Raghuveer Parthasarathy

Zebrafish are an important model organism for investigating collective behavior and neuropsychiatric disorders due to attributes such as fast development, high genetic homology with humans and a rich behavioral repertoire. Social behavior in zebrafish emerges as early as twelve days post-fertilization, building toward diverse, coordinated, and complex adult interactions. Most studies on social phenotypes, including in zebrafish, have focused on adult animals and assess isolated traits such as aggression, boldness, or social memory, leaving the critical period of early development when sociality emerges and the mechanisms underlying social behavior establishment largely unexplored. We present here a behavior analysis pipeline that identifies features describing general locomotion and social behavior based on simple geometric criteria. We apply this analysis to pairs of freely interacting two-week-old zebrafish and characterize nascent social preference. We find that young zebrafish favor a specific range of inter-fish distances, creating a social space characterized by specific modulation of locomotion features. Through quantitative analysis and modeling, we show that this proximity behavior is largely established through a turning bias toward counterparts and a socially induced suppression of the variance in turning angles over broader ranges of inter-fish distances. Furthermore, we describe how proximity fosters engagement by young zebrafish in transient, complex short-range physical interactions that include various forms of inter-fish contacts, reminiscent of patterns described in adult animals. Our approach has broad applications for understanding the ontogeny of sociality and collective behaviors and describing subtle phenotypes associated with various human disorders.

The gut microbiota influence many aspects of their host’s health and physiology including the digestion of food, and food intake in turn influences the composition of the gut microbiome. However, the ways in which food can alter the behavior of intestinal bacteria remain largely unknown, due in large part to the difficulty of assessing behavior in situ . Larval zebrafish provide a model for addressing this gap because of their optical transparency and their ability to be prepared germ-free and then associated with specific microbial species. Using light sheet fluorescence microscopy to visualize bacteria inside the intestines of live zebrafish larvae, we examine the properties of two commensal strains with markedly different physical characteristics. One is a zebrafish-commensal Enterobacter species that forms large aggregates in unfed larvae, and the other is a pathobiont Vibrio species, capable of damaging intestinal tissue, that is motile and planktonic. We use recently developed ultraviolet irradiation methods to dramatically lower the microbial content in rotifers, a common live food for larval fish, thereby enabling the assessment of feeding effects independent of the introduction of new microbes. Following host consumption of rotifers, Enterobacter clusters disintegrate into motile individuals. Vibrio remains planktonic in fed larvae but decreases the activity of its Type VI Secretion System, as revealed by a fluorescent fusion protein comprising one of the secretion apparatus proteins and green fluorescent protein, leading to a strong decrease in damage to host tissue. Our results reveal that feeding can have major impacts on bacterial behavior that should be considered in models of normal gut microbiome dynamics as well as pathogenesis.

It is well established from studies of immune cells that stimulation by a wide range of potential signals leads to motility and morphology changes. How these physical behaviors manifest inside a living animal remains unclear due to limitations of conventional imaging and analysis approaches. Here, we establish a quantitative framework for imaging and tracking neutrophil and macrophage dynamics in larval zebrafish, spanning a large fraction of the animal for multihour timescales with few-minute temporal resolution. We focus especially on the gut, examining innate immune responses to different preparations of the intestinal microbiome. Using light sheet fluorescence microscopy and trajectory analysis of hundreds of individual cells, we characterize speeds, directional persistence measures, and cellular morphology to reveal distinct population behaviors. Individual immune cells exhibit stable motility phenotypes, favoring predominantly motile or nonmotile states rather than frequent transitions between them. Gut architecture constrains migration patterns as demonstrated by preferential anterior-posterior movement and a high probability of cells remaining in the vicinity of the gut throughout the imaging duration. Macrophages display significantly reduced sphericity during motile periods compared to nonmotile periods, providing a morphological signature that may enable inference of dynamic behavior from static snapshots. Surprisingly, migration patterns remain consistent across diverse microbial conditions—germ-free, conventionally reared, and colonized by two strains of a zebrafish-native species—indicating that tissue structure exerts a stronger influence than bacterial stimuli on immune surveillance dynamics. Previously observed tissue damage by the wild-type strain, and the resulting recruitment of immune cells toward the damage site, provide the only microbe-specific cellular behavior. These findings reveal innate immune surveillance as a stereotyped process whose characteristics reflect both cellular decision-making and larger-scale anatomical structure.

The gut microbiota influence many aspects of their host’s health and physiology including the digestion of food, and food intake in turn influences the composition of the gut microbiome. However, the ways in which food can alter the behavior of intestinal bacteria remain largely unknown, due in large part to the difficulty of assessing behavior in situ . Larval zebrafish provide a model for addressing this gap because of their optical transparency and their ability to be prepared germ-free and then associated with specific microbial species. Using light sheet fluorescence microscopy to visualize bacteria inside the intestines of live zebrafish larvae, we examine the properties of two commensal strains with markedly different physical characteristics. One is a zebrafish-commensal Enterobacter species that forms large aggregates in unfed larvae, and the other is a pathobiont Vibrio species that is motile and planktonic. Following host consumption of rotifers, a common food, Enterobacter clusters disintegrate into motile individuals. Vibrio remains planktonic in fed larvae but decreases the activity of its Type VI Secretion System, leading to a strong decrease in damage to host tissue. Our results reveal that feeding can have major impacts on bacterial behavior that should be considered in models of normal gut microbiome dynamics as well as pathogenesis.

It is well established from in vitro studies of immune cells that stimulation by a wide range of potential signals leads to motility and morphology changes. How these physical behaviors manifest inside a living animal remains unclear due to limitations of conventional imaging and analysis approaches. Here, we establish a quantitative framework for imaging and tracking neutrophil and macrophage dynamics in larval zebrafish, spanning a large fraction of the animal for multi-hour timescales with few-minute temporal resolution. We focus especially on the gut, examining innate immune responses to different preparations of the intestinal microbiome. Using light sheet fluorescence microscopy and trajectory analysis of hundreds of individual cells, we characterize speeds, directional persistence measures, and cellular morphology to reveal distinct population behaviors. Individual immune cells exhibit stable motility phenotypes, favoring predominantly motile or non-motile states rather than frequent transitions between them. Gut architecture constrains migration patterns as demonstrated by preferential anterior-posterior movement and a high probability of cells remaining in the vicinity of the gut throughout the imaging duration. Macrophages display significantly reduced sphericity during motile periods compared to non-motile periods, providing a morphological signature that may enable inference of dynamic behavior from static snapshots. Surprisingly, migration patterns remain consistent across diverse microbial conditions – germ-free, conventionally reared, and colonized by two strains of a zebrafish-native Vibrio species – indicating that tissue structure exerts a stronger influence than bacterial stimuli on immune surveillance dynamics. Previously observed tissue damage by the wild-type Vibrio strain, and the resulting recruitment of immune cells towards the damage site, provided the only microbe-specific cellular behavior. These findings reveal innate immune surveillance as a stereotyped process whose characteristics reflect both cellular decision-making and larger-scale anatomical structure.

Host-associated microbial communities profoundly impact the health of humans and other animals. Zebrafish have proven to be a useful model for uncovering mechanisms of host-microbe interactions, but the difficulty of maintaining germ-free or gnotobiotic zebrafish beyond 1 week post-fertilization has limited their utility. To address this, we have developed a simple protocol using UV irradiation of rotifers, a common and nutrient-rich prey species for larval zebrafish, to reduce the bacterial load associated with the rotifers by several orders of magnitude while maintaining their motility and viability. We find that though feeding with UV-treated rotifers does not preserve the sterility of germ-free fish, it enables the maintenance of pre-existing bacterial communities. Normal feeding, in striking contrast, leads to the near-total depletion of these prior populations. We measure the abundance of single- and three-species consortia of zebrafish-commensal bacteria inoculated into initially germ-free larvae in a series of experiments extending to 8 days of feeding, or 13 days post-fertilization. We find, in fish-fed UV-treated rotifers, the persistence of bacterial populations on timescales of days, together with strong species-specific variation. In addition, re-inoculation of differently labeled strains of the same zebrafish-commensal species alongside feeding leads to colonization by the new bacteria without displacement of earlier microbes. Our method will facilitate the use of gnotobiotic zebrafish for investigations of phenomena that emerge later in animal development and for studies that probe microbiome composition fluctuations and stability over extended timescales. IMPORTANCE: All animals, including humans, are host to vast microbial communities that contribute to health and disease through mechanisms that remain largely mysterious. These microbiomes are challenging to study, spurring the use of various model organisms, including zebrafish. Zebrafish, however, are difficult to raise beyond 1 week post-fertilization under gnotobiotic conditions, in other words, germ free or with known microbial constituents, a consequence of normally feeding on live prey that brings their own, generally unknown, microbes. Therefore, we developed a simple protocol in which UV irradiation of rotifers, a widely used small-animal food for larval zebrafish, facilitates the maintenance of gnotobiotic larvae. We show that pre-existing bacterial communities in larvae are minimally affected by feeding on UV-treated rotifers, in strong contrast to feeding on untreated rotifers. We demonstrate that this feeding method allows investigations of zebrafish-associated bacterial community stability over several days, allowing investigation of previously intractable questions about microbiome stability.

Intestinal microbes, whether resident or transient, influence the physiology of their hosts, altering both the chemical and the physical characteristics of the gut. An example of the latter is the human pathogen Vibrio cholerae’s ability to induce strong mechanical contractions, discovered in zebrafish. The underlying mechanism has remained unknown, but the phenomenon requires the actin crosslinking domain (ACD) of Vibrio ’s type VI secretion system (T6SS), a multicomponent protein syringe that pierces adjacent cells and delivers toxins. By using a zebrafish-native Vibrio and imaging-based assays of host intestinal mechanics and immune responses, we find evidence that macrophages mediate the connection between the T6SS ACD and intestinal activity. Inoculation with Vibrio gives rise to strong, ACD-dependent, gut contractions whose magnitude resembles those resulting from genetic depletion of macrophages. Vibrio also induces tissue damage and macrophage activation, both ACD-dependent, recruiting macrophages to the site of tissue damage and away from their unperturbed positions near enteric neurons that line the midgut and regulate intestinal motility. Given known crosstalk between macrophages and enteric neurons, our observations suggest that macrophage redistribution forms a key link between Vibrio activity and intestinal motility. In addition to illuminating host-directed actions of the widespread T6SS protein apparatus, our findings highlight how localized bacteria-induced injury can reshape neuro-immune cellular dynamics to impact whole-organ physiology. IMPORTANCE: Gut microbes, whether beneficial, harmful, or neutral, can have dramatic effects on host activities. The human pathogen Vibrio cholerae can induce strong intestinal contractions, though how this is achieved has remained a mystery. Using a zebrafish-native Vibrio and live imaging of larval fish, we find evidence that immune cells mediate the connection between bacteria and host mechanics. A piece of Vibrio ’s type VI secretion system, a syringe-like apparatus that stabs cellular targets, induces localized tissue damage, activating macrophages and drawing them from their normal residence near neurons, whose stimulation of gut contractions they dampen, to the damage site. Our observations reveal a mechanism in which cellular rearrangements, rather than bespoke biochemical signaling, drives a dynamic neuro-immune response to bacterial activity.

Host-associated microbial communities profoundly impact the health of humans and other animals. Zebrafish have proven to be a useful model for uncovering mechanisms of host-microbe interactions, but the difficulty of maintaining germ-free or gnotobiotic zebrafish beyond one week post-fertilization has limited their utility. To address this, we have developed a simple protocol using ultraviolet (UV) irradiation of rotifers, a common and nutrient-rich prey species for larval zebrafish, to reduce the bacterial load associated with the rotifers by several orders of magnitude while maintaining their motility and viability. We find that though feeding with UV-treated rotifers does not preserve the sterility of germ-free fish, it enables the maintenance of pre-existing bacterial communities. Normal feeding, in striking contrast, leads to the near total depletion of these prior populations. We measure the abundance of single- and three-species consortia of zebrafish-commensal bacteria inoculated into initially germ-free larvae in a series of experiments extending to 8 days of feeding, or 13 days post-fertilization. We find, in fish fed UV-treated rotifers, persistence of bacterial populations on timescales of days, together with strong species-specific variation. In addition, re-inoculation of differently labeled strains of the same zebrafish-commensal species alongside feeding leads to colonization by the new bacteria without displacement of earlier microbes. Our method will facilitate the use of gnotobiotic zebrafish for investigations of phenomena that emerge later in animal development and for studies that probe microbiome composition fluctuations and stability over extended timescales.

Intestinal microbes, whether resident or transient, influence the physiology of their hosts, altering both the chemical and the physical characteristics of the gut. An example of the latter is the human pathogen Vibrio cholerae’s ability to induce strong mechanical contractions, discovered in zebrafish. The underlying mechanism has remained unknown, but the phenomenon requires the actin crosslinking domain (ACD) of Vibrio’s Type VI Secretion System (T6SS), a multicomponent protein syringe that pierces adjacent cells and delivers toxins. By using a zebrafish-native Vibrio and imaging-based assays of host intestinal mechanics and immune responses, we find that macrophages mediate the connection between the T6SS ACD and intestinal activity: ACD-dependent tissue damage activates macrophages and recruits them from their unperturbed positions near enteric neurons lining the midgut, spurring strong gut contractions resembling those resulting from genetic depletion of macrophages. In addition to illuminating host-directed actions of the widespread T6SS protein apparatus, our findings highlight how localized bacteria-induced injury can reshape neuro-immune cellular dynamics to impact whole-organ physiology.