Single-fly genome assemblies fill major phylogenomic gaps across the Drosophilidae Tree of Life.

Long-read sequencing is driving rapid progress in genome assembly across all major groups of life, including species of the family Drosophilidae, a longtime model system for genetics, genomics, and evolution. We previously developed a cost-effective hybrid Oxford Nanopore (ONT) long-read and Illumina short-read sequencing approach and used it to assemble 101 drosophilid genomes from laboratory cultures, greatly increasing the number of genome assemblies for this taxonomic group. The next major challenge is to address the laboratory culture bias in taxon sampling by sequencing genomes of species that cannot easily be reared in the lab. Here, we build upon our previous methods to perform amplification-free ONT sequencing of single wild flies obtained either directly from the field or from ethanol-preserved specimens in museum collections, greatly improving the representation of lesser studied drosophilid taxa in whole-genome data. Using Illumina Novaseq X Plus and ONT P2 sequencers with R10.4.1 chemistry, we set a new benchmark for inexpensive hybrid genome assembly at US $150 per genome while assembling genomes from as little as 35 ng of genomic DNA from a single fly. We present 183 new genome assemblies for 179 species as a resource for drosophilid systematics, phylogenetics, and comparative genomics. Of these genomes, 62 are from pooled lab strains and 121 from single adult flies. Despite the sample limitations of working with small insects, most single-fly diploid assemblies are comparable in contiguity (>1 Mb contig N50), completeness (>98% complete dipteran BUSCOs), and accuracy (>QV40 genome-wide with ONT R10.4.1) to assemblies from inbred lines. We present a well-resolved multi-locus phylogeny for 360 drosophilid and 4 outgroup species encompassing all publicly available (as of August 2023) genomes for this group. Finally, we present a Progressive Cactus whole-genome, reference-free alignment built from a subset of 298 suitably high-quality drosophilid genomes. The new assemblies and alignment, along with updated laboratory protocols and computational pipelines, are released as an open resource and as a tool for studying evolution at the scale of an entire insect family.

Arc/Arg3.1 regulates an endosomal pathway essential for activity-dependent ß-amyloid generation.

Assemblies of ß-amyloid (Aß) peptides are pathological mediators of Alzheimer's Disease (AD) and are produced by the sequential cleavages of amyloid precursor protein (APP) by ß-secretase (BACE1) and γ-secretase. The generation of Aß is coupled to neuronal activity, but the molecular basis is unknown. Here, we report that the immediate early gene Arc is required for activity-dependent generation of Aß. Arc is a postsynaptic protein that recruits endophilin2/3 and dynamin to early/recycling endosomes that traffic AMPA receptors to reduce synaptic strength in both hebbian and non-hebbian forms of plasticity. The Arc-endosome also traffics APP and BACE1, and Arc physically associates with presenilin1 (PS1) to regulate γ-secretase trafficking and confer activity dependence. Genetic deletion of Arc reduces Aß load in a transgenic mouse model of AD. In concert with the finding that patients with AD can express anomalously high levels of Arc, we hypothesize that Arc participates in the pathogenesis of AD.

A Whole-Genome Scan for Association with Invasion Success in the Fruit Fly Drosophila suzukii Using Contrasts of Allele Frequencies Corrected for Population Structure.

Evidence is accumulating that evolutionary changes are not only common during biological invasions but may also contribute directly to invasion success. The genomic basis of such changes is still largely unexplored. Yet, understanding the genomic response to invasion may help to predict the conditions under which invasiveness can be enhanced or suppressed. Here, we characterized the genome response of the spotted wing drosophila Drosophila suzukii during the worldwide invasion of this pest insect species, by conducting a genome-wide association study to identify genes involved in adaptive processes during invasion. Genomic data from 22 population samples were analyzed to detect genetic variants associated with the status (invasive versus native) of the sampled populations based on a newly developed statistic, we called C2, that contrasts allele frequencies corrected for population structure. We evaluated this new statistical framework using simulated data sets and implemented it in an upgraded version of the program BayPass. We identified a relatively small set of single-nucleotide polymorphisms that show a highly significant association with the invasive status of D. suzukii populations. In particular, two genes, RhoGEF64C and cpo, contained single-nucleotide polymorphisms significantly associated with the invasive status in the two separate main invasion routes of D. suzukii. Our methodological approaches can be applied to any other invasive species, and more generally to any evolutionary model for species characterized by nonequilibrium demographic conditions for which binary covariables of interest can be defined at the population level.

Horizontal Transfer of Bacterial Cytolethal Distending Toxin B Genes to Insects.

Horizontal gene transfer events have played a major role in the evolution of microbial species, but their importance in animals is less clear. Here, we report horizontal gene transfer of cytolethal distending toxin B (cdtB), prokaryotic genes encoding eukaryote-targeting DNase I toxins, into the genomes of vinegar flies (Diptera: Drosophilidae) and aphids (Hemiptera: Aphididae). We found insect-encoded cdtB genes are most closely related to orthologs from bacteriophage that infect Candidatus Hamiltonella defensa, a bacterial mutualistic symbiont of aphids that confers resistance to parasitoid wasps. In drosophilids, cdtB orthologs are highly expressed during the parasitoid-prone larval stage and encode a protein with ancestral DNase activity. We show that cdtB has been domesticated by diverse insects and hypothesize that it functions in defense against their natural enemies.

Varieties of the highly dispersible and hypervariable tree, Metrosideros polymorpha, differ in response to mechanical stress and light across a sharp ecotone.

PREMISE: The drivers of isolation between sympatric populations of long-lived and highly dispersible conspecific plants are not well understood. In the Hawaiian Islands, the landscape-dominant tree, Metrosideros polymorpha, displays extraordinary phenotypic differences among sympatric varieties despite high dispersibility of its pollen and seeds, thereby presenting a unique opportunity to investigate how disruptive selection alone can maintain incipient forms. Stenophyllous M. polymorpha var. newellii is a recently evolved tree endemic to the waterways of eastern Hawai'i Island that shows striking neutral genetic differentiation from its ancestor, wet-forest M. polymorpha var. glaberrima, despite sympatry of these forms. We looked for evidence for, and drivers of, differential local adaptation of these varieties across the range of M. polymorpha var. newellii. METHODS: For paired populations of these varieties, we compared seedling performance under contrasting light conditions and a strong water current characteristic of the riparian zone. We also conducted a reciprocal transplant experiment and contrasted adult leaf anatomy. RESULTS: Results suggest that the riparian zone is harsh and that selection involving the mechanical stress of rushing water, and secondarily, light, led to significant reciprocal immigrant inviability in adjacent forest and riparian environments. The strongest adaptive divergence between varieties was seen in leaves and seedlings from the site with the sharpest ecotone, coincident with the strongest genetic isolation of M. polymorpha var. newellii observed previously. CONCLUSIONS: These findings suggest that disruptive selection across a sharp ecotone contributes to the maintenance of an incipient riparian ecotype from within a continuous population of a long-lived and highly dispersible tree species.

Deciphering the Routes of invasion of Drosophila suzukii by Means of ABC Random Forest.

Deciphering invasion routes from molecular data is crucial to understanding biological invasions, including identifying bottlenecks in population size and admixture among distinct populations. Here, we unravel the invasion routes of the invasive pest Drosophila suzukii using a multi-locus microsatellite dataset (25 loci on 23 worldwide sampling locations). To do this, we use approximate Bayesian computation (ABC), which has improved the reconstruction of invasion routes, but can be computationally expensive. We use our study to illustrate the use of a new, more efficient, ABC method, ABC random forest (ABC-RF) and compare it to a standard ABC method (ABC-LDA). We find that Japan emerges as the most probable source of the earliest recorded invasion into Hawaii. Southeast China and Hawaii together are the most probable sources of populations in western North America, which then in turn served as sources for those in eastern North America. European populations are genetically more homogeneous than North American populations, and their most probable source is northeast China, with evidence of limited gene flow from the eastern US as well. All introduced populations passed through bottlenecks, and analyses reveal five distinct admixture events. These findings can inform hypotheses concerning how this species evolved between different and independent source and invasive populations. Methodological comparisons indicate that ABC-RF and ABC-LDA show concordant results if ABC-LDA is based on a large number of simulated datasets but that ABC-RF out-performs ABC-LDA when using a comparable and more manageable number of simulated datasets, especially when analyzing complex introduction scenarios.

A Test for Gene Flow among Sympatric and Allopatric Hawaiian Picture-Winged Drosophila.

The Hawaiian Drosophila are one of the most species-rich endemic groups in Hawaii and a spectacular example of adaptive radiation. Drosophila silvestris and D. heteroneura are two closely related picture-winged Drosophila species that occur sympatrically on Hawaii Island and are known to hybridize in nature, yet exhibit highly divergent behavioral and morphological traits driven largely through sexual selection. Their closest-related allopatric species, D. planitibia from Maui, exhibits hybrid male sterility and reduced behavioral reproductive isolation when crossed experimentally with D. silvestris or D. heteroneura. A modified four-taxon test for gene flow was applied to recently obtained genomes of the three Hawaiian Drosophila species. The analysis indicates recent gene flow in sympatry, but also, although less extensive, between allopatric species. This study underscores the prevalence of gene flow, even in taxonomic groups considered classic examples of allopatric speciation on islands. The potential confounding effects of gene flow in phylogenetic and population genetics inference are discussed, as well as the implications for conservation.

Mapping Genomic Scaffolds to Chromosomes Using Laser Capture Microdissection in Application to Hawaiian Picture-Winged Drosophila.

Next-generation sequencing technologies have led to a decreased cost and an increased throughput in genome sequencing. Yet, many genome assemblies based on short sequencing reads have been assembled only to the scaffold level due to the lack of sufficient chromosome mapping information. Traditional ways of mapping scaffolds to chromosomes require a large amount of laboratory work and time to generate genetic and/or physical maps. To address this problem, we conducted a rapid technique which uses laser capture microdissection and enables mapping scaffolds of de novo genome assemblies directly to chromosomes in Hawaiian picture-winged Drosophila. We isolated and sequenced intact chromosome arms from larvae of D. differens. By mapping the reads of each chromosome to the recently assembled scaffolds from 3 Hawaiian picture-winged Drosophila species, at least 67% of the scaffolds were successfully assigned to chromosome arms. Even though the scaffolds are not ordered within a chromosome, the fast-generated chromosome information allows for chromosome-related analyses after genome assembling. We utilize this new information to test the faster-X evolution effect for the first time in these Hawaiian picture-winged Drosophila species.

Effective connectivity predicts future placebo analgesic response: A dynamic causal modeling study of pain processing in healthy controls.

A better understanding of the neural mechanisms underlying pain processing and analgesia may aid in the development and personalization of effective treatments for chronic pain. Clarification of the neural predictors of individual variability in placebo analgesia (PA) could aid in this process. The present study examined whether the strength of effective connectivity (EC) among pain-related brain regions could predict future placebo analgesic response in healthy individuals. In Visit 1, fMRI data were collected from 24 healthy subjects (13 females, mean age=22.56, SD=2.94) while experiencing painful thermal stimuli. During Visit 2, subjects were conditioned to expect less pain via a surreptitiously lowered temperature applied at two of the four sites on their feet. They were subsequently scanned again using the Visit 1 (painful) temperature. Subjects used an electronic VAS to rate their pain following each stimulus. Differences in ratings at conditioned and unconditioned sites were used to measure placebo response (PA scores). Dynamic causal modeling was used to estimate the EC among a set of brain regions related to pain processing at Visit 1 (periaqueductal gray, thalamus, rostral anterior cingulate cortex, dorsolateral prefrontal cortex). Individual PA scores from Visit 2 were regressed on salient EC parameter estimates from Visit 1. Results indicate that both greater left hemisphere modulatory DLPFC➔PAG connectivity and right hemisphere, endogenous thalamus➔DLPFC connectivity were significantly predictive of future placebo response (R(2)=0.82). To our knowledge, this is the first study to identify the value of EC in understanding individual differences in PA, and may suggest the potential modifiability of endogenous pain modulation.

Rapid adaptive radiation and host plant conservation in the Hawaiian picture wing Drosophila (Diptera: Drosophilidae).

The Hawaiian picture wing Drosophila are a striking example of adaptive radiation in specialist saprophages on an island system. We use DNA sequences from five nuclear genes with a total of 4260 nucleotides to provide a comprehensive phylogeny and biogeographic analysis of 90 species in the Hawaiian Drosophila picture wing clade. The current analysis indicates that the evolution of the picture wing clade took place more recently than previously suggested. The relationships of several morphologically anomalous taxa are resolved with strong support. Biogeography and host plant analyses show two periods of rapid divergence occurred when Kauai and Oahu were the main high islands, indicating that a combination of complex topographical features of islands and development of novel host plant associations was key to the rapid diversification of these lineages. For the past 2 million years, host associations within lineages have been largely stable, and speciation has occurred primarily due to the establishment of populations on newer islands as they arose followed by divergence by isolation. The existence of several apparently relictual taxa suggests that extinction has also played a major role in assembly of the present Hawaiian Drosophila fauna.

Postzygotic barriers isolate sympatric species of Cyrtandra (Gesneriaceae) in Hawaiian montane forest understories.

PREMISE OF THE STUDY: Recent reviews of reproductive isolation (RI) in plants propose that boundaries between closely related species are maintained predominantly through prezygotic mechanisms. However, few experimental studies have explored how boundaries are maintained in long-lived species. Hawaiian Cyrtandra presents an intriguing challenge to our understanding of RI, as it comprises 60 shrub or small tree species that are almost exclusively restricted to wet forests, where sympatry of multiple species is common. METHODS: We assessed the relative strengths of pre- and postzygotic barriers among four species of Cyrtandra occurring at the extremes of the main Hawaiian Island's natural island-age gradient, Kaua'i (4.7 Myr) and Hawai'i Island (0.6 Myr), to contrast the strengths and stages of reproductive isolation among species at different stages of divergence. KEY RESULTS: A combination of F1 seed germination, F1 seedling survival, and F1 seedling growth isolated (61-91%) three of the species from sympatric relatives. In contrast, the fourth species was isolated (59%) from its sympatric relative through phenological differences alone. Significant postzygotic barriers in between-island crosses were also observed in one species. CONCLUSIONS: Results suggest that boundaries between sympatric Cyrtandra species in Hawaii are maintained predominantly through postzygotic barriers. Observations from between-island crosses indicate that postzygotic barriers can arise in allopatry, which may be important in the initial divergence of populations. Future studies of RI in Cyrtandra should include a broader range of species to determine if postzygotic isolating barriers are foremost in the maintenance of species boundaries in this large genus.

Potential use of low-copy nuclear genes in DNA barcoding: a comparison with plastid genes in two Hawaiian plant radiations.

BACKGROUND: DNA barcoding of land plants has relied traditionally on a small number of markers from the plastid genome. In contrast, low-copy nuclear genes have received little attention as DNA barcodes because of the absence of universal primers for PCR amplification. RESULTS: From pooled-species 454 transcriptome data we identified two variable intron-less nuclear loci for each of two species-rich genera of the Hawaiian flora: Clermontia (Campanulaceae) and Cyrtandra (Gesneriaceae) and compared their utility as DNA barcodes with that of plastid genes. We found that nuclear genes showed an overall greater variability, but also displayed a high level of heterozygosity, intraspecific variation, and retention of ancient alleles. Thus, nuclear genes displayed fewer species-diagnostic haplotypes compared to plastid genes and no interspecies gaps. CONCLUSIONS: The apparently greater coalescence times of nuclear genes are likely to limit their utility as barcodes, as only a small proportion of their alleles were fixed and unique to individual species. In both groups, species-diagnostic markers from either genome were scarce on the youngest island; a minimum age of ca. two million years may be needed for a species flock to be barcoded. For young plant groups, nuclear genes may not be a superior alternative to slowly evolving plastid genes.

Patterns and processes in complex landscapes: testing alternative biogeographical hypotheses through integrated analysis of phylogeography and community ecology in Hawai'i.

The Island of Hawai'i is a dynamic assemblage of five volcanoes with wet forest habitat currently existing in four distinct natural regions that vary in area, age and geographical isolation. In this complex landscape, alternative assumptions of the relative importance of specific habitat characteristics on evolutionary and ecological processes predict strikingly different general patterns of local diversity and regional similarity. In this study, we compare alternative a priori hypotheses against observed patterns within two distinct biological systems and scales: community composition of wet forest vascular plant species and mitochondrial and nuclear genes of Drosophila sproati, a wet-forest-restricted endemic. All observed patterns display strong and similar regional structuring, with the greatest local diversity found in Kohala and the windward side of Mauna Loa, the least in Ka'u and Kona, and a distinctive pattern of regional similarity that probably reflects the historical development of this habitat on the island. These observations largely corroborate a biogeographical model that integrates multiple lines of evidence, including climatic reconstruction, over those relying on single measures, such as current habitat configuration or substrate age. This method of testing alternative hypotheses across biological systems and scales is an innovative approach for understanding complex landscapes and should prove valuable in diverse biogeographical systems.

Gene discordance in phylogenomics of recent plant radiations, an example from Hawaiian Cyrtandra (Gesneriaceae).

Resolving species relationships within recent radiations requires analysis at the interface of phylogenetics and population genetics, where coalescence and hybridization may confound our understanding of relationships. We developed 18 new primer pairs for nuclear loci in Cyrtandra (Gesneriaceae), one of the largest plant radiations in the Pacific Islands, and tested the concordance of 14 loci in establishing the phylogenetic relationships of a small number of Hawaiian species. Four genes yielded tree topologies conflicting with the primary concordance tree, suggesting plastid capture and horizontal transfer via hybridization. Combining all concordant genes yielded a tree with stronger support and a different topology from the total-evidence tree. We conclude that a small number of genes may be insufficient for accurate reconstruction of the phylogenetic relationships among closely related species. Further, the combination of genes for phylogenetic analysis without preliminary concordance tests can yield an erroneous tree topology. It seems that the number of genes needed for phylogenetic analysis of closely related species is significantly greater than the small numbers commonly used, which fail to isolate coalescence, introgression and hybridization.

Altered distributions of Gemini of coiled bodies and mitochondria in motor neurons of TDP-43 transgenic mice.

TAR DNA-binding protein-43 (TDP-43), a DNA/RNA-binding protein involved in RNA transcription and splicing, has been associated with the pathophysiology of neurodegenerative diseases, including ALS. However, the function of TDP-43 in motor neurons remains undefined. Here we use both gain- and loss-of-function approaches to determine roles of TDP-43 in motor neurons. Mice expressing human TDP-43 in neurons exhibited growth retardation and premature death that are characterized by abnormal intranuclear inclusions composed of TDP-43 and fused in sarcoma/translocated in liposarcoma (FUS/TLS), and massive accumulation of mitochondria in TDP-43-negative cytoplasmic inclusions in motor neurons, lack of mitochondria in motor axon terminals, and immature neuromuscular junctions. Whereas an elevated level of TDP-43 disrupts the normal nuclear distribution of survival motor neuron (SMN)-associated Gemini of coiled bodies (GEMs) in motor neurons, its absence prevents the formation of GEMs in the nuclei of these cells. Moreover, transcriptome-wide deep sequencing analysis revealed that a decrease in abundance of neurofilament transcripts contributed to the reduction of caliber of motor axons in TDP-43 mice. In concert, our findings indicate that TDP-43 participates in pathways critical for motor neuron physiology, including those that regulate the normal distributions of SMN-associated GEMs in the nucleus and mitochondria in the cytoplasm.

Deletion of TDP-43 down-regulates Tbc1d1, a gene linked to obesity, and alters body fat metabolism.

Tat activating regulatory DNA-binding protein (Tardbp or TDP-43), a highly conserved metazoan DNA/RNA binding protein thought to be involved in RNA transcription and splicing, has been linked to the pathophysiology of amyotrophic lateral sclerosis and frontotemporal lobar degeneration and is essential for early embryonic development. However, neither the physiological role of TDP-43 in the adult nor its downstream targets are well defined. To address these questions, we developed conditional Tardbp-KO mice and embryonic stem (ES) cell models. Here, we show that postnatal deletion of Tardbp in mice caused dramatic loss of body fat followed by rapid death. Moreover, conditional Tardbp-KO ES cells failed to proliferate. Importantly, high-throughput DNA sequencing analysis on the transcriptome of ES cells lacking Tardbp revealed a set of downstream targets of TDP-43. We show that Tbc1d1, a gene known to mediate leanness and linked to obesity, is down-regulated in the absence of TDP-43. Collectively, our results establish that TDP-43 is critical for fat metabolism and ES cell survival.

Genetic diversity, structure, and effective population size of an endangered, endemic hoary bat, 'ope'ape'a, across the Hawaiian Islands.

Island bat species are disproportionately at risk of extinction, and Hawai'i's only native terrestrial land mammal, the Hawaiian hoary bat (Lasiurus semotus) locally known as 'ope'ape'a, is no exception. To effectively manage this bat species with an archipelago-wide distribution, it is important to determine the population size on each island and connectivity between islands. We used 18 nuclear microsatellite loci and one mitochondrial gene from 339 individuals collected from 1988-2020 to evaluate genetic diversity, population structure and estimate effective population size on the Islands of Hawai'i, Maui, O'ahu, and Kaua'i. Genetic differentiation occurred between Hawai'i and Maui, both of which were differentiated from O'ahu and Kaua'i. The population on Maui presents the greatest per-island genetic diversity, consistent with their hypothesized status as the original founding population. A signature of isolation by distance was detected between islands, with contemporary migration analyses indicating limited gene flow in recent generations, and male-biased sex dispersal within Maui. Historical and long-term estimates of genetic effective population sizes were generally larger than contemporary estimates, although estimates of contemporary genetic effective population size lacked upper bounds in confidence intervals for Hawai'i and Kaua'i. Contemporary genetic effective population sizes were smaller on O'ahu and Maui. We also detected evidence of past bottlenecks on all islands with the exception of Hawai'i. Our study provides population-level estimates for the genetic diversity and geographic structure of 'ope'ape'a, that could be used by agencies tasked with wildlife conservation in Hawai'i.

The Transmission Patterns of the Endosymbiont Wolbachia within the Hawaiian Drosophilidae Adaptive Radiation.

The evolution of endosymbionts and their hosts can lead to highly dynamic interactions with varying fitness effects for both the endosymbiont and host species. Wolbachia, a ubiquitous endosymbiont of arthropods and nematodes, can have both beneficial and detrimental effects on host fitness. We documented the occurrence and patterns of transmission of Wolbachia within the Hawaiian Drosophilidae and examined the potential contributions of Wolbachia to the rapid diversification of their hosts. Screens for Wolbachia infections across a minimum of 140 species of Hawaiian Drosophila and Scaptomyza revealed species-level infections of 20.0%, and across all 399 samples, a general infection rate of 10.3%. Among the 44 Wolbachia strains we identified using a modified Wolbachia multi-locus strain typing scheme, 30 (68.18%) belonged to supergroup B, five (11.36%) belonged to supergroup A, and nine (20.45%) had alleles with conflicting supergroup assignments. Co-phylogenetic reconciliation analysis indicated that Wolbachia strain diversity within their endemic Hawaiian Drosophilidae hosts can be explained by vertical (e.g., co-speciation) and horizontal (e.g., host switch) modes of transmission. Results from stochastic character trait mapping suggest that horizontal transmission is associated with the preferred oviposition substrate of the host, but not the host's plant family or island of occurrence. For Hawaiian Drosophilid species of conservation concern, with 13 species listed as endangered and 1 listed as threatened, knowledge of Wolbachia strain types, infection status, and potential for superinfection could assist with conservation breeding programs designed to bolster population sizes, especially when wild populations are supplemented with laboratory-reared, translocated individuals. Future research aimed at improving the understanding of the mechanisms of Wolbachia transmission in nature, their impact on the host, and their role in host species formation may shed light on the influence of Wolbachia as an evolutionary driver, especially in Hawaiian ecosystems.

Bacterial and fungal components of the gut microbiome have distinct, sex-specific roles in Hawaiian Drosophila reproduction.

Gut microbiomes provide numerous physiological benefits for host animals. The role of bacterial members of microbiomes in host physiology is well-documented. However, much less is known about the contributions and interactions of fungal members of the microbiome even though fungi are significant components of many microbiomes, including those of humans and insects. Here, we used antibacterial and antifungal drugs to manipulate the gut microbiome of a Hawaiian picture-wing Drosophila species, D. grimshawi, and identified distinct, sex-specific roles for the bacteria and fungi in microbiome community stability and reproduction. Female oogenesis, fecundity and mating drive were significantly diminished when fungal communities were suppressed. By contrast, male fecundity was more strongly affected by bacterial but not fungal populations. For males and females, suppression of both bacteria and fungi severely reduced fecundity and altered fatty acid levels and composition, implicating the importance of interkingdom interactions on reproduction and lipid metabolism. Overall, our results reveal that bacteria and fungi have distinct, sexually-dimorphic effects on host physiology and interkingdom dynamics in the gut help to maintain microbiome community stability and enhance reproduction.