The Cellular and Physiological Basis of Host-Symbiont Specificity in a Model Cnidarian-Dinoflagellate Symbiosis

<p>The ability of corals to form novel partnerships with symbionts that may be better suited to new environmental conditions is an important factor when assessing the ability for corals to adapt to climate change. However, relatively little attention has been given to the effects of hosting different symbiont types on holobiont physiology, competitive interactions between these symbionts, or the capacity of the host to regulate populations of different symbionts. Such factors likely play an important role in patterns of host-symbiont specificity and flexibility, and hence the potential for corals to respond to climate change. The aim of this research was to characterise the cellular and physiological events associated with hosting different symbiont species in Exaiptasia pallida (commonly referred to as ‘Aiptasia’), a model cnidarian-dinoflagellate symbiosis, and how these events might contribute to host-symbiont specificity. The specific objectives were to: (1) determine the effect of symbiont species on the population dynamics of host colonisation and holobiont physiology; (2) measure the competitiveness of the homologous symbiont versus heterologous symbionts, under both control and elevated temperatures; and quantify the ability of the host to regulate its symbiont population in response to homologous versus heterologous symbiont taxa (3) via host-cell apoptosis and (4) via symbiont cell cycle regulation. To explore this, aposymbiotic (i.e. symbiont-free) individuals of Aiptasia were first inoculated with one of five Symbiodinium taxa (the homologous S. minutum or heterologous S. microadriaticum, phylotype C3, S. trenchii or S. voratum), and the rates and patterns of colonisation assessed. Proliferation success inside the anemone was different between symbionts, with the homologous S. minutum being the most successful species, while Symbiodinium C3 and S. voratum struggled or failed to form a long-lasting symbiosis. The spatial pattern of symbiont colonisation was identical for all the successful Symbiodinium taxa, however the timing differed between these different symbionts. Symbiont identity also had an effect on holobiont fitness, as S. microadriaticum and S. trenchii were less beneficial to the host compared to S. minutum, as indicated by lower rates of photosynthesis, anemone growth and pedal laceration (i.e. asexual reproduction). The taxon-specific differences demonstrated here provided a basis for the subsequent thesis chapters, leading to questions about how the different symbionts might compete with one another and be regulated by the host. The competitiveness of the homologous symbiont relative to heterologous ones, and hence the ability of the host to ‘switch’ and ‘shuffle’ its symbiont population, was tested by inoculating aposymbiotic sea anemones either with simultaneous or sequential mixtures of thermally tolerant and sensitive Symbiodinium and exposing them to control versus elevated temperatures. The homologous species was dominant regardless of temperature, outcompeting the heterologous, thermally tolerant S. microadriaticum and S. trenchii. This result indicates that the high level of specificity seen between Aiptasia and S. minutum in the Pacific Ocean may result, in part, from a reluctance to form new symbioses, even if such associations have the potential to confer a degree of thermal tolerance that may be beneficial under future climate change. The differential success of the different symbionts was also reflected in the host’s apoptotic response to their presence in its tissues, as measured via caspase-3 activity. In particular, anemones hosting S. minutum and S. microadriaticum exhibited lower levels of caspase activity than those hosting S. trenchii and S. voratum throughout symbiosis establishment, consistent with symbiont proliferation success. The general pattern of caspase activity during the 28-days colonisation period was similar, however, with induction of caspase-3 activity upon inoculation, followed by a marked decline in activity over the subsequent week, and then an increase (either moderate or marked depending upon symbiont identity) across the remainder of the colonisation period measured. Host cell apoptosis therefore likely plays an important role in determining the compatibility and fate of different Symbiodinium taxa in a host, and the potential for establishing novel symbioses. In contrast to the apparent importance of host apoptosis, symbiont cell cycle control did not seem to play an important role in determining the different rates of symbiont colonisation observed. Flow cytometry was used to determine the relative proportion of cells in the different phases of the cell cycle (i.e. G1, G2, S, M), with all symbiont taxa exhibiting the same pattern of cell cycle progression. In particular, more cells were in the S and G2/M phases combined than in G1 during the first two weeks of colonisation, but this changed as colonisation progressed, when a greater proportion cells were in the G1 phase. This indicates that symbiont cell division becomes limited in the later stages of colonisation as symbiont density increases, consistent with increasing resource limitation. This thesis provides valuable insights into the regulation of the cnidarian-dinoflagellate symbiosis, and the events that contribute to host-symbiont specificity. In particular, it suggests that through cellular control and physiological impacts on the host (and hence the overall symbiosis), there is likely limited potential to establish new host-symbiont partnerships that allow for adaptation to our warming climate. The next step is now to further elucidate the relative importance of post-phagocytosis control mechanisms, and to test the generality of my findings by extending them from the model Aiptasia system to reef corals.</p>

The Cellular and Physiological Basis of Host-Symbiont Specificity in a Model Cnidarian-Dinoflagellate Symbiosis

<p>The ability of corals to form novel partnerships with symbionts that may be better suited to new environmental conditions is an important factor when assessing the ability for corals to adapt to climate change. However, relatively little attention has been given to the effects of hosting different symbiont types on holobiont physiology, competitive interactions between these symbionts, or the capacity of the host to regulate populations of different symbionts. Such factors likely play an important role in patterns of host-symbiont specificity and flexibility, and hence the potential for corals to respond to climate change. The aim of this research was to characterise the cellular and physiological events associated with hosting different symbiont species in Exaiptasia pallida (commonly referred to as ‘Aiptasia’), a model cnidarian-dinoflagellate symbiosis, and how these events might contribute to host-symbiont specificity. The specific objectives were to: (1) determine the effect of symbiont species on the population dynamics of host colonisation and holobiont physiology; (2) measure the competitiveness of the homologous symbiont versus heterologous symbionts, under both control and elevated temperatures; and quantify the ability of the host to regulate its symbiont population in response to homologous versus heterologous symbiont taxa (3) via host-cell apoptosis and (4) via symbiont cell cycle regulation. To explore this, aposymbiotic (i.e. symbiont-free) individuals of Aiptasia were first inoculated with one of five Symbiodinium taxa (the homologous S. minutum or heterologous S. microadriaticum, phylotype C3, S. trenchii or S. voratum), and the rates and patterns of colonisation assessed. Proliferation success inside the anemone was different between symbionts, with the homologous S. minutum being the most successful species, while Symbiodinium C3 and S. voratum struggled or failed to form a long-lasting symbiosis. The spatial pattern of symbiont colonisation was identical for all the successful Symbiodinium taxa, however the timing differed between these different symbionts. Symbiont identity also had an effect on holobiont fitness, as S. microadriaticum and S. trenchii were less beneficial to the host compared to S. minutum, as indicated by lower rates of photosynthesis, anemone growth and pedal laceration (i.e. asexual reproduction). The taxon-specific differences demonstrated here provided a basis for the subsequent thesis chapters, leading to questions about how the different symbionts might compete with one another and be regulated by the host. The competitiveness of the homologous symbiont relative to heterologous ones, and hence the ability of the host to ‘switch’ and ‘shuffle’ its symbiont population, was tested by inoculating aposymbiotic sea anemones either with simultaneous or sequential mixtures of thermally tolerant and sensitive Symbiodinium and exposing them to control versus elevated temperatures. The homologous species was dominant regardless of temperature, outcompeting the heterologous, thermally tolerant S. microadriaticum and S. trenchii. This result indicates that the high level of specificity seen between Aiptasia and S. minutum in the Pacific Ocean may result, in part, from a reluctance to form new symbioses, even if such associations have the potential to confer a degree of thermal tolerance that may be beneficial under future climate change. The differential success of the different symbionts was also reflected in the host’s apoptotic response to their presence in its tissues, as measured via caspase-3 activity. In particular, anemones hosting S. minutum and S. microadriaticum exhibited lower levels of caspase activity than those hosting S. trenchii and S. voratum throughout symbiosis establishment, consistent with symbiont proliferation success. The general pattern of caspase activity during the 28-days colonisation period was similar, however, with induction of caspase-3 activity upon inoculation, followed by a marked decline in activity over the subsequent week, and then an increase (either moderate or marked depending upon symbiont identity) across the remainder of the colonisation period measured. Host cell apoptosis therefore likely plays an important role in determining the compatibility and fate of different Symbiodinium taxa in a host, and the potential for establishing novel symbioses. In contrast to the apparent importance of host apoptosis, symbiont cell cycle control did not seem to play an important role in determining the different rates of symbiont colonisation observed. Flow cytometry was used to determine the relative proportion of cells in the different phases of the cell cycle (i.e. G1, G2, S, M), with all symbiont taxa exhibiting the same pattern of cell cycle progression. In particular, more cells were in the S and G2/M phases combined than in G1 during the first two weeks of colonisation, but this changed as colonisation progressed, when a greater proportion cells were in the G1 phase. This indicates that symbiont cell division becomes limited in the later stages of colonisation as symbiont density increases, consistent with increasing resource limitation. This thesis provides valuable insights into the regulation of the cnidarian-dinoflagellate symbiosis, and the events that contribute to host-symbiont specificity. In particular, it suggests that through cellular control and physiological impacts on the host (and hence the overall symbiosis), there is likely limited potential to establish new host-symbiont partnerships that allow for adaptation to our warming climate. The next step is now to further elucidate the relative importance of post-phagocytosis control mechanisms, and to test the generality of my findings by extending them from the model Aiptasia system to reef corals.</p>

Frequent Drivers, Occasional Passengers: Signals of Symbiont-Driven Seasonal Adaptation and Hitchhiking in the Pea Aphid, Acyrthosiphon pisum

Insects harbor a variety of maternally inherited bacterial symbionts. As such, variation in symbiont presence/absence, in the combinations of harbored symbionts, and in the genotypes of harbored symbiont species provide heritable genetic variation of potential use in the insects’ adaptive repertoires. Understanding the natural importance of symbionts is challenging but studying their dynamics over time can help to elucidate the potential for such symbiont-driven insect adaptation. Toward this end, we studied the seasonal dynamics of six maternally transferred bacterial symbiont species in the multivoltine pea aphid (Acyrthosiphon pisum). Our sampling focused on six alfalfa fields in southeastern Pennsylvania, and spanned 14 timepoints within the 2012 growing season, in addition to two overwintering periods. To test and generate hypotheses on the natural relevance of these non-essential symbionts, we examined whether symbiont dynamics correlated with any of ten measured environmental variables from the 2012 growing season, including some of known importance in the lab. We found that five symbionts changed prevalence across one or both overwintering periods, and that the same five species underwent such frequency shifts across the 2012 growing season. Intriguingly, the frequencies of these dynamic symbionts showed robust correlations with a subset of our measured environmental variables. Several of these trends supported the natural relevance of lab-discovered symbiont roles, including anti-pathogen defense. For a seventh symbiont—Hamiltonella defensa—studied previously across the same study periods, we tested whether a reported correlation between prevalence and temperature stemmed not from thermally varying host-level fitness effects, but from selection on co-infecting symbionts or on aphid-encoded alleles associated with this bacterium. In general, such “hitchhiking” effects were not evident during times with strongly correlated Hamiltonella and temperature shifts. However, we did identify at least one time period in which Hamiltonella spread was likely driven by selection on a co-infecting symbiont—Rickettsiella viridis. Recognizing the broader potential for such hitchhiking, we explored selection on co-infecting symbionts as a possible driver behind the dynamics of the remaining six species. Out of twelve examined instances of symbiont dynamics unfolding across 2-week periods or overwintering spans, we found eight in which the focal symbiont underwent parallel frequency shifts under single infection and one or more co-infection contexts. This supported the idea that phenotypic variation created by the presence/absence of individual symbionts is a direct target for selection, and that symbiont effects can be robust under co-habitation with other symbionts. Contrastingly, in two cases, we found that selection may target phenotypes emerging from symbiont co-infections, with specific species combinations driving overall trends for the focal dynamic symbionts, without correlated change under single infection. Finally, in three cases—including the one described above for Hamiltonella—our data suggested that incidental co-infection with a (dis)favored symbiont could lead to large frequency shifts for “passenger” symbionts, conferring no apparent cost or benefit. Such hitchhiking has rarely been studied in heritable symbiont systems. We propose that it is more common than appreciated, given the widespread nature of maternally inherited bacteria, and the frequency of multi-species symbiotic communities across insects.

Global biogeography of chemosynthetic symbionts reveals both localized and globally distributed symbiont groups

In the ocean, most hosts acquire their symbionts from the environment. Due to the immense spatial scales involved, our understanding of the biogeography of hosts and symbionts in marine systems is patchy, although this knowledge is essential for understanding fundamental aspects of symbiosis such as host–symbiont specificity and evolution. Lucinidae is the most species-rich and widely distributed family of marine bivalves hosting autotrophic bacterial endosymbionts. Previous molecular surveys identified location-specific symbiont types that “promiscuously” form associations with multiple divergent cooccurring host species. This flexibility of host–microbe pairings is thought to underpin their global success, as it allows hosts to form associations with locally adapted symbionts. We used metagenomics to investigate the biodiversity, functional variability, and genetic exchange among the endosymbionts of 12 lucinid host species from across the globe. We report a cosmopolitan symbiont species, Candidatus Thiodiazotropha taylori, associated with multiple lucinid host species. Ca. T. taylori has achieved more success at dispersal and establishing symbioses with lucinids than any other symbiont described thus far. This discovery challenges our understanding of symbiont dispersal and location-specific colonization and suggests both symbiont and host flexibility underpin the ecological and evolutionary success of the lucinid symbiosis.

A Phylogeny-Informed Analysis of the Global Coral-Symbiodiniaceae Interaction Network Reveals that Traits Correlated with Thermal Bleaching Are Specific to Symbiont Transmission Mode

ABSTRACT The complex network of associations between corals and their dinoflagellates (family Symbiodiniaceae) are the basis of coral reef ecosystems but are sensitive to increasing global temperatures. Coral-symbiont interactions are restricted by ecological and evolutionary determinants that constrain partner choice and influence holobiont response to environmental stress; however, little is known about how these processes shape thermal resilience of the holobiont. Here, we built a network of global coral-Symbiodiniaceae associations, mapped species traits (e.g., symbiont transmission mode and biogeography) and phylogenetic relationships of both partners onto the network, and assigned thermotolerance to both host and symbiont nodes. Using network analysis and phylogenetic comparative methods, we determined the contribution of species traits to thermal resilience of the holobiont, while accounting for evolutionary patterns among species. We found that the network shows nonrandom interactions among species, which are shaped by evolutionary history, symbiont transmission mode (horizontally transmitted [HT] or vertically transmitted [VT] corals) and biogeography. Coral phylogeny, but not Symbiodiniaceae phylogeny, symbiont transmission mode, or biogeography, was a good predictor of thermal resilience. Closely related corals have similar Symbiodiniaceae interaction patterns and bleaching susceptibilities. Nevertheless, the association patterns that explain increased host thermal resilience are not generalizable across the entire network but are instead unique to HT and VT corals. Under nonstress conditions, thermally resilient VT coral species associate with thermotolerant phylotypes and limit their number of unique symbionts and overall symbiont thermotolerance diversity, while thermally resilient HT coral species associate with a few host-specific symbiont phylotypes. IMPORTANCE Recent advances have revealed a complex network of interactions between coral and Symbiodiniaceae. Specifically, nonrandom association patterns, which are determined in part by restrictions imposed by symbiont transmission mode, increase the sensitivity of the overall network to thermal stress. However, little is known about the extent to which coral-Symbiodiniaceae network resistance to thermal stress is shaped by host and symbiont species phylogenetic relationships and host and symbiont species traits, such as symbiont transmission mode. We built a frequency-weighted global coral-Symbiodiniaceae network and used network analysis and phylogenetic comparative methods to show that evolutionary relatedness, but not transmission mode, predicts thermal resilience of the coral-Symbiodiniaceae holobiont. Consequently, thermal stress events could result in nonrandom pruning of susceptible lineages and loss of taxonomic diversity with catastrophic effects on community resilience to future events. Our results show that inclusion of the contribution of evolutionary and ecological processes will further our understanding of the fate of coral assemblages under climate change.

Interaction of Arsenophonus with Wolbachia in Nilaparvata lugens

Background Co-infection of endosymbionts in the same host is ubiquitous, and the interactions of the most common symbiont Wolbachia with other symbionts, including Spiroplasma, in invertebrate organisms have received increasing attention. However, the interactions between Wolbachia and Arsenophonus, another widely distributed symbiont in nature, are poorly understood. We tested the co-infection of Wolbachia and Arsenophonus in different populations of Nilaparvata lugens and investigated whether co-infection affected the population size of the symbionts in their host. Results A significant difference was observed in the co-infection incidence of Wolbachia and Arsenophonus among 5 populations of N. lugens from China, with nearly half of the individuals in the Zhenjiang population harbouring the two symbionts simultaneously, and the rate of occurrence was significantly higher than that of the other 4 populations. The Arsenophonus density in the superinfection line was significantly higher only in the Maanshan population compared with that of the single-infection line. Differences in the density of Wolbachia and Arsenophonus were found in all the tested double-infection lines, and the dominant symbiont species varied with the population only in the Nanjing population, with Arsenophonus the overall dominant symbiont. Conclusions Wolbachia and Arsenophonus could coexist in N. lugens, and the co-infection incidence varied with the geographic populations. Antagonistic interactions were not observed between Arsenophonus and Wolbachia, and the latter was the dominant symbiont in most populations.

Variation in Ectosymbiont Assemblages Associated with Rock Pigeons (Columba livia) from Coast to Coast in Canada

When a species colonizes a new area, it has the potential to bring with it an array of smaller-bodied symbionts. Rock Pigeons (Columba livia Gmelin) have colonized most of Canada and are found in almost every urban center. In its native range, C. livia hosts more than a dozen species of ectosymbiotic arthropods, and some of these lice and mites have been reported from Rock Pigeons in the United States. Despite being so abundant and widely distributed, there are only scattered host-symbiont records for rock pigeons in Canada. Here we sample Rock Pigeons from seven locations across Canada from the west to east (a distance of > 4000 km) to increase our knowledge of the distribution of their ectosymbionts. Additionally, because ectosymbiont abundance can be affected by temperature and humidity, we looked at meteorological variables for each location to assess whether they were correlated with ectosymbiont assemblage structure. We found eight species of mites associated with different parts of the host’s integument: the feather dwelling mites Falculifer rostratus (Buchholz), Pterophagus columbae (Sugimoto) and Diplaegidia columbae (Buchholz); the skin mites: Harpyrhynchoides gallowayi Bochkov, OConnor and Klompen, H. columbae (Fain), and Ornithocheyletia hallae Smiley; and the nasal mites Tinaminyssus melloi (Castro) and T. columbae (Crossley). We also found five species of lice: Columbicola columbae (Linnaeus), Campanulotes compar (Burmeister), Coloceras tovornikae Tendeiro, Hohorstiella lata Piaget, and Bonomiella columbae Emerson. All 13 ectosymbiont species were found in the two coastal locations of Vancouver (British Columbia) and Halifax (Nova Scotia). The symbiont species found in all sampling locations were the mites O. hallae, H. gallowayi, T. melloi and T. columbae, and the lice Colu. columbae and Camp. compar. Three local meteorological variables were significantly correlated with mite assemblage structure: annual minimum and maximum temperatures and maximum humidity in the month the pigeon was collected. Two local meteorological variables, annual maximum and average temperatures, were significantly correlated with louse assemblages. Our results suggest that milder climatic conditions may affect richness and assemblage structure of ectosymbiont assemblages associated with Rock Pigeons in Canada.

Presence and Genetic Identity of Symbiodiniaceae in the Bioeroding Sponge Genera Cliona and Spheciospongia (Clionaidae) in the Spermonde Archipelago (SW Sulawesi), Indonesia

Members of the family Symbiodiniaceae form symbiotic relationships with several metazoan groups on coral reefs, most notably scleractinian corals. However, despite their importance to the health of coral reefs, their relationship with other host organisms such as bioeroding sponges (Clionaidae) is still relatively understudied. In this study we investigate the presence and identity of Symbiodiniaceae in Clionaidae species in Indonesia and evaluate findings related to the evolution and ecology of the host-symbiont relationship. Clionaidae were collected throughout the Spermonde Archipelago in Indonesia. Morphological and molecular techniques were used to identify the sponge host (28S ribosomal DNA) and their Symbiodiniaceae symbionts (ITS2). Seven Clionaidae species were found, of which four species contained Symbiodiniaceae. Cliona aff. orientalis, Cliona thomasi and Spheciospongia maeandrina were host to Cladocopium, while Spheciospongia digitata contained Durusdinium and Freudenthalidium. In the remaining species: Cliona sp., Cliona utricularis and Spheciospongia trincomaliensis no evidence of the presence of Symbiodiniaceae was found. Our results provide the first record of Symbiodiniaceae in the sponge genus Spheciospongia. Additionally, we provide the first findings of Freudenthalidium and first molecular evidence of Durusdinium in bioeroding sponges. Our results indicate coevolution between Spheciospongia digitata, Spheciospongia maeandrina and their symbionts. We discuss that the diversity of Symbiodiniaceae within bioeroding sponges is likely far greater than currently reported in literature. Considering the threat bioeroding sponges can pose to the health of coral reefs, it is crucial to understand Symbiodiniaceae diversity within Clionaidae and their effect on the functioning of Clionaidae species. We propose that the identity of the symbiont species is mostly related to the host species, but we did observe a potential case of environmental adaptation related to environmental stressors.

Interaction of Arsenophonus with Wolbachia in Nilaparvata lugens

Background Co-infection of endosymbionts in the same host is ubiquitous, and the interactions of the most common symbiont Wolbachia with other symbionts, including Spiroplasma et al., in invertebrate organisms have received increasing attention. However, the interactions between Wolbachia and Arsenophonus, another widely distributed symbiont in nature, are poorly understood. We tested the co-infection of Wolbachia and Arsenophonus in different populations of Nilaparvata lugens and investigated whether co-infection affected the population size of the symbionts in their host.Results A significant difference was observed in the co-infection incidence of Wolbachia and Arsenophonus among 5 populations of N. lugens from China, with nearly half of the individuals in the Zhenjiang population harbouring the two symbionts simultaneously, and the rate of occurrence was significantly higher than that of the other 4 populations. The Arsenophonus density in the superinfection line was significantly higher only in the Maanshan population compared with that of the single-infection line. Differences in the density of Wolbachia and Arsenophonus were found in all the tested double-infection lines, and the dominant symbiont species varied with the population only in the Nanjing population, with Arsenophonus the overall dominant symbiont. Conclusions Wolbachia and Arsenophonus could coexist in N. lugens, and the co-infection incidence varied with the geographic populations. Antagonistic interactions were not observed between Arsenophonus and Wolbachia, and the latter was the dominant symbiont in most populations.