Symbiont Population Research Articles

Attenuation of host NO production by MAMPs potentiates development of the host in the squid-vibrio symbiosis

Bacterial pathogens typically upregulate the host's production of nitric oxide synthase (NOS) and nitric oxide (NO) as antimicrobial agents, a response that is often mediated by microbe-associated molecular patterns (MAMPs) of the pathogen. In contrast, previous studies of the beneficial Euprymna scolopes/Vibrio fischeri symbiosis demonstrated that symbiont colonization results in attenuation of host NOS/NO, which occurs in high levels in hatchling light organs. Here, we sought to determine whether V. fischeri MAMPs, specifically lipopolysaccharide (LPS) and the peptidoglycan derivative tracheal cytotoxin (TCT), attenuate NOS/NO, and whether this activity mediates the MAMPs-induced light organ morphogenesis. Using confocal microscopy, we characterized levels of NOS with immunocytochemistry and NO with a NO-specific fluorochrome. When added exogenously to seawater containing hatchling animals, V. fischeri LPS and TCT together, but not individually, induced normal NOS/NO attenuation. Further, V. fischeri mutants defective in TCT release did not. Experiments with NOS inhibitors and NO donors provided evidence that NO mediates apoptosis and morphogenesis associated with symbiont colonization. Attenuation of NOS/NO by LPS and TCT in the squid-vibrio symbiosis provides another example of how the host's response to MAMPs depends on the context. These data also provide a mechanism by which symbiont MAMPs regulate host development.

Phylogeographical patterns among Mediterranean sepiolid squids and their Vibrio symbionts: environment drives specificity among sympatric species.

Bobtail squid from the genera Sepiola and Rondeletiola (Cephalopoda: Sepiolidae) form mutualistic associations with luminous Gram-negative bacteria (Gammaproteobacteria: Vibrionaceae) from the genera Vibrio and Photobacterium. Symbiotic bacteria proliferate inside a bilobed light organ until they are actively expelled by the host into the surrounding environment on a diel basis. This event results in a dynamic symbiont population with the potential to establish the symbiosis with newly hatched sterile (axenic) juvenile sepiolids. In this study, we examined the genetic diversity found in populations of sympatric sepiolid squid species and their symbionts by the use of nested clade analysis with multiple gene analyses. Variation found in the distribution of different species of symbiotic bacteria suggests a strong influence of abiotic factors in the local environment, affecting bacterial distribution among sympatric populations of hosts. These abiotic factors include temperature differences incurred by a shallow thermocline, as well as a lack of strong coastal water movement accompanied by seasonal temperature changes in overlapping niches. Host populations are stable and do not appear to have a significant role in the formation of symbiont populations relative to their distribution across the Mediterranean Sea. Additionally, all squid species examined (Sepiola affinis, S. robusta, S. ligulata, S. intermedia, and Rondeletiola minor) are genetically distinct from one another regardless of location and demonstrate very little intraspecific variation within species. These findings suggest that physical boundaries and distance in relation to population size, and not host specificity, are important factors in limiting or defining gene flow within sympatric marine squids and their associated bacterial symbionts in the Mediterranean Sea.

Environmental Symbiont Acquisition May Not Be the Solution to Warming Seas for Reef-Building Corals

BackgroundCoral reefs worldwide are in decline. Much of the mortality can be attributed to coral bleaching (loss of the coral's intracellular photosynthetic algal symbiont) associated with global warming. How corals will respond to increasing oceanic temperatures has been an area of extensive study and debate. Recovery after a bleaching event is dependent on regaining symbionts, but the source of repopulating symbionts is poorly understood. Possibilities include recovery from the proliferation of endogenous symbionts or recovery by uptake of exogenous stress-tolerant symbionts.Methodology/Principal FindingsTo test one of these possibilities, the ability of corals to acquire exogenous symbionts, bleached colonies of Porites divaricata were exposed to symbiont types not normally found within this coral and symbiont acquisition was monitored. After three weeks exposure to exogenous symbionts, these novel symbionts were detected in some of the recovering corals, providing the first experimental evidence that scleractinian corals are capable of temporarily acquiring symbionts from the water column after bleaching. However, the acquisition was transient, indicating that the new symbioses were unstable. Only those symbiont types present before bleaching were stable upon recovery, demonstrating that recovery was from the resident in situ symbiont populations.Conclusions/SignificanceThese findings suggest that some corals do not have the ability to adjust to climate warming by acquiring and maintaining exogenous, more stress-tolerant symbionts. This has serious ramifications for the success of coral reefs and surrounding ecosystems and suggests that unless actions are taken to reverse it, climate change will lead to decreases in biodiversity and a loss of coral reefs.

Real-Time PCR Reveals Endosymbiont Titer Fluctuations inMetaseiulus occidentalis(Acari: Phytoseiidae) Colonies Held at Different Temperatures

Real-time PCR (Polymerase Chain Reaction) can estimate Wolbachia endosymbiont population density in mosquitoes (Wiwatanaratanabutr & Kittayapong 2009). The COS (?arbaryl-OP-Sul fur resistant) colony of the phytoseiid Metaseiulus (=Typhlodromus or Gcdendromus) occidentalis (Nesbitt) harbors several endosymbionts, includ ing Cardinium, Wolbachia, Enterobacter, and Bacteroidetes in their gut and reproductive tis sues (Johanowicz & Hoy 1996; Hoy & Jeyaprakash 2005). Johanowicz & Hoy (1998) ob served mating incompatibility when females from a heat-treated (33?C) COS colony (a treatment presumed to eliminate Wolbachia) was crossed with males from a room-temperature (23?C) COS colony containing Wolbachia, resulting in the pro duction of shriveled eggs that did not hatch. It was not clear whether the Wolbachia population was reduced or completely eliminated from the heat-treated mites because a standard PCR pro tocol was used, which is relatively insensitive compared to high-fidelity PCR (Jeyaprakash & Hoy 2000). In this paper, we estimate the endo symbiont population density in COS colonies of M. occidentalis held under different tempera tures in the laboratory using real-time PCR. One colony of M. occidentalis was maintained at room-temperature (23-25?C) and 2 colonies ini tiated from this line were maintained at 32 and 34?C for More than 1 year in growth chambers prior to experiments. Female mites (100) were isolated from each colony and their genomic DNA extracted by Puregene and QIAGEN methods (Jeyaprakash & Hoy 2007), and resuspended in 50 pL of sterile water. Four different plasmids carrying endosymbiont 16S rRNA sequences; pAJ233 (Cardinium), pAJ234 (Bacteroidetes), pAJ235 (Wolbachia) and pAJ239 (Enterobacter), were extracted and their yield estimated with a BioPhotometer Plus (Eppendorf AG, Hamburg, Germany). Species-specific primers were de signed with Primer 3 software (http:// frodo.wi.mit.edu/primer3/) and a probe was de signed with Primer X software (Applied Biosys tems, Foster City, CA) from the variable regions for each species (Table 1). Real-time PCR was per formed in a 20-pL reaction volume containing ge nomic DNA from 2 females (1 pL) or serially-di luted plasmid DNA (100 pg to 1 fg) or a no DNA control (1 pL), forward and reverse primers (400 pM), probe (100 pM) and TaqMan master mix (10 pL). Two linked profiles: (1) one cycle of 50?C for 2 min and 95?C for 10 min, and (2) 60 cycles consist ing of denaturation at 95?C for 15 s, annealing at 59?C (Cardinium) or 57?C (Wolbachia or Entero bacter or Bacteroidetes) for 30 s and extension at 72?C for 30 s, were used. Each treatment was rep l cated 3 times. The starting copy number of all plasmid DNA dilutions used in the real-time PCR was obtained with an open-source software (http:/

Stable isotopic records of bleaching and endolithic algae blooms in the skeleton of the boulder forming coral Montastraea faveolata

Within boulder forming corals, fixation of dissolved inorganic carbon is performed by symbiotic dinoflagellates within the coral tissue and, to a lesser extent, endolithic algae within the coral skeleton. Endolithic algae produce distinctive green bands in the coral skeleton, and their origin may be related to periods of coral bleaching due to complete loss of dinoflagellate symbionts or “paling” in which symbiont populations are patchily reduced in coral tissue. Stable carbon isotopes were analyzed in coral skeletons across a known bleaching event and 12 blooms of endolithic algae to determine whether either of these types of changes in photosynthesis had a clear isotopic signature. Stable carbon isotopes tended to be enriched in the coral skeleton during the initiation of endolith blooms, consistent with enhanced photosynthesis by endoliths. In contrast, there were no consistent δ13C patterns directly associated with bleaching, suggesting that there is no unique isotopic signature of bleaching. On the other hand, isotopic values after bleaching were lighter 92% of the time when compared to the bleaching interval. This marked drop in skeletal δ13C may reflect increased kinetic fractionation and slow symbiont recolonization for several years after bleaching.

Dual symbiosis of the vent shrimp Rimicaris exoculata with filamentous gamma‐ and epsilonproteobacteria at four Mid‐Atlantic Ridge hydrothermal vent fields

The shrimp Rimicaris exoculata from hydrothermal vents on the Mid-Atlantic Ridge (MAR) harbours bacterial epibionts on specialized appendages and the inner surfaces of its gill chamber. Using comparative 16S rRNA sequence analysis and fluorescence in situ hybridization (FISH), we examined the R. exoculata epibiosis from four vents sites along the known distribution range of the shrimp on the MAR. Our results show that R. exoculata lives in symbiosis with two types of filamentous epibionts. One belongs to the Epsilonproteobacteria, and was previously identified as the dominant symbiont of R. exoculata. The second is a novel gammaproteobacterial symbiont that belongs to a clade consisting exclusively of sequences from epibiotic bacteria of hydrothermal vent animals, with the filamentous sulfur oxidizer Leucothrix mucor as the closest free-living relative. Both the epsilon- and the gammaproteobacterial symbionts dominated the R. exoculata epibiosis at all four MAR vent sites despite striking differences between vent fluid chemistry and distances between sites of up to 8500 km, indicating that the symbiosis is highly stable and specific. Phylogenetic analyses of two mitochondrial host genes showed little to no differences between hosts from the four vent sites. In contrast, there was significant spatial structuring of both the gamma- and the epsilonproteobacterial symbiont populations based on their 16S rRNA gene sequences that was correlated with geographic distance along the MAR. We hypothesize that biogeography and host-symbiont selectivity play a role in structuring the epibiosis of R. exoculata.

Deep sequencing reveals exceptional diversity and modes of transmission for bacterial sponge symbionts

Marine sponges contain complex bacterial communities of considerable ecological and biotechnological importance, with many of these organisms postulated to be specific to sponge hosts. Testing this hypothesis in light of the recent discovery of the rare microbial biosphere, we investigated three Australian sponges by massively parallel 16S rRNA gene tag pyrosequencing. Here we show bacterial diversity that is unparalleled in an invertebrate host, with more than 250 000 sponge-derived sequence tags being assigned to 23 bacterial phyla and revealing up to 2996 operational taxonomic units (95% sequence similarity) per sponge species. Of the 33 previously described ‘sponge-specific’ clusters that were detected in this study, 48% were found exclusively in adults and larvae – implying vertical transmission of these groups. The remaining taxa, including ‘Poribacteria’, were also found at very low abundance among the 135 000 tags retrieved from surrounding seawater. Thus, members of the rare seawater biosphere may serve as seed organisms for widely occurring symbiont populations in sponges and their host association might have evolved much more recently than previously thought.

Microsatellite loci for Symbiodinium A3 (S. fitti) a common algal symbiont among Caribbean Acropora (stony corals) and Indo-Pacific giant clams (Tridacna)

We developed ten polymorphic microsatellite loci for S. fitti (type A3), and tested their utility on a Caribbean population associated with the coral Acropora palmata and an Indo-Pacific population harbored by the giant clam Tridacna maxima. Our analyses identified from 3 to 8 alleles for each haploid locus. Diversity (R) indices were 0.48 for the Indo-Pacific and 0.81 for the Caribbean. Greater than 95% of the samples possessed a single, symbiont, multilocus genotype (MLG). Among their many uses for the study of coral-algal symbioses, population genetic markers provide insight on the potential for symbiont dispersal, can be used to assess symbiont population stability/longevity in hospite, and partition symbiont diversity into reproductively isolated operational taxonomic units (i.e. species).

Host control over infection and proliferation of a cheater symbiont

Host control mechanisms are thought to be critical for selecting against cheater mutants in symbiont populations. Here, we provide the first experimental test of a legume host's ability to constrain the infection and proliferation of a native-occurring rhizobial cheater. Lotus strigosus hosts were experimentally inoculated with pairs of Bradyrhizobium strains that naturally vary in symbiotic benefit, including a cheater strain that proliferates in the roots of singly infected hosts, yet provides zero growth benefits. Within co-infected hosts, the cheater exhibited lower infection rates than competing beneficial strains and grew to smaller population sizes within those nodules. In vitro assays revealed that infection-rate differences among competing strains were not caused by variation in rhizobial growth rate or interstrain toxicity. These results can explain how a rapidly growing cheater symbiont--that exhibits a massive fitness advantage in single infections--can be prevented from sweeping through a beneficial population of symbionts.

Origins of cheating and loss of symbiosis in wildBradyrhizobium

Rhizobial bacteria nodulate legume roots and fix nitrogen in exchange for photosynthates. These symbionts are infectiously acquired from the environment and in such cases selection models predict evolutionary spread of uncooperative mutants. Uncooperative rhizobia - including nonfixing and non-nodulating strains - appear common in agriculture, yet their population biology and origins remain unknown in natural soils. Here, a phylogenetically broad sample of 62 wild-collected rhizobial isolates was experimentally inoculated onto Lotus strigosus to assess their nodulation ability and effects on host growth. A cheater strain was discovered that proliferated in host tissue while offering no benefit; its fitness was superior to that of beneficial strains. Phylogenetic reconstruction of Bradyrhizobium rDNA and transmissible symbiosis-island loci suggest that the cheater evolved via symbiotic gene transfer. Many strains were also identified that failed to nodulate L. strigosus, and it appears that nodulation ability on this host has been recurrently lost in the symbiont population. This is the first study to reveal the adaptive nature of rhizobial cheating and to trace the evolutionary origins of uncooperative rhizobial mutants.

Phenotypic Variance Predicts Symbiont Population Densities in Corals: A Modeling Approach

BackgroundWe test whether the phenotypic variance of symbionts (Symbiodinium) in corals is closely related with the capacity of corals to acclimatize to increasing seawater temperatures. Moreover, we assess whether more specialist symbionts will increase within coral hosts under ocean warming. The present study is only applicable to those corals that naturally have the capacity to support more than one type of Symbiodinium within the lifetime of a colony; for example, Montastraea annularis and Montastraea faveolata. Methodology/Principal FindingsThe population dynamics of competing Symbiodinium symbiont populations were projected through time in coral hosts using a novel, discrete time optimal–resource model. Models were run for two Atlantic Ocean localities. Four symbiont populations, with different environmental optima and phenotypic variances, were modeled to grow, divide, and compete in the corals under seasonal fluctuations in solar insolation and seawater temperature. Elevated seawater temperatures were input into the model 1.5°C above the seasonal summer average, and the symbiont population response was observed for each location. The models showed dynamic fluctuations in Symbiodinium populations densities within corals. Population density predictions for Lee Stocking Island, the Bahamas, where temperatures were relatively homogenous throughout the year, showed a dominance of both type 2, with high phenotypic variance, and type 1, a high-temperature and high-insolation specialist. Whereas the densities of Symbiodinium types 3 and 4, a high-temperature, low-insolation specialist, and a high-temperature, low-insolation generalist, remained consistently low. Predictions for Key Largo, Florida, where environmental conditions were more seasonally variable, showed the coexistence of generalists (types 2 and 4) and low densities of specialists (types 1 and 3). When elevated temperatures were input into the model, population densities in corals at Lee Stocking Island showed an emergence of high-temperature specialists. However, even under high temperatures, corals in the Florida Keys were dominated by generalists.Conclusions/SignificancePredictions at higher seawater temperatures showed endogenous shuffling and an emergence of the high-temperature Symbiodinium specialists, even though their phenotypic variance was low. The model shows that sustaining these “hidden” specialists becomes advantageous under thermal stress conditions, and shuffling symbionts may increase the corals' capacity to acclimatize but not adapt to climatechange–induced ocean warming.

Transcriptional patterns in both host and bacterium underlie a daily rhythm of anatomical and metabolic change in a beneficial symbiosis.

Mechanisms for controlling symbiont populations are critical for maintaining the associations that exist between a host and its microbial partners. We describe here the transcriptional, metabolic, and ultrastructural characteristics of a diel rhythm that occurs in the symbiosis between the squid Euprymna scolopes and the luminous bacterium Vibrio fischeri. The rhythm is driven by the host's expulsion from its light-emitting organ of most of the symbiont population each day at dawn. The transcriptomes of both the host epithelium that supports the symbionts and the symbiont population itself were characterized and compared at four times over this daily cycle. The greatest fluctuation in gene expression of both partners occurred as the day began. Most notable was an up-regulation in the host of >50 cytoskeleton-related genes just before dawn and their subsequent down-regulation within 6 h. Examination of the epithelium by TEM revealed a corresponding restructuring, characterized by effacement and blebbing of its apical surface. After the dawn expulsion, the epithelium reestablished its polarity, and the residual symbionts began growing, repopulating the light organ. Analysis of the symbiont transcriptome suggested that the bacteria respond to the effacement by up-regulating genes associated with anaerobic respiration of glycerol; supporting this finding, lipid analysis of the symbionts' membranes indicated a direct incorporation of host-derived fatty acids. After 12 h, the metabolic signature of the symbiont population shifted to one characteristic of chitin fermentation, which continued until the following dawn. Thus, the persistent maintenance of the squid-vibrio symbiosis is tied to a dynamic diel rhythm that involves both partners.

SYMBIODINIUM (DINOPHYTA) DIVERSITY AND STABILITY IN AQUARIUM CORALS(1).

Indo-Pacific reef corals growing for years in closed-system aquaria provide an alternate means to investigate host-symbiont specificity and stability. The diversity of dinoflagellate endosymbionts (Symbiodinium spp.) from coral communities in private and public aquaria was investigated using molecular-genetic analyses. Of the 29 symbiont types (i.e., species) identified, 90% belonged to the most prevalent group of Symbiodinium harbored by Indo-Pacific reef corals, Clade C, while the rest belonged to Clade D. Sixty-five percent of all types were known from field surveys conducted throughout the Pacific and Indian oceans. Because specific coral-dinoflagellate partnerships appear to have defined geographic distributions, correspondence of the same symbionts in aquarium and field-collected specimens identifies regions where particular colonies must have been collected in the wild. Symbiodinium spp. in clade D, believed to be "stress-tolerant" and/or "opportunistic," occurred in a limited number of individual colonies. The absence of a prevalent, or "weedy," symbiont suggests that conditions under which aquarium corals are grown do not favor competitive replacements of their native symbiont populations. The finding of typical and diverse assemblages of Symbiodinium spp. among aquarium corals living many years under variable chemical/physical conditions, artificial and natural light, while undergoing fragmentation periodically, indicates that individual colonies maintain stable, long-term symbiotic associations.

Life cycle and population dynamics of a protective insect symbiont reveal severe bottlenecks during vertical transmission

Insects engage in mutualistic relationships with a wide variety of microorganisms that are usually transmitted vertically to the next generation. During transmission, the symbiont populations often suffer significant bottlenecks that may entail major genetic and genomic consequences. Here we investigated the life-cycle and the severity of transmission bottlenecks in a symbiotic system with an unusual way of post-hatch vertical transmission by using quantitative PCRs and morphological 3D-reconstructions. European beewolves (Philanthus triangulum, Hymenoptera: Crabronidae) harbor symbiotic bacteria (‘Candidatus Streptomyces philanthi’) in specialized antennal gland reservoirs and secrete them into their subterranean brood cells. The symbionts are later taken up by the beewolf larva and incorporated into the cocoon material to provide protection against pathogenic microorganisms. Even after months of hibernation, the symbiont population on the cocoon is estimated to encompass around 1.4 × 105 cells. However, our results indicate that only few of these bacterial cells (about 9.7 × 102) are taken up from the cocoon by the emerging female. The symbiont population subsequently undergoes logistic growth within the antennal gland reservoirs and reaches a maximum of about 1.5 × 107 cells 3–4 days after emergence. The maximum specific growth rate is estimated to be 0.084–0.105 h−1. With a total reduction in cell numbers of about 6.7 × 10−5 during vertical transmission, the symbiont population experiences one of the most severe bottlenecks known for any symbiotic system to date. This extreme bottleneck may have significantly affected the evolution of the beewolf-Streptomyces symbiosis by increased genetic drift, an accumulation of mildly deleterious mutations and genome erosion.

Pyrosequencing analysis of endosymbiont population structure: co‐occurrence of divergent symbiont lineages in a single vesicomyid host clam

Bacteria-eukaryote endosymbioses are perhaps the most pervasive co-evolutionary associations in nature. Here, intracellular chemosynthetic symbionts of deep-sea clams (Vesicomyidae) were analysed by amplicon pyrosequencing to explore how symbiont transmission mode affects the genetic diversity of the within-host symbiont population. Vesicomyid symbionts (Gammaproteobacteria) are presumed to be obligately intracellular, to undergo nearly strict vertical transmission between host generations, and to be clonal within a host. However, recent data show that vesicomyid symbionts can be acquired laterally via horizontal transfer between hosts or uptake from the environment, potentially creating opportunities for multiple symbiont strains to occupy the same host. Here, genotype-specific PCR and direct sequencing of the bacterial internal transcribed spacer initially demonstrated the co-occurrence of two symbiont strains, symA and symB (93.5% nt identity), in 8 of 118 Vesicomya sp. clams from 3 of 7 hydrothermal vent sites on the Juan de Fuca Ridge. To confirm multiple strains within individual clams, amplicon pyrosequencing of two symbiont loci was used to obtain deep-coverage measurements (mean: approximately 1500x coverage per locus per clam) of symbiont population structure. Pyrosequencing confirmed symA-symB co-occurrence for two individuals, showing the presence of both genotypes in amplicon pools. However, in the majority of clams, the endosymbiont population was remarkably homogenous, with > 99.5% of sequences collapsing into a single symbiont genotype in each clam. These results support the hypothesis that a predominantly vertical transmission strategy leads to the fixation of a single symbiont strain in most hosts. However, mixed symbiont populations do occur in vesicomyids, potentially facilitating the exchange of genetic material between divergent symbiont lineages.

Effects of Long-Term Starvation on a Host Bivalve (Codakia orbicularis, Lucinidae) and Its Symbiont Population

The bivalve Codakia orbicularis, hosting sulfur-oxidizing gill endosymbionts, was starved (in artificial seawater filtered through a 0.22-mum-pore-size membrane) for a long-term experiment (4 months). The effects of starvation were observed using transmission electron microscopy, fluorescence in situ hybridization and catalyzed reporter deposition (CARD-FISH), and flow cytometry to monitor the anatomical and physiological modifications in the gill organization of the host and in the symbiotic population housed in bacteriocytes. The abundance of the symbiotic population decreased through starvation, with a loss of one-third of the bacterial population each month, as shown by CARD-FISH. At the same time, flow cytometry revealed significant changes in the physiology of symbiotic cells, with a decrease in cell size and modifications to the nucleic acid content, while most of the symbionts maintained a high respiratory activity (measured using the 5-cyano-2,3-ditolyl tetrazolium chloride method). Progressively, the number of symbiont subpopulations was reduced, and the subsequent multigenomic state, characteristic of this symbiont in freshly collected clams, turned into one and five equivalent genome copies for the two remaining subpopulations after 3 months. Concomitant structural modifications appeared in the gill organization. Lysosymes became visible in the bacteriocytes, while large symbionts disappeared, and bacteriocytes were gradually replaced by granule cells throughout the entire lateral zone. Those data suggested that host survival under these starvation conditions was linked to symbiont digestion as the main nutritional source.

Distributions of stress-resistant coral symbionts match environmental patterns at local but not regional scales

Distribution patterns of stress-tolerant coral symbionts suggest that maximum habitat temperatures can drive local scale adaptation of symbiont populations, but at regional scales other processes can dominate. We assayed clade membership for symbionts of 2 closely related corals from American Samoa, Fiji, the Philippines and Palmyra Atoll. Temperature stress-tolerant Clade D symbionts occur more frequently in American Samoa (83%) than in Palmyra, Fiji or the Philippines (<1%). In American Samoa, Clade D symbionts dominate habitats with higher maximum temperatures, while Clades C and D are both common under lower maximum temperatures. While corals in American Samoa show more stress-tolerant symbionts, this region does not exhibit higher sea surface temperatures, a greater record of heating anomalies or more bleaching than the other 3 regions. That these local patterns do not hold regionally suggests the importance of other factors, including host responses, other environmental correlates, within-clade physiological diversity and dispersal limitation, in driving the distribution of coral symbionts.

High genetic similarity between two geographically distinct strains of the sulfur-oxidizing symbiont ‘Candidatus Thiobios zoothamnicoli’

The giant marine ciliate Zoothamnium niveum (Ciliophora, Oligohymenophora) is obligatorily covered by a monolayer of putative chemoautotrophic sulfur-oxidizing (thiotrophic) bacteria. For Z. niveum specimens from the Caribbean Sea it has been demonstrated that this ectosymbiotic population consists of only a single pleomorphic phylotype described as Candidatus Thiobios zoothamnicoli. The goal of our study was to identify and phylogenetically analyse the ectosymbiont(s) of a recently discovered Z. niveum population from the Mediterranean Sea, and to compare marker genes encoding key enzymes of the carbon and sulfur metabolism between the two symbiont populations. We identified a single bacterial phylotype representing the ectosymbiont of Z. niveum from the Mediterranean population showing 99.7% 16S rRNA gene (99.2% intergenic spacer region) similarity to the Caribbean Z. niveum ectosymbiont. Genes encoding enzymes typical for an inorganic carbon metabolism [ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO)] and for sulfur metabolism (5'-adenylylsulfate reductase, dissimilatory sulfite reductase) were detected in both symbiotic populations. The very high amino acid sequence identity (97-100%) and the high nucleic acid sequence identity (90-98%) of these marker enzymes in two geographically distant symbiont populations suggests that the association of Z. niveum with Cand. Thiobios zoothamnicoli is very specific as well as temporally and spatially stable.

Population structure of Vibrio fischeri within the light organs of Euprymna scolopes squid from Two Oahu (Hawaii) populations.

We resolved the intraspecific diversity of Vibrio fischeri, the bioluminescent symbiont of the Hawaiian sepiolid squid Euprymna scolopes, at two previously unexplored morphological and geographical scales. These scales ranged from submillimeter regions within the host light organ to the several kilometers encompassing two host populations around Oahu. To facilitate this effort, we employed both novel and standard genetic and phenotypic assays of light-organ symbiont populations. A V. fischeri-specific fingerprinting method and five phenotypic assays were used to gauge the genetic richness of V. fischeri populations; these methods confirmed that the symbiont population present in each adult host's light organ is polyclonal. Upon statistical analysis of these genetic and phenotypic population data, we concluded that the characteristics of symbiotic populations were more similar within individual host populations than between the two distinct Oahu populations of E. scolopes, providing evidence that local geographic symbiont population structure exists. Finally, to better understand the genesis of symbiont diversity within host light organs, the process of symbiosis initiation in newly hatched juvenile squid was examined both experimentally and by mathematical modeling. We concluded that, after the juvenile hatches, only one or two cells of V. fischeri enter each of six internal epithelium-lined crypts present in the developing light organ. We hypothesize that the expansion of different, crypt-segregated, clonal populations creates the polyclonal adult light-organ population structure observed in this study. The stability of the luminous-bacterium-sepiolid squid mutualism in the presence of a polyclonal symbiont population structure is discussed in the context of contemporary evolutionary theory.

In situ photobiology of corals over large depth ranges: A multivariate analysis on the roles of environment, host, and algal symbiont

We applied a multivariate analysis to investigate the roles of host and symbiont on the in situ physiological response of genus Madracis holobionts towards light. Across a large depth gradient (5‐40 m) and for four Madracis species and three symbiont genotypes, we assessed several variables by measuring chlorophyll a fluorescence, photosynthetic pigment composition, or symbiont population descriptors. Most of the variation is explained by two major photobiological components: light‐use efficiency and symbiont cell densities. Two other minor components emphasize photoprotective pathways and light‐harvesting properties such as secondary pigments. Statistics highlight the role of irradiance on coral physiology and reveal mechanisms that are either genetically constrained, such as symbiont cell sizes, or environmentally dependent, such as photochemical efficiencies. Other parameters, such as cellular light‐harvesting and photoprotective pigment concentrations, are regulated by host, symbiont, and environment. The interaction between host and environment stresses the role of host properties in adjusting the internal environment available for the endosymbionts. Different holobiont strategies, relating to symbiont cell density, vary in their physiological optimization of light‐harvesting or photoprotective mechanisms and link to host‐species distribution and dominance over the reef slope. Symbiont functional diversity appears to have a significant role but does not explain host vertical distribution patterns per se, highlighting the importance of species‐specific morphological and physiological properties of the coral host.