB67961.

The code used in the analyses is available in the GitHub repository at

https://github.com/AnnaAntonatouPap/-Bats-Bacteria-and-Bat-Smell-V.2.0-Repeatable-Sex-specific-Differences-in-Scent-Organ-Microbiota

Competing interests

The authors declare that they have no competing interests.

Funding

This study was financially supported by the German Research Foundation (Deutsche

Forschungsgemeinschaft (doi:10.13039/501100001659) to .B. A. C. and K. S.; grant number SCHN 1584/2-1) and a Freigeist Fellowship from the Volkswagen Foundation to B. A. C.

Authors' contributions

Ö. M., B. A. C. and K. S. conceptualised the research idea. B. A. C., K. S., M. S., and I. S. collected the samples and spatial data. Ö. M. carried out laboratory experiments. S. J. performed the bioinformatic analyses. A. A. P. and Ö. M carried out the statistical analyses. The manuscript was drafted by Ö. M. and all authors approved the final manuscript.

Acknowledgements

We acknowledge the technical support of Elke Hippauf, Barbara Fuchs, Tobias Busche, Yvonne Kutter and Katharina Hanuschka during the experimental procedures. We acknowledge the financial support of the German Research Foundation (DFG) and the Open Access Publication Fund of Bielefeld University for the article processing charge. This research was inspired by the SFB-TRR 212.

Authors' information (optional)

Not applicable

Schmidt S. Shall we Really do it Again? The Powerful Concept of Replication is Neglected in the Social Sciences. Rev Gen Psychol. 2009;13:90–100.
Fidler F, Chee YE, Wintle BC, Burgman MA, McCarthy MA, Gordon A. Metaresearch for Evaluating Reproducibility in Ecology and Evolution. Bioscience. 2017;67:282–9.
Zwaan RA, Etz A, Lucas RE, Donnellan MB. Making replication mainstream. Behav Brain Sci. 2018;41:e120.
Filazzola A, Cahill J. Replication in field ecology: Identifying challenges and proposing solutions. Methods Ecol Evol. 2021;12.
Nakagawa S, Parker TH. Replicating research in ecology and evolution: feasibility, incentives, and the cost-benefit conundrum. BMC Biol. 2015;13:88.
Albone ES, Perry GC. Anal sac secretion of the red fox,Vulpes vulpes; volatile fatty acids and diamines: Implications for a fermentation hypothesis of chemical recognition. J Chem Ecol. 1976;2:101–11.
Archie EA, Theis KR. Animal behaviour meets microbial ecology. Anim Behav. 2011;82:425–36.
Gorman ML, Nedwell DB, Smith RM. An analysis of the contents of the anal scent pockets of Herpestes auropunctatus (Carnivora: Viverridae). J Zool. 1974;172:389–99.
Carthey AJR, Gillings MR, Blumstein DT. The Extended Genotype: Microbially Mediated Olfactory Communication. Trends Ecol Evol. 2018;33:885–94.
Ezenwa VO, Williams AE. Microbes and animal olfactory communication: Where do we go from here? BioEssays. 2014;36:847–54.
Ezenwa VO, Williams AE. Microbes and animal olfactory communication: Where do we go from here? BioEssays. 2014;36:847–54.
Maraci Ö, Engel K, Caspers BA. Olfactory Communication via Microbiota: What Is Known in Birds? Genes. (Basel). 2018;9:387.
Theis KR, Venkataraman A, Dycus JA, Koonter KD, Schmitt-Matzen EN, Wagner AP, et al. Symbiotic bacteria appear to mediate hyena social odors. Proc Natl Acad Sci U S A. 2013;110:19832–7.
Leclaire S, Jacob S, Greene LK, Dubay GR, Drea CM. Social odours covary with bacterial community in the anal secretions of wild meerkats. Sci Rep. 2017;7:3240.
Leclaire S, Nielsen JF, Drea CM. Bacterial communities in meerkat anal scent secretions vary with host sex, age, and group membership. Behav Ecol. 2014;25:996–1004.
Theis KR, Venkataraman A, Wagner AP, Holekamp KE, Schmidt TM. Age-Related Variation in the Scent Pouch Bacterial Communities of Striped Hyenas (Hyaena hyaena). In: Schulte BA, Goodwin TE, Ferkin MH, editors. Chemical Signals in Vertebrates 13. Cham: Springer International Publishing; 2016. pp. 87–103.
Theis KR, Schmidt TM, Holekamp KE. Evidence for a bacterial mechanism for group-specific social odors among hyenas. Sci Rep. 2012;2:615.
Ma R, Zheng W, Guo J, Hou R, Huang H, Xue F, et al. Symbiotic microbiota and odor ensure mating in time for giant pandas. Front Microbiol. 2022;13:1015513.
Voigt CC, Caspers B, Speck S. Bats, Bacteria, and Bat Smell: Sex-Specific Diversity of Microbes in a Sexually Selected Scent Organ. J Mammal. 2005;86:745–9.
Bradbury JW, Emmons LH. Social Organization of some Trinidad Bats. Z für Tierpsychologie. 1974;36:137–83.
Voigt CC, von Helversen O, Michener R, Kunz TH. The economics of harem maintenance in the sac-winged bat, Saccopteryx bilineata (Emballonuridae). Behav Ecol Sociobiol. 2001;50:31–6.
Voigt CC, Schwarzenberger F. Reproductive Endocrinology of a Small Tropical Bat (Female Saccopteryx bilineata; Emballonuridae) Monitored by Fecal Hormone Metabolites. J Mammal. 2008;89:50–7.
Tannenbaum. Reproductive strategies in the white-lined bat (PhD dissertation). Ithaca, New York, Cornell University. 1975.
Heckel G, von Helversen O. Male tactics and reproductive success in the harem polygynous bat Saccopteryx bilineata. Behav Ecol. 2002;13:750–6.
Voigt CC, Behr O, Caspers B, von Helversen O, Knörnschild M, Mayer F, et al. Songs, Scents, and Senses: Sexual Selection in the Greater Sac-Winged Bat, Saccopteryx bilineata. J Mammal. 2008;89:1401–10.
Caspers B, Franke S, Voigt CC. The Wing-Sac Odour of Male Greater Sac-Winged Bats Saccopteryx bilineata (Emballonuridae) as a Composite Trait: Seasonal and Individual Differences. In: Hurst JL, Beynon RJ, Roberts SC, Wyatt TD, editors. Chemical Signals in Vertebrates 11. New York, NY: Springer; 2008. pp. 151–60.
Schneeberger K, Schulze M, Scheffler I, Caspers BA. Evidence of female preference for odor of distant over local males in a bat with female dispersal. Behav Ecol. 2021;32:657–61.
Caspers B, Wibbelt G, Voigt CC. Histological examinations of facial glands in Saccopteryx bilineata (Chiroptera, Emballonuridae), and their potential use in territorial marking. Zoomorphology. 2009;128:37–43.
Caspers BA, Schroeder FC, Franke S, Voigt CC. Scents of adolescence: the maturation of the olfactory phenotype in a free-ranging mammal. PLoS ONE. 2011;6:e21162.
Schneeberger K, Voigt CC, Müller C, Caspers BA. Multidimensionality of Chemical Information in Male Greater Sac-Winged Bats (Saccopteryx bilineata). Front Ecol Evol. 2016;4.
Scully WM, Fenton MB, Saleuddin AS. A histological examination of the holding sacs and glandular scent organs of some bat species (Emballonuridae, Hipposideridae, Phyllostomidae, Vespertilionidae, and Molossidae). Can J Zool. 2000;78:613–23.
Voigt CC. Individual variation in perfume blending in male greater sac-winged bats. Anim Behav. 2002;63:907–13.
Voigt CC, Heckel G, Mayer F. Sexual selection favours small and symmetric males in the polygynous greater sac-winged bat Saccopteryx bilineata (Emballonuridae, Chiroptera). Behav Ecol Sociobiol. 2005;57:457–64.
Voigt CC, von Helversen O. Storage and display of odour by male Saccopteryx bilineata (Chiroptera, Emballonuridae). Behav Ecol Sociobiol. 1999;47:29–40.
Wade W. Unculturable bacteria–the uncharacterized organisms that cause oral infections. J R Soc Med. 2002;95:81–3.
Zheng J, Wittouck S, Salvetti E, Franz CMAP, Harris HMB, Mattarelli P, et al. A taxonomic note on the genus Lactobacillus: Description of 23 novel genera, emended description of the genus Lactobacillus Beijerinck 1901, and union of Lactobacillaceae and Leuconostocaceae. Int J Syst Evol Microbiol. 2020;70:2782–858.
Ritschard JS, Van Loon H, Amato L, Meile L, Schuppler M. High Prevalence of Enterobacterales in the Smear of Surface-Ripened Cheese with Contribution to Organoleptic Properties. Foods. 2022;11:361.
Nowocień K, Sokołowska B. Bacillus spp. as a new direction in biocontrol and deodorization of organic fertilizers. AIMSES. 2022;9:95–105.
Ushida K, Hashizume K, Miyazaki K, Kojima Y, Takakuwa S. Isolation of Bacillus SP. as a Volatile Sulfur-Degrading Bacterium and Its Application to Reduce the Fecal Odor of Pig. Asian-Australasian J Anim Sci. 2003;16:1795–8.
Rojas-Gätjens D, Valverde-Madrigal KS, Rojas-Jimenez K, Pereira R, Avey-Arroyo J, Chavarría M. Antibiotic-producing Micrococcales govern the microbiome that inhabits the fur of two- and three-toed sloths. Environ Microbiol. 2022;24:3148–63.
Rojas CA, Holekamp KE, Winters AD, Theis KR. Body site-specific microbiota reflect sex and age-class among wild spotted hyenas. FEMS Microbiol Ecol. 2020;96:fiaa007.
Gassett JW, Dasher KA, Miller KV, Osborn DA, Russell SM. White-tailed deer tarsal glands: sex and age-related variation in microbial flora. 2000;64:371–8.
Vila T, Rizk AM, Sultan AS, Jabra-Rizk MA. The power of saliva: Antimicrobial and beyond. PLoS Pathog. 2019;15:e1008058.
Chambers ST, Lever M. Betaines and urinary tract infections. Nephron. 1996;74:1–10.
Kucheria R, Dasgupta P, Sacks SH, Khan MS, Sheerin NS. Urinary tract infections: new insights into a common problem. Postgrad Med J. 2005;81:83–6.
Bowen M, Miles C, Hegseth R, Anderson CM, Brandon CS, Langford ML, et al. The potential interplay between the glandular microbiome and scent marking behavior in owl monkeys (Aotus nancymaae). Am J Primatol. 2021;83:e23324.
Maraci Ö, Antonatou-Papaioannou A, Jünemann S, Castillo-Gutiérrez O, Busche T, Kalinowski J et al. The Gut Microbial Composition Is Species-Specific and Individual-Specific in Two Species of Estrildid Finches, the Bengalese Finch and the Zebra Finch. Front Microbiol. 2021;12.
Magoč T, Salzberg SL. FLASH: fast length adjustment of short reads to improve genome assemblies. Bioinformatics. 2011;27:2957–63.
Martin M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J. 2011;17:10–2.
Schloss PD, Westcott SL, Ryabin T, Hall JR, Hartmann M, Hollister EB, et al. Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol. 2009;75:7537–41.
Edgar RC. Search and clustering orders of magnitude faster than BLAST. Bioinformatics. 2010;26:2460–1.
Quast C, Pruesse E, Yilmaz P, Gerken J, Schweer T, Yarza P, et al. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res. 2013;41:D590–6.
R Core Team. 2020.
DeSantis TZ, Hugenholtz P, Keller K, Brodie EL, Larsen N, Piceno YM et al. NAST: a multiple sequence alignment server for comparative analysis of 16S rRNA genes. Nucleic Acids Res. 2006;34 Web Server issue:W394–9.
Shannon CE. A mathematical theory of communication. Bell Syst Tech J. 1948;27:379–423.
Wickham H. ggplot2: Elegant Graphics for Data Analysis. New York, NY: Springer New York; 2009.
Jaccard P. The Distribution of the Flora in the Alpine Zone.1. New Phytol. 1912;11:37–50.
Bray JR, Curtis JT. An Ordination of the Upland Forest Communities of Southern Wisconsin. Ecol Monogr. 1957;27:325–49.
Lozupone C, Knight R. UniFrac: a new phylogenetic method for comparing microbial communities. Appl Environ Microbiol. 2005;71:8228–35.
Lozupone CA, Hamady M, Kelley ST, Knight R. Quantitative and qualitative beta diversity measures lead to different insights into factors that structure microbial communities. Appl Environ Microbiol. 2007;73:1576–85.
Oksanen J, Blanchet FG, Friendly M, Kindt R, Legendre P, McGlinn D et al. Package ‘vegan.’ Community ecology package, version. 2019;2.
Anderson MJ. A new method for non-parametric multivariate analysis of variance. Austral Ecol. 2001;26:32–46.
Martin BD, Witten D, Willis AD, MODELING MICROBIAL ABUNDANCES, AND DYSBIOSIS WITH BETA-BINOMIAL REGRESSION. Ann Appl Stat. 2020;14:94–115.
Benjamini Y, Hochberg Y. Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing. J Roy Stat Soc: Ser B (Methodol). 1995;57:289–300.

No competing interests reported.

Download PDF

Journal Publication

published 25 Mar, 2024

Read the published version in Microbial Ecology →

Version 1

posted

You are reading this latest preprint version

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

Status:

Journal Publication

Version 1

Abstract

Figures

Background

Results

Sex-specific differences in microbial richness, diversity and composition

Microbial communities did not differ between the populations

Discussion

Conclusions

Methods

Sample collection

DNA extraction and library preparation

Data Analyses

Abbreviations

Declarations

Ethics approval and consent to participate

Consent for publication

Availability of data and material

Competing interests

Funding

Authors' contributions

Authors' information (optional)

References

Additional Declarations

Status:

Journal Publication

Version 1