Symbiotic green algae in eggs of Hynobius nigrescens,an amphibian endemic to Japan

The egg capsules of some amphibians' eggs are known to become green colored before hatching. This is due to the increase of green symbionts in the egg capsule surrounding the embryo. The green symbionts in North American amphibian eggs were reported to be unicellular green algae in the Oophila‐clade of Volvocales, Chlorophyceae. However, it remains unclear whether this is also the case in other parts of the world. In this study, we analyzed the green symbionts in green‐colored eggs of Hynobius nigrescens, an amphibian endemic to Japan, obtained from five distinct locations. Microscopic observations revealed that the green symbionts were similar in appearance to Oophila amblystomatis, which was reported in some amphibian eggs in North America, in which non‐motile cells of the algae had thick cell walls with reticulate protuberances. PCR‐DGGE followed by phylogenetic analyses of partial 18S rRNA sequences revealed that the symbionts from the five locations were identical and most likely unialgal in each egg capsule. They formed an independent subclade within the Oophila‐clade, indicating that H. nigrescens has a unique symbiont. Our data are consistent with the previous report on North American amphibian eggs and support the specific symbiotic relationships between Oophila‐clade symbionts and the eggs of amphibians. This is the first report on the specific symbiont‐and‐host association between an Oophila‐clade symbiont and an amphibian outside of North America. We also discuss several possibilities regarding the origin of green symbionts (vertical transmission or invasion) on the basis of the discovery and detailed observation of H. nigrescens eggs without any green symbionts.     

Taxonomy and nomenclature of Oophila amblystomatis (Chlorophyceae,Chlamydomonadales)

The unicellular green alga Oophila amblystomatis was named by Lambert in 1905 based upon its association with egg masses of the spotted salamander Ambystoma maculatum. We collected algal cells from Lambert's original egg capsule preparations that were contributed to Phycotheca Boreali-Americana (PBA) in 1905 and subjected them to DNA extraction and PCR with O. amblystomatis-specific 18S rRNA gene primers. DNA amplified from these preparations was cloned and nine clones were sequenced. Along with representative sequences from the Oophila clade and Chlorophyceae, a phylogenetic tree was inferred. Seven sequences clustered within the Oophila clade and two clustered with Chlamydomonas moewusii, which is included in a sister clade to Oophila. By sequencing algal material from the egg capsules of representative type material we can unambiguously characterize O. amblystomatis and define a monophyletic clade centered on this type material. Accordingly, we reject a recent proposal that this species be transferred to Chlorococcum.     

Dynamics of the growth,life history transformation and photosynthetic capacity of Oophila amblystomatis (Chlorophyceae), a green algal symbiont associated with embryos of the northeastern yellow spotted salamander Ambystoma maculatum (Amphibia)

The recent discovery that the unicellular green alga Oophila amblystomatis, invades embryonic tissues and cells of the salamander Ambystoma maculatum prompted us to investigate the growth and life history transformations of the algal symbionts in egg capsules. During embryonic development, symbionts were first detected microscopically as a cohesive population of swimming cells in the vicinity of the blastopore around embryonic stage 17. This population of cells grew and at embryonic stage 25, a fraction of the population began to affix to the inside of the egg capsule. Cells then underwent syngamy, lost flagella, and transformed into non-motile cells. We observed a linear increase in the accumulation of such capsule-associated cells from embryonic stage 25 to 40. The population of zoospores did not grow over this period and showed a declining trend between stage 39 and 40. We verified the population growth by measuring relative chlorophyll a content and also measured quantum yield (QY) of photosystem II (PS II) using pulse amplitude modulated (PAM) fluorometry. The population, but not the cell size, of non-motile capsule membrane-bound cells increased modestly during a one-month period after hatching, and continued to contain high levels of chlorophyll a and photosynthetic capacity. We conclude that O. amblystomatis undergoes a life history transition in egg capsules and speculate that many of these symbionts become zygotes, rather than invading the embryo.     

Symbioses between salamander embryos and green algae

The symbiosis between Ambystoma maculatum (spotted salamander) embryos and green algae was initially described over 120 years ago. Algae populate the egg capsules that surround individual A. maculatum embryos, giving the intracapsular fluid a characteristic green hue. Early work established this symbiosis to be a mutualism, while subsequent studies sought to identify the material benefits of this association to both symbiont and host. These studies have shown that salamander embryos benefit from increased oxygen concentrations provided by their symbiotic algae. The algae, in turn, may benefit from ammonia excreted by the embryos. All of these early studies considered the association to be an ectosymbiotic mutualism. However our recent work has shown that algae invade both embryonic salamander cells and tissues during development. The unexpected invasion of algal cells into a salamander host changes our understanding of this symbiosis. This review will summarize the earlier research on this association in the context of these recent findings. It will also emphasize gaps in our understanding of this and other amphibian embryo-algal interactions and suggest various research avenues to address these unanswered questions.     

Endosymbiotic Bacteria in the Esophageal Organ of Glossiphoniid Leeches

We characterized the intracellular symbiotic bacteria of the hematophagous glossiphoniid leeches Placobdelloides siamensis and a Parabdella sp. These leeches have a specialized structure called an “esophageal organ,” the cells of which harbor bacterial symbionts. From the esophageal organ of each species, a 1.5-kb eubacterial 16S rRNA gene segment was amplified by PCR, cloned, and sequenced. Diagnostic PCR detected the symbiont in the esophageal organ and intestine. Phylogenetic analysis of the 16S rRNA gene(s) demonstrated that the symbionts from the leeches formed a monophyletic group in a well-defined clade containing endosymbiotic bacteria of plant sap-feeding insects in the γ-subdivision of the Proteobacteria. The nucleotide compositions of the 16S rRNA gene from the leech symbionts were highly AT biased (53.7%).     

Evolutionary Relationships of Three New Species of Enterobacteriaceae Living as Symbionts of Aphids and Other Insects

Ecological studies on three bacterial lineages symbiotic in aphids have shown that they impose a variety of effects on their hosts, including resistance to parasitoids and tolerance to heat stress. Phylogenetic analyses of partial sequences of gyrB and recA are consistent with previous analyses limited to 16S rRNA gene sequences and yield improved confidence of the evolutionary relationships of these symbionts. All three symbionts are in the Enterobacteriaceae. One of the symbionts, here given the provisional designation “Candidatus Serratia symbiotica,” is a Serratia species that has acquired a symbiotic lifestyle. The other two symbionts, here designated “Candidatus Hamiltonella defensa” and “Candidatus Regiella insecticola,” are sister groups to one another and together show a relationship to species of Photorhabdus.     

Diversity and characterization of bacterial communities of five co‐occurring species at a hydrothermal vent on the Tonga Arc

Host–symbiont relationships in hydrothermal vent ecosystems, supported by chemoautotrophic bacteria as primary producers, have been extensively studied. However, the process by which densely populated co‐occurring invertebrate hosts form symbiotic relationships with bacterial symbionts remains unclear. Here, we analyzed gill‐associated symbiotic bacteria (gill symbionts) of five co‐occurring hosts, three mollusks (“Bathymodiolus” manusensis, B. brevior, and Alviniconcha strummeri) and two crustaceans (Rimicaris variabilis and Austinograea alayseae), collected together at a single vent site in the Tonga Arc. We observed both different compositions of gill symbionts and the presence of unshared operational taxonomic units (OTUs). In addition, the total number of OTUs was greater for crustacean hosts than for mollusks. The phylogenetic relationship trees of gill symbionts suggest that γ‐proteobacterial gill symbionts have coevolved with their hosts toward reinforcement of host specificity, while campylobacterial Sulfurovum species found across various hosts and habitats are opportunistic associates. Our results confirm that gill symbiont communities differ among co‐occurring vent invertebrates and indicate that hosts are closely related with their gill symbiont communities. Considering the given resources available at a single site, differentiation of gill symbionts seems to be a useful strategy for obtaining nutrition and energy while avoiding competition among both hosts and gill symbionts.     

srDna-based taxonomic affinities of algal symbionts from a planktonic foraminifer and a solitary radiolarian

Planktonic sarcodines (acantharia, radiolaria, and planktonic foraminifera) are oceanic amoeboid protozoa that often harbor a variety of microalgae as intracellular symbionts. The identity and function of these endosymbiotic algae have intrigued and perplexed biologists for more than a century. The most conspicuous and well‐studied symbiotic algae of planktonic foraminifera and radiolaria are dinoflagellates, but a variety of nondinoflagellate taxa have also been reported. Ultrastructural features have been used to characterize some of these nondinoflagellate algae, but rarely has this led to clear taxonomic affiliations. We analyzed the nuclear small subunit ribosomal DNA (srDNA) isolated from the symbionts of the spinose planktonic foraminiferan Globigerinella siphonifera d'Orbigny (=Globigerinella aequilateralis Brady) and a solitary radiolarian (Spongodrymus sp. Haeckel) in order to determine the identity of these symbionts. The small coccoid algae isolated from G. siphonifera correspond to the Type I symbionts described by Faber et al. (1988) . Phylogenetic analysis of the srDNA sequences places these symbionts within the prymnesiophyte (haptophyte) lineage, closer to Prymnesium Conrad than to Phaeocystis Lagerheim. To our knowledge, this is the first confirmed case of a symbiotic prymnesiophyte. In addition, we were able to examine the level of sequence heterogeneity between symbionts isolated from different individuals of a single host species. The three isolates in this study had srDNA sequences that were almost identical, indicating that the three were all of the same species. Very green symbiotic algae were isolated from three solitary radiolaria identified as species of Spongodrymus. The symbiont srDNA sequences from the three individual hosts were identical to each other, again implicating a single species of algae in that symbiotic association. These symbionts are prasinophytes most closely related to the clade containing Tetaselmis convolutae Norris, Hori et Chihara. Tetraselmis convolutae is the algal symbiont of the marine flatworm, Convolutae roscoffensis Graff.     

Arsenophonus and Sodalis Symbionts in Louse Flies: an Analogy to the Wigglesworthia and Sodalis System in Tsetse Flies

Symbiosis between insects and bacteria result in a variety of arrangements, genomic modifications, and metabolic interconnections. Here, we present genomic, phylogenetic, and morphological characteristics of a symbiotic system associated with Melophagus ovinus, a member of the blood-feeding family Hippoboscidae. The system comprises four unrelated bacteria representing different stages in symbiosis evolution, from typical obligate mutualists inhabiting bacteriomes to freely associated commensals and parasites. Interestingly, the whole system provides a remarkable analogy to the association between Glossina and its symbiotic bacteria. In both, the symbiotic systems are composed of an obligate symbiont and two facultative intracellular associates, Sodalis and Wolbachia. In addition, extracellular Bartonella resides in the gut of Melophagus. However, the phylogenetic origins of the two obligate mutualist symbionts differ. In Glossina, the mutualistic Wigglesworthia appears to be a relatively isolated symbiotic lineage, whereas in Melophagus, the obligate symbiont originated within the widely distributed Arsenophonus cluster. Although phylogenetically distant, the two obligate symbionts display several remarkably similar traits (e.g., transmission via the host''s “milk glands” or similar pattern of genome reduction). To obtain better insight into the biology and possible role of the M. ovinus obligate symbiont, “Candidatus Arsenophonus melophagi,” we performed several comparisons of its gene content based on assignments of the Cluster of Orthologous Genes (COG). Using this criterion, we show that within a set of 44 primary and secondary symbionts, “Ca. Arsenophonus melophagi” is most similar to Wigglesworthia. On the other hand, these two bacteria also display interesting differences, such as absence of flagellar genes in Arsenophonus and their presence in Wigglesworthia. This finding implies that a flagellum is not essential for bacterial transmission via milk glands.     

Symbiotic Bacteria Associated with Stomach Discs of Human Lice

The symbiotic bacteria associated with the stomach disc, a large aggregate of bacteriocytes on the ventral side of the midgut, of human body and head lice were characterized. Molecular phylogenetic analysis of 16S rRNA gene sequences showed that the symbionts formed a distinct and well-defined clade in the Gammaproteobacteria. The sequences exhibited AT-biased nucleotide composition and accelerated molecular evolution. In situ hybridization revealed that in nymphs and adult males, the symbiont was localized in the stomach disc, while in adult females, the symbiont was not in the stomach disc but in the lateral oviducts and the posterior pole of the oocytes due to female-specific symbiont migration. We propose the designation “Candidatus Riesia pediculicola” for the louse symbionts.     

The Establishment of the Hydra-Chlorella Symbiosis: Recognition of Potential Algal Symbionts

The epithelial cells lining the gastric cavity of the freshwater hydra, Hydra viridis, harbor unicellular algal symbionts of the genus Cblorella. It has long been known that these hydra cells can readily phagocytose algal cells and will sequester those algae that have the potential to form a symbiotic association. In this paper the evidence is discussed for when and how recognition of potential symbionts by hydra cells occurs, i.e. during phagocytosis or during the subsequent intracellular events leading to sequestration of algal symbionts.     

α-Proteobacterial Symbionts of Marine Bryozoans in the Genus Watersipora

Bacterial symbionts that resembled mollicutes were discovered in the marine bryozoan Watersipora arcuata in the 1980s. In this study, we used PCR and sequencing of 16S rRNA genes, specific fluorescence in situ hybridization, and phylogenetic analysis to determine that the bacterial symbionts of “W. subtorquata” and “W. arcuata” from several locations along the California coast are actually closely related α-Proteobacteria, not mollicutes. We propose the names “Candidatus Endowatersipora palomitas” and “Candidatus Endowatersipora rubus” for the symbionts of “W. subtorquata” and “W. arcuata,” respectively.     

Marine algal symbionts benefit benthic invertebrate embryos deposited in gelatinous egg masses

Algae colonize the gelatinous egg masses of marine invertebrates. This study demonstrates a symbiotic relationship between marine algae and the invertebrate embryos in gelatinous egg masses found in Indian River Lagoon, FL, USA. The benefits to the embryos in this association differ among host species investigated. The embryos of the polychaete Axiothella mucosa graze on the diatom assemblage in their egg masses and the fitness of the crawl-away juveniles is improved by this food source. The tenuous egg masses of the polychaete Arenicola cristata and the mollusk Haminoea succinea are negatively buoyant when spawned and become buoyant when symbiotic algae are present. In addition to increased dispersal of their lecithotrophic larvae, the potential of the egg masses of A. cristata and H. succinea to float may reduce predation on the embryos by benthic predators such as the gastropod Nassarius vibex. Photosynthetically derived oxygen from the algae may benefit the embryos of the opisthobranch Haminoea elegans by increasing oxygen supply when crawl-away juveniles emerge from the egg mass. However, when mostly earlier stage larvae are hatched from egg masses of H. elegans, the additional oxygen supplied by the algae does not provide a substantial advantage. Algae were absent in the gelatinous egg mass core of only one of the five species examined, Haminoea antillarum. H. antillarum has both a short embryonic development time and denser egg mass gel than the other four species tested. What is not understood is whether invertebrate egg masses are an opportunistic space for algae to colonize or whether only a few microalgal species can exploit the gelatinous substrate.     

Effects of “Reduced” and “Business-As-Usual” CO2 Emission Scenarios on the Algal Territories of the Damselfish Pomacentrus wardi (Pomacentridae)

Turf algae are a very important component of coral reefs, featuring high growth and turnover rates, whilst covering large areas of substrate. As food for many organisms, turf algae have an important role in the ecosystem. Farming damselfish can modify the species composition and productivity of such algal assemblages, while defending them against intruders. Like all organisms however, turf algae and damselfishes have the potential to be affected by future changes in seawater (SW) temperature and pCO₂. In this study, algal assemblages, in the presence and absence of farming Pomacentrus wardi were exposed to two combinations of SW temperature and pCO₂ levels projected for the austral spring of 2100 (the B1 “reduced” and the A1FI “business-as-usual” CO₂ emission scenarios) at Heron Island (GBR, Australia). These assemblages were dominated by the presence of red algae and non-epiphytic cyanobacteria, i.e. cyanobacteria that grow attached to the substrate rather than on filamentous algae. The endpoint algal composition was mostly controlled by the presence/absence of farming damselfish, despite a large variability found between the algal assemblages of individual fish. Different scenarios appeared to be responsible for a mild, species specific change in community composition, observable in some brown and green algae, but only in the absence of farming fish. Farming fish appeared unaffected by the conditions to which they were exposed. Algal biomass reductions were found under “reduced” CO₂ emission, but not “business-as-usual” scenarios. This suggests that action taken to limit CO₂ emissions may, if the majority of algae behave similarly across all seasons, reduce the potential for phase shifts that lead to algal dominated communities. At the same time the availability of food resources to damselfish and other herbivores would be smaller under “reduced” emission scenarios.     

Symbiotic interactions in the Silurian of North America

There are at least 47 different symbiotic pairs of taxa and 16 symbiotic associations in the Silurian of North America. Crinoids are most common host species and they hosted variety of epibiotic and endobiotic symbionts, including Tremichnus, platyceratid gastropods, brachiopods, microconchids, cornulitids, cyclostome bryozoans and favositid tabulates. Eighteen symbiotic pairs contain at least one colonial partner. Stromatoporoids hosted the most diverse fauna of endobiotic symbionts, including cornulitids, lingulids, Chaetosalpinx, Heliocosalpinx and rugosans. Among 16 symbiotic associations of Silurian of North America, 8 are common between North America and Baltica. North American symbiotic associations involving stromatoporoid hosts are the most similar to their Baltic equivalents.     

Diversity and stability of egg‐bacterial assemblages: The role of paternal care in the glassfrog Hyalinobatrachium colymbiphyllum

Embryos of oviparous organisms must cope with harsh environments and are especially susceptible to disease, considering that many immune mechanisms do not develop until later in life. Parents may transmit symbiotic microflora to eggs, which can contribute to embryo immune defense. Despite the importance of symbiotic microbes for immune function and survival of adult amphibians, vertical transfer of symbionts in amphibians has received less attention than in other taxa. Here, we test the role of male‐only parental care in establishing and maintaining the diversity of egg‐bacterial assemblages in a Neotropical glassfrog (Centrolenidae). Previous research suggests that father Hyalinobatrachium colymbiphyllum may transfer bacterial symbionts to their eggs. We combined a male‐removal experiment in situ with 16S rRNA gene amplicon sequencing to determine whether egg attendance by father H. colymbiphyllum influences the bacterial community and survival of eggs. We found that eggs harbor a diverse and stable bacterial assemblage. Despite different host environments, we found that adult skin and eggs supported very similar bacterial assemblages—even after removing fathers. While we found overlap in the bacteria present on eggs and their fathers, our experiment reveals that extended male care does not contribute to the maintenance of egg‐bacterial communities, so there may be other potential routes of transfer. This study contributes to our understanding of the diversity and maintenance of egg microbiomes, and motivates further research on how initial bacteria are acquired and the ontogenetic development of host–symbiont communities.     

Phylogenetic placement of "zoochlorellae" (Chlorophyta), algal symbiont of the temperate sea anemone Anthopleura elegantissima

At northern latitudes the sea anemones Anthopleura elegantissima and its congener A. xanthogrammica contain unidentified green chlorophytes (zoochlorellae) in addition to dinophytes belonging to the genus Symbiodinium. This dual algal symbiosis, involving members of distinct algal phyla in one host, has been extensively studied from the perspective of the ecological and energetic consequences of hosting one symbiotic type over the other. However, the identity of the green algal symbiont has remained elusive. We determined the phylogenetic position of the marine zoochlorellae inhabiting A. elegantissima by comparing sequence data from two cellular compartments, the nuclear 18S ribosomal RNA gene region and the plastid-encoded rbcL gene. The results support the inclusion of these zoochlorellae in a clade of green algae that form symbioses with animal (Anthopleura elegantissima), fungal (the lichen genus Nephroma), and seed plant (Ginkgo) partners. This clade is distinct from the Chlorella symbionts of Hydra. The phylogenetic diversity of algal hosts observed in this clade indicates a predisposition for this group of algae to participate in symbioses. An integrative approach to the study of these algae, both within the host and in culture, should yield important clues about how algae become symbionts in other organisms.     

Microbial Communities of Three Sympatric Australian Stingless Bee Species

Bacterial symbionts of insects have received increasing attention due to their prominent role in nutrient acquisition and defense. In social bees, symbiotic bacteria can maintain colony homeostasis and fitness, and the loss or alteration of the bacterial community may be associated with the ongoing bee decline observed worldwide. However, analyses of microbiota associated with bees have been largely confined to the social honeybees (Apis mellifera) and bumblebees (Bombus spec.), revealing – among other taxa – host-specific lactic acid bacteria (LAB, genus Lactobacillus) that are not found in solitary bees. Here, we characterized the microbiota of three Australian stingless bee species (Apidae: Meliponini) of two phylogenetically distant genera (Tetragonula and Austroplebeia). Besides common plant bacteria, we find LAB in all three species, showing that LAB are shared by honeybees, bumblebees and stingless bees across geographical regions. However, while LAB of the honeybee-associated Firm4–5 clusters were present in Tetragonula, they were lacking in Austroplebeia. Instead, we found a novel clade of likely host-specific LAB in all three Australian stingless bee species which forms a sister clade to a large cluster of Halictidae-associated lactobacilli. Our findings indicate both a phylogenetic and geographical signal of host-specific LAB in stingless bees and highlight stingless bees as an interesting group to investigate the evolutionary history of the bee-LAB association.     

“Endomicrobia” and Other Bacteria Associated with the Hindgut of Dermolepida albohirtum Larvae

Symbiotic bacteria residing in the hindgut chambers of scarab beetle larvae may be useful in paratransgenic approaches to reduce larval root-feeding activities on agricultural crops. We compared the bacterial community profiles associated with the hindgut walls of individual Dermolepida albohirtum third-instar larvae over 2 years and those associated with their plant root food source among different geographic regions. Denaturing gradient gel electrophoresis analysis was used with universal and Actinobacteria-specific 16S rRNA primers to reveal a number of taxa that were found consistently in all D. albohirtum larvae but not in samples from their food source, sugarcane roots. These taxa included representatives from the “Endomicrobia,” Firmicutes, Proteobacteria, and Actinobacteria and were related to previously described bacteria from the intestines of other scarab larvae and termites. These universally distributed taxa have the potential to form vertically transmitted symbiotic associations with these insects.     

Localization of attachment area of the symbiotic Chlorella variabilis of the ciliate Paramecium bursaria during the algal removal and reinfection

Chlorella spp. and ciliate Paramecium bursaria share a mutual symbiosis. However, both alga-removed P. bursaria and isolated symbiotic algae can grow independently. Additionally, mixing them experimentally can cause algal reinfection through host phagocytosis. Although the symbiotic algal localization beneath the host cell cortex is a prerequisite phenomenon for maintenance of the relationship of their endosymbiosis, how and where the algae locate beneath the host cell cortex remains unknown. To elucidate this phenomenon, algal distribution patterns during algal removal and reinfection were observed. During algal removal, algae at the host anterior cortex were easier to remove than at the posterior and ventral or dorsal cortex areas. During algal reinfection, the algae after separation from the host digestive vacuoles tended to localize beneath the host ventral or dorsal cortex more readily than that at other cortices. Algae that reinfected trichocyst-removed paramecia didn’t show this localization. Trichocyst-discharge experiments clarified that trichocysts of the anterior cortex are difficult to remove. In 14 strains of P. bursaria, some of the paramecia lacked their symbiotic algae at the anterior cortex. These observations demonstrate that symbiotic algae of P. bursaria are difficult to localize at the anterior cortex and that they are easy to remove from the area.