Coordination and persistence of aggressive visual communication in Siamese fighting fish.

Animals coordinate their behavior with each other during both cooperative and agonistic social interactions. Such coordination often adopts the form of "turn taking", in which the interactive partners alternate the performance of a behavior. Apart from acoustic communication, how turn taking between animals is coordinated is not well understood. Furthermore, the neural substrates that regulate persistence in engaging in social interactions are poorly studied. Here, we use Siamese fighting fish ( Betta splendens ), to study visually-driven turn-taking aggressive behavior. Using encounters with conspecifics and with animations, we characterize the dynamic visual features of an opponent and the behavioral sequences that drive turn taking. Through a brain-wide screen of neuronal activity during coordinated and persistent aggressive behavior, followed by targeted brain lesions, we find that the caudal portion of the dorsomedial telencephalon, an amygdala-like region, promotes persistent participation in aggressive interactions, yet is not necessary for coordination. Our work highlights how dynamic visual cues shape the rhythm of social interactions at multiple timescales, and points to the pallial amygdala as a region controlling engagement in such interactions. These results suggest an evolutionarily conserved role of the vertebrate pallial amygdala in regulating the persistence of emotional states.

Evolution of a novel adrenal cell type that promotes parental care.

Cell types with specialized functions fundamentally regulate animal behaviour, and yet the genetic mechanisms that underlie the emergence of novel cell types and their consequences for behaviour are not well understood1. Here we show that the monogamous oldfield mouse (Peromyscus polionotus) has recently evolved a novel cell type in the adrenal gland that expresses the enzyme AKR1C18, which converts progesterone into 20α-hydroxyprogesterone. We then demonstrate that 20α-hydroxyprogesterone is more abundant in oldfield mice, where it induces monogamous-typical parental behaviours, than in the closely related promiscuous deer mice (Peromyscus maniculatus). Using quantitative trait locus mapping in a cross between these species, we ultimately find interspecific genetic variation that drives expression of the nuclear protein GADD45A and the glycoprotein tenascin N, which contribute to the emergence and function of this cell type in oldfield mice. Our results provide an example by which the recent evolution of a new cell type in a gland outside the brain contributes to the evolution of social behaviour.

Evolution of gene expression across brain regions in behaviourally divergent deer mice.

The evolution of innate behaviours is ultimately due to genetic variation likely acting in the nervous system. Gene regulation may be particularly important because it can evolve in a modular brain-region specific fashion through the concerted action of cis- and trans-regulatory changes. Here, to investigate transcriptional variation and its regulatory basis across the brain, we perform RNA sequencing (RNA-Seq) on ten brain subregions in two sister species of deer mice (Peromyscus maniculatus and P. polionotus)-which differ in a range of innate behaviours, including their social system-and their F1 hybrids. We find that most of the variation in gene expression distinguishes subregions, followed by species. Interspecific differential expression (DE) is pervasive (52-59% of expressed genes), whereas the number of DE genes between sexes is modest overall (~3%). Interestingly, the identity of DE genes varies considerably across brain regions. Much of this modularity is due to cis-regulatory divergence, and while 43% of genes were consistently assigned to the same gene regulatory class across subregions (e.g. conserved, cis- or trans-regulatory divergence), a similar number were assigned to two or more different gene regulatory classes. Together, these results highlight the modularity of gene expression differences and divergence in the brain, which may be key to explain how the evolution of brain gene expression can contribute to the astonishing diversity of animal behaviours.

The main genetic locus associated with the evolution of gamecocks is centered on ISPD.

Chickens were domesticated >4,000 years ago, probably first for fighting them and only later as a source of food. Fighting chickens, commonly known as gamecocks, continue to be bred throughout the world, but the genetic relationships among geographically diverse gamecocks and with nongame chickens are not known. Here, we sequenced the genomes of 44 geographically diverse gamecocks and 62 nongame chickens representing a variety of breeds. We combined these sequences with published genomes to generate the most diverse chicken genomes dataset yet assembled, with 307 samples. We found that gamecocks do not form a homogeneous group, yet they share genetic similarities that distinguish them from nongame chickens. Such similarities are likely the result of a common origin before their local diversification into, or mixing with nongame chickens. Particularly noteworthy is a variant in an intron of the isoprenoid synthase domain containing gene (ISPD), an extreme outlier present at a frequency of 89% in gamecocks but only 4% in nongame chickens. The ISPD locus has the strongest signal of selection in gamecocks, suggesting it is important for fighting performance. Because ISPD variants that are highly prevalent in gamecocks are still segregating in nongame chickens, selective breeding may help reduce its frequency in farm conditions in which aggression is not a desired trait. Altogether, our work provides genomic resources for agricultural genetics, uncovers a common origin for gamecocks from around the world and what distinguishes them genetically from chickens bred for purposes other than fighting, and points to ISPD as the most important locus related to fighting performance.

Noncoding genetic variation in ISPD distinguishes gamecocks from nongame chickens.

Chickens were domesticated >4,000 years ago, probably first for fighting them and only later as a source of food. Fighting chickens, commonly known as gamecocks, continue to be bred throughout the world, but the genetic relationships among geographically diverse gamecocks and with nongame chickens are not known. Here, we sequenced the genomes of 44 geographically diverse gamecocks and of 62 nongame chickens representing a variety of breeds. We combined these sequences with published genomes to generate the most diverse chicken genomes dataset yet assembled, at 307 samples. We found that gamecocks do not form a homogeneous group, yet they share genetic similarities that distinguish them from nongame chickens. Such similarities are likely the result of a common origin before their local diversification into, or mixing with, nongame chickens. Particularly noteworthy is a variant in an intron of ISPD, an extreme outlier present at a frequency of 90% in gamecocks but only 4% in nongame chickens. The ISPD locus has the strongest signal of selection in gamecocks, suggesting it is important for fighting performance. Because ISPD variants that are highly prevalent in gamecocks are still segregating in nongame chickens, selective breeding may help reduce its frequency in farm conditions in which aggression is not a desired trait. Altogether, our work provides genomic resources for agricultural genetics, uncovers a common origin for gamecocks from around the world and what distinguishes them genetically from chickens bred for purposes other than fighting, and points to ISPD as the most important locus related to fighting performance.

Genetic manipulation of betta fish.

Betta splendens, also known as Siamese fighting fish or "betta," is a freshwater fish species renowned for its astonishing morphological diversity and extreme aggressive behavior. Despite recent advances in our understanding of the genetics and neurobiology of betta, the lack of tools to manipulate their genome has hindered progress at functional and mechanistic levels. In this study, we outline the use of three genetic manipulation technologies, which we have optimized for use in betta: CRISPR/Cas9-mediated knockout, CRISPR/Cas9-mediated knockin, and Tol2-mediated transgenesis. We knocked out three genes: alkal2l, bco1l, and mitfa, and analyzed their effects on viability and pigmentation. Furthermore, we knocked in a fluorescent protein into the mitfa locus, a proof-of-principle experiment of this powerful technology in betta. Finally, we used Tol2-mediated transgenesis to create fish with ubiquitous expression of GFP, and then developed a bicistronic plasmid with heart-specific expression of a red fluorescent protein to serve as a visible marker of successful transgenesis. Our work highlights the potential for the genetic manipulation of betta, providing valuable resources for the effective use of genetic tools in this animal model.

Genetic mapping reveals Pou2af2/OCA-T1-dependent tuning of tuft cell differentiation and intestinal type 2 immunity.

Chemosensory epithelial tuft cells contribute to innate immunity at barrier surfaces, but their differentiation from epithelial progenitors is not well understood. Here, we exploited differences between inbred mouse strains to identify an epithelium-intrinsic mechanism that regulates tuft cell differentiation and tunes innate type 2 immunity in the small intestine. Balb/cJ (Balb) mice had fewer intestinal tuft cells than C57BL/6J (B6) mice and failed to respond to the tuft cell ligand succinate. Most of this differential succinate response was determined by the 50- to 67-Mb interval of chromosome 9 (Chr9), such that congenic Balb mice carrying the B6 Chr9 interval had elevated baseline numbers of tuft cells and responded to succinate. The Chr9 locus includes Pou2af2, which encodes the protein OCA-T1, a transcriptional cofactor essential for tuft cell development. Epithelial crypts expressed a previously unannotated short isoform of Pou2af2 predicted to use a distinct transcriptional start site and encode a nonfunctional protein. Low tuft cell numbers and the resulting lack of succinate response in Balb mice were explained by a preferential expression of the short isoform and could be rescued by expression of full-length Pou2af2. Physiologically, Pou2af2 isoform usage tuned innate type 2 immunity in the small intestine. Balb mice maintained responsiveness to helminth pathogens while ignoring commensal Tritrichomonas protists and reducing norovirus burdens.

Genetic manipulation of betta fish.

Betta splendens , also known as Siamese fighting fish or 'betta', are renowned for their astonishing morphological diversity and extreme aggressive behavior. Despite recent advances in our understanding of the genetics and neurobiology of betta, the lack of tools to manipulate their genome has hindered progress at functional and mechanistic levels. In this study, we outline the use of three genetic manipulation technologies, which we have optimized for use in betta: CRISPR/Cas9-mediated knockout, CRISPR/Cas9-mediated knockin, and Tol2-mediated transgenesis. We knocked out three genes: alkal2l, bco1l , and mitfa , and analyzed their effects on viability and pigmentation. Furthermore, we successfully knocked in a fluorescent protein into the mitfa locus, a proof-of-principle experiment of this powerful technology in betta. Finally, we used Tol2-mediated transgenesis to create fish with ubiquitous expression of GFP, and then developed a bicistronic plasmid with heart-specific expression of a red fluorescent protein to serve as a visible marker of successful transgenesis. Our work highlights the potential for the genetic manipulation of betta, providing valuable resources for the effective use of genetic tools in this animal model.

Post-mating parental behavior trajectories differ across four species of deer mice.

Among species, parental behaviors vary in their magnitude, onset relative to reproduction, and sexual dimorphism. In deer mice (genus Peromyscus), while most species are promiscuous with low paternal care, monogamy and biparental care have evolved at least twice under different ecological conditions. Here, in a common laboratory setting, we monitored parental behaviors of males and females of two promiscuous (eastern deer mouse P. maniculatus and white-footed mouse P. leucopus) and two monogamous (oldfield mouse P. polionotus and California mouse P. californicus) species from before mating to after giving birth. In the promiscuous species, females showed parental behaviors largely after parturition, while males showed little parental care. In contrast, both sexes of monogamous species performed parental behaviors. However, while oldfield mice began to display parental behaviors before mating, California mice showed robust parental care behaviors only postpartum. These different parental-care trajectories in the two monogamous species align with their socioecology. Oldfield mice have overlapping home ranges with relatives, so infants they encounter, even if not their own, are likely to be closely related. By contrast, California mice disperse longer distances into exclusive territories with possibly unrelated neighbors, decreasing the inclusive fitness benefits of caring for unfamiliar pups before parenthood. Together, we find that patterns of parental behaviors in Peromyscus are consistent with predictions from inclusive fitness theory.

Care and Use of Siamese Fighting Fish (Betta Splendens) for Research.

Betta splendens, also called Siamese fighting fish or 'betta,' are a popular species in the fishkeeping hobby. Native to South- east Asia, betta have been selectively bred for their fighting ability for hundreds of years, which has resulted in the species' characteristic male aggression. More recently, betta have been bred for a number of ornamental traits such as coloration, fin morphology, and body size. Betta have unique characteristics and an evolutionary history that make them a useful model for studies in the fields of behavior, endocrinology, neurobiology, genetics, development, and evolution. However, standard laboratory procedures for raising and keeping these fish are not well established, which has limited their use. Here, we briefly review the past and present use of betta in research, with a focus on their utility in behavioral, neurobiological, and evolutionary studies. We then describe effective husbandry practices for maintaining betta as a research colony.

Parent-offspring inference in inbred populations.

Genealogical relationships are fundamental components of genetic studies. However, it is often challenging to infer correct and complete pedigrees even when genome-wide information is available. For example, inbreeding can obscure genetic differences between individuals, making it difficult to even distinguish first-degree relatives such as parent-offspring from full siblings. Similarly, genotyping errors can interfere with the detection of genetic similarity between parents and their offspring. Inbreeding is common in natural, domesticated, and experimental populations and genotyping of these populations often has more errors than in human data sets, so efficient methods for building pedigrees under these conditions are necessary. Here, we present a new method for parent-offspring inference in inbred pedigrees called specific parent-offspring relationship estimation (spore). spore is vastly superior to existing pedigree-inference methods at detecting parent-offspring relationships, in particular when inbreeding is high or in the presence of genotyping errors, or both. spore therefore fills an important void in the arsenal of pedigree inference tools.

Genomic consequences of domestication of the Siamese fighting fish.

Siamese fighting (betta) fish are among the most popular and morphologically diverse pet fish, but the genetic bases of their domestication and phenotypic diversification are largely unknown. We assembled de novo the genome of a wild Betta splendens and whole-genome sequenced 98 individuals across five closely related species. We find evidence of bidirectional hybridization between domesticated ornamental betta and other wild Betta species. We discover dmrt1 as the main sex determination gene in ornamental betta and that it has lower penetrance in wild B. splendens. Furthermore, we find genes with signatures of recent, strong selection that have large effects on color in specific parts of the body or on the shape of individual fins and that most are unlinked. Our results demonstrate how simple genetic architectures paired with anatomical modularity can lead to vast phenotypic diversity generated during animal domestication and launch betta as a powerful new system for evolutionary genetics.

WHotLAMP: A simple, inexpensive, and sensitive molecular test for the detection of SARS-CoV-2 in saliva.

Despite the development of effective vaccines against SARS-CoV-2, epidemiological control of the virus is still challenging due to slow vaccine rollouts, incomplete vaccine protection to current and emerging variants, and unwillingness to get vaccinated. Therefore, frequent testing of individuals to identify early SARS-CoV-2 infections, contact-tracing and isolation strategies remain crucial to mitigate viral spread. Here, we describe WHotLAMP, a rapid molecular test to detect SARS-CoV-2 in saliva. WHotLAMP is simple to use, highly sensitive (~4 viral particles per microliter of saliva) and specific, as well as inexpensive, making it ideal for frequent screening. Moreover, WHotLAMP does not require toxic chemicals or specialized equipment and thus can be performed in point-of-care settings, and may also be adapted for resource-limited environments or home use. While applied here to SARS-CoV-2, WHotLAMP can be modified to detect other pathogens, making it adaptable for other diagnostic assays, including for use in future outbreaks.

Loop-Mediated Isothermal Amplification Detection of SARS-CoV-2 and Myriad Other Applications.

As the second year of the COVID-19 pandemic begins, it remains clear that a massive increase in the ability to test for SARS-CoV-2 infections in a myriad of settings is critical to controlling the pandemic and to preparing for future outbreaks. The current gold standard for molecular diagnostics is the polymerase chain reaction (PCR), but the extraordinary and unmet demand for testing in a variety of environments means that both complementary and supplementary testing solutions are still needed. This review highlights the role that loop-mediated isothermal amplification (LAMP) has had in filling this global testing need, providing a faster and easier means of testing, and what it can do for future applications, pathogens, and the preparation for future outbreaks. This review describes the current state of the art for research of LAMP-based SARS-CoV-2 testing, as well as its implications for other pathogens and testing. The authors represent the global LAMP (gLAMP) Consortium, an international research collective, which has regularly met to share their experiences on LAMP deployment and best practices; sections are devoted to all aspects of LAMP testing, including preanalytic sample processing, target amplification, and amplicon detection, then the hardware and software required for deployment are discussed, and finally, a summary of the current regulatory landscape is provided. Included as well are a series of first-person accounts of LAMP method development and deployment. The final discussion section provides the reader with a distillation of the most validated testing methods and their paths to implementation. This review also aims to provide practical information and insight for a range of audiences: for a research audience, to help accelerate research through sharing of best practices; for an implementation audience, to help get testing up and running quickly; and for a public health, clinical, and policy audience, to help convey the breadth of the effect that LAMP methods have to offer.

How Natural Genetic Variation Shapes Behavior.

Nervous systems allow animals to acutely respond and behaviorally adapt to changes and recurring patterns in their environment at multiple timescales-from milliseconds to years. Behavior is further shaped at intergenerational timescales by genetic variation, drift, and selection. This sophistication and flexibility of behavior makes it challenging to measure behavior consistently in individual subjects and to compare it across individuals. In spite of these challenges, careful behavioral observations in nature and controlled measurements in the laboratory, combined with modern technologies and powerful genetic approaches, have led to important discoveries about the way genetic variation shapes behavior. A critical mass of genes whose variation is known to modulate behavior in nature is finally accumulating, allowing us to recognize emerging patterns. In this review, we first discuss genetic mapping approaches useful for studying behavior. We then survey how variation acts at different levels-in environmental sensation, in internal neuronal circuits, and outside the nervous system altogether-and then discuss the sources and types of molecular variation linked to behavior and the mechanisms that shape such variation. We end by discussing remaining questions in the field.

Generation, Coordination, and Evolution of Neural Circuits for Vocal Communication.

In many species, vocal communication is essential for coordinating social behaviors including courtship, mating, parenting, rivalry, and alarm signaling. Effective communication requires accurate production, detection, and classification of signals, as well as selection of socially appropriate responses. Understanding how signals are generated and how acoustic signals are perceived is key to understanding the neurobiology of social behaviors. Here we review our long-standing research program focused on Xenopus, a frog genus which has provided valuable insights into the mechanisms and evolution of vertebrate social behaviors. In Xenopus laevis, vocal signals differ between the sexes, through development, and across the genus, reflecting evolutionary divergence in sensory and motor circuits that can be interrogated mechanistically. Using two ex vivo preparations, the isolated brain and vocal organ, we have identified essential components of the vocal production system: the sexually differentiated larynx at the periphery, and the hindbrain vocal central pattern generator (CPG) centrally, that produce sex- and species-characteristic sound pulse frequencies and temporal patterns, respectively. Within the hindbrain, we have described how intrinsic membrane properties of neurons in the vocal CPG generate species-specific vocal patterns, how vocal nuclei are connected to generate vocal patterns, as well as the roles of neurotransmitters and neuromodulators in activating the circuit. For sensorimotor integration, we identified a key forebrain node that links auditory and vocal production circuits to match socially appropriate vocal responses to acoustic features of male and female calls. The availability of a well supported phylogeny as well as reference genomes from several species now support analysis of the genetic architecture and the evolutionary divergence of neural circuits for vocal communication. Xenopus thus provides a vertebrate model in which to study vocal communication at many levels, from physiology, to behavior, and from development to evolution. As one of the most comprehensively studied phylogenetic groups within vertebrate vocal communication systems, Xenopus provides insights that can inform social communication across phyla.

The genetic basis of parental care evolution in monogamous mice.

Parental care is essential for the survival of mammals, yet the mechanisms underlying its evolution remain largely unknown. Here we show that two sister species of mice, Peromyscus polionotus and Peromyscus maniculatus, have large and heritable differences in parental behaviour. Using quantitative genetics, we identify 12 genomic regions that affect parental care, 8 of which have sex-specific effects, suggesting that parental care can evolve independently in males and females. Furthermore, some regions affect parental care broadly, whereas others affect specific behaviours, such as nest building. Of the genes linked to differences in nest-building behaviour, vasopressin is differentially expressed in the hypothalamus of the two species, with increased levels associated with less nest building. Using pharmacology in Peromyscus and chemogenetics in Mus, we show that vasopressin inhibits nest building but not other parental behaviours. Together, our results indicate that variation in an ancient neuropeptide contributes to interspecific differences in parental care.

Long-range regulatory polymorphisms affecting a GABA receptor constitute a quantitative trait locus (QTL) for social behavior in Caenorhabditis elegans.

Aggregation is a social behavior that varies between and within species, providing a model to study the genetic basis of behavioral diversity. In the nematode Caenorhabditis elegans, aggregation is regulated by environmental context and by two neuromodulatory pathways, one dependent on the neuropeptide receptor NPR-1 and one dependent on the TGF-ß family protein DAF-7. To gain further insight into the genetic regulation of aggregation, we characterize natural variation underlying behavioral differences between two wild-type C. elegans strains, N2 and CB4856. Using quantitative genetic techniques, including a survey of chromosome substitution strains and QTL analysis of recombinant inbred lines, we identify three new QTLs affecting aggregation in addition to the two known N2 mutations in npr-1 and glb-5. Fine-mapping with near-isogenic lines localized one QTL, accounting for 5%-8% of the behavioral variance between N2 and CB4856, 3' to the transcript of the GABA neurotransmitter receptor gene exp-1. Quantitative complementation tests demonstrated that this QTL affects exp-1, identifying exp-1 and GABA signaling as new regulators of aggregation. exp-1 interacts genetically with the daf-7 TGF-ß pathway, which integrates food availability and population density, and exp-1 mutations affect the level of daf-7 expression. Our results add to growing evidence that genetic variation affecting neurotransmitter receptor genes is a source of natural behavioral variation.

Genetic contributions to behavioural diversity at the gene-environment interface.

Recent work on behavioural variation within and between species has furthered our understanding of the genetic architecture of behavioural traits, the identities of relevant genes and the ways in which genetic variants affect neuronal circuits to modify behaviour. Here we review our understanding of the genetics of natural behavioural variation in non-human animals and highlight the implications of these findings for human genetics. We suggest that gene-environment interactions are central to natural genetic variation in behaviour and that genes affecting neuromodulatory pathways and sensory processing are preferred sites of naturally occurring mutations.

Catecholamine receptor polymorphisms affect decision-making in C. elegans.

Innate behaviours are flexible: they change rapidly in response to transient environmental conditions, and are modified slowly by changes in the genome. A classical flexible behaviour is the exploration-exploitation decision, which describes the time at which foraging animals choose to abandon a depleting food supply. We have used quantitative genetic analysis to examine the decision to leave a food patch in Caenorhabditis elegans. Here we show that patch-leaving is a multigenic trait regulated in part by naturally occurring non-coding polymorphisms in tyra-3 (tyramine receptor 3), which encodes a G-protein-coupled catecholamine receptor related to vertebrate adrenergic receptors. tyra-3 acts in sensory neurons that detect environmental cues, suggesting that the internal catecholamines detected by tyra-3 regulate responses to external conditions. These results indicate that genetic variation and environmental cues converge on common circuits to regulate behaviour, and suggest that catecholamines have an ancient role in regulating behavioural decisions.