Non-invasive Sampling Research Articles

Leveraging Epigenetic Alterations in Pancreatic Ductal Adenocarcinoma for Clinical Applications.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy characterized by late detection and poor prognosis. Recent research highlights the pivotal role of epigenetic alter- ations in driving PDAC development and progression. These changes, in conjunction with genetic mutations, contribute to the intricate molecular landscape of the disease. Specific modifications in DNA methylation, histone marks, and non-coding RNAs are emerging as robust predictors of disease progression and patient survival, offering the potential for more precise prognostic tools compared to conventional clinical staging. Moreover, the detection of epigenetic alterations in blood and other non-invasive samples holds promise for earlier diagnosis and improved manage- ment of PDAC. This review comprehensively summarises current epigenetic research in PDAC and identifies persisting challenges. These include the complex nature of epigenetic profiles, tumour hetero- geneity, limited access to early-stage samples, and the need for highly sensitive liquid biopsy technologies. Addressing these challenges requires the standardisation of methodologies, integra- tion of multi-omics data, and leveraging advanced computational tools such as machine learning and artificial intelligence. While resource-intensive, these efforts are essential for unravelling the functional consequences of epigenetic changes and translating this knowledge into clinical appli- cations. By overcoming these hurdles, epigenetic research has the potential to revolutionise the management of PDAC and improve patient outcomes.

Detection of porcine reproductive and respiratory syndrome virus type 2 in air emissions from grow-finish barns.

Diagnosis of porcine reproductive and respiratory syndrome virus (PRRSV) infections can be accomplished using various sample types and testing methods. The objective of this study was to evaluate the feasibility of using air emission samples to detect the onset of PRRSV type 2 infections in growing pigs. Air emissions and oral fluid samples were collected from three grow-finish barns, stocked with PRRSV-negative pigsevery 2 weeksfor 14-20 weeks. All samples were assayed for PRRSV using RT-PCR. In all barns, PRRSV was initially detected in both air emissions and oral fluid samples at the same sampling event. Compared with oral fluids, air emissions collected subsequently yielded higher cycle threshold values and showed a lower likelihood of being RT-PCR positive. Sampling was limited to three grow-finish barns. Air emission samples from pig barns are a biosecure and non-invasive sample type that is comparable to oral fluids fordetection of the onset of PRRSV infection.

Closing the air gap: the use of drones for studying wildlife ecophysiology.

Techniques for non-invasive sampling of ecophysiological data in wild animals have been developed in response to challenges associated with studying captive animals or using invasive methods. Of these, drones, also known as Unoccupied Aerial Vehicles (UAVs), and their associated sensors, have emerged as a promising tool in the ecophysiology toolkit. In this review, we synthesise research in a scoping review on the use of drones for studying wildlife ecophysiology using the PRISMA-SCr checklist and identify where efforts have been focused and where knowledge gaps remain. We use these results to explore current best practices and challenges and provide recommendations for future use. In 136 studies published since 2010, drones aided studies on wild animal body condition and morphometrics, kinematics and biomechanics, bioenergetics, and wildlife health (e.g. microbiomes, endocrinology, and disease) in both aquatic and terrestrial environments. Focal taxa are biased towards marine mammals, particularly cetaceans. While conducted globally, research is primarily led by institutions based in North America, Oceania, and Europe. The use of drones to obtain body condition and morphometric data through standard colour sensors and single camera photogrammetry predominates. Techniques such as video tracking and thermal imaging have also allowed insights into other aspects of wildlife ecophysiology, particularly when combined with external sampling techniques such as biologgers. While most studies have used commercially available multirotor platforms and standard colour sensors, the modification of drones to collect samples, and integration with external sampling techniques, have allowed multidisciplinary studies to integrate a suite of remote sensing methods more fully. We outline how technological advances for drones will play a key role in the delivery of both novel and improved wildlife ecophysiological data. We recommend that researchers prepare for the influx of drone-assisted advancements in wildlife ecophysiology through multidisciplinary and cross-institutional collaborations. We describe best practices to diversify across species and environments and use current data sources and technologies for more comprehensive results.

Comparison of whole genome sequencing performance from fish swabs and fin clips

ObjectiveFin clipping is the standard DNA sampling technique for whole genome sequencing (WGS) of small fish. The collection of fin clips requires anaesthesia or even euthanisation of the individual. Swabbing may be a less invasive, non-lethal alternative to fin-clipping. Whether skin and gill swabs are comparable to fin clips in terms of DNA extraction quality and sequence read mapping performance from WGS was tested here on Eurasian minnows (Phoxinus phoxinus).ResultsOf 49 fin clips, all met the DNA concentration threshold of 20 ng/μl, whereas 43 of 88 swabs met this requirement. Preserving swabs in ATL buffer and treatment with Proteinase K during DNA extraction consistently raised skin swab DNA concentrations above the cut-off. All samples passed the A260/A280 absorbance ratio cut-off of 1.3. Ultimately, 93.88% of the fin clips, 30.61% of the skin, and 7.69% of the gill swabs were suitable for sequencing. Mapping performances of all three tissues were comparable in reads passing quality filtering, percentage of reads mapping to the P. phoxinus reference genome, and coverage. Overall, skin swabs treated with Proteinase K during extraction, can match fin clips in WGS performance and represent a viable non-invasive DNA sampling alternative.

Chemical signatures of social information in Barbary macaques

Primates are well-known for their complex social lives and intricate social relationships, which requires them to obtain and update social knowledge about conspecifics. The sense of smell may provide access to social information that is unavailable in other sensory domains or enhance the precision and reliability of other sensory cues. However, the cognition of social information in catarrhine primates has been studied primarily in the visual and auditory domain. We assessed the social information content of body odor in a catarrhine primate, the Barbary macaque (Macaca sylvanus), in three semi-free ranging groups at Affenberg Salem, Germany. In particular, we related socially relevant attributes (identity, genetic relatedness, rank, sex, age, group membership) to chemical profiles of body odor. We applied non-invasive active sampling via thermal desorption tubes and analyzed samples by gas chromatography–mass spectrometry. We found robust evidence for individual odor signatures and limited support for kin signatures. Chemical profiles were also related to group membership, but little to rank, sex or age. The detected associations between chemical profiles and socially relevant attributes form the theoretical foundations for olfactory information transfer, highlighting the potential of body odor to provide valuable social information in this highly visually oriented primate.

Toxic metals and other elements in Svalbard reindeer: Establishing baselines and assessing non-invasive sampling for biomonitoring.

Pollutants emitted from all over the world may reach pristine areas, such as the Arctic. The Svalbard reindeer (Rangifer tarandus platyrhynchus) has been the subject of a few studies reporting toxic metal concentrations. However, these studies either date back a few decades or exclusively used non-invasive samples (e.g., faeces and fur), leaving us without an updated assessment of the concentrations in internal tissues and target organs. This study is the first to present the concentrations of the toxic metals mercury (Hg), cadmium (Cd), and lead (Pb) in both invasive and non-invasive samples from female Svalbard reindeer. The highest concentrations of both Hg and Cd were found in the kidneys (x̄=0.68μg/g and 16.3μg/g dw, respectively). The highest concentration of Pb was found in faeces (x̄=2.62μg/g dw), followed by liver (x̄=0.28μg/g dw). While both Cd and Pb concentrations in Svalbard reindeer were comparable to levels reported in other reindeer subspecies or circumpolar wildlife, Hg concentrations were lower than those reported in most other studies on reindeer. Conversely, Hg levels in Svalbard reindeer were still higher than levels reported in wildlife from central Europe, demonstrating the influence of long-range transport of Hg to the Arctic. By comparing the toxic metal concentrations in different sample types, we confirm a relationship between Hg concentrations in invasive and non-invasive samples, with faeces being the most promising proxy for soft tissue Hg concentrations. Consequently, future Hg biomonitoring efforts may be carried out with relatively simple sampling procedures and without sacrificing Svalbard reindeer.

The distribution of pigeon adenoviruses in Northern Chinese pigeon and turtledove flocks provides further evidence of viral crosstransmission.

To understand the prevalence, genetic diversity, and potential pathogenicity of adenoviruses present in pigeon and turtledove populations. Nested PCR and Sanger sequencing methods were used to identify the genotype and percentage of various adenoviruses in the feces of pigeon (Columba) and turtledove (Streptopelia) populations. In Beijing, China, a total of 194 fresh feces samples from meat-use pigeons (C livia domestica), homing pigeons (C livia domestica), wild pigeons (C livia domestica), and turtledoves (S decaocto and S chinensis) were collected using noninvasive sampling collection techniques. Their partial DNA-dependent DNA polymerase gene sequences were obtained using nested PCR and double-ended Sanger sequencing, and their genotypes were then ascertained based on sequence alignment. A total of 6 genotypes of adenovirus were detected in pigeon and turtledove flocks, including pigeon adenovirus (PiAdV)-1, PiAdV-2A, PiAdV-3, PiAdV-4, PiAdV-5, and a novel adenovirus genotype (PiAdV-6). Among them, PiAdV-1 was found widespread in flocks of pigeons exhibiting extensive presentations of hepatic necrosis. Highly conserved PiAdV-4 and PiAdV-5 were found to be nonpathogenic and extensively distributed in all pigeon and turtledove groups. These findings imply the presence of diverse PiAdVs in pigeon and turtledove flocks, and the wild pigeons and wild turtledove birds are potentially serving as natural sources of these viruses. This study provides supportive evidence of the pathogenicity of different genotypes of adenovirus in pigeon flocks and also implies that stopping the transmission of the virus brought by wild pigeons and turtledoves may be important for the prevention of diseases associated with PiAdVs.

Advances in Glioblastoma Diagnosis: Integrating Genetics, Noninvasive Sampling, and Advanced Imaging.

Glioblastoma is the most common primary brain tumor in adult patients, and despite standard-of-care treatment, median survival has remained less than two years. Advances in our understanding of molecular mutations have led to changes in the diagnostic criteria of glioblastoma, with the WHO classification integrating important mutations into the grading system in 2021. We sought to review the basics of the important genetic mutations associated with glioblastoma, including known mechanisms and roles in disease pathogenesis/treatment. We also examined new advances in image processing as well as less invasive and noninvasive diagnostic tools that can aid in the diagnosis and surveillance of those undergoing treatment for glioblastoma. Our review is intended to serve as an overview of the current state-of-the-art in the diagnosis and management of glioblastoma.

Variation in faecal testosterone levels in male gray whales on a foraging ground relative to maturity and timing.

Understanding wildlife reproductive seasonality is crucial for effective management and long-term monitoring of species. This study investigates the seasonal variability of testosterone in male Pacific Coast Feeding Group (PCFG) gray whales, using an eight-year dataset (2016-2023) of individual sightings, drone-based photogrammetry and endocrine analysis of faecal samples. We analyzed the relationship between faecal testosterone levels and total body length (TL), body condition (body area index, BAI), sexual maturity and day of the year using generalized additive mixed models. Our findings reveal a significant increase in faecal testosterone levels in mature males (MM) towards the end of the foraging season. This increase was not observed in JM, highlighting age-dependent development of sexual characteristics. No significant relationship was found between testosterone levels and TL. Additionally, BAI was not significantly associated with testosterone levels. Our results suggest that the increasing testosterone levels in MM gray whales may indicate preparation for mating before the southbound migration. These findings provide valuable insights into the reproductive biology of PCFG gray whales and underscore the importance of non-invasive faecal sampling for studying reproductive seasonality in large whales. Our approach not only provides further insights into the seasonality of male reproduction for the PCFG gray whales but also offers tools to enhance the understanding of male reproduction in baleen whales broadly with non-invasive approaches.

Genetic Rescue of the Dinaric Lynx Population: Insights for Conservation From Genetic Monitoring and Individual-Based Modelling.

Inbreeding depression poses a severe threat to small populations, leading to the fixation of deleterious mutations and decreased survival probability. While the establishment of natural gene flow between populations is an ideal long-term solution, its practical implementation is often challenging. Reinforcement of populations by translocating individuals from larger populations is a viable strategy for reducing inbreeding, increasing genetic diversity and potentially saving populations from extinction. The Dinaric population of Eurasian lynx (Lynx lynx) has faced high inbreeding levels, with effective inbreeding reaching 0.316 in 2019, endangering the population's survival. To counteract this, population reinforcement was implemented between 2019 and 2023, involving the translocation of 12 individuals from the Carpathian Mountains to the Dinaric Mountains of Slovenia and Croatia. We conducted comprehensive genetic monitoring in this area, gathering 588 non-invasive and tissue samples, which were used for individual identification and estimation of population genetic parameters. We used stochastic modelling to assess the long-term viability of the Dinaric lynx population post-translocation and formulate effective conservation strategies. The model predicts that, despite significant improvement of genetic diversity after translocations, inbreeding will return to critical levels within 45 years. Our results highlight the fact that reinforcement is just the first step and that long-term genetic management is needed to keep the population from sliding back towards extinction. The Dinaric lynx population serves as a compelling example of genetic rescue. The lessons learnt here will be essential for ensuring the viability of the Dinaric lynx in the future and also provide a useful template for conservation of other populations and species facing similar threats.

Noninvasive genetic testing for type IV collagen nephropathy using oral mucosa DNA sampling in children with haematuria

Objective Hematuria is one of the most common conditions in children, and increase the risk of chronic kidney disease. Persistent hematuria may be the earliest manifestation of type IV collagen-related nephropathy. Early diagnosis is essential for optimized therapy. Due to the invasive nature of kidney biopsy and the high cost of whole exome sequencing, its application in the diagnosis of isolated hematuria is rare. Hence, we performed noninvasive and convenient genetic testing approaches for type IV collagen-related nephropathy. Methods We used noninvasive oral mucosa sampling as an alternative method for DNA isolation for genetic testing and designed a panel targeting three type IV collagen nephropathy-related genes in children with hematuria. Children with persistent hematuria unaccompanied by clinically significant proteinuria or renal insufficiency who underwent genetic testing using a hematuria panel were enrolled. Results Thirty-seven of 112 (33.0%) patients were found to have a genetic variant in COL4A3/A4/A5. Pathogenic/likely pathogenic COL4A3/A4/A5 variants were identified in 17 of the 112 patients analyzed (15.2%), which were considered to explain their hematuria manifestations. In addition, variants of unknown significance (VUSs) were found in 17.8% (20/112) of patients. Furthermore, we observed a much greater COL4A3/A4/A5 variant detection rate in patients with a positive family history or more severe hematuria (RBC ≥ 20/HP) or with coexisting microalbuminuria (59.2% vs. 12.7%, p < 0.001; 64.0% vs. 24.1%, p < 0.001; 66.7% vs. 30.1%, p = 0.025). Conclusions We present the high prevalence of variants in COL4A genes in a multicenter pediatric cohort with hematuria, which requires close monitoring and long-term follow-up.

Comparative analysis of PFASs concentrations in fur, muscle, and liver of wild roe deer as biomonitoring matrices

BackgroundRecently, environmental pollution has become a significant concern for human, animal, and environmental health, fitting within the “One Health” framework. Among the various environmental contaminants, per- and polyfluoroalkyl substances (PFASs) have gathered substantial attention due to their persistence, bioaccumulation, and adverse health effects. This study aimed to compare the levels of 12 PFASs in the fur, liver, and muscle of wild roe deer to evaluate the feasibility of using fur as a non-invasive biomonitoring matrix.MethodsA total of 20 male and 20 female roe deer aged between 12 and 24 months were randomly sampled from a hunting area in Northern Italy. Samples of fur, muscle, and liver were collected post-mortem, and PFAS concentrations were measured using a validated UHPLC-HRMS method.Results and discussionThe results indicated significant differences in PFAS concentrations among the three matrices. Fur, although easier to sample and store, showed highly variable PFAS levels, with different detection frequencies compared to the muscle and liver. PFASs such as PFHxA were more frequently detected in fur than in the liver and muscle, while compounds such as PFBA, PFPeA, PFHpA, PFDA, PFHxS, 6-2 FTS, and 8-2 FTS were less frequently detected in fur. In conclusion, while fur presents many practical advantages for biomonitoring, such as non-invasive sampling and stability, its use is complicated by varying detection frequencies and concentration levels. These aspects, together with the use of a single sampling technique, can be considered a limitation of the study. Notably, compounds such as PFOA, PFNA, and PFOS showed partially similar detection frequencies across the matrices, suggesting potential interest for further research. This study offers new perspectives on the use of fur for environmental monitoring, highlighting the need for more extensive research to understand the relationship between PFAS concentrations in fur and other biological matrices. Future studies should focus on methodological improvements in extraction and quantification techniques for PFASs in fur to enhance their reliability as a biomonitoring tool.

Breath biopsy in inborn errors of metabolism: A proof-of-principle study in propionic acidemia.

Impaired oxidation of branched chain amino acids may give rise to volatile organic compounds (VOCs). We hypothesized that VOCs will be present in exhaled breath of participants with propionic acidemia (PA), and their relative abundance would correlate with clinical and biochemical characteristics of the disease. We enrolled 5 affected participants from a natural history study of PA (ClinicalTrials.gov ID NCT02890342) plus five age- and sex-matched unaffected controls. We collected exhaled breath using a non-invasive breath sampling platform paired with thermal desorption-gas chromatography-mass spectrometry. Clinical and biochemical parameters were correlated with the relative abundance of VOCs. Unbiased screening identified several candidate VOC biomarkers of PA. One candidate putatively identified as 3-pentanone was the most abundant (45-fold higher in cases vs. controls, p-value <0.05). 3-Pentanone abundance positively correlated with plasma propionylcarnitine (p=0.01), plasma 2-methylcitrate (p<0.05), 3-OH-propionate (p<0.01), full scale IQ (p<0.01), and showed a statistical trend with height z-scores (p=0.08). It inversely correlated with the whole-body in vivo oxidation of 1-13C-propionate (p<0.05). In a participant who received an orthotopic liver transplant, 3-pentanone levels were lower and segregated with "mild" PA. Non-invasive breath sampling is a promising method to identify and quantitate VOCs that correlate with the clinical and biochemical parameters of PA. Our proof-of-principle findings may have wide implications for the diagnosis and severity stratification of inborn errors of metabolism affecting oxidation of amino acids which might be monitored in a similar fashion. A proof-of-principle study putatively identifies 3-pentanone in exhaled breath as a correlate of the clinical and biochemical outcomes in propionic acidemia.

Fe/Pt-doped carbon nanoparticles with peroxidase-like activity for point-of-care determination of uric acid.

Apasting-3D microfluidic paper-based analytical device (P-3D μPAD) was developed. Itenabled an efficient cascade reaction between urate oxidase (UOX) and Fe/Pt-doped carbon nanoparticles (Fe/Pt-CNPs) for visual colorimetric detectionof uric acid (UA). The novel Fe/Pt-CNP nanozyme performed high peroxidase-like activity toward 3,3',5,5'-tetramethylbenzidine (TMB) and H2O2 with Michaelis - Menten constants (Km) of 0.97 and 2.30mM, respectively. The UOX-Fe/Pt-CNP system was incorporated into P-3D μPAD: the 1st layer was UOX hydrolysis reaction under alkaline pH, and the 2nd layer was Fe/Pt-CNPs catalyzing H2O2 to oxidize TMB at acidic pH. The separated two layers allowed cascade reaction under different working pH without sacrificing the activity of UOX or Fe/Pt-CNPs. The images were captured and analyzed by the camera and "Color Recognition" application using a smartphone. The linear range of P-3D μPAD for UA was 25-1000μM with a limit of detection of 10μM which met the requirements for clinical applications. The high accuracy of P-3D μPAD for UA detection in invasive (blood) and noninvasive (saliva) samples has been confirmed by the biochemical analyzer. This work offers a sensitive, flexible, affordable, and disposable tool for on-site UA level monitoring and provides new insight into natural enzyme and nanozyme tandem systems for biosensing.

Microbial and proteomic signatures of type 2 diabetes in an Arab population

BackgroundThe rising prevalence of Type 2 diabetes mellitus (T2D) in the Qatari population presents a significant public health challenge, highlighting the need for innovative approaches to early detection and management. While most efforts are centered on using blood samples for biomarker discovery, the use of saliva remains underexplored.MethodsUsing noninvasive saliva samples from 2974 Qatari subjects, we analyzed the microbial communities from diabetic, pre-diabetic, and non-diabetic participants based on their HbA1C levels. The salivary microbiota was assessed in all subjects by sequencing the V1–V3 regions of 16S rRNA gene. For the proteomics profiling, we randomly selected 50 gender and age-matched non-diabetic and diabetic subjects and compared their proteome with SOMAscan. Microbiota and proteome profiles were then integrated to reveal candidate biomarkers for T2D.ResultsOur results indicate that the salivary microbiota of pre-diabetic and diabetic individuals differs significantly from that of non-diabetic subjects. Specifically, a significant increase in the abundance of Campylobacter, Dorea, and Bacteroidales was observed in the diabetic subjects compared to their non-diabetic controls. Metabolic pathway prediction analysis for these bacteria revealed a significant overrepresentation of genes associated with fatty acid and lipid biosynthesis, as well as aromatic amino acid metabolism in the diabetic group. Additionally, we observed distinct differences in salivary proteomic profiles between diabetic and non-diabetic subjects. Notably, levels of Haptoglobin, Plexin-C1, and MCL-1 were elevated, while Osteopontin (SPP1), Histone1H3A (HIST3H2A), and Histone H1.2 were reduced in diabetic individuals. Furthermore, integrated correlation analysis of salivary proteome and microbiota data demonstrated a strong positive correlation between HIST1H3A and HIST3H2A with Porphyromonas sp., all of which were decreased in the diabetic group.ConclusionThis is the first study to assess the salivary microbiota in T2D patients from a large cohort of the Qatari population. We found significant differences in the salivary microbiota of pre-diabetic and diabetic individuals compared to non-diabetic controls. Our study is also the first to assess the salivary proteome using SOMAScan in diabetic and non-diabetic subjects. Integration of the microbiota and proteome profiles revealed a unique signature for T2D that can be used as potential T2D biomarkers.

An LC-MS/MS assay for simultaneous determination of 13 steroid hormones and two synthetic steroids in saliva: potential utility for paediatric population and beyond

Saliva samples offer the possibility to obtain stress-free non-invasive samples, also for home-testing, especially useful when blood collection is either undesirable or difficult. The aim of this work was to develop an LC-MS/MS method to determine clinically relevant steroid hormones cortisol, cortisone, 11-deoxycortisol, 21-deoxycortisol, 17OH-progesterone, aldosterone, corticosterone, deoxycorticosterone, testosterone, androstenedione, DHEAS, DHEA, 17OH-pregnenolone, betamethasone and dexamethasone. A special effort was made to adapt the method to neonatal population with respect to choice of saliva as matrix, low sample volumes, selection of analytes and multiplexing. Validation included selectivity, interferences, matrix effects, lower and upper limit of quantification, linearity of calibration, dilution of samples, trueness, within-run and total analytical repeatability, robustness, carry-over, stability and recovery from sample collection swab. Ten microliters were acceptable but 50 µL preferable sample volume, except for 21-deoxycortisol. Cortisol, cortisone, aldosterone, 11-deoxycortisol, deoxycorticosterone, dexamethasone, betamethasone, 17OH-pregnenolone and DHEAS could be determined in as little as 2-5µL saliva. Total analytical variation was <15%, except for 17OH-progesterone, deoxycorticosterone, 17OH-pregnenolone, 21-deoxycortisol, androstenedione, DHEA and betamethasone, that could only be determined semi-quantitatively or qualitatively. The minimum turnaround time was 3–4 h. Recovery from the best performing sample collection swab SalivaBio ranged from 83 to 127%. Aldosterone, cortisone and DHEA were more stable in saliva compared to serum when stored at ambient temperature for one week. Corticosterone and 17OH-progesterone needed immediate freezing. The non-invasiveness, small saliva volume requirement, on-swab stability and analytical performance make the method relevant for both research and diagnostics, above all in the setting of neonatal intensive care unit.

Non-Invasive Malaria Detection in Sub-Saharan Africa Using a DNA-Based Sensor System.

Malaria poses a serious global health problem, with half the world population being at risk. Regular screening is crucial for breaking the transmission cycle and combatting the disease spreading. However, current diagnostic tools relying on blood samples face challenges in many malaria-epidemic areas. In the present study, we demonstrate the detection of the malaria-causing Plasmodium parasite in non-invasive saliva samples (N = 61) from infected individuals by combining a DNA-based Rolling-circle-Enhanced-Enzyme-Activity-Detection (REEAD) sensor system with a chemiluminescence readout that could be detected with an in-house-developed affordable and battery-powered portable reader. We successfully transferred the technology to sub-Saharan Africa, where the malaria burden is high, and demonstrated a proof of concept in a small study (N = 40) showing significant differences (p < 0.00001) between malaria-positive individuals (N = 33) and presumed asymptomatic negative individuals (N = 7) all collected in Gabon. This is the first successful application of the REEAD sensor system for the detection of malaria in saliva in a high-epidemic area and holds promise for the potential future use of REEAD for malaria diagnosis or surveillance based on non-invasive specimens in sub-Saharan Africa.

Inter-year consistencies and discrepancies on intestinal microbiota for overwintering relict gulls: correlations with food composition and implications for environmental adaptation

The gut microbiota of migratory birds is influenced by their food choices, and exploring the potential relationship between diet composition and gut microbiota can help better protect related species. By integrating non-invasive sampling techniques, high-throughput sequencing technology, and microscopic examination technology, this study presents the first evidence on diet composition during overwintering periods as well as the potential relationship between diet composition and gut microbiota in wild relict gulls (Larus relictus). Thirty-five fecal samples from two consecutive overwintering periods (2021 and 2022 overwintering periods) in Tianjin coastal wetland were used to investigate inter-year consistencies and discrepancies on diet composition and gut microbiota in wild Larus relictus. It was found that the common dominant phyla of both 2021 and 2022 group included Firmicutes, Proteobacteria, Chloroflexi and Actinobacteriota. The common dominant genera were Catellicoccus and Ilumatobacter. The diversity of gut microbiome in 2022 group was higher, while the richness was not significantly different. Based on the high-throughput sequencing technology of 18S rDNA, the study found that the dominant classes within the diet components of Larus relictus included Polychaeta, Bivalvia, Malacostraca, Gastropoda, unclassified_p__Dinoflagellata, Dinophyceae, and Ostracoda. Among them, Bivalvia, Malacostraca, and Gastropoda were also found with microscopic examination technology from the same samples. The abundance of Fusobacteriota and Cetobacterium were positively correlated with the abundance of Bivalvia and Malacostraca; while the abundance of Psychrobacter and Breznakia were negatively correlated with the abundance of Malacostraca and Gastropoda. Findings from this study could provide scientific references for health monitoring and conservation of relict gulls.

Molecular Method Based on Hydrolysis Probe Assays to Identify the Sex Chromosomes of Iberian Desman (Galemys pyrenaicus) Using Non-Invasive Sampling.

Desmans belong to the subfamily Desmaninae, which are members of the family Talpidae. Desmans and moles show limited sexual dimorphism, making unclear sex discrimination by phenotypic assessment. The Iberian desman (Galemys pyrenaicus) is an endangered species with a severe population decline. Knowledge of sex and sex ratio is essential for conservation and management. Based on these arguments and although previous conventional PCR studies amplifying DBX/DBY genes were relatively successful in sexing the desman, high-resolution sex-specific PCR has been requested. All these reasons encouraged us to develop new species-specific RT-qPCR assays by TaqMan probes to determine the sex in desman, especially with genetic material from non-invasive samples. Accordingly, efficiency, limit of detection (LOD), specificity, and DNA analysis from faeces were verified. The target genes DBX and DBY were amplified with gDNA from both sexes, with Y-chromosome consistently absent in the female. Despite the modest efficiency, regression analysis (R2 > 0.999) indicated a linear range of the DBX and DBY assays extending from 20 to 0.2 ng/µL DNA. LOD analyses estimated that twice as much gDNA was needed in males as in females for DBX detection. Paradoxically, the Y-chromosome required three times as much gDNA as the X-chromosome using a male sample. Therefore, an unexpected dosage imbalance in the genome in favour of the X chromosome was discussed in light of an apparent multicopy nature of the DBX gene and with a sexing success rate of 49.9% of the non-invasive samples, supporting Fisher's principle for the mammalian XX/XY sex system, as expected.

Advancements in noninvasive koala monitoring through combining Chlamydia detection with a targeted koala genotyping assay

Wildlife diseases are major players in local and global extinctions. Effective disease surveillance, management and conservation strategies require accurate estimates of pathogen prevalence. Yet pathogen detection in wild animals remains challenging. Current gold standards often require samples collected through veterinary examination, but this method is costly, intensive, invasive, and requires specialised staff and equipment. Collection of non-invasive samples, such as scats, is an effective monitoring tool which can be deployed at large scale, as scats contain DNA of both host and pathogens. The koala (Phascolarctos cinereus) is listed as ‘endangered’ under the EPBC Act 1999, with chlamydial disease representing a major threat. Here, we present a new approach that combines restriction-enzyme associated sequencing and targeted-sequence-capture genotyping, namely DArTcap, to detect Chlamydia pecorum in koala scats. We found this method has similar accuracy to current gold standards (qPCR of swab samples), with a sensitivity of 91.7% and a specificity of 100%. This method can be incorporated into existing koala genetic studies using marker panels, where population attributes can be estimated alongside C. pecorum presence, using the same scat samples, with the option to add further markers of interest. Such a one-stop-shop panel would considerably reduce processing times and cost.