Host Evolution Research Articles

Comparative gut microbiome research through the lens of ecology: theoretical considerations and best practices

ABSTRACTComparative approaches in animal gut microbiome research have revealed patterns of phylosymbiosis, dietary and physiological convergences, and environment–host interactions. However, most large‐scale comparative studies, especially those that are highly cited, have focused on mammals, and efforts to integrate comparative approaches with existing ecological frameworks are lacking. While mammals serve as useful model organisms, developing generalised principles of how animal gut microbiomes are shaped and how these microbiomes interact bidirectionally with host ecology and evolution requires a more complete sampling of the animal kingdom. Here, we provide an overview of what past comparative studies have taught us about the gut microbiome, and how community ecology theory may help resolve certain contradictions in comparative gut microbiome research. We explore whether certain hypotheses are supported across clades, and how the disproportionate focus on mammals has introduced potential bias into gut microbiome theory. We then introduce a methodological solution by which public gut microbiome data of understudied hosts can be compiled and analysed in a comparative context. Our aggregation and analysis of 179 studies shows that generating data sets with rich host diversity is possible with public data and that key gut microbes associated with mammals are widespread across the animal kingdom. We also show the effects that sample size and taxonomic rank have on comparative gut microbiome studies and that results of multivariate analyses can vary significantly with these two parameters. While challenges remain in developing a universal model of the animal gut microbiome, we show that existing ecological frameworks can help bring us one step closer to integrating the gut microbiome into animal ecology and evolution.

How host-microbiome/holobiont evolution depends on whether the microbiome affects host lifespan or fecundity.

There is overwhelming evidence that the microbiome can be important to host physiology and fitness. As such, there is interest in and some theoretical work on understanding when hosts and microbiomes (co)evolve so that microbes benefit hosts and hosts favour beneficial microbes. However, the outcome of evolution likely depends on how microbes benefit hosts. Here, we use adaptive dynamics to investigate how host and symbiont evolution depend on whether symbionts increase host lifespan or host reproduction in a simple model of host and symbiont dynamics. In addition, we investigate 2 ways hosts release (and transmit) symbionts: by releasing symbionts steadily during their lifetime or by releasing them at reproduction, potentially increasing symbionts' chances of infecting the host's offspring. The former is strict horizontal transmission, whereas the latter is also a form of indirect or "pseudovertical" transmission. Our first key result is that the evolution of symbionts that benefit host fecundity requires pseudovertical transmission, while the evolution of symbionts that benefit host lifespan does not. Furthermore, our second key result is that when investing in host benefits is costly to the free-living symbiont stage, intermediate levels of pseudovertical transmission are needed for selection to favour beneficial symbionts. This is true regardless of fitness effects because release at reproduction increases the free-living symbiont population, which increases competition for hosts. Consequently, hosts could evolve away from traits that favour beneficial symbionts. Generally, our work emphasizes the importance of different forms of vertical transmission and fitness benefits in host, microbiome, and holobiont evolution as highlighted by our prediction that the evolution of fecundity-increasing symbionts requires parent-to-offspring transmission.

Prophages in Vibrio.

Although tailed bacteriophages (phages) of the class Caudoviricetes are thought to constitute the most abundant and ecologically relevant group of phages that can integrate their genome into the host chromosome, it is becoming increasingly clear that other prophages are widespread. Here, we show that prophages derived from filamentous and tailless phages with genome sizes below 16 kb make up the majority of prophages in marine bacteria of the genus Vibrio. To estimate prophage prevalence unaffected by database biases, we combined comparative genomics and chemical induction of 58 diverse Vibrio cyclitrophicus isolates, resulting in 107 well-curated prophages. Complemented with computationally predicted prophages, we obtained 1,158 prophages from 931 naturally co-existing strains of the family Vibrionaceae. Prophages resembling tailless and filamentous phages predominated, accounting for 80% of all prophages in V. cyclitrophicus and 60% across the Vibrionaceae. In our experimental model, prophages of all three viral realms actively replicated upon induction indicating their ability to transfer to new hosts. Indeed, prophages were rapidly gained and lost, as suggested by variable prophage content between closely related V. cyclitrophicus. Prophages related to filamentous and tailless phages were integrated into only three genomic locations and restored the function of their integration site. Despite their small size, they contained highly diverse accessory genes that may contribute to host fitness, such as phage defense systems. We propose that, like their well-studied tailed equivalent, tailless and filamentous temperate phages are active and highly abundant drivers of host ecology and evolution in marine Vibrio, which have been largely overlooked.

Influenza Virus Types, Subtypes and Genomic Lineage with Its Prevention and Control: A Narrative Review

Since influenza viruses continue to be a serious hazard to both humans and animals, they are still very important. Types A, B, and C influenza viruses are among the members of the Orthomyxoviridae family. A quick summary of the molecular factors is provided, which determine pathogenicity and clinical signs and symptoms of influenza. A review of host range and evolution highlights the genetic diversity of influenza A viruses and their capacity to successfully infect a variety of hosts, including avian and mammalian species. Moreover, influenza viruses may reassemble segments because of the way their segmented genome is designed. The significance of host sialic acid distribution and viral receptor-binding hemagglutinins in species-restricted virus binding is emphasized. It results in yearly outbreaks and necessitates the creation of novel vaccination formulations. This may ultimately result in the creation of a virus that can spread among humans and has unique antigenic qualities, perhaps sparking a pandemic. Current developments in our knowledge of the seasonality, transmission, and prevention of influenza viruses are outlined, along with their significance for halting the virus's spread. Journal of Monno Medical College June, 2024; 10 (1):43-51

Environment and diet shape the geography-specific Drosophila melanogaster microbiota composition.

The microbial communities of animals influence their hosts' evolution and wild fitness, but it is hard to predict and explain how the microbiota varies in wild animals. Here, we describe that the microbiota composition of wild Drosophila melanogaster can be ordered by temperature, humidity, geographic distance, diet decomposition, and diet type. We show how these determinants of microbiota variation can help explain lactic acid bacteria (LAB) abundance in the flies, including the rarity of LAB in some previous studies. Finally, we show that wild fly phenotypes segregate with the flies' diet and microbiota composition, illuminating links between the microbiota and host evolution. Together, these findings help explain how variation in microbiota compositions can shape an animal's life history.

Plant G × Microbial E: Plant Genotype Interaction with Soil Bacterial Community Shapes Rhizosphere Composition During Invasion

It is increasingly recognized that different genetic variants of hosts can uniquely shape their microbiomes. Invasive species often evolve in their introduced ranges, but little is known about the potential for their microbial associations to change during invasion as a result. We asked whether host genotype (G), microbial environment (E), or their interaction (G × E) affected the composition and diversity of host-associated microbiomes in Centaurea solstitialis (yellow starthistle), a Eurasian plant that is known to have evolved novel genotypes and phenotypes and to have altered microbial interactions, in its severe invasion of CA, USA. We conducted an experiment in which native and invading plant genotypes were inoculated with native and invaded range soil microbial communities. We used amplicon sequencing to characterize rhizosphere bacteria in both the experiment and the field soils from which they were derived. We found that native and invading plant genotypes accumulated different microbial associations at the family level in each soil community, often counter to differences in family abundance between soil communities. Root associations with potentially beneficial Streptomycetaceae were particularly interesting, as these were more abundant in the invaded range field soil and accumulated on invading genotypes. We also found that bacterial diversity is higher in invaded soils, but that invading genotypes accumulated a lower diversity of bacteria and unique microbial composition in experimental inoculations, relative to native genotypes. Thus variation in microbial associations of invaders was driven by the interaction of plant G and microbial E, and rhizosphere microbial communities appear to change in composition in response to host evolution during invasion.

Technical note: A biological anthropologist's guide for applying microbiome science to studies of human and non-human primates.

A central goal of biological anthropology is connecting environmental variation to differences in host physiology, biology, health, and evolution. The microbiome represents a valuable pathway for studying how variation in host environments impacts health outcomes. While there are many resources for learning about methods related to microbiome sample collection, laboratory analyses, and genetic sequencing, there are fewer dedicated to helping researchers navigate the dense portfolio of bioinformatics and statistical approaches for analyzing microbiome data. Those that do exist are rarely related to questions in biological anthropology and instead are often focused on human biomedicine. To address this gap, we expand on existing tutorials and provide a "road map" to aid biological anthropologists in understanding, selecting, and deploying the data analysis and visualization methods that are most appropriate for their specific research questions. Leveraging an existing dataset of fecal samples and survey data collected from wild geladas living in Simien Mountains National Park in Ethiopia (Baniel et al., 2021), this paper guides researchers toward answering three questions related to variation in the gut microbiome across host and environmental factors. By providing explanations, examples, and a reproducible workflow for different analytic methods, we move beyond the theoretical benefits of considering the microbiome within anthropological research and instead present researchers with a guide for applying microbiome science to their work. This paper makes microbiome science more accessible to biological anthropologists and paves the way for continued research into the microbiome's role in the ecology, evolution, and health of human and non-human primates.

Recent evolutionary origin and localized diversity hotspots of mammalian coronaviruses

Several coronaviruses infect humans, with three, including the SARS-CoV2, causing diseases. While coronaviruses are especially prone to induce pandemics, we know little about their evolutionary history, host-to-host transmissions, and biogeography. One of the difficulties lies in dating the origination of the family, a particularly challenging task for RNA viruses in general. Previous cophylogenetic tests of virus-host associations, including in the Coronaviridae family, have suggested a virus-host codiversification history stretching many millions of years. Here, we establish a framework for robustly testing scenarios of ancient origination and codiversification versus recent origination and diversification by host switches. Applied to coronaviruses and their mammalian hosts, our results support a scenario of recent origination of coronaviruses in bats and diversification by host switches, with preferential host switches within mammalian orders. Hotspots of coronavirus diversity, concentrated in East Asia and Europe, are consistent with this scenario of relatively recent origination and localized host switches. Spillovers from bats to other species are rare, but have the highest probability to be towards humans than to any other mammal species, implicating humans as the evolutionary intermediate host. The high host-switching rates within orders, as well as between humans, domesticated mammals, and non-flying wild mammals, indicates the potential for rapid additional spreading of coronaviruses across the world. Our results suggest that the evolutionary history of extant mammalian coronaviruses is recent, and that cases of long-term virus–host codiversification have been largely over-estimated.

Recent evolutionary origin and localized diversity hotspots of mammalian coronaviruses.

Several coronaviruses infect humans, with three, including the SARS-CoV2, causing diseases. While coronaviruses are especially prone to induce pandemics, we know little about their evolutionary history, host-to-host transmissions, and biogeography. One of the difficulties lies in dating the origination of the family, a particularly challenging task for RNA viruses in general. Previous cophylogenetic tests of virus-host associations, including in the Coronaviridae family, have suggested a virus-host codiversification history stretching many millions of years. Here, we establish a framework for robustly testing scenarios of ancient origination and codiversification versus recent origination and diversification by host switches. Applied to coronaviruses and their mammalian hosts, our results support a scenario of recent origination of coronaviruses in bats and diversification by host switches, with preferential host switches within mammalian orders. Hotspots of coronavirus diversity, concentrated in East Asia and Europe, are consistent with this scenario of relatively recent origination and localized host switches. Spillovers from bats to other species are rare, but have the highest probability to be towards humans than to any other mammal species, implicating humans as the evolutionary intermediate host. The high host-switching rates within orders, as well as between humans, domesticated mammals, and non-flying wild mammals, indicates the potential for rapid additional spreading of coronaviruses across the world. Our results suggest that the evolutionary history of extant mammalian coronaviruses is recent, and that cases of long-term virus-host codiversification have been largely over-estimated.

Host and venom evolution in parasitoid wasps: does independently adapting to the same host shape the evolution of the venom gland transcriptome?

BackgroundVenoms have repeatedly evolved over 100 occasions throughout the animal tree of life, making them excellent systems for exploring convergent evolutionary novelty. Growing evidence supports that venom evolution is predominantly driven by prey or host-related selection pressures, and the expression patterns of venom glands reflect adaptive evolution. However, it remains elusive whether the evolution of expression patterns in venom glands is likewise a convergent evolution driven by their prey/host species.ResultsWe utilized parasitoid wasps that had independently adapted to Drosophila hosts as models to investigate the convergent evolution of venom gland transcriptomes in 19 hymenopteran species spanning ~ 200 million years of evolution. Comparative transcriptome analysis reveals that the global expression patterns among the venom glands of Drosophila parasitoid wasps do not achieve higher similarity compared to non-Drosophila parasitoid wasps. Further evolutionary analyses of expression patterns at the single gene, orthogroup, and Gene Ontology (GO) term levels indicate that some orthogroups/GO terms show correlation with the Drosophila parasitoid wasps. However, these groups rarely include genes highly expressed in venom glands or putative venom genes in the Drosophila parasitoid wasps.ConclusionsOur study suggests that convergent evolution may not play a predominant force shaping gene expression levels in the venom gland of the Drosophila parasitoid wasps, offering novel insights into the co-evolution between venom and prey/host.

Bacterial Protein Signatures Identified in Porphyromonas gingivalis Containing-Autophagic Vacuoles Reveal Co-Evolution Between Oral Red/Orange Complex Bacteria and Gut Bacteria.

Modern oral bacterial species present as a concoction of commensal and opportunistic pathogens originating from their evolution in humans. Due to the intricate colonization mechanisms shared amongst oral and gut bacteria, these bacteria have likely evolved together to establish and adapt in the human oro-digestive tract, resulting in the transfer of genetic information. Our liquid chromatography-with-tandem-mass-spectrometry (LC-MS-MS) analyses have revealed protein signatures, Elongation Factor Tu, RagB/SusD nutrient uptake outer membrane protein and DnaK, specifically from Porphyromonas gingivalis -containing autophagic vacuoles isolated from the infected human primary gingival epithelial cells. Interestingly, our Mass-Spectrometry analysis reported similar proteins from closely related oral bacteria, Tannerella forsythia and Prevotella intermedia . In our phylogenetic study of these key protein signatures, we have established that pathogenic oral bacteria share extensive relatedness to each other and gut resident bacteria. We show that in the virulence factors identified from gut bacteria, Elongation Factor Tu and DnaK, there are several structural similarities and conservations with proteins from oral pathogenic bacteria. There are also major similarities in the RagB/SusD proteins of oral bacteria to prominent gut bacteria. These findings not only highlight the shared virulence mechanisms amongst oral bacterial pathogens/pathobionts but also gut bacteria and elucidate their co-evolutions in the human host.

'Drifting' Buchnera genomes track the microevolutionary trajectories of their aphid hosts.

Evolution of Buchnera-aphid host symbioses is often studied among species at macroevolutionary scales. Investigations within species offer a different perspective about how eco-evolutionary processes shape patterns of genetic variation at microevolutionary scales. Our study leverages new and publicly available whole-genome sequencing data to study Buchnera-aphid host evolution in Myzus persicae, the peach potato aphid, a globally invasive and polyphagous pest. Across 43 different asexual, clonally reproducing isofemale strains, we examined patterns of genomic covariation between Buchnera and their aphid host and considered the distribution of mutations in protein-coding regions of the Buchnera genome. We found Buchnera polymorphisms within aphid strains, suggesting the presence of genetically different Buchnera strains within the same clonal lineage. Genetic distance between pairs of Buchnera samples was positively correlated to genetic distance between their aphid hosts, indicating shared evolutionary histories. However, there was no segregation of genetic variation for both M. persicae and Buchnera with plant host (Brassicaceae and non-tobacco Solanaceae) and no associations between genetic and geographic distance at global or regional spatial scales. Abundance patterns of non-synonymous mutations were similar to synonymous mutations in the Buchnera genome, and both mutation classes had similar site frequency spectra. We hypothesize that a predominance of neutral processes results in the Buchnera of M. persicae to simply 'drift' with the evolutionary trajectory of their aphid hosts. Our study presents a unique microevolutionary characterization of Buchnera-aphid host genomic covariation across multiple aphid clones. This provides a new perspective on the eco-evolutionary processes generating and maintaining polymorphisms in a major pest aphid species and its obligate primary endosymbiont.

Domestication and evolutionary histories of specialized gut symbionts across cephalotine ants.

The evolution of animals and their gut symbionts is a complex phenomenon, obscured by lability and diversity. In social organisms, transmission of symbionts among relatives may yield systems with more stable associations. Here, we study the history of a social insect symbiosis involving cephalotine ants and their extracellular gut bacteria, which come predominantly from host-specialized lineages. We perform multi-locus phylogenetics for symbionts from nine bacterial orders, and map prior amplicon sequence data to lineage-assigned symbiont genomes, studying distributions of rigorously defined symbionts across 20 host species. Based on monophyly and additional hypothesis testing, we estimate that these specialized gut bacteria belong to 18 distinct lineages, of which 15 have been successfully isolated and cultured. Several symbiont lineages showed evidence for domestication events that occurred later in cephalotine evolutionary history, and only one lineage was ubiquitously detected in all 20 host species and 48 colonies sampled with amplicon 16S rRNA sequencing. We found evidence for phylogenetically constrained distributions in four symbionts, suggesting historical or genetic impacts on community composition. Two lineages showed evidence for frequent intra-lineage co-infections, highlighting the potential for niche divergence after initial domestication. Nearly all symbionts showed evidence for occasional host switching, but four may, more often, co-diversify with their hosts. Through our further assessment of symbiont localization and genomic functional profiles, we demonstrate distinct niches for symbionts with shared evolutionary histories, prompting further questions on the forces underlying the evolution of hosts and their gut microbiomes.

Methanotrophic Methanoperedens archaea host diverse and interacting extrachromosomal elements.

Methane emissions are mitigated by anaerobic methane-oxidizing archaea, including Methanoperedens. Some Methanoperedens host huge extrachromosomal genetic elements (ECEs) called Borgs that may modulate their activity, yet the broader diversity of Methanoperedens ECEs is understudied. Here we report small enigmatic linear ECEs, circular viruses and unclassified ECEs that are predicted to replicate within Methanoperedens. Linear ECEs have inverted terminal repeats, tandem repeats and coding patterns that are strongly reminiscent of Borgs, but they are only 52-145 kb in length. As they share proteins with Borgs and Methanoperedens, we refer to them as mini-Borgs. Mini-Borgs are genetically diverse and can be assigned to at least five family-level groups. We identify eight families of Methanoperedens viruses, some of which encode multi-haem cytochromes, and circular ECEs encoding transposon-associated TnpB genes with proximal population-heterogeneous CRISPR arrays. These ECEs exchange genetic information with each other and with Methanoperedens, probably impacting their archaeal host activity and evolution.

Experimental Evolution Studies in Φ6 Cystovirus.

Experimental evolution studies, in which biological populations are evolved in a specific environment over time, can address questions about the nature of spontaneous mutations, responses to selection, and the origins and maintenance of novel traits. Here, we review more than 30 years of experimental evolution studies using the bacteriophage (phage) Φ6 cystovirus. Similar to many lab-studied bacteriophages, Φ6 has a high mutation rate, large population size, fast generation time, and can be genetically engineered or cryogenically frozen, which facilitates its rapid evolution in the laboratory and the subsequent characterization of the effects of its mutations. Moreover, its segmented RNA genome, outer membrane, and capacity for multiple phages to coinfect a single host cell make Φ6 a good non-pathogenic model for investigating the evolution of RNA viruses that infect humans. We describe experiments that used Φ6 to address the fitness effects of spontaneous mutations, the consequences of evolution in the presence of coinfection, the evolution of host ranges, and mechanisms and consequences of the evolution of thermostability. We highlight open areas of inquiry where further experimentation on Φ6 could inform predictions for pathogenic viruses.

Prevalence and underlying mechanisms of phylosymbiosis in land plants

Abstract Phylosymbiosis, the congruence of microbiome composition with host phylogeny, is a valuable framework for investigating plant–microbe associations and their evolutionary ecology. This review assesses the prevalence of phylosymbiosis across the plant kingdom, elucidates the fundamental ecological and evolutionary processes contributing to its occurrence based on previous research and explores commonly used methods for identifying phylosymbiosis. We find that the presence of phylosymbiosis may be influenced by both phylogenetic distance and the taxonomic level at which host plants are examined, with the strength of associations potentially decreasing as the taxonomic scale becomes finer. Notably, the endophytic microbiome exhibits a stronger phylosymbiosis signal compared with the epiphytic or rhizosphere-associated microbiomes. Microorganisms such as fungi and bacteria can yield highly variable evidence for phylosymbiosis due to differences in colonization, transmission or functional characteristics. We also outline how the four community assembly processes (dispersal, selection, diversification and drift) contribute to the establishment and maintenance of host–microbe phylosymbiosis. Furthermore, we highlight the diversity of methods employed to detect phylosymbiosis, which involves three key processes: constructing host phylogenies, assessing microbial data and statistically evaluating the correlation between host phylogeny and microbial composition. Remarkably different methodologies across studies make comparisons between findings challenging. To advance our understanding, future research is expected to explore phylosymbiosis at lower taxonomic levels and investigate different microbial communities coexisting synergistically within the same host. Understanding the relative importance of community assembly processes in driving phylosymbiosis will be critical for gaining deeper insights into the ecology and evolution of host–microbe interactions.

Viruses shuttle between fungi and plants

The intimate relationships between plants and fungi provide an opportunity for the shuttling of viruses. Dai et al. recently discovered that a virus undergoes cross-kingdom transmission, and naturally spreads to both plant and fungal populations. This finding expands our understanding of viral host range, evolution, transmission, and disease management.

High parasite diversity maintained after an alga-virus coevolutionary arms race.

Arms race dynamics are a common outcome of host-parasite coevolution. While they can theoretically be maintained indefinitely, realistic arms races are expected to be finite. Once an arms race has ended, for example due to the evolution of a generalist-resistant host, the system may transition into coevolutionary dynamics that favour long-term diversity. In microbial experiments, host-parasite arms races often transition into a stable coexistence of generalist-resistant hosts, (semi-)susceptible hosts, and parasites. While long-term host diversity is implicit in these cases, parasite diversity is usually overlooked. In this study, we examined parasite diversity after the end of an experimental arms race between a unicellular alga (Chlorella variabilis) and its lytic virus (PBCV-1). First, we isolated virus genotypes from multiple time points from two replicate microcosms. A time-shift experiment confirmed that the virus isolates had escalating host ranges, i.e., that arms races had occurred. We then examined the phenotypic and genetic diversity of virus isolates from the post-arms race phase. Post-arms race virus isolates had diverse host ranges, survival probabilities, and growth rates; they also clustered into distinct genetic groups. Importantly, host range diversity was maintained throughout the post-arms race phase, and the frequency of host range phenotypes fluctuated over time. We hypothesize that this dynamic polymorphism was maintained by a combination of fluctuating selection and demographic stochasticity. Together with previous work in prokaryotic systems, our results link experimental observations of arms races to natural observations of long-term host and parasite diversity.

Influence of sipunculan (peanut worm) activity on orifice formation in scleractinian Heterocyathus for adaptation to soft substrates

Mutualism profoundly affects the morphology and ecological evolution of both hosts and symbionts involved. Heterocyathus is a solitary scleractinian coral that lives on soft substrata, and sipunculan worms live symbiotically in the tube-like cavities (orifice) inside the coral skeletons. This habitat provides protection to the sipunculan worms against predators and—owing to the mobility of the worms—prevents the coral from being buried with sediments. The orifice growth is closely related to the symbiont sipunculan worms; however, this has not been previously elucidated. Here, we clarified the growth process of scleractinian coral orifices and the influence of sipunculan activity on this. The orifices were originally formed by rapid accretion deposits. The coral soft tissue enveloping the growth edge of the orifice repeatedly retreated to the outer side due to direct damage to the soft part and/or excessive stress caused by the rubbing of the sipunculan through locomotion, excretion, and feeding behaviour. This resulted in a toppled-domino microskeletal structure appearance and maintenance of the orifice growth. These outcomes demonstrate the first example of the direct influence of symbionts on the skeletal morphogenesis of scleractinian corals. The mutualism between the two organisms is maintained by the beneficial confrontation in forming orifices.

Symbiont Diversity in Imidacloprid-Resistant and Imidacloprid-Susceptible Populations of Nilaparvata lugens (Hemiptera: Delphacidae)1

Abstract Imidacloprid, a neonicotinoid insecticide, is efficacious against hemipterans, including the brown planthopper, Nilaparvata lugens (Stål) (Hemiptera: Delphacidae). However, frequent use of this insecticide has resulted in the development of high levels of resistance among brown planthopper populations. Endosymbionts of insects have contributed to host physiology and evolution and play a role in resistance to chemical toxins. In this study, polymerase chain reaction–denaturing gradient gel electrophoresis was used to analyze the bacterial and yeast-like symbiont communities of imidacloprid-resistant and -susceptible brown planthopper populations. The Shannon-Weaver diversity index and the evenness index indicated no differences in the richness and the expression of overall species distribution of the symbiotic communities of resistant and susceptible populations. The similarity coefficients of 0.53 and 0.56 for bacterial and yeast-like symbionts, respectively, indicated the main types of differences among microorganisms in resistant and susceptible populations. Sequence comparison analysis indicated the bacterial species in the susceptible population were members of the Enterobacteriaceae and Moraxellaceae, and those in the resistant population were members of the Enterobacteriaceae, Oxalobacteriaceae, Rhodobacteriaceae, and Sphingomonadaceae. Differences also were found in the composition of yeast-like symbionts of the two populations; Cryptococcus luteolus, Pseudozyma aphidis, and Pseudozyma antarctica were detected in the susceptible population, and Cladosporium perangustum was detected in the resistant population.