Life Sciences Commons^™

Does Channel Island Acmispon (Fabaceae) Form Cohesive Evolutionary Groups?, Mitchell E. Mcglaughlin, Lynn Riley, Kaius Helenurm, Lisa A. Wallace

Biological Sciences Faculty Publications

The California Channel Islands are unique relative to other island chains due to their close proximity to the California mainland and the fact that individual islands, or groups of islands, vary in their distance to the mainland and other islands. This orientation raises questions about whether island taxa with widespread distributions form cohesive evolutionary units, or if they are actually composed of several distinct evolutionary entities, either derived from independent mainland-to-island colonization events or divergence due to prolonged allopatric isolation. The 4 northern islands are clustered in a line (6-8 km separation among islands), while the 4 southern islands are …

Nocturnally-Migrating Birds Traverse Earth's Most Light-Polluted Regions, And Bright Lights Confound Their Habitat Use En Route, Jeffrey J. Butler, James D. Mclaren, Sergio A. Cabrera-Cruz, Jaclyn A. Smolinsky, Timothy D. Schreckengost, Matthew E. Boone, E. Emiel Van Loon, Deanna K. Dawson, Eric L. Walters

Biological Sciences Faculty Publications

No abstract provided.

Towards Understanding The Role Of Small Mammals In The Transmission Of Lyme Disease In Virginia, Usa, Robert K. Rose, Laura Bitzer, Wayne Hynes, Holly D. Gaff, Robyn Nadolny, Jory Brinkerhoff

Biological Sciences Faculty Publications

Larvae and nymphs of many species of ticks select small mammals, reptiles, and birds as hosts. Since 2011, we have examined more than 1,000 small mammals of 10 species and collected a small tissue sample as well as all visible ticks; rates of infestation range from 18% in harvest mice to 53% in meadow voles. Identification of immature ticks is challenging and requires molecular or genetic methods beyond classification as being from the genus Ixodes or another genus. Ixodes ticks include Ixodes scapularis, the species that transmits the Lyme disease-causing bacterium Borrelia burgdorferi to humans. Of the approximately 2,300 ticks …

Energy-Rich Mesopelagic Fishes Revealed As A Critical Prey Resource For A Deep-Diving Predator Using Quantitative Fatty Acid Signature Analysis, Chandra Goetsch, Melinda G. Conners, Suzanne M. Budge, Yoko Mitani, William A. Walker, Jeffrey F. Bromaghin, Samantha E. Simmons, Colleen Reichmuth, Daniel P. Costa

Biological Sciences Faculty Publications

Understanding the diet of deep-diving predators can provide essential insight to the trophic structure of the mesopelagic ecosystem. Comprehensive population-level diet estimates are exceptionally difficult to obtain for elusive marine predators due to the logistical challenges involved in observing their feeding behavior and collecting samples for traditional stomach content or fecal analyses. We used quantitative fatty acid signature analysis (QFASA) to estimate the diet composition of a wide-ranging mesopelagic predator, the northern elephant seal (Mirounga angustirostris), across five years. To implement QFASA, we first compiled a library of prey fatty acid (FA) profiles from the mesopelagic eastern North Pacific. Given …

Interesting, Provocative, And Enigmatic: Morphological Observations On Southeastern Quillworts (Isoetes Isoetaceae, Lycopodiophyta, Rebecca D. Bray, Peter W. Schafran, Lytton J. Musselman

Biological Sciences Faculty Publications

The unique morphology of quillworts has received limited attention, and such studies have been limited to only a few species. Our extensive field work during the past two decades has revealed variation in the structures of the plant, including the rhizomorph, scales, phyllopodia, and abscission caps. Polarity of the axes of southeastern quillwort rhizomorphs varies from discoid (most species), to elongate, to upright with axis branching in several species. In species of intermittent streams, these branched rhizomorphs produce plantlets that break off in running water. Scales are tiny brown or black structures in alternating whorls with sporophylls (leaves). They are …

Better Living Through Chemistry: Addressing Emerging Antibiotic Resistance, Nathan P. Coussens, Ashley L. Molinaro, Kayla J. Culbertson, Tyler Peryea, Gergely Zahoránszky-Köhalmi, Matthew D. Hall, Dayle A. Daines

Biological Sciences Faculty Publications

The increasing emergence of multidrug-resistant bacteria is recognized as a major threat to human health worldwide. While the use of small molecule antibiotics has enabled many modern medical advances, it has also facilitated the development of resistant organisms. This minireview provides an overview of current small molecule drugs approved by the US Food and Drug Administration (FDA) for use in humans, the unintended consequences of antibiotic use, and the mechanisms that underlie the development of drug resistance. Promising new approaches and strategies to counter antibiotic-resistant bacteria with small molecules are highlighted. However, continued public investment in this area is critical …

Range Expansion Of Tick Disease Vectors In North America: Implications For Spread Of Tick-Borne Disease, Daniel E. Sonenshine

Biological Sciences Faculty Publications

Ticks are the major vectors of most disease-causing agents to humans, companion animals and wildlife. Moreover, ticks transmit a greater variety of pathogenic agents than any other blood-feeding arthropod. Ticks have been expanding their geographic ranges in recent decades largely due to climate change. Furthermore, tick populations in many areas of their past and even newly established localities have increased in abundance. These dynamic changes present new and increasing severe public health threats to humans, livestock and companion animals in areas where they were previously unknown or were considered to be of minor importance. Here in this review, the geographic …

Adiposity And Fat Metabolism During Combined Fasting And Lactation In Elephant Seals, Melinda Fowler, Cory Champagne, Daniel Croker

Biological Sciences Faculty Publications

Animals that fast depend on mobilizing lipid stores to power metabolism. Northern elephant seals (Mirounga angustirostris) incorporate extended fasting into several life-history stages: development, molting, breeding and lactation. The physiological processes enabling fasting and lactation are important in the context of the ecology and life history of elephant seals. The rare combination of fasting and lactation depends on the efficient mobilization of lipid from adipose stores and its direction into milk production. The mother elephant seal must ration her finite body stores to power maintenance metabolism, as well as to produce large quantities of lipid and protein rich …

Do Avian Blood Parasites Influence Hypoxia Physiology In A High Elevation Environment?, Farah Ishtiaq, Sahas Barve

Biological Sciences Faculty Publications

BACKGROUND: Montane birds which engage in elevational movements have evolved to cope with fluctuations in environmental hypoxia, through changes in physiological parameters associated with blood oxygen-carrying capacity such as haemoglobin concentration (Hb) and haematocrit (Hct). In particular, elevational migrants which winter at low elevations, encounter varying intensities of avian haemosporidian parasites as they traverse heterogeneous environments. Whilst high intensity parasite infections lead to anaemia, one can expect that the ability to cope with haemosporidian infections should be a key trait for elevational migrants that must be balanced against reducing the oxygen-carrying capacity of blood in response to high elevation. In …

Review: Application Of Tick Control Technologies For Blacklegged, Lone Star, And American Dog Ticks, Alexis White, Holly Gaff

Biological Sciences Faculty Publications

Tick population control technologies have been studied for several decades but no method is successful in all situations. The success of each technology depends on tick species identity and abundance, host species identity and abundance, phenology of both ticks and hosts, geographic region, and a multitude of other factors. Here we review current technologies, presenting an overview of each and its effect on three common tick species in the eastern United States: blacklegged ticks (Ixodes scapularis (Say; Ixodida: Ixodidae)), lone star ticks (Amblyomma americanum (Linnaeus; Ixodida: Ixodidae)), and American dog ticks (Dermacentor variabilis (Say; Ixodida: Ixodidae)). Moreover, …

The Spatial Context Of “Winning” In Mpa Network Design: Location Matters, Andrew S. Kough, Claire B. Paris, Mark J. Butler Iv

Biological Sciences Faculty Publications

(First paragraph) Chollett et al. (2017) make the case that a local network of marine protected areas (MPAs) enhances fisheries for Caribbean spiny lobster (Panulirus argus) off the coast of Honduras. However, their simulation focused on one ecoregion where self-recruitment is predicted to be among the highest in the Caribbean (Cowen, Paris, & Srinivasan, 2006). The shallow banks and scattered cays of the Honduran-Nicaraguan Rise, separating the Cayman and Colombian basins, create an obstacle to the powerful southern Caribbean jet (Richardson, 2005), fostering an ideal location for topographically steered eddies and larval retention. Local management,whether based on traditional …

Ticks, Ixodes Scapularis, Feed Repeatedly On White-Footed Mice Despite Strong Inflammatory Response: An Expanding Paradigm For Understanding Tick-Host Interactions, Jennifer M. Anderson, Ian N. Moore, Bianca M. Nagata, José M.C. Ribeiro, Jesus G. Valenzuela, Daniel E. Sonenshine

Biological Sciences Faculty Publications

Ticks transmit infectious agents including bacteria, viruses and protozoa. However, their transmission may be compromised by host resistance to repeated tick feeding. Increasing host resistance to repeated tick bites is well known in laboratory animals, including intense inflammation at the bite sites. However, it is not known whether this also occurs in wild rodents such as white-footed mice, Peromyscus leucopus, and other wildlife, or if it occurs at all. According to the "host immune incompetence" hypothesis, if these mice do not have a strong inflammatory response, they would not reject repeated tick bites by Ixodes scapularis. To test …

Modeling Virus Coinfection To Inform Management Of Maize Lethal Necrosis In Kenya, Frank H. Hilker, Linda S. J. Allen, Vrushali A. Bokil, Cheryl J. Briggs, Zhilan Feng, Karen A. Garrett, Louis J. Gross, Frédéric M. Hamelin, Michael J. Jeger, Carrie A. Manore, Alison G. Power, Margaret G. Redinbaugh, Megan A. Rúa, Nik J. Cunniffee

Biological Sciences Faculty Publications

Maize lethal necrosis (MLN) has emerged as a serious threat to food security in sub-Saharan Africa. MLN is caused by coinfection with two viruses, Maize chlorotic mottle virus and a potyvirus, often Sugarcane mosaic virus. To better understand the dynamics of MLN and to provide insight into disease management, we modeled the spread of the viruses causing MLN within and between growing seasons. The model allows for transmission via vectors, soil, and seed, as well as exogenous sources of infection. Following model parameterization, we predict how management affects disease prevalence and crop performance over multiple seasons. Resource-rich farmers with …

Human Rickettsial Pathogen Modulates Arthropod Organic Anion Transporting Polypeptide And Tryptophan Pathway For Its Survival In Ticks, Vikas Taank, Shovan Dutta, Amrita Dasgupta, Durland Fish, John F. Anderson, Hameeda Sultana, Girish Neelakanta

Biological Sciences Faculty Publications

The black-legged tick Ixodes scapularis transmits the human anaplasmosis agent, Anaplasma phagocytophilum. In this study, we show that A. phagocytophilum specifically up-regulates I. scapularis organic anion transporting polypeptide, isoatp4056 and kynurenine amino transferase (kat), a gene involved in the production of tryptophan metabolite xanthurenic acid (XA), for its survival in ticks. RNAi analysis revealed that knockdown of isoatp4056 expression had no effect on A. phagocytophilum acquisition from the murine host but affected the bacterial survival in tick cells. Knockdown of the expression of kat mRNA alone or in combination with isoatp4056 mRNA significantly affected A. phagocytophilum survival …

Urbanization Alters Communities Of Flying Arthropods In Parks And Gardens Of A Medium-Sized City, Edward Lagucki, Justin D. Burdine, Kevin E. Mccluney

Biological Sciences Faculty Publications

Urbanization transforms undeveloped landscapes into built environments, causing changes in communities and ecological processes. Flying arthropods play important roles in these processes as pollinators, decomposers, and predators, and can be important in structuring food webs. The goal of this study was to identify associations between urbanization and the composition of communities of flying (and floating) arthropods within gardens and parks in a medium-sized mesic city. We predicted that flying arthropod abundance and diversity would respond strongly to percent impervious surface and distance to city center, measurements of urbanization. Flying arthropods were sampled from 30 gardens and parks along an urbanization …

Microbial Invasion Vs. Tick Immune Regulation, Daniel E. Sonenshine, Kevin R. Macaluso

Biological Sciences Faculty Publications

Ticks transmit a greater variety of pathogenic agents that cause disease in humans and animals than any other haematophagous arthropod, including Lyme disease, Rocky Mountain spotted fever, human granulocytic anaplasmosis, babesiosis, tick-borne encephalitis, Crimean Congo haemorhagic fever, and many others (Gulia-Nuss et al., 2016). Although diverse explanations have been proposed to explain their remarkable vectorial capacity, among the most important are their blood feeding habit, their long term off-host survival, the diverse array of bioactive molecules that disrupt the host's natural hemostatic mechanisms, facilitate blood flow, pain inhibitors, and minimize inflammation to prevent immune rejection (Hajdušek et al., 2013). Moreover, …

Allergen Homologs In The Euroglyphus Maynei Draft Genome, S. Dean Rider, Majorie S. Morgan, Larry G. Arlian

Biological Sciences Faculty Publications

Euroglyphus maynei is a house dust mite commonly found in homes worldwide and is the source of allergens that sensitize and induce allergic reactions in humans. It is the source of species-specific allergens as well as allergens that are cross-reactive with the allergens from house dust mites Dermatophagoides farinae and D. pteronyssinus, and the ectoparasitic scabies mite Sarcoptes scabiei. The genomics, proteomics and molecular biology of E. maynei and its allergens have not been as extensively investigated as those of D. farinae, D. pteronyssinus, and S. scabiei where natural and recombinant allergens from these species have been characterized. Until now, …

Accommodating Mixed-Severity Fire To Restore And Maintain Ecosystem Integrity With A Focus On The Sierra Nevada Of California, Usa, Dominick A. Dellasala, Richard L. Hutto, Chad T. Hanson, Monica L. Bond, Timothy Ingalsbee, Dennis C. Odion, William L. Baker

Biological Sciences Faculty Publications

Existing fire policy encourages the maintenance of ecosystem integrity in fire management, yet this is difficult to implement on lands managed for competing economic, human safety, and air quality concerns. We discuss a fire management approach in the mid-elevations of the Sierra Nevada, California, USA, that may exemplify similar challenges in other fire-adapted regions of the western USA. We also discuss how managing for pyrodiversity through mixed-severity fires can promote ecosystem integrity in Sierran mixed conifer and ponderosa pine (Pinus ponderosa Laws) forests. To illustrate, we show how coarse-filter (landscape-level) and complementary fine-filter (species-level) approaches can enhance forest management …

Anaplasma Phagocytophilum Infection Modulates Expression Of Megakaryocyte Cell Cycle Genes Through Phosphatidylinositol-3-Kinase Signaling, Supreet Khanal, Hameeda Sultana, John D. Catravas, Jason A. Carlyon, Girish Neelakanta

Biological Sciences Faculty Publications

Anaplasma phagocytophilum, the agent of human granulocytic anaplasmosis infects neutrophils and other cells from hematopoietic origin. Using human megakaryocytic cell line, MEG-01, we show that expression of cell cycle genes in these cells are altered upon A. phagocytophilum infection. Expression of several cell cycle genes in MEG-01 cells was significantly up regulated at early and then down regulated at later stages of A. phagocytophilum infection. Lactate dehydrogenase (LDH) assays revealed reduced cellular cytotoxicity in MEG-01 cells upon A. phagocytophilum infection. The levels of both PI3KCA (p110 alpha, catalytic subunit) and PI3KR1 (p85, regulatory subunit) of Class …

Large Marine Protected Areas Represent Biodiversity Now And Under Climate Change, T. E. Davies, S. M. Maxwell, K. Kaschner, C. Garilao, N. C. Ban

Biological Sciences Faculty Publications

Large marine protected areas (>30,000 km²) have a high profile in marine conservation, yet their contribution to conservation is contested. Assessing the overlap of large marine protected areas with 14,172 species, we found large marine protected areas cover 4.4% of the ocean and at least some portion of the range of 83.3% of the species assessed. Of all species within large marine protected areas, 26.9% had at least 10% of their range represented, and this was projected to increase to 40.1% in 2100. Cumulative impacts were significantly higher within large marine protected areas than outside, refuting the …

Reply To Marques Et Al. (2017): How To Best Handle Potential Detectability Bias, Richard L. Hutto

Biological Sciences Faculty Publications

Marques et al. (2017) write in response to a communications paper in which I (Hutto 2016) question whether a model-based approach is the only way to deal with the detectability problem inherent in bird survey work. The model-based approach to dealing with potential detectability bias is widely viewed as the operational gold standard, and I welcome the comments from experts in this field. The authors did uncover some errors on my part, which I address below, but they also misrepresented my most important points, so I am glad to have the opportunity to respond here. [1st paragraph]

Population-Level Coordination Of Pigment Response In Individual Cyanobacterial Cells Under Altered Nitrogen Levels, Jaclyn Murton, Aparna Nagarajan, Amelia Y. Nguyen, Michelle Liberton, Harmony A. Hancock, Himadri B. Pakrasi, Jerilyn A. Timlin

Biological Sciences Faculty Publications

Cyanobacterial phycobilisome (PBS) pigment-protein complexes harvest light and transfer the energy to reaction centers. Previous ensemble studies have shown that cyanobacteria respond to changes in nutrient availability by modifying the structure of PBS complexes, but this process has not been visualized for individual pigments at the single-cell level due to spectral overlap. We characterized the response of four key photosynthetic pigments to nitrogen depletion and repletion at the subcellular level in individual, live Synechocystis sp. PCC 6803 cells using hyperspectral confocal fluorescence microscopy and multivariate image analysis. Our results revealed that PBS degradation and re-synthesis comprise a rapid response to …

Identification Of Antigenic Sarcoptes Scabiei Proteins For Use In A Diagnostic Test And Of Non-Antigenic Proteins That May Be Immunomodulatory, Majorie S. Morgan, S. Dean Rider, Larry G. Arlian

Biological Sciences Faculty Publications

Background

Scabies, caused by the mite, Sarcoptes scabiei, infects millions of humans, and many wild and domestic mammals. Scabies mites burrow in the lower stratum corneum of the epidermis of the skin and are the source of substances that are antigenic or modulate aspects of the protective response of the host. Ordinary scabies is a difficult disease to diagnose.

Objective

The goal of this project was to identify S. scabiei proteins that may be candidate antigens for use in a diagnostic test or may be used by the mite to modulate the host’s protective response.

Methods

An aqueous …

Animals And Their Epibiota As Net Autotrophs: Size Scaling Of Epibiotic Metabolism On Snail Shells, Nicole Lukens, Benjamin Kraemer, Vanessa Constant, Ellen J. Hamann, Ellinor Michel, Anne M. Socci, Yvonne Vadeboncoeur, Peter B. Mcintyre

Biological Sciences Faculty Publications

Animals are heterotrophic by definition, but species from many taxonomic groups are hosts to epibiota that may alter their net metabolism. We tested the degree to which snail-shell epibiota can generate net ecosystem productivity for snails and their epibiota (snail–epibiota ecosystems; SEEs) after accounting for snail respiration. We focused on 3 species from the Lavigeria snail assemblage in Africa’s Lake Tanganyika and quantified the scaling of SEE metabolism with shell size under light and dark conditions. The metabolism of snails and their epibiota shifted significantly across the size gradient. SEEs of large snails (>20 mm) were consistently autotrophic during …

A Variant Pfcrt Isoform Can Contribute To Plasmodium Falciparum Resistance To The First-Line Partner Drug Piperaquine, Satish K. Dhingra, Devasha Redhi, Jill M. Combrinck, Tomas Yeo, John Okombo, Philipp P. Henrich, Annie N. Cowell, Purva Gupta, Matthew L. Stegman, Jonathan M. Hoke, Roland A. Cooper, Elizabeth Winzeler, Sachel Mok, Timothy J. Egan, David A. Fidock

Biological Sciences Faculty Publications

Current efforts to reduce the global burden of malaria are threatened by the rapid spread throughout Asia of Plasmodium falciparum resistance to artemisinin-based combination therapies, which includes increasing rates of clinical failure with dihydroartemisinin plus piperaquine (PPQ) in Cambodia. Using zinc finger nuclease-based gene editing, we report that addition of the C101F mutation to the chloroquine (CQ) resistance-conferring PfCRT Dd2 isoform common to Asia can confer PPQ resistance to cultured parasites. Resistance was demonstrated as significantly higher PPQ concentrations causing 90% inhibition of parasite growth (IC₉₀) or 50% parasite killing (50% lethal dose [LD₅₀]). This mutation …

Turning Performance Of Brief Squid Lolliguncula Brevis During Attacks On Shrimp And Fish, Rachel A. Jastrebsky, Ian K. Bartol, Paul S. Krueger

Biological Sciences Faculty Publications

Although squid are generally considered to be effective predators, little is currently known of how squid maneuver and position themselves during prey strikes. In this study, high-speed video and kinematic analyses were used to study attacks by the brief squid Lolliguncula brevis on both shrimp and fish. Squid attack successwas high (>80%) and three behavioral phases were identified: (1) approach, (2) strike and (3) recoil. Lolliguncula brevis demonstrated greater maneuverability (i.e. a smaller length-specific turning radius) and employed more body adjustments (i.e. mantle angle posturing) during approaches toward shrimp versus fish. Squid exhibited higher linear approach/strike velocities and accelerations …

Ticks Elicit Variable Fibrinogenolytic Activities Upon Feeding On Hosts With Different Immune Backgrounds, Ashish Vora, Vikas Taank, John F. Anderson, Durland Fish, Daniel E. Sonenshine, John D. Catravas, Hameeda Sultana, Girish Neelakanta

Biological Sciences Faculty Publications

Ticks secrete several anti-hemostatic factors in their saliva to suppress the host innate and acquired immune defenses against infestations. Using Ixodes scapularis ticks and age-matched mice purchased from two independent commercial vendors with two different immune backgrounds as a model, we show that ticks fed on immunodeficient animals demonstrate decreased fibrinogenolytic activity in comparison to ticks fed on immunocompetent animals. Reduced levels of D-dimer (fibrin degradation product) were evident in ticks fed on immunodeficient animals in comparison to ticks fed on immunocompetent animals. Increased engorgement weights were noted for ticks fed on immunodeficient animals in comparison to ticks fed on …

Adaptation To Blue Light In Marine Synechococcus Requires Mpeu, An Enzyme With Similarity To Phycoerythrobilin Lyase Isomerases, Wendy M. Schluchter, Rania Mohamed Mahmoud, Joseph Sanfilippo, Adam A. Nguyen, Johann A. Strnat, Frédéric Partensky, Laurence Garczarek, Nabil Kassem, David M. Kehoe

Biological Sciences Faculty Publications

Marine Synechococcus has successfully adapted to environments with different light colors, which likely contributes to this genus being the second most abundant group of microorganisms worldwide. Populations of Synechococcus that grow in deep, blue ocean waters contain large amounts of the blue-light absorbing chromophore phycourobilin (PUB) in their light harvesting complexes (phycobilisomes). Here, we show that all Synechococcus strains adapted to blue light possess a gene called mpeU. MpeU is structurally similar to phycobilin lyases, enzymes that ligate chromophores to phycobiliproteins. Interruption of mpeU caused a reduction in PUB content, impaired phycobilisome assembly and reduced growth rate more strongly in …

Blubber Transciptome Response To Acute Stress Axis Activation Involves Transient Charges In Adipogenesis And Lipolysis In Fast-Adapted Marine Mammal, J. I. Khudyakov, C. D. Champagne, L. M. Meneghetti, D. E. Crocker

Biological Sciences Faculty Publications

Stress can compromise an animal's ability to conserve metabolic stores and participate in energy-demanding activities that are critical for fitness. Understanding how wild animals, especially those already experiencing physiological extremes (e.g. fasting), regulate stress responses is critical for evaluating the impacts of anthropogenic disturbance on physiology and fitness, key challenges for conservation. However, studies of stress in wildlife are often limited to baseline endocrine measurements and few have investigated stress effects in fasting-adapted species. We examined downstream molecular consequences of hypothalamic-pituitary-adrenal (HPA) axis activation by exogenous adrenocorticotropic hormone (ACTH) in blubber of northern elephant seals due to the ease of …

The Behavioral And Physiological Effects Of High-Fat Diet And Alcohol Consumption: Sex Differences In C57bl6/J Mice, Rachel R. Gelineau, Nicole L. Arruda, Jasmin A. Hicks, Isabella Monteiro De Pina, Aikaterini Hatzidis, Joseph A. Seggio

Biological Sciences Faculty Publications

Background and Objective: Animal studies can be a great tool to investigate sex differences in a variety of different ways, including behavioral and physiological responses to drug treatments and different “lifestyle variables” such as diets. Consumption of both high-fat diets and alcohol is known to affect anxiety behaviors and overall health. This project investigated how high-fat diet and alcohol access and its combination affected the behavior and physiology of male and female C57BL/6J mice.

Method: Mice were separated into three food groups: high-fat diet, 10% fat diet, and regular chow, and each group was paired with either water or 10% …