From left to right all through the night: Characteristics of lying rest in zoo elephants.

Despite increased research during the past years, many characteristics of resting behavior in elephants are still unknown. For example, there is only limited data suggesting elephants express longer lying bouts and increased total nightly lying durations on soft substrates as compared to hard surfaces. Additionally, it has not been investigated how frequently elephants change body sides between lying bouts. Here we present these characteristics based on observations of nighttime lying behavior in 10 zoo elephants (5 African Loxodonta africana and 5 Asian Elephas maximus elephants) living in five different European facilities. We found that elephants housed on soft substrates have significantly increased total lying durations per night and longer average lying bouts. Furthermore, at 70%-85% of all bouts, a consistently higher frequency of side change between lying bouts occurred on soft substrates, leading to an overall equal laterality in resting behavior. Deviations from this pattern became evident in elephants living on nonsand flooring or/and in nondominant individuals of nonfamily groups, respectively. Based on our findings, we consider elephants to normally have several lying bouts per night with frequent side changes, given an appropriate substrate and healthy social environment. We encourage elephant-keeping facilities to monitor these characteristics in their elephants' nighttime behavior to determine opportunities for further improvements and detect alterations putatively indicating social or health problems in individual elephants at an early stage.

The historical development of zoo elephant survivorship.

In the discussion about zoo elephant husbandry, the report of Clubb et al. (2008, Science 322: 1649) that zoo elephants had a "compromised survivorship" compared to certain non-zoo populations is a grave argument, and was possibly one of the triggers of a large variety of investigations into zoo elephant welfare, and changes in zoo elephant management. A side observation of that report was that whereas survivorship in African elephants (Loxodonta africana) improved since 1960, this was not the case in Asian elephants (Elephas maximus). We used historical data (based on the Species360 database) to revisit this aspect, including recent developments since 2008. Assessing the North American and European populations from 1910 until today, there were significant improvements of adult (≥10 years) survivorship in both species. For the period from 1960 until today, survivorship improvement was significant for African elephants and close to a significant improvement in Asian elephants; Asian elephants generally had a higher survivorship than Africans. Juvenile (<10 years) survivorship did not change significantly since 1960 and was higher in African elephants, most likely due to the effect of elephant herpes virus on Asian elephants. Current zoo elephant survivorship is higher than some, and lower than some other non-zoo populations. We discuss that in our view, the shape of the survivorship curve, and its change over time, are more relevant than comparisons with specific populations. Zoo elephant survivorship should be monitored continuously, and the expectation of a continuous trend towards improvement should be met.

Reconstruction of evolutionary changes in fat and toxin consumption reveals associations with gene losses in mammals: A case study for the lipase inhibitor PNLIPRP1 and the xenobiotic receptor NR1I3.

The inactivation of ancestral protein-coding genes (gene loss) can be associated with phenotypic modifications. Within placental mammals, repeated losses of PNLIPRP1 (gene inhibiting fat digestion) occurred preferentially in strictly herbivorous species, whereas repeated NR1I3 losses (gene involved in detoxification) occurred preferentially in strictly carnivorous species. It was hypothesized that lower fat contents of herbivorous diets and lower toxin contents of carnivorous diets cause relaxed selection pressure on these genes, resulting in the accumulation of mutations and ultimately to convergent gene losses. However, because herbivorous and carnivorous diets differ vastly in their composition, a fine-grained analysis is required for hypothesis testing. We generated a trait matrix recording diet and semi-quantitative estimates of fat and toxin consumption for 52 placental species. By including data from 31 fossil taxa, we reconstructed the ancestral diets in major lineages (grundplan reconstruction). We found support that PNLIPRP1 loss is primarily associated with low levels of fat intake and not simply with herbivory/carnivory. In particular, PNLIPRP1 loss also occurred in carnivorous lineages feeding on a fat-poor diet, suggesting that the loss of this gene may be beneficial for occupying ecological niches characterized by fat-poor food resources. Similarly, we demonstrated that carnivorous species are indeed less exposed to diet-related toxins, suggesting that the loss of NR1I3 and related genes (NR1I2 and UGT1A6) resulted from relaxed selection pressure. This study illustrates the need of detailed phenotype studies to obtain a deeper understanding of factors underlying gene losses and to progress in understanding genomic causes of phenotypic variation in mammals.

Mammalian intestinal allometry, phylogeny, trophic level and climate.

An often-stated ecomorphological assumption that has the status of 'textbook knowledge' is that the dimensions of the digestive tract correlate with diet, where herbivores-consuming diets of lower digestibility-have longer intestinal tracts than faunivores-consuming diets of higher digestibility. However, statistical approaches have so far failed to demonstrate this link. Here, we collated data on the length of intestinal sections and body mass of 519 mammal species, and test for various relationships with trophic, climatic and other biological characteristics. All models showed a strong phylogenetic signal. Scaling relationships with body mass showed positive allometry at exponents greater than 0.33, except for the caecum, which is particularly large in smaller species. Body mass was more tightly linked to small intestine than to large intestine length. Adding a diet proxy to the relationships increased model fit for all intestinal sections, except for the small intestine when accounting for phylogeny. Thus, the diet has a main effect on the components of the large intestine, with longer measures in herbivores. Additionally, measures of habitat aridity had a positive relationship with large intestine length. The small intestine was longer in species from colder habitats at higher latitudes, possibly facilitating the processing of peak intake rates during the growing season. This study corroborates intuitive expectations on digestive tract anatomy, while the dependence of significant results on large sample sizes and inclusion of specific taxonomic groups indicates that the relationships cannot be considered fixed biological laws.

Soft and persistent-The influence of sand-flooring and calves on the resting behavior of a zoo-kept African elephant (Loxodonta africana) group.

Caring for all aspects of zoo elephants' well-being is considered a major challenge. Providing an appropriate flooring substrate to facilitate lying rest presents a meaningful part of a holistic management concept. Investigating the impact of a new sand flooring on the nocturnal resting behavior of a breeding group of seven African elephants living at one zoo revealed more total lying rest, longer bouts of lying rest and a reduced side preference in the adult females. With an average total daily lying rest of about 1.5-2.0 hrs, the investigated zoo elephants expressed longer lying rest compared to recently reported data from free-ranging individuals in Botswana. In addition, the presence of nursing calves in the observed elephant group seemed to impact the resting pattern of all group members, with around 60% of all lying bouts being discontinued after interruption by the youngsters. With respect to observed nursing during leaning rest, we encourage the installation of appropriate horizontal structures in breeding facilities to support leaning rest behavior of their female elephants. In doing so, zoos may improve rest quality of nursing females, and, in general, the welfare aspect of sleep for their elephants.

Weigh and see-Body mass recordings versus body condition scoring in European zoo elephants (Loxodonta africana and Elephas maximus).

Regular body mass (BM) monitoring plays a key role in preventative health care of zoo animals. In some species, including African (Loxodonta africana) and Asian elephants (Elephas maximus), the process of weighing can be challenging, and alternative methods such as visual body condition scoring (BCS) have been developed. We investigated the temporal development of both parameters regarding correlation patterns between them, and their suitability as monitoring measures in dependence of an elephant's life stage. While BM is more suitable in calves and juveniles under the age of 8 years, both BM and BCS are considered equally reliable in adult elephants. In elephants over the age of 40 years, BCS might be more suitable for assessing the physical status. Independent of species and sex, juvenile zoo elephants grow in BM nearly linearly with age, and reach a higher BM at an earlier age compared with conspecifics of free-ranging and semi-captive populations in the countries of origin. The BCS typically remains constant during this life stage, seemingly unaffected by growth. In adult animals, breeding females have a lower BM and BCS than nonbreeders, and BM and BCS typically indicate fluctuations in the same direction. In geriatric elephants (>40 years) a drop in BCS occurs commonly, while BM may even increase in this life stage. We recommend regular body mass recording in zoo elephants to enhance our knowledge of body mass development and allow the formulation of objective practical recommendations. BCS presents a valuable and simple tool for complementary monitoring of an elephant's condition, especially in adult and geriatric individuals.

Influencing factors on the foot health of captive Asian elephants (Elephas maximus) in European zoos.

Pathological lesions of feet occur frequently in captive elephant populations. To improve foot health, it is important to identify risk factors associated with such pathologies. Several previous studies have analyzed potentially influencing factors but were limited, for example, by small sample sizes. This study analyzed the relationship between 87 independent variables and the foot health score of 204 Asian elephants (Elephas maximus) in European zoos using bivariate correlation, multivariable regression models, and principal component analysis (PCA). Correlation and regression tests revealed significant results for 30 different variables, mainly with small effect sizes. Only three variables were significant in more than one test: sex, time spent indoors, and time spent on hard ground, with lower scores (i.e. less or less severe pathological lesions) in females, and when less time is spent indoors or on hard ground. Due to small effect sizes and differing results of the statistical tests, it is difficult to determine which risk factors are most important. Instead, a holistic consideration appears more appropriate. A biplot of the PCA shows that factors representing more advanced husbandry conditions (e.g. large areas, high proportions of sand flooring) were associated with each other and with decreased foot scores, whereas indicators of more limited conditions (e.g. high proportions of hard ground, much time spent indoors) were also associated with each other but increased the foot score. In conclusion, instead of resulting from just one or two factors, reduced foot health might be an indicator of a generally poorer husbandry system.

FOOT HEALTH OF ASIAN ELEPHANTS (ELEPHAS MAXIMUS) IN EUROPEAN ZOOS.

Foot problems are a common concern in elephant husbandry. Studies on this topic with sample sizes greater than 100 animals have only been carried out in North America. We investigated foot health of 243 Asian elephants (Elephas maximus) in 69 European institutions. During on-site visits between August 2016 and July 2017, standardized pictures were taken of each elephant's nails and pads. The pictures were analyzed with respect to pathological lesions (i.e. nail cracks, abscesses), care issues (i.e. minor abnormalities, which are easily resolvable with routine foot work), and pad structure. Of all analyzed nails and pads, 35.6% revealed varying degrees of pathological lesions, with minor nail cracks and overgrown cuticles with attachment to the nails being most frequently observed. The most lateral nail (N5) on both front feet demonstrated the highest percentage of pathological lesions, providing support to a separate study showing that the mean peak pressure of an elephant's foot occurs along the most lateral digits; however, this was not observed along the most lateral nail (N5) of the rear feet. Three (of 243) elephants did not show any pathological lesions in their feet. The most common issues requiring foot care were fissures in the nail sole. The structure of the pads was categorized in four grades reflecting the percentage of surface marked by sulci. These four grades occurred at nearly equal frequency. Pearson product moment correlations revealed no significant association between the frequency of care issues and pathological lesions per nail. Despite this finding, it may be prudent to implement husbandry protocols that could alleviate commonly observed pathological and care foot issues in captive Asian elephants. A standardized approach to evaluate elephant foot health will provide a more objective way to monitor responses to management and medical decisions and ultimately contribute to the overall wellbeing of elephants in human care.

When elephants fall asleep: A literature review on elephant rest with case studies on elephant falling bouts, and practical solutions for zoo elephants.

Little attention has been paid to the resting and sleeping behavior of zoo elephants so far. An important concern is when elephants avoid lying down, due to degenerative joint and foot disease, social structure, or stressful environmental changes. Inability or unwillingness to lie down for resting is an important welfare issue, as it may impair sleep. We emphasize the importance of satisfying rest in elephants by reviewing the literature on resting behavior in elephants (Loxodonta africana and Elephas maximus) as well as the documentation of four cases from European zoos and our own direct observations in a zoo group of four female African elephants during 12 entire days. The common denominator in the case reports is the occurrence of a falling bout out of a standing position subsequently to a cessation of lying rest for different periods of time. Although well-known in horses as "episodic collapse" or "excessive drowsiness," this syndrome has not been described in elephants before. To enable its detection, we recommend nocturnal video monitoring for elephant-keeping institutions. The literature evaluation as well as own observational data suggest an inverse relationship between lying rest and standing rest. Preventative measures consist of enclosure modifications that facilitate lying rest (e.g., sand hills) or standing rest in a leaning position as a substitute. Anecdotal observations suggest that the provision of appropriate horizontal environmental structures may encourage safe, sleep-conducive standing rest. We provide drawings on how to install such structures. Effects of providing such structures should be evaluated in the future.

Identification of a multi-protein reductive dehalogenase complex in Dehalococcoides mccartyi strain CBDB1 suggests a protein-dependent respiratory electron transport chain obviating quinone involvement.

Dehalococcoides mccartyi strain CBDB1 is an obligate organohalide-respiring bacterium using only hydrogen as electron donor and halogenated organics as electron acceptor. Here, we studied proteins involved in the respiratory chain under non-denaturing conditions. Using blue native gel electrophoresis (BN-PAGE), gel filtration and ultrafiltration an active dehalogenating protein complex with a molecular mass of 250-270 kDa was identified. The active subunit of reductive dehalogenase (RdhA) colocalised with a complex iron-sulfur molybdoenzyme (CISM) subunit (CbdbA195) and an iron-sulfur cluster containing subunit (CbdbA131) of the hydrogen uptake hydrogenase (Hup). No colocalisation between the catalytically active subunits of hydrogenase and reductive dehalogenase was found. By two-dimensional BN/SDS-PAGE the stability of the complex towards detergents was assessed, demonstrating stepwise disintegration with increasing detergent concentrations. Chemical cross-linking confirmed the presence of a higher molecular mass reductive dehalogenase protein complex composed of RdhA, CISM I and Hup hydrogenase and proved to be a potential tool for stabilising protein-protein interactions of the dehalogenating complex prior to membrane solubilisation. Taken together, the identification of the respiratory dehalogenase protein complex and the absence of indications for quinone participation in the respiration suggest a quinone-independent protein-based respiratory electron transfer chain in D. mccartyi.

Dehalococcoides mccartyi strain DCMB5 Respires a broad spectrum of chlorinated aromatic compounds.

Polyhalogenated aromatic compounds are harmful environmental contaminants and tend to persist in anoxic soils and sediments. Dehalococcoides mccartyi strain DCMB5, a strain originating from dioxin-polluted river sediment, was examined for its capacity to dehalogenate diverse chloroaromatic compounds. Strain DCMB5 used hexachlorobenzenes, pentachlorobenzenes, all three tetrachlorobenzenes, and 1,2,3-trichlorobenzene as well as 1,2,3,4-tetra- and 1,2,4-trichlorodibenzo-p-dioxin as electron acceptors for organohalide respiration. In addition, 1,2,3-trichlorodibenzo-p-dioxin and 1,3-, 1,2-, and 1,4-dichlorodibenzo-p-dioxin were dechlorinated, the latter to the nonchlorinated congener with a remarkably short lag phase of 1 to 4 days following transfer. Strain DCMB5 also dechlorinated pentachlorophenol and almost all tetra- and trichlorophenols. Tetrachloroethene was dechlorinated to trichloroethene and served as an electron acceptor for growth. To relate selected dechlorination activities to the expression of specific reductive dehalogenase genes, the proteomes of 1,2,3-trichlorobenzene-, pentachlorobenzene-, and tetrachloroethene-dechlorinating cultures were analyzed. Dcmb_86, an ortholog of the chlorobenzene reductive dehalogenase CbrA, was the most abundant reductive dehalogenase during growth with each electron acceptor, suggesting its pivotal role in organohalide respiration of strain DCMB5. Dcmb_1041 was specifically induced, however, by both chlorobenzenes, whereas 3 putative reductive dehalogenases, Dcmb_1434, Dcmb_1339, and Dcmb_1383, were detected only in tetrachloroethene-grown cells. The proteomes also harbored a type IV pilus protein and the components for its assembly, disassembly, and secretion. In addition, transmission electron microscopy of DCMB5 revealed an irregular mode of cell division as well as the presence of pili, indicating that pilus formation is a feature of D. mccartyi during organohalide respiration.

Comparison of targeted peptide quantification assays for reductive dehalogenases by selective reaction monitoring (SRM) and precursor reaction monitoring (PRM).

Targeted absolute protein quantification yields valuable information about physiological adaptation of organisms and is thereby of high interest. Especially for this purpose, two proteomic mass spectrometry-based techniques namely selective reaction monitoring (SRM) and precursor reaction monitoring (PRM) are commonly applied. The objective of this study was to establish an optimal quantification assay for proteins with the focus on those involved in housekeeping functions and putative reductive dehalogenase proteins from the strictly anaerobic bacterium Dehalococcoides mccartyi strain CBDB1. This microbe is small and slow-growing; hence, it provides little biomass for comprehensive proteomic analysis. We therefore compared SRM and PRM techniques. Eleven peptides were successfully quantified by both methods. In addition, six peptides were solely quantified by SRM and four by PRM, respectively. Peptides were spiked into a background of Escherichia coli lysate and the majority of peptides were quantifiable down to 500 amol absolute on column by both methods. Peptide quantification in CBDB1 lysate resulted in the detection of 15 peptides using SRM and 14 peptides with the PRM assay. Resulting quantification of five dehalogenases revealed copy numbers of <10 to 115 protein molecules per cell indicating clear differences in abundance of RdhA proteins during growth on hexachlorobenzene. Our results indicated that both methods show comparable sensitivity and that the combination of the mass spectrometry assays resulted in higher peptide coverage and thus more reliable protein quantification.

Epigenetic gene expression links heart failure to memory impairment.

In current clinical practice, care of diseased patients is often restricted to separated disciplines. However, such an organ-centered approach is not always suitable. For example, cognitive dysfunction is a severe burden in heart failure patients. Moreover, these patients have an increased risk for age-associated dementias. The underlying molecular mechanisms are presently unknown, and thus, corresponding therapeutic strategies to improve cognition in heart failure patients are missing. Using mice as model organisms, we show that heart failure leads to specific changes in hippocampal gene expression, a brain region intimately linked to cognition. These changes reflect increased cellular stress pathways which eventually lead to loss of neuronal euchromatin and reduced expression of a hippocampal gene cluster essential for cognition. Consequently, mice suffering from heart failure exhibit impaired memory function. These pathological changes are ameliorated via the administration of a drug that promotes neuronal euchromatin formation. Our study provides first insight to the molecular processes by which heart failure contributes to neuronal dysfunction and point to novel therapeutic avenues to treat cognitive defects in heart failure patients.

Reductive tetrachloroethene dehalogenation in the presence of oxygen by Sulfurospirillum multivorans: physiological studies and proteome analysis.

Reductive dehalogenation of organohalides is carried out by organohalide-respiring bacteria (OHRB) in anoxic environments. The tetrachloroethene (PCE)-respiring Epsilonproteobacterium Sulfurospirillum multivorans is one of few OHRB able to respire oxygen. Therefore, we investigated the organism's capacity to dehalogenate PCE in the presence of oxygen, which would broaden the applicability to use S. multivorans, unlike other commonly oxygen-sensitive OHRB, for bioremediation, e.g. at oxic/anoxic interphases. Additionally, this has an impact on our understanding of the global halogen cycle. Sulfurospirillum multivorans performs dehalogenation of PCE to cis-1,2-dichloroethene at oxygen concentrations below 0.19 mg/L. The redox potential of the medium electrochemically adjusted up to +400 mV had no influence on reductive dehalogenation by S. multivorans in our experiments, suggesting that higher levels of oxygen impair PCE dechlorination by inhibiting or inactivating involved enzymes. The PCE reductive dehalogenase remained active in cell extracts of S. multivorans exposed to 0.37 mg/L oxygen for more than 96 h. Analysis of the proteome revealed that superoxide reductase and cytochrome peroxidase amounts increased with 5% oxygen in the gas phase, while the response to atmospheric oxygen concentrations involved catalase and hydrogen peroxide reductase. Taken together, our results demonstrate that reductive dehalogenation by OHRB is not limited to anoxic conditions.

Proteomic data set of the organohalide-respiring Epsilonproteobacterium Sulfurospirillum multivorans adapted to tetrachloroethene and other energy substrates.

Sulfurospirillum multivorans is a free-living, physiologically versatile Epsilonproteobacterium able to couple the reductive dehalogenation of chlorinated and brominated ethenes to growth (organohalide respiration). We present proteomic data of S. multivorans grown with different electron donors (formate or pyruvate) and electron acceptors (fumarate, nitrate, or tetrachloroethene [PCE]). To obtain information on the cellular localization of proteins, membrane extracts and soluble fractions were separated before data collection from both fractions. The proteome analysis of S. multivorans was performed by mass spectrometry (nanoLC-MS/MS). Raw data have been deposited at ProteomeXchange, "ProteomeXchange provides globally coordinated proteomics data submission and dissemination" [1], via the PRIDE partner repository with the dataset identifier PRIDE: PXD004011. The data might support further research in organohalide respiration and in the general metabolism of free-living Epsilonproteobacteria. The dataset is associated with a previously published study "Proteomics of the organohalide-respiring Epsilonproteobacterium S. multivorans adapted to tetrachloroethene and other energy substrates" [2].

Proteomic dataset of the organohalide-respiring bacterium Dehalococcoides mccartyi strain CBDB1 grown on hexachlorobenzene as electron acceptor.

The proteome of the anaerobic organohalide-respiring bacterium Dehalococcoides mccartyi strain CBDB1 was analyzed by nano liquid chromatography coupled to mass spectrometry (LC-MS/MS). Two different preparation methods, (i) in-solution and (ii) in-gel proteolytic digestion were assessed to elucidate the core and the functional proteome of bacterial cultures grown in synthetic anaerobic medium with hexachlorobenzene as sole electron acceptor. A detailed analysis of the data presented is available (Schiffmann et al., 2014) [1].

Proteomics of the organohalide-respiring Epsilonproteobacterium Sulfurospirillum multivorans adapted to tetrachloroethene and other energy substrates.

Organohalide respiration is an environmentally important but poorly characterized type of anaerobic respiration. We compared the global proteome of the versatile organohalide-respiring Epsilonproteobacterium Sulfurospirillum multivorans grown with different electron acceptors (fumarate, nitrate, or tetrachloroethene [PCE]). The most significant differences in protein abundance were found for gene products of the organohalide respiration region. This genomic region encodes the corrinoid and FeS cluster containing PCE reductive dehalogenase PceA and other proteins putatively involved in PCE metabolism such as those involved in corrinoid biosynthesis. The latter gene products as well as PceA and a putative quinol dehydrogenase were almost exclusively detected in cells grown with PCE. This finding suggests an electron flow from the electron donor such as formate or pyruvate via the quinone pool and a quinol dehydrogenase to PceA and the terminal electron acceptor PCE. Two putative accessory proteins, an IscU-like protein and a peroxidase-like protein, were detected with PCE only and might be involved in PceA maturation. The proteome of cells grown with pyruvate instead of formate as electron donor indicates a route of electrons from reduced ferredoxin via an Epsilonproteobacterial complex I and the quinone pool to PCE.

Proteome profile and proteogenomics of the organohalide-respiring bacterium Dehalococcoides mccartyi strain CBDB1 grown on hexachlorobenzene as electron acceptor.

Dehalococcoides mccartyi strain CBDB1 is an obligate organohalide-respiring anaerobic bacterium that is able to transform a wide range of highly relevant halogenated organic contaminants and has been detected in natural and engineered environments. In order to understand the molecular principles, we generated a protein profile of CBDB1 cultivated in a synthetic anaerobic medium with hexachlorobenzene as the sole electron acceptor. The proteome characterization through two different proteomic techniques resulted in 8491 distinct peptides corresponding to 1023 proteins that covered 70% of the annotated 1458 protein-coding sequences. From the 32 annotated reductive dehalogenases homologous genes (rdhA) we were able to detect 16 RdhA proteins. High-quality MS spectra were further used to build a spectra library to provide a platform for precise and fast proteomic searches. In addition, the derived proteome data set was used to apply a proteogenomic approach that led to a refinement of genome annotation of CBDB1. This was mostly due to protein prolongation (13 cases) and detection of translated sequences without an obvious link to existing annotations (5 cases). These data go beyond the current knowledge of the bacterial proteome and provide an expanded platform for a better understanding of the functional cellular. BIOLOGICAL SIGNIFICANCE: Anaerobic reductive dehalogenation of chlorinated organic molecules is a key process for the recycling of halogenated organic substances in natural habitats and highly relevant for groundwater bioremediation. The ability to couple reductive dehalogenation of halogenated compounds to ATP-generation via a respiratory chain is one remarkable capability prevalent in the model organism D. mccartyi strain CBDB1. Therefore, a reference proteome map was generated by comprehensive LC-MS analysis and converted into a SpectraST library to provide a platform for precise and fast shotgun proteomic searches. A proteogenomics approach led to a refinement of gene annotation of CBDB1 and will extend the current knowledge of the bacterial proteome. The approach provides an expanded platform for a better understanding of the functional cellular metabolism and thereby constitutes a tool for comparative studies of protein expression as well as further research on organohalide respiration.