Temporal matches between monarch butterfly and milkweed population changes over the past 25,000 years.

In intimate ecological interactions, the interdependency of species may result in correlated demographic histories. For species of conservation concern, understanding the long-term dynamics of such interactions may shed light on the drivers of population decline. Here, we address the demographic history of the monarch butterfly, Danaus plexippus, and its dominant host plant, the common milkweed Asclepias syriaca (A. syriaca), using broad-scale sampling and genomic inference. Because genetic resources for milkweed have lagged behind those for monarchs, we first release a chromosome-level genome assembly and annotation for common milkweed. Next, we show that despite its enormous geographic range across eastern North America, A. syriaca is best characterized as a single, roughly panmictic population. Using approximate Bayesian computation with random forests (ABC-RF), a machine learning method for reconstructing demographic histories, we show that both monarchs and milkweed experienced population expansion during the most recent recession of North American glaciers 10,000-20,000 years ago. Our data also identify concurrent population expansions in both species during the large-scale clearing of eastern forests (∼200 years ago). Finally, we find no evidence that either species experienced a reduction in effective population size over the past 75 years. Thus, the well-documented decline of monarch abundance over the past 40 years is not visible in our genomic dataset, reflecting a possible mismatch of the overwintering census population to effective population size in this species.

Herbivory, plant traits and nectar chemistry interact to affect the community of insect visitors and pollination in common milkweed, Asclepias syriaca.

Herbivory can alter plant fitness directly through changing reproductive allocation and indirectly through changing pollinator identity or behavior. Common milkweed is a plant of conservation concern with an inducible chemical defense that is also an important nectar resource. In this study, we aim to understand how herbivory severity and plant traits, including morphology and nectar chemistry, interact to affect insect visitation and pod production in common milkweed. We conducted pollinator watches on plants with experimentally varied herbivory severity and quantified insect frequency and visit length as a response to nectar chemistry, ramet height, number of inflorescences, number of flowers per inflorescence and percent tissue removed. We also quantified pollinator effectiveness and importance. Increased herbivory severity reduced floral displays, including fewer inflorescences and fewer flowers per inflorescence. A reduced floral display was correlated with reduced sucrose, fructose and glucose and resulted in a reduced number and species richness of insect visitors. Fewer flowers per inflorescence reduced the frequency of bumble bee and fly visitors, which were two important pollinators. Although honeybees, flies, small bees, soldier beetles and bumble bees were equally effective pollinators, only bumble bee frequency was positively correlated with pod production. The differences in pollinator visitation have the potential to create diversifying selection on plant floral traits, many of which are also affected by herbivores. This research demonstrates potentially conflicting selection pressures between native and non-native pollinators as well as non-native herbivores.

Range-wide variations in common milkweed traits and their effect on monarch larvae.

PREMISE: Leaf economic spectrum (LES) theory has historically been employed to inform vegetation models of ecosystem processes, but largely neglects intraspecific variation and biotic interactions. We attempt to integrate across environment-plant trait-herbivore interactions within a species at a range-wide scale. METHODS: We measured traits in 53 populations spanning the range of common milkweed (Asclepias syriaca) and used a common garden to determine the role of environment in driving patterns of intraspecific variation. We used a feeding trial to determine the role of plant traits in monarch (Danaus plexippus) larval development. RESULTS: Trait-trait relationships largely followed interspecific patterns in LES theory and persisted in a common garden when individual traits change. Common milkweed showed intraspecific variation and biogeographic clines in traits. Clines did not persist in a common garden. Larvae ate more and grew larger when fed plants with more nitrogen. A longitudinal environmental gradient in precipitation corresponded to a resource gradient in plant nitrogen, which produces a gradient in larval performance. CONCLUSIONS: Biogeographic patterns in common milkweed traits can sometimes be predicted from LES, are largely driven by environmental conditions, and have consequences for monarch larval performance. Changes to nutrient dynamics of landscapes with common milkweed could potentially influence monarch population dynamics. We show how biogeographic trends in intraspecific variation can influence key ecological interactions, especially in common species with large distributions.

The demographic effects of functional traits: an integral projection model approach reveals population-level consequences of reproduction-defence trade-offs.

Quantitatively linking individual variation in functional traits to demography is a necessary step to advance our understanding of trait-based ecological processes. We constructed a population model for Asclepias syriaca to identify how functional traits affect vital rates and population growth and whether trade-offs in chemical defence and demography alter population growth. Plants with higher foliar cardenolides had lower fibre, cellulose and lignin levels, as well as decreased sexual and clonal reproduction. Average cardenolide concentrations had the strongest effect on population growth. In both the sexual and clonal pathway, the trade-off between reproduction and defence affected population growth. We found that both increasing the mean of the distribution of individual plant values for cardenolides and herbivory decreased population growth. However, increasing the variance in both defence and herbivory increased population growth. Functional traits can impact population growth and quantifying individual-level variation in traits should be included in assessments of population-level processes.

Long- and short-term responses of Asclepias species differ in respect to fire, grazing, and nutrient addition.

PREMISE OF THE STUDY: The tallgrass prairie ecosystem has experienced a dramatic reduction over the past 150 yr. This reduction has impacted the abundance of native grassland species, including milkweeds (Asclepias). METHODS: We used two long-term (27 yr) data sets to examine how fire, grazing, and nutrient addition shape milkweed abundance in tallgrass prairie. We compared these results to those of a greenhouse experiment that varied nutrient levels in the absence of competition, herbivory, and mutualistic relationships. KEY RESULTS: Asclepias species exhibited broad patterns in response to burning regimes that did not include grazing, but experienced more species-specific patterns in other combinations. Asclepias syriaca was the only species to increase in abundance in plots that included burning and nutrient addition. In the greenhouse we found that nitrogen significantly increased biomass, while no effect of phosphorus was detected. CONCLUSIONS: These results indicate that A. syriaca will do best in settings with high nutrient loads, low competition, and no grazers. These characteristics define a small portion of the tallgrass prairie but exemplify modern agricultural settings, which have replaced prairies. However, other milkweeds examined did not share this pattern, which indicates that milkweed species will respond differently when exposed to agricultural settings, with some less able to cope with land conversion to pasture or row-crop agriculture.

Environmental variation shifts the relationship between trees and scatterhoarders along the continuum from mutualism to antagonism.

The conditional mutualism between scatterhoarders and trees varies on a continuum from mutualism to antagonism and can change across time and space, and among species. We examined 4 tree species (red oak [Quercus rubra], white oak [Quercus alba], American chestnut [Castanea dentata] and hybrid chestnut [C. dentata × Castanea mollissima) across 5 sites and 3 years to quantify the variability in this conditional mutualism. We used a published model to compare the rates of seed emergence with and without burial to the probability that seeds will be cached and left uneaten by scatterhoarders to quantify variation in the conditional mutualism that can be explained by environmental variation among sites, years, species, and seed provenance within species. All species tested had increased emergence when buried. However, comparing benefits of burial to the probability of caching by scatterhoarders indicated a mutualism in red oak, while white oak was nearly always antagonistic. Chestnut was variable around the boundary between mutualism and antagonism, indicating a high degree of context dependence in the relationship with scatterhoarders. We found that different seed provenances did not vary in their potential for mutualism. Temperature did not explain microsite differences in seed emergence in any of the species tested. In hybrid chestnut only, emergence on the surface declined with soil moisture in the fall. By quantifying the variation in the conditional mutualism that was not caused by changes in scatterhoarder behavior, we show that environmental conditions and seed traits are an important and underappreciated component of the variation in the relationship between trees and scatterhoarders.

A conceptual framework for restoration of threatened plants: the effective model of American chestnut (Castanea dentata) reintroduction.

We propose a conceptual framework for restoration of threatened plant species that encourages integration of technological, ecological, and social spheres. A sphere encompasses ideas relevant to restoration and the people working within similar areas of influence or expertise. Increased capacity within a sphere and a higher degree of coalescing among spheres predict a greater probability of successful restoration. We illustrate this with Castanea dentata, a foundation forest tree in North America that was annihilated by an introduced pathogen; the species is a model that effectively merges biotechnology, reintroduction biology, and restoration ecology. Because of C. dentata's ecological and social importance, scientists have aggressively pursued blight resistance through various approaches. We summarize recent advancements in tree breeding and biotechnology that have emerged from C. dentata research, and describe their potential to bring new tools to bear on socio-ecological restoration problems. Successful reintroduction of C. dentata will also depend upon an enhanced understanding of its ecology within contemporary forests. We identify a critical need for a deeper understanding of societal influences that may affect setting and achieving realistic restoration goals. Castanea dentata may serve as an important model to inform reintroduction of threatened plant species in general and foundation forest trees in particular.

Climate influences the demography of three dominant sagebrush steppe plants.

Climate change could alter the population growth of dominant species, leading to profound effects on community structure and ecosystem dynamics. Understanding the links between historical variation in climate and population vital rates (survival, growth, recruitment) is one way to predict the impact of future climate change. Using a unique, long-term data set from eastern Idaho, USA, we parameterized integral projection models (IPMs) for Pseudoroegneria spicata, Hesperostipa comata, and Artemisia tripartita to identify the demographic rates and climate variables most important for population growth. We described survival, growth, and recruitment as a function of genet size using mixed-effect regression models that incorporated climate variables. Elasticites for the survival + growth portion of the kernel were larger than the recruitment portion for all three species, with survival + growth accounting for 87-95% of the total elasticity. The genet sizes with the highest elasticity values in each species were very close to the genet size threshold where survival approached 100%. We found strong effects of climate on the population growth rate of two of our three species. In H. comata, a 1% decrease in previous year's precipitation would lead to a 0.6% decrease in population growth. In A. tripartita, a 1% increase in summer temperature would result in a 1.3% increase in population growth. In both H. comata and A. tripartita, climate influenced population growth by affecting genet growth more than survival or recruitment. Late-winter snow was the most important climate variable for P. spicata, but its effect on population growth was smaller than the climate effects we found in H. comata or A. tripartita. For all three species, demographic responses lagged climate by at least one year. Our analysis indicates that understanding climate effects on genet growth may be crucial for anticipating future changes in the structure and function of sagebrush steppe vegetation.

A multifunctional androgen receptor screening assay using the high-throughput Hypercyt flow cytometry system.

The androgen receptor (AR) is a steroid hormone receptor which regulates transcription of androgen-sensitive genes and is responsible for the development and maintenance of male secondary sexual characteristics. Chemicals that interfere with AR activity may lead to pathological conditions in androgen-sensitive tissues. A variety of reporter systems have been developed, driven by androgen-sensitive promoters, which screen for chemicals that modulate androgenic activity. We have developed a flexible, high-throughput AR transcriptional activation assay, designated the Multifunctional Androgen Receptor Screening (MARS) assay, to facilitate the identification of novel modulators of AR transcriptional activity using flow cytometry. Androgen-independent human prostate cancer-derived PC3 cells were transiently cotransfected with an expression vector for the wild-type human AR and an androgen-sensitive promoter regulating the expression of destabilized enhanced GFP (dsEGFP). The transfected cells were stimulated with established androgenic and antiandrogenic compounds and assessed for increased or decreased dsEGFP expression. To screen for antagonists of AR transcription, the AR agonist R1881 was coadministered at submaximal concentrations with potential AR antagonists. The assay was formatted for high-throughput screening using the HyperCyt flow cytometry system. Agents with established androgenic and antiandrogenic activity were used for validation of the MARS assay. AR agonists were found to potently induce dsEGFP. Furthermore, AR agonists induced dsEGFP expression in a dose-dependent manner. Alternatively, AR antagonists blocked dsEGFP expression when coadministered with low-dose R1881, which also occurred in a dose-dependent manner. Modulators of AR transcriptional activity can be successfully identified by the MARS assay, utilizing a rapid, flexible, sensitive, and high-throughput format. Dose-response curves can be successfully generated for these compounds, allowing for an assessment of potency. Because of its simplicity and high-throughput compatibility, the MARS assay and HyperCyt system combined with flow cytometric analysis represents a valuable and novel addition to the current repertoire of AR transcriptional activation screening assays.

Responses of two bunchgrasses to nitrogen addition in tallgrass prairie: the role of bud bank demography.

Growth of tallgrass prairie plants, many of which maintain substantial bud banks, can be limited by nitrogen (N), water, and/or light. We hypothesized that tallgrass prairie plants respond to increases in N through demographic effects on the bud bank. We tested the effects of a pulse of N on (1) bud bank demography, (2) plant reproductive allocation, and (3) ramet size. We parameterized matrix models, considering each genet as a population of plant parts. Nitrogen addition significantly impacted bud bank demography in two subdominant species of bunchgrass: Sporobolus heterolepis (a C(4) grass) and Koeleria macrantha (a C(3) grass), but had no effect on the size of individual ramets. Emergence from the bud bank and ramet population growth rates (λ) were significantly higher in S. heterolepis genets that received supplemental N. Nitrogen addition also affected the bud demography of K. macrantha, but N addition decreased rather than increased λ. Prospective and retrospective demographic analyses indicated that bud bank dynamics were the most important demographic processes driving plant responses to nutrient availability. Thus, the variation in productivity in these tallgrass prairie species is driven principally by the demography of the bud bank rather than by the physiology and growth of aboveground tillers. Improved understanding of bud bank dynamics may lead to improved predictive models of grassland responses to environmental changes such as altered N deposition and precipitation.

Below-ground bud banks increase along a precipitation gradient of the North American Great Plains: a test of the meristem limitation hypothesis.

In perennial grasslands, the below-ground population of meristems (bud bank) plays a fundamental role in plant population dynamics. Here, we tested the 'meristem limitation hypothesis' prediction - that bud banks increase along an increasing precipitation/productivity gradient in North American grasslands - and assessed the seasonal dynamics of bud banks. We sampled bud and stem populations quarterly at six sites across a 1100 km gradient in central North America. Bud banks increased with average annual precipitation, which explained 80% of the variability between the sites. In addition, seasonal changes in grass bud banks were surprisingly similar across a 2.5-fold range in precipitation and a 4-fold range of productivity: densities peaked in March, decreased in June and increased slightly in September. Increasing meristem limitation may constrain vegetation responses to inter-annual changes in resources. An important consequence of this is that biomes with large bud banks may be the most responsive to environmental change. If meristem limitation represents an important constraint on productivity responses to environmental variability, then bud banks must be considered in developing predictive models for grassland responses to environmental change.

Clusterin contributes to caspase-3-independent brain injury following neonatal hypoxia-ischemia.

Clusterin, also known as apolipoprotein J, is a ubiquitously expressed molecule thought to influence a variety of processes including cell death. In the brain, it accumulates in dying neurons following seizures and hypoxic-ischemic (H-I) injury. Despite this, in vivo evidence that clusterin directly influences cell death is lacking. Following neonatal H-I brain injury in mice (a model of cerebral palsy), there was evidence of apoptotic changes (neuronal caspase-3 activation), as well as accumulation of clusterin in dying neurons. Clusterin-deficient mice had 50% less brain injury following neonatal H-I. Surprisingly, the absence of clusterin had no effect on caspase-3 activation, and clusterin accumulation and caspase-3 activation did not colocalize to the same cells. Studies with cultured cortical neurons demonstrated that exogenous purified astrocyte-secreted clusterin exacerbated oxygen/glucose-deprivation-induced necrotic death. These results indicate that clusterin may be a new therapeutic target to modulate non-caspase-dependent neuronal death following acute brain injury.

Apolipoprotein J/clusterin limits the severity of murine autoimmune myocarditis.

Apolipoprotein J/clusterin (apoJ/clusterin), an intriguing protein with unknown function, is induced in myocarditis and numerous other inflammatory injuries. To test its ability to modify myosin-induced autoimmune myocarditis, we generated apoJ-deficient mice. ApoJ-deficient and wild-type mice exhibited similar initial onset of myocarditis, as evidenced by the induction of two early markers of the T cell-mediated immune response, MHC-II and TNF receptor p55. Furthermore, autoantibodies against the primary antigen cardiac myosin were induced to the same extent. Although the same proportion of challenged animals exhibited some degree of inflammatory infiltrate, inflammation was more severe in apoJ-deficient animals. Inflammatory lesions were more diffuse and extensive in apoJ-deficient mice, particularly in females. In marked contrast to wild-type animals, the development of a strong generalized secondary response against cardiac antigens in apoJ-deficient mice was predictive of severe myocarditis. Wild-type mice with a strong Ab response to secondary antigens appeared to be protected from severe inflammation. After resolution of inflammation, apoJ-deficient, but not wild-type, mice exhibited cardiac function impairment and severe myocardial scarring. These results suggest that apoJ limits progression of autoimmune myocarditis and protects the heart from postinflammatory tissue destruction.

Targeted disruption of the murine lecithin:cholesterol acyltransferase gene is associated with reductions in plasma paraoxonase and platelet-activating factor acetylhydrolase activities but not in apolipoprotein J concentration.

Lecithin:cholesteryl acyltransferase (LCAT) deficiency resulting from targeted disruption of the Lcat gene in the mouse is associated with dramatic decreases in HDL concentration and the accumulation of nascent HDL in the plasma. We examined whether LCAT deficiency in mice is associated with a concomitant decrease in two antioxidative enzymes, paraoxonase (PON) and platelet-activating factor acetylhydrolase (PAF-AH). In control Lcat (+/+) mice both these enzymes are transported on HDL. Compared to Lcat (+/+) mice, HDL-cholesterol is reduced 94% and apoA-I, 90%, in Lcat (-/-) mice; this reduction in HDL is paralleled by a 71% decrease in PAF-AH activity and in a 58% decrease in PON activity. Apolipoprotein J (apoJ) levels, rather than being decreased, were significantly (P = 0.01) higher (36%) in Lcat (-/-) than in Lcat (+/+) mice, and the apo J/PON ratio was 3-fold greater in Lcat (-/-) than in Lcat (+/+) animals. Even though apolipoprotein A-I (apoA-I) concentration and PON activity were drastically reduced, there was no reduction in apoA-I and PON liver mRNA levels suggesting that post-transcriptional events are responsible for the reduction of plasma PON and apoA-I levels. Fast protein liquid chromatography (FPLC) revealed that in Lcat (+/+) mice both PON and PAF-AH activity is associated with large, apoA-I-containing HDL particles (9.7 nm by non-denaturing gradient gel electrophoresis) while in Lcat (-/-) mice both enzymes are associated with small 8.2 nm particles. We conclude that the concomitant reduction in HDL and apoA-I concentrations and PON and PAF-AH activities is best explained by rapid clearance of the small HDL particles found in LCAT deficiency.

Identification and immunohistochemical localization of protein precursors to human axillary odors in apocrine glands and secretions.

OBJECTIVES: To determine the cellular localization in male and female axillary tissue for apocrine secretion odor-binding proteins 1 (ASOB1) and 2 (ASOB2) and the electrophoretic pattern of female apocrine proteins and to begin characterization of the ASOB1 protein. DESIGN: Immunohistochemical techniques were used with biopsy samples from axillary tissue of male and female subjects. Immunological techniques and microsequencing were used to characterize several of the proteins in male and female apocrine secretions. SETTING: A university medical center. PARTICIPANTS: Healthy male and female volunteers who donated apocrine secretions and/or axillary tissue. RESULTS: Specific immunoreactivity was localized only to the apocrine glands in both sexes. Furthermore, only preabsorption with a mixed apocrine secretion sample eliminated all immunoreactivity. The electrophoretic pattern of proteins in female apocrine secretions is similar to that in male secretions. Western blotting of the separated proteins from female samples using serum samples containing antibodies to ASOB1 and ASOB2 yielded identical results to those found with separated proteins from male samples. Partial sequence data obtained from the N-terminus of ASOB1 suggested that it shares homology with the alpha-chain of apolipoprotein J (Apo J). Apocrine secretion odor-binding protein 1 is not immunologically similar to ApoJ, but 2 other apocrine secretion proteins are. CONCLUSIONS: Male and female subjects appear to have the same glycoprotein carriers for (E)-3-methyl-2-hexenoic acid localized to the apocrine glands. The N-terminal sequence for ASOB1 may be homologous to Apo J, but it is not immunologically similar to it. However, 2 other proteins in the apocrine secretion appear to be the monomer and dimer forms of Apo J.

Neurotoxicity of the 22 kDa thrombin-cleavage fragment of apolipoprotein E and related synthetic peptides is receptor-mediated.

Potent neurotoxicity is associated with both apolipoprotein E (apoE)-related synthetic peptides and the 22 kDa N-terminal thrombin-cleavage fragment of apoE. Furthermore, the E4 isoform of the 22 kDa fragment is significantly more toxic than the same fragment derived from the E3 isoform, suggesting the possibility of a direct role of apoE-associated neurotoxicity in the pathophysiology of Alzheimer's disease. In the present study, the potential role of cell surface receptors in mediating neurotoxicity was assessed by using a variety of agents that should block the heparin-binding and receptor-binding activity of apoE. Effective inhibitors of neurotoxicity of both the apoE peptides and the apoE fragment include heparin, heparan sulfate, sodium chlorate and heparinase, the low-density lipoprotein (LDL) receptor-related protein receptor-associated protein, and a polyclonal anti-LDL receptor-related protein antibody. These results suggest that the neurotoxicity of the 22 kDa thrombin cleavage fragment of apoE and related peptides is receptor-mediated, and that the most likely candidate receptor is a heparan sulfate proteoglycan-LDL receptor-related protein complex.

A thrombin cleavage fragment of apolipoprotein E exhibits isoform-specific neurotoxicity.

A 22 kDa fragment of apoE containing a putative cytotoxi domain was identified in postmortem human brain tissue and fresh CSF. This fragment is apparently equivalent to the major apoE thrombin cleavage product. In vitro toxicity assays demonstrate that the corresponding fragment derived from recombinantly expressed human apoE is toxic to primary neurons in culture and that the E4-derived fragment is significantly more toxic than the fragment derived from the E3 isoform. These results suggest that proteolytic fragments of apoE may play a direct role in the pathology associated with AD and other diseases in which apoE has been implicated.

Apolipoprotein J/clusterin expression defines distinct stages of blastocyst implantation in the mouse uterus.

The endometrium is a dynamic tissue that responds to hormonal cues and growth factors to accommodate, regulate, and nurture developing embryos. To provide clues about the molecular mechanisms underlying the responsiveness of this tissue, we have begun to identify genes that are expressed at specific stages of early pregnancy. One such gene, apolipoprotein J (apoJ), encodes a secretory glycoprotein capable of binding lipids and membrane-active proteins. Uterine apoJ gene activity was not detected immediately following fertilization, but glandular epithelial expression of apoJ mRNA appeared just before the time of blastocyst implantation and persisted postimplantation. During implantation, uterine luminal epithelial cells also expressed apoJ, but expression was excluded from luminal cells adjacent to the sites of attached blastocysts. ApoJ protein accumulated in the glandular and uterine lumens in proximity to the epithelial cells that expressed apoJ mRNA. We suggest that apoJ expression is a marker of uterine receptivity to blastocyst implantation. Subsequent expression of apoJ message in uterine stromal cell types and in circular muscle myocytes coincided with the onset of decidualization. During this period the myocytes of the longitudinal muscle layer showed no evidence of apoJ mRNA. ApoJ protein was localized to nondecidualized tissue but was not evident in decidualized cells. In contrast, the protein was dispersed throughout both the circular and longitudinal myometrium. In the uteri of hormone-treated females stimulated with oil, apoJ was also expressed during decidualization in stromal cells and in circular myocytes, indicating that signals specifically transmitted from the embryo itself are not responsible for apoJ mRNA accumulation.