A global reptile assessment highlights shared conservation needs of tetrapods.

Comprehensive assessments of species' extinction risks have documented the extinction crisis1 and underpinned strategies for reducing those risks2. Global assessments reveal that, among tetrapods, 40.7% of amphibians, 25.4% of mammals and 13.6% of birds are threatened with extinction3. Because global assessments have been lacking, reptiles have been omitted from conservation-prioritization analyses that encompass other tetrapods4-7. Reptiles are unusually diverse in arid regions, suggesting that they may have different conservation needs6. Here we provide a comprehensive extinction-risk assessment of reptiles and show that at least 1,829 out of 10,196 species (21.1%) are threatened-confirming a previous extrapolation8 and representing 15.6 billion years of phylogenetic diversity. Reptiles are threatened by the same major factors that threaten other tetrapods-agriculture, logging, urban development and invasive species-although the threat posed by climate change remains uncertain. Reptiles inhabiting forests, where these threats are strongest, are more threatened than those in arid habitats, contrary to our prediction. Birds, mammals and amphibians are unexpectedly good surrogates for the conservation of reptiles, although threatened reptiles with the smallest ranges tend to be isolated from other threatened tetrapods. Although some reptiles-including most species of crocodiles and turtles-require urgent, targeted action to prevent extinctions, efforts to protect other tetrapods, such as habitat preservation and control of trade and invasive species, will probably also benefit many reptiles.

Retinopathy in Greyhound dogs: Prevalence, fundoscopic, and histopathological findings.

OBJECTIVES: To determine the prevalence of retinal lesions and describe the fundoscopic findings of retinopathy in Greyhound dogs in the Manawatu/Whanganui region of New Zealand. To examine possible associations between sex, age, and racing variables with retinopathy in the study population. To describe retinal histologic findings in seven Greyhounds with retinopathy in New Zealand. METHODS: Two hundred Greyhound dogs from the Manawatu/Whanganui region of New Zealand underwent fundoscopy and fundic photography to identify and score the degree of retinopathy. Associations between retinopathy and age, sex, as well as racing variables, were examined. Histologic examination of the retina was undertaken on the eyes of seven Greyhounds from the Manawatu and Canterbury regions previously diagnosed with retinopathy by fundoscopy. RESULTS: Fifty dogs (25.1%) were identified with retinopathy of varying degrees of severity. In at least one eye, 7.5% of dogs had mild retinopathy, 11.6% moderate retinopathy, and 6.0% severe retinopathy. Males were more likely to be affected in both eyes and with moderate or severe grades, than females. Increasing age was not associated with increased prevalence of retinopathy, nor increased grade of severity. Retinal histology identified multifocal retinal detachment in 5 of the 7 cases examined and other common lesions included choroidal necrosis and outer to full-thickness retinal atrophy in the absence of significant inflammation. CONCLUSIONS: Retinopathy is prevalent in Greyhounds in the Manawatu/Whanganui region of New Zealand, but more research is required to elucidate the etiopathogenesis. Consideration should be made to include mandatory eye health examination in racing Greyhound dogs.

The covalent complex of Jo-In results from a long-lived, non-covalent intermediate state with near-native structure.

Covalent protein complexes have been used to assemble enzymes in large scaffolds for biotechnology purposes. Although the catalytic mechanism of the covalent linking of such proteins is well known, the recognition and overall structural mechanisms driving the association are far less understood but could help further functional engineering of these complexes. Here, we study the Jo-In complex by NMR spectroscopy and molecular modelling. We characterize a transient non-covalent complex, with structural elements close to those in the final covalent complex. Using site specific mutagenesis, we further show that this non-covalent association is essential for the covalent complex to form.

Improved NMR Detection of Phospho-Metabolites in a Complex Mixture.

Phosphorylated metabolites are omnipresent in cells, but their analytical characterization faces several technical hurdles. Here, we detail an improved NMR workflow aimed at assigning the high-resolution subspectrum of the phospho-metabolites in a complex mixture. Combining a pure absorption J-resolved spectrum (Pell, A. J.; J. Magn. Reson. 2007, 189 (2), 293-299) with alternate on- and off-switching of the 31P coupling interaction during the t1 evolution with a pure in-phase (PIP) HSQMBC experiment (Castañar, L.; Angew. Chem., Int. Ed. 2014, 53 (32), 8379-8382) without or with total correlation spectroscopy (TOCSY) transfer during the insensitive nuclei enhancement by polarization transfer (INEPT) gives access to selective identification of the individual subspectra of the phosphorylated metabolites. Returning to the initial J-res spectra, we can extract with optimal resolution the full trace for the individual phospho-metabolites, which can then be transposed on the high-resolution quantitative one dimensional spectrum.

Integrated pH Measurement during Reaction Monitoring with Dual-Reception 1H-31P NMR Spectroscopy.

Simultaneous detection of 1H and 31P NMR signals through a dual-detection scheme with two receivers allows monitoring of both the signals of a molecule and the pH of the solution through the resonance of the inorganic phosphate. We evaluate here the method in terms of sensitivity and ease of implementation and show that the additional information obtained without any loss of information or increase in measuring time can be of practical importance in a number of biochemical systems.

Stepping behavior and muscle activity of dairy cattle standing on concrete or rubber flooring for 1 or 3 hours.

The type of flooring in dairy cattle systems influences cows' health and welfare. Although concrete is common, the use of more compressible flooring, such as rubber, is increasing. Cows prefer to stand and walk on rubber surfaces than on concrete; however, it is largely unknown how walking and standing for longer periods of time influence muscle activity and fatigue. Therefore, we used measures of behavior and muscle activity to investigate the potential benefits of providing a rubber flooring surface to dairy cattle. Sixteen lactating Holstein cows were forced to stand on either concrete or rubber flooring for 1 or 3 h in a 2 × 2 crossover design. Surface electromyograms (SEMG) and skin surface temperature were used to evaluate muscle activity, fatigue, and movement of muscle activity between the hind legs. Activity of 2 muscles, the bicep femoris and middle gluteal, was assessed during both static contractions, when cows transferred weight to each hind leg, before and after 1 and 3 h of standing, and dynamic contractions, associated with steps and with shifts in weight without steps. In addition, we evaluated stepping rate, time between each step, feeding behavior, skin surface temperature, and latency to lie down after standing. Standing duration influenced both the behavior and muscle activity of cows. Stepping rate increased with standing time for cows on both flooring types. Static muscle activity parameters of the bicep femoris muscle were higher after 3 h of standing for cows standing on both flooring types (2.3 and 3.6% increases in median amplitude and median power frequency, respectively) compared with the change after 1 h and compared with baseline values before the standing treatment. Flooring type influenced the behavior and muscle activity of the cows, particularly during the first hour of exposure; cows standing on rubber had a higher stepping rate, shorter interval between steps, and higher number of SEMG shifts (muscle activity shifts with or without visible steps) than cows on concrete. There was no difference in skin surface temperature, feeding behavior, or latency to lie down between the treatments. The results show that standing on a rubber flooring caused a different initial behavioral response compared with standing on concrete; however, possible reasons for these changes are unclear. Standing for 3 h resulted in an increase in stepping rate and in some muscle activity parameters; however, the results regarding muscle fatigue in relation to flooring type are inconclusive.

Mitochondria and vesicles differ between adult and prepubertal sheep oocytes during IVM.

Oocytes from prepubertal animals have a reduced ability to undergo normal embryo development and produce viable offspring. The correct quantity, activity and cytoplasmic distribution of oocyte organelles are essential for oocyte maturation, fertilisation and subsequent embryo development. The aim of this study was to quantify the ultrastructural differences between oocytes from prepubertal lamb and adult ewes using electron microscopy and stereology. We also determined whether quantitative polymerase chain reaction (qPCR) methods give comparable estimates of mitochondrial number to stereology. Mean storage vesicle volume was greater in adult compared with lamb oocytes before IVM and decreased during maturation in both adult and lamb oocytes. Mitochondrial volume and number increased in adult oocytes during maturation; however, no increase was observed in lamb oocytes. Mitochondrial DNA copy number measured by qPCR showed no differences between adult and lamb oocytes. A different distribution of mitochondria was observed in lamb oocytes before maturation, while the percentage of hooded mitochondria increased during maturation in adult oocytes and decreased in the lamb. In conclusion, the present study has identified differences in the vesicles and mitochondria between adult and lamb oocytes from ewes that may contribute to reduced developmental competence in prepubertal oocytes.

Effects of space allowance on the behavior and physiology of cattle temporarily managed on rubber mats.

Dairy cattle managed in some pasture-based systems, such as in New Zealand, are predominantly kept outdoors all year around but are taken off pasture for periods, especially in wet weather to avoid soil damage. The use of rubber matting for such stand-off practices is becoming more common to improve animal welfare, and our objective was to investigate the effects of different space allowances on cow behavior and physiology when managed temporarily on rubber mats during a weather-induced stand-off period. Thirty pregnant, nonlactating Holstein-Friesian dairy cows were divided into 6 groups of 5 and exposed to 6 treatments following a Williams designed 6×6 Latin square. The treatments consisted of 6 space allowances on a 24-mm rubber surface during a simulated weather-induced stand-off period: 3.0, 4.5, 6.0, 7.5, 9.0, and 10.5 m(2)/cow. The stand-off period consisted of 18 h in the treatment pens followed by 6 h at pasture to allow for their daily feed intake (no feed was available during stand-off, following normal farm practice), for 3 consecutive days, with 6 d of recovery on pasture between treatments. When cows had more space available during the stand-off period, they spent more time lying on the rubber mats and less time lying on pasture during their daily 6-h feed break. Mean lying times (24 h, pasture and rubber mats combined) for the different space allowances were for 3.0 m(2)=7.5 h, 4.5 m(2)=10.2 h, 6.0 m(2)=11.9 h, 7.5 m(2)=12.4 h, and 10.5 m(2)=13.8 h. At 6.0 m(2) of space allowance per cow, the animals spent similar times lying per 24 h as when the cows were on recovery on pasture in between treatments (11.9 and 11.2 h, respectively). Aggressive interactions and nonaggressive lying disturbances were more frequent at lower space allowances (aggressive interactions decreased by 35% from 3.0 to 4.5 m(2)/cow, with a slower decline thereafter). Cows were dirtier after the stand-off period, particularly at lower space allowances. All cows had higher gait scores after the stand-off period; however, this change was unaffected by space allowance and very minor. Stride length, plasma cortisol, and body weight were all unaffected by the stand-off period and space allowance. The results suggest that to reduce aggressive behavior and maintain adequate lying times, dairy cattle managed temporarily on rubber matting for up to 18 h per day, without feed, should have a space allowance of at least 4.5 to 6.0 m(2) per cow.

A multi-taxon analysis of European Red Lists reveals major threats to biodiversity.

Biodiversity loss is a major global challenge and minimizing extinction rates is the goal of several multilateral environmental agreements. Policy decisions require comprehensive, spatially explicit information on species' distributions and threats. We present an analysis of the conservation status of 14,669 European terrestrial, freshwater and marine species (ca. 10% of the continental fauna and flora), including all vertebrates and selected groups of invertebrates and plants. Our results reveal that 19% of European species are threatened with extinction, with higher extinction risks for plants (27%) and invertebrates (24%) compared to vertebrates (18%). These numbers exceed recent IPBES (Intergovernmental Platform on Biodiversity and Ecosystem Services) assumptions of extinction risk. Changes in agricultural practices and associated habitat loss, overharvesting, pollution and development are major threats to biodiversity. Maintaining and restoring sustainable land and water use practices is crucial to minimize future biodiversity declines.

Virtual decoupling to break the simplification versus resolution trade-off in nuclear magnetic resonance of complex metabolic mixtures.

The heteronuclear single quantum correlation (HSQC) experiment developed by Bodenhausen and Ruben (1980) in the early days of modern nuclear magnetic resonance (NMR) is without a doubt one of the most widely used experiments, with applications in almost every aspect of NMR including metabolomics. Acquiring this experiment, however, always implies a trade-off: simplification versus resolution. Here, we present a method that artificially lifts this barrier and demonstrate its application towards metabolite identification in a complex mixture. Based on the measurement of clean in-phase and clean anti-phase (CLIP/CLAP) HSQC spectra (Enthart et al., 2008), we construct a virtually decoupled HSQC (vd-HSQC) spectrum that maintains the highest possible resolution in the proton dimension. Combining this vd-HSQC spectrum with a J-resolved spectrum (Pell and Keeler, 2007) provides useful information for the one-dimensional proton spectrum assignment and for the identification of metabolites in Dreissena polymorpha (Prud'homme et al., 2020).

Automated Collection and Analysis of Infrared Thermograms for Measuring Eye and Cheek Temperatures in Calves.

As the reliance upon automated systems in the livestock industry increases, technologies need to be developed which can be incorporated into these systems to monitor animal health and welfare. Infrared thermography (IRT) is one such technology that has been used for monitoring animal health and welfare and, through automation, has the potential to be integrated into automated systems on-farm. This study reports on an automated system for collecting thermal infrared images of calves and on the development and validation of an algorithm capable of automatically detecting and analysing the eye and cheek regions from those images. Thermal infrared images were collected using an infrared camera integrated into an automated calf feeder. Images were analysed automatically using an algorithm developed to determine the maximum eye and cheek (3 × 3-pixel and 9 × 9-pixel areas) temperatures in a given image. Additionally, the algorithm determined the maximum temperature of the entire image (image maximum temperature). In order to validate the algorithm, a subset of 350 images analysed using the algorithm were also analysed manually. Images analysed using the algorithm were highly correlated with manually analysed images for maximum image (R2 = 1.00), eye (R2 = 0.99), cheek 3 × 3-pixel (R2 = 0.85) and cheek 9 × 9-pixel (R2 = 0.90) temperatures. These findings demonstrate the algorithm to be a suitable method of analysing the eye and cheek regions from thermal infrared images. Validated as a suitable method for automatically detecting and analysing the eye and cheek regions from thermal infrared images, the integration of IRT into automated on-farm systems has the potential to be implemented as an automated method of monitoring calf health and welfare.

The Zebra Mussel (Dreissena polymorpha) as a Model Organism for Ecotoxicological Studies: A Prior 1H NMR Spectrum Interpretation of a Whole Body Extract for Metabolism Monitoring.

The zebra mussel (Dreissena polymorpha) represents a useful reference organism for the ecotoxicological study of inland waters, especially for the characterization of the disturbances induced by human activities. A nuclear magnetic resonance (NMR)-based metabolomic approach was developed on this species. The investigation of its informative potential required the prior interpretation of a reference 1H NMR spectrum of a lipid-free zebra mussel extract. After the extraction of polar metabolites from a pool of whole-body D. polymorpha powder, the resulting highly complex 1D 1H NMR spectrum was interpreted and annotated through the analysis of the corresponding 2D homonuclear and heteronuclear NMR spectra. The spectrum interpretation was completed and validated by means of sample spiking with 24 commercial compounds. Among the 238 detected 1H signals, 53% were assigned, resulting in the identification of 37 metabolites with certainty or high confidence, while 5 metabolites were only putatively identified. The description of such a reference spectrum and its annotation are expected to speed up future analyses and interpretations of NMR-based metabolomic studies on D. polymorpha and to facilitate further explorations of the impact of environmental changes on its physiological state, more particularly in the context of large-scale ecological and ecotoxicological studies.

A structural basis for the reduced toxicity of dinophysistoxin-2.

Okadaic acid (OA), dinophysistoxin-1 (DTX-1), and dinophysistoxin-2 (DTX-2) are algal toxins that can accumulate in shellfish and cause diarrhetic shellfish poisoning. Recent studies indicate that DTX-2 is about half as toxic and has about half the affinity for protein phosphatase 2A (PP2A) as OA. NMR structural studies showed that DTX-1 possessed an equatorial 35-methyl group but that DTX-2 had an axial 35-methyl group. Molecular modeling studies indicated that an axial 35-methyl could exhibit unfavorable interactions in the PP2A binding site, and this has been proposed as the reason for the reduced toxicity of DTX-2. Statistical analyses of published data indicate that the affinity of PP2A for DTX-1 is 1.6-fold higher, and for DTX-2 is 2-fold lower, than for OA. We obtained X-ray crystal structures of DTX-1 and DTX-2 bound to PP2A. The crystal structures independently confirm the C-35 stereochemistries determined in the earlier NMR study. The structure for the DTX-1 complex was virtually identical to that of the OA-PP2A complex, except for the presence of the equatorial 35-methyl on the ligand. The favorable placement of the equatorial 35-methyl group of DTX-1 against the aromatic pi-bonds of His191 may account for the increased affinity of PP2A toward DTX-1. In contrast, the axial 35-methyl of DTX-2 caused the side chain of His191 to rotate 140 degrees so that it pointed toward the solvent, thereby opening one end of the hydrophobic binding cage. This rearrangement to accommodate the unfavorable interaction from the axial 35-methyl of DTX-2 reduces the binding energy and appears to be responsible for the reduced affinity of PP2A for DTX-2. These results highlight the potential of molecular modeling studies for understanding the relative toxicity of analogues once the binding site at the molecular target has been properly characterized.

Infrared Thermography-A Non-Invasive Method of Measuring Respiration Rate in Calves.

Respiration rate (RR) is a common measure of cattle health and welfare. Traditionally, measuring RR involves counting flank movements as the animal inhales and exhales with each breath. This method is often considered difficult, labour-intensive and impractical. We validated the use of infrared thermography (IRT) as an alternative method of non-invasively measuring RR in young calves. RR was simultaneously recorded in two ways: (1) by observing flank movements from video recordings; and (2) by observing thermal fluctuations around the nostrils during inhalations and exhalations from infrared recordings. For each method, the time taken to complete five consecutive breaths (a breath being a complete inhalation/exhalation cycle) was recorded and used to calculate RR (breaths/min). From a group of five calves, a total of 12 video recordings and 12 infrared recordings were collected. For each procedure, 47 sets of five consecutive breaths were assessed. The RRs measured from video recordings of flank movements and thermal fluctuations around the nostrils from infrared recordings were highly correlated (R2 = 0.93). Validated as a suitable method for recording RR, future research can now focus on the development of algorithms to automate the use of IRT to support its integration into existing automated systems to remotely monitor calf health and welfare.

A standard lexicon for biodiversity conservation: unified classifications of threats and actions.

An essential foundation of any science is a standard lexicon. Any given conservation project can be described in terms of the biodiversity targets, direct threats, contributing factors at the project site, and the conservation actions that the project team is employing to change the situation. These common elements can be linked in a causal chain, which represents a theory of change about how the conservation actions are intended to bring about desired project outcomes. If project teams want to describe and share their work and learn from one another, they need a standard and precise lexicon to specifically describe each node along this chain. To date, there have been several independent efforts to develop standard classifications for the direct threats that affect biodiversity and the conservation actions required to counteract these threats. Recognizing that it is far more effective to have only one accepted global scheme, we merged these separate efforts into unified classifications of threats and actions, which we present here. Each classification is a hierarchical listing of terms and associated definitions. The classifications are comprehensive and exclusive at the upper levels of the hierarchy, expandable at the lower levels, and simple, consistent, and scalable at all levels. We tested these classifications by applying them post hoc to 1191 threatened bird species and 737 conservation projects. Almost all threats and actions could be assigned to the new classification systems, save for some cases lacking detailed information. Furthermore, the new classification systems provided an improved way of analyzing and comparing information across projects when compared with earlier systems. We believe that widespread adoption of these classifications will help practitioners more systematically identify threats and appropriate actions, managers to more efficiently set priorities and allocate resources, and most important, facilitate cross-project learning and the development of a systematic science of conservation.

A literally blinded trial of palpation in dermatologic diagnosis.

BACKGROUND: As an adjunct to visual inspection, dermatologists routinely palpate the skin during clinical examination. The value of palpation in isolation is unproven. METHODS: To determine whether palpation alone could distinguish between two common dermatoses, atopic dermatitis and plaque psoriasis, a cohort of 16 patients were examined by a dermatologist using touch alone, with screens to prevent visualisation of the lesions. RESULTS: The diagnosis was correctly made in 14 of 16 cases (P = .012). LIMITATIONS: Patients were not consecutive as availability of the dermatologist and nursing staff was required, and several exclusion criteria applied. There was no formal power calculation. The study aimed to prove a concept about dissociating sensory modalities and does not prove that palpation would necessarily have provided "added value" to a visual diagnosis in the cases selected. CONCLUSION: This study demonstrates that dissociation between sensory inputs that are usually concurrent (in this case, visual and tactile) is a feasible experiment in dermatology, and that such dissociation did not remove the diagnostic input of the sole sensory modality that was permitted-in this case, confirming that palpation alone does have some importance in dermatologic diagnosis. It may explain some of the reservations about accuracy of teledermatology diagnoses of dermatoses, where palpation is not possible.

Conservation needs of amphibians in China: a review.

The conservation status of all the amphibians in China is analyzed, and the country is shown to be a global priority for conservation in comparison to many other countries of the world. Three Chinese regions are particularly rich in amphibian diversity: Hengduan, Nanling, and Wuyi mountains. Salamanders are more threatened than frogs and toads. Several smaller families show a high propensity to become seriously threatened: Bombinatoridae, Cryptobranchidae, Hynobiidae and Salamandridae. Like other parts of the world, stream-breeding, high-elevation forest amphibians have a much higher likelihood of being seriously threatened. Habitat loss, pollution, and over-harvesting are the most serious threats to Chinese amphibians. Over-harvesting is a less pervasive threat than habitat loss, but it is more likely to drive a species into rapid decline. Five conservation challenges are mentioned with recommendations for the highest priority research and conservation actions.

Aploneura lentisci (Homoptera: Aphididae) and Its Interactions with Fungal Endophytes in Perennial Ryegrass (Lolium perenne).

Aploneura lentisci Pass. is endemic to the Mediterranean region where it is holocyclic, forming galls on its primary host, Pistacia lentiscus and alternating over a 2-year period between Pistacia and secondary hosts, principally species of Gramineae. This aphid is widely distributed in Australia and New Zealand on the roots of the common forage grasses, ryegrass (Lolium spp.) and tall fescue (Schedonorus phoenix) where it exists as permanent, anholocyclic, parthenogenetic populations. Previous studies have indicated that infestations of A. lentisci significantly reduce plant growth and may account for differences in field performance of Lolium perenne infected with different strains of the fungal endophyte Epichloë festucae var. lolii. These obligate biotrophs protect their host grasses from herbivory via the production of alkaloids. To confirm the hypothesis that growth of L. perenne is associated with the effect of different endophyte strains on aphid populations, herbage and root growth were measured over time in two pot trials that compared three fungal endophyte strains with an endophyte-free control. In both pot trials, aphid numbers were lowest on plants infected with endophyte strain AR37 at all sampling times. In plants infected with a common toxic strain naturalized in New Zealand, aphid numbers overall were lower than on uninfected plants or those infected with strain AR1, but numbers did not always differ significantly from these treatments. Populations on AR1-infected plants were occasionally significantly higher than those on endophyte-free. Cumulative foliar growth was reduced in AR1 and Nil treatments relative to AR37 in association with population differences of A. lentisci in both trials and root dry weight was reduced in one trial. In four Petri dish experiments survival of A. lentisci on plants infected with AR37 declined to low levels after an initial phase of up to 19 days during which time aphids fed and populations were similar to those on plants without endophyte. Aphids on AR37-infected plants became uncoordinated in their movement and developed tremors before dying suggesting a neurotoxin was responsible for their mortality. Results support the hypothesis that differences in A. lentisci populations due to endophyte infection status and strain affects plant growth.