Fox (Vulpes vulpes) involvement identified in a series of cat carcass mutilations.

This study was designed to identify the cause of mutilation and death in 32 cats, part of a larger cohort found dead in Greater London, the United Kingdom, between 2016 and 2018. At the time, discussion in the media led to concerns of a human serial cat killer (dubbed The Croydon Cat Killer) pursuing domestic cats, causing a state of disquietude. Given the link between animal abuse and domestic violence, human intervention had to be ruled out. Using a combination of DNA testing, computed tomography imaging, and postmortem examination, no evidence was found to support any human involvement. Instead, a significant association between cat carcass mutilation and the presence of fox DNA was demonstrated. Gross examination identified shared characteristics including the pattern of mutilation, level of limb or vertebral disarticulation, wet fur, wound edges with shortened fur, and smooth or irregular contours, and marks in the skin, muscle, and bone consistent with damage from carnivore teeth. Together these findings supported the theory that the cause of mutilation was postmortem scavenging by red foxes (Vulpes vulpes). The probable cause of death was established in 26/32 (81%) carcasses: 10 were predated, 8 died from cardiorespiratory failure, 6 from blunt force trauma, one from ethylene glycol toxicity, and another from liver failure. In 6 carcasses a cause of death was not established due to autolysis and/or extensive mutilation. In summary, this study highlights the value of a multidisciplinary approach to fully investigate cases of suspected human-inflicted mutilation of animals.

DNA barcoding validates species labelling of certified seafood.

Seafood is one of the most traded food commodities in the world with demand steadily increasing [1]. There is, however, a rising concern over the vulnerability of seafood supply chains to species mislabelling and fraud [1,2]. DNA methods have been widely used to detect species mislabelling and a recent meta-analysis of 4500 seafood product tests from 51 publications found an average of 30 percent were not the species stated on the label or menu [3]. This high rate poses a serious threat to consumer trust, reputations of seafood businesses and the sustainability of fishery resources. Seafood certification schemes may help reduce this problem. Here, we use DNA barcoding [4] to validate the species identity of 1402 certified seafood products derived from 27 species across 18 countries and find that in over 99% of cases species labelling was correct.

An internationally standardized species identification test for use on suspected seized rhinoceros horn in the illegal wildlife trade.

Rhinoceros (rhino) numbers have dwindled substantially over the past century. As a result, three of the five species are now considered to be critically endangered, one species is vulnerable and one species is near-threatened. Poaching has increased dramatically over the past decade due to a growing demand for rhino horn products, primarily in Asia. Improved wildlife forensic techniques, such as validated tests for species identification of seized horns, are critical to aid current enforcement and prosecution efforts and provide a deterrent to future rhino horn trafficking. Here, we present an internationally standardized species identification test based on a 230 base pair cytochrome-b region. This test improves on previous nested PCR protocols and can be used for the discrimination of samples with <20pg of template DNA, thus suitable for DNA extracted from horn products. The assay was designed to amplify water buffalo samples, a common 'rhino horn' substitute, but to exclude human DNA, a common contaminant. Phylogenetic analyses using this partial cytochrome-b region resolved the five extant rhino species. Testing successfully returned a sequence and correct identification for all of the known rhino horn samples and vouchered rhino samples from museum and zoo collections, and provided species level identification for 47 out of 52 unknown samples from seizures. Validation and standardization was carried out across five different laboratories, in four different countries, demonstrating it to be an effective and reproducible test, robust to inter laboratory variation in equipment and consumables (such as PCR reagents). This is one of the first species identification tests to be internationally standardized to produce data for evidential proceedings and the first published validated test for rhinos, one of the flagship species groups of the illegal wildlife trade and for which forensic tools are urgently required. This study serves as a model for how species identification tests should be standardized and disseminated for wildlife forensic testing.

Quantifying fenbendazole and its metabolites in self-medicating wild red grouse Lagopus lagopus scoticus using an HPLC-MS-MS approach.

On red grouse estates in the UK the nematode parasite Trichostrongylus tenuis is often controlled by application of grit medicated with the anthelmintic fenbendazole (FBZ). To date, assessment of the efficacy has been inhibited by the inability to quantify uptake of FBZ by the birds. We have developed a simple and sensitive HPLC-MS-MS method for detecting and quantifying FBZ and its metabolites from a 300 mg sample of red grouse liver. This method could be used to improve the efficacy of medicated grit treatment by allowing the identification of conditions and application methods that optimize the uptake of FBZ. With the necessary modifications, our method will also be applicable to other wildlife species where self-medication is used for parasite control.

Characterising functionally important and ecologically meaningful genetic diversity using a candidate gene approach.

Over the past two decades the fields of molecular ecology and population genetics have been dominated by the use of putatively neutral DNA markers, primarily to resolve spatio-temporal patterns of genetic variation to inform our understanding of population structure, gene flow and pedigree. Recent emphasis in comparative functional genomics, however, has fuelled a resurgence of interest in functionally important genetic variation that underpins phenotypic traits of adaptive or ecological significance. It may prove a major challenge to transfer genomics information from classical model species to examine functional diversity in non-model species in natural populations, but already multiple gene-targeted candidate loci with major effect on phenotype and fitness have been identified. Here we briefly describe some of the research strategies used for isolating and characterising functional genetic diversity at candidate gene-targeted loci, and illustrate the efficacy of some of these approaches using our own studies on red grouse (Lagopus lagopus scoticus). We then review how candidate gene markers have been used to: (1) quantify genetic diversity among populations to identify those depauperate in genetic diversity and requiring specific management action; (2) identify the strength and mode of selection operating on individuals within natural populations; and (3) understand direct mechanistic links between allelic variation at single genes and variance in individual fitness.

Physiological stress mediates the honesty of social signals.

BACKGROUND: Extravagant ornaments used as social signals evolved to advertise their bearers' quality. The Immunocompetence Handicap Hypothesis proposes that testosterone-dependent ornaments reliably signal health and parasite resistance; however, empirical studies have shown mixed support. Alternatively, immune function and parasite resistance may be indirectly or directly related to glucocorticoid stress hormones. We propose that an understanding of the interplay between the individual and its environment, particularly how they cope with stressors, is crucial for understanding the honesty of social signals. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed corticosterone deposited in growing feathers as an integrated measure of hypothalamic-pituitary-adrenal activity in a wild territorial bird, the red grouse Lagopus lagopus scoticus. We manipulated two key, interrelated components, parasites and testosterone, which influence both ornamentation and fitness. Birds were initially purged of parasites, and later challenged with parasites or not, while at the same time being given testosterone or control implants, using a factorial experimental design. At the treatment level, testosterone enhanced ornamentation, while parasites reduced it, but only in males not implanted with testosterone. Among individuals, the degree to which both parasites and testosterone had an effect was strongly dependent on the amount of corticosterone in the feather grown during the experiment. The more stressors birds had experienced (i.e., higher corticosterone), the more parasites developed, and the less testosterone enhanced ornamentation. CONCLUSIONS/SIGNIFICANCE: With this unique focus on the individual, and a novel, integrative, measure of response to stressors, we show that ornamentation is ultimately a product of the cumulative physiological response to environmental challenges. These findings lead toward a more realistic concept of honesty in signaling as well as a broader discussion of the concept of stress.

Honest sexual signalling mediated by parasite and testosterone effects on oxidative balance.

Extravagant ornaments evolved to advertise their bearers' quality, the honesty of the signal being ensured by the cost paid to produce or maintain it. The oxidation handicap hypothesis (OHH) proposes that a main cost of testosterone-dependent ornamentation is oxidative stress, a condition whereby the production of reactive oxygen and nitrogen species (ROS/RNS) overwhelms the capacity of antioxidant defences. ROS/RNS are unstable, very reactive by-products of normal metabolic processes that can cause extensive damage to key biomolecules (cellular proteins, lipids and DNA). Oxidative stress has been implicated in the aetiology of many diseases and could link ornamentation and genetic variation in fitness-related traits. We tested the OHH in a free-living bird, the red grouse. We show that elevated testosterone enhanced ornamentation and increased circulating antioxidant levels, but caused oxidative damage. Males with smaller ornaments suffered more oxidative damage than those with larger ornaments when forced to increase testosterone levels, consistent with a handicap mechanism. Parasites depleted antioxidant defences, caused oxidative damage and reduced ornament expression. Oxidative damage extent and the ability of males to increase antioxidant defences also explained the impacts of testosterone and parasites on ornamentation within treatment groups. Because oxidative stress is intimately linked to immune function, parasite resistance and fitness, it provides a reliable currency in the trade-off between individual health and ornamentation. The costs induced by oxidative stress can apply to a wide range of signals, which are testosterone-dependent or coloured by pigments with antioxidant properties.

Cross-species utility of microsatellite markers in trichostrongyloid nematodes.

The development of microsatellite markers for parasitic nematodes has been hampered by technical difficulties in isolation and PCR amplification. We have investigated the potential for circumventing these problems using microsatellites from 3 trichostrongyloid species on a panel of 7 species. Ten of the 22 PCR primer pairs tested amplified in species other than the target species, usually in closely related species, and 2 new variable loci were discovered in the sheep parasite Trichostrongylus vitrinus. This study provides evidence that cross-species testing of microsatellite primers can be an effective alternative to isolation de novo.

Absence of three known benzimidazole resistance mutations in Trichostrongylus tenuis, a nematode parasite of avian hosts.

Benzimidazole (BZ) resistance is widespread in nematode parasites of livestock, but very little is known about the levels of BZ resistance in parasites with avian hosts. We investigated BZ resistance in Trichostrongylus tenuis, a nematode parasite of red grouse, Lagopus lagopus scotica. BZ anthelmintics had been in use in this system for up to 15 years, yet existing phenotypic evidence for resistance was inconclusive. We screened 1530 individuals from 14 populations at the principal beta-tubulin locus for BZ resistance (isotype 1, residue 200) and 940 of these at two further resistance sites (isotype 1, residue 167; isotype 2, residue 200). No BZ resistant genotypes were found. Alternative mechanisms may be responsible for BZ resistance in this system, or the method and timing of treatments may reduce selection pressure for BZ resistance by creating substantial refugia for susceptible genotypes.

Macrogeographic population structure in a parasitic nematode with avian hosts.

Much remains to be discovered about the population genetic structure of parasites, despite the importance of such knowledge to understanding the processes involved in the spread of drug resistance through populations. Here we present a study of population genetic diversity in Trichostrongylus tenuis, an avian parasitic nematode infecting both poultry and game birds, where anthelmintic use is common. We examined diversity of nicotinamide dehydrogenase subunit 4 (nad4) mtDNA sequences within and between seven locations: five in the UK (red grouse hosts), one in Iceland (domestic goose) and one in Norway (willow grouse). Within-UK comparisons showed high nucleotide diversity (pi=0.015, n=23) but no structure between locations (Phi(ST)=0.022, P=0.27), with over 97% of variation distributed within-hosts. The highest diversity was found in Iceland (pi=0.043, n=4), and the lowest in Norway (pi=0.003, n=4). Differentiation between countries was considerable (Phi(CT)=0.44, P<0.05), in spite of the potential mixing effects of gene flow via migrating wild hosts and the poultry trade. However, significant pairwise F(ST) values were found only between Norway and the other locations. Phylogenetic analysis provided statistical support for a separate clade for Norwegian samples only, with unresolved diversity leading to a star-shaped relationship between Icelandic and UK haplotypes. These results suggest that Norwegian T. tenuis are isolated, but that there is some connectivity between UK and Icelandic populations. Although anthelmintic resistance has not yet been reported for T. tenuis, the population structure is such that emerging resistance has the potential to spread by gene flow over a large geographic scale.

Abundant variation in microsatellites of the parasitic nematode Trichostrongylus tenuis and linkage to a tandem repeat.

An understanding of how genes move between and within populations of parasitic nematodes is important in combating the evolution and spread of anthelmintic resistance. Much has been learned by studying mitochondrial DNA markers, but autosomal markers such as microsatellites have been applied to only a few nematode species, despite their many advantages for studying gene flow in eukaryotes. Here, we describe the isolation of 307 microsatellites from Trichostrongylus tenuis, an intestinal nematode of red grouse. High levels of variation were revealed at sixteen microsatellite loci (including three sex-lined loci) in 111 male T. tenuis nematodes collected from four hosts at a single grouse estate in Scotland (average He = 0.708; mean number of alleles = 12.2). A population genetic analysis detected no deviation from panmixia either between (F(ST) = 0.00) or within hosts (F(IS) = 0.015). We discuss the feasibility of developing microsatellites in parasitic nematodes and the problem of null alleles. We also describe a novel 146-bp repeat element, TteREP1, which is linked to two-thirds of the microsatellites sequenced and is associated with marker development failure. The sequence of TteREP1 is related to the TcREP-class of repeats found in several other trichostrongyloid species including Trichostrongylus colubriformis and Haemonchus contortus.

Molecular systematics of Acarus siro s. lat., a complex of stored food pests.

The astigmatid mite Acarus siro (Linnaeus 1758) is an important agricultural pest and environmental allergen. However, it is likely that many mites described in the literature as A. siro, collected from both outdoor and stored product habitats, may belong to one of its sibling species, A. farris [Ent. Ber. Amst. 2 (26) (1905) 20] or A. immobilis [Bull. Br. Mus. Nat. Hist. 11 (1964a) 413; Acarologia. 6 (Suppl) (1964) 101]. The three species are difficult to separate morphologically, gene exchange between some of them is possible and, although each species displays environmental preferences, they occur together in some environments. This raises a question about their separate species status. In a pilot study, we investigated whether genetic data supported the separate species status of these forms. Both nuclear (the second internal transcribed spacer region [ITS-2] of the ribosomal cistron) and mitochondrial (cytochrome oxidase subunit I, mtcoxI hereafter) loci were employed for this purpose. Mtcox1 data does not conflict the differentiation into three separate species and while the ITS2 data were problematic for this group of mites it suggested that a congener, Acarus gracilis [Ann. Mag. Nat. Hist. 10 (1957) 753], is basal to the A. siro species complex.