Experimental Manipulation Research Articles

Sense of control and noise sensitivity affect frustration from interfering noise.

In order to develop effective strategies to address noise annoyance, it is essential to develop an explanatory model of the psychological mechanism(s) by which noise becomes annoying. Two online studies were conducted in which a total of 193 participants completed speech perception tasks with varying degrees of background noise. Signal-to-noise ratio levels ranged from -2 to -10 dB. The crucial manipulation in both experiments concerned participants' sense of control over the noise level in the task. Dependent measures were task performance, a self-reported measure of frustration with the task, and self-reported sensitivity (trait) to noise. Results showed small but significant effects of noise sensitivity and sense of control on subjective frustration. In both experiments, more noise-sensitive individuals expressed greater frustration than did those reporting less noise sensitivity. In addition, in experiment 2 there was a significant interaction between sense of control and noise level. Listeners under the higher noise-level conditions expressed relatively similar degrees of frustration irrespective of their sense of control, while those under the lower noise-level condition showed lower frustration with greater sense of control. Results support Stallen's [(1999). Noise Health 1(3), 69-79] theoretical framework of noise annoyance, but also suggest the need for further research under more ecologically plausible conditions.

Pitchfork, Race, Collaboration, and the Making of “Indie Rap”

This essay historicizes the moment in popular music when rap music and indie rock began conversing with one another. It shows how Pitchfork received, what is referred to as, “indie rap” by drawing a particular line of influence: from indie rock to rap music. Pitchfork is thereby shown to have contributed to the historical whitening of Black music. Drawing on the work of Fred Moten, this essay argues that Pitchfork established the “white avant-gardism” of indie rock through the construction of a series of binary oppositions that associated Black rap music with pastness and the commercial and “white” indie rock with futurity and the experimental. Such a critical bifurcation is revealed through a close reading of Pitchfork’s reception of the artist most representative of “indie rap:” Kanye West. This essay shows how Pitchfork understood West as needing to appropriate “European” music to move from the past into the future and become experimental. It counters this antagonistic racialization by situating West’s use of Auto-Tune within a lineage of Black radical aesthetics that includes the experimental vocal manipulations of artists ranging from Stevie Wonder to Drake. This essay seeks to recover the collaborative spirit that defined “indie rap.” It focuses on West’s collaborations with Justin Vernon (of Bon Iver), showing how they labored together to produce an “indie rap” that crossed the musical and racial lines established by Pitchfork. This essay concludes that, if West epitomized an ethos of collaboration before his reactionary turn, it is Vernon who now carries forward this collaborative spirit.

Predation scars provide a new method to distinguish native and invasive crab predation on mollusc prey.

Crab species are increasingly important socioeconomic resources that are threatened by human exploitation, climate change, and invasive species, such as European green crabs (Carcinus maenas). However, the continued health of their populations is often uncertain given the limited long-term population data, necessitating alternate approaches to ensure their continued viability. Furthermore, C. maenas are one of the most highly invasive and destructive marine species globally, posing a threat to local ecosystems and species, including socioeconomically important crabs and their mollusc prey. Improved understanding of C. maenas invasions and their impacts on local crab and mollusc resources is therefore vitally important. Here, we present a new method for identifying species-level presence and relative abundances of important crab species, including invasive C. maenas, from the scars they leave on their prey. We conducted controlled manipulative feeding experiments in which individuals of Dungeness crabs (Metacarcinus magister), red rock crabs (Cancer productus), and C. maenas, were allowed to attack snails (Tegula funebralis) and produce sublethal shell damage. Resulting shell damage was photographed and landmarked for geometric morphometric analyses to determine any differences in the shape of shell damage between crab species. There were statistically significant differences between the shape of shell damage created by all three crab species (p < .0001). Shell damage formed a gradient from narrow/deep (C. productus) to shallow/wide (C. maenas) with M. magister as an intermediate form. Our method provides a novel, cost-effective tool for long-term species-specific reconstructions of crab populations and assessing the broader ecological impacts of C. maenas invasions that can inform management and mitigation for these three important crab species.

SynBot is an open-source image analysis software for automated quantification of synapses

The formation of precise numbers of neuronal connections, known as synapses, is crucial for brain function. Therefore, synaptogenesis mechanisms have been one of the main focuses of neuroscience. Immunohistochemistry is a common tool for visualizing synapses. Thus, quantifying the numbers of synapses from light microscopy images enables screening the impacts of experimental manipulations on synapse development. Despite its utility, this approach is paired with low-throughput analysis methods that are challenging to learn, and the results are variable between experimenters, especially when analyzing noisy images of brain tissue. We developed an open-source ImageJ-based software, SynBot, to address these technical bottlenecks by automating the analysis. SynBot incorporates the advanced algorithms ilastik and SynQuant for accurate thresholding for synaptic puncta identification, and the code can easily be modified by users. The use of this software will allow for rapid and reproducible screening of synaptic phenotypes in healthy and diseased nervous systems.

Nutrient availability influences the thermal response of marine diatoms

AbstractUnderstanding how phytoplankton growth responds to temperature is critical for forecasting marine productivity in a warming ocean. While previous laboratory studies have shown that phytoplankton thermal traits such as optimal temperature (Topt) can be affected by nutrient availability, it is unclear whether this can be extrapolated to natural communities. To address this, we tested the impacts of nutrient availability on the thermal responses of two cosmopolitan diatom genera, Pseudo‐nitzschia and Leptocylindrus, through a series of in situ manipulation experiments on natural phytoplankton communities. Analysis of the thermal performance curves revealed that nutrient limitation during summer not only limited the growth of these two genera but also reduced their Topt and the maximum growth rates (μmax). Topt was close to or lower than in situ temperature under ambient nutrient conditions, suggesting that further warming may have a detrimental effect on their growth. However, increasing nutrient supply could counteract this by enhancing Topt and μmax. To further confirm the interactive effects of nutrients and temperature on diatoms, we analyzed a 20‐yr monitoring dataset on Pseudo‐nitzschia, Leptocylindrus, and the whole diatom assembly in Hong Kong coastal waters. We found that the abundances of marine diatoms were significantly higher at high temperatures under nutrient‐rich environments while relatively low under low nutrient concentrations. Findings on natural diatom cell density align with the growth performance derived from in situ manipulation experiments, suggesting that abundant nutrients bolster marine diatoms in coping with warming. Our results highlight the importance of considering the influence of nutrient availability on thermal response of phytoplankton growth, which sheds light on how marine primary production may change under climate warming.

Elemental and isotopic analysis of leaves predicts nitrogen-fixing phenotypes

Nitrogen (N)-fixing symbiosis is critical to terrestrial ecosystems, yet possession of this trait is known for few plant species. Broader presence of the symbiosis is often indirectly determined by phylogenetic relatedness to taxa investigated via manipulative experiments. This data gap may ultimately underestimate phylogenetic, spatial, and temporal variation in N-fixing symbiosis. Still needed are simpler field or collections-based approaches for inferring symbiotic status. N-fixing plants differ from non-N-fixing plants in elemental and isotopic composition, but previous investigations have not tested predictive accuracy using such proxies. Here we develop a regional field study and demonstrate a simple classification model for fixer status using nitrogen and carbon content measurements, and stable isotope ratios (δ15N and δ13C), from field-collected leaves. We used mixed models and classification approaches to demonstrate that N-fixing phenotypes can be used to predict symbiotic status; the best model required all predictors and was 80–94% accurate. Predictions were robust to environmental context variation, but we identified significant variation due to native vs. non-native (exotic) status and phylogenetic affinity. Surprisingly, N content—not δ15N—was the strongest predictor, suggesting that future efforts combine elemental and isotopic information. These results are valuable for understudied taxa and ecosystems, potentially allowing higher-throughput field-based N-fixer assessments.

Euphemism as a powerful framing device that influences moral judgments and punitive responses after wrongdoing.

Euphemism-that is, softening words or phrases substituted for more direct language-has become pervasive in our everyday personal and professional lives. Leveraging theory and research on construal and framing effects, we conceptualize euphemism as a linguistic framing device that influences how observers construe situations and the people, groups, objects, and events within them. We then experimentally investigate the effects of euphemism as a linguistic framing device on third-party judgments about moral transgressions (i.e., bribery, fraud). Across studies (total N = 3,081) we find consistent evidence that employing euphemistic labels (relative to their noneuphemistic analogs) reduces the perceived severity of moral transgressions and, as a result, also reduces third-party motivations to punish transgressors. Overt experimental manipulations to reconstrue euphemistic labels into their noneuphemistic forms reduced, but did not entirely eliminate, the effects on moral severity and punishment judgments. Participants did not sufficiently adjust their judgments. These findings underscore the power of simple linguistic manipulations in influencing public opinion, and they have important implications for the possibility of creating a more just and fair society. (PsycInfo Database Record (c) 2024 APA, all rights reserved).

Creativity in motion: examining the impact of meaningful movement on creative cognition

This study examined the link between creative cognition and meaningful physical movement among university students (N = 151) from a cognitive offloading perspective. A linear mixed model, repeated measures design examined whether divergent thinking on three Alternative Uses Tasks (AUTs; within-subjects factor) was influenced by movement in three independent experimental groups: (1) gesture as meaningful movement (n = 51), (2) meaningless movement (n = 50), and (3) restricted movement (n = 50). Linear mixed model results indicated that changes in AUT fluency, flexibility, and originality across trials did not significantly interact with group. However, baseline gesture frequency was positively associated with all four creativity outcome measures and moderated group-level changes in originality across trials. Infrequent baseline gesturers demonstrated lower originality scores in the gesture as meaningful movement group compared to those in the restricted movement group. More frequent baseline gesturers experienced higher originality in both the gesture as meaningful movement and meaningless movement groups compared to those in the restricted movement group. This study demonstrates that the cognitive processes involved in novel idea generation may be differentially impacted by movement and may be more sensitive to individual differences in movement predisposition than experimental movement manipulation.

Sexism in esports: How male and female players evaluate each others’ performance and agency

While equality and inclusivity are promoted in the realm of video gaming, most gamer communities remain male-dominated, which may give rise to prejudices against female players and their skill among gamer and the general public. In this study, we conducted two experiments where we measured how participants evaluate players’ performance and agency attributions. We used two distinct experimental manipulations—one utilizing videos of players playing (n = 312) and the other using narrative vignettes (n = 338). We found that performance of male players’, to a limited extent, can be attributed to the gaming outcomes compared pre-existing social preconceptions about them and other variables. Female players’ performance assessment was mostly strongly predicted by pre-existing social sex-related prejudice against females, albeit not directly, but through percieved agency of the female players’. These results contribute to the literature on gaming-related stereotypes by enhancing the understanding of societal prejudices in the evaluation of players’ skills.

Activated sludge microbial community assembly: the role of influent microbial community immigration.

Wastewater treatment plants (WWTPs) are host to diverse microbial communities and receive a constant influx of microbes from influent wastewater. However, the impact of immigrants on the structure and activities of the activated sludge (AS) microbial community remains unclear. To gain insight on this phenomenon known as perpetual community coalescence, the current study utilized controlled manipulative experiments that decoupled the influent wastewater composition from the microbial populations to reveal the fundamental mechanisms involved in immigration between sewers and AS-WWTP. The immigration dynamics of heterotrophs were analyzed by harvesting wastewater biomass solids from three different sewer systems and adding to synthetic wastewater. Immigrating influent populations were observed to contribute up to 14% of the sequencing reads in the AS. By modeling the net growth rate of taxa, it was revealed that immigrants primarily exhibited low or negative net growth rates. By developing a protocol to reproducibly grow AS-WWTP communities in the lab, we have laid down the foundational principles for the testing of operational factors creating community variations with low noise and appropriate replication. Understanding the processes that drive microbial community diversity and assembly is a key question in microbial ecology. In the future, this knowledge can be used to manipulate the structure of microbial communities and improve system performance in WWTPs.IMPORTANCEIn biological wastewater treatment processes, the microbial community composition is essential in the performance and stability of the system. This study developed a reproducible protocol to investigate the impact of influent immigration (or perpetual coalescence of the sewer and activated sludge communities) with appropriate reproducibility and controls, allowing intrinsic definitions of core and immigrant populations to be established. The method developed herein will allow sequential manipulative experiments to be performed to test specific hypothesis and optimize wastewater treatment processes to meet new treatment goals.

Ecological filtering drives rapid spatiotemporal dynamics in fish skin microbiomes.

Skin microbiomes provide vital functions, yet knowledge about the drivers and processes structuring their species assemblages is limited-especially for non-model organisms. In this study, fish skin microbiome was assessed by high throughput sequencing of amplicon sequence variants from metabarcoding of V3-V4 regions in the 16S rRNA gene on fish hosts subjected to the following experimental manipulations: (i) translocation between fresh and brackish water habitats to investigate the role of environment; (ii) treatment with an antibacterial disinfectant to reboot the microbiome and investigate community assembly and priority effects; and (iii) maintained alone or in pairs to study the role of social environment and inter-host dispersal of microbes. The results revealed that fish skin microbiomes harbour a highly dynamic microbial composition that was distinct from bacterioplankton communities in the ambient water. Microbiome composition first diverged as an effect of translocation to either the brackish or freshwater habitat. When the freshwater individuals were translocated back to brackish water, their microbiome composition converged towards the fish microbiomes in the brackish habitat. In summary, external environmental conditions and individual-specific factors jointly determined the community composition dynamics, whereas inter-host dispersal had negligible effects. The dynamics of the microbiome composition was seemingly non-affected by reboot treatment, pointing towards high resilience to disturbance. The results emphasised the role of inter-individual variability for the unexplained variation found in many host-microbiome systems, although the mechanistic underpinnings remain to be identified.

Precipitation changes alter plant dominant species and functional groups by changing soil salinity in a coastal salt marsh

Specific mechanisms of precipitation change due to global climate variability on plant communities in coastal salt marsh ecosystems remain unknown. Hence, a field manipulative precipitation experiment was established in 2014 and 5 years of field surveys of vegetation from 2017 to 2021 to explore the effects of precipitation changes on plant community composition. The results showed that changes in plant community composition were driven by dominant species, and that the dominance of key species changed significantly with precipitation gradient and time, and that these changes ultimately altered plant community traits (i.e., community density, height, and species richness). Community height increased but community density decreased with more precipitation averaged five years. Furthermore, changes in precipitation altered dominant species composition and functional groups mainly by influencing soil salinity. Salinity stress caused by decreased precipitation shifted species composition from a dominance of taller perennials and grasses to dwarf annuals and forbs, while the species richness decreased. Conversely, soil desalination caused by increased precipitation increased species richness, especially increasing in the dominance of grasses and perennials. Specifically, Apocynaceae became dominance from rare while Amaranthaceae decreased in response to increased precipitation, but Poaceae was always in a position of dominance. Meanwhile, the dominance of grasses and perennials has the cumulative effect of years and their proportion increased under the increased 60% of ambient precipitation throughout the years. However, the annual forb Suaeda glauca was gradually losing its dominance or even becoming extinct over years. Our study highlights that the differences in plant salinity tolerance are key to the effects of precipitation changes on plant communities in coastal salt marsh. These findings aim to provide a theoretical basis for predicting vegetation dynamics and developing ecological management strategies to adapt to future precipitation changes.

The Role of Charismatic Leadership Tactics and Inconsistent Messages in Processing and Evaluating Climate Change Information

Climate change poses an unprecedented challenge to humanity, and persuasive environmental communication is a crucial step for global leaders to counteract its consequences. Charismatic leaders are especially effective in communicating information. Interestingly, in recent years, there have been frequent incidences where charismatic leaders made inconsistent environmental statements. How people process such inconsistencies can provide key insights into understanding the processing of climate information. In two preregistered online studies (N = 668), we examined how people read and evaluate pro- and contra-environmentalism messages from an environmental, charismatic leader. We found persistent effects of message inconsistencies, leading to longer reading times and lower convincingness ratings for information. However, we failed to find an interaction effect of the experimental manipulations or a main effect of charisma on reading times for environmental messages. In Study 2, we detected an interaction effect of leader charisma and message inconsistency on how convincing participants rated text messages. Participants rated contra-environmental messages as more convincing when following displays of charismatic rather than neutral leader behavior. We discuss future research avenues to explore charismatic leader influences on processing climate change information.

Mistletoes benefit from initiating cascading effects in a cactus–stem borers–arthropods system: A positive feedback

Abstract Parasitic plants may indirectly modify arthropod community structure by inducing resistance/susceptibility in host plants. In the Atacama Desert, the mistletoe Tristerix aphyllus initiates cascading effects on its cactus host, Echinopsis chiloensis, inducing susceptibility to stem‐borer beetles, whose brood chambers are colonized by a diverse arthropod community. We evaluated whether those cascading effects initiated by Tristerix generate a positive or negative feedback loop on this parasitic plant. A positive or negative feedback loop would occur if the mistletoe‐elicited indirect effects on arthropod community result in increased or decreased mistletoe fitness, respectively. Specifically, by way of field sampling, experimental manipulation of number of brood chambers, and structural equation modelling, we assessed the indirect impact of number of brood chambers on Tristerix fitness mediated by its influence on the abundance of arthropod trophic guilds (omnivores, nectarivores, herbivores and predators). We found that the number of brood chambers had a positive indirect effect on mistletoe fitness, likely mediated by the increased abundance of nectarivores. The number of brood chambers was positively associated with the abundance of predators, nectarivores and herbivores. However, nectarivores were the only trophic guild showing a positive association with both number of brood chambers and Tristerix fitness. This positive feedback loop, together with the known pattern of specific disperser‐mediated cyclic reinfection of cacti by Tristerix, would indicate that mistletoe infection may ultimately threaten cactus survival.

Placing the leading limb closer to an obstacle reduces collision of the trailing limb: an investigation in a virtual environment.

When walking and stepping over an obstacle of a certain height, tripping occurs more frequently with the trailing limb than the leading limb. The present study was designed to address whether collisions involving the trailing limb can be improved with experimental manipulation of the placement of the leading limb after stepping over an obstacle. We used an immersive, virtual obstacle-crossing task to ensure that the collision was not improved simply due to the experience of physical collision with an obstacle. Fourteen young participants (12 males and 2 females, 28.7 ± 3.5 years) were required to walk and step over a virtual horizontal pole under one of four conditions. In three conditions, participants were required to place their leading foot on a square target located along their walking path after crossing the obstacle. The target was positioned so that it was relatively close to the obstacle (10 cm from the obstacle, referred to hereafter as the closer condition), at a position that would naturally be stepped on in successful trials without a collision (20 cm from the obstacle, the middle condition), or relatively far from the obstacle (40 cm from the obstacle, the farther condition). For the fourth condition, participants were free to select where they would step after stepping over the obstacle (the control condition). The results showed that the collision rate of the trailing limb was significantly lower under the closer condition than under the other three conditions. Compared to the control condition, under the closer condition the movement of the trailing limb was modified so that obstacle crossing was performed at approximately the moment when the height of the toe of the trailing limb was higher, and the walking speed was slower. These findings suggest that placing the foot of the leading limb closer to the obstacle after crossing the obstacle may ensure safe obstacle avoidance by the trailing limb.

Effects of a waiting list control design on alcohol consumption among online help-seekers: A randomised controlled trial

BackgroundIndirect evidence suggests that using waiting list control designs in behavioural research may have unintended consequences. The aim of this study was to estimate the effects of a waiting list design on alcohol consumption among individuals who had looked online for help. MethodsA two-arm randomised controlled trial was employed. The intervention group was informed that they belonged to the intervention group and would receive immediate access to a digital alcohol intervention. The waiting list control group was informed that they belonged to the group that had to wait four weeks to be given access to the intervention and in the meantime, they would be given a summary of their drinking. However, both groups received immediate access to the same digital alcohol intervention; the experimental contrast was thus between being told to wait or not. ResultsWe randomised 3388 participants (intervention: 1692, waiting list: 1696). Data were available for 954 participants at 1-month follow-up. We found no strong evidence that alcohol consumption differed between groups, but the evidence pointed towards the intervention group reporting lowering weekly alcohol consumption compared to the waiting list control group (IRR = 0.95, 95 % CI = 0.83; 1.08, probability of effect = 78.8 %). ConclusionWe found no strong evidence that being informed that access to an intervention would be delayed produced differential self-reported alcohol consumption compared to being informed that access would be immediate. We did find a difference in engagement with the intervention materials, indicating that the experimental manipulation was successful.

Sex, flies and flower trap: Trapping trichomes and their function in pollination

Abstract Trapping trichomes, a common trait found in protogynous trap flowers, are thought to enable interaction with pollinators, promoting both pollen removal and fruit set. However, this hypothesis has yet to be empirically confirmed. In this study, we experimentally tested whether trapping trichomes favour male and female functions in trap flowers. Using protogynous South American Aristolochia esperanzae trap flowers, we observed the temporal variation in both sexual functions and associated them with the wilting of the flower and trapping trichomes. Next, we described the trapping trichomes features associated with the capture, entrapment and release of pollinators. Finally, we used manipulative experiments involving trapping trichome removal to quantify their influence on pollinator entrapment, pollen removal and fruit set. While flowers wilted after the end of the female phase, the trapping trichomes only wilted some hours after pollen presentation, promoting a delay in the exit of pollinators, so they carried pollen. Trapping trichomes features, such as a density gradient towards the interior of the tube, unidirectional orientation and epicuticular waxes, may be important in the capture and entrapment of pollinators. Trapping trichome removal decreased drastically the probability of pollinator entrapment and pollen removal, leading to no fruit set. These results indicate that the evolution and maintenance of trapping trichomes in trap flowers probably occurred through selective pressures from both sexual functions and highlight their role in the functioning of this highly phenotypically specialized pollination system. Read the free Plain Language Summary for this article on the Journal blog.

Unpacking the dark side of positive online destination brand engagement: effects on stress, disengagement, and switching intention

ABSTRACT The brand engagement has always been a key objective for destination marketers due to its positive outcomes. However, using the Job Demands-Resources (JD-R) theory, this study delves into an unexplored area by investigating the detrimental effects of positive online destination brand engagement (DBE) on perceived stress, disengagement, and destination-switching intention. The study also delves into the sequential mediation effects of perceived stress and consumer disengagement between online DBE and destination-switching intention. The authors conducted an experimental study with three levels of engagement (low, moderate, and high) in an online DBE scenario. They analysed data from 278 consumers using SmartPLS. The study's findings reveal that online DBE positively impacts perceived stress, which in turn leads to consumer disengagement, ultimately resulting in destination-switching intention. The results validate sequential mediation, indicating that the online DBE's indirect effect on destination-switching intention, as mediated through perceived stress and consumer disengagement. Importantly, the experimental manipulation demonstrated that a moderate level of engagement with destination brands yields more favourable outcomes compared to low and high levels, thereby enhancing the study's practical relevance. The study's findings offer valuable insights for destination marketers, underlining the need to manage online brand engagement effectively to avoid negative consequences.

Palatability of ascidians: a meta-analysis of the predation effect on ascidians

Ascidians are marine sessile animals that have evolved many strategies to reduce predation. Previous manipulative experiments with ascidian tissues or pellets have shown that they have chemical defenses that render them unpalatable. However, predation-exclusion experiments on a community scale have shown that ascidians are almost entirely eaten when exposed to predators. Based on these contrasting results, we performed a meta-analysis to assess the importance of study site, experiment design, ascidian sociability, and predator identity to the efficacy of the ascidian defense. Our study is the first quantitative review of predation on ascidians, and it emphasizes the importance of ecological interactions beyond the specific defense tactics of the organisms. We found that multiple factors can interfere with the effectiveness of the ascidian defense. Palatability studies have shown evidence for ascidian defense mechanisms; however, they depend on the identity of the predators (e.g. fish, crab, amphipod). We did not find evidence of ascidian defense in community studies. There is a lack of field experiments, mainly on solitary ascidians, that evaluate their predation risk in communities. Research on ascidian defense mechanisms is also geographically biased toward the temperate region in the Northern Hemisphere. The commonly held belief that ascidians possess active defenses may be overestimated, and the defenses that do exist are probably restricted to only a few species. This misconception has been caused mainly by methodological and geographical bias that has resulted in tests being performed only on species with previous evidence of defenses. Therefore, we need more worldwide studies focusing on the ecological relationships between ascidians and predators, specifically in natural communities under field conditions.

Chromosome structure in Drosophila is determined by boundary pairing not loop extrusion.

Two different models have been proposed to explain how the endpoints of chromatin looped domains ('TADs') in eukaryotic chromosomes are determined. In the first, a cohesin complex extrudes a loop until it encounters a boundary element roadblock, generating a stem-loop. In this model, boundaries are functionally autonomous: they have an intrinsic ability to halt the movement of incoming cohesin complexes that is independent of the properties of neighboring boundaries. In the second, loops are generated by boundary:boundary pairing. In this model, boundaries are functionally non-autonomous, and their ability to form a loop depends upon how well they match with their neighbors. Moreover, unlike the loop-extrusion model, pairing interactions can generate both stem-loops and circle-loops. We have used a combination of MicroC to analyze how TADs are organized, and experimental manipulations of the even skipped TAD boundary, homie, to test the predictions of the 'loop-extrusion' and the 'boundary-pairing' models. Our findings are incompatible with the loop-extrusion model, and instead suggest that the endpoints of TADs in flies are determined by a mechanism in which boundary elements physically pair with their partners, either head-to-head or head-to-tail, with varying degrees of specificity. Although our experiments do not address how partners find each other, the mechanism is unlikely to require loop extrusion.