Search

Neotropical army ants of the genus Eciton are top arthropod predators in tropical rainforests. Microbial symbionts, including Unclassified Firmicutes (UF) and Unclassified Entomoplasmatales (UE), are associated with this genus and likely play a significant role in the biology of these ants. While previous work focused on associations of army ants and gut microbes across large geographic scales, here we report a community survey of the gut microbes colonizing the six sympatric Eciton army ant species in a single Costa Rican location. Furthermore, we characterized the gut microbiota associated with different army ant castes in the swarm-raiding species Eciton burchellii. We employed a combination of 16S ribosomal RNA (rRNA) amplicon sequencing as well as fluorescence and electron microscopy to identify gut microbes and to verify their presence in ant guts. We also measured the diversity and interaction specificity of the ant-gut microbe interaction network. The two most dominant operational taxonomic unit (OTU) phylotypes in all species were related to UF and UE previously found in army ants, followed by OTUs assigned to the genus Weissella. Furthermore, the worker castes of E. burchellii shared similar gut microbiota, also dominated by UF and UE phylotypes. Overall, we found a low diversity of gut microbes and a low interaction specificity between army ants and microbes at the community level, mainly because most microbe strains were detected in various Eciton species. The fluorescence in-situ hybridization analyses documented the presence of the two dominant phylotypes within ant guts, and electron microscopy located bacterial biofilms in the hindgut near the microvilli. Their morphology suggests that these bacteria probably belong to the dominant phylotypes UF and UE. Taken together, our results confirm that the Eciton gut microbiome is consistently dominated by a few species of specialized bacteria that may improve nutrient uptake efficiency of host ants. Further research should employ multi-omics and culture-dependent strategies to fully understand the role of these potential symbionts in ant ecophysiology.

A new species of spiroplasmid, Spiroplasma burmanica sp. nov. (Mollicutes: Entomoplasmatales: Spiroplasmataceae) is described from the body cavity of a fossil plant louse (Psylloidea: Sternorrhyncha) in Burmese amber. The new species is pleomorphic with body shapes varying from oval to helical. The majority of the helical cells occur in the head, thorax (including leg cavities) and abdomen of the fossil psyllid. The association between S. burmanica and the psyllid is considered to be a case of symbiosis, similar to extant relationships. This discovery of the first fossil spiroplasmid shows that psyllids carried these microorganisms some 100 million years ago.

BackgroundVarious endosymbiotic bacteria, includingWolbachiaof the Alphaproteobacteria, infect a wide range of insects and are capable of inducing reproductive abnormalities to their hosts such as cytoplasmic incompatibility (CI), parthenogenesis, feminization and male-killing. These extended phenotypes can be potentially exploited in enhancing environmentally friendly methods, such as the sterile insect technique (SIT), for controlling natural populations of agricultural pests. The goal of the present study is to investigate the presence ofWolbachia,Spiroplasma,ArsenophonusandCardiniumamongBactrocera,DacusandZeugodacusflies of Southeast Asian populations, and to genotype any detectedWolbachiastrains.ResultsA specific 16SrRNAPCR assay was used to investigate the presence of reproductive parasites in natural populations of nine different tephritid species originating from three Asian countries, Bangladesh, China and India.Wolbachiainfections were identified inBactrocera dorsalis,B. correcta,B. scutellarisandB. zonata, with 12.2–42.9% occurrence, Entomoplasmatales inB. dorsalis,B. correcta,B. scutellaris,B. zonata,Zeugodacus cucurbitaeandZ. tau(0.8–14.3%) andCardiniuminB. dorsalisandZ. tau(0.9–5.8%), while none of the species tested, harbored infections withArsenophonus. Infected populations showed a medium (between 10 and 90%) or low (Wolbachia, 2 to 33% for Entomoplasmatales and 5 to 45% forCardinium.Wolbachiaand Entomoplasmatales infections were found both in tropical and subtropical populations, the former mostly in India and the latter in various regions of India and Bangladesh.Cardiniuminfections were identified in both countries but only in subtropical populations. Phylogenetic analysis revealed the presence ofWolbachiawith some strains belonging either to supergroup B or supergroup A. Sequence analysis revealed deletions of variable length and nucleotide variation in threeWolbachiagenes.Spiroplasmastrains were characterized as citri–chrysopicola–mirum and ixodetis strains while the remaining Entomoplasmatales to the Mycoides–Entomoplasmataceae clade.Cardiniumstrains were characterized as group A, similar to strains infectingEncarsia pergandiella.ConclusionsOur results indicated that in the Southeast natural populations examined, supergroup AWolbachiastrain infections were the most common, followed by Entomoplasmatales andCardinium. In terms of diversity, most strains of each bacterial genus detected clustered in a common group. Interestingly, the deletions detected in threeWolbachiagenes were either new or similar to those of previously identified pseudogenes that were integrated in the host genome indicating putative horizontal gene transfer events inB. dorsalis,B. correctaandB. zonata.

Mollicutes, a widespread class of bacteria associated with animals and plants, were recently identified as abundant abdominal endosymbionts in healthy workers of attine fungus-farming leaf-cutting ants. We obtained draft genomes of the two most common strains harbored by Panamanian fungus-growing ants. Reconstructions of their functional significance showed that they are independently acquired symbionts, most likely to decompose excess arginine consistent with the farmed fungal cultivars providing this nitrogen-rich amino-acid in variable quantities. Across the attine lineages, the relative abundances of the two Mollicutes strains are associated with the substrate types that foraging workers offer to fungus gardens. One of the symbionts is specific to the leaf-cutting ants and has special genomic machinery to catabolize citrate/glucose into acetate, which appears to deliver direct metabolic energy to the ant workers. Unlike other Mollicutes associated with insect hosts, both attine ant strains have complete phage-defense systems, underlining that they are actively maintained as mutualistic symbionts., eLife digest Bacteria live inside the gut of most creatures. Some are harmful, some beneficial, and some have no clear effects. Studying the genetic material (the genome) of gut bacteria has revealed how they can improve the health, efficiency, and reproductive success of their hosts. For example, studies show that insects with low nutrient diets often benefit from gut bacteria that make vitamins or help them convert food into energy. Panamanian leafcutter ants live in large colonies and farm fungus for food. They harvest leaves to feed their fungus farms and many are major crop pests in Latin America. How they evolved to be so successful is unclear. Recent studies have shown that huge numbers of bacteria called Mollicutes live in the leafcutter ants’ guts. These bacteria do not make the ants sick, so they were thought to be somehow beneficial. Now, Sapountzis et al. show that the two most common types of Mollicutes found in leafcutter ants evolved to make fungus farming more efficient. The complete genomes of two Mollicutes strains were analyzed and compared to the ones found in other insects. The results showed that both types of Mollicutes can turn excess quantities of the amino acid arginine into a nitrogen-rich fertilizer the ants deposit on their fungal gardens as feces. This helps the ants produce more food. One of the two types can also decompose citrate from plant sap and fruit juice consumed by the ants. This produces acetate, which supplements the ants’ fungal diets and provides extra energy. The unique energy-producing Mollicutes may explain why leafcutter ants evolved larger colonies and sustain higher levels of worker activity than other species of fungus-growing ants. The genome data also showed that both types of Mollicutes have costly defense systems to protect themselves against bacteria-destroying viruses. Many bacteria do not invest is such systems, but the Mollicutes may be able to afford them because their ant hosts provide them with plenty of food. This suggests that both the ants and the Mollicutes benefit from their symbiotic relationship.

Microbiome surveys provide clues for the functional roles of symbiotic microbial communities and their hosts. In this study, we elucidated bacterial microbiomes associated with the vertically transmitted fungal inocula (pellets) used by foundress queens of the leaf-cutting ant Atta texana as starter-cultures for new gardens. As reference microbiomes, we also surveyed bacterial microbiomes of foundress queens, gardens and brood of incipient nests. Pseudomonas, Acinetobacter, Propionibacterium and Corynebacterium were consistently present in high abundance in microbiomes. Some pellet and ant samples contained abundant bacteria from an Entomoplasmatales-clade, and a separate PCR-based survey of Entomoplasmatales bacteria in eight attine ant-genera from Brazil placed these bacteria in a monophyletic clade within the bacterial genus Mesoplasma. The attine ant-Mesoplasma association parallels a similar association between a closely related, monophyletic Entomoplasmatales-clade and army ants. Of thirteen A. texana nests surveyed, three nests with exceptionally high Mesoplasma abundance died, whereas the other nests survived. It is unclear whether Mesoplasma was the primary cause of mortality, or Mesoplasma became abundant in moribund nests for non-pathogenic reasons. However, the consistent and geographically widespread presence of Mesoplasma suggests an important functional role in the association with attine ants.

The "Spiroplasma cluster" is a taxonomically heterogeneous assemblage within the phylum Tenericutes encompassing different Entomoplasmatales species as well as the genus Mycoplasma, type genus of the order Mycoplasmatales. Within this cluster, the family Entomoplasmataceae contains two non-cohesive genera Entomoplasma and Mesoplasma with their members exhibiting extensive polyphyletic branching; additionally, the genus Mycoplasma is also embedded within this family. Genome sequences are now available for all 19 Entomoplasmataceae species with validly published names, as well as 6 of the 7 species from the genus Mycoplasma. With the aim of developing a reliable phylogenetic and taxonomic framework for the family Entomoplasmataceae, exhaustive phylogenetic and comparative genomic studies were carried out on these genome sequences. Phylogenetic trees were constructed based on concatenated sequences of 121 core proteins for this cluster, 67 conserved proteins shared with the phylum Firmicutes, 40 ribosomal proteins, three major subunits of RNA polymerase (RpoA, B and C) by different means and also for the 16S rRNA gene sequences. The interspecies relationships as well as different species groups observed in these trees were identical and robustly resolved. In all of these trees, members of the genera Mesoplasma and Entomoplasma formed three and two distinct clades, respectively, which were interspersed among the members of the other genus. The observed species groupings in the phylogenetic trees are independently strongly supported by our identification of 103 novel molecular markers or synapomorphies in the forms of conserved signature indels and conserved signature proteins, which are uniquely shared by the members of different observed species clades. To account for the different observed species clades, we are proposing a division of the genus Mesoplasma into an emended genus Mesoplasma and two new genera Tullyiplasma gen. nov. and Edwardiiplasma gen. nov. Likewise, to recognize the distinct species groupings of Entomoplasma, we are proposing its division into an emended genus Entomoplasma and a new genus Williamsoniiplasma gen. nov. Lastly, to rectify the long-existing taxonomic anomaly caused by the presence of genus Mycoplasma (order Mycoplasmatales) within the Entomoplasmatales, we are proposing an emendation of the family Mycoplasmataceae to include both Entomoplasmataceae plus Mycoplasma species and an emendation of the order Mycoplasmatales, which now comprises of the emended family Mycoplasmataceae and the family Spiroplasmataceae. The taxonomic reclassifications proposed here accurately reflect the species relationships within this group of Tenericutes and they should lead to a better understanding of their biological and pathogenic characteristics.

Phytoplasmas are a diverse but phylogenetically coherent group of cell-wall-less bacteria affiliated with the class Mollicutes . Due to difficulties in establishing axenic culture, phytoplasmas were assigned to a provisional genus, ‘Candidatus Phytoplasma’, and the genus was embraced within the order Acholeplasmatales . However, phytoplasmas differ significantly from species of the genus Acholeplasma in their habitat specificities, modes of life, metabolic capabilities, genomic architectures, and phylogenetic positions. This communication describes the unique ecological, nutritional, biochemical, genomic and phylogenetic properties that distinguish phytoplasmas from species of the genus Acholeplasma and all other taxa in the class Mollicutes . Since such distinguishing properties of the phytoplasmas are not referable to the descriptions of the order Acholeplasmatales and of all other existing orders, namely Mycoplasmatales , Entomoplasmatales and Anaeroplasmatales , this communication raises the question of whether ‘ Candidatus Phytoplasma ’ should be retained in the order Acholeplasmatales or whether a novel provisional order and family should be created to accommodate the genus ‘ Ca. Phytoplasma ’.

Made available in DSpace on 2018-12-11T17:13:28Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-10-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Bacterial endosymbionts are common in all insects, and symbiosis has played an integral role in ant evolution. Atta sexdens rubropilosa leaf-cutting ants cultivate their symbiotic fungus using fresh leaves. They need to defend themselves and their brood against diseases, but they also need to defend their obligate fungus gardens, their primary food source, from infection, parasitism, and usurpation by competitors. This study aimed to characterize the microbial communities in whole workers and different tissues of A. sexdens rubropilosa queens using Ion Torrent NGS. Our results showed that the microbial community in the midgut differs in abundance and diversity from the communities in the postpharyngeal gland of the queen and in whole workers. The main microbial orders in whole workers were Lactobacillales, Clostridiales, Enterobacteriales, Actinomycetales, Burkholderiales, and Bacillales. In the tissues of the queens, the main orders were Burkholderiales, Clostridiales, Syntrophobacterales, Lactobacillales, Bacillales, and Actinomycetales (midgut) and Entomoplasmatales, unclassified γ-proteobacteria, and Actinomycetales (postpharyngeal glands). The high abundance of Entomoplasmatales in the postpharyngeal glands (77%) of the queens was an unprecedented finding. We discuss the role of microbial communities in different tissues and castes. Bacteria are likely to play a role in nutrition and immune defense as well as helping antimicrobial defense in this ant species. Centro de Estudos de Insetos Sociais UNESP - Univ Estadual Paulista, Campus Rio Claro, Av. 24A, 1515, Bela Vista Universidade Federal do Piauí - Campus Ministro Reis Velloso, Av. São Sebastião, 2819 Centro de Estudos de Insetos Sociais UNESP - Univ Estadual Paulista, Campus Rio Claro, Av. 24A, 1515, Bela Vista CAPES: 007343/2014-00 CNPq: 157837/2015-7 FAPESP: 2012/12541-3 CNPq: 306910/2011-0

Symbiotic bacteria play important roles in the biology of their arthropod hosts. Yet the microbiota of many diverse and influential groups remain understudied, resulting in a paucity of information on the fidelities and histories of these associations. Motivated by prior findings from a smaller scale, 16S rRNA-based study, we conducted a broad phylogenetic and geographical survey of microbial communities in the ecologically dominant New World army ants (Formicidae: Dorylinae). Amplicon sequencing of the 16S rRNA gene across 28 species spanning the five New World genera showed that the microbial communities of army ants consist of very few common and abundant bacterial species. The two most abundant microbes, referred to as Unclassified Firmicutes and Unclassified Entomoplasmatales, appear to be specialized army ant associates that dominate microbial communities in the gut lumen of three host genera, Eciton, Labidus and Nomamyrmex. Both are present in other army ant genera, including those from the Old World, suggesting that army ant symbioses date back to the Cretaceous. Extensive sequencing of bacterial protein-coding genes revealed multiple strains of these symbionts co-existing within colonies, but seldom within the same individual ant. Bacterial strains formed multiple host species-specific lineages on phylogenies, which often grouped strains from distant geographic locations. These patterns deviate from those seen in other social insects, and raise intriguing questions about the influence of army ant colony swarm-founding and within-colony genetic diversity on strain co-existence, and the effects of hosting a diverse suite of symbiont strains on colony ecology.

The impact of different diets on the gut microbiota of reared Nephrops norvegicus was investigated based on bacterial 16S rRNA gene diversity. Specimens were collected from Pagasitikos Gulf (Greece) and kept in experimental rearing tanks, under in situ conditions, for 6months. Treatments included three diets: frozen natural (mussel) food (M), dry formulated pellet (P) and starvation (S). Gut samples were collected at the initiation of the experiment, and after 3 and 6months. Tank water and diet samples were also analyzed for bacterial 16S rRNA gene diversity. Statistical analysis separated the two groups fed or starved (M and P vs. S samples). Most gut bacteria were not related to the water or diet bacteria, while bacterial diversity was higher in the starvation samples. M and P samples were dominated by Gammaproteobacteria, Epsilonproteobacteria and Tenericutes. Phylotypes clustering in Photobacterium leiognathi, Shewanella sp. and Entomoplasmatales had high frequencies in the M and P samples but low sequence frequencies in S samples. The study showed that feeding resulted in the selection of specific species, which also occurs in the natural population, and might be associated with the animal's nutrition.

In the present study, we compared communities of bacteria in two jellyfish species (the ‘golden’ jellyfish Mastigias cf. papua and the box jellyfish Tripedalia cf. cystophora) and water in three marine lakes located in the Berau region of northeastern Borneo, Indonesia. Jellyfish-associated bacterial communities were compositionally distinct and less diverse than bacterioplankton communities. Alphaproteobacteria, Gammaproteobacteria, Synechococcophycidae and Flavobacteriia were the most abundant classes in water. Jellyfish-associated bacterial communities were dominated by OTUs assigned to the Gammaproteobacteria (family Endozoicimonaceae), Mollicutes, Spirochaetes and Alphaproteobacteria (orders Kiloniellales and Rhodobacterales). Mollicutes were mainly restricted to Mastigias whereas Spirochaetes and the order Kiloniellales were most abundant in Tripedalia hosts. The most abundant OTU overall in jellyfish hosts was assigned to the family Endozoicimonaceae and was highly similar to organisms in Genbank obtained from various hosts including an octocoral, bivalve and fish species. Other abundant OTUs included an OTU assigned to the order Entomoplasmatales and mainly found in Mastigias hosts and OTUs assigned to the Spirochaetes and order Kiloniellales and mainly found in Tripedalia hosts. The low sequence similarity of the Entomoplasmatales OTU to sequences in Genbank suggests that it may be a novel lineage inhabiting Mastigias and possibly restricted to marine lakes

In this article, we describe the distributions of Entomoplasmatales bacteria across the ants, identifying a novel lineage of gut bacteria that is unique to the army ants. While our findings indicate that the Entomoplasmatales are not essential for growth or development, molecular analyses suggest that this relationship is host specific and potentially ancient. The documented trends add to a growing body of literature that hints at a diversity of undiscovered associations between ants and bacterial symbionts.

Arbuscular mycorrhizal fungi (AMF) have been symbionts of land plants for at least 450 Myr. It is known that some AMF host in their cytoplasm Gram-positive endobacteria called bacterium-like organisms (BLOs), of unknown phylogenetic origin. In this study, an extensive inventory of 28 cultured AMF, from diverse evolutionary lineages and four continents, indicated that most of the AMF species investigated possess BLOs. Analyzing the 16S ribosomal DNA (rDNA) as a phylogenetic marker revealed that BLO sequences from divergent lineages all clustered in a well-supported monophyletic clade. Unexpectedly, the cell-walled BLOs were shown to likely represent a sister clade of the Mycoplasmatales and Entomoplasmatales, within the Mollicutes, whose members are lacking cell walls and show symbiotic or parasitic lifestyles. Perhaps BLOs maintained the Gram-positive trait whereas the sister groups lost it. The intracellular location of BLOs was revealed by fluorescent in situ hybridization (FISH), and confirmed by pyrosequencing. BLO DNA could only be amplified from AMF spores and not from spore washings. As highly divergent BLO sequences were found within individual fungal spores, amplicon libraries derived from Glomus etunicatum isolates from different geographic regions were pyrosequenced; they revealed distinct sequence compositions in different isolates. Our results show a vertically inherited, monophyletic and globally distributed lineage of endobacteria thriving in AMF cytoplasm. These bacteria split from their sister groups more than 400 Myr ago, colonizing their fungal hosts already before main AMF lineages separated. The BLO-AMF symbiosis can, therefore, be dated back at least to the time when AMF formed the ancestral symbiosis with emergent land plants.

We investigated the diversity of the bacterial 16S rRNA genes occurring on the abdominal setal tufts and in the emptied midgut of the marine mudshrimp Pestarella tyrrhena (Decapoda: Thalassinidea). There were no dominant phylotypes on the setal tufts. The majority of the phylotypes belonged to the phylum Bacteroidetes, frequently occurring in the water column. The rest of the phylotypes were related to anoxygenic photosynthetic α-Proteobacteria and to Actinobacteria. This bacterial profile seems more of a marine assemblage rather than a specific one suggesting that no specific microbial process can be inferred on the setal tufts. In the emptied midgut, 64 clones were attributed to 16 unique phylotypes with the majority (40.6%) belonging to the γ-Proteobacteria, specifically to the genus Vibrio, a marine group with known symbionts of decapods. The next most abundant group was the ɛ-Proteobacteria (28.1%), with members as likely symbionts related to the processes involving redox reactions occurring in the midgut. In addition, phylotypes related to the Spirochaetes (10.9%) were also present, with relatives capable of symbiosis conducting a nitrite associated metabolism. Entomoplasmatales, Bacteroidetes and Actinobacteria related phylotypes were also found. These results indicate a specific bacterial community dominated by putative symbiotic Bacteria within the P. tyrrhena’s midgut.

Bacterial symbionts are important fitness determinants of insects. Some hosts have independently acquired taxonomically related microbes to meet similar challenges, but whether distantly related hosts that live in tight symbiosis can maintain similar microbial communities has not been investigated. Varying degrees of nest sharing between Megalomyrmex social parasites (Solenopsidini) and their fungus‐growing ant hosts (Attini) from the genera Cyphomyrmex, Trachymyrmex and Sericomyrmex allowed us to address this question, as both ant lineages rely on the same fungal diet, interact in varying intensities and are distantly related. We used tag‐encoded FLX 454 pyrosequencing and diagnostic PCR to map bacterial symbiont diversity across the Megalomyrmex phylogenetic tree, which also contains free‐living generalist predators. We show that social parasites and hosts share a subset of bacterial symbionts, primarily consisting of Entomoplasmatales, Bartonellaceae, Acinetobacter, Wolbachia and Pseudonocardia and that Entomoplasmatales and Bartonellaceae can co‐infect specifically associated combinations of hosts and social parasites with identical 16S rRNA genotypes. We reconstructed in more detail the population‐level infection dynamics for Entomoplasmatales and Bartonellaceae in Megalomyrmex symmetochus guest ants and their Sericomyrmex amabilis hosts. We further assessed the stability of the bacterial communities through a diet manipulation experiment and evaluated possible transmission modes in shared nests such as consumption of the same fungus garden food, eating of host brood by social parasites, trophallaxis and grooming interactions between the ants, or parallel acquisition from the same nest environment. Our results imply that cohabiting ant social parasites and hosts may obtain functional benefits from bacterial symbiont transfer even when they are not closely related.

Ants and termites have independently evolved obligate fungus-farming mutualisms, but their gardening procedures are fundamentally different, as the termites predigest their plant substrate whereas the ants deposit it directly on the fungus garden. Fungus-growing termites retained diverse gut microbiota, but bacterial gut communities in fungus-growing leaf-cutting ants have not been investigated, so it is unknown whether and how they are specialized on an exclusively fungal diet. Here we characterized the gut bacterial community of Panamanian Acromyrmex species, which are dominated by only four bacterial taxa: Wolbachia , Rhizobiales , and two Entomoplasmatales taxa. We show that the Entomoplasmatales can be both intracellular and extracellular across different gut tissues, Wolbachia is mainly but not exclusively intracellular, and the Rhizobiales species is strictly extracellular and confined to the gut lumen, where it forms biofilms along the hindgut cuticle supported by an adhesive matrix of polysaccharides. Tetracycline diets eliminated the Entomoplasmatales symbionts but hardly affected Wolbachia and only moderately reduced the Rhizobiales , suggesting that the latter are protected by the biofilm matrix. We show that the Rhizobiales symbiont produces bacterial NifH proteins that have been associated with the fixation of nitrogen, suggesting that these compartmentalized hindgut symbionts alleviate nutritional constraints emanating from an exclusive fungus garden diet reared on a substrate of leaves.

Uncultivated bacteria that densely colonize the midgut glands (hepatopancreas) of the terrestrial isopod Porcellio scaber (Crustacea: Isopoda) were identified by cloning and sequencing of their 16S rRNA genes. Phylogenetic analysis revealed that these symbionts represent a novel lineage of the Mollicutes and are only distantly related (Mycoplasmatales and Entomoplasmatales . Fluorescence in situ hybridization with a specific oligonucleotide probe confirmed that the amplified 16S rRNA gene sequences indeed originated from a homogeneous population of symbionts intimately associated with the epithelial surface of the hepatopancreas. The same probe also detected morphotypically identical symbionts in other crinochete isopods. Scanning and transmission electron microscopy revealed uniform spherical bacterial cells without a cell wall, sometimes interacting with the microvilli of the brush border by means of stalk-like cytoplasmic appendages, which also appeared to be involved in cell division through budding. Based on the isolated phylogenetic position and unique cytological properties, the provisional name “ Candidatus Hepatoplasma crinochetorum” is proposed for this new taxon of Mollicutes colonizing the hepatopancreas of P. scaber .

The genus Spiroplasma (helical mollicutes: Bacteria: Firmicutes: Mollicutes: Entomoplasmatales: Spiroplasmataceae) is associated primarily with insects. The Mycoplasma mycoides cluster (sensu Weisburg et al. 1989 and Johansson and Pettersson 2002) is a group of mollicutes that includes the type species - Mycoplasma mycoides - of Mycoplasmatales, Mycoplasmataceae and Mycoplasma. This cluster, associated solely with ruminants, contains five other species and subspecies. Earlier phylogenetic reconstructions based on partial 16S rDNA sequences and a limited sample of Spiroplasma and Mycoplasma sequences suggested that the genus Mycoplasma was polyphyletic, as the M. mycoides cluster and the grouping that consisted of the hominis and pneumoniae groups of Mycoplasma species were widely separated phylogenetically and the M. mycoides cluster was allied with Spiroplasma. It is shown here that the M. mycoides cluster arose from Spiroplasma through an intermediate group of non-helical spiroplasmal descendants - the Entomoplasmataceae. As this conclusion has profound implications in the taxonomy of Mollicutes, a detailed phylogenetic study of Spiroplasma and its non-helical descendants was undertaken. These analyses, done with maximum-parsimony, provide cladistic status; a new nomenclature is introduced here, based on 'bottom-up' rather than 'top-down' clade classification. The order Entomoplasmatales consists of four major clades: (i) the Mycoides-Entomoplasmataceae clade, which contains M. mycoides and its allies and Entomoplasma and Mesoplasma species and is a sister lineage to (ii) the Apis clade of Spiroplasma. Spiroplasma and the Entomoplasmataceae are paraphyletic, but this status does not diminish their phylogenetic usefulness. Five species that were previously unclassified phylogenetically are basal to the Apis clade sensu strictu and to the Mycoides clade. One of these species, Spiroplasma sp. TIUS-1, has very poor helicity and a very small genome (840 kbp); this putative species can be envisioned as a 'missing link' in the evolution of the Mycoides-Entomoplasmataceae clade. The other two Spiroplasma clades are: (iii) the Citri-Chrysopicola-Mirum clade (serogroups I, II, V and VIII) and (iv) the ixodetis clade (serogroup VI). As Mesoplasma lactucae represents a basal divergence within the Mycoides-Entomoplasmataceae clade, and as Entomoplasma freundtii is basal to the Mycoides clade, M. mycoides and its allies must have arisen from an ancestor in the Entomoplasmataceae. The paraphyletic grouping that consists of the Hominis and Pneumoniae groups (sensu Johansson & Pettersson 2002) of Mycoplasma species contains the ancestral roots of Ureaplasma spp. and haemoplasmas. This clade is a sister lineage to the Entomoplasmatales clade. Serological classifications of spiroplasma are very highly supported by the trees presented. Genome size and G+C content of micro-organismal DNA were moderately conserved, but there have been frequent and polyphyletically distributed genome reductions. Sterol requirements were polyphyletic, as was the ability to grow in the presence of polyoxyethylene sorbitan-supplemented, but not serum-supplemented, media. As this character is not phylogenetically distributed, Mesoplasma and Entomoplasma should be combined into a single genus. The phylogenetic trees presented here confirm previous reports of polyphyly of the genus Mycoplasma. As both clades of Mycoplasma contain several species of great practical importance, a change of the genus name for species in either clade would have immense practical implications. In addition, a change of the genus name for M. mycoides would have to be approved by the Judicial Commission. For these reasons, the Linnaean and phylogenetic classifications of Mycoplasma must for now be discrepant.

Traditionally, isolation, maintenance, and testing of Spiroplasma species (Mollicutes: Entomoplasmatales) from horse flies (Tabanus spp.) and deer flies (Chrysops spp.) (Diptera: Tabanidae) have been accomplished in the complex M1D medium. A relatively inexpensive, simplified medium for tabanid spiroplasmas could expedite procedures that require large quantities of growth medium. Nine strains of spiroplasmas, eight from tabanids and one from mosquitoes, were cultured in three simplified broth media, R2, R8-1, and C-3G, and in M1D. There was no significant difference in the rate of spiroplasma growth in M1D and the three simplified media. R2medium supported the growth of tabanid spiroplasmas more consistently and with better morphology through 10 subcultures than did the other simplified media. Primary isolations were made in R2medium from tabanids collected (i) in Georgia, U.S.A., with 10 isolations from 10 flies and (ii) in coastal Costa Rica, with isolation rates of 70% (28/40) and 73% (27/37), respectively, for R2and M1D. Of the seven group VIII field isolates from Costa Rica, four were capable of sustained growth in R2, and three were triply cloned in this simplified medium. These results suggest that the simplified medium R2is suitable for many procedures with tabanid spiroplasmas.Key words: simplified media, Mollicutes, spiroplasma, tabanid, horse fly.

Social insects live in cooperative colonies, often in high densities and with closely related individuals, and interact using social contact behaviours. Compared to solitary insects, social insects have evolved multi-level immunity that includes immune responses common to holometabolous insects, and social immunity, which is exclusive to social taxa. This suggests that social insects may be subject to high pathogen pressure, yet relatively little is known about the range of symbiotic and pathogenic microbial communities that associate with social insects. In this study we examined transcriptome data generated from the ant Formica exsecta for sequences identifying as microbes (or other organisms potentially of non-ant origin). Sequences showing homology to two viruses and several other potentially or obligate intracellular organisms, such as Wolbachia, Arsenophonus, Entomoplasmatales and Microsporidia, were present in the transcriptome data. These homologous sequence matches correspond to genera/species that have previously been associated with a variety of insects, including social insects. There were also sequences with identity to several other microbes such as common moulds and soil bacteria. We conclude that this sequence data provides a starting point for a deeper understanding of the biological interactions between a species of ant and the micro- and macrobiotic communities that it potentially encounters.

Spiroplasma strain TALS-2T from the viscera of the striped horsefly, Tabanus lineola, collected in Georgia was serologically distinct from other Spiroplasma species, groups, putative groups, and subgroups. Light and electron microscopy of cells of strain TALS-2T revealed helical motile cells surrounded only by a single cytoplasmic membrane. The organism grew in M1D and SP-4 liquid media. Growth also occurred in 1% serum fraction medium and in conventional horse serum medium. Growth in liquid media was serum dependent. The strain passed through 220-nm filter pores, but was retained in filters with 100-nm pores. The optimum temperature for growth was 30 degrees C. Multiplication occurred at temperatures from 20 to 37 degrees C, with a doubling time at the optimum temperature of 5.6 h in M1D broth. Strain TALS-2T catabolized glucose but hydrolyzed neither arginine nor urea. The guanine-plus-cytosine content of the DNA was 25 +/- 1 mol%. The genome size was 1,390 kbp. Six isolates serologically similar to strain TALS-2T were obtained from the same host in coastal Georgia. Three strains closely related to strain TALS-2T were isolated from the horsefly Poeciloderas quadripunctatus in Costa Rica. Strain TALS-2T (= ATCC 51749), a representative of group XXVII, is designated the type strain of a new species, Spiroplasma lineolae (Mollicutes: Entomoplasmatales).

Many tabanid flies (Diptera: Tabanidae) are infected with spiroplasmas (Mollicutes: Spiroplasmataceae). Naturally-infected Tabanus gladiator Stone and T. sulcifrons Maquart flies were restrained and fed 10% sucrose to determine the exit points of Spiroplasma from tabanid flies. The flies were allowed to feed for 24 h, and the resulting oral and anal specks were cultured in MID broth. Spiroplasmas were isolated from 21 of 51 oral specks but not from 23 anal specks deposited on plastic. In contrast, when anal specks were deposited in a sucrose solution, 9 of 28 anal specks in sucrose yielded spiroplasma cultures. Tabanus limola F. and T. longiusculus Hine were offered a culture of Spiroplasma strain EC-1 on a stewed raisin or in 5% sucrose in the form of a hanging drop. After 4 d, the minced abdominal viscera of each fly were incubated in MID broth and 25 of 32 tabanids yielded cultures of Spiroplasma.

The phylogenetic diversity of the gut microbial population of the hydrothermal shrimp Rimicaris exoculata was determined. The presence of micro-organisms in the hindgut of the shrimp was determined, and their morphology illustrated for the first time by transmission electron microscopy. DNA was extracted from the fore-, mid- and hindgut of shrimps collected at the Rainbow site, at the Mid-Atlantic Ridge, and the sequences of the small-subunit rRNA (16S rDNA) were analyzed. Seven different bacterial phylotypes were identified from the 90 clones screened. The sequences were affiliated to three major groups: epsilon-Proteobacteria (48,8%), Entomoplasmatales (23.3%) and representatives of the new phylum Deferribacteres (26.6%). These results show that the diversity in the shrimp gut is very low compared to that of the surrounding medium. Furthermore, the presence of groups that are not found in the external medium (Spiroplasma sp. and Geovibrio sp.) suggests the existence of a local microflora. The potential roles of these bacteria are discussed, involving the shrimp diet and metal bioremediation.

Organisms of the class Mollicutes (meaning soft-skin) have regressively evolved, by genome reduction, from Gram-positive bacterial ancestors, namely certain clostria (1). The taxonomy of the class Mollicutes, containing four orders, five familes, and eight genera, is shown in Table 1 (2) The term "mollicute" is sometimes trivially used to describe any organism in the class. The term "mycoplasma" might be used best to describe any member of the genus Mycoplasma, but is also used, as in this chapter, in a trivial way to refer to any organism in the class. Table 1 Taxonomy and Properties of Mycoplasmas (class Mollicutes; Adapted from ref. 2 ) Classification Current no. of Recognized species Genome size(kbp) Mel%G+C content Distinctive properties Habitat Order I: Mycoplasmatales Family I:Mycoplasmataceae Genus I:Mycoplasma 100 580-l300 23-41 Optimum growth at 37°C Humans,animals,plants,insects Genus II Ureaplasma 6 730-1160 27-30 Urease positive Humans,animals Order II Entomoplasmatales Family I Entomoplasmataceae Genus I Entomoplasma 5 790-l140 27-29 Optimum growth Plants,insects at 30°C Genus II Mesoplasma 12 870-1100 27-30 Plants,insects Family II Spiroplasmataceae Genus I:Spiroplasma 17 940-2240 25-31 Helical filaments Arthropods(includ- ing insects), plants Order III Acholeplasmatales Family I Acholeplasmataceae Genus I Acholeplasma 13 About 1600 27-36 Animals,plants, insects Order IV Anaeroplasmatales Family I Anaeroplasmataceae Genus I.Anaeroplasma 4 About 1600 29-33 Oxygen-sensitive Bovine-ovine rumen obligate anaerobes Genus II Asteroleplasma 1 About 1600 40 Oxygen-sensitrve Bovine-ovine rumen obligate anaerobes Uncultivated,unclassified MLOs 506-1185 23-29 Uncultivated as yet Plants,insects.

On the basis of recent phylogenetic studies of 47 species within the class Mollicutes and recent molecular and physiologic findings that distinguish a new group of non-sterol-requiring insect and plant mollicutes from Acholeplasma species, we propose a revised taxonomy of the class Mollicutes. Order I (Mycoplasmatales) is retained as described earlier (S. Razin and E. A. Freundt, p. 740-742, in N. R. Krieg and J. G. Holt, ed., Bergey's Manual of Systematic Bacteriology, vol. 1, 1984), with provision for a single family (Mycoplasmataceae) and two genera (Mycoplasma and Ureaplasma) for classification of sterol-requiring mollicutes primarily associated with vertebrates. Emended descriptions of the order and family for the above taxa are hereby included. Proposed order II (Entomoplasmatales ord. nov.) contains subtaxa for either nonhelical (Entomoplasmataceae fam. nov.) or helical (Spiroplasmataceae) mollicutes; the latter family is hereby transferred from the order Mycoplasmatales. The new order and family permit designation and classification of a monophyletic cluster of mollicute strains associated primarily with arthropods. It is proposed that nonhelical, sterolrequiring insect and plant mollicutes previously designated Mycoplasma ellychniae (Tully et al., 1989), Mycoplasma melaleucae (Tully et al., 1990), Mycoplasma somnilux (Williamson et al., 1990), Mycoplasma luminosum (Williamson et al., 1990), and Mycoplasma lucivorax (Williamson et al., 1990) be transferred to Entomoplasma gen. nov., family Entomoplasmataceae fam. nov., as Entomoplasma ellychniae comb. nov., Entomoplasma melaleucae comb. nov., Entomoplasma somnilux comb. nov, Entomoplasma luminosum comb. nov., and Entomoplasma lucivorax comb. nov. Furthermore, the genus Mesoplasma gen. nov. within the family Entomoplasmataceae fam. nov. is proposed for four non-sterol-requiring insect and plant mollicutes. This designation requires the transfer of Acholeplasma florum (McCoy et al., 1984), Acholeplasma entomophilum (Tully et al., 1988), Acholeplasma seiffertii (Bonnet et al., 1991), and Mycoplasma lactucae (Rose et al., 1990) to Mesoplasma florum comb. nov., Mesoplasma entomophilum comb. nov., Mesoplasma seiffertii comb. nov., and Mesoplasma lactucae comb. nov., respectively. Previously established orders Acholeplasmatales and Anaeroplasmatales within the class Mollicutes are designated orders III and IV, respectively, in the proposed revision. Classification and characteristics of lower taxa within these two orders remain as previously given. The proposal outlined here does not consider the large cluster of nonculturable, arthropod-associated, plant-pathogenic mollicutes (“mycoplasma-like organisms”) recently shown to be more closely related to members of the order Acholeplasmatales.