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Primary and Secondary Symbionts, So Similar, Yet So Different Fabrice Vavre and Henk R. Braig Proteobacteria as Primary Endosymbionts of Arthropods Abdelaziz Heddi and Roy Gross The Bacteroidetes Blattabacterium and Sulcia as Primary Endosymbionts of Arthropods M. Montagna, L. Sacchi, N. Lo, E. Clementi, D. Daffonchio, A. Alma, D. Sassera, and C. Bandi Secondary Symbionts of Insects: Acetic Acid Bacteria Elena Crotti, Elena Gonella, Irene Ricci, Emanuela Clementi, Mauro Mandrioli, Luciano Sacchi, Guido Favia, Alberto Alma, Kostas Bourtzis, Ameur Cherif, Claudio Bandi, and Daniele Daffonchio Facultative Tenants from the Enterobacteriaceae within Phloem-Feeding Insects T.L. Wilkinson Stammerula and Other Symbiotic Bacteria within the Fruit Flies Inhabiting Asteraceae Flowerheads Luca Mazzon, Isabel Martinez Sanudo, Claudia Savio, Mauro Simonato, and Andrea Squartini Candidatus Midichloria mitochondrii: Symbiont or Parasite of Tick Mitochondria? D. Pistone, L. Sacchi, N. Lo, S. Epis, M. Pajoro, G. Favia, M. Mandrioli, C. Bandi, and D. Sassera Rickettsiella, Intracellular Pathogens of Arthropods Didier Bouchon, Richard Cordaux, and Pierre Greve Arthropods Shopping for Wolbachia Daniela Schneider, Wolfgang J. Miller, and Markus Riegler Host and Symbiont Adaptations Provide Tolerance to Beneficial Microbes: Sodalis and Wigglesworthia Symbioses in Tsetse Flies Brian L. Weiss, Jingwen Wang, Geoffrey M. Attardo, and Serap Aksoy Rickettsia Get Around Yuval Gottlieb, Steve J. Perlman, Elad Chiel, and Einat Zchori-Fein Cardinium: The Next Addition to the Family of Reproductive Parasites J.A.J. Breeuwer, V.I.D. Ros, and T.V.M. Groot The Genus Arsenophonus Timothy E. Wilkes, Olivier Duron, Alistair C. Darby, Vaclav Hypsa, Eva Novakova, and Gregory D. D. Hurst Index

Tsetse harbors an obligate symbiont, Wigglesworthia glossinidia, that must be present during larval maturation for the fly's immune system to develop and function properly during adulthood., Beneficial microbial symbionts serve important functions within their hosts, including dietary supplementation and maintenance of immune system homeostasis. Little is known about the mechanisms that enable these bacteria to induce specific host phenotypes during development and into adulthood. Here we used the tsetse fly, Glossina morsitans, and its obligate mutualist, Wigglesworthia glossinidia, to investigate the co-evolutionary adaptations that influence the development of host physiological processes. Wigglesworthia is maternally transmitted to tsetse's intrauterine larvae through milk gland secretions. We can produce flies that lack Wigglesworthia (GmmWgm −) yet retain their other symbiotic microbes. Such offspring give rise to adults that exhibit a largely normal phenotype, with the exception being that they are reproductively sterile. Our results indicate that when reared under normal environmental conditions GmmWgm − adults are also immuno-compromised and highly susceptible to hemocoelic E. coli infections while age-matched wild-type individuals are refractory. Adults that lack Wigglesworthia during larval development exhibit exceptionally compromised cellular and humoral immune responses following microbial challenge, including reduced expression of genes that encode antimicrobial peptides (cecropin and attacin), hemocyte-mediated processes (thioester-containing proteins 2 and 4 and prophenoloxidase), and signal-mediating molecules (inducible nitric oxide synthase). Furthermore, GmmWgm − adults harbor a reduced population of sessile and circulating hemocytes, a phenomenon that likely results from a significant decrease in larval expression of serpent and lozenge, both of which are associated with the process of early hemocyte differentiation. Our results demonstrate that Wigglesworthia must be present during the development of immature progeny in order for the immune system to function properly in adult tsetse. This phenomenon provides evidence of yet another important physiological adaptation that further anchors the obligate symbiosis between tsetse and Wigglesworthia., Author Summary Beneficial bacterial symbionts, which are ubiquitous in nature, are often characterized by the extent to which they interact with the host. In the case of mutualistic symbioses, both partners benefit so that each one can inhabit diverse ecological niches where neither could survive on its own. Unfortunately, little is known about the functional mechanisms that underlie mutualistic relationships. Insects represent a group of advanced multi-cellular organisms that harbor well-documented symbiotic associations. One such insect, the tsetse fly, harbors a maternally transmitted bacterial mutualist called Wigglesworthia that provides its host with essential metabolites missing from its vertebrate blood-specific diet. In this study, we further examine the relationship between tsetse and Wigglesworthia by investigating the interaction between this bacterium and its host's immune system. We have found that when Wigglesworthia is absent from tsetse during the maturation of immature larval stages, subsequent adults are characterized by an underdeveloped cellular immune system and thus highly susceptible to infection with a normally non-pathogenic foreign microbe. These findings represent an additional adaptation that further anchors the steadfast relationship shared between tsetse and its obligate symbiont.

Tsetse flies are vectors of the protozoan parasite African trypanosomes, which cause sleeping sickness disease in humans and nagana in livestock. Although there are no effective vaccines and efficacious drugs against this parasite, vector reduction methods have been successful in curbing the disease, especially for nagana. Potential vector control methods that do not involve use of chemicals is a genetic modification approach where flies engineered to be parasite resistant are allowed to replace their susceptible natural counterparts, and Sterile Insect technique (SIT) where males sterilized by chemical means are released to suppress female fecundity. The success of genetic modification approaches requires identification of strong drive systems to spread the desirable traits and the efficacy of SIT can be enhanced by identification of natural mating incompatibility. One such drive mechanism results from the cytoplasmic incompatibility (CI) phenomenon induced by the symbiont Wolbachia. CI can also be used to induce natural mating incompatibility between release males and natural populations. Although Wolbachia infections have been reported in tsetse, it has been a challenge to understand their functional biology as attempts to cure tsetse of Wolbachia infections by antibiotic treatment damages the obligate mutualistic symbiont (Wigglesworthia), without which the flies are sterile. Here, we developed aposymbiotic (symbiont-free) and fertile tsetse lines by dietary provisioning of tetracycline supplemented blood meals with yeast extract, which rescues Wigglesworthia-induced sterility. Our results reveal that Wolbachia infections confer strong CI during embryogenesis in Wolbachia-free (GmmApo) females when mated with Wolbachia-infected (GmmWt) males. These results are the first demonstration of the biological significance of Wolbachia infections in tsetse. Furthermore, when incorporated into a mathematical model, our results confirm that Wolbachia can be used successfully as a gene driver. This lays the foundation for new disease control methods including a population replacement approach with parasite resistant flies. Alternatively, the availability of males that are reproductively incompatible with natural populations can enhance the efficacy of the ongoing sterile insect technique (SIT) applications by eliminating the need for chemical irradiation., Author Summary Infections with the parasitic bacterium Wolbachia are widespread in insects and cause a number of reproductive modifications, including cytoplasmic incompatibility (CI). There is growing interest in Wolbachia, as CI may be able to drive desired phenotypes such as disease resistance traits, into natural populations. Although Wolbachia infections had been reported in the medically and agriculturally important tsetse, their functional role was unknown. This is because attempts to cure tsetse of Wolbachia by antibiotic treatment damages the obligate mutualist Wigglesworthia, without which the flies are sterile. Here we have succeeded in the development of Wolbachia free and still fertile tsetse lines. Mating experiments for the first time provides evidence of strong CI in tsetse. We have incorporated our empirical data in a mathematical model and show that Wolbachia infections can be harnessed in tsetse to drive desirable phenotypes into natural populations in few generations. This finding provides additional support for the application of genetic approaches, which aim to spread parasite resistance traits in natural populations as a novel disease control method. Alternatively, releasing Wolbachia infected males can enhance Sterile Insect applications, as this will reduce the fecundity of natural females either uninfected or carrying a different strain of Wolbachia.

Some of the world's most devastating diseases are transmitted by arthropod vectors. Attempts to control these arthropods are currently being challenged by the widespread appearance of insecticide resistance. It is therefore desirable to develop alternative strategies to complement existing methods of vector control. In this review, Charles Beard, Scott O'Neill, Robert Tesh, Frank Richards and Serap Aksoy present an approach for introducing foreign genes into insects in order to confer refractoriness to vector populations, ie. the inability to transmit disease-causing agents. This approach aims to express foreign anti-parasitic or anti-viral gene products in symbiotic bacteria harbored by insects. The potential use of naturally occurring symbiont-based mechanisms in the spread of such refractory phenotypes is also discussed.

Two isolates of bacterial endosymbionts, GP01 and GM02, were established in cell free medium from haemolymph of the tsetse, Glossina pallidipes and G. morsitans. These microorganisms appear similar to rickettsia-like organisms reported previously from various tsetse species. The 16S rRNA sequence analysis, however, placed them within the gamma subdivision of the Proteobacteria, phylogenetically distinct from most members of the Rickettsiaceae which align with the alpha subdivision. Distinct multiple endogenous plasmids are harboured by GP01 and GM02, suggesting that the two isolates are different. Restriction mapping analysis showed that one of the conserved plasmids is present in high copy number and is at least 80 kb in size. A heterologous plasmid pSUP204, which contains the broad host range oriV replication origin, was used to transfect bacterial cultures. The symbiont GM02 was transformed, and it expressed plasmid encoded resistance to the antibiotics ampicillin, tetracycline and chloramphenicol. Transformation of these symbionts may provide a novel means for expressing anti-parasitic genes within tsetse populations.

Teneral Glossina morsitans morsitans from outbred and susceptible stocks infected with Trypanosoma (Nannomonas) congolense developed, respectively, three and six times higher midgut infection rates than flies of the same stock which had previously taken a bloodmeal. Non-teneral G. m. morsitans remained relatively refractory to infection when infected at subsequent feeds. Differences in susceptibility to midgut infection between teneral flies from susceptible and outbred lines of G. m. morsitans disappeared in non-teneral flies, showing that maternally inherited susceptibility to midgut infection is a phenomenon restricted to the teneral state of the fly. Laboratory reared G. m. morsitans were found to have become significantly more susceptible to trypanosome infection than wild flies from the population from which the colony was derived. The likely role of rickettsia-like organisms (RLO) in potentiating teneral susceptibility to midgut infection is discussed. The addition of the specific midgut lectin inhibitor D-glucosamine to the infective feed of non-teneral flies increased midgut infection rates to levels comparable with those achieved in teneral flies. It is concluded that the peritrophic membrane does not act as a barrier preventing non-teneral flies becoming infected. The relative refractoriness of non-teneral flies suggests that they do not play a significant part in the epidemiology of Trypanozoon or T. congolense infections.

This paper reports the first evidence of the presence of bacteria, other than the three previously described as symbionts, Wigglesworthia glossinidia, Wolbachia, and Sodalis glossinidius, in the midgut of Glossina palpalis palpalis, the tsetse fly, a vector of the chronic form of human African trypanosomiasis in sub-Saharan African countries. Based on the morphological, nutritional, physiological, and phylogenetic results, we identified Enterobacter, Enterococcus, and Acinetobacter spp. as inhabitants of the midgut of the tsetse fly from Angola. Enterobacter spp. was the most frequently isolated. The role of these bacteria in the gut, in terms of vector competence of the tsetse fly, is discussed, as is the possibility of using these bacteria to produce in situ trypanolytic molecules.

Tsetse flies (Diptera: Glossinidae) are vectors for trypanosome parasites, the agents of the deadly sleeping sickness disease in Africa. Tsetse also harbor two maternally transmitted enteric mutualist endosymbionts: the primary intracellular obligate Wigglesworthia glossinidia and the secondary commensal Sodalis glossinidius . Both endosymbionts are transmitted to the intrauterine progeny through the milk gland secretions of the viviparous female. We administered various antibiotics either continuously by per os supplementation of the host blood meal diet or discretely by hemocoelic injections into fertile females in an effort to selectively eliminate the symbionts to study their individual functions. A symbiont-specific PCR amplification assay and fluorescence in situ hybridization analysis were used to evaluate symbiont infection outcomes. Tetracycline and rifampin treatments eliminated all tsetse symbionts but reduced the fecundity of the treated females. Ampicillin treatments did not affect the intracellular Wigglesworthia localized in the bacteriome organ and retained female fecundity. The resulting progeny of ampicillin-treated females, however, lacked Wigglesworthia but still harbored the commensal Sodalis . Our results confirm the presence of two physiologically distinct Wigglesworthia populations: the bacteriome-localized Wigglesworthia involved with nutritional symbiosis and free-living Wigglesworthia in the milk gland organ responsible for maternal transmission to the progeny. We evaluated the reproductive fitness, longevity, digestion, and vectorial competence of flies that were devoid of Wigglesworthia . The absence of Wigglesworthia completely abolished the fertility of females but not that of males. Both the male and female Wigglesworthia -free adult progeny displayed longevity costs and were significantly compromised in their blood meal digestion ability. Finally, while the vectorial competence of the young newly hatched adults without Wigglesworthia was comparable to that of their wild-type counterparts, older flies displayed higher susceptibility to trypanosome infections, indicating a role for the mutualistic symbiosis in host immunobiology. The ability to rear adult tsetse that lack the obligate Wigglesworthia endosymbionts will now enable functional investigations into this ancient symbiosis.

1-ACL (articles avec comité de lecture); A key process in the tsetse reproductive cycle is the transfer of essential nutrients and bacterial symbionts from mother to intrauterine offspring. The tissue mediating this transfer is the milk gland. This work focuses upon the localization and function of two milk proteins (milk gland protein (GmmMGP) and transferrin (GmmTsf)) and the tsetse endosymbionts (Sodalis and Wigglesworthia), in the context of milk gland physiology. Fluorescent in situ hybridization (FISH) and immunohistochemical analysis confirm that the milk gland secretory cells synthesize and secrete milk gland protein and transferrin. Knockdown of gmmmgp by double stranded RNA (dsRNA) mediated RNA interference results in reduction of tsetse fecundity, demonstrating its functional importance in larval nutrition and development. Bacterial species-specific in situ hybridizations of milk gland sections reveal large numbers of Sodalis and Wigglesworthia within the lumen of the milk gland. Sodalis is also localized within the cytoplasm of the secretory cells. Within the lumen, Wigglesworthia localize close to the channels leading to the milk storage reservoir of the milk gland secretory cells. We discuss the significance of the milk gland in larval nutrition and in transmission of symbiotic bacteria to developing offspring.

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 fly's 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., Author Summary In many cases, parasites adapt to their hosts' biology over time and the extent of their harmful effects gradually diminishes. Insect-transmitted parasites such as African trypanosomes, however, are unusually pathogenic for their mammalian hosts because they rely on their invertebrate hosts for transmission to the next mammalian host. To ensure their maximum transmission, it is essential that parasite infections do not compromise insect host's fitness traits, including longevity and host-finding ability. Our results in tsetse indicate that, as theory predicts, trypanosome infections do not reduce host longevity. Instead, they divert host resources from reproduction and can reduce reproductive output by as much as 30%. Such loss of reproductive fitness occurs as a result of the induction of tsetse's immune responses. A closely related non-immunogenic parasite line does not induce host responses and does not compromise host fecundity. It is possible that host immune responses are needed in the case of the immunogenic line to control the parasite density to prevent excessive host damage. Because tsetse are viviparous and each adult female typically gives rise to only few progeny during their lifetime, even modest costs on reproduction can have a significant impact on host abundance. Our model predicts that if the prevalence of immunogenic parasite infections in tsetse populations reaches over 26%, they begin to have a negative impact on population growth rate. Infection rates as high as 30% have been reported with trypanosomes in the field. Our laboratory findings coupled with our modeling studies now provide a framework to investigate the status of co-infections, host immune activation processes, fecundity outcomes, transmission dynamics, and host virulence phenotypes in natural tsetse–trypanosome populations.

Reductive evolution in mitochondria and obligate intracellular microbes has led to a significant reduction in their genome size and guanine plus cytosine content (GC). We show that genome shrinkage during reductive evolution in prokaryotes follows an exponential decay pattern and provide a method to predict the extent of this decay on an evolutionary timescale. We validated predictions by comparison with estimated extents of genome reduction known to have occurred in mitochondria and Buchnera aphidicola, through comparative genomics and by drawing on available fossil evidences. The model shows how the mitochondrial ancestor would have quickly shed most of its genome, shortly after its incorporation into the protoeukaryotic cell and prior to codivergence subsequent to the split of eukaryotic lineages. It also predicts that the primary rickettsial parasitic event would have occurred between 180 and 425 million years ago (MYA), an event of relatively recent evolutionary origin considering the fact that Rickettsia and mitochondria evolved from a common alphaproteobacterial ancestor. This suggests that the symbiotic events of Rickettsia and mitochondria originated at different time points. Moreover, our model results predict that the ancestor of Wigglesworthia glossinidia brevipalpis, dated around the time of origin of its symbiotic association with the tsetse fly (50-100 MYA), was likely to have been an endosymbiont itself, thus supporting an earlier proposition that Wigglesworthia, which is currently a maternally inherited primary endosymbiont, evolved from a secondary endosymbiont.

Tsetse flies transmit African trypanosomes, responsible for sleeping sickness in humans and nagana in animals. This disease affects many people with considerable impact on public health and economy in sub-Saharan Africa, whereas trypanosomes' resistance to drugs is rising. The symbiont Sodalis glossinidius is considered to play a role in the ability of the fly to acquire trypanosomes. Different species of Glossina were shown to harbor genetically distinct populations of S. glossinidius. We therefore investigated whether vector competence for a given trypanosome species could be linked to the presence of specific genotypes of S. glossinidius. Glossina palpalis gambiensis individuals were fed on blood infected either with Trypanosoma brucei gambiense or Trypanosoma brucei brucei. The genetic diversity of S. glossinidius strains isolated from infected and noninfected dissected flies was investigated using amplified fragment length polymorphism markers. Correspondence between occurrence of these markers and parasite establishment was analyzed using multivariate analysis. Sodalis glossinidius strains isolated from T. brucei gambiense-infected flies clustered differently than that isolated from T. brucei brucei-infected individuals. The ability of T. brucei gambiense and T. brucei brucei to establish in G. palpalis gambiensis insect midgut is statistically linked to the presence of specific genotypes of S. glossinidius. This could explain variations in Glossina vector competence in the wild. Then, assessment of the prevalence of specific S. glossinidius genotypes could lead to novel risk management strategies.

Tsetse flies ( Glossina spp.) can harbor up to three distinct species of endosymbiotic bacteria that exhibit unique modes of transmission and evolutionary histories with their host. Two mutualist enterics, Wigglesworthia and Sodalis , are transmitted maternally to tsetse flies' intrauterine larvae. The third symbiont, from the genus Wolbachia , parasitizes developing oocytes. In this study, we determined that Sodalis isolates from several tsetse fly species are virtually identical based on a phylogenetic analysis of their ftsZ gene sequences. Furthermore, restriction fragment-length polymorphism analysis revealed little variation in the genomes of Sodalis isolates from tsetse fly species within different subgenera ( Glossina fuscipes fuscipes and Glossina morsitans morsitans ). We also examined the impact on host fitness of transinfecting G. fuscipes fuscipes and G. morsitans morsitans flies with reciprocal Sodalis strains. Tsetse flies cleared of their native Sodalis symbionts were successfully repopulated with the Sodalis species isolated from a different tsetse fly species. These transinfected flies effectively transmitted the novel symbionts to their offspring and experienced no detrimental fitness effects compared to their wild-type counterparts, as measured by longevity and fecundity. Quantitative PCR analysis revealed that transinfected flies maintained their Sodalis populations at densities comparable to those in flies harboring native symbionts. Our ability to transinfect tsetse flies is indicative of Sodalis ' recent evolutionary history with its tsetse fly host and demonstrates that this procedure may be used as a means of streamlining future paratransgenesis experiments.

Though scientists recently downsized estimates of insect species from over 30 million to around 4 million to 6 million, it's safe to say that insects are among Earth's most numerous and diverse group of organisms. At least some of this success derives from mutually beneficial interactions with bacteria, established as long as 270 million years ago, that allow insects to thrive in otherwise unsuitable niches. These symbiotic bacteria—called endosymbionts because they live inside cells—synthesize essential nutrients for their hosts, while the insects provide an ecological niche for the bacteria. In a new study, Dongying Wu, Jonathan Eisen, Nancy Moran, and colleagues used comparative genome analysis to investigate the inner life of a widespread agricultural pest, the glassy-winged sharpshooter (Homalodisca coagulata). As the vector of Pierce disease (caused by the Xylella fastidiosa bacterium), the sharpshooter poses a major threat to California crops, having already destroyed an estimated $14 million of Southern California grapevines. It lives on xylem sap—which distributes salts and water throughout the plant but provides very few organic nutrients to the insect—and houses two endosymbionts, Baumannia cicadellinicola and Sulcia muelleri. These two unrelated endosymbionts, they show, play distinct, complementary roles that supplement their host's diet. Endosymbionts were first described through microscopy in the early 20th century, but their metabolic secrets have only recently come to light by making inferences about gene function based on genome analysis. After sequencing the B. cicadellinicola genome, Wu et al. built a genome-based evolutionary tree (called a phylogenetic tree) for B. cicadellinicola and related endosymbiont species. B. cicadellinicola was grouped together with endosymbionts of aphids (Buchnera), tsetse flies (Wigglesworthia), and ants (Blochmania), in keeping with other genomic studies, but was “the deepest branching symbiont,” suggesting it had separated from the group earlier. The endosymbiont genomes shared a number of features, including reduced genome size, fewer guanine-cytosine (G-C) than adenine-thymine (A-T) base pairs, and rapidly evolving proteins. These trends can be found in endosymbionts arising across all the major bacterial groups, challenging biologists to figure out the mechanisms driving them. One explanation centers on changes that arise by chance in small populations (called drift); the other centers on a higher rate of mutations stemming from the loss of DNA repair systems. Interestingly, though some researchers favor one hypothesis over the other, analysis of the B. cicadellinicola genome suggests both are important, but act at different levels. The differences between endosymbionts and free-living species appear to be due to genetic drift. Differences among symbionts, however, appear to be due to differential loss of DNA repair genes. The researchers argue that the nature of some of B. cicadellinicola's other genome features (such as G-C content) and position on the evolutionary tree can help fill in missing gaps between free-living and intracellular species. And because its proteins are evolving more slowly than those of other endosymbionts—making it more likely that similar sequences really do reflect evolutionary relatedness rather than artifacts that occur when only fast-evolving sequences are used—inferences about the evolutionary events promoting intracelluarity can be made with more confidence. As for B. cicadellinicola's metabolic capabilities, the researchers found a “relatively limited repertoire.” It lacks the necessary genes to support sugar metabolism, and probably does not use sugar as energy source since almost none is present in the xylem sap diet. Energy likely comes from using the more abundant amino acids, one of the main types of organic compounds present in xylem. In return, it provides the insect with a range of vitamins and cofactors (required for enzyme activity). But surprisingly, B. cicadellinicola lacks key enzymes needed to synthesize essential amino acids. Obviously, the insect must get the missing essential nutrients from somewhere, and the most likely candidate, they reasoned, was the other bacterium known to reside in its cells, S. muelleri. They went back to the sequences that didn't assemble with B. cicadellinicola, realizing they might contain part of the S. muelleri genome, and found genes required for the synthesis of several essential amino acids. Though some of the sequences belonged to other bacterial species, the vast majority required for amino acid synthesis belonged to S. muelleri. Since the sample of this bacterium's genome contained very few genes related to vitamin or cofactor synthesis, Wu et al. concluded that the two endosymbionts perform nonredundant, complementary services for their host. B. cicadellinicola synthesizes most of the sharpshooter's vitamins and cofactors, while S. muelleri appears to provide essential amino acids. How three unrelated organisms manage to integrate such complex operations as metabolism and gene expression is a question for future study. But targeting any number of these essential bacterial pathways could prove a promising strategy for containing the spread of a potentially devastating pest—a risk perceived as so great that the sharpshooter is the only insect listed as a potential bioterrorism agent. And the researchers make a strong case that endosymbiont systems can help shed light on the molecular and evolutionary changes that allowed free-living organisms to take up residence in the cells of others—a path echoing the evolution of eukaryotes, whose cells contain mitochondria and chloroplasts, the descendants of ancient free-living bacteria.

The application of recombinant DNA technologies for molecular genetic approaches promises to bring about new strategies for control of vector-borne-diseases. These approaches can also enhance the existing tools. Here, one application of this technology is presented where parasite refractory insects are engineered that can then be spread to replace their susceptible counterparts in the field to reduce disease transmission. The approach presented here utilizes the symbiotic bacteria that are naturally harbored in tsetse to express foreign genes. As these naturally harbored organisms reside in the same tissues as trypanosomes, the expression of anti-trypanosoma1 products in these bacterial symbionts can adversely effect parasite biology. The use of Wolbachia symbionts, which are known to induce phenomena such as cytoplasmic incompatibility, is discussed as a potential gene driving system.

Genetic diversity among Sodalis glossinidius populations was investigated using amplified fragment length polymorphism markers. Strains collected from Glossina palpalis gambiensis and Glossina morsitans morsitans flies group into separate clusters, being differentially structured. This differential structuring may reflect different host-related selection pressures and may be related to the different vector competences of Glossina spp.

It is common enough to read a paper that uses genome-sequence data to make conclusions about how an organism lives. Less common is the approach used by Pal et al. who have generated gene networks for a group of related organisms with similar lifestyles, and used those to infer gene content of another member of the group. With the genome of E. coli as starting point, they predicted the metabolism of Buchnera, an intracellular symbiont with a heavily reduced genome, that was derived from an ancestor of E. coli. This work has implications for the search for a ‘minimal genome’, and already indicates that the concept of a ‘non-essential gene’ can be spurious, as a gene can easily become essential in changing genomic contexts. Rather than using genome sequence data to make conclusions about how an organism lives, the reverse approach can be adopted — using gene networks generated for a group of related organisms with similar lifestyles to infer the gene content of another member of the group. It is possible to infer aspects of an organism's lifestyle from its gene content1. Can the reverse also be done? Here we consider this issue by modelling evolution of the reduced genomes of endosymbiotic bacteria. The diversity of gene content in these bacteria may reflect both variation in selective forces and contingency-dependent loss of alternative pathways. Using an in silico representation of the metabolic network of Escherichia coli, we examine the role of contingency by repeatedly simulating the successive loss of genes while controlling for the environment. The minimal networks that result are variable in both gene content and number. Partially different metabolisms can thus evolve owing to contingency alone. The simulation outcomes do preserve a core metabolism, however, which is over-represented in strict intracellular bacteria. Moreover, differences between minimal networks based on lifestyle are predictable: by simulating their respective environmental conditions, we can model evolution of the gene content in Buchnera aphidicola and Wigglesworthia glossinidia with over 80% accuracy. We conclude that, at least for the particular cases considered here, gene content of an organism can be predicted with knowledge of its distant ancestors and its current lifestyle.

International audience; Most endosymbiotic bacteria have extremely reduced genomes, accelerated evolutionary rates, and strong AT base compositional bias thought to reflect reduced efficacy of selection and increased mutational pressure. Here, we present a comparative study of evolutionary forces shaping five fully sequenced bacterial endosymbionts of insects. The results of this study were three-fold: (i) Stronger conservation of high expression genes at not just nonsynonymous, but also synonymous, sites. (ii) Variation in amino acid usage strongly correlates with GC content and expression level of genes. This pattern is largely explained by greater conservation of high expression genes, leading to their higher GC content. However, we also found indication of selection favoring GC-rich amino acids that contrasts with former studies. (iii) Although the specific nutritional requirements of the insect host are known to affect gene content of endosymbionts, we found no detectable influence on substitu! tion rates, amino acid usage, or codon usage of bacterial genes involved in host nutrition.

Insect-borne diseases exact a high public health burden and have a devastating impact on livestock and agriculture. To date, control has proved to be exceedingly difficult. One such disease that has plagued sub-Saharan Africa is caused by the protozoan African trypanosomes (Trypanosoma species) and transmitted by tsetse flies (Diptera: Glossinidae). This presentation describes the biology of the tsetse fly and its interactions with trypanosomes as well as its symbionts. Tsetse can harbor up to three distinct microbial symbionts, including two enterics (Wigglesworthia glossinidia and Sodalis glossinidius) as well as facultative Wolbachia infections, which influence host physiology. Recent investigations into the genome of the obligate symbiont Wigglesworthia have revealed characteristics indicative of its long co-evolutionary history with the tsetse host species. Comparative analysis of the commensal-like Sodalis with free-living enterics provides examples of adaptations to the host environment (physiology and ecology), reflecting genomic tailoring events during the process of transitioning into a symbiotic lifestyle. From an applied perspective, the extensive knowledge accumulated on the genomic and developmental biology of the symbionts coupled with our ability to both express foreign genes in these microbes in vitro and repopulate tsetse midguts with these engineered microbes now provides a means to interfere with the host physiological traits which contribute to vector competence promising a novel tool for disease management.

SUMMARY Mutualistic associations of obligate intracellular bacteria and insects have attracted much interest in the past few years due to the evolutionary consequences for their genome structure. However, much less attention has been paid to the metabolic ramifications for these endosymbiotic microorganisms, which have to compete with but also to adapt to another metabolism—that of the host cell. This review attempts to provide insights into the complex physiological interactions and the evolution of metabolic pathways of several mutualistic bacteria of aphids, ants, and tsetse flies and their insect hosts.

Tsetse flies (Diptera:Glossinidae) are vectors of African trypanosomes, the protozoan agents of devastating diseases in humans and animals. Prior studies in trypanosome infected Glossina morsitans morsitans have shown induced expression and synthesis of several antimicrobial peptides in fat body tissue. Here, we have expressed one of these peptides, Attacin (GmAttA1) in Drosophila (S2) cells in vitro. We show that the purified recombinant protein (recGmAttA1) has strong antimicrobial activity against Escherichia coli-K12, but not against the enteric gram-negative symbiont of tsetse, Sodalis glossinidius. The recGmAttA1 also demonstrated inhibitory effects against both the mammalian bloodstream form and the insect stage Trypanosoma brucei in vitro (minimal inhibitory concentration MIC50 0.075 microM). When blood meals were supplemented with purified recGmAttA1 during the course of parasite infection, the prevalence of trypanosome infections in tsetse midgut was significantly reduced. Feeding fertile females GmAttA1 did not affect the fecundity or the longevity of mothers, nor did it affect the hatchability of their offspring. We discuss a paratransgenic strategy, which involves the expression of trypanocidal molecules such as recGmAttA1 in the midgut symbiont Sodalis in vivo to reduce trypanosome transmission.

Wigglesworthia glossinidiabrevipalpis, the obligate bacterial endosymbiont of the tsetse flyGlossina brevipalpis, is characterized by extreme genome reduction and AT nucleotide composition bias. Here, multivariate statistical analyses are used to test the hypothesis that mutational bias and genetic drift shape synonymous codon usage and amino acid usage ofWigglesworthia. The results show that synonymous codon usage patterns vary little across the genome and do not distinguish genes of putative high and low expression levels, thus indicating a lack of translational selection. Extreme AT composition bias across the genome also drives relative amino acid usage, but predicted high-expression genes (ribosomal proteins and chaperonins) use GC-rich amino acids more frequently than do low-expression genes. The levels and configuration of amino acid differences betweenWigglesworthiaandEscherichia coliwere compared to test the hypothesis that the relatively GC-rich amino acid profiles of high-expression genes reflect greater amino acid conservation at these loci. This hypothesis is supported by reduced levels of protein divergence at predicted high-expressionWigglesworthiagenes and similar configurations of amino acid changes across expression categories. Combined, the results suggest that codon and amino acid usage in theWigglesworthiagenome reflect a strong AT mutational bias and elevated levels of genetic drift, consistent with expected effects of an endosymbiotic lifestyle and repeated population bottlenecks. However, these impacts of mutation and drift are apparently attenuated by selection on amino acid composition at high-expression genes.

Through understanding the mechanisms by which tsetse endosymbionts potentiate trypanosome susceptibility in tsetse, it may be possible to engineer modified endosymbionts which, when introduced into tsetse, render these insects incapable of transmitting parasites. In this study we have assayed the effect of three different antibiotics on the endosymbiotic microflora of tsetse (Glossina morsitans morsitans). We showed that the broad-spectrum antibiotics, ampicillin and tetracycline, have a dramatic impact on tsetse fecundity and pupal emergence, effectively rendering these insects sterile. This results from the loss of the tsetse primary endosymbiont, Wigglesworthia glossinidia, which is eradicated by ampicillin and tetracycline treatment. Using the sugar analogue and antibiotic, streptozotocin, we demonstrated specific elimination of the tsetse secondary endosymbiont, Sodalis glossinidius, with no observed detrimental effect upon W. glossinidia. The specific eradication of S. glossinidius had a negligible effect upon the reproductive capability of tsetse but did effect a significant reduction in fly longevity. Furthermore, elimination of S. glossinidius resulted in increased refractoriness to trypanosome infection in tsetse, providing further evidence that S. glossinidius plays an important role in potentiating trypanosome susceptibility in this important disease vector. In the light of these findings, we highlight progress made towards developing recombinant Sodalis strains engineered to avoid potentiating trypanosome susceptibility in tsetse. In particular, we focus on the chitinase/N-acetyl-D-glucosamine catabolic machinery of Sodalis which has previously been implicated in causing immune inhibition in tsetse.

Tsetse flies Glossina spp. (Diptera: Glossinidae) harbor three different symbiotic microorganisms, one being an intracellular Rickettsia of the genus Wolbachia. This bacterium infects a wide range of arthropods, where it causes a variety of reproductive abnormalities, one of which is termed cytoplasmic incompatibility (CI) that, when expressed, results in embryonic death due to disruptions in fertilization events. We report here that in colonized flies, Wolbachia infections can be detected in 100% of sampled individuals, while infections vary significantly in field populations. Based on Wolbachia Surface Protein (wsp) gene sequence analysis, the infections associated with different fly species are all unique within the A group of the Wolbachia pipientis clade. In addition to being present in germ-line tissues, Wolbachia infections have been found in somatic tissues of several insects. Using a Wolbachia-specific PCR-based assay, the tissue tropism of infections in Glossina morsitans morsitans Westwood, Glossina brevipalpis Newstead and Glossina austeni Newstead were analysed. While infections in G. m. morsitans and G. brevipalpis were limited to reproductive tissues, in G. austeni, Wolbachia could be detected in various somatic tissues.

A secondary intracellular symbiotic bacterium was isolated from the haemolymph of the tsetse fly Glossina morsitans morsitans and cultured in Aedes albopictus cell line C6/36. Pure-culture isolation of this bacterium was achieved through the use of solid-phase culture under a microaerobic atmosphere. After isolation of strain M1T, a range of tests was performed to determine the phenotypic properties of this bacterium. Considering the results of these tests, along with the phylogenetic position of this micro-organism, it is proposed that this intracellular symbiont from G. m. morsitans should be classified in a new genus Sodalis gen. nov., as Sodalis glossinidius gen. nov., sp. nov. Strain M1T is the type strain for this new species.

Many tsetse species (Diptera: Glossinidae) harbour two morphologically different intracellular endosymbiotic microorganisms associated with gut tissue: primary (P) and secondary (S) endosymbionts. The P-endosymbionts of tsetse (Wigglesworthia glossinidia) are sequestered in specialized epithelial cells, bacteriocytes, which form a structure (bacteriome) in the anterior portion of the gut. Phylogenetic characterization of P-endsymbionts from the three subgenera of genus Glossina has shown that these organisms constitute a distinct lineage within the gamma-subdivision of Proteobacteria and have evolved concordantly with their insect host species, suggesting an evolutionarily ancient association for this symbiosis. The S-endosymbiont is a smaller (1-2 micron) gram-negative rod and is harboured in midgut epithelial cells. Its phylogenetic characterization from Glossina morsitans morsitans had shown that it is a member of the family Enterobacteriaceae within the gamma-3 subdivision of the Proteobacteria, closely related to enteric bacteria. Some tsetse species harbour a third bacterium in their reproductive tissue, which was shown phylogenetically to belong to to the Wolbachia pipientis assemblage of microorganisms. Here, we show that S-endosymbionts from five tsetse species, representing all three subgenera, form a cluster of closely related microorganisms, based on their almost identical 16S rRNA gene sequences. The high similarity provides strong evidence of recent independent acquisition of S-endosymbionts by individual tsetse species, unlike Wigglesworthia which displays concordant evolution with host insect species. A PCR-based assay and restriction fragment length polymorphism (RFLP) analysis was developed to localize the S-endosymbionts and Wigglesworthia in ovary, egg, milk-gland and spermatheca tissues in order to investigate the potential routes for the vertical transmission of these symbionts to the intrauterine larvae. Only S-endosymbionts were found to infect milk gland tissue, suggesting that milk gland secretions represent a route of transmission for these symbionts into the developing larva. The ovary tissue was found to harbour only Wolbachia, confirming its transovarial transmission, whereas the mode of transmission of Wigglesworthia remains unknown.

Tsetse flies (Glossina spp.) vector pathogenic African trypanosomes, which cause sleeping sickness in humans and nagana in domesticated animals. Additionally, tsetse harbors 3 maternally transmitted endosymbiotic bacteria that modulate their host's physiology. Tsetse is highly resistant to infection with trypanosomes, and this phenotype depends on multiple physiological factors at the time of challenge. These factors include host age, density of maternally-derived trypanolytic effector molecules present in the gut, and symbiont status during development. In this study, we investigated the molecular mechanisms that result in tsetse's resistance to trypanosomes. We found that following parasite challenge, young susceptible tsetse present a highly attenuated immune response. In contrast, mature refractory flies express higher levels of genes associated with humoral (attacin and pgrp-lb) and epithelial (inducible nitric oxide synthase and dual oxidase) immunity. Additionally, we discovered that tsetse must harbor its endogenous microbiome during intrauterine larval development in order to present a parasite refractory phenotype during adulthood. Interestingly, mature aposymbiotic flies (Gmm Apo) present a strong immune response earlier in the infection process than do WT flies that harbor symbiotic bacteria throughout their entire lifecycle. However, this early response fails to confer significant resistance to trypanosomes. Gmm Apo adults present a structurally compromised peritrophic matrix (PM), which lines the fly midgut and serves as a physical barrier that separates luminal contents from immune responsive epithelial cells. We propose that the early immune response we observe in Gmm Apo flies following parasite challenge results from the premature exposure of gut epithelia to parasite-derived immunogens in the absence of a robust PM. Thus, tsetse's PM appears to regulate the timing of host immune induction following parasite challenge. Our results document a novel finding, which is the existence of a positive correlation between tsetse's larval microbiome and the integrity of the emerging adult PM gut immune barrier., Author Summary Tsetse flies serve as a host to many micro-organisms. Specifically, this fly houses beneficial endosymbiotic bacteria, and can also serve as a vector of pathogenic trypanosomes across much of sub-Saharan Africa. Although flies feed on parasite-infected reservoir hosts, only a small proportion (1–5%) of individuals that acquire an infectious meal become infected and subsequently transmit disease to a naïve host. Several physiological factors, including tsetse's age, nutritional status and innate immune mechanisms, contribute to trypanosome infection outcomes in the fly. We demonstrate that tsetse's endogenous microbiome also impacts the fly's resistance to parasites. Specifically, we show that tsetse must harbor it's symbiotic bacteria during larval development in order to present a trypanosome-refractory phenotype during adulthood. These microbes appear to indirectly regulate the fly's ability to immunologically detect and respond to the presence of trypanosomes. One of the mechanisms by which these microbes regulate parasite transmission involves modulating the formation of a physical barrier (called the ‘peritrophic matrix’) in their host's gut. Our findings are indicative of the complex functional association that exists between tsetse's symbiotic microbes and host immune mechanisms that regulate trypanosome infection outcomes.

The primary endosymbionts (P-endosymbionts) of tsetse flies (Diptera: Glossinidae) are harbored inside specialized cells (mycetocytes) in the anterior region of the gut, and these specialized cells form a white, U-shaped organelle called mycetome. The P-endosymbionts of five tsetse fly species belonging to the Glossinidae have been characterized morphologically, and their 16S ribosomal DNA sequences have been determined for phylogenetic analysis. These organisms were found to belong to a distinct lineage related to the family Enterobacteriaceae in the gamma subdivision of Proteobacteria, which includes the secondary endosymbionts of various insects and Escherichia coli. These bacteria are also related to the P-endosymbionts of aphids, Buchnera aphidicola. Signature sequences in the 16S ribosomal DNA and genomic organizational differences which distinguish the tsetse fly P-endosymbionts from members of the Enterobacteriaceae and from the genus Buchnera are described in this paper. I propose that the P-endosymbionts of tsetse flies should be classified in a new genus, the genus Wigglesworthia, and a new species, Wigglesworthia glossinidia. The P-endosymbiont found in the mycetocytes of Glossina morsitans morsitans is designated the type strain of this species.

Tsetse flies (Diptera: Glossinidae) harbour two morphologically different endosymbionts intracellularly associated with gut tissue: a primary (P) and a secondary (S) organism. The P-endosymbiont is a gram-negative rod, 8-10 microns in size, and resides intracellularly within specialized cells, mycetocytes which are organized into an organelle (mycetome), in the anterior portion of the gut. The S-endosymbiont is a smaller (1-2 microns) gram-negative rod and is harboured in the epithelial sheath cells in midgut. Phylogenetic characterization of S-endosymbionts from taxonomically distant insects including tsetse flies has shown that they are related to the free-living bacterium, Escherichia coli, and are members of the family Enterobacteriaceae within the gamma-3 subdivision of Proteobacteria. In this study, a polymerase chain reaction (PCR) based assay was designed utilizing the conserved sequences of 16S rDNA in order to phylogenetically characterize the mycetome-associated P-endosymbionts directly from tsetse mycetome tissue. Analysis from five species of flies representing the three major subgenera of genus Glossina indicates that P-endosymbionts constitute a distinct lineage within the gamma-3 subdivision of Proteobacteria. Mycetome endosymbiont phylogeny appears to parallel the classic taxonomic assignments independently developed for their insect host species. This suggests an ancient association for this symbiosis, which may have subsequently radiated with time, giving rise to the current species of tsetse flies and their modern-day endosymbionts. Based on endosymbiont phylogeny, the fusca flies constitute the most ancient subgenus, followed by the morsitans and palpalis groups.

Many blood-feeding insects, including tsetse flies (Diptera: Glossinidae), harbour intracellular bacterial symbionts. Using isolates from tissues of several Glossina species and diagnostic DNA oligonucleotide primers, a polymerase chain reaction (PCR) based assay was designed to identify symbiotic bacteria. Those inhabiting the midgut of Glossina were found to belong to the gamma subdivision, whereas ovarian Proteobacteria were of the alpha subdivision - probably genus Wolbachia (Rickettsiaceae). The presence of Wolbachia-like Rickettsia in the ovaries of G. morsitans subspecies may help to explain the maternally inherited incompatibility of some crosses within this species.

SUMMARYMaintaining the puparial stage of successive generations of a population of tsetse 3 °C lower than normal reduced the numbers of rickettsia-like organisms (RLO) carried by emerging flies. The susceptibility of these flies to midgut infection with Trypanosoma congolense was also significantly reduced compared with control flies held at normal temperature. These results support the view that the relationship between RLO and susceptibility is quantitative – teneral flies with heavier RLO infections being more susceptible to trypanosome infection.

Tsetse flies fed on rabbits which previously had been immunized with whole flies without symbionts showed an increase in mortality and only a small decrease in fecundity. Flies fed on rabbits immunized with symbionts only became aposymbiotic. Their fecundity decreased drastically while their longevity was not affected. The antibodies found in the flies were specifically attached to the tissues which had been used as antigens.

THE uterine or milk glands in tsetse flies (Glossina spp.) are modified female accessory reproductive glands which elaborate and release a nutritive liquid of proteinaceous and lipoid nature for the maturing intrauterine larva1–3. The multi-branched tubules of the gland converge into a pair of efferent ducts which fuse inside the oviductal shelf and open into the lumen of the uterus just posterior to the opening of the oviduct4,5. Cytological details of the milk gland and their modulations in relation to the state of pregnancy of the female have been described6 (W-C. M., D. L. D., D. S. Smith and U. Jarlfors, in preparation). Earlier work has suggested that milk is released directly into the lumen of the gland by apocrine secretion4. Our observations on the structure of the milk gland do not support such a mechanism, but rather, a novel type of exocrine discharge in which secretion is stored in an extracellular reservoir and released into the lumen through a dense cuticular network. At the points of milk release the lumen is frequently inhabited by bacteria which have not previously been described in milk glands. Our examination is based on milk glands from G. morsitans morsitans West-wood, but a comparative study using G. austeni Newstead and G. longipalpis pallidipes Austen has shown no essential differences among the three species.

A method is described for the in vitro cultivation of the rickettsia-like-organisms (RLO) from Glossina spp. which are believed to be associated with susceptibility to trypanosome infection. Cultures of RLO were established by infecting a mosquito cell line (Aedes albopictus) with haemolymph taken from teneral flies. RLO from nine species of Glossina have been isolated and maintained in continuous culture using this technique.

Tsetse flies fed on blood containing oxytetracycline, sulphaquinoxaline or lysozyme do not reproduce. It could be proved that primarily the symbionts in flies are damaged, which secondarily leads to sterility.

The tsetse fly is the insect vector for the Trypanosoma brucei parasite, the causative agent of human African trypanosomiasis. The colonization and spread of the trypanosome correlate positively with the presence of a secondary symbiotic bacterium, Sodalis glossinidius The metabolic requirements and interactions of the bacterium with its host are poorly understood, and herein we describe a metabolic model of S. glossinidius metabolism. The model enabled the design and experimental verification of a defined medium that supports S. glossinidius growth ex vivo This has been used subsequently to analyze in vitro aspects of S. glossinidius metabolism, revealing multiple unique adaptations of the symbiont to its environment. Continued dependence on a sugar, and the importance of the chitin monomer N-acetyl-d-glucosamine as a carbon and energy source, suggests adaptation to host-derived molecules. Adaptation to the amino acid-rich blood diet is revealed by a strong dependence on l-glutamate as a source of carbon and nitrogen and by the ability to rescue a predicted l-arginine auxotrophy. Finally, the selective loss of thiamine biosynthesis, a vitamin provided to the host by the primary symbiont Wigglesworthia glossinidia, reveals an intersymbiont dependence. The reductive evolution of S. glossinidius to exploit environmentally derived metabolites has resulted in multiple weaknesses in the metabolic network. These weaknesses may become targets for reagents that inhibit S. glossinidius growth and aid the reduction of trypanosomal transmission.IMPORTANCE Human African trypanosomiasis is caused by the Trypanosoma brucei parasite. The tsetse fly vector is of interest for its potential to prevent disease spread, as it is essential for T. brucei life cycle progression and transmission. The tsetse's mutualistic endosymbiont Sodalis glossinidius has a link to trypanosome establishment, providing a disease control target. Here, we describe a new, experimentally verified model of S. glossinidius metabolism. This model has enabled the development of a defined growth medium that was used successfully to test aspects of S. glossinidius metabolism. We present S. glossinidius as uniquely adapted to life in the tsetse, through its reliance on the blood diet and host-derived sugars. Additionally, S. glossinidius has adapted to the tsetse's obligate symbiont Wigglesworthia glossinidia by scavenging a vitamin it produces for the insect. This work highlights the use of metabolic modeling to design defined growth media for symbiotic bacteria and may provide novel inhibitory targets to block trypanosome transmission.

Three recent contributions in Current Biology[1xEndosymbiosis: Double-take on plastid origins. Archibald, J.M. Curr. Biol. 2006; 16: R690–R692Abstract | Full Text | Full Text PDF | PubMed | Scopus (17)See all References, 2xMinimal plastid evolution in the Paulinella endosymbiont. Yoon, H.S., Reyes-Prieto, A., Melkonian, M., and Bhattacharya, D. Curr. Biol. 2006; 16: R670–R672Abstract | Full Text | Full Text PDF | PubMed | Scopus (65)See all References, 3xPlastid origin: Replaying the tape. Rodriguez-Ezpeleta, N. and Philippe, H. Curr. Biol. 2006; 16: R54–R56Abstract | Full Text | Full Text PDF | Scopus (22)See all References] have addressed new findings on the classical cyanobacterial endosymbiont of Paulinella chromatophora, but refer to the endosymbiont as a ‘plastid’. Yoon et al.[2xMinimal plastid evolution in the Paulinella endosymbiont. Yoon, H.S., Reyes-Prieto, A., Melkonian, M., and Bhattacharya, D. Curr. Biol. 2006; 16: R670–R672Abstract | Full Text | Full Text PDF | PubMed | Scopus (65)See all References[2] even opine that Paulinella “has the honor of being the only known case of an independent primary (cyanobacterial) plastid acquisition.” Others have called the Paulinella endosymbiont a “photosynthetic organelle” [4xA plastid in the making: Evidence for a second primary endosymbiosis. Marin, B., Nowack, E.C.M., and Melkonian, M. Protist. 2005; Vol. 156: 425–432Crossref | Scopus (132)See all References[4] instead.Endosymbionts are organisms that live within other organisms. Many endosymbionts are obligate — they cannot live outside their hosts [5xMetabolic interdependence of obligate intracellular bacteria and their insect hosts. Zientz, E., Dandekar, T., and Gross, R. Microbiol. Mol. Biol. Rev. 2004; 68: 745–770Crossref | PubMed | Scopus (143)See all References[5] — as also reported for Paulinella chromatophora[2xMinimal plastid evolution in the Paulinella endosymbiont. Yoon, H.S., Reyes-Prieto, A., Melkonian, M., and Bhattacharya, D. Curr. Biol. 2006; 16: R670–R672Abstract | Full Text | Full Text PDF | PubMed | Scopus (65)See all References[2]. And many obligate endosymbionts are essential for their hosts as well [5xMetabolic interdependence of obligate intracellular bacteria and their insect hosts. Zientz, E., Dandekar, T., and Gross, R. Microbiol. Mol. Biol. Rev. 2004; 68: 745–770Crossref | PubMed | Scopus (143)See all References[5], for example Buchnera aphidicola, which supplies amino acids for its aphid host [6xGenome sequence of the endocellular bacterial symbiont of aphids Buchnera sp. APS. Shigenobu, S., Watanabe, H., Hattori, M., Sakaki, Y., and Ishikawa, H. Nature. 2000; 407: 81–86Crossref | PubMed | Scopus (739)See all References[6].Plastids, such as mitochondria, are not endosymbionts; they are organelles. They once were endosymbionts, but they now are double membrane-bounded organelles, compartments of eukaryotic cells.All of the functional proteins in the cytosol of an endosymbiont are encoded by its own genome. By contrast, only a very small fraction of the proteins that function in organelles are encoded by organellar DNA. The majority of organellar proteins are encoded by the nuclear DNA, translated on cytosolic ribosomes and imported into the organelle with the help of a protein import apparatus [7xEvolution of the molecular machines for protein import into mitochondria. Dolezal, P., Likic, V., Tachezy, J., and Lithgow, T. Science. 2006; 313: 314–318Crossref | PubMed | Scopus (320)See all References, 8xProtein import into chloroplasts. Soll, J. and Schleiff, E. Nat. Rev. Mol. Cell Biol. 2004; 5: 198–208Crossref | PubMed | Scopus (277)See all References].This evolutionarily and functionally sharp distinction between organelles and endosymbionts — protein import, or not — was crisply articulated by Cavalier-Smith and Lee [9xProtozoa as hosts for endosymbioses and the conversion of symbionts into organelles. Cavalier-Smith, T. and Lee, J.J. J. Protozool. 1985; 32: 376–379CrossrefSee all References[9]. It has proven to be exquisitely robust.Unless the Paulinella endosymbiont can be shown to possess a protein import apparatus, it is just another member in a long list of known cases of endosymbionts: the proteobacterial endosymbionts of insects such as Buchnera, Wigglesworthia, and Wolbachia[5xMetabolic interdependence of obligate intracellular bacteria and their insect hosts. Zientz, E., Dandekar, T., and Gross, R. Microbiol. Mol. Biol. Rev. 2004; 68: 745–770Crossref | PubMed | Scopus (143)See all References, 6xGenome sequence of the endocellular bacterial symbiont of aphids Buchnera sp. APS. Shigenobu, S., Watanabe, H., Hattori, M., Sakaki, Y., and Ishikawa, H. Nature. 2000; 407: 81–86Crossref | PubMed | Scopus (739)See all References, 10xThe genome sequence and evolution of the reproductive parasite Wolbachia pipientis wMel: a streamlined -Proteobacterium massively infected with mobile genetic elements. Wu, M., Sun, L., Vamathevan, J., Riegler, M., Deboy, R., Brownlie, J., McGraw, E., Mohamoud, Y., Lee, P., Berry, K. et al. PLoS Biol. 2004; 2: 327–341Crossref | Scopus (180)See all References], the methanogenic endosymbionts of anaerobic ciliates [11xSystematic and morphological diversity of endosymbiotic methanogens in anaerobic ciliates. Embley, T.M. and Finlay, B.J. Antonie Van Leeuwenhoek. 1993; 64: 261–271Crossref | PubMed | Scopus (24)See all References[11], the nitrogen-fixing symbionts in the diatom Rhopalodia[12xIntracellular spheroid bodies of Rhopalodia gibba have nitrogen-fixing apparatus of cyanobacterial origin. Prechtl, J., Kneip, C., Lockhart, P., Wenderoth, K., and Maier, U.G. Mol. Biol. Evol. 2004; 21: 1477–1481Crossref | PubMed | Scopus (58)See all References[12], the chemosynthetic endosymbiont consortia of gutless tubeworms [13xSymbiosis insights through metagenomic analysis of a microbial consortium. Woyke, T., Teeling, H., Ivanova, N.N., Hunteman, M., Richter, M., Gloeckner, F.O., Boffelli, D., Anderson, I.J., Barry, K.W., Shapiro et al. Nature. 2006; 443: 950–955Crossref | PubMed | Scopus (240)See all References[13], the cyanobacterial endosymbionts of sponges [14xImpacts of shading on sponge-Cyanobacteria symbioses: A comparison between host-specific and generalist associations. Thacker, R.W. Integr. Comp. Biol. 2005; 45: 369–376Crossref | PubMed | Scopus (3)See all References[14], and endosymbionts that live within other prokaryotes [15xIntracellular bacteria in the blue-green-alga Pleurocapsa minor. Wujek, D.E. Trans. Am. Micros. Soc. 1979; 98: 143–145CrossrefSee all References[15] — to name just very few examples.The rate-limiting step in the transition from endosymbionts to organelles would appear to be the origin of the protein import machinery itself [9xProtozoa as hosts for endosymbioses and the conversion of symbionts into organelles. Cavalier-Smith, T. and Lee, J.J. J. Protozool. 1985; 32: 376–379CrossrefSee all References[9]: the TIM and TOM complexes of mitochondria [7xEvolution of the molecular machines for protein import into mitochondria. Dolezal, P., Likic, V., Tachezy, J., and Lithgow, T. Science. 2006; 313: 314–318Crossref | PubMed | Scopus (320)See all References[7] and the TIC and TOC complexes of plastids [8xProtein import into chloroplasts. Soll, J. and Schleiff, E. Nat. Rev. Mol. Cell Biol. 2004; 5: 198–208Crossref | PubMed | Scopus (277)See all References[8].The origin of those complexes allowed each organelle to specifically import proteins synthesized in the host's cytosol, thereby allowing the endosymbionts to relinquish their prokaryotic genes without relinquishing their prokaryotic biochemistry.Calling the Paulinella endosymbiont a plastid or an organelle might make a story more exciting, but at the cost of scientific accuracy. Some proteobacterial endosymbionts of aphids have genomes smaller than those of some plastids [16xThe 160-kilobase genome of the bacterial endosymbiont Carsonella. Nakabachi, A., Yamashita, A., Toh, H., Ishikawa, H., Dunbar, H.E., Moran, N.A., and Hattori, M. Science. 2006; 314: 267Crossref | PubMed | Scopus (286)See all References[16]. Would anyone call those endosymbionts ‘mitochondria’? Hardly.For the same reasons, we should not call the Paulinella endosymbionts ‘plastids’ any more than we should say that sponges [14xImpacts of shading on sponge-Cyanobacteria symbioses: A comparison between host-specific and generalist associations. Thacker, R.W. Integr. Comp. Biol. 2005; 45: 369–376Crossref | PubMed | Scopus (3)See all References[14] have ‘plastids’. There is a difference between endosymbionts and organelles.

Background The bacterial family Enterobacteriaceae gave rise to a variety of symbiotic forms, from the loosely associated commensals, often designated as secondary (S) symbionts, to obligate mutualists, called primary (P) symbionts. Determination of the evolutionary processes behind this phenomenon has long been hampered by the unreliability of phylogenetic reconstructions within this group of bacteria. The main reasons have been the absence of sufficient data, the highly derived nature of the symbiont genomes and lack of appropriate phylogenetic methods. Due to the extremely aberrant nature of their DNA, the symbiotic lineages within Enterobacteriaceae form long branches and tend to cluster as a monophyletic group. This state of phylogenetic uncertainty is now improving with an increasing number of complete bacterial genomes and development of new methods. In this study, we address the monophyly versus polyphyly of enterobacterial symbionts by exploring a multigene matrix within a complex phylogenetic framework. Results We assembled the richest taxon sampling of Enterobacteriaceae to date (50 taxa, 69 orthologous genes with no missing data) and analyzed both nucleic and amino acid data sets using several probabilistic methods. We particularly focused on the long-branch attraction-reducing methods, such as a nucleotide and amino acid data recoding and exclusion (including our new approach and slow-fast analysis), taxa exclusion and usage of complex evolutionary models, such as nonhomogeneous model and models accounting for site-specific features of protein evolution (CAT and CAT+GTR). Our data strongly suggest independent origins of four symbiotic clusters; the first is formed by Hamiltonella and Regiella (S-symbionts) placed as a sister clade to Yersinia, the second comprises Arsenophonus and Riesia (S- and P-symbionts) as a sister clade to Proteus, the third Sodalis, Baumannia, Blochmannia and Wigglesworthia (S- and P-symbionts) as a sister or paraphyletic clade to the Pectobacterium and Dickeya clade and, finally, Buchnera species and Ishikawaella (P-symbionts) clustering with the Erwinia and Pantoea clade. Conclusions The results of this study confirm the efficiency of several artifact-reducing methods and strongly point towards the polyphyly of P-symbionts within Enterobacteriaceae. Interestingly, the model species of symbiotic bacteria research, Buchnera and Wigglesworthia, originated from closely related, but different, ancestors. The possible origins of intracellular symbiotic bacteria from gut-associated or pathogenic bacteria are suggested, as well as the role of facultative secondary symbionts as a source of bacteria that can gradually become obligate maternally transferred symbionts.