Host-symbiont co-speciation and reductive genome evolution in gut symbiotic bacteria of acanthosomatid stinkbugs

Background  

Host-symbiont co-speciation and reductive genome evolution have been commonly observed among obligate endocellular insect symbionts, while such examples have rarely been identified among extracellular ones, the only case reported being from gut symbiotic bacteria of stinkbugs of the family Plataspidae. Considering that gut symbiotic communities are vulnerable to invasion of foreign microbes, gut symbiotic associations have been thought to be evolutionarily not stable. Stinkbugs of the family Acanthosomatidae harbor a bacterial symbiont in the midgut crypts, the lumen of which is completely sealed off from the midgut main tract, thereby retaining the symbiont in the isolated cryptic cavities. We investigated histological, ecological, phylogenetic, and genomic aspects of the unique gut symbiosis of the acanthosomatid stinkbugs.     

The Ruminococci: key symbionts of the gut ecosystem

Mammalian gut microbial communities form intricate mutualisms with their hosts, which have profound implications on overall health. One group of important gut microbial mutualists are bacteria in the genus Ruminococcus, which serve to degrade and convert complex polysaccharides into a variety of nutrients for their hosts. Isolated decades ago from the bovine rumen, ruminococci have since been cultured from other ruminant and non-ruminant sources, and next-generation sequencing has further shown their distribution to be widespread in a diversity of animal hosts. While most ruminococci that have been studied are those capable of degrading cellulose, much less is known about non-cellulolytic, nonruminant-associated species, such as those found in humans. Furthermore, a mechanistic understanding of the role of Ruminococcus spp. in their respective hosts is still a work in progress. This review highlights the broad work done on species within the genus Ruminococcus with respect to their physiology, phylogenetic relatedness, and their potential impact on host health.     

Transformed bacterial symbionts re-introduced to and detected in host gut

Strains of Enterobacter agglomerans and Klebsiella pneumoniae isolated from Rhagoletis completa Cresson were engineered to express transgenic fluorescent proteins (ECFP, DsRed). These bacteria were introduced into flies by feeding the flies a sucrose solution in which the bacteria were suspended. The transgenic and heterologous marker protein was expressed and visible in the bacteria after they were ingested by WHF and while they were in the fly gut. We describe the plasmids used to transform these bacteria and demonstrate expression of heterologous proteins from the transforming plasmids and discuss the implications for future pest control strategies. Received: 14 September 2001 / Accepted: 22 October 2001     

Thermal niches of specialized gut symbionts: the case of social bees

Responses to climate change are particularly complicated in species that engage in symbioses, as the niche of one partner may be modified by that of the other. We explored thermal traits in gut symbionts of honeybees and bumblebees, which are vulnerable to rising temperatures. In vitro assays of symbiont strains isolated from 16 host species revealed variation in thermal niches. Strains from bumblebees tended to be less heat-tolerant than those from honeybees, possibly due to bumblebees maintaining cooler nests or inhabiting cooler climates. Overall, however, bee symbionts grew at temperatures up to 44°C and withstood temperatures up to 52°C, at or above the upper thermal limits of their hosts. While heat-tolerant, most strains of the symbiont Snodgrassella grew relatively slowly below 35°C, perhaps because of adaptation to the elevated body temperatures that bees maintain through thermoregulation. In a gnotobiotic bumblebee experiment, Snodgrassella was unable to consistently colonize bees reared at 29°C under conditions that limit thermoregulation. Thus, host thermoregulatory behaviour appears important in creating a warm microenvironment for symbiont establishment. Bee–microbiome–temperature interactions could affect host health and pollination services, and inform research on the thermal biology of other specialized gut symbionts.     

Tetraponera ants have gut symbionts related to nitrogen-fixing root-nodule bacteria

Some Tetraponera ants (Formicidae, Pseudomyrmecinae) subsist almost entirely on amino acid deficient honeydew secretions of pseudococcids and harbour a dense aggregation of bacterial symbionts in a unique pouch-shaped organ at the junction of the midgut and the intestine. The organ is surrounded by a network of intruding tracheae and Malpighian tubules, suggesting that these bacteria are involved in the oxidative recycling of nitrogen-rich metabolic waste. We have examined the ultrastructure of these bacteria and have amplified, cloned and sequenced ribosomal RNA-encoding genes, showing that the ant pouch contains a series of close relatives of Flavobacteria and Rhizobium, Methylobacterium, Burkholderia and Pseudomonas nitrogen-fixing root-nodule bacteria. We argue that pouch bacteria have been repeatedly 'domesticated' by the ants as nitrogen-recycling endosymbionts. This ant-associated community of mutualists is, to our knowledge, the first finding of symbionts related to root-nodule bacteria in animals.     

Polyphyly of the fern family Tectariaceae sensu Ching: Insights from cpDNA sequence data

Tectariaceae are a pantropical fern family of about 20 genera, among which 8 are distributed in China. The morphological distinctiveness of the family is widely recognized, yet relatively little systematic research has been conducted on members of Tectariaceae. Phylogenetic analyses of chloroplast DNA sequence data (rbcL and atpB) from 15 species representing all 8 genera in China were carried out under parsimony criteria and Bayesian inference. The phylogenetic reconstructions indicated that the fern family Tectariaceae as traditionally circumscribed are polyphyletic. Ctenitis, Dryopsis, Lastreopsis clustered with and should be included within the newly-defined Dryopteridaceae, and Pleocnemia is also tentatively assigned to it. A narrowly monophyletic Tectariaceae is identified, which includes Ctenitopsis, Hemigramma, Pteridrys, Quercifilix, and Tectaria. In the single rbcL analysis, Arthropteris clustered with the above-mentioned monophyletic Tectariaceae. Although further investigations are still needed to identify infrafamilial relationships within the monophyletic Tectariaceae and to redefine several problematic genera, we propose a working concept here that better reflects the inferred evolu- tionary history of this group.     

Polyphyly of the fern family Tectariaceae sensu Ching: insights from cpDNA sequence data

Tectariaceae are a pantropical fern family of about 20 genera, among which 8 are distributed in China. The morphological distinctiveness of the family is widely recognized, yet relatively little systematic research has been conducted on members of Tectariaceae. Phylogenetic analyses of chloroplast DNA sequence data (rbcL and atpB) from 15 species representing all 8 genera in China were carried out under parsimony criteria and Bayesian inference. The phylogenetic reconstructions indicated that the fern family Tectariaceae as traditionally circumscribed are polyphyletic. Ctenitis, Dryopsis, Lastreopsis clustered with and should be included within the newly-defined Dryopteridaceae, and Pleocnemia is also tentatively assigned to it. A narrowly monophyletic Tectariaceae is identified, which includes Ctenitopsis, Hemigramma, Pteridrys, Quercifilix, and Tectaria. In the single rbcL analysis, Arthropteris clustered with the above-mentioned monophyletic Tectariaceae. Although further investigations are still needed to identify infrafamilial relationships within the monophyletic Tectariaceae and to redefine several problematic genera, we propose a working concept here that better reflects the inferred evolutionary history of this group.     

The structured diversity of specialized gut symbionts of the New World army ants

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 geographic 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 coexisting 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 coexistence, and the effects of hosting a diverse suite of symbiont strains on colony ecology.     

Extinction of anciently associated gut bacterial symbionts in a clade of stingless bees

Animal-microbe symbioses are often stable for millions of years. An example is the clade consisting of social corbiculate bees—honeybees, bumblebees, and stingless bees—in which a shared ancestor acquired specialized gut bacteria that subsequently diversified with hosts. This model may be incomplete, however, as few microbiomes have been characterized for stingless bees, which are diverse and ecologically dominant pollinators in the tropics. We surveyed gut microbiomes of Brazilian stingless bees, focusing on the genus Melipona, for which we sampled multiple species and biomes. Strikingly, Melipona lacks Snodgrassella and Gilliamella, bacterial symbionts ubiquitous in other social corbiculate bees. Instead, Melipona species harbor more environmental bacteria and bee-specific Starmerella yeasts. Loss of Snodgrassella and Gilliamella may stem from ecological shifts in Melipona or the acquisition of new symbionts as functional replacements. Our findings demonstrate the value of broadly sampling microbiome biodiversity and show that even ancient symbioses can be lost.Subject terms: Symbiosis, Microbiome, Microbial ecology, Metagenomics, Microbial ecology     

Intranuclear verrucomicrobial symbionts and evidence of lateral gene transfer to the host protist in the termite gut

In 1944, Harold Kirby described microorganisms living within nuclei of the protists Trichonympha in guts of termites; however, their taxonomic assignment remains to be accomplished. Here, we identified intranuclear symbionts of Trichonympha agilis in the gut of the termite Reticulitermes speratus. We isolated single nuclei of T. agilis, performed whole-genome amplification, and obtained bacterial 16S rRNA genes by PCR. Unexpectedly, however, all of the analyzed clones were from pseudogenes of 16S rRNA with large deletions and numerous sequence variations even within a single-nucleus sample. Authentic 16S rRNA gene sequences were finally recovered by digesting the nuclear DNA; these pseudogenes were present on the host Trichonympha genome. The authentic sequences represented two distinct bacterial species belonging to the phylum Verrucomicrobia, and the pseudogenes have originated from each of the two species. Fluorescence in situ hybridization confirmed that both species are specifically localized, and occasionally co-localized, within nuclei of T. agilis. Transmission electron microscopy revealed that they are distorted cocci with characteristic electron-dense and lucent regions, which resemble the intranuclear symbionts illustrated by Kirby. For these symbionts, we propose a novel genus and species, ‘Candidatus Nucleococcus trichonymphae'' and ‘Candidatus Nucleococcus kirbyi''. These formed a termite-specific cluster with database sequences, other members of which were also detected within nuclei of various gut protists, including both parabasalids and oxymonads. We suggest that this group is widely distributed as intranuclear symbionts of diverse protists in termite guts and that they might have affected the evolution of the host genome through lateral gene transfer.     

Repeated evolution of bacteriocytes in lygaeoid stinkbugs

Host–microbe symbioses often evolved highly complex developmental processes and colonization mechanisms for establishment of stable associations. It has long been recognized that many insects harbour beneficial bacteria inside specific symbiotic cells (bacteriocytes) or organs (bacteriomes). However, the evolutionary origin and mechanisms underlying bacterial colonization in bacteriocyte/bacteriome formation have been poorly understood. In order to uncover the origin of such evolutionary novelties, we studied the development of symbiotic organs in five stinkbug species representing the superfamily Lygaeoidea in which diverse bacteriocyte/bacteriome systems have evolved. We tracked the symbiont movement within the eggs during the embryonic development and determined crucial stages at which symbiont infection and bacteriocyte formation occur, using whole-mount fluorescence in situ hybridization. In summary, three distinct developmental patterns were observed: two different modes of symbiont transfer from initial symbiont cluster (symbiont ball) to presumptive bacteriocytes in the embryonic abdomen, and direct incorporation of the symbiont ball without translocation of bacterial cells. Across the host taxa, only closely related species seemed to have evolved relatively conserved types of bacteriome development, suggesting repeated evolution of host symbiotic cells and organs from multiple independent origins.     

Detection and decay rates of prey and prey symbionts in the gut of a predator through metagenomics

DNA methods are useful to identify ingested prey items from the gut of predators, but reliable detection is hampered by low amounts of degraded DNA. PCR‐based methods can retrieve minute amounts of starting material but suffer from amplification biases and cross‐reactions with the predator and related species genomes. Here, we use PCR‐free direct shotgun sequencing of total DNA isolated from the gut of the harlequin ladybird Harmonia axyridis at five time points after feeding on a single pea aphid Acyrthosiphon pisum. Sequence reads were matched to three reference databases: Insecta mitogenomes of 587 species, including H. axyridis sequenced here; A. pisum nuclear genome scaffolds; and scaffolds and complete genomes of 13 potential bacterial symbionts. Immediately after feeding, multicopy mtDNA of A. pisum was detected in tens of reads, while hundreds of matches to nuclear scaffolds were detected. Aphid nuclear DNA and mtDNA decayed at similar rates (0.281 and 0.11 h^?1 respectively), and the detectability periods were 32.7 and 23.1 h. Metagenomic sequencing also revealed thousands of reads of the obligate Buchnera aphidicola and facultative Regiella insecticola aphid symbionts, which showed exponential decay rates significantly faster than aphid DNA (0.694 and 0.80 h^?1, respectively). However, the facultative aphid symbionts Hamiltonella defensa, Arsenophonus spp. and Serratia symbiotica showed an unexpected temporary increase in population size by 1–2 orders of magnitude in the predator guts before declining. Metagenomics is a powerful tool that can reveal complex relationships and the dynamics of interactions among predators, prey and their symbionts.     

白蚁肠道共生微生物的作用:酵母,一个“隐身”的特别菌群

长期以来,白蚁对木质纤维素的降解能力令人惊叹,毫无疑问,其在全球碳循环中扮演着一个十分重要的角色。这一强大功能的实现极大地依赖于一种特别的肠道"消化液(digestome)",它的构成不仅包括了来自白蚁自身产生的木质纤维素降解酶系统,还来源于独特与多样的肠道共生微生物的贡献(包括了古细菌、细菌、酵母以及其他真核生物),它们的协同作用能有效地将木质纤维素生物质高效转化为乙酸、甲烷、二氧化碳、氢气等物质。然而,到目前为止,我们对这类昆虫的独特肠道生物转化系统的认识还很不深入,特别是针对肠道内的那些各类共生微生物菌群的功能、白蚁与共生微生物间的相互关系、以及潜在的科学与应用价值还无法给予明确的科学解释,更不用说针对其肠道中的共生酵母菌群,一类通常被忽略的独特微生物。近20多年来,越来越多的研究证据表明,白蚁肠道共生酵母在与寄主的关系中表现了不可或缺的重要性与独特功能,已被证明广泛分布于不同白蚁及许多其他昆虫的肠道中。随着近20年来越来越多昆虫肠道共生微生物酵母群被发现和鉴定,他们潜在的功能以及与寄主的共生机制被逐步解析,这些研究结果进一步揭示了"隐身"的昆虫肠道酵母类微生物菌群与寄主的营养、关键生物质转化过程中的重要酶系统、转化过程中的关键中间产物的转化与利用、抵御外源性的重要病原物,甚至对白蚁种群繁衍的远缘交配等方面均可能发挥了重要和不可缺少的作用。本文将试图归纳相关研究的最新进展,系统总结与解析白蚁肠道来源共生酵母的重要科学价值及其在不同领域的潜在应用前景。     

Evolution of symbiotic organs and endosymbionts in lygaeid stinkbugs

We investigated seed bugs of the genus Nysius (Insecta: Hemiptera: Lygaeidae) for their symbiotic bacteria. From all the samples representing 4 species, 18 populations and 281 individuals, specific bacterial 16S rRNA gene sequences were consistently identified, which formed a distinct clade in the Gammaproteobacteria. In situ hybridization showed that the bacterium was endocellularly localized in a pair of large bacteriomes that were amorphous in shape, deep red in color, and in association with gonads. In the ovary of adult females, the endosymbiont was also localized in the ‘infection zone'' in the middle of each germarium and in the ‘symbiont ball'' at the anterior pole of each oocyte, indicating vertical transmission of the endosymbiont through the ovarial passage. Phylogenetic analyses based on bacterial 16S rRNA, groEL and gyrB genes consistently supported a coherent monophyly of the Nysius endosymbionts. The possibility of a sister relationship to ‘Candidatus Kleidoceria schneideri'', the bacteriome-associated endosymbiont of a lygaeid bug Kleidocerys resedae, was statistically rejected, indicating independent evolutionary origins of the endosymbionts in the Lygaeidae. The endosymbiont genes consistently exhibited AT-biased nucleotide compositions and accelerated rates of molecular evolution, and the endosymbiont genome was only 0.6 Mb in size. The endosymbiont phylogeny was congruent with the host insect phylogeny, suggesting strict vertical transmission and host–symbiont co-speciation over evolutionary time. Based on these results, we discuss the evolution of bacteriomes and endosymbionts in the Heteroptera, most members of which are associated with gut symbiotic bacteria. The designation ‘Candidatus Schneideria nysicola'' is proposed for the endosymbiont clade.     

"Endomicrobia": cytoplasmic symbionts of termite gut protozoa form a separate phylum of prokaryotes

Lignocellulose digestion by wood-feeding termites depends on the mutualistic interaction of unusual, flagellate protists located in their hindgut. Most of the flagellates harbor numerous prokaryotic endosymbionts of so-far-unknown identity and function. Using a full-cycle molecular approach, we show here that the endosymbionts of the larger gut flagellates of Reticulitermes santonensis belong to the so-called termite group 1 (TG-1) bacteria, a group of clones previously obtained exclusively from gut homogenates of Reticulitermes speratus that are only distantly related to other bacteria and are considered a novel bacterial phylum based on their 16S rRNA gene sequences. Fluorescence in situ hybridization with specifically designed oligonucleotide probes confirmed that TG-1 bacteria are indeed located within the flagellate cells and demonstrated that Trichonympha agilis (Hypermastigida) and Pyrsonympha vertens (Oxymonadida) harbor phylogenetically distinct populations of symbionts (<95% sequence similarity). Transmission electron microscopy revealed that the symbionts are small, spindle-shaped cells (0.6 microm in length and 0.3 microm in diameter) surrounded by two membranes and located within the cytoplasm of their hosts. The symbionts of the two flagellates are described as candidate species in the candidate genus "Endomicrobium." Moreover, we provide evidence that the members of the TG-1 phylum, for which we propose the candidate name "Endomicrobia," are phylogenetically extremely diverse and are present in and also restricted to the guts of all lower termites and wood-feeding cockroaches of the genus Cryptocercus, the only insects that are in an exclusive, obligately mutualistic association with such unique cellulose-fermenting protists.     

Host and gut microbiota symbiotic factors: lessons from inflammatory bowel disease and successful symbionts

Humans are colonized by a diverse collection of microbes, the largest numbers of which reside in the distal gut. The vast majority of humans coexist in a beneficial equilibrium with these microbes. However, disruption of this mutualistic relationship can manifest itself in human diseases such as inflammatory bowel disease. Thus the study of inflammatory bowel disease and its genetics can provide insight into host pathways that mediate host-microbiota symbiosis. Bacteria of the human intestinal ecosystem face numerous challenges imposed by human dietary intake, the mucosal immune system, competition from fellow members of the gut microbiota, transient ingested microbes and invading pathogens. Considering features of human resident gut bacteria provides the opportunity to understand how microbes have achieved their symbiont status. While model symbionts have provided perspective into host-microbial homeostasis, high-throughput approaches are becoming increasingly practical for functionally characterizing the gut microbiota as a community.     

Hydrogen production by termite gut protists: characterization of iron hydrogenases of Parabasalian symbionts of the termite Coptotermes formosanus

Cellulolytic flagellated protists in the guts of termites produce molecular hydrogen (H(2)) that is emitted by the termites; however, little is known about the physiology and biochemistry of H(2) production from cellulose in the gut symbiotic protists due to their formidable unculturability. In order to understand the molecular basis for H(2) production, we here identified two genes encoding proteins homologous to iron-only hydrogenases (Fe hydrogenases) in Pseudotrichonympha grassii, a large cellulolytic symbiont in the phylum Parabasalia, in the gut of the termite Coptotermes formosanus. The two Fe hydrogenases were phylogenetically distinct and had different N-terminal accessory domains. The long-form protein represented a phylogenetic lineage unique among eukaryotic Fe hydrogenases, whereas the short form was monophyletic with those of other parabasalids. Active recombinant enzyme forms of these two Fe hydrogenases were successfully obtained without the specific auxiliary maturases. Although they differed in their extent of specific activity and optimal pH, both enzymes preferentially catalyzed H(2) evolution rather than H(2) uptake. H(2) evolution, at least that associated with the short-form enzyme, was still active even under high hydrogen partial pressure. H(2) evolution activity was detected in the hydrogenosomal fraction of P. grassii cells; however, the vigorous H(2) uptake activity of the endosymbiotic bacteria compensated for the strong H(2) evolution activity of the host protists. The results suggest that termite gut symbionts are a rich reservoir of novel Fe hydrogenases whose properties are adapted to the gut environment and that the potential of H(2) production in termite guts has been largely underestimated.