A new C-type lectin similar to the human immunoreceptor DC-SIGN mediates symbiont acquisition by a marine nematode

Although thiotrophic symbioses have been intensively studied for the last three decades, nothing is known about the molecular mechanisms of symbiont acquisition. We used the symbiosis between the marine nematode Laxus oneistus and sulfur-oxidizing bacteria to study this process. In this association a monolayer of symbionts covers the whole cuticle of the nematode, except its anterior-most region. Here, we identify a novel Ca(2+)-dependent mannose-specific lectin that was exclusively secreted onto the posterior, bacterium-associated region of L. oneistus cuticle. A recombinant form of this lectin induced symbiont aggregation in seawater and was able to compete with the native lectin for symbiont binding in vivo. Surprisingly, the carbohydrate recognition domain of this mannose-binding protein was similar both structurally and functionally to a human dendritic cell-specific immunoreceptor. Our results provide a molecular link between bacterial symbionts and host-secreted mucus in a marine symbiosis and suggest conservation in the mechanisms of host-microbe interactions throughout the animal kingdom.     

Sequence variability of the pattern recognition receptor Mermaid mediates specificity of marine nematode symbioses

Selection of a specific microbial partner by the host is an all-important process. It guarantees the persistence of highly specific symbioses throughout host generations. The cuticle of the marine nematode Laxus oneistus is covered by a single phylotype of sulfur-oxidizing bacteria. They are embedded in a layer of host-secreted mucus containing the mannose-binding protein Mermaid. This Ca²⁺-dependent lectin mediates symbiont aggregation and attachment to the nematode. Here, we show that Stilbonema majum—a symbiotic nematode co-occurring with L. oneistus in shallow water sediment—is covered by bacteria phylogenetically distinct to those covering L. oneistus. Mermaid cDNA analysis revealed extensive protein sequence variability in both the nematode species. We expressed three recombinant Mermaid isoforms, which based on the structural predictions display the most different carbohydrate recognition domains (CRDs). We show that the three CRDs (DNT, DDA and GDA types) possess different affinities for L. oneistus and S. majum symbionts. In particular, the GDA type, exclusively expressed by S. majum, displays highest agglutination activity towards its symbionts and lowest towards its L. oneistus symbionts. Moreover, incubation of L. oneistus in the GDA type does not result in complete symbiont detachment, whereas incubation in the other types does. This indicates that the presence of particular Mermaid isoforms on the nematode surface has a role in the attachment of specific symbionts. This is the first report of the functional role of sequence variability in a microbe-associated molecular patterns receptor in a beneficial association.     

A Lipidomic Approach to Understanding Free Fatty Acid Lipogenesis Derived from Dissolved Inorganic Carbon within Cnidarian-Dinoflagellate Symbiosis

The cnidarian-dinoflagellate symbiosis is arguably one of the most important within the marine environment in that it is integral to the formation of coral reefs. However, the regulatory processes that perpetuate this symbiosis remain unresolved. It is essential to understand these processes, if we are to elucidate the mechanisms that support growth and resource accumulation by coral host, and conversely, recently observed reduction and/or mortality of corals in response to rapid environmental change. This study specifically focused on one area of metabolic activity within the symbiosis, that of free fatty acid synthesis within both the dinoflagellate symbionts and cnidarian host. The main model system used was Aiptasia pulchella and Symbiodinium sp. in combination with aposymbiotic A. pulchella, the symbiotic coral Acropora millepora system and dinoflagellate culture. Fatty acids (FAs) were selected because of their multiple essential roles inclusive of energy storage (resource accumulation), membrane structure fluidity and cell signaling. The study addressed free FA lipogenesis by using a new method of enriched stable isotopic (¹³C) incorporation from dissolved inorganic carbon (DI¹³C) combined with HPLC-MS. FAs derived from DI¹³C aligned with a mixture of known lipogenesis pathways with the addition of some unusual FAs. After 120 hr, ¹³C-enriched FA synthesis rates were attributed to only a complex integration of both n–3 and n–6 lipogenesis pathways within the dinoflagellate symbionts. Furthermore, there was no detectible evidence of symbiont derived enriched isotope fatty acids, catabolized ¹³C derivatives or DI¹³C being directly utilized, in host late n–6 pathway long-chain FA lipogenesis. These findings do not align with a popular mutualistic translocation model with respect to the use of translocated symbiont photoassimilates in host long-chain FA lipogenesis, which has important connotations for linking nutrient sources with metabolite production and the dynamic regulation of this symbiosis.     

Specific Midgut Region Controlling the Symbiont Population in an Insect-Microbe Gut Symbiotic Association

Many insects possess symbiotic bacteria that affect the biology of the host. The level of the symbiont population in the host is a pivotal factor that modulates the biological outcome of the symbiotic association. Hence, the symbiont population should be maintained at a proper level by the host''s control mechanisms. Several mechanisms for controlling intracellular symbionts of insects have been reported, while mechanisms for controlling extracellular gut symbionts of insects are poorly understood. The bean bug Riptortus pedestris harbors a betaproteobacterial extracellular symbiont of the genus Burkholderia in the midgut symbiotic organ designated the M4 region. We found that the M4B region, which is directly connected to the M4 region, also harbors Burkholderia symbiont cells, but the symbionts therein are mostly dead. A series of experiments demonstrated that the M4B region exhibits antimicrobial activity, and the antimicrobial activity is specifically potent against the Burkholderia symbiont but not the cultured Burkholderia and other bacteria. The antimicrobial activity of the M4B region was detected in symbiotic host insects, reaching its highest point at the fifth instar, but not in aposymbiotic host insects, which suggests the possibility of symbiont-mediated induction of the antimicrobial activity. This antimicrobial activity was not associated with upregulation of antimicrobial peptides of the host. Based on these results, we propose that the M4B region is a specialized gut region of R. pedestris that plays a critical role in controlling the population of the Burkholderia gut symbiont. The molecular basis of the antimicrobial activity is of great interest and deserves future study.     

Cell surface carbohydrates of symbiotic dinoflagellates and their role in the establishment of cnidarian–dinoflagellate symbiosis

Symbiodiniaceae algae are often photosymbionts of reef-building corals. The establishment of their symbiosis resembles a microbial infection where eukaryotic pattern recognition receptors (e.g. lectins) are thought to recognize a specific range of taxon-specific microbial-associated molecular patterns (e.g. glycans). The present study used the sea anemone, Exaiptasia diaphana and three species of Symbiodiniaceae (the homologous Breviolum minutum, the heterologous-compatible Cladocopium goreaui and the heterologous-incompatible Fugacium kawagutii) to compare the surface glycomes of three symbionts and explore the role of glycan–lectin interactions in host–symbiont recognition and establishment of symbiosis. We identified the nucleotide sugars of the algal cells, then examined glycans on the cell wall of the three symbiont species with monosaccharide analysis, lectin array technology and fluorescence microscopy of the algal cell decorated with fluorescently tagged lectins. Armed with this inventory of possible glycan moieties, we then assayed the ability of the three Symbiodiniaceae to colonize aposymbiotic E. diaphana after modifying the surface of one of the two partners. The Symbiodiniaceae cell-surface glycome varies among algal species. Trypsin treatment of the alga changed the rate of B. minutum and C. goreaui uptake, suggesting that a protein-based moiety is an essential part of compatible symbiont recognition. Our data strongly support the importance of D-galactose (in particular β-D-galactose) residues in the establishment of the cnidarian–dinoflagellate symbiosis, and we propose a potential involvement of L-fucose, D-xylose and D-galacturonic acid in the early steps of this mutualism.Subject terms: Cellular microbiology, Molecular biology     

Bacterial Symbiont Transmission in the Wood-Boring Shipworm Bankia setacea (Bivalvia: Teredinidae)

The Teredinidae (shipworms) are a morphologically diverse group of marine wood-boring bivalves that are responsible each year for millions of dollars of damage to wooden structures in estuarine and marine habitats worldwide. They exist in a symbiosis with cellulolytic nitrogen-fixing bacteria that provide the host with the necessary enzymes for survival on a diet of wood cellulose. These symbiotic bacteria reside in distinct structures lining the interlamellar junctions of the gill. This study investigated the mode by which these nutritionally essential bacterial symbionts are acquired in the teredinid Bankia setacea. Through 16S ribosomal DNA (rDNA) sequencing, the symbiont residing within the B. setacea gill was phylogenetically characterized and shown to be distinct from previously described shipworm symbionts. In situ hybridization using symbiont-specific 16S rRNA-directed probes bound to bacterial ribosome targets located within the host gill coincident with the known location of the gill symbionts. These specific probes were then used as primers in a PCR-based assay which consistently detected bacterial rDNA in host gill (symbiont containing), gonad tissue, and recently spawned eggs, demonstrating the presence of symbiont cells in host ovary and offspring. These results suggest that B. setacea ensures successful inoculation of offspring through a vertical mode of symbiont transmission and thereby enables a broad distribution of larval settlement.     

Insect Gut Symbiont Susceptibility to Host Antimicrobial Peptides Caused by Alteration of the Bacterial Cell Envelope

The molecular characterization of symbionts is pivotal for understanding the cross-talk between symbionts and hosts. In addition to valuable knowledge obtained from symbiont genomic studies, the biochemical characterization of symbionts is important to fully understand symbiotic interactions. The bean bug (Riptortus pedestris) has been recognized as a useful experimental insect gut symbiosis model system because of its cultivatable Burkholderia symbionts. This system is greatly advantageous because it allows the acquisition of a large quantity of homogeneous symbionts from the host midgut. Using these naïve gut symbionts, it is possible to directly compare in vivo symbiotic cells with in vitro cultured cells using biochemical approaches. With the goal of understanding molecular changes that occur in Burkholderia cells as they adapt to the Riptortus gut environment, we first elucidated that symbiotic Burkholderia cells are highly susceptible to purified Riptortus antimicrobial peptides. In search of the mechanisms of the increased immunosusceptibility of symbionts, we found striking differences in cell envelope structures between cultured and symbiotic Burkholderia cells. The bacterial lipopolysaccharide O antigen was absent from symbiotic cells examined by gel electrophoretic and mass spectrometric analyses, and their membranes were more sensitive to detergent lysis. These changes in the cell envelope were responsible for the increased susceptibility of the Burkholderia symbionts to host innate immunity. Our results suggest that the symbiotic interactions between the Riptortus host and Burkholderia gut symbionts induce bacterial cell envelope changes to achieve successful gut symbiosis.     

Can maternally inherited endosymbionts adapt to a novel host? Direct costs of Spiroplasma infection,but not vertical transmission efficiency,evolve rapidly after horizontal transfer into D. melanogaster

Maternally inherited symbionts are common in arthropods and many have important roles in host adaptation. The observation that specific symbiont lineages infect distantly related host species implies new interactions are commonly established by lateral transfer events. However, studies have shown that symbionts often perform poorly in novel hosts. We hypothesized selection on the symbiont may be sufficiently rapid that poor performance in a novel host environment is rapidly ameliorated, permitting symbiont maintenance. Here, we test this prediction for a Spiroplasma strain transinfected into the novel host Drosophila melanogaster. In the generations immediately following transinfection, the symbiont had low transmission efficiency to offspring and imposed severe fitness costs on its host. We observed that effects on host fitness evolved rapidly, being undetectable after 17 generations in the novel host, whereas vertical transmission efficiency was poorly responsive over this period. Our results suggest that long-term symbiosis may more readily be established in cases where symbionts perform poorly in just one aspect of symbiosis.     

Genomic versatility and functional variation between two dominant heterotrophic symbionts of deep-sea Osedax worms

An unusual symbiosis, first observed at ∼3000 m depth in the Monterey Submarine Canyon, involves gutless marine polychaetes of the genus Osedax and intracellular endosymbionts belonging to the order Oceanospirillales. Ecologically, these worms and their microbial symbionts have a substantial role in the cycling of carbon from deep-sea whale fall carcasses. Microheterogeneity exists among the Osedax symbionts examined so far, and in the present study the genomes of the two dominant symbionts, Rs1 and Rs2, were sequenced. The genomes revealed heterotrophic versatility in carbon, phosphate and iron uptake, strategies for intracellular survival, evidence for an independent existence, and numerous potential virulence capabilities. The presence of specific permeases and peptidases (of glycine, proline and hydroxyproline), and numerous peptide transporters, suggests the use of degraded proteins, likely originating from collagenous bone matter, by the Osedax symbionts. ¹³C tracer experiments confirmed the assimilation of glycine/proline, as well as monosaccharides, by Osedax. The Rs1 and Rs2 symbionts are genomically distinct in carbon and sulfur metabolism, respiration, and cell wall composition, among others. Differences between Rs1 and Rs2 and phylogenetic analysis of chemotaxis-related genes within individuals of symbiont Rs1 revealed the influence of the relative age of the whale fall environment and support possible local niche adaptation of ‘free-living'' lifestages. Future genomic examinations of other horizontally-propogated intracellular symbionts will likely enhance our understanding of the contribution of intraspecific symbiont diversity to the ecological diversification of the intact association, as well as the maintenance of host diversity.     

Trans-generational specificity within a cnidarian–algal symbiosis

Ocean warming and other anthropogenic stresses threaten the symbiosis between tropical reef cnidarians and their dinoflagellate endosymbionts (Symbiodinium). Offspring of many cnidarians acquire their algal symbionts from the environment, and such flexibility could allow corals to respond to environmental changes between generations. To investigate the effect of both habitat and host genotype on symbiont acquisition, we transplanted aposymbiotic offspring of the common Caribbean octocoral Briareum asbestinum to (1) an environmentally different habitat that lacked B. asbestinum and (2) an environmentally similar habitat where local adults harbored Symbiodinium phylotypes that differed from parental colonies. Symbiont acquisition and establishment of symbioses over time was followed using a within-clade DNA marker (23S chloroplast rDNA) and a within-phylotype marker (unique alleles at a single microsatellite locus). Early in the symbiosis, B. asbestinum juveniles harbored multiple symbiont phylotypes, regardless of source (parent or site). However, with time (~4 yr), offspring established symbioses with the symbiont phylotype dominant in the parental colonies, regardless of transplant location. Within-phylotype analyses of the symbionts revealed a similar pattern, with offspring acquiring the allelic variant common in symbionts in the parental population regardless of the environment in which the offspring was reared. These data suggest that in this host species, host–symbiont specificity is a genetically determined trait. If this level of specificity is widespread among other symbiotic cnidarians, many cnidarian–algal symbioses may not be able to respond to rapid, climate change-associated environmental changes by means of between-generation switching of symbionts.     

Dual Symbiosis in a Bathymodiolus sp. Mussel from a Methane Seep on the Gabon Continental Margin (Southeast Atlantic): 16S rRNA Phylogeny and Distribution of the Symbionts in Gills

Deep-sea mussels of the genus Bathymodiolus (Bivalvia: Mytilidae) harbor symbiotic bacteria in their gills and are among the dominant invertebrate species at cold seeps and hydrothermal vents. An undescribed Bathymodiolus species was collected at a depth of 3,150 m in a newly discovered cold seep area on the southeast Atlantic margin, close to the Zaire channel. Transmission electron microscopy, comparative 16S rRNA analysis, and fluorescence in situ hybridization indicated that this Bathymodiolus sp. lives in a dual symbiosis with sulfide- and methane-oxidizing bacteria. A distinct distribution pattern of the symbiotic bacteria in the gill epithelium was observed, with the thiotrophic symbiont dominating the apical region and the methanotrophic symbiont more abundant in the basal region of the bacteriocytes. No variations in this distribution pattern or in the relative abundances of the two symbionts were observed in mussels collected from three different mussel beds with methane concentrations ranging from 0.7 to 33.7 μM. The 16S rRNA sequence of the methanotrophic symbiont is most closely related to those of known methanotrophic symbionts from other bathymodiolid mussels. Surprisingly, the thiotrophic Bathymodiolus sp. 16S rRNA sequence does not fall into the monophyletic group of sequences from thiotrophic symbionts of all other Bathymodiolus hosts. While these mussel species all come from vents, this study describes the first thiotrophic sequence from a seep mussel and shows that it is most closely related (99% sequence identity) to an environmental clone sequence obtained from a hydrothermal plume near Japan.     

Diversification of endosymbiosis: replacements,co-speciation and promiscuity of bacteriocyte symbionts in weevils

The processes and mechanisms underlying the diversification of host–microbe endosymbiotic associations are of evolutionary interest. Here we investigated the bacteriocyte-associated primary symbionts of weevils wherein the ancient symbiont Nardonella has experienced two independent replacement events: once by Curculioniphilus symbiont in the lineage of Curculio and allied weevils of the tribe Curculionini, and once by Sodalis-allied symbiont in the lineage of grain weevils of the genus Sitophilus. The Curculioniphilus symbiont was detected from 27 of 36 Curculionini species examined, the symbiont phylogeny was congruent with the host weevil phylogeny, and the symbiont gene sequences exhibited AT-biased nucleotide compositions and accelerated molecular evolution. These results suggest that the Curculioniphilus symbiont was acquired by an ancestor of the tribe Curculionini, replaced the original symbiont Nardonella, and has co-speciated with the host weevils over evolutionary time, but has been occasionally lost in several host lineages. By contrast, the Sodalis-allied symbiont of Sitophilus weevils exhibited no host–symbiont co-speciation, no AT-biased nucleotide compositions and only moderately accelerated molecular evolution. These results suggest that the Sodalis-allied symbiont was certainly acquired by an ancestor of the Sitophilus weevils and replaced the original Nardonella symbiont, but the symbiotic association must have experienced occasional re-associations such as new acquisitions, horizontal transfers, replacements and/or losses. We detected Sodalis-allied facultative symbionts in populations of the Curculionini weevils, which might represent potential evolutionary sources of the Sodalis-allied primary symbionts. Comparison of these newcomer bacteriocyte-associated symbiont lineages highlights potential evolutionary trajectories and consequences of novel symbionts after independent replacements of the same ancient symbiont.     

Phylogenetic Analysis and Fluorescence <Emphasis Type="Italic">In Situ</Emphasis> Hybridization Detection of Archaeal and Bacterial Endosymbionts in the Anaerobic Ciliate <Emphasis Type="Italic">Trimyema Compressum</Emphasis>

The anaerobic free-living ciliate, Trimyema compressum, is known to harbor both methanogenic archaeal and bacterial symbionts in the cytoplasm. To clarify their phylogenetic belongings, a full-cycle rRNA approach was applied to this symbiosis. Phylogenetic analysis showed that the methanogenic symbiont was related to Methanobrevibacter arboriphilicus, which was distantly related to symbionts found in other Trimyema species. This result suggested that Trimyema species do not require very specific methanogenic symbionts, and symbiont replacement could have occurred in the history of Trimyema species. On the other hand, the bacterial symbiont was located near the lineage of the family Syntrophomonadaceae in the phylum Firmicutes. The sequence similarity between the bacterial symbiont and the nearest species was 85%, indicating that bacterial symbionts may be specific to the Trimyema species. The elimination of bacterial symbionts from the ciliate cell by antibiotic treatment resulted in considerably decreased host growth. However, it was not restored by stigmasterol addition (<2 μg ml⁻¹), which was different from the previous report that showed that the symbiont-free strain required exogenous sterols for growth. In addition, the decline of host growth was not accompanied by host metabolism shift toward the formation of more reduced products, which suggested that the contribution of bacterial symbionts to the host ciliate was not a dispose of excessive reducing equivalent arising from the host’s fermentative metabolism as methanogenic symbionts do. This study showed that bacterial symbionts make a significant contribution to the host ciliate by an unknown function and suggested that interactions between bacterial symbionts and T. compressum are more complicated than hitherto proposed.     

Characterization of chemoautotrophic bacterial symbionts in a gutless marine worm Oligochaeta, Annelida) by phylogenetic 16S rRNA sequence analysis and in situ hybridization.

The phylogenetic relationships of chemoautotrophic endosymbionts in the gutless marine oligochaete Inanidrilus leukodermatus to chemoautotrophic ecto- and endosymbionts from other host phyla and to free-living bacteria were determined by comparative 16S rRNA sequence analysis. Fluorescent in situ hybridization confirmed that the 16S rRNA sequence obtained from these worms originated from the symbionts. The symbiont sequence is unique to I. leukodermatus. In phylogenetic trees inferred by both distance and parsimony methods, the oligochaete symbiont is peripherally associated with one of two clusters of chemoautotrophic symbionts that belong to the gamma subdivision of the Proteobacteria. The endosymbionts of this oligochaete form a monophyletic group with chemoautotrophic ectosymbionts of a marine nematode. The oligochaete and nematode symbionts are very closely related, although their hosts belong to separate, unrelated animal phyla. Thus, cospeciation between the nematode and oligochaete hosts and their symbionts could not have occurred. Instead, the similar geographic locations and habitats of the hosts may have influenced the establishment of these symbioses.     

Molecular evidence of symbiotic activity between <Emphasis Type="Italic">Symbiodinium</Emphasis> and <Emphasis Type="Italic">Tridacna maxima</Emphasis> larvae

Dinoflagellates in the genus Symbiodinium (zooxanthellae) provide the photosynthesis that sustains the majority of primary production in coral reefs. They occur symbiotically with several phyla, including mollusks such as giant clams (Tridacna spp.). This mutualistic association is obligatory for the giant clams, but the exact point in which this symbiosis is established and the main translocated photosynthate are unknown. In this study, we tracked the expression of specific genes for symbiosis and glycerol synthesis during a time course experiment. Giant clam larvae were raised until 75 h post-fertilization and then infected with cultured isolates of Symbiodinium clade A3. Expression of symbiosis-specific and housekeeping genes was monitored at four time points. The expression of H⁺-ATPase, a symbiosis-specific gene in Symbiodinium, was observed at 24 h after symbiont acquisition by the clam larvae. The expression of an enzyme responsible for glycerol synthesis was also observed. Together, these results show that the symbiotic relationship was already in place 24 h after Symbiodinium acquisition, during veliger larval stage. This is the first report using a molecular symbiosis-specific marker that supports symbiotic activity between Symbiodinium and a metazoan larva of an organism that acquires symbionts horizontally. From the expression of the glycerol-synthesizing gene, it was qualitatively determined that Symbiodinium cells may produce glycerol regardless of whether they are free-living or in symbiosis.     

Flexibility of nutritional strategies within a mutualism: food availability affects algal symbiont productivity in two congeneric sea anemone species

Mutualistic symbioses are common, especially in nutrient-poor environments where an association between hosts and symbionts can allow the symbiotic partners to persist and collectively out-compete non-symbiotic species. Usually these mutualisms are built on an intimate transfer of energy and nutrients (e.g. carbon and nitrogen) between host and symbiont. However, resource availability is not consistent, and the benefit of the symbiotic association can depend on the availability of resources to mutualists. We manipulated the diets of two temperate sea anemone species in the genus Anthopleura in the field and recorded the responses of sea anemones and algal symbionts in the family Symbiodiniaceae to our treatments. Algal symbiont density, symbiont volume and photosynthetic efficiency of symbionts responded to changes in sea anemone diet, but the responses depended on the species of sea anemone. We suggest that temperate sea anemones and their symbionts can respond to changes in anemone diet, modifying the balance between heterotrophy and autotrophy in the symbiosis. Our data support the hypothesis that symbionts are upregulated or downregulated based on food availability, allowing for a flexible nutritional strategy based on external resources.     

Investigating the causes and consequences of symbiont shuffling in a multi-partner reef coral symbiosis under environmental change

Dynamic symbioses may critically mediate impacts of climate change on diverse organisms, with repercussions for ecosystem persistence in some cases. On coral reefs, increases in heat-tolerant symbionts after thermal bleaching can reduce coral susceptibility to future stress. However, the relevance of this adaptive response is equivocal owing to conflicting reports of symbiont stability and change. We help reconcile this conflict by showing that change in symbiont community composition (symbiont shuffling) in Orbicella faveolata depends on the disturbance severity and recovery environment. The proportion of heat-tolerant symbionts dramatically increased following severe experimental bleaching, especially in a warmer recovery environment, but tended to decrease if bleaching was less severe. These patterns can be explained by variation in symbiont performance in the changing microenvironments created by differentially bleached host tissues. Furthermore, higher proportions of heat-tolerant symbionts linearly increased bleaching resistance but reduced photochemical efficiency, suggesting that any change in community structure oppositely impacts performance and stress tolerance. Therefore, even minor symbiont shuffling can adaptively benefit corals, although fitness effects of resulting trade-offs are difficult to predict. This work helps elucidate causes and consequences of dynamism in symbiosis, which is critical to predicting responses of multi-partner symbioses such as O. faveolata to environmental change.     

Intraspecific variation in immune gene expression and heritable symbiont density

Host genetic variation plays an important role in the structure and function of heritable microbial communities. Recent studies have shown that insects use immune mechanisms to regulate heritable symbionts. Here we test the hypothesis that variation in symbiont density among hosts is linked to intraspecific differences in the immune response to harboring symbionts. We show that pea aphids (Acyrthosiphon pisum) harboring the bacterial endosymbiont Regiella insecticola (but not all other species of symbionts) downregulate expression of key immune genes. We then functionally link immune expression with symbiont density using RNAi. The pea aphid species complex is comprised of multiple reproductively-isolated host plant-adapted populations. These ‘biotypes’ have distinct patterns of symbiont infections: for example, aphids from the Trifolium biotype are strongly associated with Regiella. Using RNAseq, we compare patterns of gene expression in response to Regiella in aphid genotypes from multiple biotypes, and we show that Trifolium aphids experience no downregulation of immune gene expression while hosting Regiella and harbor symbionts at lower densities. Using F1 hybrids between two biotypes, we find that symbiont density and immune gene expression are both intermediate in hybrids. We propose that in this system, Regiella symbionts are suppressing aphid immune mechanisms to increase their density, but that some hosts have adapted to prevent immune suppression in order to control symbiont numbers. This work therefore suggests that antagonistic coevolution can play a role in host-microbe interactions even when symbionts are transmitted vertically and provide a clear benefit to their hosts. The specific immune mechanisms that we find are downregulated in the presence of Regiella have been previously shown to combat pathogens in aphids, and thus this work also highlights the immune system’s complex dual role in interacting with both beneficial and harmful microbes.     

Bacterial Endosymbiosis in a Chordate Host: Long-Term Co-Evolution and Conservation of Secondary Metabolism

Intracellular symbiosis is known to be widespread in insects, but there are few described examples in other types of host. These symbionts carry out useful activities such as synthesizing nutrients and conferring resistance against adverse events such as parasitism. Such symbionts persist through host speciation events, being passed down through vertical transmission. Due to various evolutionary forces, symbionts go through a process of genome reduction, eventually resulting in tiny genomes where only those genes essential to immediate survival and those beneficial to the host remain. In the marine environment, invertebrates such as tunicates are known to harbor complex microbiomes implicated in the production of natural products that are toxic and probably serve a defensive function. Here, we show that the intracellular symbiont Candidatus Endolissoclinum faulkneri is a long-standing symbiont of the tunicate Lissoclinum patella, that has persisted through cryptic speciation of the host. In contrast to the known examples of insect symbionts, which tend to be either relatively recent or ancient relationships, the genome of Ca. E. faulkneri has a very low coding density but very few recognizable pseudogenes. The almost complete degradation of intergenic regions and stable gene inventory of extant strains of Ca. E. faulkneri show that further degradation and deletion is happening very slowly. This is a novel stage of genome reduction and provides insight into how tiny genomes are formed. The ptz pathway, which produces the defensive patellazoles, is shown to date to before the divergence of Ca. E. faulkneri strains, reinforcing its importance in this symbiotic relationship. Lastly, as in insects we show that stable symbionts can be lost, as we describe an L. patella animal where Ca. E. faulkneri is displaced by a likely intracellular pathogen. Our results suggest that intracellular symbionts may be an important source of ecologically significant natural products in animals.     

Breaking the language barrier: experimental evolution of non-native <Emphasis Type="Italic">Vibrio fischeri</Emphasis> in squid tailors luminescence to the host

Although most Vibrio fischeri isolates are capable of symbiosis, the coevolution of certain strains with the Hawaiian bobtail squid, Euprymna scolopes, has led to specific adaptation to this partnership. For instance, strains from different hosts or from a planktonic environment are ineffective squid colonists. Even though bioluminescence is a symbiotic requirement, curiously, symbionts of E. scolopes are dim in culture relative to fish symbionts and free-living isolates. It is unclear whether this dim phenotype is related to the symbiosis or simply coincidental. To further explore the basis of symbiont specificity, we developed an experimental evolution model that utilizes the daily light organ venting behavior of the squid and horizontal acquisition of symbionts for serial passage of cultures. We passaged six populations each derived from the squid-naïve strains of V. fischeri MJ11 (a fish symbiont) and WH1 (a free-living isolate) through a series of juvenile squid light organs. After 15 serially colonized squid for each population, or an estimated 290–360 bacterial generations, we isolated representatives of the light organ populations and characterized their bioluminescence. Multiple evolved lines of both strains produced significantly less bioluminescence both in vitro and in vivo. This reduction in bioluminescence did not correlate with reduced quorum sensing for most isolates tested. The remarkable phenotypic convergence with squid symbionts further emphasizes the importance of bioluminescence in this symbiosis, and suggests that reduced light production is a specific adaptation to the squid.