Predicting the outcomes of a tri‐trophic interaction between an indigenous parasitoid and an exotic herbivorous pest and its host plants

Understanding the novel ecological interactions that result from biological invasions is a critical issue in modern ecology and evolution as well as pest management. Introduced herbivorous insects may interact with native plants and indigenous natural enemies, creating novel tri‐trophic interactions. To help predict the potential outcomes of novel interactions, we investigated the behavioural and physiological responses of an indigenous generalist parasitoid (Habrobracon gelechiae) to an introduced generalist herbivore (the light brown apple moth, Epiphyas postvittana) and its new host plants in California. We first examined the parasitoid's host location and acceptance on a range of nine common host plants of the moth representing distinctly different geographic origins and morphologies (to examine the effect of a known toxic plant on the parasitoid's performance, an additional toxic plant species was also tested that the moth consumes in the laboratory but does not naturally attack). The parasitoid was able to locate the host larvae on all plants equally well, although clutch size was affected by host plant. We then determined fitness of the moth and the parasitoid on four representative plants. The moth larvae suffered higher mortality and a slower developmental rate on the known toxic plant than on the other three plants, but the parasitoid's fitness correlates did not differ between the host food plants. These results show a high level of plasticity in the indigenous generalist parasitoid in its ability to exploit the exotic host on a wide range of host plants, generating an invasion‐driven novel tri‐trophic interaction.     

A dendro‐ecological study of forest overstorey productivity following the invasion of the non‐indigenous shrub Lonicera maackii

Question: Will a non‐indigenous, invasive, understorey shrub, such as Lonicera maackii (Amur honeysuckle) have an impact on the productivity of overstorey trees in hardwood forests? Location: Trees from 12 invaded and four non‐invaded sites were sampled in hardwood forests of southwestern Ohio, US. Methods: Changes in radial and basal area tree growth in the ten years prior to L. maackii invasion vs. ten years after invasion were examined using dendrochronological techniques. Intervention analysis was used to detect growth changes 25 years prior to and 25 years following invasion, and estimates of load impacts for L. maackii population and biomass were also calculated. Results: We found that the rate of radial and basal area growth of overstorey trees was reduced significantly in eleven out of twelve invaded sites. Non‐invaded sites did not exhibit this consistent pattern of reduced growth. For invaded vs. non‐invaded sites, the mean basal area growth was reduced by 15.8%, and the overall rate of basal area growth was reduced by 53.1%. Intervention analysis revealed that the first significant growth reductions were 6.25 ± 1.24 (mean ± SE) years after invasion with the greatest frequency of negative growth changes occurring 20 years after invasion. In invaded stands, 41% of trees experienced negative growth changes. In terms of invasive load estimates per 1000 L. maackii individuals, radial tree growth was reduced by 0.56 mm.a^?1, and basal area growth was reduced by 0.74 cm².a^?1, Given these findings, significant economic losses could occur in hardwood forests of Ohio. Conclusions: To our knowledge, this is the first study using dendrochronological techniques to investigate the impact of a non‐indigenous, understorey plant on overstorey tree growth. Active management will likely be needed to maintain forest productivity in L. maackii impacted landscapes.     

Co‐adapting societal and ecological interactions following large disturbances in urban park woodlands

The responses of urban park woodlands to large disturbances provide the opportunity to identify and examine linkages in social‐ecological systems in urban landscapes. We propose that the Panarchy model consisting of hierarchically nested adaptive cycles provides a useful framework to evaluate those linkages. We use two case studies as examples – Cherokee Park in Louisville, Kentucky, USA and Tijuca Forest in Rio de Janeiro, Brazil. In the Cherokee Park case study, the disturbance, a destructive tornado, triggered bottom‐up societal responses that created new institutions that influenced park management and shifted the woodland community from a successional pathway dominated by invasive exotic plants to one where native plant species are regaining importance. In the Tijuca Forest example, the disturbance, large scale land‐use changes during the 18th century, triggered primarily top‐down societal responses to create the world's largest urban forest through a transformative programme of intensive multispecies forest replanting and management. Currently, fine scale disturbances – primarily anthropogenically caused fire – threaten portions of Tijuca Forest through loss of forest structure and establishment of flammable invasive plants, and again elicited top‐down societal responses to stop further destruction and promote greater native plant regeneration. These case studies illustrate that either natural or anthropogenic disturbances to natural systems can alter the direction and magnitude of interactions between social and natural domains in urban landscapes in a co‐adaptive manner that alters structures and processes in both system components.     

Beyond ecosystem modeling: A roadmap to community cyberinfrastructure for ecological data‐model integration

In an era of rapid global change, our ability to understand and predict Earth's natural systems is lagging behind our ability to monitor and measure changes in the biosphere. Bottlenecks to informing models with observations have reduced our capacity to fully exploit the growing volume and variety of available data. Here, we take a critical look at the information infrastructure that connects ecosystem modeling and measurement efforts, and propose a roadmap to community cyberinfrastructure development that can reduce the divisions between empirical research and modeling and accelerate the pace of discovery. A new era of data‐model integration requires investment in accessible, scalable, and transparent tools that integrate the expertise of the whole community, including both modelers and empiricists. This roadmap focuses on five key opportunities for community tools: the underlying foundations of community cyberinfrastructure; data ingest; calibration of models to data; model‐data benchmarking; and data assimilation and ecological forecasting. This community‐driven approach is a key to meeting the pressing needs of science and society in the 21st century.     

Egyptian tortoise conservation: A community‐based,field research program developed from a study on a captive population

Local community participation and ex situ conservation has the potential to assist the recovery of the endangered Egyptian tortoise, Testudo kleinmanni. We initiated an in situ community‐based conservation and research program from a captive population of T. kleinmanni. We used a captive population of the Egyptian tortoise to train a member of the local community as a research technician and used his indigenous tracking skills and knowledge of the area to collect activity and dietary data on 28 captive tortoises. We overcame problems with illiteracy by creating a data sheet based on symbols and numbers. This data sheet allowed us to use the indigenous knowledge of various people from the community, and employ them in the future. Our local community approach to data collection, in conjunction with a craft program, made the conservation of the Egyptian tortoise more rewarding to the local community by providing a more sustainable form of income than collecting animals for the pet trade. Our multidimensional approach (local community participation as research technicians, craft program, and trust building) for gaining local support eventually led to the rediscovery of wild Egyptian tortoises in North Sinai, which was significant, as this species was presumed extinct in Egypt. We have now shifted our focus to in situ conservation, using the research and local capacity building template developed from this captive population study. Our template can be used by zoos and conservation organizations with small budgets and collections of native species in natural habitats to create similar captive research programs that can be applied to in situ conservation. Zoo Biol 26:397–406, 2007. © 2007 Wiley‐Liss, Inc.     

Protective effects of isoquercitrin on streptozotocin‐induced neurotoxicity

Alzheimer's disease (AD) is characterized by irreversible and progressive memory loss and has no effective treatment. Recently, many small molecule nature products have been identified with neuroprotective functions and shown beneficial effects to AD patients. In the current study, we thus performed a small scale screening to determine the protective effects of natural compounds on streptozotocin (STZ)‐induced neurotoxicity and Alzheimer's disease (AD). We found that a lead flavonoid compound, isoquercitrin (ISO) display the most effective anti‐cytotoxic activities via inhibiting STZ‐induced apoptosis, mitochondria dysfunction and oxidative stress. Treatment with ISO largely rescues STZ‐induced differentiation inhibition and enhances neurite outgrowth of Neuro2a (N2a) cells in vitro. Moreover, oral administration of ISO protects hippocampal neurons from STZ‐induced neurotoxicity and significantly improves the cognitive and behavioural impairment in STZ‐induced AD rats. In general, our screening identifies ISO as an effective therapeutic candidate against STZ‐induced neurotoxicity and AD‐like changes.     

A socio‐ecological model for predicting impacts of land‐use and climate change on regional plant diversity in the Austrian Alps

Climate and land‐use change jointly affect the future of biodiversity. Yet, biodiversity scenarios have so far concentrated on climatic effects because forecasts of land use are rarely available at appropriate spatial and thematic scales. Agent‐based models (ABMs) represent a potentially powerful but little explored tool for establishing thematically and spatially fine‐grained land‐use scenarios. Here, we use an ABM parameterized for 1,329 agents, mostly farmers, in a Central European model region, and simulate the changes to land‐use patterns resulting from their response to three scenarios of changing socio‐economic conditions and three scenarios of climate change until the mid of the century. Subsequently, we use species distribution models to, first, analyse relationships between the realized niches of 832 plant species and climatic gradients or land‐use types, respectively, and, second, to project consequent changes in potential regional ranges of these species as triggered by changes in both the altered land‐use patterns and the changing climate. We find that both drivers determine the realized niches of the studied plants, with land use having a stronger effect than any single climatic variable in the model. Nevertheless, the plants' future distributions appear much more responsive to climate than to land‐use changes because alternative future socio‐economic backgrounds have only modest impact on land‐use decisions in the model region. However, relative effects of climate and land‐use changes on biodiversity may differ drastically in other regions, especially where landscapes are still dominated by natural or semi‐natural habitat. We conclude that agent‐based modelling of land use is able to provide scenarios at scales relevant to individual species distribution and suggest that coupling ABMs with models of species' range change should be intensified to provide more realistic biodiversity forecasts.     

Host‐specific variation in off‐host performance of a temperate ectoparasite

Antagonistic host–parasite interactions are rarely considered from an ecological perspective of the parasite. We used a blood‐feeding ectoparasite of boreal cervids, the deer ked (Lipoptena cervi L., Hippoboscidae), to study host‐dependent variation in a parasite's ability to cope with an abiotic environment during the free‐living stage(s) in two allopatric Fennoscandian populations. We found that a strongly host‐specific deer ked population in eastern Fennoscandia, exploiting only moose (Alces alces), produced the largest offspring that were the most cold‐tolerant and emerged the earliest as adults, when compared with the western Fennoscandian population that exploited two hosts efficiently. Within the western population, however, offspring produced on roe deer (Capreolus capreolus) were significantly larger, more cold‐tolerant, and had higher survival than those produced on moose in the same area. We discuss potential causes for both host‐specific and geographical differences in off‐host performance: (1) maternal host directly affects the offspring survival prospects; (2) divergent co‐evolution with local main host(s) has shaped the parasite's life history; and/or (3) off‐host performance is shaped by adaptation to the local abiotic environment. In conclusion, this study increases our understanding of the evolution of host–parasite interactions by demonstrating how geographical differences in host exploitation may result in differences in survival prospects outside the host.     

Pollen metabarcoding as a tool for tracking long‐distance insect migrations

Insects account for a large portion of Earth's biodiversity and are key players for ecosystems, notably as pollinators. While insect migration is suspected to represent a natural phenomenon of major importance, remarkably little is known about it, except for a few flagship species. The reason for this situation is mainly due to technical limitations in the study of insect movement. Here, we propose using metabarcoding of pollen carried by insects as a method for tracking their migrations. We developed a flexible and simple protocol allowing efficient multiplexing and not requiring DNA extraction, one of the most time‐consuming part of metabarcoding protocols, and apply this method to the study of the long‐distance migration of the butterfly Vanessa cardui, an emerging model for insect migration. We collected 47 butterfly samples along the Mediterranean coast of Spain in spring and performed metabarcoding of pollen collected from their bodies to test for potential arrivals from the African continent. In total, we detected 157 plant species from 23 orders, most of which (82.8%) were insect‐pollinated. Taxa present in Africa–Arabia represented 73.2% of our data set, and 19.1% were endemic to this region, strongly supporting the hypothesis that migratory butterflies colonize southern Europe from Africa in spring. Moreover, our data suggest that a northwards trans‐Saharan migration in spring is plausible for early arrivals (February) into Europe, as shown by the presence of Saharan floristic elements. Our results demonstrate the possibility of regular insect‐mediated transcontinental pollination, with potential implications for ecosystem functioning, agriculture and plant phylogeography.     

Cost‐effective ecological restoration

Ecological restoration is a multibillion dollar industry critical for improving degraded habitat. However, most restoration is conducted without clearly defined success measures or analysis of costs. Outcomes are influenced by environmental conditions that vary across space and time, yet such variation is rarely considered in restoration planning. Here, we present a cost‐effectiveness analysis of terrestrial restoration methods to determine how practitioners may restore the highest native plant cover per dollar spent. We recorded costs of 120 distinct methods and described success in terms of native versus non‐native plant germination, growth, cover, and density. We assessed effectiveness using a basic, commonly used metric (% native plant cover) and developed an index of cost‐effectiveness (% native cover per dollar spent on restoration). We then evaluated success of multiple methods, given environmental variation across topography and multiple years, and found that the most successful method for restoring high native plant cover is often different from the method that results in the largest area restored per dollar expended, given fixed mitigation budgets. Based on our results, we developed decision‐making trees to guide practitioners through established phases of restoration—site preparation, seeding and planting, and maintenance. We also highlight where additional research could inform restoration practice, such as improved seasonal weather forecasts optimizing allocation of funds in time or valuation practices that include costs of specific outcomes in the collection of in lieu fees.     

The river echoes with laughter: a child‐centred analysis of social change in Amazonia

This article examines radical social, cultural, and political changes taking place in Amazonia from the perspective of indigenous children and youth: a group who, despite their demographic prevalence, have received limited attention in the regional literature. Drawing on fieldwork with Matses people in Peru, I consider how children and youth are playing a critical role in the transition from a hunter‐gatherer, forest‐based society towards a riverine lifestyle that is increasingly engaged in trade, the market economy, and exchanges with chotac, or non‐indigenous people. I argue that by engaging with their surroundings through playing and working, Matses children are becoming affectively attached to some parts of the world rather than others. This represents a purposeful shift from the lifestyle and worldviews of older generations and highlights how children are active agents who shape possible future directions of Matses society and transform the community's relationships with the world. Accordingly, I propose a child‐centred view of social change that seeks to demonstrate the implications of children's creativity and agency for society at large and its future development.     

Fifty years of co‐evolution and beyond: integrating co‐evolution from molecules to species

Fifty years after Ehrlich and Raven's seminal paper, the idea of co‐evolution continues to grow as a key concept in our understanding of organic evolution. This concept has not only provided a compelling synthesis between evolutionary biology and community ecology, but has also inspired research that extends beyond its original scope. In this article, we identify unresolved questions about the co‐evolutionary process and advocate for the integration of co‐evolutionary research from molecular to interspecific interactions. We address two basic questions: (i) What is co‐evolution and how common is it? (ii) What is the unit of co‐evolution? Both questions aim to explore the heart of the co‐evolutionary process. Despite the claim that co‐evolution is ubiquitous, we argue that there is in fact little evidence to support the view that reciprocal natural selection and coadaptation are common in nature. We also challenge the traditional view that co‐evolution only occurs between traits of interacting species. Co‐evolution has the potential to explain evolutionary processes and patterns that result from intra‐ and intermolecular biochemical interactions within cells, intergenomic interactions (e.g. nuclear‐cytoplasmic) within species, as well as intergenomic interactions mediated by phenotypic traits between species. Research that bridges across these levels of organization will help to advance our understanding of the importance of the co‐evolutionary processes in shaping the diversity of life on Earth.     

Value of long‐term ecological studies

Long‐term ecological studies are critical for providing key insights in ecology, environmental change, natural resource management and biodiversity conservation. In this paper, we briefly discuss five key values of such studies. These are: (1) quantifying ecological responses to drivers of ecosystem change; (2) understanding complex ecosystem processes that occur over prolonged periods; (3) providing core ecological data that may be used to develop theoretical ecological models and to parameterize and validate simulation models; (4) acting as platforms for collaborative studies, thus promoting multidisciplinary research; and (5) providing data and understanding at scales relevant to management, and hence critically supporting evidence‐based policy, decision making and the management of ecosystems. We suggest that the ecological research community needs to put higher priority on communicating the benefits of long‐term ecological studies to resource managers, policy makers and the general public. Long‐term research will be especially important for tackling large‐scale emerging problems confronting humanity such as resource management for a rapidly increasing human population, mass species extinction, and climate change detection, mitigation and adaptation. While some ecologically relevant, long‐term data sets are now becoming more generally available, these are exceptions. This deficiency occurs because ecological studies can be difficult to maintain for long periods as they exceed the length of government administrations and funding cycles. We argue that the ecological research community will need to coordinate ongoing efforts in an open and collaborative way, to ensure that discoverable long‐term ecological studies do not become a long‐term deficiency. It is important to maintain publishing outlets for empirical field‐based ecology, while simultaneously developing new systems of recognition that reward ecologists for the use and collaborative sharing of their long‐term data sets. Funding schemes must be re‐crafted to emphasize collaborative partnerships between field‐based ecologists, theoreticians and modellers, and to provide financial support that is committed over commensurate time frames.     

Evaluating the performance of neutrality tests of a local community using a niche‐structured simulation model

Understanding the processes that underlie species diversity and abundance in a community is a fundamental issue in community ecology. While the species abundance distributions (SADs) of various natural communities may be well explained by Hubbell's neutral model, it has been repeatedly pointed out that Hubbell's SAD‐fitting approach lacks the ability to detect the effects of non‐neutral factors such as niche differentiation; however, our understanding of its quantitative effect is limited. Herein, we conducted extensive simulations to quantitatively evaluate the performance of the SAD‐fitting method and other recently developed tests. For simulations, we developed a niche model that incorporates the random stochastic demography of individuals and the nonrandom replacements of those individuals, i.e. niche differentiation. It therefore allows us to explore situations with various degrees of niche differentiation. We found that niche differentiation has strong effects on SADs and the number of species in the community under this model. We then examined the performance of these neutrality tests, including Hubbell's SAD‐fitting method, using extensive simulations. It was demonstrated that all these tests have relatively poor performance except for the cases with very strong niche structure, which is in accordance with previous studies. This is likely because two important parameters in Hubbell's model are usually unknown and are commonly estimated from the data to be tested. To demonstrate this point, we showed that the precise estimation of the two parameters substantially improved the performance of these neutrality tests, indicating that poor performance can be owed to overfitting Hubbell's neutral model with unrealistic parameters. Our results therefore emphasize the importance of accurate parameter estimation, which should be obtained from data independent of the local community to be tested.     

Identifying refugia from climate change using coupled ecological and genetic data in a transitional Mediterranean‐temperate tree species

Populations occurring in areas of overlap between the current and future distribution of a species are particularly important because they can represent “refugia from climate change”. We coupled ecological and range‐wide genetic variation data to detect such areas and to evaluate the impacts of habitat suitability changes on the genetic diversity of the transitional Mediterranean‐temperate tree Fraxinus angustifolia. We sampled and genotyped 38 natural populations comprising 1006 individuals from across Europe. We found the highest genetic diversity in western and northern Mediterranean populations, as well as a significant west to east decline in genetic diversity. Areas of potential refugia that correspond to approximately 70% of the suitable habitat may support the persistence of more than 90% of the total number of alleles in the future. Moreover, based on correlations between Bayesian genetic assignment and climate, climate change may favour the westward spread of the Black Sea gene pool in the long term. Overall, our results suggest that the northerly core areas of the current distribution contain the most important part of the genetic variation for this species and may serve as in situ macrorefugia from ongoing climate change. However, rear‐edge populations of the southern Mediterranean may be exposed to a potential loss of unique genetic diversity owing to habitat suitability changes unless populations can persist in microrefugia that have facilitated such persistence in the past.     

Front‐Loading Natural‐Product‐Screening Libraries for log P: Background,Development, and Implementation

In the period from January 1981 to December 2010, 1068 small‐molecule new chemical entities (NCEs) were introduced, of which ca. 34% are either a natural product or a close analogue. While this metric reflects the impact natural products have played in delivering new chemical starting points (leads) for the pharmaceutical industry, it does not capture the decline this approach has suffered over the last 20 years as the high‐throughput screening (HTS) of pure compound libraries has become more popular. An impediment to natural‐product drug discovery in the HTS paradigm is the lack of a clear strategy that enables front‐loading of an extract or fraction's chemical constituents so that they are compliant with lead‐ and drug‐like chemical space. To address this imbalance, an approach based on lipophilicity, as measured by clog P has been developed that, together with advances being made in isolation and structural elucidation, can afford natural product leads in timelines compatible with pure compound screening.     

A new approach for mealybug management: recruiting an indigenous,but ‘non‐natural’ enemy for biological control using an attractant

We previously discovered that (2,4,4‐trimethyl‐2‐cyclohexenyl)‐methyl butyrate (cyclolavandulyl butyrate, CLB) is an attractant for the mealybug‐parasitic wasp Anagyrus sawadai Ishii (Hymenoptera: Encyrtidae: Anagyrini). This wasp is not likely to parasitize the Japanese mealybug, Planococcus kraunhiae (Kuwana) (Hemiptera: Pseudococcidae), under natural conditions. In this study, we showed that this ‘non‐natural’ enemy wasp can parasitize P. kraunhiae in the presence of CLB in field experiments. Laboratory‐reared mealybugs placed on persimmon trees with CLB‐impregnated rubber septa were parasitized significantly more often by endoparasitic wasps than those on non‐treatment trees (18.1–40.3 vs. 0–6.3%). Anagyrus sawadai accounted for 20% of the wasps that emerged from mealybugs placed on CLB‐treated trees. Moreover, CLB attracted another minor parasitoid, Leptomastix dactylopii Howard (Hymenoptera: Encyrtidae: Anagyrini), which also parasitized more P. kraunhiae in the presence of CLB. All wasps that emerged from the mealybugs on control trees were Anagyrus fujikona Tachikawa, a major parasitoid of P. kraunhiae around the test location. These results demonstrated that CLB can recruit an indigenous, but ‘non‐natural’ enemy that does not typically attack P. kraunhiae under natural conditions, as well as a minor natural enemy, for biological control of this mealybug species.     

The Usefulness of 4‐Amino‐3‐hydroxyphenylalanine as a Specific Marker of Pheomelanin

Reductive hydrolysis of pheomelanin with hydriodic acid (HI) gives two aminohydroxyphenylalanine isomers, 4‐amino‐3‐hydroxyphenylalanine (`specific AHP') and 3‐amino‐4‐hydroxyphenylalanine (3‐aminotyrosine, AT), which derive from the oxidative polymerization of 5‐S‐cysteinyldopa, and 2‐S‐cysteinyldopa, respectively. Since we first introduced this analytical method, the combined amount of AHP and AT (`total AHP') has been extensively used as a marker of pheomelanin. However, one problem with using total AHP as a marker is that background levels originate from precursors other than pheomelanin. Considerable and variable amounts of background AT are produced from other sources, most likely nitrotyrosine residues in proteins. In order to overcome this problem, we developed HPLC conditions which enable the direct injection of the HI reduction products into the HPLC system allowing good separation of AHP and AT. In this way we could study the importance of both degradation products separately and their specificity as markers for pheomelanin. The usefulness of the present method is validated using human hair samples of various colours which were divided into dark, fair or red colours. The combined amount of specific AHP and AT shows an excellent correlation with total AHP, and the amount of specific AHP also correlates with the amount of total AHP. We also examined total AHP and specific AHP values against pyrrole‐2,3,5‐tricarboxylic acid (PTCA) values in the human hair samples. These results show that specific AHP measurement gives a more prominent segregation for the ratio of specific AHP to PTCA among hairs of various colours than the ratio of total AHP to PTCA. Thus, we conclude that `specific AHP' is a more specific marker of pheomelanin than is `total AHP'.     

Accounting for tagging‐to‐harvest mortality in a Brownie tag‐recovery model by incorporating radio‐telemetry data

The Brownie tag‐recovery model is useful for estimating harvest rates but assumes all tagged individuals survive to the first hunting season; otherwise, mortality between time of tagging and the hunting season will cause the Brownie estimator to be negatively biased. Alternatively, fitting animals with radio transmitters can be used to accurately estimate harvest rate but may be more costly. We developed a joint model to estimate harvest and annual survival rates that combines known‐fate data from animals fitted with transmitters to estimate the probability of surviving the period from capture to the first hunting season, and data from reward‐tagged animals in a Brownie tag‐recovery model. We evaluated bias and precision of the joint estimator, and how to optimally allocate effort between animals fitted with radio transmitters and inexpensive ear tags or leg bands. Tagging‐to‐harvest survival rates from >20 individuals with radio transmitters combined with 50–100 reward tags resulted in an unbiased and precise estimator of harvest rates. In addition, the joint model can test whether transmitters affect an individual's probability of being harvested. We illustrate application of the model using data from wild turkey, Meleagris gallapavo, to estimate harvest rates, and data from white‐tailed deer, Odocoileus virginianus, to evaluate whether the presence of a visible radio transmitter is related to the probability of a deer being harvested. The joint known‐fate tag‐recovery model eliminates the requirement to capture and mark animals immediately prior to the hunting season to obtain accurate and precise estimates of harvest rate. In addition, the joint model can assess whether marking animals with radio transmitters affects the individual's probability of being harvested, caused by hunter selectivity or changes in a marked animal's behavior.