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Factors such as host species, phylogeny, diet, and both timing and location of sampling are thought to influence the composition of gut-associated bacteria in insects. In this study, we compared the faecal-associated bacterial taxa for three Coenagrion and one Enallagma damselfly species. We expected high overlap in representation of bacterial taxa due to the shared ecology and diet of these species. Using metabarcoding based on the 16S rRNA gene, we identified 1513 sequence variants, representing distinct bacterial ‘taxa’. Intriguingly, the damselfly species showed somewhat different magnitudes of richness of ZOTUs, ranging from 480 to 914 ZOTUs. In total, 921 (or 60.8% of the 1513) distinct ZOTUs were non-shared, each found only in one species, and then most often in only a single individual. There was a surfeit of these non-shared incidental ZOTUs in the Enallagma species accounting for it showing the highest bacterial richness and accounting for a sample-wide pattern of more single-species ZOTUs than expected, based on comparisons to the null model. Future studies should address the extent to which faecal bacteria represent non-incidental gut bacteria and whether abundant and shared taxa are true gut symbionts. Pictures of odonates adopted from Norske Art databank under Creative Commons License (CC BY 4.0). [ABSTRACT FROM AUTHOR]

Gathering ecological knowledge about species with cryptic behaviour can be challenging and strongly depends on human, financial and logistical resources. Yet, this is essential for the conservation of threatened and/or elusive species and wildlife management. The use of DNA tools is emerging as an efficient solution to overcome these constraints. Here, we developed specific primers and a droplet digital PCR (ddPCR) protocol for the quantitative detection of the endemic amphibian Calotriton asper in freshwater and fish faeces. After successful in silico validation of the developed primers, we tested their validity in the wild. First, using an environmental DNA approach, we confirmed the presence of C. asper in eight out of 13 sites deemed climatically suitable for the species, although it had never been observed there before. Then, we sampled brown trout (Salmo trutta) living in sympatry with C. asper and, through fish faeces analyses, confirmed that brown trout are predators of C. asper. These molecular tools proved efficient for monitoring (i) elusive species such as C. asper in the wild, allowing for better characterization of their current distribution and habitat use, and (ii) predation events by fish, which might be a significant pressure on low‐density and isolated C. asper populations. We provide a valuable, low‐cost and complementary tool for environmental managers and scientists that should help improve the development of fine‐tuned conservation and management plans for C. asper. [ABSTRACT FROM AUTHOR]

Dietary analysis of nocturnal aerial insectivores such as bats is not possible by direct visual observation of predation, therefore diet is traditionally determined by analysing dietary remains in scats. Analysing DNA in scats through metabarcoding can reveal dietary items not preserved in faeces but also resolve dietary preferences to species level. DNA metabarcoding analysis of scats collected from six individual long-tailed bats (Chalinolobus tuberculatus) from Whirinaki Forest Park revealed a predominance of aquatic insects (Ephemeroptera and Trichoptera) and small-bodied moths (Lepidoptera). This contrasts with earlier studies of faecal remains in this species which revealed a strong preference for Dipterans and Coleopterans. The use of DNA metabarcoding may provide a better understanding of diets, as soft-bodied insects can be detected even when not apparent in physical examinations of scat, and thus will allow for more robust comparison of differences in diets between populations and seasons. The application of DNA metabarcoding is a simple but high-resolution tool to allow further study on dietary preference in this endangered species. [ABSTRACT FROM AUTHOR]

DNA metabarcoding is a contemporary technique in diet composition studies and stands to fill key knowledge gaps left by traditional diet analysis methods. For endangered species such as the African wild dog (Lycaon pictus), the fulfilment of these knowledge gaps presents an opportunity for improved management practices and vulnerability assessments. There are an estimated ~600 African wild dogs remaining in South Africa. These dogs are generally understood to prey upon impala (Aepyceros melampus) and other medium‐sized ungulates. Here, we present the first assessment of DNA metabarcoding as a valuable method for diet composition analysis of this highly social carnivore. DNA from faecal samples collected across seven landscape types in the Kruger National Park (KNP) was extracted and used to determine the presence of seven unique prey taxa, including novel species such as the Cape hare (Lepus capensis). Impala was identified as a prey item in all landscape types, complementing the diet preference prediction made with stable isotope analysis using the same samples and existing understanding of wild dog diet. Given recommended improvements, the application of DNA metabarcoding in wild dog diet analysis shows promising prospects for identifying novel prey species and validating previous records of this endangered canids diet. [ABSTRACT FROM AUTHOR]

North American river otters (Lontra canadensis) are semiaquatic mammals distributed throughout the North American continent. They are generalist carnivores with a high potential to affect community structure and dynamics. However, habitat degradation and ecosystem changes have the potential to impact their trophic interactions. Therefore, investigating their diet is critical to understand how anthropogenic disturbances can affect their ecological roles, particularly in the urbanised ecosystems in Florida, where such diet information is currently unavailable. Due to their elusive nature, direct observation of feeding events is a major challenge. Here, we have applied DNA metabarcoding of faecal matter (fDNA), opportunistically collected from river otters killed by road traffic in eastern Florida. Two mitochondrial markers were used to identify prey species' DNA—one for fish and one for vertebrates. A total of 23 unique exact sequence variants (ESVs) were identified, the majority belonging not only to teleosts but also to amphibians, reptiles, birds and mammals. The prey species identified are known dietary resources for river otters and the recovered dietary profiles provided independently by the fish and vertebrate assays were similar. Among the identified teleost prey, three fish species are considered invasive. Here, we show that when sufficient reference sequences are available, fDNA metabarcoding provides high prey species resolution. The collection and metabarcoding of faecal samples is particularly advantageous for scaling diet studies, both geographically and longitudinally, highlighting the potential of fDNA metabarcoding for the investigation of the diet of predators. This can provide foundational information on the organisation of ecological networks, which is vital for implementing successful ecosystem‐based conservation management. [ABSTRACT FROM AUTHOR]

Understanding species coexistence requires information about how species interact and partition their ecological niche. Niches of closely related species tend to be quite similar and thereby, when syntopic, overlap can result in competition when shared resources are limiting. Brocket deer of tropical forests are ecologically similar species due to convergence or relatedness. As a group, what little is known about their ecology suggests spatial and temporal niche separation while dietary differences among species remain largely unknown. The possibility of competition has been suggested, as it is rare for more than two species to co‐occur. If competition is important, then it may limit syntopy of more species and its importance should increase with relatedness between species. Based on analyses of faecal samples, camera traps, and stable isotopes and multidimensional analysis of niche space, we test spatial, temporal and dietary niche differences and compare niche breadth in two syntopic species pairs: Mazama rufa with M. nana (both of subtribe Odocoileina) and M. jucunda (Odocoileina) with Subulo gouazoubira (Blastocerina). In 2007–2008 and 2017–2019, 240 faecal samples and 375 records were collected in the Brazilian Atlantic Forest. Spatial partitioning among both species pairs was found, with S. gouazoubira and M. nana having greater niche breadth, which may permit or facilitate coexistence. Odocoileina tend to be nocturnal, while Blastocerina tend to be diurnal, which suggests that phylogeny may influence species coexistence. Furthermore, the species tended to overlap with respect to diet. These results suggest interference competition avoidance by niche partitioning at the level of habitat and time, allowing coexistence. Thereby, despite the morphological similarities, brocket deer species of evolutionarily convergent subtribes preserve differences in ecological niche dimensions that facilitate the coexistence of their populations. [ABSTRACT FROM AUTHOR]

Context. In south-eastern Australia, the abundance and distribution of non-native sambar deer (Cervus unicolor) has increased dramatically in alpine environments. As a result, significant concern surrounds the potential for the species to impact rare plant species and vegetation communities through browsing. Aims. We aimed to determine the diversity of the plant species eaten by sambar deerin the Alpine National Park and to understand any spatial and temporal variation in deer diets. Methods. We collected 90 sambar deer faecal pellet samples over a 3-month flowering period across two contrasting study sites with differing elevation, vegetation and underlying geology. We performed DNA sequencing using the ITS2 gene region and assigned dietary items to the lowest taxonomic level possible. The frequency of occurrence and sequencing read depth of each dietary item was calculated to investigate the diet of sambar deer at spatial and temporal scales, and dietary preferencing was assessed by comparing the frequency of occurrence of dietary items to the observation records for each dietary item in the study area. Key results. We detected a total of 369 unique plant Operational Taxonomic Units (OTUs) from sambar deer faecal samples, representing 35 families and 80 genera. Considerable variation in the diet was observed over small spatial scales, and evidence of temporal diet variation was noted in one of the study sites. We detected Silky Snow-daisy (Celmisia sericophylla), which is currently listed as critically endangered under the Victorian Flora and Fauna Guarantee Act 1988, and Hawkweed (Pilosella spp.), a highly invasive, non-native taxon that is sparingly established in Alpine ecosystems. Conclusions. Sambar deer displayed an intermediate feeder behaviour in alpine environments, foraging on a variety of forbs and shrubs, but forbs were the dominant dietary items. The spatial variation observed in the diet of sambar deer suggests that individual deer are unlikely to be dispersing widely while foraging. Implications. Our results emphasise the need for careful evaluation of sambar deer impacts within individual sites and at small spatial scales. The detection of species of conservation significance in the diet indicates that the presence of sambar deer should be considered a significantrisk to biodiversity in areas of high conservation value. [ABSTRACT FROM AUTHOR]

The animal gut microbiome has been implicated in a number of key biological processes, ranging from digestion to behaviour, and has also been suggested to facilitate local adaptation. Yet studies in wild animals rarely compare multiple populations that differ ecologically, which is the level at which local adaptation may occur. Further, few studies simultaneously characterize diet and gut microbiome from the same sample, despite their probable interdependence. Here, we investigate the interplay between diet and gut microbiome in three geographically isolated populations of the critically endangered Grauer's gorilla (Gorilla beringei graueri), which we show to be genetically differentiated. We find population‐ and social group‐specific dietary and gut microbial profiles and covariation between diet and gut microbiome, despite the presence of core microbial taxa. There was no detectable effect of age, and only marginal effects of sex and genetic relatedness on the microbiome. Diet differed considerably across populations, with the high‐altitude population consuming a lower diversity of plants compared to low‐altitude populations, consistent with plant availability constraining dietary choices. The observed pattern of covariation between diet and gut microbiome is probably a result of long‐term social and environmental factors. Our study suggests that the gut microbiome is sufficiently plastic to support flexible food selection and hence contribute to local adaptation. [ABSTRACT FROM AUTHOR]

Abstract Background Veterinary diagnostics aid intervention strategies, track zoonoses, and direct selective breeding programs in livestock. In ruminants, gastrointestinal nematode (GIN) parasites are a major cause of production losses, but morphologically similar species limit our understanding of how specific GIN co-infections impact health in resource-limited settings. To estimate the presence and relative abundance of GINs and other helminths at the species level, we sought to develop a low-cost and low-resource molecular toolkit applied to goats from rural Malawi smallholdings. Methods Goats were subjected to health scoring and faecal sampling on smallholdings in Lilongwe district, Malawi. Infection intensities were estimated by faecal nematode egg counts with a faecal subsample desiccated for DNA analysis. Two DNA extraction methods were tested (low-resource magbead kit vs high-resource spin-column kit), with resulting DNA screened by endpoint polymerase chain reaction (PCR), semi-quantitative PCR, quantitative PCR (qPCR), high-resolution melt curve analysis (HRMC), and ‘nemabiome’ internal transcribed spacer 2 (ITS-2) amplicon sequencing. Results Both DNA isolation methods yielded comparable results despite poorer DNA purity and faecal contaminant carryover from the low-resource magbead method. GINs were detected in 100% of samples regardless of infection intensity. Co-infections with GINs and coccidia (Eimeria spp.) were present in most goats, with GIN populations dominated by Haemonchus contortus, Trichostrongylus colubriformis, Trichostrongylus axei, and Oesophagostomum columbianum. Both multiplex PCR and qPCR were highly predictive of GIN species proportions obtained using nemabiome amplicon sequencing; however, HRMC was less reliable than PCR in predicting the presence of particular species. Conclusions These data represent the first ‘nemabiome’ sequencing of GINs from naturally infected smallholder goats in Africa and show the variable nature of GIN co-infections between individual animals. A similar level of granularity was detected by semi-quantitative PCR methods, which provided an accurate summary of species composition. Assessing GIN co-infections is therefore possible using cost-efficient low-resource DNA extraction and PCR approaches that can increase the capacity of molecular resources in areas where sequencing platforms are not available; and also open the door to affordable molecular GIN diagnostics. Given the diverse nature of infections in livestock and wildlife, these approaches have potential for disease surveillance in other areas. Graphical Abstract

Understanding the influence of non‐native herbivores on ecosystems by means of dietary foraging and seed dispersal is important for understanding how non‐native species can alter an invaded landscape, yet requires multiple methodologies. In south‐eastern Australia, introduced sambar deer (Rusa unicolor) are rapidly expanding in range and placing native ecosystems at risk through browsing and as vectors for seed dispersal. We simultaneously investigated sambar deer dietary composition and seed dispersal using DNA sequencing and germination trials, from faecal pellets collected in alpine and wet forest ecosystems. This allowed us to contrast the dietary impacts of introduced sambar deer in different environments, and to explore the potential for habitat‐specific variation in diet. DNA sequencing of the trnL, ITS2 and rbcL gene regions revealed a diverse plant species dietary composition comprising 1003 operational taxonomic units (OTUs). Sambar deer exhibited intermediate feeder behaviours dominated by forbs in alpine and shrubs in wet forest ecosystems. A large proportion of plant OTUs were considered likely to be native, however, the proportion of exotic species in the diet in both ecosystems was greater than would be expected based on the proportion of exotic species in each of the two landscapes. Seed germination trials indicated that sambar deer can disperse a substantial number of native and exotic species in both alpine and wet forest ecosystems. In alpine ecosystems, an individual sambar deer was estimated to disperse on average 816 (±193) seeds per day during the study period, of which 652 (±176) were exotic. Synthesis and applications. Our results suggest that native plant species comprise the majority of sambar deer diets in Australian ecosystems and that the introduced species is dispersing both native and exotic plant species via endozoochory. However, exotic species seedling germination numbers were significantly higher in alpine ecosystems, and given the large daily movements of sambar deer, represents a significant vector for the spread of exotic plant species. Management of native plant species and vegetation communities of conservation significance, or at risk to sambar deer browsing is of high priority, through either the removal of sambar deer or implementation of exclusion‐based methods. [ABSTRACT FROM AUTHOR]

Context: Climate change is considered an important factor affecting the distribution and genetic diversity of species. While many studies have described the influence of climate change on population structure at various scales, little is known about the genetic consequences of a changing climate on endemic species. Objectives: To assess possible changes in the distribution and genetic structure of the endangered Nilgiri tahr (Nilgiritragus hylocrius), which is endemic to the Western Ghats in India, under climate change and human disturbances. Methods: We integrated tahr occurrence and nuclear DNA data with environmental geo-datasets to project the response of tahr populations to future climate change with respect to its distribution, genetic diversity and population structure. We screened the environmental variables using MaxEnt to identify a manageable set of predictors to be used in an ensemble approach, based on ten species distribution modelling techniques, to quantify the current tahr distribution. We then projected the distribution and genetic structure under two climate change scenarios. Results: We found that suitable habitat for tahr (9,605 km2) is determined predominantly by a combination of climatic, human disturbance and topographic factors that result in a highly fragmented habitat throughout its distribution range in the Western Ghats. Under the severe high emissions RCP8.5 scenario tahr populations may lose more than half of their available habitat (55.5%) by 2070. Application of spatial Bayesian clustering suggests that their current genetic structure comprise four genetic clusters, with three of them reflecting a clear geographic structure. However, under climate change, two of these clusters may be lost, and in the future a homogenization of the genetic background of the remaining populations may arise due to prevalence of one gene pool cluster in the remaining populations. Conclusions: Our interdisciplinary approach that combines niche modelling and genetic data identified the climate refugia (i.e., the remaining stable habitats that overlap with the current suitable areas), where the tahr populations would be restricted to small, isolated and fragmented areas. Essential factors to avert local extinctions of vulnerable tahr populations are a reduction of human disturbances, dispersal of tahr between fragmented populations, and the availability of corridors. [ABSTRACT FROM AUTHOR]

Background: Veterinary diagnostics aid intervention strategies, track zoonoses, and direct selective breeding programs in livestock. In ruminants, gastrointestinal nematode (GIN) parasites are a major cause of production losses, but morphologically similar species limit our understanding of how specific GIN co-infections impact health in resource-limited settings. To estimate the presence and relative abundance of GINs and other helminths at the species level, we sought to develop a low-cost and low-resource molecular toolkit applied to goats from rural Malawi smallholdings. Methods: Goats were subjected to health scoring and faecal sampling on smallholdings in Lilongwe district, Malawi. Infection intensities were estimated by faecal nematode egg counts with a faecal subsample desiccated for DNA analysis. Two DNA extraction methods were tested (low-resource magbead kit vs high-resource spin-column kit), with resulting DNA screened by endpoint polymerase chain reaction (PCR), semi-quantitative PCR, quantitative PCR (qPCR), high-resolution melt curve analysis (HRMC), and 'nemabiome' internal transcribed spacer 2 (ITS-2) amplicon sequencing. Results: Both DNA isolation methods yielded comparable results despite poorer DNA purity and faecal contaminant carryover from the low-resource magbead method. GINs were detected in 100% of samples regardless of infection intensity. Co-infections with GINs and coccidia (Eimeria spp.) were present in most goats, with GIN populations dominated by Haemonchus contortus, Trichostrongylus colubriformis, Trichostrongylus axei, and Oesophagostomum columbianum. Both multiplex PCR and qPCR were highly predictive of GIN species proportions obtained using nemabiome amplicon sequencing; however, HRMC was less reliable than PCR in predicting the presence of particular species. Conclusions: These data represent the first 'nemabiome' sequencing of GINs from naturally infected smallholder goats in Africa and show the variable nature of GIN co-infections between individual animals. A similar level of granularity was detected by semi-quantitative PCR methods, which provided an accurate summary of species composition. Assessing GIN co-infections is therefore possible using cost-efficient low-resource DNA extraction and PCR approaches that can increase the capacity of molecular resources in areas where sequencing platforms are not available; and also open the door to affordable molecular GIN diagnostics. Given the diverse nature of infections in livestock and wildlife, these approaches have potential for disease surveillance in other areas. [ABSTRACT FROM AUTHOR]

Animal diet and health influence fitness, making individual variation in these markers essential for understanding how individuals and populations respond to their environments. Faecal and hair samples provide a record of this information and can be non-invasively collected from animals in the field. However, physiology, diet, and susceptibility to parasitic infections vary within individuals, requiring repeated samples from individuals. We developed a technique using biotelemetry data for individual identification of non-invasive faecal material and hair sampled from female elk (Cervus canadensis). We non-invasively collected individually genotyped faecal and hair samples from resting sites, then compared the accuracy of supervised machine learning models to predict the individual identities of the samples. We found both the tightness of global positioning system point clusters and activity level surrounding the sample allowed us to positively identify samples belonging to specific individuals with 77% accuracy. Our approach can be applied to other populations for which biotelemetry data are available and is potentially adaptable for other species. Furthermore, application of our approach will reduce the need for individual identification of non-invasive samples using genetic analysis, which is costly and prone to low recovery success. Increased access to physiological, dietary, and health information obtainable from individual non-invasive samples will strengthen our understanding of animal responses to their environments. [ABSTRACT FROM AUTHOR]

Antibiotic resistance is a major public health concern worldwide. The gut microbiota harbours multiple antibiotic resistant genes (ARGs) that contribute to the existing and future microbial population in a community or ecosystem. This study aimed to investigate the prevalence of 35 antibiotic resistance genes (ARGs) in the gut microbiota of the tribal people of Nabarangpur, Odisha, India. A total of 83 faecal samples were collected from three different tribes (Bhatra, Gond, and Paraja). Total faecal DNA was extracted, and the simplex polymerase chain reaction was performed to detect selected ARGs. Further analysis was done to estimate the incidence of these ARGs across these tribes based on alcohol consumption habits. We identified a higher prevalence of tetracycline resistance genes (tetW, tetQ and tetM) in the gut microbiota among three populations. Furthermore, a significant (P = 0·024) difference in ARG prevalence against vancomycin in individuals with and without alcohol consumption habits was noticed. The overall distribution of ARGs among the three major tribes of this location was found to be very similar. Together, irrespective of the tribes, the people of this location have gut microbiota harbouring different kinds of ARGs and tetracycline‐resistant genes are the most commonly found ARGs. [ABSTRACT FROM AUTHOR]

Conservation genomics is an important tool to manage threatened species under current biodiversity loss. Recent advances in sequencing technology mean that we can now use whole genomes to investigate demographic history, local adaptation, inbreeding, and more in unprecedented detail. However, for many rare and elusive species only non-invasive samples such as faeces can be obtained, making it difficult to take advantage of whole genome data. We present a method to extract DNA from the mucosal layer of faecal samples to re-sequence high coverage whole genomes using standard laboratory techniques. We use wild collected faecal pellets collected from caribou (Rangifer tarandus), a species undergoing declines in many parts of its range in Canada and subject to comprehensive conservation and population monitoring measures. We compare four faecal genomes to two tissue genomes sequenced in the same run. Quality metrics were similar between faecal and tissue samples with the main difference being the alignment success of raw reads to the reference genome due to differences in low quality and endogenous DNA content, affecting overall coverage. One of our faecal genomes was only re-sequenced at low coverage (1.6 ×), however the other three obtained between 7 and 15 ×, compared to 19 and 25 × for the tissue samples. We successfully re-sequenced high-quality whole genomes from faecal DNA and are one of the first to obtain genome-wide data from wildlife faecal DNA in a non-primate species. Our work represents an important advancement for non-invasive conservation genomics. [ABSTRACT FROM AUTHOR]

In the European Union, all bat species are strictly protected and member states must ensure their conservation. However, if populations are genetically structured, conservation units that correspond to whole countries may be too large, putting small populations with specific conservation requirements at risk. Geoffroy's bat (Myotis emarginatus) has undergone well-documented declines at its north-western European range edge between the 1960 and 1990s and is considered to be negatively affected by habitat fragmentation. Here we analysed the species' genetic population structure and diversity to identify subpopulations with reduced genetic diversity and to scientifically inform conservation management. We generated 811 microsatellite-based genetic profiles obtained from 42 European nursery colonies and analysed a total of 932 sequences of the hypervariable region II of the mitochondrial control region sampled from across Europe. While two geographically widespread genetic populations were inferred to be present in north-western Europe, both nuclear and mitochondrial genetic diversity were lowest in the areas that had experienced a decline during the last century. A microsatellite-based analysis of demographic history did not permit, however, to unequivocally link that reduced genetic diversity to the population contraction event. Given the large geographic extent of the genetic populations, preserving the connectivity of mating sites requires concerted conservation efforts across multiple political jurisdictions. Genetic monitoring ought to be done on a regular basis to ensure that large-scale connectivity is maintained and further loss of genetic diversity is prevented. [ABSTRACT FROM AUTHOR]

The hypothesis that predation is the cause of the regular small rodent population oscillations observed in boreal and Arctic regions has long been debated. Within this hypothesis, it is proposed that the most likely predators to cause these destabilizing effects are sedentary specialists, with small mustelids being possible candidates. One such case would be the highly specialized least weasel (Mustela nivalis) driving the Norwegian lemming (Lemmus lemmus) cycle in Fennoscandia. These predators are often elusive and therefore distribution data can only be based on field signs, which is problematic when various mustelid species are sympatric, such as weasels and stoats (Mustela erminea). Here we present the results of using mustelid faeces in predated winter lemming nests to correctly identify the predator and thus discern which species exerts the strongest predation pressure on lemming winter populations. Samples were obtained during different phases in the lemming cycle, spanning 6 years, to account for different prey densities. Faecal mitochondrial DNA extraction and amplification of a 400-bp fragment was successful in 92/114 samples (81%); the sequencing of these samples proved that most predation occurrences (83%) could be attributed to the least weasel. These findings support the hypothesis that weasels in particular show high specificity in predation and could therefore be candidates to driving the lemming cycle in this area. We conclude that DNA analysis of faecal remains around predated nests can be a useful tool for further investigations concerning predator–prey interactions in the tundra. [ABSTRACT FROM AUTHOR]

American mink Neovison vison is a semi-aquatic mustelid that has been introduced throughout Europe. Several attempts to eradicate this invasive species have been carried out since its spread. In the Iberian Peninsula, an eradication program has started since 2001, as part of the national action plan for the conservation of European mink Mustela lutreola. Proper detection is crucial for management, both for monitoring variation in mink abundance and prevent the recolonization of cleared areas. Mink scats can be confounded with those of several same-sized mustelids, and with the spraints of Eurasian otter Lutra lutra, which also occur along watercourses. However, these difficulties can be overcome by non-invasive genetic methods. With the aim of providing a cost-effective and reliable technique for differentiating mink scats from those of the largest possible number of species with morphologically similar scats, we tested the effectiveness of an already available faecal mtDNA-based PCR–RFLP method for distinguishing mink- and otter faeces. We then applied the method for analysing scats collected and identified in the field by trained surveyors for monitoring the success of mink control operations. The PCR–RFLP method clearly differentiated mink scats from those of otters, as restriction enzymes produced well-defined different patterns. Eighty-five percent of 94 faecal samples could be assigned to a mustelid species. Only three out of seven putative mink samples were confirmed, suggesting that the accuracy of the visual identification of scats is questionable and genetic methods are needed for the reliable assessment of the success of eradication programs. [ABSTRACT FROM AUTHOR]

Noninvasive genotyping methods have become key elements of wildlife research over the last two decades, but their widespread adoption is limited by high costs, low success rates and high error rates. The information lost when genotyping success is low may lead to decreased precision in animal population densities, which could misguide conservation and management actions. Single nucleotide polymorphisms (SNPs) provide a promising alternative to traditionally used microsatellites as SNPs allow amplification of shorter DNA fragments, are less prone to genotyping errors and produce results that are easily shared among laboratories. Here, we outline a detailed protocol for cost‐effective and accurate noninvasive SNP genotyping using multiplexed amplicon sequencing optimized for degraded DNA. We validated this method for individual identification by genotyping 216 scats, 18 hairs and 15 tissues from coyotes (Canis latrans) using 26 SNPs. Our genotyping success rate for scat samples was 93%, and 100% for hair and tissue, representing a substantial increase compared to previous microsatellite‐based studies while remaining at a low cost of under $5 per PCR replicate (excluding labour). The accuracy of the genotypes was further corroborated in that genotypes from scats matching known, GPS‐collared coyotes were always located within the territory of the known individual. We also show that different levels of multiplexing produced similar results, but that PCR product cleanup strategies can have substantial effects on genotyping success. By making noninvasive genotyping more affordable, accurate and efficient, this research may allow for a substantial increase in the use of noninvasive methods to monitor and conserve free‐ranging wildlife populations. [ABSTRACT FROM AUTHOR]

The Sage Grouse Centrocercus urophasianus is a species of conservation concern throughout its range in western North America. Since the 1950s, the high count of males at leks has been used as an index for monitoring populations. However, the relationship between this lek‐count index and population size is unclear, and its reliability for assessing population trends has been questioned. We used non‐invasive genetic mark‐recapture analysis of faecal and feather samples to estimate pre‐breeding population size for the Parachute‐Piceance‐Roan, a small, geographically isolated population of Sage Grouse in western Colorado, during two consecutive winters from 2012 to 2014. We estimated total pre‐breeding population size as 335 (95% confidence interval (CI): 287–382) in the first winter and 745 (95% CI: 627–864) in the second, an approximate doubling in abundance between years. Although we also observed a large increase in the spring lek‐count index between those years, high male count data poorly represented mark‐recapture estimates of male abundance in each year. Our data suggest that lek counts are useful for detecting the direction and magnitude of large changes in Sage Grouse abundance over time but they may not reliably reflect small changes in abundance that may be relevant to small populations of conservation concern. [ABSTRACT FROM AUTHOR]

Pinna nobilis (Linnaeus, 1758) is the largest bivalve endemic to the Mediterranean. It is distributed in a wide range of coastal environments, including estuaries. Pinna nobilis has recently become a critically endangered species (with almost 100% mortality) along the entire Spanish Mediterranean coast. This may be due to coinfections caused by Haplosporidium pinnae and bacterial pathogens such as Mycobacterium spp. We extensively sampled P. nobilis from Mar Menor lagoon (SE Spain), a site where individuals still survive. Using conventional PCR, we found Haplosporidium spp. in 7.1% of mantle and faecal DNA samples in different individuals of P. nobilis. We identified and quantified Haplosporidium pinnae in P. nobilis using Sanger sequencing and qPCR. Faecal H. pinnae detection is non-invasive, unlike biopsies. Therefore, this non-lethal and non-invasive sampling method could contribute to the welfare of living populations, particularly in eutrophic environments, where they are prone to septicaemia. The use of faecal DNA analysis could be a major advance in epidemiology and recovery assessment studies of P. nobilis.

Outbreaks of the processionary moth Thaumetopoea pityocampa (Denis & Schiffermüller, 1775), a forest pest from the Palearctic, are thought to induce a behavioral response of bats, but up to now the moth has been seldom identified as bats’ prey. Studies on bat diets suggest moths with cyclical outbreaks attract a wide array of bat species from different foraging guilds. We test whether bats feed upon T. pityocampa in the Iberian Peninsula irrespective of the predator’s ecological and morphological features. We found that seven out of ten bat species belonging to different foraging guilds contained T. pityocampa DNA in their faeces and no difference was found in the foraging frequency among foraging guilds. A different size of the typical prey or the lack of fondness for moths can explain the absence of the pest in some bat species. Moreover, the intraspecific foraging frequency of T. pityocampa also changed with the sampling site likely representing differential availability of the moth. Lack of information on flight and dispersal behavior or the tympanate nature of the adult moth complicates understanding how different foraging guilds of bats prey upon the same prey. Our data suggests that T. pityocampa is a remarkable food source for many thousands of individual bats in the study area and we anticipate that more bats besides the species studied here are consuming this moth.

Outbreaks of the processionary moth Thaumetopoea pityocampa (Denis & Schiffermüller, 1775), a forest pest from the Palearctic, are thought to induce a behavioral response of bats, but up to now the moth has been seldom identified as bats’ prey. Studies on bat diets suggest moths with cyclical outbreaks attract a wide array of bat species from different foraging guilds. We test whether bats feed upon T. pityocampa in the Iberian Peninsula irrespective of the predator’s ecological and morphological features. We found that seven out of ten bat species belonging to different foraging guilds contained T. pityocampa DNA in their faeces and no difference was found in the foraging frequency among foraging guilds. A different size of the typical prey or the lack of fondness for moths can explain the absence of the pest in some bat species. Moreover, the intraspecific foraging frequency of T. pityocampa also changed with the sampling site likely representing differential availability of the moth. Lack of information on flight and dispersal behavior or the tympanate nature of the adult moth complicates understanding how different foraging guilds of bats prey upon the same prey. Our data suggests that T. pityocampa is a remarkable food source for many thousands of individual bats in the study area and we anticipate that more bats besides the species studied here are consuming this moth. [ABSTRACT FROM AUTHOR]

Abstract Background Bats are among the most eco-epidemiologically important mammals, owing to their presence in human settlements and animal keeping facilities. Roosting of bats in buildings may bring pathogens of veterinary-medical importance into the environment of domestic animals and humans. In this context bats have long been studied as carriers of various pathogen groups. However, despite their close association with arthropods (both in their food and as their ectoparasites), only a few molecular surveys have been published on their role as carriers of vector-borne protozoa. The aim of the present study was to compensate for this scarcity of information. Findings Altogether 221 (mostly individual) bat faecal samples were collected in Hungary and the Netherlands. The DNA was extracted, and analysed with PCR and sequencing for the presence of arthropod-borne apicomplexan protozoa. Babesia canis canis (with 99-100 % homology) was identified in five samples, all from Hungary. Because it was excluded with an Ixodidae-specific PCR that the relevant bats consumed ticks, these sequences derive either from insect carriers of Ba. canis, or from the infection of bats. In one bat faecal sample from the Netherlands a sequence having the highest (99 %) homology to Besnoitia besnoiti was amplified. Conclusions These findings suggest that some aspects of the epidemiology of canine babesiosis are underestimated or unknown, i.e. the potential role of insect-borne mechanical transmission and/or the susceptibility of bats to Ba. canis. In addition, bats need to be added to future studies in the quest for the final host of Be. besnoiti.

Asiatic wild dog (Cuon alpinus) or dhole is an endangered canid with fragmented distribution in South, East and Southeast Asia. The remaining populations of this species face severe conservation challenges from anthropogenic interventions, but only limited information is available at population and demography levels. Here, we describe the novel molecular approaches for unambiguous species and sex identification from noninvasively collected dhole samples. We successfully tested these assays on 130 field-collected dhole faecal samples from the Vidarbha part of central Indian tiger landscape that resulted in 97 and 77% success rates in species and sex identification, respectively. These accurate, fast and cheap molecular approaches prove the efficacy of such methods in gathering ecological data from this elusive, endangered canid and show their application in generating population level information from noninvasive samples. [ABSTRACT FROM AUTHOR]

The DNA detection of wildlife from environmental samples has the potential to contribute significantly to wildlife management and ecological research. In terrestrial ecosystems, much work has focused on the identification of mammal predators from faecal (scat) samples. However, the relatively high time and financial costs of collecting and analysing scat DNA remain barriers to more widespread implementation of such DNA detection methods, especially for high-throughput surveys. Here, we evaluate methods used for DNA extraction from scats, as applied to detection of the Australian red fox, an introduced predator. We compare the relative costs of two approaches: the method previously used to screen thousands of scat samples in surveys over several years, and a modified version which involves swabbing scats at the time of collection and using a mechanised liquid handling platform to extract DNA from the swabs. We demonstrate that mechanised DNA extraction from swabs is more efficient than manual DNA extraction from whole scats, in terms of both time and resources. This provides a means for rapid, high-throughput screening of scats for the presence of mammal predators, enabling time-effective management responses to non-invasive surveys. [ABSTRACT FROM AUTHOR]

Abstract: Light pollution has been increasing around the globe and threatens to disturb natural rhythms of wildlife species. Artificial light impacts the behaviour of insectivorous bats in numerous ways, including foraging behaviour, which may in turn lead to altered prey selection. In a manipulative field experiment, we collected faecal samples from six species of insectivorous bats in naturally dark and artificially lit conditions, and identified prey items using molecular methods to investigate effects of light pollution on prey selection. Proportional differences in identified prey were not consistent and appeared to be species specific. Red bats, little brown bats and grey bats exhibited expected increases in moths at lit sites. Beetle‐specialist big brown bats had a sizeable increase in beetle consumption around lights, while tri‐coloured bats and evening bats showed little change in moth consumption between experimental conditions. Dietary overlap was high between experimental conditions within each species, and dietary breadth only changed significantly between experimental conditions in one species, the little brown bat. Policy implications. Our results, building on others, demonstrate that bat–insect interactions may be more nuanced than the common assertion that moth consumption increases around lights. They highlight the need for a greater mechanistic understanding of bat–light interactions to predict which species will be most affected by light pollution. Given differences in bat and insect communities, we advocate biologists, land stewards and civil planners work collaboratively to determine lighting solutions that minimize changes in foraging behaviour of species in the local bat community. Such efforts may allow stakeholders to more effectively craft management strategies to minimize unnatural shifts in prey selection caused by artificial lights. [ABSTRACT FROM AUTHOR]

Noninvasive genetic sampling allows studying wildlife without having to catch, handle or even observe individuals. In this study, factors which may affect the quality of noninvasive samples of Asiatic black bear (Ursus thibetanus) in the subtropical areas were identified. We collected hair and faecal samples from captive Asiatic black bears and quantitatively evaluated the effects of hair age (from fresh to 60 days), faeces age (from fresh to 14 days), faeces sampling locations (i.e. sample collected from either the surface, inside or a mixture of both the surface and inside of faeces), and faeces preservation methods (frozen or kept at room temperature in 95% ethanol) on amplification success rates of mitochondrial DNA fragments of different sizes (450bp, 900bp, and 1600bp). The results showed that the amplification success rates decreased with sample age and amplicon size in both hair and faecal DNA. In subtropical environment, there was no significant difference between amplification success of DNA extracted from fresh and 7-day-old samples of either the hair or faeces. The amplification success rates were not influenced by sampling location of faeces. For faeces preserved in 95% ethanol, the amplification success appeared unaffected by frozen at -20 °C or kept at room temperature in shorter mtDNA fragments, but was significantly influenced when amplicon size was 1600bp. The results of this study will reinforce the optimization of noninvasive sampling approaches in Asiatic black bear research, especially in the subtropics. [ABSTRACT FROM AUTHOR]

Microsatellite markers have played a major role in ecological, evolutionary and conservation research during the past 20 years. However, technical constrains related to the use of capillary electrophoresis and a recent technological revolution that has impacted other marker types have brought to question the continued use of microsatellites for certain applications. We present a study for improving microsatellite genotyping in ecology using high-throughput sequencing ( HTS). This approach entails selection of short markers suitable for HTS, sequencing PCR-amplified microsatellites on an Illumina platform and bioinformatic treatment of the sequence data to obtain multilocus genotypes. It takes advantage of the fact that HTS gives direct access to microsatellite sequences, allowing unambiguous allele identification and enabling automation of the genotyping process through bioinformatics. In addition, the massive parallel sequencing abilities expand the information content of single experimental runs far beyond capillary electrophoresis. We illustrated the method by genotyping brown bear samples amplified with a multiplex PCR of 13 new microsatellite markers and a sex marker. HTS of microsatellites provided accurate individual identification and parentage assignment and resulted in a significant improvement of genotyping success (84%) of faecal degraded DNA and costs reduction compared to capillary electrophoresis. The HTS approach holds vast potential for improving success, accuracy, efficiency and standardization of microsatellite genotyping in ecological and conservation applications, especially those that rely on profiling of low-quantity/quality DNA and on the construction of genetic databases. We discuss and give perspectives for the implementation of the method in the light of the challenges encountered in wildlife studies. [ABSTRACT FROM AUTHOR]

Aim: To study the genome profile of Chital deer (A.axis) by microsatellite markers using faecal DNA & to assess its usefulness as non invasive method of genotyping. Materials and Methods: DNA was isolated from 44 animals from faeces and blood, out of which 26 (3 from blood and 23 from faeces) were subjected for genotyping. The PCR products of all the ten microsatellite loci were subjected to 6 % Urea-PAGE to differentiate allelic size. Result: Total 57 alleles and 49 genotypes were found with their frequencies ranging from 0.022 to 0.457 and 0.043 to 0.696 respectively. The observed number of alleles ranged from 4 -8, (Av. = 5.7 per locus). The PIC and Shannon's information index values for all loci ranged between 0.6645 to 0.8270 (Av. = 0.7442 per locus) and 1.1870 to 1.8852 (Av. = 1.5122 per locus) respectively. Conclusion: Faecal DNA can be effectively used in genotyping wild animals, however the method and time of collection & storage as well as the type of faecal material largely influences the quality & quantity of DNA. [Vet World 2013; 6(2.000): 83-86]

Disagreement exists within the scientific community with regards to the level of competition for feed between sheep and kangaroos in the Australian rangelands. The greatest challenge to solving this debate is finding effective means of determining the composition of the diets of these potential grazing competitors. An option is to adopt a non-invasive approach that combines faecal collection and molecular techniques that focus on faecal DNA as the primary source of dietary information. As proof-of-concept, we show that a DNA reference data bank on plant species can be established. This DNA reference data bank was then used as a library to identify plant species in kangaroo faeces collected in the southern rangelands of Western Australia. To enhance the method development and to begin the investigation of competitive grazing between sheep and kangaroos, 16 plant species known to be palatable to sheep were initially targeted for collection. To ensure that only plant sequences were studied, PCR amplification was performed using a universal primer pair previously shown to be specific to the chloroplast transfer RNA leucine (trnL) UAA gene intron. Overall, genus-specific, single and differently sized amplicons were reliably and reproducibly generated; enabling the differentiation of reference plants by PCR product length heterogeneity. However, there were a few plants that could not be clearly differentiated on the basis of size alone. This prompted the adoption of a post-PCR step that enabled further differentiation according to base sequence variation. Restriction endonucleases make sequence-specific cleavages on DNA to produce discrete and reproducible fragments having unique sizes and base compositions. Their availability, affordability and simplicity-of-use put restriction enzyme sequence (RES) profiling as a logical post-PCR step for confirming plant species identity. We demonstrate that PCR–RES profiling of plant and faecal matter is useful for the identification of plants included in the diet of kangaroos. The limitations, potential and the opportunities created for researchers interested in investigating the diet of competing herbivores in the rangelands are discussed.

Blood-feeding is one of the most specialized foraging habits, as it demands extreme morphological, physiological, and behavioral adaptations. Three species of vampire bats ( Desmodus rotundus, Diaemus youngi, and Diphylla ecaudata) rely on blood as their only food. The first two are considered less specialized, whereas D. ecaudata is frequently pointed out as a bird-specialist. We assessed what prey D. ecaudata consumes in the Caatinga dry forests of northeastern Brazil, a highly modified biome. How the species would behave in a situation of scarcity of wild birds and increase in the availability of domestic animals? Could Diphylla have been induced to include also mammals in its diet? Using PCR-amplification of DNA fragments in the feces of D. ecaudata, we detected the regular consumption of chicken blood and human blood - a novel prey for this species. Our results suggest that the diet of D. ecaudata is more flexible than expected. The record of humans as prey and the absence of blood from native species may reflect a low availability of wild birds in the study site, reinforcing the impact of human activities on local ecological processes. This also opens a range of research possibilities on vampire bats in the Caatinga, both on the species' biology and the consequences for public health, considering the potential increase in the transmission of rabies in the region. [ABSTRACT FROM AUTHOR]

Amplicon sequencing on a High Throughput Sequencing platform (custom barcoding) was used to detect and characterise myxosporean communities in environmental DNA samples from marine and freshwater environments and in faeces of animals that may serve as hosts or whose prey may host myxosporean infections. A diversity of myxozoans in filtered water samples and in faeces of piscivores (otters and great cormorants) was detected, demonstrating the suitability of lineage-specific amplicons for characterising otherwise difficult to sample parasite communities. The importance of using this approach was highlighted by the lack of myxosporean detection using commonly employed, broadly targeted eukaryotic primers. These results suggest that, despite being frequently present in eDNA samples, myxozoans have been generally overlooked in “eukaryote-wide” surveys. Lineage-specific primers, in contrast, detected 107 operational taxonomic units that were assigned to both the “freshwater” and “marine” myxosporean lineages. Only 7% of these OTUs clustered with sequences in GenBank, providing evidence for substantial undescribed myxosporean diversity. Many new operational taxonomic units, including those found in otter faeces, clustered with a clade of myxosporeans previously characterised by sequences from invertebrate hosts and water samples only. Because myxozoan species identification is heavily reliant on molecular signatures, lineage-specific amplicon sequencing offers an effective and non-destructive means of improving our knowledge of myxozoan diversity. In addition, the analysis of myxozoan DNA in faeces of piscivores offers a potentially efficient method of sampling for diversity and revealing life cycles as piscivore activities may integrate myxozoan infections in fish over relatively broad spatial scales. [ABSTRACT FROM AUTHOR]

Background: Bats are among the most eco-epidemiologically important mammals, owing to their presence in human settlements and animal keeping facilities. Roosting of bats in buildings may bring pathogens of veterinary-medical importance into the environment of domestic animals and humans. In this context bats have long been studied as carriers of various pathogen groups. However, despite their close association with arthropods (both in their food and as their ectoparasites), only a few molecular surveys have been published on their role as carriers of vector-borne protozoa. The aim of the present study was to compensate for this scarcity of information. Findings: Altogether 221 (mostly individual) bat faecal samples were collected in Hungary and the Netherlands. The DNA was extracted, and analysed with PCR and sequencing for the presence of arthropod-borne apicomplexan protozoa. Babesia canis canis (with 99-100 % homology) was identified in five samples, all from Hungary. Because it was excluded with an Ixodidae-specific PCR that the relevant bats consumed ticks, these sequences derive either from insect carriers of Ba. canis, or from the infection of bats. In one bat faecal sample from the Netherlands a sequence having the highest (99 %) homology to Besnoitia besnoiti was amplified. Conclusions: These findings suggest that some aspects of the epidemiology of canine babesiosis are underestimated or unknown, i.e. the potential role of insect-borne mechanical transmission and/or the susceptibility of bats to Ba. canis. In addition, bats need to be added to future studies in the quest for the final host of Be. besnoiti. [ABSTRACT FROM AUTHOR]

Earlier studies on savannah elephants ( Loxodonta africana) investigated the genetic structure of fragmented or isolated populations. Contrastingly, this study aimed to determine if there was genetic evidence for spatial structuring in a continuous elephant population in the Kavango-Zambezi Transfrontier Conservation Area (KAZA-TFCA). We sequenced one mtDNA gene region for 88 individuals and genotyped 100 individuals for ten nuclear microsatellite loci. Bayesian Clustering Algorithms in Geneland identified groups of genetically similar individuals. An analysis of molecular variance determined if these groups (sub-populations) were significantly differentiated. We identified geographic areas with high genetic divergence (genetic barriers) between samples using a GIS landscape genetic toolbox. There were three significantly differentiated mtDNA sub-populations (F = 0.787) and two nDNA sub-populations that were not significantly differentiated (Fst = −0.02; Rst = −0.045), implying obstructed mtDNA, but high nDNA gene flow across the study region. The KAZA-TFCA population has a genetic diversity (mtDNA pairwise number of differences ( π) = 2.59; nDNA mean alleles/locus and H = 7.5, 0.71) higher than other southern African populations, and inter-population movements may be responsible for maintaining this genetic diversity. We discount anthropogenic and geographic barriers as the primary drivers of genetic structuring in the KAZA-TFCA population and suggest that future studies should consider the influence of intrinsic factors (resource dependencies and social variables that limit movement) when investigating the genetic structure of elephant populations. We recommend continued support for conservation initiatives that aim at maintaining and restoring connectivity between populations, which in so doing may ensure inter-population gene flow and uphold the current genetic state of the KAZA-TFCA population. [ABSTRACT FROM AUTHOR]

The location of pine marten records in northern Italy suggests that main rivers may play the role of natural corridors favouring this species' colonisation of cultivated lowlands. We assessed the distribution and habitat use by the pine marten on a 35 km long stretch of the River Ticino. Surveys were carried out between October 2011 and June 2012 along linear transects in a 2 × 2 km grid. Using the variation in marking intensity as an indicator of habitat use, habitat selection was assessed at two landscape levels-at transect-scale by the χ test with Bonferroni's confidence intervals for the proportion of use, and at grid-scale by multiple linear regression. By a polymerase chain reaction-restriction fragment length polymorphism method, 91 faecal samples were assigned to the pine marten. Faeces were mainly located in wooded areas, while fields were avoided. At the grid-scale of analysis, marking intensity was positively related to the mean area of wooded patches and negatively to their mean perimeter-area ratio. This suggests that pine marten relative abundance may partially depend on the degree of fragmentation and structure of residual woods. The survey protocol allowed to assess the probability of detection. Occupancy models outlined that heterogeneity in detection probability may arise as a result of variation in marking intensity, i.e. the number of marking individuals. Our results suggest that the availability of both woodland corridors and wood patches are major factors shaping pine marten distribution in intensively cultivated plains and that non-invasive genetic surveys are a cost-effective method for future studies at a broader scale. [ABSTRACT FROM AUTHOR]

Non-invasive genetic sampling using scats has a well established role in conservation biology, but has rarely been applied to reptiles. Using scats from captive and wild Egernia stokesii (Squamata, Scincidae) we evaluated two storage and six DNA-extraction methods and the reliability of subsequent genotype and sequence data. Accurate genotype and sequence data were obtained from frozen and dried captive lizard scat DNA extracted using a QIAamp® DNA Stool Mini Kit and a modified Gentra® Puregene® method, but success rates were reduced for wild lizard scats. Wild E. stokesii eat more plants than their captive counterparts, possibly resulting in scat DNA extracts containing plant compounds that inhibit PCR-amplifications. Notably, reliable genotypes and sequences were obtained from wild E. stokesii scat DNA extracted using a Qiagen DNeasy® Plant Mini Kit, a method designed to remove plant inhibitory compounds. Results highlight the opportunity for using scat-derived DNA in lizard studies, particularly for species that deposit scats in piles. [ABSTRACT FROM AUTHOR]

Non-invasive genetics is a powerful tool in wildlife research and monitoring, especially when dealing with elusive and rare species such as the Eurasian otter ( Lutra lutra). Nevertheless, otter DNA obtained from scats and anal secretions appears to be exposed to very quick degradation processes, and the success rate in DNA amplification is lower than in other carnivores. We collected 191 samples from April to September 2011 along the river Sangro basin (Italy) which was recently re-colonized by the Eurasian otter. Using two sets of microsatellite loci (six Lut and seven OT loci), we investigated the influence of sample type and age, collection time, storage time, temperature and humidity on genotyping success and amplification success. We also tested the efficacy of different DNA extraction kits and storage buffer mediums. Finally, we compared amplification success rate, allelic dropout and false allele rates for each locus. We obtained a mean amplification success rate of 79.0 % and a genotyping success rate of 35.1 %. Fresh pure jellies yielded the highest amplification success and genotyping rate. Six microsatellite loci should be theoretically sufficient to distinguish the individual unrelated otters (PID = 0.001), while 13 loci were needed to distinguish sibling otters (PID = 0.002) in our population. We identified 11 otters, and molecular sexing ascertained the presence of five males, four females and two uncertain individuals. Generalized linear models highlighted a significant influence of sample type and age, temperature and humidity both on genotyping and amplification success. [ABSTRACT FROM AUTHOR]

Abstract Riassunto Monitoraggio genetico non invasivo della martora (Martes martes) nella pianura padana occidentale: risultati preliminari. Tra il 2003 e il 2004, due esemplari di martora (Martes martes) sono stati investiti da automezzi sulla sponda sinistra del fiume Po (R. N. Garzaia di Valenza, Piemonte). Per accertare la presenza stabile della specie nella Riserva e lungo il tratto vercellese-alessandrino del Parco fluviale del Po, è stato applicato un metodo PCR-RFLP per l’analisi di mtDNA di origine fecale. A partire da gennaio 2007, sono state raccolte 212 feci attribuibili al genere Martes in base a forma e dimensioni. L’analisi di un campione di 117 feci (55%) ha permesso di identificare 10 feci di martora e 15 di faina (M. foina), confermando la presenza della martora nella Riserva e accertandola per la prima volta in un’area collinare in sponda destra del Po (Camino, AL), circa 32 km a monte della Riserva. Resta da valutare se la scarsa resa dell’amplificazione del DNA (21,4%) sia dovuta alla degradazione del materiale genetico o ad un numero elevato di feci di volpe (Vulpes vulpes) erroneamente attribuite a Martes durante la fase di raccolta.

Abstract: The presence of gastrointestinal nematode eggs in faecal samples is diagnostic of infection by these parasites. However, this technique cannot be used to distinguish between species of importance. The faecal culture technique and subsequent microscopic analysis of developed larvae is currently used to determine which parasite species are present in the samples, but these techniques take a week to perform and have inherent limitations. To overcome these parasite detection and identification problems, we have developed a DNA extraction method for sheep faeces, and a quantitative multiplex PCR (qPCR) test which can both enumerate and identify Haemonchus, Trichostrongylus and Teladorsagia. We demonstrate that the technique is sensitive to 10 eggs per gram and that dilution of DNA to 0.1 fold can overcome PCR inhibition issues for samples obtained from the field, while maintaining assay sensitivity. Further development of these tests for commercial use is warranted, given their potential to provide consistently faster and more accurate diagnoses of these parasites using simple sample collection and laboratory methods which can be easily adapted for the detection of a variety of pathogens from the same faecal sample. [Copyright &y& Elsevier]

Abstract: Diagnostic tests used for Johne's disease in sheep either have poor sensitivity and specificity or only detect disease in later stages of infection. Predicting which of the infected sheep are likely to become infectious later in life is currently not feasible and continues to be a major hindrance in disease control. We conducted this longitudinal study to investigate if a suite of diagnostic tests conducted in Mycobacterium avium subspecies paratuberculosis (MAP) exposed lambs at 4 months post infection can accurately predict their clinical status at 12 months post infection. We tracked cellular and humoral responses and quantity of MAP shedding for up to 12 months post challenge in 20 controls and 37 exposed sheep. Infection was defined at necropsy by tissue culture and disease spectrum by lesion type. Data were analysed using univariable and multivariable logistic regression models and a subset of variables from the earliest period post inoculation (4 months) was selected for predicting disease outcomes later on (12 months). Sensitivity and specificity of tests and their combinations in series and parallel were determined. Early elevation in faecal MAP DNA quantity and a lower interferon gamma (IFNγ) response were significantly associated with sheep becoming infectious as well as progressing to severe disease. Conversely, early low faecal MAP DNA and higher interleukin-10 responses were significantly associated with an exposed animal developing protective immunity. Combination of early elevated faecal MAP DNA or lower IFNγ response had the highest sensitivity (75%) and specificity (81%) for identifying sheep that would become infectious. Collectively, these results highlight the potential for combined test interpretation to aid in the early prediction of sheep susceptibility to MAP infection. [Copyright &y& Elsevier]

Non-invasive genetic analysis has been frequently employed to estimate ecological and population parameters for many secretive and/or threatened species. However, Neotropical carnivores have so far been scarcely targeted by such studies. The Neotropical otter ( Lontra longicaudis) is a poorly-known species for which local levels of genetic diversity and demographic parameters are virtually absent. We employed non-invasive sampling and amplification of microsatellite loci to investigate population size and density, spatial organization, and relatedness of a wild Neotropical otter population in an Atlantic forest area in southern Brazil. We directly identified 28 individuals and estimate a rather high population density at the study site. Spatial organization analysis indicated that male cumulative displacement was higher than that of females, with the latter sex showing evidence of philopatric behaviour. Also, the reconstruction of genealogical relationships suggests that spatial organization in this otter appears to be influenced by relatedness. By allowing the testing of specific hypothesis targeting these issues, our results provided important glimpses into the Neotropical otter's population biology. Moreover, the findings of the present study reaffirm the power of non-invasive genetics to investigate the biology of this elusive species, and open up new avenues for ecological and demographic studies of other Neotropical carnivores. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 109, 932-948. [ABSTRACT FROM AUTHOR]

Acquiring reliable estimates for an elusive species' distribution and population size can be problematic. For cryptic species such as the Eurasian otter (Lutra lutra), traditional monitoring approaches rely heavily on identifying field signs that may under or overestimate population sizes. Increasingly, non-invasive genetic sampling is effectively applied to assess the abundance and population structure of otters by genotyping faeces (spraints). Here we present the results of a non-invasive survey conducted in Cork City, Ireland, which aimed to estimate otter population size, sex ratio and genetic diversity. We incorporated a citizen science approach by training members of the public in spraint collection, thus increasing our search effort and sample detection rate. From October 2011 to May 2012, 199 spraints were collected and 187 (94%) were genetically identified as otter. Of these positive otter samples, 13 spraints (7%) yielded genetic information identifying 11 individuals (5 female and 6 male) using nine microsatellite loci. The results indicate that the urban environment does not prevent otters from using the area and we consider the implications based upon contemporary knowledge on otter spatial behaviour. This study demonstrates that non-invasive survey techniques combined with a citizen science approach can effectively reveal otter population parameters and increase urban otter awareness within the community. [ABSTRACT FROM AUTHOR]