Using Head Patch Pattern as a Reliable Biometric Character for Noninvasive Individual Recognition of an Endangered Pitviper Protobothrops mangshanensis

Mangshan pitviper, Protobothrops mangshanensis(formerly Zhaoermia mangshanensis) is endemic to China. Unfortunately, due to the decreasing size of its wild populations, this snake has been listed as critically endangered. Research carried out on the Mangshan pitviper's population ecology and captive reproduction has revealed that the unique head patch patterns of different individuals may potentially be used as a noninvasive recognition biometric character. We collected head patch pattern images of 40 individuals of P. mangshanensis between 1994 and 2011. By comparing each pitviper's head patch pattern, we found that the head patch pattern of individual snakes was different and unique. Additionally, we observed and recorded the head patch pattern characters of four adults and five juveniles before and after ecdysis. Our findings confirmed that head patch patterns of Mangshan pitvipers are unique and stable, remaining unchanged after ecdysis. Thus, individuals can be quickly identified by examining the head patch pattern within a specific recognition area on the head. This method may be useful for noninvasive individual recognition in many other species that display color patch pattern variations, especially in studies of endangered species where the use of invasive marking techniques is undesirable.     

Monitoring vegetation change caused by trampling: a study in the Cairngorms,Scotland

Summary

A study was made in the Cairngorms, Scotland to make recommendations for a monitoring scheme capable of detecting changes in the vegetation caused by recreational pressure following the development of a funicular railway. Four methods were used in field trials to assess percentage cover of plant species and gravel, rock and bare ground, where appropriate, in two vegetation types (open and closed). The methods used were visual estimates in 50 × 40 cm quadrats (Q), the mean of visual estimates in twenty 10 × 10 cm sub-quadrats of the 50 × 40 cm quadrats (Q₂₀), a modified point intercept method (RL) and photography. Variances between observers and between-quadrats were estimated for the different methods. The sampling design for detecting change was based on a model of variance, constructed from field trial data.

Between-observer and between-quadrat variances were related to mean percentage cover and approximated to a binomial distribution. The between-quadrat variance was larger than observer variance. The Q₂₀ method achieved appreciably better precision than the other methods. Analysis of half of the 10 × 10 cmsub-quadrats (1/2Q₂₀) selected in a checker board design achieved a relative efficiency of 78% compared with the Q₂₀. This result suggests that comparable precision to the Q₂₀ method could be achieved by choosing about 14 sub-quadrats in a larger quadrat, thus saving some time. Variation between quadrats also suggested that the Q₂₀ method was the one of choice for maximising precision. The precision of the photographic method was based on fewer data points, so is less accurate than other estimates.

Minimum sample sizes were estimated for detecting a 10% relative change of a species in open vegetation with 30% cover (i.e. a change from 30% to <27 or to >33% cover). With a 10 % Type II error rate and 5 % Type I error rate the minimum sample sizes were 47 quadrats for Q, 18 for Q ₂₀, 43 for RL, and 23 for the means of ten 10 × 10 cm sub-quadrats in open vegetation.

The most time-efficient field recording appeared to be the use of Q despite the required sample size being 2.6 times higher than that of Q₂₀. The far lower time requirement per quadrat, however, compensated for the higher numbers. The number of quadrats would depend on the specified change in percentage cover and on the statistical significance level used. For example, to detect a 10% absolute change in cover (i.e. from 30% to either <20 % or >40 % cover) at 95 % probability the net effective recording time is estimated at 5 h per vegetation type while to detect a 5 % change at 99 % probability would require c. 25 h. Larger samples may be required for other species or for species with a low initial cover.     

A universal and reliable assay for molecular sex identification of three‐spined sticklebacks (Gasterosteus aculeatus)

In heterogametic species, biological differences between the two sexes are ubiquitous, and hence, errors in sex identification can be a significant source of noise and bias in studies where sex‐related sources of variation are of interest or need to be controlled for. We developed and validated a universal multimarker assay for reliable sex identification of three‐spined sticklebacks (Gasterosteus aculeatus). The assay makes use of genotype scores from three sex‐linked loci and utilizes Bayesian probabilistic inference to identify sex of the genotyped individuals. The results, validated with 286 phenotypically sexed individuals from six populations of sticklebacks representing all major genetic lineages (cf. Pacific, Atlantic and Japan Sea), indicate that in contrast to commonly used single‐marker‐based sex identification assays, the developed multimarker assay should be 100% accurate. As the markers in the assay can be scored from agarose gels, it provides a quick and cost‐efficient tool for universal sex identification of three‐spined sticklebacks. The general principle of combining information from multiple markers to improve the reliability of sex identification is transferable and can be utilized to develop and validate similar assays for other species.     

Testosterone and cortisol concentrations vary with reproductive status in wild female red deer

Although hormones are key regulators of many fitness and life history traits, the causes of individual level variation in hormones, particularly in wild systems, remain understudied. Whilst we know that androgen and glucocorticoid levels vary within and among individuals in mammalian populations, how this relates to key reproductive processes such as gestation and lactation, and their effects on a female''s measurable hormone levels are poorly understood in wild systems. Using fecal samples collected from females in a wild red deer population between 2001 and 2013, we explore how fecal androgen (FAM) and cortisol (FCM) metabolite concentrations change with age and season, and how individual differences relate to variation in reproductive state. Both FAM and FCM levels increase toward parturition, although this only affects FCM levels in older females. FCM levels are also higher when females suckle a male rather than a female calf, possibly due to the higher energetic costs of raising a son. This illustrates the importance of accounting for a female''s life history and current reproductive status, as well as temporal variation, when examining individual differences in hormone levels. We discuss these findings in relation to other studies of mammalian systems and in particular to the relatively scarce information on variation in natural levels of hormones in wild populations.     

When can we measure stress noninvasively? Postdeposition effects on a fecal stress metric confound a multiregional assessment

Measurement of stress hormone metabolites in fecal samples has become a common method to assess physiological stress in wildlife populations. Glucocorticoid metabolite (GCM) measurements can be collected noninvasively, and studies relating this stress metric to anthropogenic disturbance are increasing. However, environmental characteristics (e.g., temperature) can alter measured GCM concentration when fecal samples cannot be collected immediately after defecation. This effect can confound efforts to separate environmental factors causing predeposition physiological stress in an individual from those acting on a fecal sample postdeposition. We used fecal samples from American pikas (Ochotona princeps) to examine the influence of environmental conditions on GCM concentration by (1) comparing GCM concentration measured in freshly collected control samples to those placed in natural habitats for timed exposure, and (2) relating GCM concentration in samples collected noninvasively throughout the western United States to local environmental characteristics measured before and after deposition. Our timed‐exposure trials clarified the spatial scale at which exposure to environmental factors postdeposition influences GCM concentration in pika feces. Also, fecal samples collected from occupied pika habitats throughout the species' range revealed significant relationships between GCM and metrics of climate during the postdeposition period (maximum temperature, minimum temperature, and precipitation during the month of sample collection). Conversely, we found no such relationships between GCM and metrics of climate during the predeposition period (prior to the month of sample collection). Together, these results indicate that noninvasive measurement of physiological stress in pikas across the western US may be confounded by climatic conditions in the postdeposition environment when samples cannot be collected immediately after defecation. Our results reiterate the importance of considering postdeposition environmental influences on this stress metric, especially in multiregional comparisons. However, measurements of fecal GCM concentration should prove useful for population monitoring within an eco‐region or when postdeposition exposure can be minimized.     

Proteomic changes in response to crystal formation in Drosophila Malpighian tubules

Kidney stone disease is a major health burden with a complex and poorly understood pathophysiology. Drosophila Malpighian tubules have been shown to resemble human renal tubules in their physiological function. Herein, we have used Drosophila as a model to study the proteomic response to crystal formation induced by dietary manipulation in Malpighian tubules. Wild-type male flies were reared in parallel groups on standard medium supplemented with lithogenic agents: control, Sodium Oxalate (NaOx) and Ethylene Glycol (EG). Malpighian tubules were dissected after 2 weeks to visualize crystals with polarized light microscopy. The parallel group was dissected for protein extraction. A new method of Gel Assisted Sample Preparation (GASP) was used for protein extraction. Differentially abundant proteins (p<0.05) were identified by label-free quantitative proteomic analysis in flies fed with NaOx and EG diet compared with control. Their molecular functions were further screened for transmembrane ion transporter, calcium or zinc ion binder. Among these, 11 candidate proteins were shortlisted in NaOx diet and 16 proteins in EG diet. We concluded that GASP is a proteomic sample preparation method that can be applied to individual Drosophila Malpighian tubules. Our results may further increase the understanding of the pathophysiology of human kidney stone disease.     

Bulked sample analysis in genetics,genomics and crop improvement

Biological assay has been based on analysis of all individuals collected from sample populations. Bulked sample analysis (BSA), which works with selected and pooled individuals, has been extensively used in gene mapping through bulked segregant analysis with biparental populations, mapping by sequencing with major gene mutants and pooled genomewide association study using extreme variants. Compared to conventional entire population analysis, BSA significantly reduces the scale and cost by simplifying the procedure. The bulks can be built by selection of extremes or representative samples from any populations and all types of segregants and variants that represent wide ranges of phenotypic variation for the target trait. Methods and procedures for sampling, bulking and multiplexing are described. The samples can be analysed using individual markers, microarrays and high‐throughput sequencing at all levels of DNA, RNA and protein. The power of BSA is affected by population size, selection of extreme individuals, sequencing strategies, genetic architecture of the trait and marker density. BSA will facilitate plant breeding through development of diagnostic and constitutive markers, agronomic genomics, marker‐assisted selection and selective phenotyping. Applications of BSA in genetics, genomics and crop improvement are discussed with their future perspectives.     

Factor scoring models of the Pittsburgh Sleep Quality Index: a comparative confirmatory factor analysis

The Pittsburgh Sleep Quality Index (PSQI) is a rigorously validated questionnaire with extensive use in sleep assessment. Findings from numerous factor analytic studies of the PSQI have been interpreted to support a heterogeneous factor structure model for the test. Nevertheless, the literature continues to lack a focused evaluation of whether this heterogeneous factor structure is justified. A consideration of this issue led to a conclusion that a closer analysis of the PSQI’s factor structure was merited. To address this need a comparative confirmatory factor analysis for assessing the performance of the accepted factors models of the PSQI was conducted. A sample of university students (n = 418), age = 20.92 ± 1.81 years, BMI = 23.30 ± 2.57 kg/m² completed the multi-structured sleep survey at Jamia Millia Islamia, New Delhi, India. Seventeen putative factor structures (three 1-Factor, eight 2-Factor, and six 3-Factor) of the PSQI from the existing literature were selected for analysis. Fourteen models (82.35%) had almost similar values for model fit indices. Two models were misfits, and one model was a poor fit. The two misfit models incorporated gender and age as covariates. The third poor fit model was used to produce a unique path diagram, which made it distinct from the remaining 16 models. The overlapping values in the fit range of the model fit indices did not support the often projected heterogeneous factor structures of the PSQI for the vast majority of the models.