Influence of scat ageing on the gut microbiome: how old is too old?

BACKGROUND: The study of the host-microbiome by the collection of non-invasive samples has the potential to become a powerful tool for conservation monitoring and surveillance of wildlife. However, multiple factors can bias the quality of data recovered from scats, particularly when field-collected samples are used given that the time of defecation is unknown. Previous studies using scats have shown that the impact of aerobic exposure on the microbial composition is species-specific, leading to different rates of change in microbial communities. However, the impact that this aging process has on the relationship between the bacterial and fungal composition has yet to be explored. In this study, we measured the effects of time post-defecation on bacterial and fungal compositions in a controlled experiment using scat samples from the endangered koala (Phascolarctos cinereus). RESULTS: We found that the bacterial composition remained stable through the scat aging process, while the fungal composition did not. The absence of an increase in facultative anaerobes and the stable population of obligate anaerobic bacteria were likely due to our sampling from the inner portion of the scat. We report a cluster of fungal taxa that colonises scats after defecation which can dilute the genetic material from the autochthonous mycoflora and inhibit recovery. CONCLUSION: We emphasize the need to preserve the integrity of scat samples collected in the wild and combat the effects of time and provide strategies for doing so.

Characterization of the internal working-life exposome using minimally and non-invasive sampling methods - a narrative review.

During recent years, we are moving away from the 'one exposure, one disease'-approach in occupational settings and towards a more comprehensive approach, taking into account the totality of exposures during a life course by using an exposome approach. Taking an exposome approach however is accompanied by many challenges, one of which, for example, relates to the collection of biological samples. Methods used for sample collection in occupational exposome studies should ideally be minimally invasive, while at the same time sensitive, and enable meaningful repeated sampling in a large population and over a longer time period. This might be hampered in specific situations e.g., people working in remote areas, during pandemics or with flexible work hours. In these situations, using self-sampling techniques might offer a solution. Therefore, our aim was to identify existing self-sampling techniques and to evaluate the applicability of these techniques in an occupational exposome context by conducting a literature review. We here present an overview of current self-sampling methodologies used to characterize the internal exposome. In addition, the use of different biological matrices was evaluated and subdivided based on their level of invasiveness and applicability in an occupational exposome context. In conclusion, this review and the overview of self-sampling techniques presented herein can serve as a guide in the design of future (occupational) exposome studies while circumventing sample collection challenges associated with exposome studies.

Development of Genetic Markers for Sex and Individual Identification of the Japanese Giant Flying Squirrel (Petaurista leucogenys) by an Efficient Method Using High-Throughput DNA Sequencing.

DNA markers that detect differences in the number of microsatellite repeats can be highly effective for genotyping individuals that lack differences in external morphology. However, isolation of sequences with different microsatellite repeat numbers between individuals has been a time-consuming process in the development of DNA markers. Individual identification of Japanese giant flying squirrels (Petaurista leucogenys) has been challenging because this species is arboreal and nocturnal and exhibits little to no morphological variation between individuals. In this study, we developed DNA markers for sex and individual identification of this species by an efficient method using high-throughput DNA sequence data. Paired-end 5 Gb (2 × 250 bp) and 15 Gb (2 × 150 bp) genome sequences were determined from a female and a male Japanese giant flying squirrel, respectively. We searched SRY and XIST genes located on Y and X chromosomes, respectively, from high-throughput sequence data and designed primers to amplify these genes. Using these primer sets, we succeeded to identify the sex of individuals. In addition, we selected 12 loci containing microsatellites with different numbers of repeats between two individuals from the same data set, and designed primers to amplify these sequences. Twenty individuals from nine different locations were discriminated using these primer sets. Furthermore, both sex and microsatellite markers were amplified from DNA extracted non-invasively from single fecal pellet samples. Based on our results for flying squirrels, we expect our efficient method for developing non-invasive high-resolution individual- and sex-specific genotyping to be applicable to a diversity of mammalian species.

Simultaneous investigation of urinary and faecal glucocorticoid metabolite concentrations reveals short- versus long-term drivers of HPA-axis activity in a wild primate (Papio ursinus).

Glucocorticoids (GCs), a class of steroid hormones released through activation of the hypothalamic-pituitary-adrenal (HPA) axis, perform many vital functions essential for survival, including orchestrating an organism's response to stressors by modulating physiological and behavioural responses. Assessing changes and variation in GC metabolites from faecal or urine samples allows for the non-invasive monitoring of HPA-axis activity across vertebrates. The time lag of hormone excretion differs between these sample matrices, which has implications for their suitability for studying effects of different temporal nature on HPA-axis activity. However, simultaneous comparisons of predictors of faecal and urinary GC metabolites (fGCs and uGCs, respectively) are lacking. To address this gap, we employ frequent non-invasive sampling to investigate correlates of fGCs and uGCs in wild chacma baboons (Papio ursinus) (n = 17), including long-term (dominance rank, season, female reproductive state) and short-term (time of day, daily weather conditions) factors. Correlated with increasing day length, fGCs gradually decreased from winter to summer. No seasonal effect on uGCs was found but 'rain days' were associated with increased uGCs. Pregnant females had significantly higher fGCs compared to cycling and lactating females, whereas uGCs were not statistically different across reproductive states. A circadian effect was observed in uGCs but not in fGCs. Dominance rank did not affect either fGCs or uGCs. Our study highlights the difference in inherent fluctuation between uGCs and fGCs and its potential consequences for HPA-axis activity monitoring. While uGCs offer the opportunity to study short-term effects, they undergo more pronounced fluctuations, reducing their ability to capture long-term effects. Given the increasing use of urine for biological monitoring, knowledge of this potential limitation is crucial. Where possible, uGCs and fGCs should be monitored in tandem to obtain a comprehensive understanding of short- and long-term drivers of HPA-axis activity.

Study of feces of neotropical otters (Lontra longicaudis) in the Ayuquila-Armería basin, Mexico as biomonitors of the spatiotemporal distribution of pesticides.

The pesticides used have contributed to increasing food production; it has also caused them to be found in most ecosystems and have negative effects on biota. The neotropical otter (Lontra longicaudis) is vulnerable to pesticide accumulation and is characterized by being elusive, so it is necessary to address the use of indirect techniques that evaluate its populations' state in an efficient, logistically simple, and non-invasive way. This study aimed to determine the concentration of 20 pesticides in neotropical otter feces in the Ayuquila-Armería basin and to describe the spatiotemporal variation of these pesticides. The presence of 11 pesticides was determined. Imazalil, picloram, and malathion the pesticides with the highest concentrations; emamectin, λ-cyhalothrin, methomyl, and picloram were present in all samples. Emamectin was the only pesticide that presented significant differences concerning the temporality of the samplings, presenting higher concentrations in the wet season. Molinate concentrations showed significant differences concerning the location of the sampling sections in the basin; the lower part of the basin presented higher concentrations. The distribution of the populations of L. longicaudis in the Ayuquila-Armería basin does not respond to the degree of contamination by pesticides in surface waters or to the proximity to agricultural activities, and this in places with evident chemical and organic contamination and human presence. The use of otter feces for pesticide monitoring is an accepted non-invasive method to assess the degree of exposure and can be used to determine sites with pollution problems.

Reliable wolf-dog hybrid detection in Europe using a reduced SNP panel developed for non-invasively collected samples.

BACKGROUND: Understanding the processes that lead to hybridization of wolves and dogs is of scientific and management importance, particularly over large geographical scales, as wolves can disperse great distances. However, a method to efficiently detect hybrids in routine wolf monitoring is lacking. Microsatellites offer only limited resolution due to the low number of markers showing distinctive allele frequencies between wolves and dogs. Moreover, calibration across laboratories is time-consuming and costly. In this study, we selected a panel of 96 ancestry informative markers for wolves and dogs, derived from the Illumina CanineHD Whole-Genome BeadChip (174 K). We designed very short amplicons for genotyping on a microfluidic array, thus making the method suitable also for non-invasively collected samples. RESULTS: Genotypes based on 93 SNPs from wolves sampled throughout Europe, purebred and non-pedigree dogs, and suspected hybrids showed that the new panel accurately identifies parental individuals, first-generation hybrids and first-generation backcrosses to wolves, while second- and third-generation backcrosses to wolves were identified as advanced hybrids in almost all cases. Our results support the hybrid identity of suspect individuals and the non-hybrid status of individuals regarded as wolves. We also show the adequacy of these markers to assess hybridization at a European-wide scale and the importance of including samples from reference populations. CONCLUSIONS: We showed that the proposed SNP panel is an efficient tool for detecting hybrids up to the third-generation backcrosses to wolves across Europe. Notably, the proposed genotyping method is suitable for a variety of samples, including non-invasive and museum samples, making this panel useful for wolf-dog hybrid assessments and wolf monitoring at both continental and different temporal scales.

Profiling inflammatory response in lesions of cutaneous leishmaniasis patients using a non-invasive sampling method combined with a high-throughput protein detection assay.

BACKGROUND: Cutaneous leishmaniasis (CL) is an infection caused by Leishmania (L.) protozoa transmitted through the bite of infected sand fly. Previously, invasive sampling of blood and skin along with low throughput methods were used for determination of inflammatory response in CL patients. AIMS/METHODOLOGY: We established a novel approach based on a non-invasive adhesive tape-disc sampling combined with a powerful multiplexing technique called proximity extension assay for profiling 92 inflammatory cytokines, chemokines and surface molecules in the lesions of CL patients infected with L. tropica. Sample collection was done non-invasively by using adhesive tape-discs from lesion and normal skin of 33 L. tropica positive patients. RESULTS: Out of 92 inflammatory proteins, the level of 34 proteins was significantly increased in the lesions of CL patients compared to their normal skin. This includes the chemokines CCL2, CCL3, CCL4, CXCL1, CXCL5, CXCL9, CXCL10 and CXCL11, together with the interleukins IL-6, IL-8, IL-18, LIF and OSM. The remaining significantly changed inflammatory proteins include 7 surface molecules and receptors: CD5, CD40, CDCP1, 4E-BP1, TNFRSF9, IL-18R1 and OPG as well as 16 other cytokines and proteins: MMP-1, CSF-1, VEGFA, uPA, EN-RAGE, LAP TGF-ß1, HGF, MMP-10, CASP-8, TNFSF14, STAMPB, ADA, TRAIL and ST1A1. Further, 13 proteins showed an increasing trend, albeit not statistically significant, in the CL lesions, including TGF-α, CCL23, MCP-2, IL-12B, CXCL6, IL-24, FGF-19, TNFß, CD6, TRANCE, IL10, SIR2 and CCL20. CONCLUSION: We herein report a novel approach based on a non-invasive sampling method combined with the high-throughput protein assay for profiling inflammatory proteins in CL lesions. Using this approach, we could profile inflammatory proteins in the lesions from CL patients. This new non-invasive approach may have implications for studying skin inflammatory mediators in CL and other skin disorders.

Chemical profiling of the human skin surface for malaria vector control via a non-invasive sorptive sampler with GC×GC-TOFMS.

Volatile organic compounds (VOCs) and semi-VOCs detected on the human skin surface are of great interest to researchers in the fields of metabolomics, diagnostics, and skin microbiota and in the study of anthropophilic vector mosquitoes. Mosquitoes use chemical cues to find their host, and humans can be ranked for attractiveness to mosquitoes based on their skin chemical profile. Additionally, mosquitoes show a preference to bite certain regions on the human host. In this study, the chemical differences in the skin surface profiles of 20 human volunteers were compared based on inter-human attractiveness to mosquitoes, as well as inter- and intra-human mosquito biting site preference. A passive, non-invasive approach was followed to sample the wrist and ankle skin surface region. An in-house developed polydimethylsiloxane (PDMS) passive sampler was used to concentrate skin VOCs and semi-VOCs prior to thermal desorption directly in the GC inlet with comprehensive gas chromatography coupled to time-of-flight mass spectrometry (GC×GC-TOFMS). Compounds from a broad range of chemical classes were detected and identified as contributing to the differences in the surface skin chemical profiles. 5-Ethyl-1,2,3,4-tetrahydronaphthalene, 1,1'-oxybisoctane, 2-(dodecyloxy)ethanol, α,α-dimethylbenzene methanol, methyl salicylate, 2,6,10,14-tetramethylhexadecane, 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester, 4-methylbenzaldehyde, 2,6-diisopropylnaphthalene, n-hexadecanoic acid, and γ-oxobenzenebutanoic acid ethyl ester were closely associated with individuals who perceived themselves as attractive for mosquitoes. Additionally, biological lead compounds as potential attractants or repellants in vector control strategies were tentatively identified. Results augment current knowledge on human skin chemical profiles and show the potential of using a non-invasive sampling approach to investigate anthropophilic mosquito-host interactions. Graphical abstract.

Assessment of ecotoxicological risks to river otters from ingestion of invasive red swamp crayfish in metal contaminated areas: Use of feces to estimate dietary exposure.

The invasive red swamp crayfish (Procambarus clarkii) has become a major food resource for Eurasian otters (Lutra lutra) in the Iberian Peninsula. Crayfish accumulate large amounts of metals, and hence otters could be at risk of exposure and intoxication through crayfish consumption. We conducted a food safety risk assessment for otters inhabiting two historical mining areas in central Spain affected by lead (Pb) and mercury (Hg) pollution. Estimated daily intakes (EDI) of Pb and Hg were non-invasively calculated from the proportion of crayfish remains and metal levels in otter feces. We considered that the abdominal muscle and the carcass of crayfish differ significantly in relative weight, total metal content and bioavailability of metals to reduce the uncertainty of risk characterization. Fecal concentrations of Hg and Pb in the polluted areas were 1.878 and 6.554 µg/g d. w., respectively (13-fold and 7-fold higher compared to a non-polluted area). EDI of Hg and Pb in the polluted areas were 66.02 and 78.26 µg/kg-day, respectively (14- and 8-fold higher than in the reference area). EDI from the Hg area were above minimum levels susceptible to cause neurotoxicity in mustelids, and 6.3% were above levels susceptible to cause histopathological lessions. In the Pb area, 16.7% of EDI were consistent with levels causing reproductive effects. Metal exposure through crayfish consumption might prevent or slow the recovey of otters in these polluted environments, thus this factor should be considered in management strategies aimed to protect otter populations.

Non-invasive sorptive extraction for the separation of human skin surface chemicals using comprehensive gas chromatography coupled to time-of-flight mass spectrometry: A mosquito-host biting site investigation.

Variation in inter-human attractiveness to mosquitoes, and the preference of mosquitoes to bite certain regions on the human host, are possible avenues for identifying lead compounds as potential mosquito attractants or repellents. We report a practical, non-invasive method for the separation and detection of skin surface chemical compounds and comparison of skin chemical profiles between the ankle and wrist skin surface area sampled over a 5-day period of a human volunteer using comprehensive gas chromatography coupled to time-of-flight mass spectrometry. An in-house made polydimethylsiloxane passive mini-sampler, worn as an anklet or a bracelet, was used to concentrate skin volatiles and semi-volatiles prior to thermal desorption directly in the gas chromatography. A novel method for the addition of an internal standard to sorptive samplers was introduced through solvent modification. This approach enabled a more reliable comparison of human skin surface chemical profiles. Compounds that were closely associated with the wrist included 6-methyl-1-heptanol, 3-(4-isopropylphenyl)-2-methylpropionaldehyde, 2-phenoxyethyl isobutyrate, and 2,4,6-trimethyl-pyridine. Conversely, compounds only detected on the ankle region included 2-butoxyethanol phosphate, 2-heptanone, and p-menthan-8-ol. In addition to known human skin compounds we report two compounds, carvone and (E)-2-decenal, not previously reported. Limits of detection ranged from 1 pg (carvone) to 362 pg (indole).

Antarctophthirus microchir infestation in synanthropic South American sea lion (Otaria flavescens) males diagnosed by a novel non-invasive method.

Antarctophthirus microchir is a sucking louse species belonging to the family Echinophthiriidae and has been reported to parasitize all species of the subfamily Otariinae, the sea lions. Former studies on this ectoparasite mainly required fixation, immobilization, or death of host species and especially examinations of adult male sea lions are still very rare. Between March and May 2018, adult individuals of a unique "urban" bachelor group of South American sea lions (Otaria flavescens) living directly in the city of Valdivia, Chile, were studied regarding their ectoparasite infestation status. For first time, a non-invasive method in the form of a lice comb screwed on a telescopic rod and grounded with adhesive tape was used for sample taking process. Overall, during combing different stages of A. microchir were detected in 4/5 O. flavescens individuals, especially at the junction between the back and hind flippers. Our findings represent the first report of A. microchir infesting individuals of this synanthropic colony and fulfilling complete life cycle in a sea lion group despite inhabiting freshwater and in absence of females/pups. Our "telescopic lice comb apparatus" offers a new strategy to collect different stages of ectoparasites and a range of epidermal material, such as fur coat hair and superficial skin tissue for a broad spectrum of research fields in wildlife sciences in an unmolested and stress reduced manner.

Non-lethal sampling for stable isotope analysis of pike Esox lucius: how mucus, scale and fin tissue compare to muscle.

Stable isotope analysis (SIA) was used to examine the isotopic relationships between dorsal muscle and fin, scale and epidermal mucus in pike Esox lucius. δ13 C and δ15 N varied predictably within each tissue pairing, with conversion factors calculated for the surrogate tissues, enabling their application to the non-lethal sampling of E. lucius for SIA.

Determination of fatty acid and sphingoid base composition of eleven ceramide subclasses in stratum corneum by UHPLC/scheduled-MRM.

INTRODUCTION: Ceramides play a key role in skin barrier function in homeostatic and pathological conditions and can be sampled non-invasively through stratum corneum collection. OBJECTIVES: To develop a novel UHPLC/Scheduled MRM method for the identification and relative distribution of eleven classes of ceramides, which are separated by UHPLC and determined by their specific retention times. The precise composition of the fatty acid and sphingoid base parts of each individual ceramide is determined via mass fragmentation. METHODS: More than 1000 human and pig ceramides were identified. Three human and minipig ceramide classes, CER[AS], CER[NS] and CER[EOS] have been investigated in depth. RESULTS: Sphingoid bases were characterized by a prevalence of chain lengths with sizes from C16 to C22, whereas fatty acids were mainly observed in the range of C22-C26. Overall, the ceramide profiles between human and minipig stratum corneum were similar. Differences in the CER[AS] and CER[NS] classes included a more homogeneous distribution of fatty acids (16-30 carbon atoms) in minipig, whereas in human longer fatty acid chains (> 24 carbon atoms) predominated. CONCLUSION: The method will be useful for the analysis of healthy and pathological skin in various specie, and the measurement of the relative distribution of ceramides as biomarkers for pharmacodynamic studies.

Genome sequencing and conservation genomics in the Scandinavian wolverine population.

Genetic approaches have proved valuable to the study and conservation of endangered populations, especially for monitoring programs, and there is potential for further developments in this direction by extending analyses to the genomic level. We assembled the genome of the wolverine (Gulo gulo), a mustelid that in Scandinavia has recently recovered from a significant population decline, and obtained a 2.42 Gb draft sequence representing >85% of the genome and including >21,000 protein-coding genes. We then performed whole-genome resequencing of 10 Scandinavian wolverines for population genomic and demographic analyses. Genetic diversity was among the lowest detected in a red-listed population (mean genome-wide nucleotide diversity of 0.05%). Results of the demographic analyses indicated a long-term decline of the effective population size (Ne ) from 10,000 well before the last glaciation to <500 after this period. Current Ne appeared even lower. The genome-wide FIS level was 0.089 (possibly signaling inbreeding), but this effect was not observed when analyzing a set of highly variable SNP markers, illustrating that such markers can give a biased picture of the overall character of genetic diversity. We found significant population structure, which has implications for population connectivity and conservation. We used an integrated microfluidic circuit chip technology to develop an SNP-array consisting of 96 highly informative markers that, together with a multiplex pre-amplification step, was successfully applied to low-quality DNA from scat samples. Our findings will inform management, conservation, and genetic monitoring of wolverines and serve as a genomic roadmap that can be applied to other endangered species. The approach used here can be generally utilized in other systems, but we acknowledge the trade-off between investing in genomic resources and direct conservation actions.

Sweat lipid mediator profiling: a noninvasive approach for cutaneous research.

Recent advances in analytical and sweat collection techniques provide new opportunities to identify noninvasive biomarkers for the study of skin inflammation and repair. This study aims to characterize the lipid mediator profile including oxygenated lipids, endocannabinoids, and ceramides/sphingoid bases in sweat and identify differences in these profiles between sweat collected from nonlesional sites on the unflared volar forearm of subjects with and without atopic dermatitis (AD). Adapting routine procedures developed for plasma analysis, over 100 lipid mediators were profiled using LC-MS/MS and 58 lipid mediators were detected in sweat. Lipid mediator concentrations were not affected by sampling or storage conditions. Increases in concentrations of C30-C40 [NS] and [NdS] ceramides, and C18:1 sphingosine, were observed in the sweat of study participants with AD despite no differences being observed in transepidermal water loss between study groups, and this effect was strongest in men (P < 0.05, one-way ANOVA with Tukey's post hoc HSD). No differences in oxylipins and endocannabinoids were observed between study groups. Sweat mediator profiling may therefore provide a noninvasive diagnostic for AD prior to the presentation of clinical signs.

Equine behavioral enrichment toys as tools for non-invasive recovery of viral and host DNA.

Direct collection of samples from wildlife can be difficult and sometimes impossible. Non-invasive remote sampling for the purpose of DNA extraction is a potential tool for monitoring the presence of wildlife at the individual level, and for identifying the pathogens shed by wildlife. Equine herpesviruses (EHV) are common pathogens of equids that can be fatal if transmitted to other mammals. Transmission usually occurs by nasal aerosol discharge from virus-shedding individuals. The aim of this study was to validate a simple, non-invasive method to track EHV shedding in zebras and to establish an efficient protocol for genotyping individual zebras from environmental DNA (eDNA). A commercially available horse enrichment toy was deployed in captive Grévy's, mountain, and plains zebra enclosures and swabbed after 4-24 hr. Using eDNA extracted from these swabs four EHV strains (EHV-1, EHV-7, wild ass herpesvirus and zebra herpesvirus) were detected by PCR and confirmed by sequencing, and 12 of 16 zebras present in the enclosures were identified as having interacted with the enrichment toy by mitochondrial DNA amplification and sequencing. We conclude that, when direct sampling is difficult or prohibited, non-invasive sampling of eDNA can be a useful tool to determine the genetics of individuals or populations and for detecting pathogen shedding in captive wildlife.

Comparing skin swabs, buccal swabs, and toe clips for amphibian genetic sampling, a case study with a small anuran (Acris blanchardi).

Multiple methods for collecting genetic samples from amphibians exist, each with their own implications for study design, animal welfare, and costs. Toe clipping is one common method, but there is ongoing debate regarding its potential detriment. Less invasive methods should be implemented, if efficacious, as amphibians are a particularly vulnerable vertebrate group. Skin and buccal swabbing are less invasive methods for genetic sampling, but the potential for contamination and a lower yield of DNA may exist. To compare these methods, we gathered skin swabs, buccal swabs, and toe clips from the same individuals of a relatively small anuran species, Blanchard's Cricket Frog (Acris blanchardi). We then compared DNA yield, DNA purity, amplification success rate, and genotypic data quality among sample types. We found toe clips and buccal swabs generated similar DNA yield and purity, with skin swabs yielding significantly less DNA of significantly lower purity than the other sample types. Amplification success rate was significantly higher using toe clips compared to the other sample types, though buccal swab samples amplified more readily than skin swabs. Genotypic data from toe clips and buccal swabs did not differ significantly in quality, but skin swab data quality was significantly lowest among sample types. Thus, skin swabbing could produce erroneous data in some situations, but buccal swabbing is likely an effective substitute to toe clipping, even for small species. Our results can help future researchers select which genetic sampling method might best suit their research needs.

Are fecal samples an appropriate proxy for amphibian intestinal microbiota?

The intestinal microbiota, an invisible organ supporting a host's survival, has essential roles in metabolism, immunity, growth, and development. Since intestinal microbiota influences a host's biology, application of such data to wildlife conservation has gained interest. There are standard protocols for studying the human intestinal microbiota, but no equivalent for wildlife. A major challenge is sampling the intestinal microbiota in an effective, unbiased way. Fecal samples are a popular proxy for intestinal microbiota because collection is non-invasive and allows for longitudinal sampling. Yet it is unclear whether the fecal microbiota is representative of the intestinal microbiota. In wildlife studies, research on the sampling methodology is limited. In this study focusing on amphibians, we characterize and compare the microbiota (small intestine, large intestine, and feces) of two Hong Kong stream-dwelling frog species: Lesser Spiny Frog (Quasipaa exilispinosa) and Hong Kong Cascade Frog (Amolops hongkongensis). We found that the microbiota of both species are similar at the level of phylum and family, but diverge at the level of genus. When we assessed the performance of fecal microbiota in representing the intestinal microbiota in these two species, we found that (1) the microbiota of the small and large intestine differs significantly, (2) feces are not an appropriate proxy of either intestinal sections, and (3) a set of microbial taxa significantly differs between sample types. Our findings raise caution equating fecal and intestinal microbiota in stream-dwelling frogs. Sampling feces can avoid sacrifice of an animal, but researchers should avoid over-extrapolation and interpret results carefully.

Advancing the analytical toolkit in the investigation of vector mosquito host biting site selection.

High-resolution mass spectrometry and ion mobility spectrometry provide additional confidence in biological marker discovery and elucidation by adding additional peak capacity through physiochemical separation orthogonal to chromatography. Sophisticated analytical techniques have proved valuable in the identification of human skin surface chemicals used by vector mosquitoes to find their human host. Polydimethylsiloxane (PDMS) was used as a non-invasive passive wearable sampler to concentrate skin surface non-volatile and semi-volatile compounds prior to solvent desorption directly in an LC vial, thereby simplifying the link between extraction and analysis. Ultra-performance liquid chromatography with ion mobility spectrometry coupled with high-resolution mass spectrometry (UPLC-IMS-HRMS) was used for compound separation and detection. A comparison of the skin chemical profiles between the ankle and wrist skin surface region sampled over a 5-day period for a human volunteer was done. Twenty-three biomarkers were tentatively identified with the aid of a collision cross-section (CCS) prediction tool, seven associated with the ankle skin surface region and 16 closely associated with the wrist skin surface. Ten amino acids were detected and unequivocally identified on the human skin surface for the first time. Furthermore, 22 previously unreported skin surface compounds were tentatively identified on the human skin surface using accurate mass, CCS values and fragmentation patterns. Method limits of detection for the passive skin sampling method ranged from 8.7 (sulfadimethoxine) to 95 ng (taurine). This approach enabled the detection and identification of as-yet unknown human skin surface compounds and provided corresponding CCS values.

Hair Cortisol and Testosterone Concentrations in Relation to Maturity and Breeding Status of Male Feral Horses.

Steroid-hormone concentrations from non-invasively obtained biomarkers, like hair, can provide a representation of circulating hormones diffused over relatively long time periods (e.g., weeks or months). The hormone cortisol is often associated with physiological or even psychological stress, while testosterone is strongly associated with male development and reproductive success. Increasingly, studies are using hormone levels derived from hair to make inferences among both domestic animals and wildlife. For horses, all previous hair hormone analysis has been done on companion or working animals. We evaluated the levels of hair cortisol (n = 153) and testosterone (n = 48) from 136 feral horses living on Sable Island, Canada that have been part of a long-term individual-based study since 2008. This population has been undisturbed and unmanaged for over 50 years, and exhibits the natural social organization for horses, harem defense polygyny. Hair samples were collected in mid to late summer and the segment analyzed corresponds with hair grown during, and following, the peak of the reproductive season. Social position was determined based on the male's role as either a dominant breeding Stallion (Stallion), a non-breeding subordinate male (tag), adult Bachelor (5 years old or older), or Immature male (2-4 years of age). While there was no difference in hair-cortisol concentration among any class of adult males (i.e., Stallion, tag, or Bachelor), Immature males had significantly lower hair cortisol concentrations than the other groups (p = 0.001). Hair testosterone levels among the four social positions were significantly higher among Stallions (p = 0.04). Hair testosterone concentration was also significantly related to the probability of a male being either a Bachelor or Stallion and was the only variable remaining in AICc model selection (p = 0.016, AICc = 32.3, Null AICc = 38.8). While not a significant relationship, Stallions had a negative correlation between hair cortisol concentrations and testosterone (R2 = -0.20, p = 0.383), and Bachelors, conversely, had a positive association (R2 = 0.43, p = 0.246). Our observations of hormone concentrations in relation to physiological, social, or reproductive parameters in this population suggest trends that are similar to what has been established using blood or other matrices.