Rita V. M. Rio

Insight into the Transmission Biology and Species-Specific Functional Capabilities of Tsetse (Diptera: Glossinidae) Obligate Symbiont Wigglesworthia

ABSTRACT Ancient endosymbionts have been associated with extreme genome structural stability with little differentiation in gene inventory between sister species. Tsetse flies (Diptera: Glossinidae) harbor an obligate endosymbiont, Wigglesworthia, which has coevolved with the Glossina radiation. We report on the ~720-kb Wigglesworthia genome and its associated plasmid from Glossina morsitans morsitans and compare them to those of the symbiont from Glossina brevipalpis. While there was overall high synteny between the two genomes, a large inversion was noted. Furthermore, symbiont transcriptional analyses demonstrated host tissue and development-specific gene expression supporting robust transcriptional regulation in Wigglesworthia, an unprecedented observation in other obligate mutualist endosymbionts. Expression and immunohistochemistry confirmed the role of flagella during the vertical transmission process from mother to intrauterine progeny. The expression of nutrient provisioning genes (thiC and hemH) suggests that Wigglesworthia may function in dietary supplementation tailored toward host development. Furthermore, despite extensive conservation, unique genes were identified within both symbiont genomes that may result in distinct metabolomes impacting host physiology. One of these differences involves the chorismate, phenylalanine, and folate biosynthetic pathways, which are uniquely present in Wigglesworthia morsitans. Interestingly, African trypanosomes are auxotrophs for phenylalanine and folate and salvage both exogenously. It is possible that W. morsitans contributes to the higher parasite susceptibility of its host species. IMPORTANCE Genomic stasis has historically been associated with obligate endosymbionts and their sister species. Here we characterize the Wigglesworthia genome of the tsetse fly species Glossina morsitans and compare it to its sister genome within G. brevipalpis. The similarity and variation between the genomes enabled specific hypotheses regarding functional biology. Expression analyses indicate significant levels of transcriptional regulation and support development- and tissue-specific functional roles for the symbiosis previously not observed in obligate mutualist symbionts. Retention of the genetically expensive flagella within these small genomes was demonstrated to be significant in symbiont transmission and tailored to the unique tsetse fly reproductive biology. Distinctions in metabolomes were also observed. We speculate an additional role for Wigglesworthia symbiosis where infections with pathogenic trypanosomes may depend upon symbiont species-specific metabolic products and thus influence the vector competence traits of different tsetse fly host species. Genomic stasis has historically been associated with obligate endosymbionts and their sister species. Here we characterize the Wigglesworthia genome of the tsetse fly species Glossina morsitans and compare it to its sister genome within G. brevipalpis. The similarity and variation between the genomes enabled specific hypotheses regarding functional biology. Expression analyses indicate significant levels of transcriptional regulation and support development- and tissue-specific functional roles for the symbiosis previously not observed in obligate mutualist symbionts. Retention of the genetically expensive flagella within these small genomes was demonstrated to be significant in symbiont transmission and tailored to the unique tsetse fly reproductive biology. Distinctions in metabolomes were also observed. We speculate an additional role for Wigglesworthia symbiosis where infections with pathogenic trypanosomes may depend upon symbiont species-specific metabolic products and thus influence the vector competence traits of different tsetse fly host species.

Comparative Genomics of Insect-Symbiotic Bacteria: Influence of Host Environment on Microbial Genome Composition

ABSTRACT Commensal symbionts, thought to be intermediary amid obligate mutualists and facultative parasites, offer insight into forces driving the evolutionary transition into mutualism. Using macroarrays developed for a close relative, Escherichia coli, we utilized a heterologous array hybridization approach to infer the genomic compositions of a clade of bacteria that have recently established symbiotic associations: Sodalis glossinidius with the tsetse fly (Diptera, Glossina spp.) and Sitophilus oryzae primary endosymbiont (SOPE) with the rice weevil (Coleoptera, Sitophilus oryzae). Functional biologies within their hosts currently reflect different forms of symbiotic associations. Their hosts, members of distant insect taxa, occupy distinct ecological niches and have evolved to survive on restricted diets of blood for tsetse and cereal for the rice weevil. Comparison of genome contents between the two microbes indicates statistically significant differences in the retention of genes involved in carbon compound catabolism, energy metabolism, fatty acid metabolism, and transport. The greatest reductions have occurred in carbon catabolism, membrane proteins, and cell structure-related genes for Sodalis and in genes involved in cellular processes (i.e., adaptations towards cellular conditions) for SOPE. Modifications in metabolic pathways, in the form of functional losses complementing particularities in host physiology and ecology, may have occurred upon initial entry from a free-living to a symbiotic state. It is possible that these adaptations, streamlining genomes, act to make a free-living state no longer feasible for the harnessed microbe.

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.

Genome size determination and coding capacity of Sodalis glossinidius, an enteric symbiont of tsetse flies, as revealed by hybridization to Escherichia coli gene arrays.

Recent molecular characterization of various microbial genomes has revealed differences in genome size and coding capacity between obligate symbionts and intracellular pathogens versus free-living organisms. Multiple symbiotic microorganisms have evolved with tsetse fly, the vector of African trypanosomes, over long evolutionary times. Although these symbionts are indispensable for tsetse fecundity, the biochemical and molecular basis of their functional significance is unknown. Here, we report on the genomic aspects of the secondary symbiont Sodalis glossinidius. The genome size of Sodalis is approximately 2 Mb. Its DNA is subject to extensive methylation and based on some of its conserved gene sequences has an A+T content of only 45%, compared to the typically AT-rich genomes of endosymbionts. Sodalis also harbors an extrachromosomal plasmid about 134 kb in size. We used a novel approach to gain insight into Sodalis genomic contents, i.e., hybridizing its DNA to macroarrays developed for Escherichia coli, a closely related enteric bacterium. In this analysis we detected 1,800 orthologous genes, corresponding to about 85% of the Sodalis genome. The Sodalis genome has apparently retained its genes for DNA replication, transcription, translation, transport, and the biosynthesis of amino acids, nucleic acids, vitamins, and cofactors. However, many genes involved in energy metabolism and carbon compound assimilation are apparently missing, which may indicate an adaptation to the energy sources available in the only nutrient of the tsetse host, blood. We present gene arrays as a rapid tool for comparative genomics in the absence of whole genome sequence to advance our understanding of closely related bacteria.

Infections with Immunogenic Trypanosomes Reduce Tsetse Reproductive Fitness: Potential Impact of Different Parasite Strains on Vector Population Structure

The parasite Trypanosoma brucei rhodesiense and its insect vector Glossina morsitans morsitans were used to evaluate the effect of parasite clearance (resistance) as well as the cost of midgut infections on tsetse host fitness. Tsetse flies are viviparous and have a low reproductive capacity, giving birth to only 6–8 progeny during their lifetime. Thus, small perturbations to their reproductive fitness can have a major impact on population densities. We measured the fecundity (number of larval progeny deposited) and mortality in parasite-resistant tsetse females and untreated controls and found no differences. There was, however, a typanosome-specific impact on midgut infections. Infections with an immunogenic parasite line that resulted in prolonged activation of the tsetse immune system delayed intrauterine larval development resulting in the production of fewer progeny over the flys lifetime. In contrast, parasitism with a second line that failed to activate the immune system did not impose a fecundity cost. Coinfections favored the establishment of the immunogenic parasites in the midgut. We show that a decrease in the synthesis of Glossina Milk gland protein (GmmMgp), a major female accessory gland protein associated with larvagenesis, likely contributed to the reproductive lag observed in infected flies. Mathematical analysis of our empirical results indicated that infection with the immunogenic trypanosomes reduced tsetse fecundity by 30% relative to infections with the non-immunogenic strain. We estimate that a moderate infection prevalence of about 26% with immunogenic parasites has the potential to reduce tsetse populations. Potential repercussions for vector population growth, parasite–host coevolution, and disease prevalence are discussed.

A 454 survey reveals the community composition and core microbiome of the common bed bug (Cimex lectularius) across an Urban Landscape.

Elucidating the spatial dynamic and core constituents of the microbial communities found in association with arthropod hosts is of crucial importance for insects that may vector human or agricultural pathogens. The hematophagous Cimex lectularius (Hemiptera: Cimicidae), known as the human bed bug, has made a recent resurgence in North America, as well as worldwide, potentially owing to increased travel, climate change and resistance to insecticides. A comprehensive survey of the bed bug microbiome has not been performed to date, nor has an assessment of the spatial dynamics of its microbiome. Here we present a survey of internal and external bed bug microbial communities by amplifying the V4–V6 hypervariable region of the 16S rDNA gene region followed by 454 Titanium sequencing using 31 individuals from eight distinct collection locations obtained from residences in Cincinnati, OH. Across all samples, 97% of the microbial community is made up of two dominant OTUs, previously identified as the α-proteobacterium Wolbachia and an unnamed γ-proteobacterium from the Enterobacteriaceae. Microbial communities varied among host locations for measures of community diversity and exhibited structure according to collection location. This broad survey represents the most in-depth assessment, to date, of the microbes that associate with bed bugs.

The phylogeny of Sodalis-like symbionts as reconstructed using surface-encoding loci.

Phylogenetic analyses of 16S rRNA support close relationships between the Gammaproteobacteria Sodalis glossinidius, a tsetse (Diptera: Glossinidae) symbiont, and bacteria infecting diverse insect orders. To further examine the evolutionary relationships of these Sodalis-like symbionts, phylogenetic trees were constructed for a subset of putative surface-encoding genes (i.e. ompA, spr, slyB, rcsF, ycfM, and ompC). The ompA and ompC loci were used toward examining the intra- and interspecific diversity of Sodalis within tsetse, respectively. Intraspecific analyses of ompA support elevated nonsynonymous (dN) polymorphism with an excess of singletons, indicating diversifying selection, specifically within the tsetse Glossina morsitans. Additionally, interspecific ompC comparisons between Sodalis and Escherichia coli demonstrate deviation from neutrality, with higher fixed dN observed at sites associated with extracellular loops. Surface-encoding genes varied in their phylogenetic resolution of Sodalis and related bacteria, suggesting conserved vs. host-specific roles. Moreover, Sodalis and its close relatives exhibit genetic divergence at the rcsF, ompA, and ompC loci, indicative of initial molecular divergence. The application of outer membrane genes as markers for further delineating the systematics of recently diverged bacteria is discussed. These results increase our understanding of insect symbiont evolution, while also identifying early genome alterations occurring upon integration of microorganisms with eukaryotic hosts.

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. Proteins 2014; 82:2797–2811.

Symbiont Succession during Embryonic Development of the European Medicinal Leech, Hirudo verbana

ABSTRACT 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.

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.