Breaking the spell: Changes in the behavior of two zoo-kept tigers (Panthera tigris) after exposure to a distinct feast-and-fast feeding regime.

The behavior of zoo carnivores has received intense attention due to their propensity for locomotor stereotypies. We observed two adult male tiger (Panthera tigris) siblings kept together for the duration of 104 days by round-the-clock video observation. The period consisted of three baseline periods with the zoo's regular feeding regime of five feeding days per week interrupted by two individual fasting days, with feeding occurring in the evening (B1-B3 of 14 days each). These periods were interrupted by two intervention periods (I1: randomized feeding times, 28 days; I2: gorge-feeding with three 10-day fasting periods, 34 days). As expected, day and night-time behavior was different, with the majority of sleep occurring at night. Pacing, which was mainly considered anticipatory, significantly decreased from 88 ± 132 min/day during B1 to 20 ± 33 min/day during B3. Pacing did not increase during the fasting days of I2. Over the course of whole study, lying time decreased and nonpacing locomotion increased. A major difference was observed between gorge-feeding and the subsequent first fasting days: during gorge-feeding, tigers spent a large part of the day feeding and locomoting (and less sleeping); on the subsequent day, they locomoted about 4.5 h less and slept about 4.3 h more. We suggest that interrupting routines by fasting periods of several days may be effective for reducing regular anticipatory behavior and creates an across-day structure that may correspond to the evolved psychological disposition of large carnivores.

Nocturnal behavioral patterns of African ungulates in zoos.

Currently, most studies on ungulates' behavior are conducted during the daylight hours, but their nocturnal behavior patterns differ from those shown during day. Therefore, it is necessary to observe ungulates' behavior also overnight. Detailed analyses of nocturnal behavior have only been conducted for very prominent ungulates such as Giraffes (Giraffa camelopardalis), African Elephants (Loxodonta africana), or livestock (e.g., domesticated cattle, sheep, or pigs), and the nocturnal rhythms exhibited by many ungulates remain unknown. In the present study, the nocturnal rhythms of 192 individuals of 18 ungulate species from 20 European zoos are studied with respect to the behavioral positions standing, lying-head up, and lying-head down (the typical REM sleep position). Differences between individuals of different age were found, but no differences with respect to the sex were seen. Most species showed a significant increase in the proportion of lying during the night. In addition, the time between two events of "lying down" was studied in detail. A high degree of rhythmicity with respect to this quantity was found in all species. The proportion of lying in such a period was greater in Artiodactyla than in Perissodactyla, and greater in juveniles than in adults.

Time series cluster analysis reveals individual assignment of microbiota in captive tiger (Panthera tigris) and wildebeest (Connochaetes taurinus).

Fecal microbiota variability and individuality are well studied in humans and also in farm animals (related to diet- or disease-specific influences), but very little is known for exotic zoo-housed animals. This includes a wide range of species that differ greatly in microbiota composition and variation. For example, herbivorous species show a very similar and constant fecal microbiota over time, whereas carnivorous species appear to be highly variable in fecal microbial diversity and composition. Our objective was to determine whether species-specific and individual-specific clustering patterns were observed in the fecal microbiota of wildebeest (Connochaetes taurinus) and tigers (Panthera tigris). We collected 95 fecal samples of 11 animal individuals that were each sampled over eight consecutive days and analyzed those with Illumina MiSeq sequencing of the V3-V4 region of the 16SrRNA gene. In order to identify species or individual clusters, we applied two different agglomerative hierarchical clustering algorithms - a community detection algorithm and Ward's linkage. Our results showed that both, species-specific and individual-specific clustering is possible, but more reliable results were achieved when applying dynamic time warping which finds the optimal alignment between different time series. Furthermore, the bacterial families that distinguish individuals from each other in both species included daily occurring core bacteria (e.g., Acidaminococcaceae in wildebeests or Clostridiaceae in tigers) as well as individual dependent and more fluctuating bacterial families. Our results suggest that while it is necessary to consider multiple consecutive samples per individual, it is then possible to characterize individual abundance patterns in fecal microbiota in both herbivorous and carnivorous species. This would allow establishing individual microbiota profiles of animals housed in zoos, which is a basic prerequisite to quickly detect deviations and use microbiome analysis as a non-invasive and cost-effective tool in animal welfare.

Development of A Tool for Assessing the Reputation of Zoos: The Zoo Ethical Reputation Survey (ZERS).

Nowadays, most zoos have taken prominent and active positions in endangered species conservation and educating visitors about the value of biodiversity. However, to be effective and trusted in their mission, they must act ethically and have a good reputation. Yet, the drivers that can influence their reputation are still little investigated, and there are still few studies focused on assessing the reputation of these institutions. In the present work, we report the development of a tool, the Zoo Ethical Reputation Survey (ZERS), and its pilot application to assess the opinions of the visitors of two zoos, one in Italy and one in Germany, on drivers that may influence the ethical reputation of zoos. Preliminary results based on the answers of 274 respondents show that visitors' opinions on zoos acting with ethical responsibility are correlated with emotional appeal and familiarity with these institutions. The application of ZERS can help zoos identify weaknesses in their reputation and develop new strategies to improve people's attitudes towards them, bringing many benefits to the individual zoo and zoological institutions in general.

Diurnal and Nocturnal Behaviour of Cheetahs (Acinonyx jubatus) and Lions (Panthera leo) in Zoos.

Mammals are constantly exposed to exogenous and endogenous influences that affect their behaviour and daily activity. Light and temperature, as well as anthropogenic factors such as husbandry routines, visitors, and feeding schedules are potential influences on animals in zoological gardens. In order to investigate the effects of some of these factors on animal behaviour, observational studies based on the analyses of activity budgets can be used. In this study, the daily and nightly activity budgets of six lions (Panthera leo) and five cheetahs (Acinonyx jubatus) from four EAZA institutions were investigated. Focused on the influencing factor light and feeding, we analysed these activity budgets descriptively. Behaviour was recorded and analysed during the winter months over an observation period of 14 days and 14 nights using infrared-sensitive cameras. Our results show that lions and cheetahs exhibit activity peaks at crepuscular and feeding times, regardless of husbandry. Thus, lions in captivity shift nocturnal behaviour familiar from the wild to crepuscular and diurnal times. In cheetahs, in contrast, captive and wild individuals show similar 24 h behavioural rhythms. The resting behaviour of both species is more pronounced at night, with cheetahs having a shorter overall sleep duration than lions. This study describes the results of the examined animals and is not predictive. Nevertheless, the results of this study make an important contribution to gaining knowledge about possible factors influencing the behaviour of lions and cheetahs in zoos and offer implications that could be useful for improving husbandry and management.

Introducing the Software CASE (Cluster and Analyze Sound Events) by Comparing Different Clustering Methods and Audio Transformation Techniques Using Animal Vocalizations.

Unsupervised clustering algorithms are widely used in ecology and conservation to classify animal sounds, but also offer several advantages in basic bioacoustics research. Consequently, it is important to overcome the existing challenges. A common practice is extracting the acoustic features of vocalizations one-dimensionally, only extracting an average value for a given feature for the entire vocalization. With frequency-modulated vocalizations, whose acoustic features can change over time, this can lead to insufficient characterization. Whether the necessary parameters have been set correctly and the obtained clustering result reliably classifies the vocalizations subsequently often remains unclear. The presented software, CASE, is intended to overcome these challenges. Established and new unsupervised clustering methods (community detection, affinity propagation, HDBSCAN, and fuzzy clustering) are tested in combination with various classifiers (k-nearest neighbor, dynamic time-warping, and cross-correlation) using differently transformed animal vocalizations. These methods are compared with predefined clusters to determine their strengths and weaknesses. In addition, a multidimensional data transformation procedure is presented that better represents the course of multiple acoustic features. The results suggest that, especially with frequency-modulated vocalizations, clustering is more applicable with multidimensional feature extraction compared with one-dimensional feature extraction. The characterization and clustering of vocalizations in multidimensional space offer great potential for future bioacoustic studies. The software CASE includes the developed method of multidimensional feature extraction, as well as all used clustering methods. It allows quickly applying several clustering algorithms to one data set to compare their results and to verify their reliability based on their consistency. Moreover, the software CASE determines the optimal values of most of the necessary parameters automatically. To take advantage of these benefits, the software CASE is provided for free download.

BOVIDS: A deep learning-based software package for pose estimation to evaluate nightly behavior and its application to common elands (Tragelaphus oryx) in zoos.

Only a few studies on the nocturnal behavior of African ungulates exist so far, with mostly small sample sizes. For a comprehensive understanding of nocturnal behavior, the data basis needs to be expanded. Results obtained by observing zoo animals can provide clues for the study of wild animals and furthermore contribute to a better understanding of animal welfare and better husbandry conditions in zoos. The current contribution reduces the lack of data in two ways. First, we present a stand-alone open-source software package based on deep learning techniques, named Behavioral Observations by Videos and Images using Deep-Learning Software (BOVIDS). It can be used to identify ungulates in their enclosure and to determine the three behavioral poses "Standing," "Lying-head up," and "Lying-head down" on 11,411 h of video material with an accuracy of 99.4%. Second, BOVIDS is used to conduct a case study on 25 common elands (Tragelaphus oryx) out of 5 EAZA zoos with a total of 822 nights, yielding the first detailed description of the nightly behavior of common elands. Our results indicate that age and sex are influencing factors on the nocturnal activity budget, the length of behavioral phases as well as the number of phases per behavioral state during the night while the keeping zoo has no significant influence. It is found that males spend more time in REM sleep posture than females while young animals spend more time in this position than adult ones. Finally, the results suggest a rhythm between the Standing and Lying phases among common elands that opens future research directions.

Nature Interest Scale - Development and Evaluation of a Measurement Instrument for Individual Interest in Nature.

Interest is an important factor for successful learning that has been the subject of intensive research for decades. Although interest in nature is of great importance for environmental education, to date there is no valid and reliable measurement tool. Therefore, the purpose of this study was to develop and test a scale for interest in nature, the Nature Interest Scale (NIS). In study 1, nine items were selected based on the three dimensions of the psychological interest construct to represent interest in nature. The factor structure of this new measurement instrument, was tested using confirmatory factor analyses. The results show that the instrument represents the three dimensions of the interest construct well. In study 2 the validity (discriminant and convergent validity) as well as the reliability (internal consistency, composite reliability, test-retest reliability) of the NIS were demonstrated. In study 3, the applicability of the NIS was tested with a different target group, students with learning disabilities. The results of this factor analysis also confirm the factor structure of the scale. Thus, this study provides a valid and reliable measurement tool for individual interest in nature that can be used for future research.

Effects of Positive Reinforcement Training and Novel Object Exposure on Salivary Cortisol Levels under Consideration of Individual Variation in Captive African Elephants (Loxodonta africana).

Dealing with potential stress in species that have high husbandry requirements, such as elephants, is a challenge for zoos. The objective of the present study was to determine whether positive reinforcement training (PRT) and exposure to a novel object (NOV) for enrichment induced a salivary cortisol response indicative of activation of the hypothalamic-pituitary-adrenal (HPA) axis and which factors determine individual variation in this regard in captive African elephants. We repeatedly sampled the saliva of ten animals (three zoos) for the analysis of cortisol (SACort) before and up to 60 min (in 10-15 min intervals) after the onset of PRT (three repeats) or NOV (nine repeats), which lasted 10 min. There was considerable individual variation in SACort in response to PRT or NOV. Using mixed models, we were able to control these and to reveal that PRT was associated with high SACort before and relatively low SACort after PRT, while NOV induced a moderate SACort increase. The individual differences in SACort were related to age and sex (NOV), while the effects of zoo, handling method (free vs. protected contact) and reproductive and social status were variable. We conclude that positive affective states, such as anticipation or arousal, should be taken into account when interpreting the differences in the SACort responses between PRT and NOV. In addition, understanding the individuality of stress will support management decisions aimed at promoting captive elephant welfare.

Discrimination of Acoustic Stimuli and Maintenance of Graded Alarm Call Structure in Captive Meerkats.

Animals living in human care for several generations face the risk of losing natural behaviors, which can lead to reduced animal welfare. The goal of this study is to demonstrate that meerkats (Suricata suricatta) living in zoos can assess potential danger and respond naturally based on acoustic signals only. This includes that the graded information of urgency in alarm calls as well as a response to those alarm calls is retained in captivity. To test the response to acoustic signals with different threat potential, meerkats were played calls of various animals differing in size and threat (e.g., robin, raven, buzzard, jackal) while their behavior was observed. The emitted alarm calls were recorded and examined for their graded structure on the one hand and played back to them on the other hand by means of a playback experiment to see whether the animals react to their own alarm calls even in the absence of danger. A fuzzy clustering algorithm was used to analyze and classify the alarm calls. Subsequently, the features that best described the graded structure were isolated using the LASSO algorithm and compared to features already known from wild meerkats. The results show that the graded structure is maintained in captivity and can be described by features such as noise and duration. The animals respond to new threats and can distinguish animal calls that are dangerous to them from those that are not, indicating the preservation of natural cooperative behavior. In addition, the playback experiments show that the meerkats respond to their own alarm calls with vigilance and escape behavior. The findings can be used to draw conclusions about the intensity of alertness in captive meerkats and to adapt husbandry conditions to appropriate welfare.

Unraveling differences in fecal microbiota stability in mammals: from high variable carnivores and consistently stable herbivores.

BACKGROUND: Through the rapid development in DNA sequencing methods and tools, microbiome studies on a various number of species were performed during the last decade. This advance makes it possible to analyze hundreds of samples from different species at the same time in order to obtain a general overview of the microbiota. However, there is still uncertainty on the variability of the microbiota of different animal orders and on whether certain bacteria within a species are subject to greater fluctuations than others. This is largely due to the fact that the analysis in most extensive comparative studies is based on only a few samples per species or per study site. In our study, we aim to close this knowledge gap by analyzing multiple individual samples per species including two carnivore suborders Canoidea and Feloidea as well as the orders of herbivore Perissodactyla and Artiodactyla held in different zoos. To assess microbial diversity, 621 fecal samples from 31 species were characterized by sequencing the V3-V4 region of the 16S rRNA gene using Illumina MiSeq. RESULTS: We found significant differences in the consistency of microbiota composition and in fecal microbial diversity between carnivore and herbivore species. Whereas the microbiota of Carnivora is highly variable and inconsistent within and between species, Perissodactyla and Ruminantia show fewer differences across species boundaries. Furthermore, low-abundance bacterial families show higher fluctuations in the fecal microbiota than high-abundance ones. CONCLUSIONS: Our data suggest that microbial diversity is significantly higher in herbivores than in carnivores, whereas the microbiota in carnivores, unlike in herbivores, varies widely even within species. This high variability has methodological implications and underlines the need to analyze a minimum amount of about 10 samples per species. In our study, we found considerable differences in the occurrence of different bacterial families when looking at just three and six samples. However, from a sample number of 10 onwards, these within-species fluctuations balanced out in most cases and led to constant and more reliable results.

Deep learning-based pose estimation for African ungulates in zoos.

The description and analysis of animal behavior over long periods of time is one of the most important challenges in ecology. However, most of these studies are limited due to the time and cost required by human observers. The collection of data via video recordings allows observation periods to be extended. However, their evaluation by human observers is very time-consuming. Progress in automated evaluation, using suitable deep learning methods, seems to be a forward-looking approach to analyze even large amounts of video data in an adequate time frame.In this study, we present a multistep convolutional neural network system for detecting three typical stances of African ungulates in zoo enclosures which works with high accuracy. An important aspect of our approach is the introduction of model averaging and postprocessing rules to make the system robust to outliers.Our trained system achieves an in-domain classification accuracy of >0.92, which is improved to >0.96 by a postprocessing step. In addition, the whole system performs even well in an out-of-domain classification task with two unknown types, achieving an average accuracy of 0.93. We provide our system at https://github.com/Klimroth/Video-Action-Classifier-for-African-Ungulates-in-Zoos/tree/main/mrcnn_based so that interested users can train their own models to classify images and conduct behavioral studies of wildlife.The use of a multistep convolutional neural network for fast and accurate classification of wildlife behavior facilitates the evaluation of large amounts of image data in ecological studies and reduces the effort of manual analysis of images to a high degree. Our system also shows that postprocessing rules are a suitable way to make species-specific adjustments and substantially increase the accuracy of the description of single behavioral phases (number, duration). The results in the out-of-domain classification strongly suggest that our system is robust and achieves a high degree of accuracy even for new species, so that other settings (e.g., field studies) can be considered.

Biological and environmental factors as sources of variation in nocturnal behavior of giraffe.

Upon a drastic decline of the giraffe population in the wild, conservation efforts and therefore the role of zoos have become more important than ever. With their unique opportunities, zoos provide excellent conditions to study animal behavior, expanding the knowledge about the giraffe's behavior repertoire and their ability to adapt. This study therefore examined the nocturnal behavior of 63 giraffe living in 13 different EAZA zoos across Germany and the Netherlands. Giraffe were observed and videos recorded via infrared sensitive cameras during the winter seasons 2015-2018. The observation period spanned nightly from 17:00 to 7:00. Thus, 198 nights, with a total of 2772 h were recorded and analyzed. Linear mixed models were then used to assess potential biological and environmental factors influencing behavior during the dark phase. Results show that individual variables such as age, subspecies and motherhood determined nocturnal activity and sleep behavior most. Among the variables studied, husbandry conditions and environmental factors complying with EAZA standards had no influence on the giraffe's nocturnal behavior. By combining nocturnal activity analyses and an assessment of potential influencing factors, our findings present a holistic approach to a better understanding of captive giraffe behavior and allow for management implications.

Impact of biological education and gender on students' connection to nature and relational values.

The new concept of relational values (RVs) is gaining more and more attention in environmental research, but empirical analyses are still rare. However, this type of research is necessary because the RVs have an influence on environmental behavior. To evaluate the impact of biological education on attributing higher importance to RVs and connectedness to nature, we compared the connection to nature scores (using the inclusion of nature scale (INS) and connectedness to nature scale (CNS)) and RV scores of biologically interested high school students (n = 417) with first year (n = 593) and advanced biology (n = 223) students. While high school students showed significant lower connection to nature scores than university students, there was no significant difference in RVs between the test groups. These results suggest that there is a lack of factors in the university study of biology that can change RVs. The gender comparison of RVs and connection to nature showed a significant higher RV score for females while INS and CNS did not show a gender difference. Thus, the study makes an important contribution to the research, as it was able to prove that gender has an influence on a person's RVs but not on their connection to nature.

Connecting High School Students With Nature - How Different Guided Tours in the Zoo Influence the Success of Extracurricular Educational Programs.

Zoos attract millions of visitors every year, many of whom are schoolchildren. For this reason, zoos are important institutions for the environmental education of future generations. Empirical studies on the educational impact of environmental education programs in zoos are still rare. To address this issue, we conducted two studies: In study 1, we investigated students' interests in different biological topics, including zoos (n = 1,587). Data analysis of individual topics revealed large differences of interest, with advanced students showing less interest in zoos. In study 2, we invited school classes of this age group to visit different guided tours at the zoo and tested connection to nature before and after each educational intervention (n = 608). The results showed that the guided tours are an effective tool to raise students' connection to nature. Add-on components have the potential to further promote connection to nature. The education programs are most effective with students with a low initial nature connection.

Exploring the Construct of Relational Values: An Empirical Approach.

In recent environmental research, relational values (RVs) have emerged as a new group of values to explain environmental behavior. Although this new concept is attracting attention, empirical studies on the subject are still rare. On this basis, we have conducted three studies to analyze an existing measurement tool for RVs and compared the construct with the concept of connection to nature. In study 1, we confirmed convergent and discriminant validity of the RV scale by comparing it with the Two Factor Model of Environmental Values (2-MEV) model using a sample of n = 350 university students. Additionally, study 1 verified reliability using test-retest reliability on three different groups of students (n 1 = 53; n 2 = 37; n 3 = 48). In study 2, principal component analyses were performed to examine the structure of RVs and to compare it to the concept of connection to nature by reusing the sample 350 university students from study 1. The results show that RVs and connection to nature are not fundamentally distinct constructs, but overlap. However, if the structure of the RV measurement is forced to a single factor, no perfect fit is found, making a multidimensional solution more likely. A third study was conducted to review the results from study 2 using confirmatory factor analysis on a new sample of 878 university and high school students. Study 3 confirmed RVs as a multidimensional construct with three factors: care, community, and connection. It also proved the overlap of the connection to nature and RV concepts to some extent.

Nightly selection of resting sites and group behavior reveal antipredator strategies in giraffe.

This study presents the first findings on nocturnal behavior patterns of wild Angolan giraffe. We characterized their nocturnal behavior and analyzed the influence of ecological factors such as group size, season, and habitat use. Giraffe were observed using night vision systems and thermal imaging cameras on Okapuka Ranch, Namibia. A total of 77 giraffe were observed during 24 nights over two distinct periods-July-August 2016 (dry season) and February-March 2017 (wet season). Photoperiod had a marked influence on their activity and moving behavior. At dusk, giraffe reduced the time spent moving and increasingly lay down and slept at the onset of darkness. Body postures that likely correspond to rapid eye movement (REM) sleep posture (RSP) were observed 15.8 ± 18.3 min after giraffe sat down. Season had a significant effect with longer RSP phases during the dry season (dry: 155.2 ± 191.1 s, n = 79; wet: 85.8 ± 94.9 s, n = 73). Further analyses of the influence of social behavior patterns did not show an effect of group size on RSP lengths. When a group of giraffe spent time at a specific resting site, several individuals were alert (vigilant) while other group members sat down or took up RSP. Simultaneous RSP events within a group were rarely observed. Resting sites were characterized by single trees or sparse bushes on open areas allowing for good visibility in a relatively sheltered location.

Diurnal variation of salivary cortisol in captive African elephants (Loxodonta africana) under routine management conditions and in relation to a translocation event.

The present study assessed the diurnal variation in salivary cortisol in captive African elephants during routine management (baseline) and in relation to a potential stressor (translocation) to evaluate to what extent acute stress may affect diurnal cortisol patterns. Under baseline conditions, we collected morning and afternoon saliva samples of 10 animals (three zoos) on different days in two study periods (n = 3-10 per animal, daytime and period). Under stress conditions, we sampled the transported cow (newcomer) and the two cows of the destination zoo before and after the transport in the morning and afternoon (n = 3-9 per animal, daytime and transport phase), as well as after the first introduction of the newcomer to the bull (n = 1 per animal). Cortisol was measured in unextracted samples by enzyme immunoassay. Under baseline conditions, we observed the expected diurnal variation with higher cortisol levels in the morning than in the afternoon. Under stress conditions, neither a significant difference between pre- and posttransport, nor between morning and afternoon levels was found. The percentage difference between morning and afternoon cortisol after the transport, however, was remarkably lower than before the transport in the newcomer potentially indicating a stress response to familiarization. Saliva samples taken immediately after the introduction of the newcomer to the bull revealed a marked cortisol increase. Our findings indicate that stressors may disturb the diurnal cortisol rhythm. Furthermore, provided that samples can be collected promptly, salivary cortisol is a useful minimally invasive measure of physiological stress in the African elephant.

Phasic GABAA -receptor activation is required to suppress epileptiform activity in the CA3 region of the immature rat hippocampus.

PURPOSE: Despite the consistent observation that γ-aminobutyric acid A (GABA(A) ) receptors mediate excitatory responses at perinatal stages, the role of the GABAergic system in the generation of neonatal epileptiform activity remains controversial. Therefore, we analyzed whether tonic and phasic GABAergic transmission had differential effects on neuronal excitability during early development. METHODS: We performed whole cell patch-clamp and field potential recordings in the CA3 region of hippocampal slices from immature (postnatal day 4-7) rats to analyze the effect of specific antagonists and modulators of tonic and phasic GABAergic components on neuronal excitability. KEY FINDINGS: The GABAergic antagonists gabazine (3 µm) and picrotoxin (100 µm) induced epileptiform discharges, whereas activation of GABA(A) receptors attenuated epileptiform discharges. Under low-Mg(2+) conditions, 100 nm gabazine and 1 µm picrotoxin were sufficient to provoke epileptiform activity in 63.2% (n = 19) and 53.8% (n = 26) of the slices, respectively. Whole-cell patch-clamp experiments revealed that these concentrations significantly reduced the amplitude of phasic GABAergic postsynaptic currents but had no effect on tonic currents. In contrast, 1-µm 4,5,6,7-tetrahydroisoxaz-olo[5,4-c]-pyridin-3-ol (THIP) induced a tonic current of -12 ± 2.5 pA (n = 6) and provoked epileptiform discharges in 57% (n = 21) of the slices. SIGNIFICANCE: We conclude from these results that in the early postnatal rat hippocampus a constant phasic synaptic activity is required to control excitability and prevent epileptiform activity, whereas tonic GABAergic currents can mediate excitatory responses. Pharmacologic intervention at comparable human developmental stages should consider these ambivalent GABAergic actions.