Draft genome sequence of Wigglesworthia glossinidia "palpalis gambiensis" isolate.

The 0.719 Mb genome of the tsetse endosymbiont, Wigglesworthia glossinidia, from Glossina palpalis gambiensis is presented. This Wigglesworthia genome retains 611 protein-coding sequences and a 25.3% GC content. A cryptic plasmid is conserved, between Wigglesworthia isolates, suggesting functional significance. This genome adds a further dimension to characterize Wigglesworthia lineage-based differences.

Symbiosis preservation: Putative regulation of fatty acyl-CoA reductase by miR-31a within the symbiont harboring bacteriome through tsetse evolution.

Tsetse flies are the sole vectors of African trypanosomes. In addition to trypanosomes, tsetse harbor obligate Wigglesworthia glossinidia bacteria that are essential to tsetse biology. The absence of Wigglesworthia results in fly sterility, thus offering promise for population control strategies. Here, microRNA (miRNAs) and mRNA expression are characterized and compared between the exclusive Wigglesworthia-containing bacteriome and adjacent aposymbiotic tissue in females of two evolutionarily distant tsetse species (Glossina brevipalpis and G. morsitans). A total of 193 miRNAs were expressed in either species, with 188 of these expressed in both species, 166 of these were novel to Glossinidae, and 41 miRNAs exhibited comparable expression levels between species. Within bacteriomes, 83 homologous mRNAs demonstrated differential expression between G. morsitans aposymbiotic and bacteriome tissues, with 21 of these having conserved interspecific expression. A large proportion of these differentially expressed genes are involved in amino acid metabolism and transport, symbolizing the essential nutritional role of the symbiosis. Further bioinformatic analyses identified a sole conserved miRNA::mRNA interaction (miR-31a::fatty acyl-CoA reductase) within bacteriomes likely catalyzing the reduction of fatty acids to alcohols which comprise components of esters and lipids involved in structural maintenance. The Glossina fatty acyl-CoA reductase gene family is characterized here through phylogenetic analyses to further understand its evolutionary diversification and the functional roles of members. Further research to characterize the nature of the miR-31a::fatty acyl-CoA reductase interaction may find novel contributions to the symbiosis to be exploited for vector control.

Microbe Profile: Wigglesworthia glossinidia: the tsetse fly's significant other.

Wigglesworthia glossinidia is an obligate, maternally transmitted endosymbiont of tsetse flies. The ancient association between these two organisms accounts for many of their unique physiological adaptations. Similar to other obligate mutualists, Wigglesworthia's genome is dramatically reduced in size, yet it has retained the capacity to produce many B-vitamins that are found at inadequate quantities in the fly's vertebrate blood-specific diet. These Wigglesworthia-derived B-vitamins play essential nutritional roles to maintain tsetse's physiological homeostasis as well as that of other members of the fly's microbiota. In addition to its nutritional role, Wigglesworthia contributes towards the development of tsetse's immune system during the larval period. Tsetse produce amidases that degrade symbiotic peptidoglycans and prevent activation of antimicrobial responses that can damage Wigglesworthia. These amidases in turn exhibit antiparasitic activity and decrease tsetse's ability to be colonized with parasitic trypanosomes, which reduce host fitness. Thus, the Wigglesworthia symbiosis represents a fine-tuned association in which both partners actively contribute towards achieving optimal fitness outcomes.

The Tsetse Metabolic Gambit: Living on Blood by Relying on Symbionts Demands Synchronization.

Tsetse flies have socioeconomic significance as the obligate vector of multiple Trypanosoma parasites, the causative agents of Human and Animal African Trypanosomiases. Like many animals subsisting on a limited diet, microbial symbiosis is key to supplementing nutrient deficiencies necessary for metabolic, reproductive, and immune functions. Extensive studies on the microbiota in parallel to tsetse biology have unraveled the many dependencies partners have for one another. But far less is known mechanistically on how products are swapped between partners and how these metabolic exchanges are regulated, especially to address changing physiological needs. More specifically, how do metabolites contributed by one partner get to the right place at the right time and in the right amounts to the other partner? Epigenetics is the study of molecules and mechanisms that regulate the inheritance, gene activity and expression of traits that are not due to DNA sequence alone. The roles that epigenetics provide as a mechanistic link between host phenotype, metabolism and microbiota (both in composition and activity) is relatively unknown and represents a frontier of exploration. Here, we take a closer look at blood feeding insects with emphasis on the tsetse fly, to specifically propose roles for microRNAs (miRNA) and DNA methylation, in maintaining insect-microbiota functional homeostasis. We provide empirical details to addressing these hypotheses and advancing these studies. Deciphering how microbiota and host activity are harmonized may foster multiple applications toward manipulating host health, including identifying novel targets for innovative vector control strategies to counter insidious pests such as tsetse.

The holobiont transcriptome of teneral tsetse fly species of varying vector competence.

BACKGROUND: Tsetse flies are the obligate vectors of African trypanosomes, which cause Human and Animal African Trypanosomiasis. Teneral flies (newly eclosed adults) are especially susceptible to parasite establishment and development, yet our understanding of why remains fragmentary. The tsetse gut microbiome is dominated by two Gammaproteobacteria, an essential and ancient mutualist Wigglesworthia glossinidia and a commensal Sodalis glossinidius. Here, we characterize and compare the metatranscriptome of teneral Glossina morsitans to that of G. brevipalpis and describe unique immunological, physiological, and metabolic landscapes that may impact vector competence differences between these two species. RESULTS: An active expression profile was observed for Wigglesworthia immediately following host adult metamorphosis. Specifically, 'translation, ribosomal structure and biogenesis' followed by 'coenzyme transport and metabolism' were the most enriched clusters of orthologous genes (COGs), highlighting the importance of nutrient transport and metabolism even following host species diversification. Despite the significantly smaller Wigglesworthia genome more differentially expressed genes (DEGs) were identified between interspecific isolates (n = 326, ~ 55% of protein coding genes) than between the corresponding Sodalis isolates (n = 235, ~ 5% of protein coding genes) likely reflecting distinctions in host co-evolution and adaptation. DEGs between Sodalis isolates included genes involved in chitin degradation that may contribute towards trypanosome susceptibility by compromising the immunological protection provided by the peritrophic matrix. Lastly, G. brevipalpis tenerals demonstrate a more immunologically robust background with significant upregulation of IMD and melanization pathways. CONCLUSIONS: These transcriptomic differences may collectively contribute to vector competence differences between tsetse species and offers translational relevance towards the design of novel vector control strategies.

Quorum sensing sets the stage for the establishment and vertical transmission of Sodalis praecaptivus in tsetse flies.

Bacterial virulence factors facilitate host colonization and set the stage for the evolution of parasitic and mutualistic interactions. The Sodalis-allied clade of bacteria exhibit striking diversity in the range of both plant and animal feeding insects they inhabit, suggesting the appropriation of universal molecular mechanisms that facilitate establishment. Here, we report on the infection of the tsetse fly by free-living Sodalis praecaptivus, a close relative of many Sodalis-allied symbionts. Key genes involved in quorum sensing, including the homoserine lactone synthase (ypeI) and response regulators (yenR and ypeR) are integral for the benign colonization of S. praecaptivus. Mutants lacking ypeI, yenR and ypeR compromised tsetse survival as a consequence of their inability to repress virulence. Genes under quorum sensing, including homologs of the binary insecticidal toxin PirAB and a putative symbiosis-promoting factor CpmAJ, demonstrated negative and positive impacts, respectively, on tsetse survival. Taken together with results obtained from experiments involving weevils, this work shows that quorum sensing virulence suppression plays an integral role in facilitating the establishment of Sodalis-allied symbionts in diverse insect hosts. This knowledge contributes to the understanding of the early evolutionary steps involved in the formation of insect-bacterial symbiosis. Further, despite having no established history of interaction with tsetse, S. praecaptivus can infect reproductive tissues, enabling vertical transmission through adenotrophic viviparity within a single host generation. This creates an option for the use of S. praecaptivus in the biocontrol of insect disease vectors via paratransgenesis.

Diversity and function of fungi associated with the fungivorous millipede, Brachycybe lecontii.

Fungivorous millipedes (subterclass Colobognatha) likely represent some of the earliest known mycophagous terrestrial arthropods, yet their fungal partners remain elusive. Here we describe relationships between fungi and the fungivorous millipede, Brachycybe lecontii. Their fungal community is surprisingly diverse, including 176 genera, 39 orders, four phyla, and several undescribed species. Of particular interest are twelve genera conserved across wood substrates and millipede clades that comprise the core fungal community of B. lecontii. Wood decay fungi, long speculated to serve as the primary food source for Brachycybe species, were absent from this core assemblage and proved lethal to millipedes in pathogenicity assays while entomopathogenic Hypocreales were more common in the core but had little effect on millipede health. This study represents the first survey of fungal communities associated with any colobognath millipede, and these results offer a glimpse into the complexity of millipede fungal communities.

Comparative genomic analysis of six Glossina genomes, vectors of African trypanosomes.

BACKGROUND: Tsetse flies (Glossina sp.) are the vectors of human and animal trypanosomiasis throughout sub-Saharan Africa. Tsetse flies are distinguished from other Diptera by unique adaptations, including lactation and the birthing of live young (obligate viviparity), a vertebrate blood-specific diet by both sexes, and obligate bacterial symbiosis. This work describes the comparative analysis of six Glossina genomes representing three sub-genera: Morsitans (G. morsitans morsitans, G. pallidipes, G. austeni), Palpalis (G. palpalis, G. fuscipes), and Fusca (G. brevipalpis) which represent different habitats, host preferences, and vectorial capacity. RESULTS: Genomic analyses validate established evolutionary relationships and sub-genera. Syntenic analysis of Glossina relative to Drosophila melanogaster shows reduced structural conservation across the sex-linked X chromosome. Sex-linked scaffolds show increased rates of female-specific gene expression and lower evolutionary rates relative to autosome associated genes. Tsetse-specific genes are enriched in protease, odorant-binding, and helicase activities. Lactation-associated genes are conserved across all Glossina species while male seminal proteins are rapidly evolving. Olfactory and gustatory genes are reduced across the genus relative to other insects. Vision-associated Rhodopsin genes show conservation of motion detection/tracking functions and variance in the Rhodopsin detecting colors in the blue wavelength ranges. CONCLUSIONS: Expanded genomic discoveries reveal the genetics underlying Glossina biology and provide a rich body of knowledge for basic science and disease control. They also provide insight into the evolutionary biology underlying novel adaptations and are relevant to applied aspects of vector control such as trap design and discovery of novel pest and disease control strategies.

Mutualist-Provisioned Resources Impact Vector Competency.

Many symbionts supplement their host's diet with essential nutrients. However, whether these nutrients also enhance parasitism is unknown. In this study, we investigated whether folate (vitamin B9) production by the tsetse fly (Glossina spp.) essential mutualist, Wigglesworthia, aids auxotrophic African trypanosomes in completing their life cycle within this obligate vector. We show that the expression of Wigglesworthia folate biosynthesis genes changes with the progression of trypanosome infection within tsetse. The disruption of Wigglesworthia folate production caused a reduction in the percentage of flies that housed midgut (MG) trypanosome infections. However, decreased folate did not prevent MG trypanosomes from migrating to and establishing an infection in the fly's salivary glands, thus suggesting that nutrient requirements vary throughout the trypanosome life cycle. We further substantiated that trypanosomes rely on symbiont-generated folate by feeding this vitamin to Glossina brevipalpis, which exhibits low trypanosome vector competency and houses Wigglesworthia incapable of producing folate. Folate-supplemented G. brevipalpis flies were significantly more susceptible to trypanosome infection, further demonstrating that this vitamin facilitates parasite infection establishment. Our cumulative results provide evidence that Wigglesworthia provides a key metabolite (folate) that is "hijacked" by trypanosomes to enhance their infectivity, thus indirectly impacting tsetse species vector competency. Parasite dependence on symbiont-derived micronutrients, which likely also occurs in other arthropod vectors, represents a relationship that may be exploited to reduce disease transmission.IMPORTANCE Parasites elicit several physiological changes in their host to enhance transmission. Little is known about the functional association between parasitism and microbiota-provisioned resources typically dedicated to animal hosts and how these goods may be rerouted to optimize parasite development. This study is the first to identify a specific symbiont-generated metabolite that impacts insect vector competence by facilitating parasite establishment and, thus, eventual transmission. Specifically, we demonstrate that the tsetse fly obligate mutualist Wigglesworthia provisions folate (vitamin B9) that pathogenic African trypanosomes exploit in an effort to successfully establish an infection in the vector's MG. This process is essential for the parasite to complete its life cycle and be transmitted to a new vertebrate host. Disrupting metabolic contributions provided by the microbiota of arthropod disease vectors may fuel future innovative control strategies while also offering minimal nontarget effects.

Into the Wild: Parallel Transcriptomics of the Tsetse-Wigglesworthia Mutualism within Kenyan Populations.

Tsetse flies (Diptera: Glossinidae) have medical significance as the obligate vectors of African trypanosomes. In addition, tsetse harbor a simple gut microbiota. A predominant gut microbiota member, the Gammaproteobacterium Wigglesworthia spp., has coevolved with tsetse for a significant portion of Glossina radiation proving critical to tsetse fitness. Although multiple roles have been described for Wigglesworthia within colony flies, little research has been dedicated towards functional characterization within wild tsetse. Here, dual RNA-Seq was performed to characterize the tsetse-Wigglesworthia symbiosis within flies captured in Nguruman, Kenya. A significant correlation in Gene Ontology (GO) distribution between tsetse and Wigglesworthia was observed, with homogeneous enrichment in metabolic and transport categories, likely supporting a hallmark of the symbiosis-bidirectional metabolic exchange. Within field flies, highly transcribed Wigglesworthia loci included those involved in B vitamin synthesis and in substrate translocation, including amino acid transporters and multidrug efflux pumps, providing a molecular means for interaction. The universal expression of several Wigglesworthia and G. pallidipes orthologs, putatively involved in nutrient provisioning and resource allocation, was confirmed in sister tsetse species. These transcriptional profiles varied through host age and mating status likely addressing varying symbiont demands and also confirming their global importance within Glossina. This study, not only supports symbiont nutrient provisioning roles, but also serves as a foundation for insight into novel roles and molecular mechanisms associated with vector-microbiota interactions. The role of symbiont B vitamin provisioning towards impacting host epigenetics is discussed. Knowledge of vector-microbiota interactions may lead to the discovery of novel targets in pest control.

Don't Bite the Hand that Feeds You.

Eukaryotic-bacterial symbioses are ubiquitous in nature. Pathogens and symbionts employ similar machinery, yet symbionts can minimize host damage. In this issue of Cell Host & Microbe, Enomoto et al. (2017) demonstrate how quorum sensing regulates expression of virulence genes at appropriate times, thereby enabling symbiont retention throughout the host lifespan.

Grandeur Alliances: Symbiont Metabolic Integration and Obligate Arthropod Hematophagy.

Several arthropod taxa live exclusively on vertebrate blood. This food source lacks essential metabolites required for the maintenance of metabolic homeostasis, and as such, these arthropods have formed symbioses with nutrient-supplementing microbes that facilitate their host's 'hematophagous' feeding ecology. Herein we highlight metabolic contributions of bacterial symbionts that reside within tsetse flies, bed bugs, lice, reduviid bugs, and ticks, with specific emphasis on B vitamin and cofactor biosynthesis. Importantly, these arthropods can transmit pathogens of medical and veterinary relevance and/or cause infestations that induce psychological and dermatological distress. Microbial metabolites, and the biochemical pathways that generate them, can serve as specific targets of novel control mechanisms aimed at disrupting the metabolism of hematophagous arthropods, thus combatting pest invasion and vector-borne pathogen transmission.

A Tale of Transmission: Aeromonas veronii Activity within Leech-Exuded Mucus.

Transmission, critical to the establishment and persistence of host-associated microbiotas, also exposes symbionts to new environmental conditions. With horizontal transmission, these different conditions represent major lifestyle shifts. Yet genome-wide analyses of how microbes adjust their transcriptomes toward these dramatic shifts remain understudied. Here, we provide a comprehensive and comparative analysis of the global transcriptional profiles of a symbiont as it shifts between lifestyles during transmission. The gammaproteobacterium Aeromonas veronii is transmitted from the gut of the medicinal leech to other hosts via host mucosal castings, yet A. veronii can also transition from mucosal habitancy to a free-living lifestyle. These three lifestyles are characterized by distinct physiological constraints and consequently lifestyle-specific changes in the expression of stress-response genes. Mucus-bound A. veronii had the greatest expression in terms of both the number of loci and levels of transcription of stress-response mechanisms. However, these bacteria are still capable of proliferating within the mucus, suggesting the availability of nutrients within this environment. We found that A. veronii alters transcription of loci in a synthetic pathway that obtains and incorporates N-acetylglucosamine (NAG; a major component of mucus) into the bacterial cell wall, enabling proliferation. Our results demonstrate that symbionts undergo dramatic local adaptation, demonstrated by widespread transcriptional changes, throughout the process of transmission that allows them to thrive while they encounter new environments which further shape their ecology and evolution.

Draft Genome Sequence of Pedobacter sp. Strain Hv1, an Isolate from Medicinal Leech Mucosal Castings.

The Pedobacter sp. Hv1 strain was isolated from the medicinal leech, Hirudo verbana, mucosal castings. These mucosal sheds have been demonstrated to play a role in horizontal symbiont transmission. Here, we report the draft 4.9 Mbp genome sequence of Pedobacter sp. strain Hv1.

A Unique Set of the Burkholderia Collagen-Like Proteins Provides Insight into Pathogenesis, Genome Evolution and Niche Adaptation, and Infection Detection.

Burkholderia pseudomallei and Burkholderia mallei, classified as category B priority pathogens, are significant human and animal pathogens that are highly infectious and broad-spectrum antibiotic resistant. Currently, the pathogenicity mechanisms utilized by Burkholderia are not fully understood, and correct diagnosis of B. pseudomallei and B. mallei infection remains a challenge due to limited detection methods. Here, we provide a comprehensive analysis of a set of 13 novel Burkholderia collagen-like proteins (Bucl) that were identified among B. pseudomallei and B. mallei select agents. We infer that several Bucl proteins participate in pathogenesis based on their noncollagenous domains that are associated with the components of a type III secretion apparatus and membrane transport systems. Homology modeling of the outer membrane efflux domain of Bucl8 points to a role in multi-drug resistance. We determined that bucl genes are widespread in B. pseudomallei and B. mallei; Fischer's exact test and Cramer's V2 values indicate that the majority of bucl genes are highly associated with these pathogenic species versus nonpathogenic B. thailandensis. We designed a bucl-based quantitative PCR assay which was able to detect B. pseudomallei infection in a mouse with a detection limit of 50 CFU. Finally, chromosomal mapping and phylogenetic analysis of bucl loci revealed considerable genomic plasticity and adaptation of Burkholderia spp. to host and environmental niches. In this study, we identified a large set of phylogenetically unrelated bucl genes commonly found in Burkholderia select agents, encoding predicted pathogenicity factors, detection targets, and vaccine candidates.

"Wigglesworthia morsitans" Folate (Vitamin B9) Biosynthesis Contributes to Tsetse Host Fitness.

Closely related ancient endosymbionts may retain minor genomic distinctions through evolutionary time, yet the biological relevance of these small pockets of unique loci remains unknown. The tsetse fly (Diptera: Glossinidae), the sole vector of lethal African trypanosomes (Trypanosoma spp.), maintains an ancient and obligate mutualism with species belonging to the gammaproteobacterium Wigglesworthia. Extensive concordant evolution with associated Wigglesworthia species has occurred through tsetse species radiation. Accordingly, the retention of unique symbiont loci between Wigglesworthia genomes may prove instrumental toward host species-specific biological traits. Genome distinctions between "Wigglesworthia morsitans" (harbored within Glossina morsitans bacteriomes) and the basal species Wigglesworthia glossinidia (harbored within Glossina brevipalpis bacteriomes) include the retention of chorismate and downstream folate (vitamin B9) biosynthesis capabilities, contributing to distinct symbiont metabolomes. Here, we demonstrate that these W. morsitans pathways remain functionally intact, with folate likely being systemically disseminated through a synchronously expressed tsetse folate transporter within bacteriomes. The folate produced by W. morsitans is demonstrated to be pivotal for G. morsitans sexual maturation and reproduction. Modest differences between ancient symbiont genomes may still play key roles in the evolution of their host species, particularly if loci are involved in shaping host physiology and ecology. Enhanced knowledge of the Wigglesworthia-tsetse mutualism may also provide novel and specific avenues for vector control.

Expansion of the APC superfamily of secondary carriers.

The amino acid-polyamine-organoCation (APC) superfamily is the second largest superfamily of secondary carriers currently known. In this study, we establish homology between previously recognized APC superfamily members and proteins of seven new families. These families include the PAAP (Putative Amino Acid Permease), LIVCS (Branched Chain Amino Acid:Cation Symporter), NRAMP (Natural Resistance-Associated Macrophage Protein), CstA (Carbon starvation A protein), KUP (K⁺ Uptake Permease), BenE (Benzoate:H⁺ Virginia Symporter), and AE (Anion Exchanger). The topology of the well-characterized human Anion Exchanger 1 (AE1) conforms to a UraA-like topology of 14 TMSs (12 α-helical TMSs and 2 mixed coil/helical TMSs). All functionally characterized members of the APC superfamily use cation symport for substrate accumulation except for some members of the AE family which frequently use anion:anion exchange. We show how the different topologies fit into the framework of the common LeuT-like fold, defined earlier (Proteins. 2014 Feb;82(2):336-46), and determine that some of the new members contain previously undocumented topological variations. All new entries contain the two 5 or 7 TMS APC superfamily repeat units, sometimes with extra TMSs at the ends, the variations being greatest within the CstA family. New, functionally characterized members transport amino acids, peptides, and inorganic anions or cations. Except for anions, these are typical substrates of established APC superfamily members. Active site TMSs are rich in glycyl residues in variable but conserved constellations. This work expands the APC superfamily and our understanding of its topological variations.

Hitchhiking of host biology by beneficial symbionts enhances transmission.

Transmission plays a key role in the evolution of symbiosis. Mixed mode transmission combines horizontal and vertical mechanisms for symbiont acquisition. However, features that enable mixed transmission are poorly understood. Here, we determine the mechanistic basis for the recruitment of the beneficial bacterium, Aeromonas veronii by the leech, Hirudo verbana. We demonstrate that host mucosal secretions complement imperfect symbiont vertical transmission. First, we show that the A. veronii population within secretions originates from the host digestive tract and proliferates synchronously with shedding frequency, demonstrating the coupling of partner biology. Furthermore, leeches are attracted to these castings with oral contact proving sufficient for symbiont transmission. Leech attraction to mucus is not affected by the symbiont state of either the host or mucus, suggesting that A. veronii exploits preexisting host behavior and physiological traits. A dual transmission mode, integrating multiple layers of host contributions, may prove evolutionarily advantageous for a wide range of symbioses. Using such a strategy, host infection is ensured, while also providing access to a higher genetic diversity of symbionts. Countless host-associated microbes exhibit mixed mode transmission, supporting the use of the leech symbiosis as a model for enhancing our understanding of the specificity, establishment and persistence of microbiotas.

Characterization of shed medicinal leech mucus reveals a diverse microbiota.

Microbial transmission through mucosal-mediated mechanisms is widespread throughout the animal kingdom. One example of this occurs with Hirudo verbana, the medicinal leech, where host attraction to shed conspecific mucus facilitates horizontal transmission of a predominant gut symbiont, the Gammaproteobacterium Aeromonas veronii. However, whether this mucus may harbor other bacteria has not been examined. Here, we characterize the microbiota of shed leech mucus through Illumina deep sequencing of the V3-V4 hypervariable region of the 16S rRNA gene. Additionally, Restriction Fragment Length Polymorphism (RFLP) typing with subsequent Sanger Sequencing of a 16S rRNA gene clone library provided qualitative confirmation of the microbial composition. Phylogenetic analyses of full-length 16S rRNA sequences were performed to examine microbial taxonomic distribution. Analyses using both technologies indicate the dominance of the Bacteroidetes and Proteobacteria phyla within the mucus microbiota. We determined the presence of other previously described leech symbionts, in addition to a number of putative novel leech-associated bacteria. A second predominant gut symbiont, the Rikenella-like bacteria, was also identified within mucus and exhibited similar population dynamics to A. veronii, suggesting persistence in syntrophy beyond the gut. Interestingly, the most abundant bacterial genus belonged to Pedobacter, which includes members capable of producing heparinase, an enzyme that degrades the anticoagulant, heparin. Additionally, bacteria associated with denitrification and sulfate cycling were observed, indicating an abundance of these anions within mucus, likely originating from the leech excretory system. A diverse microbiota harbored within shed mucus has significant potential implications for the evolution of microbiomes, including opportunities for gene transfer and utility in host capture of a diverse group of symbionts.