Transmission of cyanobacterial symbionts during embryogenesis in the coral reef ascidians Trididemnum nubilum and T. clinides (Didemnidae, Ascidiacea, Chordata)

Vertical transmission of cyanobacterial symbionts occurs in didemnid ascidians harboring Prochloron as an obligate symbiont; the photosymbionts are transferred from the parental ascidian colony to the offspring in various ways depending on host species. Although several didemnids harbor non-Prochloron cyanobacteria in their tunics, few studies have reported the processes of vertical transmission in these didemnids. Here we describe the histological processes of the transmission of cyanobacteria in two didemnids, Trididemnum nubilum harboring Synechocystis and T. clinides harboring three cyanobacterial species. In both species, the photosymbionts in the tunic of the parent colony were apparently captured by the tunic cells of the host and transferred to the embryos brooded in the tunic. The symbiont cells were then incorporated into the inner tunic of the embryo. This mode of transmission is essentially the same as that of T. miniatum harboring Prochloron in the tunic, although there are some differences among species in the timing of the release of the symbionts from the tunic cells. We suggest that the similar modes of vertical transmission are an example of convergent evolution caused by constraints in the distribution patterns of symbiont cells in the host colony.     

Cyanobacterial diversity and a new acaryochloris-like symbiont from Bahamian sea-squirts

Symbiotic interactions between ascidians (sea-squirts) and microbes are poorly understood. Here we characterized the cyanobacteria in the tissues of 8 distinct didemnid taxa from shallow-water marine habitats in the Bahamas Islands by sequencing a fragment of the cyanobacterial 16S rRNA gene and the entire 16S-23S rRNA internal transcribed spacer region (ITS) and by examining symbiont morphology with transmission electron (TEM) and confocal microscopy (CM). As described previously for other species, Trididemnum spp. mostly contained symbionts associated with the Prochloron-Synechocystis group. However, sequence analysis of the symbionts in Lissoclinum revealed two unique clades. The first contained a novel cyanobacterial clade, while the second clade was closely associated with Acaryochloris marina. CM revealed the presence of chlorophyll d (chl d) and phycobiliproteins (PBPs) within these symbiont cells, as is characteristic of Acaryochloris species. The presence of symbionts was also observed by TEM inside the tunic of both the adult and larvae of L. fragile, indicating vertical transmission to progeny. Based on molecular phylogenetic and microscopic analyses, Candidatus Acaryochloris bahamiensis nov. sp. is proposed for this symbiotic cyanobacterium. Our results support the hypothesis that photosymbiont communities in ascidians are structured by host phylogeny, but in some cases, also by sampling location.     

A new didemnid ascidian Lissoclinum midui sp. nov. from Kumejima Island (Okinawa, Japan), with remarks on the absence of a common cloacal system and the presence of an unknown organ

A new photosymbiotic didemnid, Lissoclinum midui sp. nov., is described from coral reefs in the Ryukyu Archipelago, Japan. Colonies of the didemnid are green due to Prochloron algal symbionts, which are distributed solely in the tunic. The new species is placed in Lissoclinum because of its uncoiled vas deferens and the presence of globular spicules. However, two unique characters distinguish this species from all other didemnid ascidians: the absence of a common cloacal system, and the presence of an unknown organ in the bottom wall of the branchial sac. In the phylogenetic trees inferred from partial sequences of cytochrome c oxidase subunit I (COI) gene, the new species diverged at the basal point of the clade of four photosymbiotic Lissoclinum species analyzed here.     

Localization of symbiotic cyanobacteria in the colonial ascidian Trididemnum miniatum (Didemnidae, Ascidiacea)

Trididemnum miniatum is a colonial ascidian harboring the photosymbiotic prokaryote Prochloron sp. These bacterial cells are located in the tunic of the host animal. The present study revealed, by ultrastructural analysis, that the Prochloron cells were exclusively distributed and proliferated in the tunic. They were shown to be embedded in the tunic matrix and to have no direct contact with ascidian cells. Some tunic cells of the ascidians, however, did phagocytize and digest the symbiont. Round cell masses were sometimes found in the tunic and appeared to consist of disintegrating cyanobacterial cells. The thoracic epidermis of ascidian zooids was often digitated, and the epidermal cells extended microvilli into the tunic. Since there were no Prochloron cells in the alimentary tract of the ascidian zooids, the photosymbionts would not be considered part of the typical diet of the host ascidians. Thin layer chromatography showed that the symbionts possessed both chlorophyll a and b, while a 16S rRNA gene phylogeny supported the identification of the photosymbiont of T. miniatum as Prochloron sp.     

Multiple Symbiont Acquisition Strategies as an Adaptive Mechanism in the Coral Stylophora pistillata

In obligate symbioses, the host’s survival relies on the successful acquisition and maintenance of symbionts. Symbionts can either be transferred from parent to offspring via direct inheritance (vertical transmission) or acquired anew each generation from the environment (horizontal transmission). With vertical symbiont transmission, progeny benefit by not having to search for their obligate symbionts, and, with symbiont inheritance, a mechanism exists for perpetuating advantageous symbionts. But, if the progeny encounter an environment that differs from that of their parent, they may be disadvantaged if the inherited symbionts prove suboptimal. Conversely, while in horizontal symbiont acquisition host survival hinges on an unpredictable symbiont source, an individual host may acquire genetically diverse symbionts well suited to any given environment. In horizontal acquisition, however, a potentially advantageous symbiont will not be transmitted to subsequent generations. Adaptation in obligate symbioses may require mechanisms for both novel symbiont acquisition and symbiont inheritance. Using denaturing-gradient gel electrophoresis and real-time PCR, we identified the dinoflagellate symbionts (genus Symbiodinium) hosted by the Red Sea coral Stylophora pistillata throughout its ontogenesis and over depth. We present evidence that S. pistillata juvenile colonies may utilize both vertical and horizontal symbiont acquisition strategies. By releasing progeny with maternally derived symbionts, that are also capable of subsequent horizontal symbiont acquisition, coral colonies may acquire physiologically advantageous novel symbionts that are then perpetuated via vertical transmission to subsequent generations. With symbiont inheritance, natural selection can act upon the symbiotic variability, providing a mechanism for coral adaptation.     

Down under the tunic: bacterial biodiversity hotspots and widespread ammonia-oxidizing archaea in coral reef ascidians

Ascidians are ecologically important components of marine ecosystems yet the ascidian microbiota remains largely unexplored beyond a few model species. We used 16S rRNA gene tag pyrosequencing to provide a comprehensive characterization of microbial symbionts in the tunic of 42 Great Barrier Reef ascidian samples representing 25 species. Results revealed high bacterial biodiversity (3 217 unique operational taxonomic units (OTU_0.03) from 19 described and 14 candidate phyla) and the widespread occurrence of ammonia-oxidizing Thaumarchaeota in coral reef ascidians (24 of 25 host species). The ascidian microbiota was clearly differentiated from seawater microbial communities and included symbiont lineages shared with other invertebrate hosts as well as unique, ascidian-specific phylotypes. Several rare seawater microbes were markedly enriched (200–700 fold) in the ascidian tunic, suggesting that the rare biosphere of seawater may act as a conduit for horizontal symbiont transfer. However, most OTUs (71%) were rare and specific to single hosts and a significant correlation between host relatedness and symbiont community similarity was detected, indicating a high degree of host-specificity and potential role of vertical transmission in structuring these communities. We hypothesize that the complex ascidian microbiota revealed herein is maintained by the dynamic microenvironments within the ascidian tunic, offering optimal conditions for different metabolic pathways such as ample chemical substrate (ammonia-rich host waste) and physical habitat (high oxygen, low irradiance) for nitrification. Thus, ascidian hosts provide unique and fertile niches for diverse microorganisms and may represent an important and previously unrecognized habitat for nitrite/nitrate regeneration in coral reef ecosystems.     

Host specificity and phylogeography of the prochlorophyte Prochloron sp., an obligate symbiont in didemnid ascidians

Prochloron is an oxygenic photosynthetic bacterium that lives in obligate symbiosis with didemnid ascidians, such as Diplosoma spp., Lissoclinum spp. and Trididemnum spp. This study investigated the genetic diversity of the genus Prochloron by constructing a phylogenetic tree based on the 16S rRNA gene sequences of 27 isolates from 11 species of didemnid ascidians collected from Japan, Australia and the USA. The 27 isolates formed three phylogenetic groups: 22 of the samples were identified to be closely related members of Prochloron. Two samples, isolated from Trididemnum nubilum and Trididemnum clinides, were found to belong to the species Synechocystis trididemni, the closest relative of Prochloron. Three isolates formed a separate group from both Prochloron sp. and S. trididemni, potentially indicating a new symbiotic phylotype. Genomic polymorphism analysis, employing cyanobacterium-specific highly iterative palindrome 1 repeats, could not delineate the isolates further. For the Prochloron sp. isolates, the phylogenetic outcome was independent of host species and geographic origin of the sample indicating a low level of host specificity, low genetic variation within the taxon and possibly a lack of a host-symbiont relationship during reproductive dispersal. This study contributes significantly to the understanding of Prochloron diversity and phylogeny, and implications for the evolutionary relationship of prochlorophytes, cyanobacteria and chloroplasts are also discussed.     

Symbionts in excess? No correlation between symbiont density and the ability of mealybug hosts to exploit plant species or tolerate insecticide stress

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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.     

Symbiosis lost: imperfect vertical transmission of fungal endophytes in grasses

Vertically transmitted symbionts associate with some of the most ecologically dominant species on Earth, and their fixation has led to major evolutionary transitions (e.g., the development of mitochondria). Theory predicts that exclusive vertical transmission should favor mutualism and generate high frequencies of symbiosis in host populations. However, host populations often support lower-than-expected symbiont frequencies. Imperfect transmission (i.e., symbiont is not transmitted to all offspring) can reduce symbiont frequency, but for most beneficial symbionts it is unknown whether vertical transmission can be imperfect or during which life-history stage the symbiont is lost. Using quantitative natural history surveys of fungal endophytes in grasses, we show that transmission was imperfect in at least one stage for all seven host species examined. Endophytes were lost at all possible stages: within adult plants, from adult tillers to seeds, and from seeds to seedlings. Despite this loss, uninfected seeds failed to germinate in some species, resulting in perfect transmission to seedlings. The type and degree of loss differed among host populations and species and between endophyte genera. Populations with lower endophyte frequencies had higher rates of loss. Our results indicate new directions for understanding cooperation and conflict in symbioses and suggest mechanisms for host sanctions against costly symbionts.     

Host hybridization as a potential mechanism of lateral symbiont transfer in deep‐sea vesicomyid clams

Deep‐sea vesicomyid clams live in mutualistic symbiosis with chemosynthetic bacteria that are inherited through the maternal germ line. On evolutionary timescales, strictly vertical transmission should lead to cospeciation of host mitochondrial and symbiont lineages; nonetheless, examples of incongruent phylogenies have been reported, suggesting that symbionts are occasionally horizontally transmitted between host species. The current paradigm for vesicomyid clams holds that direct transfers cause host shifts or mixtures of symbionts. An alternative hypothesis suggests that hybridization between host species might explain symbiont transfers. Two clam species, Archivesica gigas and Phreagena soyoae, frequently co‐occur at deep‐sea hydrocarbon seeps in the eastern Pacific Ocean. Although the two species typically host gammaproteobacterial symbiont lineages marked by divergent 16S rRNA phylotypes, we identified a number of clams with the A. gigas mitotype that hosted symbionts with the P. soyoae phylotype. Demographic inference models based on genome‐wide SNP data and three Sanger sequenced gene markers provided evidence that A. gigas and P. soyoae hybridized in the past, supporting the hypothesis that hybridization might be a viable mechanism of interspecific symbiont transfer. These findings provide new perspectives on the evolution of vertically transmitted symbionts and their hosts in deep‐sea chemosynthetic environments.     

The Biogeography and Phylogeny of Unicellular Cyanobacterial Symbionts in Sponges from Australia and the Mediterranean

The distribution, host associations, and phylogenetic relationships of the unicellular cyanobacterial symbionts of selected marine sponges were investigated with direct 16s rDNA sequencing. The results indicate that the symbionts of the marine sponges Aplysina aerophoba, Ircinia variabilis, and Petrosia ficiformis from the Mediterranean, four Chondrilla species from Australia and the Mediterranean, and Haliclona sp. from Australia support a diversity of symbionts comprising at least four closely related species of Synechococcus. These include the symbionts presently described as Aphanocapsa feldmannii from P. ficiformis and Chondrilla nucula. A fifth symbiont from Cymbastela marshae in Australia is an undescribed symbiont of sponges, related to Oscillatoria rosea. One symbiont, Candidatus Synechococcus spongiarum, was found in diverse sponge genera in the Mediterranean Sea and the Indian, Pacific, and Southern oceans, whereas others were apparently more restricted in host association and distribution. These results are discussed in terms of the biodiversity and biogeographic distributions of cyanobacterial symbionts.This revised version was published online in November 2004 with corrections to Volume 48.     

Evolutionary changes in symbiont community structure in ticks

Ecological specialization to restricted diet niches is driven by obligate, and often maternally inherited, symbionts in many arthropod lineages. These heritable symbionts typically form evolutionarily stable associations with arthropods that can last for millions of years. Ticks were recently found to harbour such an obligate symbiont, Coxiella‐LE, that synthesizes B vitamins and cofactors not obtained in sufficient quantities from blood diet. In this study, the examination of 81 tick species shows that some Coxiella‐LE symbioses are evolutionarily stable with an ancient acquisition followed by codiversification as observed in ticks belonging to the Rhipicephalus genus. However, many other Coxiella‐LE symbioses are characterized by low evolutionary stability with frequent host shifts and extinction events. Further examination revealed the presence of nine other genera of maternally inherited bacteria in ticks. Although these nine symbionts were primarily thought to be facultative, their distribution among tick species rather suggests that at least four may have independently replaced Coxiella‐LE and likely represent alternative obligate symbionts. Phylogenetic evidence otherwise indicates that cocladogenesis is globally rare in these symbioses as most originate via horizontal transfer of an existing symbiont between unrelated tick species. As a result, the structure of these symbiont communities is not fixed and stable across the tick phylogeny. Most importantly, the symbiont communities commonly reach high levels of diversity with up to six unrelated maternally inherited bacteria coexisting within host species. We further conjecture that interactions among coexisting symbionts are pivotal drivers of community structure both among and within tick species.     

Natural combinatorial peptide libraries in cyanobacterial symbionts of marine ascidians

A large family of cytotoxic cyclic peptides exemplified by the patellamides has been isolated from ascidians harboring the obligate cyanobacterial symbionts Prochloron spp.. Genome sequence analysis of these symbionts has revealed that Prochloron spp. synthesize patellamides by a ribosomal pathway. To understand how this pathway evolved to produce a suite of related metabolites, we analyzed 46 prochloron-containing ascidians from the tropical Pacific Ocean for the presence of patellamide biosynthetic genes and taxonomic markers. Here, we show that Prochloron spp. generate a diverse library of patellamides using small, hypervariable cassettes within a conserved genetic background. Each symbiont strain contains a single pathway, and mixtures of symbionts within ascidians lead to the accumulation of chemical libraries. We used this information to engineer the production of a new cyclic peptide in Escherichia coli, thereby demonstrating the power of comparative analysis of closely related symbiotic pathways to direct the genetic synthesis of new molecules.     

Incorporating the process of vertical transmission into understanding of host–symbiont dynamics

Variation exists in the frequency of obligate, vertically transmitted symbiotic organisms within and among host populations; however, these patterns have not been adequately explained by variable fitness effects of symbionts on their hosts. In this forum, we call attention to another equally important, but overlooked mechanism to maintain variation in the frequency of symbioses in nature: the rate of vertical transmission. On ecological time scales, vertical transmission can affect the equilibrium frequencies of symbionts in host populations, with potential consequences for population and community dynamics. In addition, vertical transmission has the potential to influence the evolution of symbiosis, by affecting the probability of fixation of symbiosis (and therefore the evolution of complexity) and by allowing hosts to sanction against costly symbionts. Here we use grass–epichloae symbioses as a model system to explore the causes and consequences of variation in vertical transmission rates. We identify critical points for symbiont transmission that emerge from considering the host growth cycle devoted to reproduction (asexual vs sexual) and the host capability to maintain homeostasis. We also use information on the process of transmission to predict the environmental factors that would most likely affect transmission rates. Altogether, we aim to highlight the vertical transmission rate as an important process for understanding the ecology and evolution of symbiosis, using grass–epichloae interactions as a case study.     

Ultraviolet-light-absorbing tunic cells in didemnid ascidians hosting a symbiotic photo-oxygenic prokaryote,Prochloron

Coral reef invertebrates that host phototrophic symbionts are thought to protect themselves and their symbionts with mycosporine-like amino acids (MAAs)-UV-absorbing substances that act as sunscreens (Dunlap, W. C., and J. M. Shick, 1998. J. Phycol. 34: 418-430). However, the histological distribution of MAAs in the host tissues has not yet been visualized. We have localized the UV-absorbing substances in the tissues of two colonial didemnid ascidians-Lissoclinum patella and Diplosoma sp.-that contain the symbiotic photo-oxygenic prokaryote Prochloron sp. Cross-sections of unfixed tissue from these ascidians were examined by UV-light microscopy at 320 or 330 nm, wavelengths at which UV light is absorbed by MAAs. Within the tunic, the gelatinous integument of the colony, UV light was exclusively absorbed by a particular type of cell, the tunic bladder cell. Tunic bladder cells with strong UV absorption were denser in the upper tunic, which lies over a colony's zooids, than in the basal tunic underlying the zooid. In the upper tunic, those cells with strong UV absorption were most dense near the surface. The tunic bladder cell is highly vacuolated, and the vacuole contains strong acid, which destabilizes MAAs. Furthermore, the UV-absorbing portion of tunic bladder cells seemed to be cup-shaped, indicating that the MAAs are not localized in the vacuole, but in the cytoplasm. These results strongly suggest that didemnid ascidians accumulate MAAs in tunic bladder cells as a protection against UV radiation.     

Buchnera has changed flatmate but the repeated replacement of co‐obligate symbionts is not associated with the ecological expansions of their aphid hosts

Symbiotic associations with bacteria have facilitated important evolutionary transitions in insects and resulted in long‐term obligate interactions. Recent evidence suggests that these associations are not always evolutionarily stable and that symbiont replacement, and/or supplementation of an obligate symbiosis by an additional bacterium, has occurred during the history of many insect groups. Yet, the factors favouring one symbiont over another in this evolutionary dynamic are not well understood; progress has been hindered by our incomplete understanding of the distribution of symbionts across phylogenetic and ecological contexts. While many aphids are engaged into an obligate symbiosis with a single Gammaproteobacterium, Buchnera aphidicola, in species of the Lachninae subfamily, this relationship has evolved into a ‘ménage à trois’, in which Buchnera is complemented by a cosymbiont, usually Serratia symbiotica. Using deep sequencing of 16S rRNA bacterial genes from 128 species of Cinara (the most diverse Lachninae genus), we reveal a highly dynamic dual symbiotic system in this aphid lineage. Most species host both Serratia and Buchnera but, in several clades, endosymbionts related to Sodalis, Erwinia or an unnamed member of the Enterobacteriaceae have replaced Serratia. Endosymbiont genome sequences from four aphid species confirm that these coresident symbionts fulfil essential metabolic functions not ensured by Buchnera. We further demonstrate through comparative phylogenetic analyses that cosymbiont replacement is not associated with the adaptation of aphids to new ecological conditions. We propose that symbiont succession was driven by factors intrinsic to the phenomenon of endosymbiosis, such as rapid genome deterioration or competitive interactions between bacteria with similar metabolic capabilities.     

Vertical transmission as the key to the colonization of Madagascar by fungus-growing termites?

The mutualism between fungus-growing termites (Macrotermitinae) and their mutualistic fungi (Termitomyces) began in Africa. The fungus-growing termites have secondarily colonized Madagascar and only a subset of the genera found in Africa is found on this isolated island. Successful long-distance colonization may have been severely constrained by the obligate interaction of the termites with fungal symbionts and the need to acquire these symbionts secondarily from the environment for most species (horizontal symbiont transmission). Consistent with this hypothesis, we show that all extant species of fungus-growing termites of Madagascar are the result of a single colonization event of termites belonging to one of the only two groups with vertical symbiont transmission, and we date this event at approximately 13 Mya (Middle/Upper Miocene). Vertical symbiont transmission may therefore have facilitated long-distance dispersal since both partners disperse together. In contrast to their termite hosts, the fungal symbionts have colonized Madagascar multiple times, suggesting that the presence of fungus-growing termites may have facilitated secondary colonizations of the symbiont. Our findings indicate that the absence of the right symbionts in a new environment can prevent long-distance dispersal of symbioses relying on horizontal symbiont acquisition.     

The Combined Effects of Bacterial Symbionts and Aging on Life History Traits in the Pea Aphid,Acyrthosiphon pisum

While many endosymbionts have beneficial effects on hosts under specific ecological conditions, there can also be associated costs. In order to maximize their own fitness, hosts must facilitate symbiont persistence while preventing symbiont exploitation of resources, which may require tight regulation of symbiont populations. As a host ages, the ability to invest in such mechanisms may lessen or be traded off with demands of other life history traits, such as survival and reproduction. Using the pea aphid, Acyrthosiphon pisum, we measured survival, lifetime fecundity, and immune cell counts (hemocytes, a measure of immune capacity) in the presence of facultative secondary symbionts. Additionally, we quantified the densities of the obligate primary bacterial symbiont, Buchnera aphidicola, and secondary symbionts across the host''s lifetime. We found life history costs to harboring some secondary symbiont species. Secondary symbiont populations were found to increase with host age, while Buchnera populations exhibited a more complicated pattern. Immune cell counts peaked at the midreproductive stage before declining in the oldest aphids. The combined effects of immunosenescence and symbiont population growth may have important consequences for symbiont transmission and maintenance within a host population.     

Genomic signatures of obligate host dependence in the luminous bacterial symbiont of a vertebrate

The majority of bacteria engaged in bioluminescent symbiosis are environmentally acquired and facultatively symbiotic. A few enigmatic bioluminescent symbionts have not been successfully cultured, which has led to speculation that they may be obligately dependent on their hosts. Here, we report the draft genome of the uncultured luminous symbiont of an anomalopid flashlight fish, ‘Candidatus Photodesmus katoptron’. The genome of the anomalopid symbiont is reduced by 80% compared with close relatives and lacks almost all genes necessary for amino acid synthesis and for metabolism of energy sources other than glucose, supporting obligate dependence on the host for growth. ‘Candidatus Photodesmus katoptron’ is the first described obligate mutualistic symbiont of a vertebrate. Unlike most other obligate mutualists, the anomalopid symbiont genome has retained complete pathways for chemotaxis and motility as well as most genes involved in cell wall production, consistent with the hypothesis that these bacteria may be transmitted environmentally during an extra‐host phase.