Coexistence of bacterial sulfide oxidizers, sulfate reducers, and spirochetes in a gutless worm (Oligochaeta) from the Peru margin

Olavius crassitunicatus is a small symbiont-bearing worm that occurs at high abundance in oxygen-deficient sediments in the East Pacific Ocean. Using comparative 16S rRNA sequence analysis and fluorescence in situ hybridization, we examined the diversity and phylogeny of bacterial symbionts in two geographically distant O. crassitunicatus populations (separated by 385 km) on the Peru margin (water depth, approximately 300 m). Five distinct bacterial phylotypes co-occurred in all specimens from both sites: two members of the gamma-Proteobacteria (Gamma 1 and 2 symbionts), two members of the delta-Proteobacteria (Delta 1 and 2 symbionts), and one spirochete. A sixth phylotype belonging to the delta-Proteobacteria (Delta 3 symbiont) was found in only one of the two host populations. Three of the O. crassitunicatus bacterial phylotypes are closely related to symbionts of other gutless oligochaete species; the Gamma 1 phylotype is closely related to sulfide-oxidizing symbionts of Olavius algarvensis, Olavius loisae, and Inanidrilus leukodermatus, the Delta 1 phylotype is closely related to sulfate-reducing symbionts of O. algarvensis, and the spirochete is closely related to spirochetal symbionts of O. loisae. In contrast, the Gamma 2 phylotype and the Delta 2 and 3 phylotypes belong to novel lineages that are not related to other bacterial symbionts. Such a phylogenetically diverse yet highly specific and stable association in which multiple bacterial phylotypes coexist within a single host has not been described previously for marine invertebrates.     

Coexistence of Bacterial Sulfide Oxidizers, Sulfate Reducers, and Spirochetes in a Gutless Worm (Oligochaeta) from the Peru Margin

Olavius crassitunicatus is a small symbiont-bearing worm that occurs at high abundance in oxygen-deficient sediments in the East Pacific Ocean. Using comparative 16S rRNA sequence analysis and fluorescence in situ hybridization, we examined the diversity and phylogeny of bacterial symbionts in two geographically distant O. crassitunicatus populations (separated by 385 km) on the Peru margin (water depth, ~300 m). Five distinct bacterial phylotypes co-occurred in all specimens from both sites: two members of the γ-Proteobacteria (Gamma 1 and 2 symbionts), two members of the δ-Proteobacteria (Delta 1 and 2 symbionts), and one spirochete. A sixth phylotype belonging to the δ-Proteobacteria (Delta 3 symbiont) was found in only one of the two host populations. Three of the O. crassitunicatus bacterial phylotypes are closely related to symbionts of other gutless oligochaete species; the Gamma 1 phylotype is closely related to sulfide-oxidizing symbionts of Olavius algarvensis, Olavius loisae, and Inanidrilus leukodermatus, the Delta 1 phylotype is closely related to sulfate-reducing symbionts of O. algarvensis, and the spirochete is closely related to spirochetal symbionts of O. loisae. In contrast, the Gamma 2 phylotype and the Delta 2 and 3 phylotypes belong to novel lineages that are not related to other bacterial symbionts. Such a phylogenetically diverse yet highly specific and stable association in which multiple bacterial phylotypes coexist within a single host has not been described previously for marine invertebrates.     

New gammaproteobacteria associated with blood-feeding leeches and a broad phylogenetic analysis of leech endosymbionts

Many monophagous animals have coevolutionary relationships with bacteria that provide unavailable nutrients to the host. Frequently, these microbial partners are vertically inherited and reside in specialized structures or tissues. Here we report three new lineages of bacterial symbionts of blood-feeding leeches, one from the giant Amazonian leech, Haementeria ghilianii, and two others from Placobdelloides species. These hosts each possess a different mycetome or esophageal organ morphology where the bacterial cells are located. DNA sequencing of the bacterial 16S rRNA genes and fluorescent in situ hybridization placed these symbionts in two separate clades in the class Gammaproteobacteria. We also conducted a broad phylogenetic analysis of the herein-reported DNA sequences as well as others from bacterial symbionts reported elsewhere in the literature, including alphaproteobacterial symbionts from the leech genus Placobdella as well as Aeromonas veronii from the medicinal leech, Hirudo medicinalis, and a Rickettsia sp. detected in Hemiclepsis marginata. Combined, these results indicate that blood-feeding leeches have forged bacterial partnerships at least five times during their evolutionary history.     

Specificity of the mutualistic association between actinomycete bacteria and two sympatric species of Acromyrmex leaf-cutting ants

Acromyrmex leaf-cutting ants maintain two highly specialized, vertically transmitted mutualistic ectosymbionts: basidiomycete fungi that are cultivated for food in underground gardens and actinomycete Pseudonocardia bacteria that are reared on the cuticle to produce antibiotics that suppress the growth of Escovopsis parasites of the fungus garden. Mutualism stability has been hypothesized to benefit from genetic uniformity of symbionts, as multiple coexisting strains are expected to compete and, thus, reduce the benefit of the symbiosis. However, the Pseudonocardia symbionts are likely to be involved in Red-Queen-like antagonistic co-evolution with Escovopsis so that multiple strains per host might be favoured by selection provided the cost of competition between bacterial strains is low. We examined the genetic uniformity of the Pseudonocardia symbionts of two sympatric species of Acromyrmex ants by comparing partial sequences of the nuclear Elongation Factor-Tu gene. We find no genetic variation in Pseudonocardia symbionts among nest mate workers, neither in Acromyrmex octospinosus, where colonies are founded by a single queen, nor in Acromyrmex echinatior, where mixing of bacterial lineages might happen when unrelated queens cofound a colony. We further show that the two ant species maintain the same pool of Pseudonocardia symbionts, indicating that horizontal transmission occasionally occurs, and that this pool consists of two distinct clades of closely related Pseudonocardia strains. Our finding that individual colonies cultivate a single actinomycete strain is in agreement with predictions from evolutionary theory on host-symbiont conflict over symbiont mixing, but indicates that there may be constraints on the effectiveness of the bacterial symbionts on an evolutionary timescale.     

Phylogeny of 16S rRNA, ribulose 1,5-bisphosphate carboxylase/oxygenase, and adenosine 5'-phosphosulfate reductase genes from gamma- and alphaproteobacterial symbionts in gutless marine worms (oligochaeta) from Bermuda and the Bahamas

Gutless oligochaetes are small marine worms that live in obligate associations with bacterial endosymbionts. While symbionts from several host species belonging to the genus Olavius have been described, little is known of the symbionts from the host genus Inanidrilus. In this study, the diversity of bacterial endosymbionts in Inanidrilus leukodermatus from Bermuda and Inanidrilus makropetalos from the Bahamas was investigated using comparative sequence analysis of the 16S rRNA gene and fluorescence in situ hybridization. As in all other gutless oligochaetes examined to date, I. leukodermatus and I. makropetalos harbor large, oval bacteria identified as Gamma 1 symbionts. The presence of genes coding for ribulose-1,5-bisphosphate carboxylase/oxygenase form I (cbbL) and adenosine 5'-phosphosulfate reductase (aprA) supports earlier studies indicating that these symbionts are chemoautotrophic sulfur oxidizers. Alphaproteobacteria, previously identified only in the gutless oligochaete Olavius loisae from the southwest Pacific Ocean, coexist with the Gamma 1 symbionts in both I. leukodermatus and I. makropetalos, with the former harboring four and the latter two alphaproteobacterial phylotypes. The presence of these symbionts in hosts from such geographically distant oceans as the Atlantic and Pacific suggests that symbioses with alphaproteobacterial symbionts may be widespread in gutless oligochaetes. The high phylogenetic diversity of bacterial endosymbionts in two species of the genus Inanidrilus, previously known only from members of the genus Olavius, shows that the stable coexistence of multiple symbionts is a common feature in gutless oligochaetes.     

Diversity and expression of nitrogen fixation genes in bacterial symbionts of marine sponges

Marine sponges contain complex assemblages of bacterial symbionts, the roles of which remain largely unknown. We identified diverse bacterial nifH genes within sponges and found that nifH genes are expressed in sponges. This is the first demonstration of the expression of any protein-coding bacterial gene within a sponge. Two sponges Ircinia strobilina and Mycale laxissima were collected from Key Largo, Florida and had delta(15)N values of c. 0-1 per thousand and 3-4 per thousand respectively. The potential for nitrogen fixation by symbionts was assessed by amplification of nifH genes. Diverse nifH genes affiliated with Proteobacteria and Cyanobacteria were detected, and expression of nifH genes affiliated with those from cyanobacteria was detected. The nifH genes from surrounding seawater were similar to those of Trichodesmium and clearly different from the cyanobacterial nifH genes detected in the two sponges. This study advances understanding of the role of bacterial symbionts in sponges and suggests that provision of fixed nitrogen is a means whereby symbionts benefit sponges in nutrient-limited reef environments. Nitrogen fixation by sponge symbionts is possibly an important source of new nitrogen to the reef environment that heretofore has been neglected and warrants further investigation.     

Inherited Fungal and Bacterial Endosymbionts of a Parasitic Wasp and Its Cockroach Host

Bacterial endosymbionts of insects are increasingly being recognized as common, diverse, and integral to the biology of their hosts. Inherited fungal symbionts have been largely overlooked, however, even though insect guts appear to be a key habitat for an incredible array of fungal diversity. Like bacteria, fungal symbionts also likely play important roles in the ecology and evolution of their insect associates. The objective of this study was to lay the foundations for understanding the roles of the vertically transmitted fungal and bacterial associates of both the brownbanded cockroach, Supella longipalpa, and its parasitic wasp, Comperia merceti. We used culture-dependent and culture-independent molecular methods and phylogenetic analyses in order to identify the symbionts. Two fungal associates of brownbanded cockroaches were found. To our knowledge, this is the first record of vertically transmitted fungal symbionts in the order Blattaria. The wasp was found to house a close relative of one of the cockroach fungi but no bacterial symbionts. Finally, the brownbanded cockroaches also harbored three lineages of bacterial symbionts: Blattabacterium and two lineages of Wolbachia, indicating the number of vertically transmitted symbionts in this insect may be as many as five.     

Are we underestimating the diversity and incidence of insect bacterial symbionts? A case study in ladybird beetles

Vertically transmitted bacterial symbionts are common in arthropods. However, estimates of their incidence and diversity are based on studies that test for a single bacterial genus and often only include small samples of each host species. Focussing on ladybird beetles, we collected large samples from 21 species and tested them for four different bacterial symbionts. Over half the species were infected, and there were often multiple symbionts in the same population. In most cases, more females than males were infected, suggesting that the symbionts may be sex ratio distorters. Many of these infections would have been missed in previous studies as they only infect a small proportion of the population. Furthermore, 11 out of the 17 symbionts discovered by us were either in the genus Rickettsia or Spiroplasma, which are rarely sampled. Our results suggest that the true incidence and diversity of bacterial symbionts in insects may be far greater than previously thought.     

A specific mix of generalists: bacterial symbionts in Mediterranean Ircinia spp

Microbial symbionts form abundant and diverse components of marine sponge holobionts, yet the ecological and evolutionary factors that dictate their community structure are unresolved. Here, we characterized the bacterial symbiont communities of three sympatric host species in the genus Ircinia from the NW Mediterranean Sea, using electron microscopy and replicated 16S rRNA gene sequence clone libraries. All Ircinia host species harbored abundant and phylogenetically diverse symbiont consortia, comprised primarily of sequences related to other sponge-derived microorganisms. Community-level analyses of bacterial symbionts revealed host species-specific genetic differentiation and structuring of Ircinia-associated microbiota. Phylogenetic analyses of host sponges showed a close evolutionary relationship between Ircinia fasciculata and Ircinia variabilis, the two host species exhibiting more similar symbiont communities. In addition, several bacterial operational taxonomic units were shared between I.?variabilis and Ircinia oros, the two host species inhabiting semi-sciophilous communities in more cryptic benthic habitats, and absent in I.?fasciculata, which occurs in exposed, high-irradiance habitats. The generalist nature of individual symbionts and host-specific structure of entire communities suggest that: (1) a 'specific mix of generalists' framework applies to bacterial symbionts in Ircinia hosts and (2) factors specific to each host species contribute to the distinct symbiont mix observed in Ircinia hosts.     

Infection of algae-free Climacostomum virens with symbiotic Chlorella sp. isolated from algae-containing C. virens

The ciliate Climacostomum virens normally contains algae as symbionts in its cytoplasm and retains them over many generations. An aposymbiotic strain of C. virens which cannot re-establish a new symbiotic association by ingestion of algae derived from green Climacostomum was recently isolated in our laboratory. Results of infection experiments showed that all newly ingested, potentially symbiotic algae were digested in food vacuoles. To clarify whether these ciliates have completely lost their ability to sustain symbiosis with algae or whether this ability can eventually be re-established, infection experiments were performed using a microinjection technique. We have achieved successful infection of algae-free Climacostomum using this method. The endosymbiont population was established in ciliates from as few as 3-5 injected algae, which have retained an intact perialgal vacuole membrane around them. To our knowledge, this is the first evidence of successful infection of aposymbiotic ciliates with algae by microinjection.     

Phylogeny of 16S rRNA, Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase, and Adenosine 5′-Phosphosulfate Reductase Genes from Gamma- and Alphaproteobacterial Symbionts in Gutless Marine Worms (Oligochaeta) from Bermuda and the Bahamas

Gutless oligochaetes are small marine worms that live in obligate associations with bacterial endosymbionts. While symbionts from several host species belonging to the genus Olavius have been described, little is known of the symbionts from the host genus Inanidrilus. In this study, the diversity of bacterial endosymbionts in Inanidrilus leukodermatus from Bermuda and Inanidrilus makropetalos from the Bahamas was investigated using comparative sequence analysis of the 16S rRNA gene and fluorescence in situ hybridization. As in all other gutless oligochaetes examined to date, I. leukodermatus and I. makropetalos harbor large, oval bacteria identified as Gamma 1 symbionts. The presence of genes coding for ribulose-1,5-bisphosphate carboxylase/oxygenase form I (cbbL) and adenosine 5′-phosphosulfate reductase (aprA) supports earlier studies indicating that these symbionts are chemoautotrophic sulfur oxidizers. Alphaproteobacteria, previously identified only in the gutless oligochaete Olavius loisae from the southwest Pacific Ocean, coexist with the Gamma 1 symbionts in both I. leukodermatus and I. makropetalos, with the former harboring four and the latter two alphaproteobacterial phylotypes. The presence of these symbionts in hosts from such geographically distant oceans as the Atlantic and Pacific suggests that symbioses with alphaproteobacterial symbionts may be widespread in gutless oligochaetes. The high phylogenetic diversity of bacterial endosymbionts in two species of the genus Inanidrilus, previously known only from members of the genus Olavius, shows that the stable coexistence of multiple symbionts is a common feature in gutless oligochaetes.     

Bacterial communities of two parthenogenetic aphid species cocolonizing two host plants across the Hawaiian Islands

Aphids (Hemiptera: Aphididae) have been the focus of several studies with respect to their interactions with inherited symbionts, but bacterial communities of most aphid species are still poorly characterized. In this research, we used bar-coded pyrosequencing to characterize bacterial communities in aphids. Specifically, we examined the diversity of bacteria in two obligately parthenogenetic aphid species (the melon aphid, Aphis gossypii, and the cardamom aphid, Pentalonia caladii) cocolonizing two plant species (taro, Colocasia esculenta, and ginger, Alpinia purpurata) across four Hawaiian Islands (Hawaii, Kauai, Maui, and Oahu). Results from this study revealed that heritable symbionts dominated the bacterial communities for both aphid species. The bacterial communities differed significantly between the two species, and A. gossypii harbored a more diverse bacterial community than P. caladii. The bacterial communities also differed across aphid populations sampled from the different islands; however, communities did not differ between aphids collected from the two host plants.     

Lucinidae/sulfur-oxidizing bacteria: ancestral heritage or opportunistic association? Further insights from the Bohol Sea (the Philippines)

The first studies of the 16S rRNA gene diversity of the bacterial symbionts found in lucinid clams did not clarify how symbiotic associations had evolved in this group. Indeed, although species-specific associations deriving from a putative ancestral symbiotic association have been described (coevolution scenario), associations between the same bacterial species and various host species (opportunistic scenario) have also been described. Here, we carried out a comparative molecular analysis of hosts, based on 18S and 28S rRNA gene sequences, and of symbionts, based on 16S rRNA gene sequences, to determine as to which evolutionary scenario led to modern lucinid/symbiont associations. For all sequences analyzed, we found only three bacterial symbiont species, two of which are harbored by lucinids colonizing mangrove swamps. The last symbiont is the most common and was found to be independent of biotope or depth. Another interesting feature is the similarity of ctenidial organization of lucinids from the Philippines to those described previously, with the exception that two bacterial morphotypes were observed in two different species (Gloverina rectangularis and Myrtea flabelliformis). Thus, there is apparently no specific association between Lucinidae and their symbionts, the association taking place according to which bacterial species is present in the environment.     

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.     

Bacterial Communities Associated with Host-Adapted Populations of Pea Aphids Revealed by Deep Sequencing of 16S Ribosomal DNA

Associations between microbes and animals are ubiquitous and hosts may benefit from harbouring microbial communities through improved resource exploitation or resistance to environmental stress. The pea aphid, Acyrthosiphon pisum, is the host of heritable bacterial symbionts, including the obligate endosymbiont Buchnera aphidicola and several facultative symbionts. While obligate symbionts supply aphids with key nutrients, facultative symbionts influence their hosts in many ways such as protection against natural enemies, heat tolerance, color change and reproduction alteration. The pea aphid also encompasses multiple plant-specialized biotypes, each adapted to one or a few legume species. Facultative symbiont communities differ strongly between biotypes, although bacterial involvement in plant specialization is uncertain. Here, we analyse the diversity of bacterial communities associated with nine biotypes of the pea aphid complex using amplicon pyrosequencing of 16S rRNA genes. Combined clustering and phylogenetic analyses of 16S sequences allowed identifying 21 bacterial OTUs (Operational Taxonomic Unit). More than 98% of the sequencing reads were assigned to known pea aphid symbionts. The presence of Wolbachia was confirmed in A. pisum while Erwinia and Pantoea, two gut associates, were detected in multiple samples. The diversity of bacterial communities harboured by pea aphid biotypes was very low, ranging from 3 to 11 OTUs across samples. Bacterial communities differed more between than within biotypes but this difference did not correlate with the genetic divergence between biotypes. Altogether, these results confirm that the aphid microbiota is dominated by a few heritable symbionts and that plant specialization is an important structuring factor of bacterial communities associated with the pea aphid complex. However, since we examined the microbiota of aphid samples kept a few generations in controlled conditions, it may be that bacterial diversity was underestimated due to the possible loss of environmental or transient taxa.     

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

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

Host-symbiont recognition in the environmentally transmitted sepiolid squid-Vibrio mutualism

Associations between environmentally transmitted symbionts and their hosts provide a unique opportunity to study the evolution of specificity and subsequent radiation of tightly coupled host-symbiont assemblages [3, 8, 24]. The evidence provided here from the environmentally transmitted bacterial symbiont Vibrio fischeri and its sepiolid squid host (Sepiolidae: Euprymna) demonstrates how host-symbiont specificity can still evolve without vertical transmission of the symbiont [1]. Infection by intraspecific V. fischeri symbionts exhibited preferential colonization over interspecific V. fischeri symbionts, indicating a high degree of specificity for the native symbiotic strains. Inoculation with symbiotic bacteria from other taxa (monocentrid fish and loliginid squids) produced little or no colonization in two species of Euprymna, despite their presence in the same or similar habitats as these squids. These findings of host specificity between native Vibrios and sepiolid squids provides evidence that the presence of multiple strains of symbionts does not dictate the composition of bacterial symbionts in the host.     

Bacterial endosymbionts: genome reduction in a hot spot

Prokaryotic symbionts are common in invertebrates and play an essential metabolic role in deep-sea hydrothermal vent communities. Complete genome sequences of bacterial endosymbionts of two deep-sea clams are providing new insights into evolutionary genome reduction.     

The Effect of Co-Culture of Two Coral Species on Their Bacterial Composition Under Captive Environments

Marine Biotechnology - Coral symbionts are important members of the coral holobiont, and coral bacterial flora are essential in host health maintenance and coral conservation. Coral symbionts are...     

Phylogenetically Diverse Denitrifying and Ammonia-Oxidizing Bacteria in Corals Alcyonium gracillimum and Tubastraea coccinea

To date, the association of coral–bacteria and the ecological roles of bacterial symbionts in corals remain largely unknown. In particular, little is known about the community components of bacterial symbionts of corals involved in the process of denitrification and ammonia oxidation. In this study, the nitrite reductase (nirS and nirK) and ammonia monooxygenase subunit A (amoA) genes were used as functional markers. Diverse bacteria with the potential to be active as denitrifiers and ammonia-oxidizing bacteria (AOB) were found in two East China Sea corals: stony coral Alcyonium gracillimum and soft coral Tubastraea coccinea. The 16S rRNA gene library analysis demonstrated different communities of bacterial symbionts in these two corals of the same location. Nitrite reductase nirK gene was found only in T. coccinea, while both nirK and nirS genes were detected in A. gracillimum, which might be the result of the presence of different bacterial symbionts in these two corals. AOB rather than ammonia-oxidizing archaea were detected in both corals, suggesting that AOB might play an important role in the ammonia oxidation process of the corals. This study indicates that the coral bacterial symbionts with the potential for nitrite reduction and ammonia oxidation might have multiple ecological roles in the coral holobiont, which promotes our understanding of bacteria-mediated nitrogen cycling in corals. To our knowledge, this study is the first assessment of the community structure and phylogenetic diversity of denitrifying bacteria and AOB in corals based on nirK, nirS, and amoA gene library analysis.