Broad scale functional connectivity for Asian elephants in the Nepal-India transboundary region.

The Nepal-India transboundary region hosts one of Asia's most complex large mammal assemblages, including a small (but growing) population of Asian elephants (Elephas maximus). These elephants occur in four widespread and geographically disjunct subpopulations, and some of them undergo seasonal transboundary movements. We conducted a broad-scale evaluation of the amount and quality of elephant habitat available in the region and of functional landscape connectivity between and within subpopulations using Maxent, circuit theory, and least-cost path analysis. Habitat suitability was highly influenced by abiotic geographical factors (altitude and precipitation) and less by ecological factors (habitat heterogeneity, plant productivity) and human disturbance (distance to settlements). The region had a relatively small amount of high and optimal suitability habitat (12.6% out of 93,700 km2) but all subpopulations seem to be far from carrying capacity, suggesting ample potential for further population growth. Landscape connectivity was higher between and within the west and far-west subpopulations, which should be considered a single subpopulation. The central and ea st subpopulations, however, had low to very low between-subpopulation connectivity. Conservation priorities include maintaining the current connectivity in the west subpopulation and across the border in the east, and protecting high-quality habitats in eastern Nepal. Restoring connectivity between the central and other subpopulations is possible if the number of elephants continues growing, and it should be a long-term conservation aspiration. Maintaining and enhancing landscape connectivity in this region requires transboundary cooperation and coordination between Nepali and Indian authorities. If successful, it will bring considerable benefits for the conservation of elephants and other wildlife.

Biomarker Metabolites Discriminate between Physiological States of Field, Cave and White-nose Syndrome Diseased Bats.

Analysis of volatile organic compound (VOC) emissions using electronic-nose (e-nose) devices has shown promise for early detection of white-nose syndrome (WNS) in bats. Tricolored bats, Perimyotis subflavus, from three separate sampling groups defined by environmental conditions, levels of physical activity, and WNS-disease status were captured temporarily for collection of VOC emissions to determine relationships between these combinations of factors and physiological states, Pseudogymnoascus destructans (Pd)-infection status, and metabolic conditions. Physiologically active (non-torpid) healthy individuals were captured outside of caves in Arkansas and Louisiana. In addition, healthy and WNS-diseased torpid bats were sampled within caves in Arkansas. Whole-body VOC emissions from bats were collected using portable air-collection and sampling-chamber devices in tandem. Electronic aroma-detection data using three-dimensional Principal Component Analysis provided strong evidence that the three groups of bats had significantly different e-nose aroma signatures, indicative of different VOC profiles. This was confirmed by differences in peak numbers, peak areas, and tentative chemical identities indicated by chromatograms from dual-column GC-analyses. The numbers and quantities of VOCs present in whole-body emissions from physiologically active healthy field bats were significantly greater than those of torpid healthy and diseased cave bats. Specific VOCs were identified as chemical biomarkers of healthy and diseased states, environmental conditions (outside and inside of caves), and levels of physiological activity. These results suggest that GC/E-nose dual-technologies based on VOC-detection and analyses of physiological states, provide noninvasive alternative means for early assessments of Pd-infection, WNS-disease status, and other physiological states.

Disease recovery in bats affected by white-nose syndrome.

Processes associated with recovery of survivors are understudied components of wildlife infectious diseases. White-nose syndrome (WNS) in bats provides an opportunity to study recovery of disease survivors, understand implications of recovery for individual energetics, and assess the role of survivors in pathogen transmission. We documented temporal patterns of recovery from WNS in little brown bats (Myotis lucifugus) following hibernation to test the hypotheses that: (1) recovery of wing structure from WNS matches a rapid time scale (i.e. approximately 30 days) suggested by data from free-ranging bats; (2) torpor expression plays a role in recovery; (3) wing physiological function returns to normal alongside structural recovery; and (4) pathogen loads decline quickly during recovery. We collected naturally infected bats at the end of hibernation, brought them into captivity, and quantified recovery over 40 days by monitoring body mass, wing damage, thermoregulation, histopathology of wing biopsies, skin surface lipids and fungal load. Most metrics returned to normal within 30 days, although wing damage was still detectable at the end of the study. Torpor expression declined overall throughout the study, but bats expressed relatively shallow torpor bouts - with a plateau in minimum skin temperature - during intensive healing between approximately days 8 and 15. Pathogen loads were nearly undetectable after the first week of the study, but some bats were still detectably infected at day 40. Our results suggest that healing bats face a severe energetic imbalance during early recovery from direct costs of healing and reduced foraging efficiency. Management of WNS should not rely solely on actions during winter, but should also aim to support energy balance of recovering bats during spring and summer.

Assessment of the Portable C-320 Electronic Nose for Discrimination of Nine Insectivorous Bat Species: Implications for Monitoring White-Nose Syndrome.

The development of new C-320 electronic-nose (e-nose) methods for pre-symptomatic detection of White-Nose Syndrome (WNS) in bats has required efficacy studies of instrument capabilities to discriminate between major sources of volatile organic compounds (VOCs) derived from clinical samples. In this phase-2 study, we further tested this e-nose for capabilities to distinguish between bat species based on differences in whole-body VOC emissions. Live healthy individuals of nine bat species were temporarily captured outside of caves in Arkansas and Louisiana. VOC emissions from bats were collected using newly developed portable air collection and sampling-chamber devices in tandem. Sensor-array output responses to bat VOC emissions were compared to those of 22 pure VOC analytical standards from five chemical classes. Distinct smellprint signatures were produced from e-nose analyses of VOC metabolites derived from individual bat species. Smellprint patterns were analyzed using 2-dimensional and 3-dimensional Principal Component Analysis (PCA) to produce aroma map plots showing effective discrimination between bat species with high statistical significance. These results demonstrate potential instrument efficacy for distinguishing between species-specific, bat-derived VOC metabolite emissions as major components of clinical samples collected from bats in caves for disease detection prior to symptom development. This study provided additional information required to fully test the efficacy of a portable e-nose instrument for diagnostic applications in subsequent phase-3 testing of noninvasive, early WNS disease detection in intra-cave hibernating bats.

Glycerophospholipid Profiles of Bats with White-Nose Syndrome.

Pseudogymnoascus destructans is an ascomycetous fungus responsible for the disease dubbed white-nose syndrome (WNS) and massive mortalities of cave-dwelling bats. The fungus infects bat epidermal tissue, causing damage to integumentary cells and pilosebaceous units. Differences in epidermal lipid composition caused by P. destructans infection could have drastic consequences for a variety of physiological functions, including innate immune efficiency and water retention. While bat surface lipid and stratum corneum lipid composition have been described, the differences in epidermal lipid content between healthy tissue and P. destructans-infected tissue have not been documented. In this study, we analyzed the effect of wing damage from P. destructans infection on the epidermal polar lipid composition (glycerophospholipids [GPs] and sphingomyelin) of little brown bats (Myotis lucifugus). We hypothesized that infection would lead to lower levels of total lipid or higher oxidized lipid product proportions. Polar lipids from three damaged and three healthy wing samples were profiled by electrospray ionization tandem mass spectrometry. We found lower total broad lipid levels in damaged tissue, specifically ether-linked phospholipids, lysophospholipids, phosphatidylcholine, and phosphatidylethanolamine. Thirteen individual GP species from four broad GP classes were present in higher amounts in healthy tissue. Six unsaturated GP species were absent in damaged tissue. Our results confirm that P. destructans infection leads to altered lipid profiles. Clinical signs of WNS may include lower lipid levels and lower proportions of unsaturated lipids due to cellular and glandular damage.

Isolation and identification of an extracellular subtilisin-like serine protease secreted by the bat pathogen Pseudogymnoascus destructans.

White nose syndrome (WNS) is a cutaneous fungal disease of bats. WNS is responsible for unprecedented mortalities in North American cave bat populations. There have been few descriptions of enzyme activities that may function in WNS host/pathogen interactions, while no study has isolated and described secreted proteases. To address the hypothesis that Pseudogymnoascus destructans secretes extracellular proteases that function in wing necrosis during WNS infection, the object of this study was to culture P. destructans on various media, then isolate and structurally identify those proteases accumulated stably in the culture medium. We found a single dominant protease activity on minimal nutrient broth enriched with protein substrates, which was strongly inhibited by phenylmethylsulfonyl fluoride. This P. destructans serine protease (PdSP1) was isolated by preparative isoelectric focusing and concanavalin A lectin affinity chromatography. PdSP1 showed a molecular weight 27,900 (estimated by SDS-PAGE), broad pH optimum 6-8, and temperature optimum 60°C. Structural characterization of PdSP1 by MALDI-TOF MS, Orbitrap MS/MS, and Edman amino-terminal peptide sequencing matched it directly to a hypothetical protein accession from the sequenced P. destructans genome that is further identified as a MEROPS family S8A subtilisin-like serine peptidase. Two additional isoforms, PdSP2 and PdSP3, were identified in the P. destructans genome with 90% and 53% homology, respectively. P. destructans S8A serine proteases showed closer sequence conservation to P. pannorum and plant pathogenic fungi than to human pathogenic dermatophytes. Peptide-specific polyclonal antibodies developed from the PdSP1 sequence detected the protein in western blots. These subtilisin-like serine proteases are candidates for further functional studies in WNS host-pathogen interaction.

Fatty acid methyl ester profiles of bat wing surface lipids.

Sebocytes are specialized epithelial cells that rupture to secrete sebaceous lipids (sebum) across the mammalian integument. Sebum protects the integument from UV radiation, and maintains host microbial communities among other functions. Native glandular sebum is composed primarily of triacylglycerides (TAG) and wax esters (WE). Upon secretion (mature sebum), these lipids combine with minor cellular membrane components comprising total surface lipids. TAG and WE are further cleaved to smaller molecules through oxidation or host enzymatic digestion, resulting in a complex mixture of glycerolipids (e.g., TAG), sterols, unesterified fatty acids (FFA), WE, cholesteryl esters, and squalene comprising surface lipid. We are interested if fatty acid methyl ester (FAME) profiling of bat surface lipid could predict species specificity to the cutaneous fungal disease, white nose syndrome (WNS). We collected sebaceous secretions from 13 bat spp. using Sebutape(®) and converted them to FAME with an acid catalyzed transesterification. We found that Sebutape(®) adhesive patches removed ~6× more total lipid than Sebutape(®) indicator strips. Juvenile eastern red bats (Lasiurus borealis) had significantly higher 18:1 than adults, but 14:0, 16:1, and 20:0 were higher in adults. FAME profiles among several bat species were similar. We concluded that bat surface lipid FAME profiling does not provide a robust model predicting species susceptibility to WNS. However, these results provide baseline data that can be used for lipid roles in future ecological studies, such as life history, diet, or migration.

Triacylglyceride composition and fatty acyl saturation profile of a psychrophilic and psychrotolerant fungal species grown at different temperatures.

Pseudogymnoascus destructans is a psychrophilic fungus that infects cutaneous tissues in cave dwelling bats, and it is the causal agent for white nose syndrome (WNS) in North American (NA) bat populations. Geomyces pannorum is a related psychrotolerant keratinolytic species that is rarely a pathogen of mammals. In this study, we grew P. destructans and G. pannorum in static liquid cultures at favourable and suboptimal temperatures to: 1) determine if triacylglyceride profiles are species-specific, and 2) determine if there are differences in fatty acyl (FA) saturation levels with respect to temperature. Total lipids isolated from both fungal spp. were separated by thin-layer chromatography and determined to be primarily sterols (∼15 %), free fatty acids (FFAs) (∼45 %), and triacylglycerides (TAGs) (∼50 %), with minor amounts of mono-/diacylglycerides and sterol esters. TAG compositions were profiled by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF). Total fatty acid methyl esters (FAMEs) and acyl lipid unsaturation levels were determined by gas chromatography-mass spectrometry (GC-MS). Pseudogymnoascus destructans produced higher proportions of unsaturated 18C fatty acids and TAGs than G. pannorum. Pseudogymnoascus destructans and G. pannorum produced up to a two-fold increase in 18:3 fatty acids at 5 °C than at higher temperatures. TAG proportion for P. destructans at upper and lower temperature growth limits was greater than 50 % of total dried mycelia mass. These results indicate fungal spp. alter acyl lipid unsaturation as a strategy to adapt to cold temperatures. Differences between their glycerolipid profiles also provide evidence for a different metabolic strategy to support psychrophilic growth, which may influence P. destructans' pathogenicity to bats.

Glycerophospholipid analysis of Eastern red bat (Lasiurus borealis) hair by electrospray ionization tandem mass spectrometry.

Pilosebaceous units found in the mammalian integument are composed of a hair follicle, the proximal portion of the hair shaft, a sebaceous gland, and the erector pili muscle. Pilosebaceous units release protective oils, or sebum, by holocrine secretion onto skin and hair through rupturing of sebocytes. Sebum is composed largely of polar and neutral lipids including glycerolipids, free fatty acids, sterols, wax esters, sterol esters, and squalene. In addition to these lipid classes, there is a small proportion of ionic/anionic glycerophospholipids (GPs). Composition of GPs on hair is rarely addressed despite their broad biological activities as signaling molecules and membrane stability. Furthermore, knowledge on GP composition in bats is lacking. Bat GP composition is important to document due to GP roles ranging from decreasing drag during migration to interaction with the integumentary microbiome. In this study, we analyzed GP molecular composition with liquid chromatography electrospray ionization tandem mass spectrometry and compared GP content to previous literature. A total of 152 GPs were detected. Broad GP classes identified include lysophosphatidylcholine, phosphatidylcholine (PC), lysophosphatidylethanolamine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidic acid, and phosphatidylglycerol, with PC being the most abundant class. The acyl components were consistent with fatty acid methyl esters and triacylglyceride moieties found in Eastern red bat sebum. Glycerophospholipid proportions of the hair surface were different from a previous study on bat lung surfactants. This study determined the broad class and molecular species of bat sebum GPs that may be used in future ecological studies in vespertilionid bats.

Sebaceous lipid profiling of bat integumentary tissues: quantitative analysis of free Fatty acids, monoacylglycerides, squalene, and sterols.

White-nose syndrome (WNS) is a fungal disease caused by Pseudogymnoascus destructans and is devastating North American bat populations. Sebaceous lipids secreted from host integumentary tissues are implicated in the initial attachment and recognition of host tissues by pathogenic fungi. We are interested in determining if ratios of lipid classes in sebum can be used as biomarkers to diagnose severity of fungal infection in bats. To first establish lipid compositions in bats, we isolated secreted and integral lipid fractions from the hair and wing tissues of three species: big brown bats (Eptesicus fuscus), Eastern red bats (Lasiurus borealis), and evening bats (Nycticeius humeralis). Sterols, FFAs, MAGs, and squalene were derivatized as trimethylsilyl esters, separated by gas chromatography, and identified by mass spectrometry. Ratios of sterol to squalene in different tissues were determined, and cholesterol as a disease biomarker was assessed. Free sterol was the dominant lipid class of bat integument. Squalene/sterol ratio is highest in wing sebum. Secreted wing lipid contained higher proportions of saturated FFAs and MAGs than integral wing or secreted hair lipid. These compounds are targets for investigating responses of P. destructans to specific host lipid compounds and as biomarkers to diagnose WNS.

Profiling the triacylglyceride contents in bat integumentary lipids by preparative thin layer chromatography and MALDI-TOF mass spectrometry.

The mammalian integument includes sebaceous glands that secrete an oily material onto the skin surface. Sebum production is part of the innate immune system that is protective against pathogenic microbes. Abnormal sebum production and chemical composition are also a clinical symptom of specific skin diseases. Sebum contains a complex mixture of lipids, including triacylglycerides, which is species-specific. The broad chemical properties exhibited by diverse lipid classes hinder the specific determination of sebum composition. Analytical techniques for lipids typically require chemical derivatizations that are labor-intensive and increase sample preparation costs. This paper describes how to extract lipids from mammalian integument, separate broad lipid classes by thin-layer chromatography, and profile the triacylglyceride contents using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. This robust method enables a direct determination of the triacylglyceride profiles among species and individuals, and it can be readily applied to any taxonomic group of mammals.

Variation in litter size: a test of hypotheses in Richardson's ground squirrels.

We studied litter size variation in a population of Richardson's ground squirrels (Spermophilus richardsonii) in Alberta, Canada, from 1987 to 2004. Litter size at first emergence of juveniles from the natal burrow ranged from 1 to 14; the most common litter sizes, collectively accounting for 41.0% of 999 litters, were 6 and 7. The number of offspring surviving to adulthood (attained on emergence from hibernation as yearlings) increased with increasing litter size, a result that was not predicted by Lack's "optimal litter size" hypothesis, Mountford's "cliff-edge" effect, or the "bad-years" effect. Contrary to the negative effects predicted by the "cost of reproduction" hypothesis, litter size had no significant influence on survival of mothers to the subsequent year or on the size of the subsequent litter. Rather, our results best fit the predictions of the "individual optimization" hypothesis, which suggests that litter size is determined by the body condition and environmental circumstances of each mother. Supporting this hypothesis, survival of individual offspring was not significantly associated with litter size. Additionally, year-to-year changes in maternal body mass at mating were positively associated with concurrent changes in litter size (r = 0.56), suggesting that litter size depends on the body condition of the mother. Because the mean number of recruits to adulthood increased as litter size increased (r2 = 0.96) and litter size increased with maternal condition, offspring productivity was greater for mothers in better body condition.