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.

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 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.

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.

"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.

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.

Interwoven biology of the tsetse holobiont.

Microbial symbionts can be instrumental to the evolutionary success of their hosts. Here, we discuss medically significant tsetse flies (Diptera: Glossinidae), a group comprised of over 30 species, and their use as a valuable model system to study the evolution of the holobiont (i.e., the host and associated microbes). We first describe the tsetse microbiota, which, despite its simplicity, harbors a diverse range of associations. The maternally transmitted microbes consistently include two Gammaproteobacteria, the obligate mutualists Wigglesworthia spp. and the commensal Sodalis glossinidius, along with the parasitic Alphaproteobacteria Wolbachia. These associations differ in their establishment times, making them unique and distinct from previously characterized symbioses, where multiple microbial partners have associated with their host for a significant portion of its evolution. We then expand into discussing the functional roles and intracommunity dynamics within this holobiont, which enhances our understanding of tsetse biology to encompass the vital functions and interactions of the microbial community. Potential disturbances influencing the tsetse microbiome, including salivary gland hypertrophy virus and trypanosome infections, are highlighted. While previous studies have described evolutionary consequences of host association for symbionts, the initial steps facilitating their incorporation into a holobiont and integration of partner biology have only begun to be explored. Research on the tsetse holobiont will contribute to the understanding of how microbial metabolic integration and interdependency initially may develop within hosts, elucidating mechanisms driving adaptations leading to cooperation and coresidence within the microbial community. Lastly, increased knowledge of the tsetse holobiont may also contribute to generating novel African trypanosomiasis disease control strategies.

Aspergillus collagen-like genes (acl): identification, sequence polymorphism, and assessment for PCR-based pathogen detection.

The genus Aspergillus is a burden to public health due to its ubiquitous presence in the environment, its production of allergens, and wide demographic susceptibility among cystic fibrosis, asthmatic, and immunosuppressed patients. Current methods of detection of Aspergillus colonization and infection rely on lengthy morphological characterization or nonstandardized serological assays that are restricted to identifying a fungal etiology. Collagen-like genes have been shown to exhibit species-specific conservation across the noncollagenous regions as well as strain-specific polymorphism in the collagen-like regions. Here we assess the conserved region of the Aspergillus collagen-like (acl) genes and explore the application of PCR amplicon size-based discrimination among the five most common etiologic species of the Aspergillus genus, including Aspergillus fumigatus, A. flavus, A. nidulans, A. niger, and A. terreus. Genetic polymorphism and phylogenetic analysis of the aclF1 gene were additionally examined among the available strains. Furthermore, the applicability of the PCR-based assay to identification of these five species in cultures derived from sputum and bronchoalveolar fluid from 19 clinical samples was explored. Application of capillary electrophoresis on nanogels was additionally demonstrated to improve the discrimination between Aspergillus species. Overall, this study demonstrated that Aspergillus acl genes could be used as PCR targets to discriminate between clinically relevant Aspergillus species. Future studies aim to utilize the detection of Aspergillus acl genes in PCR and microfluidic applications to determine the sensitivity and specificity for the identification of Aspergillus colonization and invasive aspergillosis in immunocompromised subjects.

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.

The tsetse fly obligate mutualist Wigglesworthia morsitans alters gene expression and population density via exogenous nutrient provisioning.

The obligate mutualist Wigglesworthia morsitans provisions nutrients to tsetse flies. The symbiont's response to thiamine (B(1)) supplementation of blood meals, specifically towards the regulation of thiamine biosynthesis and population density, is described. Despite an ancient symbiosis and associated genome tailoring, Wigglesworthia responds to nutrient availability, potentially accommodating a decreased need.

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.

Regulation of high-affinity iron acquisition homologues in the tsetse fly symbiont Sodalis glossinidius.

Sodalis glossinidius is a facultative intracellular bacterium that is a secondary symbiont of the tsetse fly (Diptera: Glossinidae). Since studies with other facultative intracellular bacteria have shown that high-affinity iron acquisition genes are upregulated in vivo, we investigated the regulation of several Sodalis genes that encode putative iron acquisition systems. These genes, SG1538 (hemT) and SG1516 (sitA), are homologous to genes encoding periplasmic heme and iron/manganese transporters, respectively. hemT promoter- and sitA promoter-gfp fusions were constructed, and in both Escherichia coli and Sodalis backgrounds, expression levels of these fusions were higher when the bacteria were grown in iron-limiting media than when the bacteria were grown in iron-replete media. The Sodalis promoters were tested for iron regulation in an E. coli strain that lacks the fur gene, which encodes the iron-responsive transcriptional repressor Fur. Expression of the promoter-gfp fusions in the E. coli fur mutant was constitutively high in both iron-replete and iron-deplete media, and addition of either Shigella flexneri fur or Sodalis fur to a plasmid restored normal regulation. A Sodalis fur mutant was constructed by intron mutagenesis, and semiquantitative reverse transcription-PCR (RT-PCR) showed that iron repression of sitA expression was also abolished in this strain. In vivo expression analysis showed that hemT and sitA are expressed when Sodalis is within tsetse fly hosts, suggesting a biological role for these genes when Sodalis is within the tsetse fly.

Nutrient provisioning facilitates homeostasis between tsetse fly (Diptera: Glossinidae) symbionts.

Host-associated microbial interactions may involve genome complementation, driving-enhanced communal efficiency and stability. The tsetse fly (Diptera: Glossinidae), the obligate vector of African trypanosomes (Trypanosoma brucei subspp.), harbours two enteric Gammaproteobacteria symbionts: Wigglesworthia glossinidia and Sodalis glossinidius. Host coevolution has streamlined the Wigglesworthia genome to complement the exclusively sanguivorous tsetse lifestyle. Comparative genomics reveal that the Sodalis genome contains the majority of Wigglesworthia genes. This significant genomic overlap calls into question why tsetse maintains the coresidence of both symbionts and, furthermore, how symbiont homeostasis is maintained. One of the few distinctions between the Wigglesworthia and Sodalis genomes lies in thiamine biosynthesis. While Wigglesworthia can synthesize thiamine, Sodalis lacks this capability but retains a thiamine ABC transporter (tbpAthiPQ) believed to salvage thiamine. This genetic complementation may represent the early convergence of metabolic pathways that may act to retain Wigglesworthia and evade species antagonism. We show that thiamine monophosphate, the specific thiamine derivative putatively synthesized by Wigglesworthia, impacts Sodalis thiamine transporter expression, proliferation and intracellular localization. A greater understanding of tsetse symbiont interactions may generate alternative control strategies for this significant medical and agricultural pest, while also providing insight into the evolution of microbial associations within hosts.

Symbiont succession during embryonic development of the European medicinal leech, Hirudo verbana.

The European medicinal leech, Hirudo verbana, harbors simple microbial communities in the digestive tract and bladder. The colonization history, infection frequency, and growth dynamics of symbionts through host embryogenesis are described using diagnostic PCR and quantitative PCR. Symbiont species displayed diversity in temporal establishment and proliferation through leech development.

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.

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.

Leeches and their microbiota: naturally simple symbiosis models.

Strictly blood-feeding leeches and their limited microbiota provide natural and powerful model systems to examine symbiosis. Blood is devoid of essential nutrients and it is thought that symbiotic bacteria synthesize these for the host. In this review, three distinct leech-microbe associations are described: (i) the mycetome, which is the large symbiont-containing organ associated with the esophagus; (ii) the nephridia and bladders that form the excretory system; and (iii) the digestive tract, where two bacterial species dominate the microbiota. The current knowledge and features of leech biology that promote the investigation of interspecific interactions (host-microbe and microbe-microbe) and their evolution are highlighted.