Bi-allelic Loss-of-Function Variants in NUP188 Cause a Recognizable Syndrome Characterized by Neurologic, Ocular, and Cardiac Abnormalities.

Nucleoporins (NUPs) are an essential component of the nuclear-pore complex, which regulates nucleocytoplasmic transport of macromolecules. Pathogenic variants in NUP genes have been linked to several inherited human diseases, including a number with progressive neurological degeneration. We present six affected individuals with bi-allelic truncating variants in NUP188 and strikingly similar phenotypes and clinical courses, representing a recognizable genetic syndrome; the individuals are from four unrelated families. Key clinical features include congenital cataracts, hypotonia, prenatal-onset ventriculomegaly, white-matter abnormalities, hypoplastic corpus callosum, congenital heart defects, and central hypoventilation. Characteristic dysmorphic features include small palpebral fissures, a wide nasal bridge and nose, micrognathia, and digital anomalies. All affected individuals died as a result of respiratory failure, and five of them died within the first year of life. Nuclear import of proteins was decreased in affected individuals' fibroblasts, supporting a possible disease mechanism. CRISPR-mediated knockout of NUP188 in Drosophila revealed motor deficits and seizure susceptibility, partially recapitulating the neurological phenotype seen in affected individuals. Removal of NUP188 also resulted in aberrant dendrite tiling, suggesting a potential role of NUP188 in dendritic development. Two of the NUP188 pathogenic variants are enriched in the Ashkenazi Jewish population in gnomAD, a finding we confirmed with a separate targeted population screen of an international sampling of 3,225 healthy Ashkenazi Jewish individuals. Taken together, our results implicate bi-allelic loss-of-function NUP188 variants in a recessive syndrome characterized by a distinct neurologic, ophthalmologic, and facial phenotype.

Comparative Observation and Analysis of Preference Behavior Based on Three Types of Taxes and Locomotor Activity in the Goldfish, Carassius auratus.

Psychophysiological studies in vertebrates have focused on taxes as indicators of behavioral change. Actually, a considerable number of studies about anxiety-like and anti-anxiety-like behaviors involving geotaxis, scototaxis, and thigmotaxis have been conducted on fish. However, few analyses considering these behaviors based on taxes in fish have been conducted. Here, using goldfish, we measured the time spent in the bright or dark area of a horizontally long rectangular tank (HLRT), in the upper or lower area of a vertically long rectangular tank (VLRT), and in the central or edge area of a circular tank (CT), respectively, for the first 30 min and the last 30 min in a 3-h period after fish had been introduced to tanks. Dark, lower, and edge preference behaviors were observed for the first 30 min in all tanks. While dark and edge preference behaviors were maintained even for the last 30 min, the lower preference was lost. Swimming distance and the number of area crossings in each tank were also compared between the first 30 min and the last 30 min. Both decreased significantly or tended to decrease in the last 30 min in the HLRT and the CT, but no change was observed in the VLRT. These results suggest that, in goldfish, preference behavior is stable for a short time, and that environmental habituation may depend on the shape of the tank and the elapsed time.

Sustained positive consequences of genetic rescue of fitness and behavioural traits in inbred populations of Drosophila melanogaster.

One solution to alleviate the detrimental genetic effects associated with reductions in population size and fragmentation is to introduce immigrants from other populations. While the effects of this genetic rescue on fitness traits are fairly well known, it is less clear to what extent inbreeding depression and subsequent genetic rescue affect behavioural traits. In this study, replicated crosses between inbred lines of Drosophila melanogaster were performed in order to investigate the effects of inbreeding and genetic rescue on egg-to-adult viability and negative geotaxis behaviour-a locomotor response used to measure, e.g. the effects of physiological ageing. Transgenerational effects of outcrossing were investigated by examining the fitness consequences in both the F1 and F4  generation. The majority of inbred lines showed evidence for inbreeding depression for both egg-to-adult viability and behavioural performance (95% and 66% of lines, respectively), with inbreeding depression being more pronounced for viability compared with the locomotor response. Subsequent outcrossing with immigrants led to an alleviation of the negative effects for both viability and geotaxis response resulting in inbred lines being similar to the outbred controls, with beneficial effects persisting from F1 to F4 . Overall, the results clearly show that genetic rescue can provide transgenerational rescue of small, inbred populations by rapidly improving population fitness components. Thus, we show that even the negative effects of inbreeding on behaviour, similar to that of neurodegeneration associated with physiological ageing, can be reversed by genetic rescue.

Differential analysis of negative geotaxis climbing trajectories in Drosophila under different conditions.

The decline of Drosophila climbing behavior is one of the common phenomena of Drosophila aging. The so-called negative geotaxis refers to the natural upward climbing behavior of Drosophila melanogaster after it oscillates to the bottom of the test tube. The strength of climbing ability is regarded as the index of aging change of D. melanogaster. At present, many laboratories use the percentage of 10 fruit flies climbing a specific height in 5 s as a general indicator of the climbing ability of fruit flies. This group research index ignores the climbing performance of a single fruit fly, and the climbing height belongs to the concept of vertical distance in physics, which cannot truly and effectively reflect the concept of curve distance in the actual climbing process of fruit flies. Therefore, based on the image processing algorithm, we added an experimental method to draw the climbing trajectory of a single fruit fly. By comparing the differences in climbing behavior of fruit flies under different sex, group or single, oscillation condition or rotation inversion condition, we can find that the K-Nearest Neighbor target detection algorithm has good applicability in fruit fly climbing experiment, and the climbing ability of fruit flies decreases with age. Under the same experimental conditions, the climbing ability of female fruit flies was greater than that of male fruit flies. The climbing track length of a single fruit fly can better reflect the climbing process of a fruit fly.

A locomotor assay reveals deficits in heterozygous Parkinson's disease model and proprioceptive mutants in adult Drosophila.

Severe locomotor impairment is a common phenotype of neurodegenerative disorders such as Parkinson's disease (PD). Drosophila models of PD, studied for more than a decade, have helped in understanding the interaction between various genetic factors, such as parkin and PINK1, in this disease. To characterize locomotor behavioral phenotypes for these genes, fly climbing assays have been widely used. While these simple current assays for locomotor defects in Drosophila mutants measure some locomotor phenotypes well, it is possible that detection of subtle changes in behavior is important to understand the manifestation of locomotor disorders. We introduce a climbing behavior assay which provides such fine-scale behavioral data and tests this proposition for the Drosophila model. We use this inexpensive, fully automated assay to quantitatively characterize the climbing behavior at high parametric resolution in 3 contexts. First, we characterize wild-type flies and uncover a hitherto unknown sexual dimorphism in climbing behavior. Second, we study climbing behavior of heterozygous mutants of genes implicated in the fly PD model and reveal previously unreported prominent locomotor defects in some of these heterozygous fly lines. Finally, we study locomotor defects in a homozygous proprioceptory mutation (Trp-γ1 ) known to affect fine motor control in Drosophila Moreover, we identify aberrant geotactic behavior in Trp-γ1 mutants, thereby opening up a finer assay for geotaxis and its genetic basis. Our assay is therefore a cost-effective, general tool for measuring locomotor behaviors of wild-type and mutant flies in fine detail and can reveal subtle motor defects.

FreeClimber: automated quantification of climbing performance in Drosophila.

Negative geotaxis (climbing) performance is a useful metric for quantifying Drosophila health. Manual methods to quantify climbing performance are tedious and often biased, while many available computational methods have challenging hardware or software requirements. We present an alternative: FreeClimber. This open source, Python-based platform subtracts a video's static background to improve detection for flies moving across heterogeneous backgrounds. FreeClimber calculates a cohort's velocity as the slope of the most linear portion of a mean vertical position versus time curve. It can run from a graphical user interface for optimization or a command line interface for high-throughput and automated batch processing, improving accessibility for users with different expertise. FreeClimber outputs calculated slopes, spot locations for follow-up analyses (e.g. tracking), and several visualizations and plots. We demonstrate FreeClimber's utility in a longitudinal study for endurance exercise performance in Drosophila mitonuclear genotypes using six distinct mitochondrial haplotypes paired with a common D. melanogaster nuclear background.

Is SGSH heterozygosity a risk factor for early-onset neurodegenerative disease?

Lysosomal dysfunction may be an important factor in the pathogenesis of neurodegenerative disorders such as Parkinson's disease (PD). Heterozygous mutations in the gene encoding the lysosomal enzyme glucocerebrosidase (GBA1) have been found in PD patients, and some but not all mutations in other lysosomal enzyme genes, for example, NPC1 and MCOLN1 have been associated with PD. We have examined the behaviour and brain structure of mice carrying a D31N mutation in the sulphamidase (Sgsh) gene which encodes a lysosomal sulphatase. Female heterozygotes and wildtype mice aged 12-, 15-, 18- and 21-months of age underwent motor phenotyping and the brain was comprehensively evaluated for disease-associated lesions. Heterozygous mice exhibited impaired performance in the negative geotaxis test when compared with wildtype mice. Whilst the brain of Sgsh heterozygotes aged up to 21-months did not exhibit any of the gross features of PD, Alzheimer's disease or the neurodegenerative lysosomal storage disorders, for example, loss of striatal dopamine, reduced GBA activity, α-synuclein-positive inclusions, perturbation of lipid synthesis, or cerebellar Purkinje cell drop-out, we noted discrete structural aberrations in the dendritic tree of cortical pyramidal neurons in 21-month old animals. The overt disease lesions and resultant phenotypic changes previously described in individuals with heterozygous mutations in lysosomal enzyme genes such as glucocerebrosidase may be enzyme dependent. By better understanding why deficiency in, or mutant forms of some but not all lysosomal proteins leads to heightened risk or earlier onset of classical neurodegenerative disorders, novel disease-causing mechanisms may be identified.

Atg2, Atg9 and Atg18 in mitochondrial integrity, cardiac function and healthspan in Drosophila.

In yeast, the Atg2-Atg18 complex regulates Atg9 recycling from phagophore assembly site during autophagy; their function in higher eukaryotes remains largely unknown. In a targeted screening in Drosophila melanogaster, we show that Mef2-GAL4-RNAi-mediated knockdown of Atg2, Atg9 or Atg18 in the heart and indirect flight muscles led to shortened healthspan (declined locomotive function) and lifespan. These flies displayed an accelerated age-dependent loss of cardiac function along with cardiac hypertrophy (increased heart tube wall thickness) and structural abnormality (distortion of the lumen surface). Using the Mef2-GAL4-MitoTimer mitochondrial reporter system and transmission electron microscopy, we observed significant elongation of mitochondria and reduced number of lysosome-targeted autophagosomes containing mitochondria in the heart tube but exaggerated mitochondrial fragmentation and reduced mitochondrial density in indirect flight muscles. These findings provide the first direct evidence of the importance of Atg2-Atg18/Atg9 autophagy complex in the maintenance of mitochondrial integrity and, regulation of heart and muscle functions in Drosophila, raising the possibility of augmenting Atg2-Atg18/Atg9 activity in promoting mitochondrial health and, muscle and heart function.

Continuous tracking of startled Drosophila as an alternative to the negative geotaxis climbing assay.

The fruit fly, Drosophila, is commonly used to study late-onset neurodegenerative diseases due to the combination of powerful genetic tools, cheap and simple husbandry and short lifespan. One widely-used measure of disease progression is the age-dependent decline in motor performance that manifests in most Drosophila neurodegeneration models. This is usually quantified using a simple climbing assay. However, the standard climbing assay lacks sensitivity and suffers from high variability meaning large numbers of flies are needed or bespoke apparatus and software solutions. Here, we present a modification of the open-source, MATLAB-based, DART software to measure the decline in "startle response" with age. We demonstrate that the DART setup is more sensitive to the motor performance decline induced by adult-onset neuronal expression of amyloid beta (Aß) peptides than a traditional climbing assay despite using smaller cohorts of flies. DART also has the potential to generate multiple metrics of motor behaviour during the startle response. The software requires no coding skills to operate and the required apparatus can be purchased commercially. Therefore, DART is a more useful method than the climbing assay for longitudinal assays of motor performance and will enable higher-throughput screen for genetic and pharmacological modifiers of neurodegeneration. In our proof-of-concept screen for modifiers of Aß-dependent phenotypes, we identified that in vivo knock-down of p53 in adult neurons is neuroprotective. This supports recent work targeting p53 in vitro and demonstrates the potential for DART to be used to screen for targets that ameliorate neurodegeneration.

Testing the parasite mass burden effect on alteration of host behaviour in the Schistocephalus-stickleback system.

Many parasites with complex life cycles modify the behaviour of their intermediate host, which has been proposed to increase transmission to their definitive host. This behavioural change could result from the parasite actively manipulating its host, but could also be explained by a mechanical effect, where the physical presence of the parasite affects host behaviour. We created an artificial internal parasite using silicone injections in the body cavity to test this mechanical effect hypothesis. We used the Schistocephalus solidus and threespine stickleback (Gasterosteus aculeatus) system, as this cestode can reach up to 92% of its fish host mass. Our results suggest that the mass burden brought by this macroparasite alone is not sufficient to cause behavioural changes in its host. Furthermore, our results show that wall-hugging (thigmotaxis), a measure of anxiety in vertebrates, is significantly reduced in Schistocephalus-infected sticklebacks, unveiling a new altered component of behaviour that may result from manipulation by this macroparasite.

Behavioral analysis of Microphallus turgidus cercariae in relation to microhabitat of two host grass shrimp species (Palaemonetes spp.).

The behavior of Microphallus turgidus cercariae was examined and compared to microhabitat selection of the second intermediate hosts of the parasite, Palaemonetes spp. grass shrimp. Cercariae were tested for photokinetic and geotactic responses, and a behavioral ethogram was established for cercariae in control and grass shrimp-conditioned brackish water. Photokinesis trials were performed using a half-covered Petri dish, and geotaxis trials used a graduated cylinder. Both photokinesis and geotaxis trials were performed in lighted and unlighted conditions. In 9 of 10 photokinesis experiments, over half of the cercariae swam horizontally under the covered half of a Petri dish in both the lighted and the unlighted trials. However, movement of the cercariae to the covered half of the dish was highest (81.4%) when the parasites were exposed to light. In the geotaxis study, most cercariae were found in the bottom third of a graduated cylinder water column in both the lighted and unlighted trials. The most frequently observed activity of individual cercariae in a lighted Petri dish was swimming on the bottom of the dish. Activity patterns of the cercariae were not affected by shrimp-conditioned water. Movement of the cercariae away from light into dark, active swimming at or near the bottom of the water column, and a lack of response to host odors suggest that the cercariae utilize search patterns that place the parasite in the preferred microhabitat of the principle second intermediate host, the grass shrimp P. pugio.

The Hillary Climber trumps manual testing: an automatic system for studying Drosophila climbing.

Climbing or negative geotaxis is an innate behavior of the fruit fly Drosophila melanogaster. There has been considerable interest in using this simple behavior to gain insights into the changes in brain function associated with aging, influence of drugs, mutated genes, and human neurological disorders. At present, most climbing tests are conducted manually and there is a lack of a simple and automatic device for repeatable and quantitative analysis of fly climbing behavior. Here we present an automatic fly climbing system, named the Hillary Climber (after Sir Edmund Hillary), that can replace the human manual tapping of vials with a mechanical tapping mechanism to provide more consistent force and reduce variability between the users and trials. Following tapping the HC records fly climbing, tracks the fly climbing path, and analyzes the velocity of individual flies and the percentage of successful climbers. The system is relatively simple to build, easy to operate, and efficient and reliable for climbing tests.

Circadian and Geotactic Behaviors: Genetic Pleiotropy in Drosophila Melanogaster.

Data presented in this paper test the hypotheses that Hirsch's positive geotaxis (Lo) and negative geotaxis (Hi5) strains of Drosophila melanogaster (fruit fly) differ in length of the free-running circadian activity period (tau) as well as adult geotaxis. Several genes have been shown to alter geotaxis in Drosophila. Two of these genes, cryptochrome (cry) and Pigment-dispersing-factor (Pdf) are integral to the function of biological clocks. Pdf plays a crucial role in maintaining free-running circadian periods. The cry gene alters blue-light (<420 nm) phototransduction which affects biological clocks, spatial orientation and taxis relative to gravity, magnetic fields, solar, lunar, and celestial radiation in several species. The cry gene is involved in phase resetting (entrainment) of the circadian clock by blue light (<420 nm). Geotaxis involves spatial orientation, so it might be expected that geotaxis is linked genetically with other forms of spatial orientation. The association between geotaxis and biological clocks is less intuitive. The data and the literature presented here show that genes, physiology and behavioural aspects of geotaxis, biological clocks, magnetosensitivity and other types of spatial orientation, are complex, intriguing and interrelated.

Tactile cues compensate for unbalanced vestibular cues during progression on inclined surfaces.

A previous study demonstrated that rodents on an inclined square platform traveled straight vertically or horizontally and avoided diagonal travel. Through behavior they aligned their head with the horizontal plane, acquiring similar bilateral vestibular cues - a basic requirement for spatial orientation and a salient feature of animals in motion. This behavior had previously been shown to be conspicuous in Tristram's jirds. Here, therefore jirds were challenged by testing their travel behavior on a circular arena inclined at 0°-75°. Our hypothesis was that if, as typical to rodents, the jirds would follow the curved arena wall, they would need to display a compensating mechanism to enable traveling in such a path shape, which involves a tilted frontal head axis and unbalanced bilateral vestibular cues. We found that with the increase in inclination, the jirds remained more in the lower section of the arena (geotaxis). When tested on the steep inclinations, however, their travel away from the arena wall was strictly straight up or down, in contrast to the curved paths that followed the circular arena wall. We suggest that traveling along a circular path while maintaining contact with the wall (thigmotaxis), provided tactile information that compensated for the unbalanced bilateral vestibular cues present when traveling along such curved inclined paths. In the latter case, the frontal plane of the head was in a diagonal posture in relation to gravity, a posture that was avoided when traveling away from the wall.

Adverse neurobehavioral changes with reduced blood and brain cholinesterase activities in mice treated with statins.

Background and Aim: Pleiotropic effects of hypolipidemic statins with behavioral outcomes have been suggested in humans and laboratory animals. There is limited information on the neurobehavioral effects of statins in mice. The aim of the present study was to examine changes in neurobehavioral performance and cholinesterase (ChE) activity in mice after high doses of three commonly used statins (atorvastatin, simvastatin, and rosuvastatin). Materials and Methods: Two hours after vehicle (control) or statin dosing at 250, 500, 750, or 1000 mg/kg orally, each mouse was subjected to 5 min open-field activity, negative geotaxis at an angle of 45°/60 s, 5 min head pocking, and forced swimming endurance. Plasma, erythrocyte, and brain ChE activities were determined spectrophotometrically 2 and 24 h after oral dosing of statins at 500 and 1000 mg/kg. Results: The statins variably, but dose-dependently and significantly (p < 0.05) delayed the latency to move in the open-field arena, decreased locomotion and rearing, reduced head pocking, and delayed negative geotaxis performance. However, statins significantly increased the duration of forced swimming and decreased the duration of immobility in the swimming tank. Statins significantly and dose-dependently decreased plasma, erythrocyte, and brain ChE activity 2 and 24 h after dosing. Plasma and brain ChE activities recovered by 5%-32.9% and 5.7%-14.4% 24 h later from the 2 h ChE values, respectively. Conclusion: High doses of statins differentially modulate neurobehavioral outcomes in mice in association with reduced plasma, erythrocyte, and brain ChE activity. Plasma or erythrocyte ChE may be used for biomonitoring of the adverse/therapeutic effects of statins.

Systemic Effects of Photoactivated 5,10,15,20-tetrakis(N-methylpyridinium-3-yl) Porphyrin on Healthy Drosophila melanogaster.

Porphyrins are frequently employed in photodynamic therapy (PDT), a non-invasive technique primarily utilized to treat subcutaneous cancers, as photosensitizing agents (PAs). The development of a new PA with improved tissue selectivity and efficacy is crucial for expanding the application of PDT for the management of diverse cancers. We investigated the systemic effects of 5,10,15,20-tetrakis(N-methylpyridinium-3-yl)-porphyrin (TMPyP3) using Drosophila melanogaster adult males. We established the oral administration schedule and demonstrated that TMPyP3 was absorbed and stored higher in neuronal than in non-neuronal extracts. Twenty-four hours after oral TMPyP3 photoactivation, the quantity of hydrogen peroxide (H2O2) increased, but exclusively in the head extracts. Regardless of photoactivation, TMPyP3 resulted in a reduced concentration of H2O2 after 7 days, and this was linked with a decreased capacity to climb, as indicated by negative geotaxis. The findings imply that systemic TMPyP3 therapy may disrupt redox regulation, impairing cellular signaling and behavioral outcomes in the process. To determine the disruptive effect of porphyrins on redox homeostasis, its duration, and the mechanistic variations in retention across various tissues, more research is required.

Neonatal Behavioral Screen for Mouse Models of Neurodevelopmental Disorders.

Behavioral phenotyping approaches for neonatal mice are important for investigating early alterations in brain development and function, relevant to neurodevelopmental disorders in humans. This chapter describes a behavioral screen that can provide an overall profile of function across the neonatal and preweaning period while also minimizing pup stress and disturbance of the maternal environment. Testing begins when mice are between 6 and 8 days in age, with additional evaluations at discrete time points until postnatal day (PD) 20-21, using tests for negative geotaxis, surface righting reflex, activity in an open field, acoustic startle responses and sensorimotor gating, and limb clasp.

Distinct sex-dependent effects of maternal preconception nicotine and enrichment on the early development of rat offspring brain and behavior.

Developmental nicotine exposure is harmful to offspring. Whereas much is known about the consequences of prenatal nicotine exposure, relatively little is understood about how maternal preconception nicotine impacts the next generation. Positive experiences, such as environmental enrichment/complexity, have considerable potential to improve developmental outcomes and even treat and prevent drug addiction. Therefore, the current study sought to identify how maternal exposure to moderate levels of nicotine prior to conception impacts offspring development, and if the presumably negative consequence of nicotine could be reversed by concurrent exposure to an enriched environment. We treated female Long Evans rats with nicotine in their drinking water (15 mg nicotine salt/L) for seven weeks while residing in either standard or enriched conditions. Both experiences occurred exclusively prior to mating. Nicotine exposure reduced dam fertility by ~20% (p = .06). Females reared their own litters, and offspring were tested in two assessments of early development: negative geotaxis and open field. Offspring were euthanized at weaning (P21), and their brains were processed with Golgi-Cox solution to allow quantification of dendritic spine density. Results indicate that neither maternal nicotine or enrichment had an impact on maternal care, but male offspring were impaired at negative geotaxis due to maternal nicotine, female offspring showed altered open field exploration due to maternal enrichment, and offspring of both sexes had increased spine density in OFC due to maternal enrichment. Therefore, this experiment provides novel insights into the unique, sex-dependent consequences of maternal preconception nicotine and enrichment on the early development of rat behavior and brain.

ShK-Domain-Containing Protein from a Parasitic Nematode Modulates Drosophila melanogaster Immunity.

A key component to understanding host-parasite interactions is the molecular crosstalk between host and parasite. Excreted/secreted products (ESPs) released by parasitic nematodes play an important role in parasitism. They can directly damage host tissue and modulate host defense. Steinernema carpocapsae, a well-studied parasite of insects releases approximately 500 venom proteins as part of the infection process. Though the identity of these proteins is known, few have been studied in detail. One protein family present in the ESPs released by these nematodes is the ShK family. We studied the most abundant ShK-domain-containing protein in S. carpocapsae ESPs, Sc-ShK-1, to investigate its effects in a fruit fly model. We found that Sc-ShK-1 is toxic under high stress conditions and negatively affects the health of fruit flies. We have shown that Sc-ShK-1 contributes to host immunomodulation in bacterial co-infections resulting in increased mortality and microbial growth. This study provides an insight on ShK-domain-containing proteins from nematodes and suggests these proteins may play an important role in host-parasite interactions.

Collective Emotional Contagion in Zebrafish.

Seeking to match our emotional state with one of those around us is known as emotional contagion-a fundamental biological process that underlies social behavior across several species and taxa. While emotional contagion has been traditionally considered to be a prerogative of mammals and birds, recent findings are demonstrating otherwise. Here, we investigate emotional contagion in groups of zebrafish, a freshwater model species which is gaining momentum in preclinical studies. Zebrafish have high genetic homology to humans, and they exhibit a complex behavioral repertoire amenable to study social behavior. To investigate whether individual emotional states can be transmitted to group members, we pharmacologically modulated anxiety-related behaviors of a single fish through Citalopram administration and we assessed whether the altered emotional state spread to a group of four untreated conspecifics. By capitalizing upon our in-house developed tracking algorithm, we successfully preserved the identity of all the subjects and thoroughly described their individual and social behavioral phenotypes. In accordance with our predictions, we observed that Citalopram administration consistently reduced behavioral anxiety of the treated individual, in the form of reduced geotaxis, and that such a behavioral pattern readily generalized to the untreated subjects. A transfer entropy analysis of causal interactions within the group revealed that emotional contagion was directional, whereby the treated individual influenced untreated subjects, but not vice-versa. This study offers additional evidence that emotional contagion is biologically preserved in simpler living organisms amenable to preclinical investigations.