Infection of algae-free Climacostomum virens with symbiotic Chlorella sp. isolated from algae-containing C. virens

The ciliate Climacostomum virens normally contains algae as symbionts in its cytoplasm and retains them over many generations. An aposymbiotic strain of C. virens which cannot re-establish a new symbiotic association by ingestion of algae derived from green Climacostomum was recently isolated in our laboratory. Results of infection experiments showed that all newly ingested, potentially symbiotic algae were digested in food vacuoles. To clarify whether these ciliates have completely lost their ability to sustain symbiosis with algae or whether this ability can eventually be re-established, infection experiments were performed using a microinjection technique. We have achieved successful infection of algae-free Climacostomum using this method. The endosymbiont population was established in ciliates from as few as 3-5 injected algae, which have retained an intact perialgal vacuole membrane around them. To our knowledge, this is the first evidence of successful infection of aposymbiotic ciliates with algae by microinjection.     

Droplet digital PCR as a tool for investigating dynamics of cryptic symbionts

Interactions among symbiotic organisms and their hosts are major drivers of ecological and evolutionary processes. Monitoring the infection patterns among natural populations and identifying factors affecting these interactions are critical for understanding symbiont–host relationships. However, many of these interactions remain understudied since the knowledge about the symbiont species is lacking, which hinders the development of appropriate tools. In this study, we developed a digital droplet PCR (ddPCR) assay based on apicomplexan COX1 gene to detect an undescribed agamococcidian symbiont. We show that the method gives precise and reproducible results and enables detecting cryptic symbionts in low target concentration. We further exemplify the assay''s use to survey seasonally sampled natural host (Pygospio elegans) populations for symbiont infection dynamics. We found that symbiont prevalence differs spatially but does not show seasonal changes. Infection load differed between populations and was low in spring and significantly increased towards fall in all populations. We also found that the symbiont prevalence is affected by host length and population density. Larger hosts were more likely to be infected, and high host densities were found to have a lower probability of infection. The observed variations could be due to characteristics of both symbiont and host biology, especially the seasonal variation in encounter rates. Our findings show that the developed ddPCR assay is a robust tool for detecting undescribed symbionts that are otherwise difficult to quantify, enabling further insight into the impact cryptic symbionts have on their hosts.     

Ecology and evolution of symbiosis in metapopulations

We present a model for symbionts in plant host metapopulation. Symbionts are assumed not only to form a systemic infection throughout the host and pass into the host seeds, but also to reproduce and infect new plants by spores. Thus, we study a metapopulation of qualitatively identical patches coupled through seeds and spores dispersal. Symbionts that are only vertically inherited cannot persist in such a uniform environment if they lower the host's fitness. They have to be beneficial in order to coexist with the host if they are not perfectly transmitted to the seeds; but evolution selects for 100% fidelity of infection inheritance. In this model we want to see how mixed strategies (both vertical and horizontal infection) affect the coexistence of uninfected and infected plants at equilibrium; also, what would evolution do for the host, for the symbionts and for their association. We present a detailed classification of the possible equilibria with examples. The stability of the steady states is rigorously proved for the first time in a metapopulation set-up.     

Characterization of the Xanthomonas AvrXv4 effector, a SUMO protease translocated into plant cells

Homologs of the Yersinia virulence factor YopJ are found in both animal and plant bacterial pathogens, as well as in plant symbionts. The conservation of this effector family indicates that several pathogens may use YopJ-like proteins to regulate bacteria-host interactions during infection. YopJ and YopJ-like proteins share structural homology with cysteine proteases and are hypothesized to functionally mimic small ubiquitin-like modifier (SUMO) proteases in eukaryotic cells. Strains of the phytopathogenic bacterium Xanthomonas campestris pv. vesicatoria are known to possess four YopJ-like proteins, AvrXv4, AvrBsT, AvrRxv, and XopJ. In this work, we have characterized AvrXv4 to determine if AvrXv4 functions like a SUMO protease in planta during Xanthomonas-plant interactions. We provide evidence that X. campestris pv. vesicatoria secretes and translocates the AvrXv4 protein into plant cells during infection in a type III-dependent manner. Once inside the plant cell, AvrXv4 is localized to the plant cytoplasm. By performing AvrXv4 deletion and mutational analysis, we have identified amino acids required for type III delivery and for host recognition. We show that AvrXv4 recognition by resistant plants requires a functional protease catalytic core, the domain that is conserved in all of the putative YopJ-like cysteine proteases. We also show that AvrXv4 expression in planta leads to a reduction in SUMO-modified proteins, demonstrating that AvrXv4 possesses SUMO isopeptidase activity. Overall, our studies reveal that the YopJ-like effector AvrXv4 encodes a type III SUMO protease effector that is active in the cytoplasmic compartment of plant cells.     

Bacterial symbiosis and paratransgenic control of vector-borne Chagas disease

The triatomine vectors of Chagas disease are obligate haematophagous insects, feeding on vertebrate blood throughout their entire developmental cycle. As a result of obtaining their nutrition from a single food source, their diet is devoid of certain vitamins and nutrients. Consequently, these insects harbour populations of bacterial symbionts within their intestinal tract, which provide the required nutrients that are lacking from their diet. We have isolated and characterised symbiont cultures from various triatomine species and developed a method for genetically transforming them. We can then reintroduce them into their original host species, thereby producing stable paratransgenic insects in which we are able to express heterologous gene products. Using this methodology, we have generated paratransgenic Rhodnius prolixus that are refractory for infection with Trypanosoma cruzi. Two examples of potentially refractory genes are currently being expressed in paratransgenic insects. These include the insect immune peptide cecropin A and active single chain antibody fragments. We have also developed an approach that would allow introduction of genetically modified bacterial symbionts into natural populations of Chagas disease vectors. This approach utilises the coprophagic behaviour of these insects, which is the way in which the symbionts are transmitted among bug populations in nature. The production and ultimate release of transgenic or paratransgenic insects for public health applications is potentially very promising but also worthy of much careful consideration with respect to environmental, political, and human safety concerns.     

Ecology and evolution of symbiosis in metapopulations

We present a model for symbionts in plant host metapopulation. Symbionts are assumed not only to form a systemic infection throughout the host and pass into the host seeds, but also to reproduce and infect new plants by spores. Thus, we study a metapopulation of qualitatively identical patches coupled through seeds and spores dispersal. Symbionts that are only vertically inherited cannot persist in such a uniform environment if they lower the host's fitness. They have to be beneficial in order to coexist with the host if they are not perfectly transmitted to the seeds; but evolution selects for 100% fidelity of infection inheritance. In this model we want to see how mixed strategies (both vertical and horizontal infection) affect the coexistence of uninfected and infected plants at equilibrium; also, what would evolution do for the host, for the symbionts and for their association. We present a detailed classification of the possible equilibria with examples. The stability of the steady states is rigorously proved for the first time in a metapopulation set-up.     

Multiple origins of the symbioses in Paramecium bursaria

Many organisms have symbioses with photosynthetic algae as typified by corals, clams, lichens, and some protozoa. Paramecium bursaria contains green algal symbionts and this unicellular ciliate is a textbook example used for microscopic observation in junior high school science projects. We have determined molecular phylogenies for the green algal symbionts. The symbiotic algae are the main constituent of the Paramecium cytoplasm, and we have recognized a total of four species, of which two were newly discovered in the present study. One should be regarded genetically as Chlorella vulgaris, and it belongs phylogenetically to the Chlorella clade (Chlorellaceae, Trebouxiophyceae) as well as "American" and "European" groups, which we previously introduced. Their genetic dissimilarities are 0.50-0.83% in 18S rDNA comparisons, but those of the internal transcribed spacer 2 (ITS2) reach an unambiguous level (22.6-26.6%). These dissimilarities suggest that they are equivalent to discrete species derived from multiple origins as paramecian symbionts. Another newcomer was clearly separated from the Chlorellaceae, and this alga clustered with Coccomyxa spp. in ITS2 analyses. These symbiotic relations indicate multiple origins of symbionts.     

昆虫共生菌的垂直传播

共生菌普遍存在于昆虫体内,它们能够为宿主昆虫提供生长发育所必需的氨基酸、固醇类等营养物质,还能提高昆虫适应高温、寄生虫、病毒等不利环境因素的能力,昆虫则为共生菌提供稳定的生存环境和营养物质,昆虫与共生菌相互依存。多数情况下,共生菌通过垂直传播在宿主代次间进行传播,即共生菌由母代传递给子代。结合最近几年相关研究,本文综述了不同昆虫共生菌的垂直传播模式。除极少数肠道共生菌通过污染卵壳被宿主幼虫取食得以垂直传播外,垂直传播的共生菌多为经卵传播。根据侵染时期的不同,共生菌经卵传播模式多数可分为以下4种：侵染宿主昆虫幼虫中的生殖干细胞、侵染宿主昆虫年轻雌成虫中的生殖干细胞、侵染宿主昆虫雌成虫中的成熟卵母细胞以及侵染宿主昆虫囊胚期胚胎。其中,有些共生菌是以共生菌菌胞整体侵染的方式进入到宿主卵巢。另外,少数肠道共生菌也通过卵巢进行垂直传播,此类共生菌先侵染卵巢侧输卵管并在侧输卵管聚集,待卵排放至侧输卵管时再进入到卵中。在文中,我们也探讨了昆虫共生菌垂直传播过程中的细胞机制和免疫机制,包括共生菌避开宿主免疫反应、共生菌通过内吞作用进入卵巢以及不同共生菌间的协同作用等。     

Coexistence of novel gammaproteobacterial and Arsenophonus symbionts in the scale insect Greenisca brachypodii (Hemiptera,Coccomorpha: Eriococcidae)

Scale insects are commonly associated with obligate, intracellular microorganisms which play important roles in complementing their hosts with essential nutrients. Here we characterized the symbiotic system of Greenisca brachypodii, a member of the family Eriococcidae. Histological and ultrastructural analyses have indicated that G. brachypodii is stably associated with coccoid and rod‐shaped bacteria. Phylogenetic analyses have revealed that the coccoid bacteria represent a sister group to the secondary symbiont of the mealybug Melanococcus albizziae, whereas the rod‐shaped symbionts are close relatives of Arsenophonus symbionts in insects – to our knowledge, this is the first report of the presence of Arsenophonus bacterium in scale insects. As a comparison of 16S and 23S rRNA genes sequences of the G. brachypodii coccoid symbiont with other gammaprotebacterial sequences showed only low similarity (～90%), we propose the name ‘Candidatus Kotejella greeniscae’ for its tentative classification. Both symbionts are transovarially transmitted from one generation to the next. The infection takes place in the neck region of the ovariole. The bacteria migrate between follicular cells, as well as through the cytoplasm of those cells to the perivitelline space, where they form a characteristic ‘symbiont ball’. Our findings provide evidence for a polyphyletic origin of symbionts of Eriococcidae.     

Host-symbiont associations of polycystine Radiolaria: epifluorescence microscopic observation of living Radiolaria

In all 29 polycystine radiolarian species were obtained from surface seawater on May 28, 1999, using a plankton-net at one station (Site 990528; 26°37′18″N, 127°47′35″E) approximately 5 km northwest of Okinawa Island, Japan. In most polycystine radiolarians of the orders Nassellarida and Spumellarida symbiotic algae were observed under light microscopy. The light microscopic (LM) images of the symbionts, however, varied in clarity among individuals because of the variations in microanatomy of the host radiolarian cells. On the other hand, epifluorescence microscopic (EFM) observation easily detected and confirmed the existence of the algal symbionts within the host cytoplasm even in radiolarians such as Dictyocoryne truncatum (Ehrenberg) that include algal symbionts in the depth of the cytoplasm. The chloroplasts of the algal symbionts emitted autofluorescence in ultraviolet irradiation and they appeared red. That is, the autofluorescence images of the chloroplasts can be used to recognize the existence of the algal symbionts within the host radiolarians. Moreover, staining of the symbiont cells with 4′,6-diamido-2-phenylindle permitted visualization of the nucleus in the center of the symbiont cell, confirming the existence of living endosymbiotic algae within the polycystine radiolarians. Both the LM and EFM observations of eight polycystine radiolarian species revealed the specific patterns of various host-symbiont associations. (1) The investigated polycystine radiolarians all possess algal symbionts, except for one species, i.e. Dictyocoryne profunda Ehrenberg. (2) The size of the algal symbionts depends on the radiolarian species. The symbionts are largely classified into two types based on the size of their diameters, i.e. about 8–10 μm for the larger group and about 5 μm for the smaller one. (3) The algal symbionts show a variety of locations within the host radiolarian cytoplasm. The types of distribution of algal symbionts may be a useful characteristic for radiolarian taxonomy.     

The Molecular Biology of a Bacterial Endosymbiont1

ABSTRACT. The marine ciliate Parauronema acutum harbors a group of symbiotic bacteria, termed xenosomes, which reside exclusively in the cytoplasm where they grow and divide in a remarkable synchronism with the host. When released into the ambient culture medium by gentle rupture of the protozoans, the symbionts can infect homologous as well as heterologous Parauronema stocks and, oddly, Miamiensis avidus, a distantly related marine ciliate. Xenosomes from certain Parauronema stocks can kill other marine ciliates, particularly those of the genus Uronema. Our principal aims with this host-symbiont system have been to study, at the molecular level, the nature of the interaction of the xenosome with the host, infection and the killer effect. Our most recent investigations have been directed toward establishing the phylogenetic origins of the symbionts using molecular approaches. This paper summarizes our previous work and updates our more recent studies on the association of a bacterial symbiont with its protozoan host. The reader is referred to previous reviews for more details on the subject [4, 5].     

Pole cells of Drosophila paulistorum: embryologic differentiation with symbionts.

The pole cells of young D. paulistorum embryos are destined to form the germinal cells of both male and female imagoes. In addition, specialized portions of the midgut may be derived from pole cell progenitors. In this initial study of their embryogenesis by means of electron microscopy, various stages of pole cell development are shown in both non-hybrid (potentially fertile) and intersemispecific hybrid (potentially sterile as males) materials. Originally, approximately 5 or 6 cells emerge to form the early polar cap and subsequently divide asynchronously until the 35-50 cells of the late polar cap are derived. Unlike other Drosophila species, however, mycoplasma-like symbionts, apparently an hereditary infection, have been traced to locations within the cytoplasm of these pole cells. They are depicted as arriving there after transmission via the egg cytoplasm, implicating this as their probable route of entry into the future germinal tissues of adult flies. It is postulated that these microorganisms function as an infectious reproductive isolating mechanism fostering hybrid male sterility between D. paulistorum semispecies.     

Magnetosome-containing bacteria living as symbionts of bivalves

Bacteria containing magnetosomes (protein-bound nanoparticles of magnetite or greigite) are common to many sedimentary habitats, but have never been found before to live within another organism. Here, we show that octahedral inclusions in the extracellular symbionts of the marine bivalve Thyasira cf. gouldi contain iron, can exhibit magnetic contrast and are most likely magnetosomes. Based on 16S rRNA sequence analysis, T. cf. gouldi symbionts group with symbiotic and free-living sulfur-oxidizing, chemolithoautotrophic gammaproteobacteria, including the symbionts of other thyasirids. T. cf. gouldi symbionts occur both among the microvilli of gill epithelial cells and in sediments surrounding the bivalves, and are therefore facultative. We propose that free-living T. cf. gouldi symbionts use magnetotaxis as a means of locating the oxic–anoxic interface, an optimal microhabitat for chemolithoautotrophy. T. cf. gouldi could acquire their symbionts from near-burrow sediments (where oxic–anoxic interfaces likely develop due to the host''s bioirrigating behavior) using their superextensile feet, which could transfer symbionts to gill surfaces upon retraction into the mantle cavity. Once associated with their host, however, symbionts need not maintain structures for magnetotaxis as the host makes oxygen and reduced sulfur available via bioirrigation and sulfur-mining behaviors. Indeed, we show that within the host, symbionts lose the integrity of their magnetosome chain (and possibly their flagellum). Symbionts are eventually endocytosed and digested in host epithelial cells, and magnetosomes accumulate in host cytoplasm. Both host and symbiont behaviors appear important to symbiosis establishment in thyasirids.     

Embryonic pole cells and mycoplasmalike symbionts in Drosophila paulistorum

Mycoplasmalike intracellular symbionts have been located in the pole cells of Drosophila paulistorum embryos. These cells are destined to form the germ cells of both sexes. The symbionts had been previously localized in larval and adult developing and mature ovaries and testes. It is via the egg cytoplasm that these microorganisms are transmitted between generations to apparently cause an infectious and hereditary hybrid male sterility.     

Ultrastructure of the symbionts and mycetocytes of bird cherry aphid (Rhopalosiphum padi)

As is known for other aphids, the symbiont cytoplasm in Rhopalosiphum padi is differentiated into a central eccentric and a cortical region which contains electron-lucent nucleoplasm and DNA fibrils. The cytoplasm is surrounded by a plasma membrane, cell wall and perisymbiotic membrane which are separated by periplasmic spaces. The periplasmic space between the cell wall and perisymbiotic membrane contains small vesicles. The symbionts are packed into mycetocyte cells which contain a large nucleus and all the normal cell organelles. For the first time, however microtubules and microfilaments have been identified, but only at high magnification and evidence has been found which makes it possible to propose a mechanism of vesicular transport.     

Endosymbiosis of Aphids with Microorganisms: a Model Case of Dynamic Endosymbiotic Evolution

Abstract Almost all aphids harbor prokaryotic intracellular symbionts in the cytoplasm of mycetocytes, huge cells in the abdomen specialized for this purpose. The aphids and their intracellular symbionts are in close mutualistic association and unable to live without their partner. The intracellular symbionts of various aphids are of a single origin; they are descendants of a prokaryote that was acquired by the common ancestor of the present aphids. The date of establishment of the symbiotic association is estimated to be 160–280 million years ago using 16S rRNA molecular clock calibrated by aphid fossils. Molecular phylogeny indicates that the intracellular symbiont belongs to a group of gut bacteria, suggesting the possibility that it was derived from a gut microbe of aphids. While the in-tracellular symbionts are universal and highly conserved amongst aphids, other types of symbiotic microorganisms are also present. In various aphids, bacterial “secondary” intracellular symbionts are found in addition to the standard symbionts. They are thought to be acquired many times in various lineages independently. Some Cerataphidini aphids do not have intracellular symbiotic system but harbor yeast-like extracellular symbionts in the hemocoel. In a lineage of this group, symbiont replacement from intracellular prokaryote to extracellular yeast must have occurred. The diversity of the endosymbiotic system of aphids illuminates a dynamic aspect of endosymbiotic evolution.     

Population genetics of rhizobia: construction and analysis of an "Infection and Release" model

A mathematical model is created to assess the inputs of sym gene transfer of in planta multiplication and of interstrain competition into dynamics of the rhizobia populations. Their microevolution is presented as a series of the "infection and release" cycles; each cycle includes transfer of sym genes from virulent initial symbionts to avirulent local bacteria yielding the virulent novel symbionts; competition between initial symbionts and novel symbionts for the host nodulation; multiplication of initial symbionts and novel symbionts in planta and their release into soil; competition between the released novel symbionts and resident local bacteria for ex planta survival. A recurrent equation is created to determine the number of novel symbionts at each cycle of evolution of the closed bacteria-plant system. Its analysis demonstrates that under certain, really allowable values of the introduced parameters two major effects may occur: (a) rapid multiplication of novel symbionts arisen from sym gene transfer; and (b) increase of frequency of rare local bacteria genotypes after acquisition of virulence. Multiplication of very rare strains (p<10(-19)) in the plant-associated bacteria population is possible at certain parameters of the system. Variation of the sizes of bacteria populations and of the parameters for interstrain competition may influence the evolutionary rate of the bacteria population. The "infection and release" model represents a selective mechanism which may be responsible for a high taxonomic diversity of rhizobia and for a panmictic structure of their populations.     

Complex symbiotic systems of two treehopper species: Centrotus cornutus (Linnaeus, 1758) and Gargara genistae (Fabricius, 1775) (Hemiptera: Cicadomorpha: Membracoidea: Membracidae)

The aim of the conducted study was to describe the symbiotic systems (the types of symbionts, distribution in the body of the host insect, the transovarial transmission between generations) of two treehoppers: Centrotus cornutus and Gargara genistae by means of microscopic and molecular techniques. We found that each of them is host to four species of bacteriome-inhabiting symbionts. In C. cornutus, ancestral bacterial symbionts Sulcia and Nasuia are accompanied by an additional symbiont—the bacterium Arsenophonus. In the bacteriomes of G. genistae, apart from Sulcia and Nasuia, bacterium Serratia is present. To our knowledge, this is the first report regarding the occurrence of Serratia as a symbiont in Hemiptera: Auchenorrhyncha. Bacteria Sulcia and Nasuia are harbored in their own bacteriocytes, whereas Arsenophonus and Serratia both inhabit their own bacteriocytes and also co-reside with bacteria Nasuia. We observed that both bacteria Arsenophonus and Serratia undergo autophagic degradation. We found that in both of the species examined, in the cytoplasm and nuclei of all of the cells of the bacteriome, bacteria Rickettsia are present. Our histological and ultrastructural observations revealed that all the bacteriome-associated symbionts of C. cornutus and G. genistae are transovarially transmitted from mother to offspring.     

Facultative symbiont infections affect aphid reproduction

Some bacterial symbionts alter their hosts reproduction through various mechanisms that enhance their transmission in the host population. In addition to its obligatory symbiont Buchnera aphidicola, the pea aphid Acyrthosiphon pisum harbors several facultative symbionts influencing several aspects of host ecology. Aphids reproduce by cyclical parthenogenesis whereby clonal and sexual reproduction alternate within the annual life cycle. Many species, including the pea aphid, also show variation in their reproductive mode at the population level, with some lineages reproducing by cyclical parthenogenesis and others by permanent parthenogenesis. While the role of facultative symbionts has been well studied during the parthenogenetic phase of their aphid hosts, very little is known on their possible influence during the sexual phase. Here we investigated whether facultative symbionts modulate the capacity to produce sexual forms in various genetic backgrounds of the pea aphid with controlled symbiont composition and also in different aphid genotypes from natural populations with previously characterized infection status and reproductive mode. We found that most facultative symbionts exhibited detrimental effects on their hosts fitness under sex-inducing conditions in comparison with the reference lines. We also showed that the loss of sexual phase in permanently parthenogenetic lineages of A. pisum was not explained by facultative symbionts. Finally, we demonstrated that Spiroplasma infection annihilated the production of males in the host progeny by inducing a male-killing phenotype, an unexpected result for organisms such as aphids that reproduce primarily through clonal reproduction.     

Vertically transmitted symbionts in structured host metapopulations

We develop a structured metapopulation model for vertically transmitted symbionts in natural host populations. We focus primarily on two questions: Are mutualism and high transmission probability prerequisites for the survival of symbionts in structured host metapopulations? What are the ecological conditions under which coexistence of infected and uninfected hosts is possible? We start with studying in depth the case of qualitatively identical patches and derive conditions for invasion and coexistence of uninfected and infected hosts. Our model predicts that, in a qualitatively uniform environment, coexistence is possible only if the symbionts increase the fitness of their host, so the mutualism is indeed needed for coexistence. We also prove that evolution selects for 100% infection frequency in the metapopulation. Then we generalize the model for different patch qualities and get conditions for invasion in a virgin environment.