Host genomic variation shapes gut microbiome diversity in threespine stickleback fish.

IMPORTANCE: A major focus of host-microbe research is to understand how genetic differences, of various magnitudes, among hosts translate to differences in their microbiomes. This has been challenging for animal hosts, including humans, because it is difficult to control environmental variables tightly enough to isolate direct genetic effects on the microbiome. Our work in stickleback fish is a significant contribution because our experimental approach allowed strict control over environmental factors, including standardization of the microbiome from the earliest stage of development and unrestricted co-housing of fish in a truly common environment. Furthermore, we measured host genetic variation over 2,000 regions of the stickleback genome, comparing this information and microbiome composition data among fish from very similar and very different genetic backgrounds. Our findings highlight how differences in the host genome influence microbiome diversity and make a case for future manipulative microbiome experiments that use host systems with naturally occurring genetic variation.

Genome-wide analysis facilitates estimation of the amount of male contribution in meiotic gynogenetic three-spined stickleback (Gasterosteus aculeatus).

Gynogenetic embryos - those inheriting only maternal DNA - can be experimentally created by fertilizing eggs with radiation-treated sperm containing inactivated paternal chromosomes. Diploidy in the zygotes can be maintained through prevention of the second meiosis or restored by preventing the first mitosis after the maternal chromosome complement has been replicated. These gynogenetic organisms are useful in many fields including aquaculture, evolutionary biology and genomics. Although gynogenetic organisms have been created in numerous species, the completeness of uni-parental inheritance has often been assumed rather than thoroughly quantified across the genome. Instead, when tests of uni-parental inheritance occur, they typically rely on well-studied genetically determined phenotypes that represent a very small sub-set of the genome. Only assessing small genomic regions for paternal inheritance leaves the question of whether some paternal contributions to offspring might still have occurred. In this study, the authors quantify the efficacy of creating gynogenetic diploid three-spined stickleback fish (Gasterosteus aculeatus). To this end, the authors mirrored previous assessments of paternal contribution using well-studied genetically determined phenotypes including sex and genetically dominant morphological traits but expanded on previous studies using dense restriction site-associated DNA sequencing (RAD-seq) markers in parents and offspring to assess paternal inheritance genome-wide. In the gynogenetic diploids, the authors found no male genotypes underlying their phenotypes of interest - sex and dominant phenotypic traits. Using genome-wide assessments of paternal contribution, nevertheless, the authors found evidence of a small, yet potentially important, amount of paternally "leaked" genetic material. The application of this genome-wide approach identifies the need for more widespread assessment of paternal contributions to gynogenetic animals and promises benefits for many aspects of aquaculture, evolutionary biology and genomics.

Effectiveness of a COVID-19 Testing Outreach Intervention for Latinx Communities: A Cluster Randomized Trial.

Importance: Latinx individuals have been disproportionately affected during the COVID-19 pandemic caused by the spread of SARS-CoV-2. It is imperative to evaluate newly developed preventive interventions to assess their effect on COVID-19 health disparities. Objective: To examine the effectiveness of a culturally tailored outreach intervention designed to increase SARS-CoV-2 testing rates among Latinx populations. Design, Setting, and Participants: In this cluster randomized trial performed from February 1 to August 31, 2021, in community settings in 9 Oregon counties, 38 sites were randomized a priori (19 to the community health promoters intervention and 19 to outreach as usual wait-listed controls). Thirty-three sites were activated. A total of 394 SARS-CoV-2 testing events were held and 1851 diagnostic samples collected, of which 919 were from Latinx persons. Interventions: A culturally informed outreach program was developed that made use of promotores de salud (community health promoters) to increase Latinx SARS-CoV-2 testing. Strategies addressed barriers by disseminating information on testing events in English and Spanish, mitigating misinformation, and increasing trust. Main Outcomes and Measures: The primary outcomes were the count of sample tests from Latinx persons and the sampled proportion of the Latinx populace. Site-level covariates included census tract Latinx populace, nativity (number of US-born individuals per 100 population), median age, and income inequality. Time-varying covariates included number of new weekly SARS-CoV-2-positive cases and percentage of vaccine coverage at the county level. Results: A total of 15 clusters (sites) were randomized to the control group and 18 to the community health promoters group. A total of 1851 test samples were collected, of which 995 (53.8%) were from female participants and 919 (49.6%) were from Latinx individuals. The intervention tested 3.84 (95% CI, 2.47-5.97) times more Latinx individuals per event than controls (incident rate ratio, 0.79; 95% CI, 0.46-1.34; Cohen d = 0.74; P < .001). The intervention was associated with a 0.28 increase in the proportion of Latinx populace being tested compared with control sites for the dependent variable scaled as the proportion of the Latinx populace ×100, or a 0.003 proportion of the raw populace count. The use of a standardized scaling of the proportion of Latinx individuals showed that the relative percentage increase was 0.53 (95% CI, 0.21-0.86) in the intervention sites compared with controls, representing a medium effect size. Conclusions and Relevance: To our knowledge, this was the first randomized evaluation of an outreach intervention designed to increase SARS-CoV-2 testing among Latinx populations. Findings could be used to implement strategies to reduce other health disparities experienced by these groups. Trial Registration: ClinicalTrials.gov Identifier: NCT04793464.

Leafy and weedy seadragon genomes connect genic and repetitive DNA features to the extravagant biology of syngnathid fishes.

Seadragons are a remarkable lineage of teleost fishes in the family Syngnathidae, renowned for having evolved male pregnancy. Comprising three known species, seadragons are widely recognized and admired for their fantastical body forms and coloration, and their specific habitat requirements have made them flagship representatives for marine conservation and natural history interests. Until recently, a gap has been the lack of significant genomic resources for seadragons. We have produced gene-annotated, chromosome-scale genome models for the leafy and weedy seadragon to advance investigations of evolutionary innovation and elaboration of morphological traits in seadragons as well as their pipefish and seahorse relatives. We identified several interesting features specific to seadragon genomes, including divergent noncoding regions near a developmental gene important for integumentary outgrowth, a high genome-wide density of repetitive DNA, and recent expansions of transposable elements and a vesicular trafficking gene family. Surprisingly, comparative analyses leveraging the seadragon genomes and additional syngnathid and outgroup genomes revealed striking, syngnathid-specific losses in the family of fibroblast growth factors (FGFs), which likely involve reorganization of highly conserved gene regulatory networks in ways that have not previously been documented in natural populations. The resources presented here serve as important tools for future evolutionary studies of developmental processes in syngnathids and hold value for conservation of the extravagant seadragons and their relatives.

Advancing human disease research with fish evolutionary mutant models.

Model organism research is essential to understand disease mechanisms. However, laboratory-induced genetic models can lack genetic variation and often fail to mimic the spectrum of disease severity. Evolutionary mutant models (EMMs) are species with evolved phenotypes that mimic human disease. EMMs complement traditional laboratory models by providing unique avenues to study gene-by-environment interactions, modular mutations in noncoding regions, and their evolved compensations. EMMs have improved our understanding of complex diseases, including cancer, diabetes, and aging, and illuminated mechanisms in many organs. Rapid advancements of sequencing and genome-editing technologies have catapulted the utility of EMMs, particularly in fish. Fish are the most diverse group of vertebrates, exhibiting a kaleidoscope of specialized phenotypes, many that would be pathogenic in humans but are adaptive in the species' specialized habitat. Importantly, evolved compensations can suggest avenues for novel disease therapies. This review summarizes current research using fish EMMs to advance our understanding of human disease.

QTL Mapping of Intestinal Neutrophil Variation in Threespine Stickleback Reveals Possible Gene Targets Connecting Intestinal Inflammation and Systemic Health.

Selection, via host immunity, is often required to foster beneficial microbial symbionts and suppress deleterious pathogens. In animals, the host immune system is at the center of this relationship. Failed host immune system-microbial interactions can result in a persistent inflammatory response in which the immune system indiscriminately attacks resident microbes, and at times the host cells themselves, leading to diseases such as Ulcerative Colitis, Crohn's Disease, and Psoriasis. Host genetic variation has been linked to both microbiome diversity and to severity of such inflammatory disease states in humans. However, the microbiome and inflammatory states manifest as quantitative traits, which encompass many genes interacting with one another and the environment. The mechanistic relationships among all of these interacting components are still not clear. Developing natural genetic models of host-microbe interactions is therefore fundamental to understanding the complex genetics of these and other diseases. Threespine stickleback (Gasterosteus aculeatus) fish are a tractable model for attacking this problem because of abundant population-level genetic and phenotypic variation in the gut inflammatory response. Previous work in our laboratory identified genetically divergent stickleback populations exhibiting differences in intestinal neutrophil activity. We took advantage of this diversity to genetically map variation in an emblematic element of gut inflammation - intestinal neutrophil recruitment - using an F2-intercross mapping framework. We identified two regions of the genome associated with increased intestinal inflammation containing several promising candidate genes. Within these regions we found candidates in the Coagulation/Complement System, NFkB and MAPK pathways along with several genes associated with intestinal diseases and neurological diseases commonly accompanying intestinal inflammation as a secondary symptom. These findings highlight the utility of using naturally genetically diverse 'evolutionary mutant models' such as threespine stickleback to better understand interactions among host genetic diversity and microbiome variation in health and disease states.

Population Genetic Divergence and Environment Influence the Gut Microbiome in Oregon Threespine Stickleback.

Much of animal-associated microbiome research has been conducted in species for which little is known of their natural ecology and evolution. Microbiome studies that combine population genetic, environment, and geographic data for wild organisms can be very informative, especially in situations where host genetic variation and the environment both influence microbiome variation. The few studies that have related population genetic and microbiome variation in wild populations have been constrained by observation-based kinship data or incomplete genomic information. Here we integrate population genomic and microbiome analyses in wild threespine stickleback fish distributed throughout western Oregon, USA. We found that gut microbiome diversity and composition partitioned more among than within wild host populations and was better explained by host population genetic divergence than by environment and geography. We also identified gut microbial taxa that were most differentially abundant across environments and across genetically divergent populations. Our findings highlight the benefits of studies that investigate host-associated microbiomes in wild organisms.

Genetic divergence outpaces phenotypic evolution among threespine stickleback populations in old freshwater habitats.

Species such as threespine stickleback (Gasterosteus aculeatus) that inhabit divergent selective environments and that have diversified on different time scales can be of value for understanding evolutionary processes. Here we synthesize high-resolution genotypic and phenotypic data to explore a largely unstudied distribution of threespine stickleback populations living in oceanic and freshwater habitats along coastal and inland regions of Oregon. Many inland aquatic habitats of Oregon remained unglaciated during the last ice age, meaning that some extant Oregon lake and river stickleback may have descended from freshwater populations established long before more well-studied, post-glacial freshwater populations. To address the degree of congruence between genetic and phenotypic divergence, we directly compared Oregon stickleback to much younger (post-glacial) Alaskan populations. We found phenotypic variation in Oregon stickleback to be primarily partitioned between oceanic and freshwater habitats, as has been documented in other stickleback systems. However, the main axis of genetic divergence was between coastal and inland regions regardless of habitat type. Furthermore, when comparing patterns between Oregon and Alaska we found similar levels of phenotypic divergence, but much greater genetic divergence among Oregon's populations. The Oregon stickleback system therefore appears well suited for future studies linking genotypic and phenotypic change, further extending the utility of this small fish to provide general insights into evolutionary processes.

Developmental timing differences underlie armor loss across threespine stickleback populations.

Comparing ontogenetic patterns within a well-described evolutionary context aids in inferring mechanisms of change, including heterochronies or deletion of developmental pathways. Because selection acts on phenotypes throughout ontogeny, any within-taxon developmental variation has implications for evolvability. We compare ontogenetic order and timing of locomotion and defensive traits in three populations of threespine stickleback that have evolutionarily divergent adult forms. This analysis adds to the growing understanding of developmental genetic mechanisms of adaptive change in this evolutionary model species by delineating when chondrogenesis and osteogenesis in two derived populations begin to deviate from the developmental pattern in their immediate ancestors. We found that differences in adult defensive morphologies arise through abolished or delayed initiation of these traits rather than via an overall heterochronic shift, that intra-population ontogenetic variation is increased for some derived traits, and that altered armor developmental timing differentiates the derived populations from each other despite parallels in adult lateral plate armor phenotypes. We found that changes in ossified elements of the pelvic armor are linked to delayed and incomplete development of an early-forming pelvic cartilage, and that this disruption likely presages the variable pelvic vestiges documented in many derived populations.

Rapid SNP discovery and genetic mapping using sequenced RAD markers.

Single nucleotide polymorphism (SNP) discovery and genotyping are essential to genetic mapping. There remains a need for a simple, inexpensive platform that allows high-density SNP discovery and genotyping in large populations. Here we describe the sequencing of restriction-site associated DNA (RAD) tags, which identified more than 13,000 SNPs, and mapped three traits in two model organisms, using less than half the capacity of one Illumina sequencing run. We demonstrated that different marker densities can be attained by choice of restriction enzyme. Furthermore, we developed a barcoding system for sample multiplexing and fine mapped the genetic basis of lateral plate armor loss in threespine stickleback by identifying recombinant breakpoints in F(2) individuals. Barcoding also facilitated mapping of a second trait, a reduction of pelvic structure, by in silico re-sorting of individuals. To further demonstrate the ease of the RAD sequencing approach we identified polymorphic markers and mapped an induced mutation in Neurospora crassa. Sequencing of RAD markers is an integrated platform for SNP discovery and genotyping. This approach should be widely applicable to genetic mapping in a variety of organisms.