Wednesday, March 10, 2010

Defining the Relationship

C. pennsylvanicus photo courtesy the excellent Alexander Wild

Having dealt last week with the termites of the sea, it would seem reasonable to leave the littoral and move along to honest-to-goodness termites of the land. But we’re not (termites will come later, I promise!). Like termites and shipworms, this week’s host organism can frequently be found boring holes in wood, and has caused consternation to many a homeowner. Unlike those aforementioned mutualists, though, this animal doesn’t consume the wood or digest any cellulose -- it just chews holes through it to make a nest. 
Carpenter ants should be familiar to pretty much everyone who’s ever seen an ant; the species commonly found here in the Eastern United States (Camponotus pennsylvanicus) is, at up to 2 cm in length, fairly hard to ignore. The ‘tribe’ to which carpenter ants belong, the Camponotini, is one of the largest and most successful groups of ants, composed of over a thousand species distributed across the globe. With such a common host species, it’s little surprise that their symbiotic bacteria were one of the first such associations to be discovered: endosymbiotic microbes were first described in the gut epithelium and ovaries of Camponotus way back in 1882 by F. Blochmann. What is surprising is that it’s taken scientists over a century to figure out what the heck the bacteria are doing there.

Partly that delay has to do with the availability of tools; it’s the same reason the story on shipworm symbiosis changed so drastically in the mid ‘90s, and in large part why I’m writing about microbial symbiosis at all: prior to the development of molecular techniques such as oligonucleotide hybridization microscopy and PCR, it was really hard to say anything at all about microbes you couldn’t grow in a beaker. Consequently, research into the symbionts of Camponotus really started to pick up in 1996, when Schroder et al. published the first paper to describe the relationship using modern molecular techniques. They were able to show, using a combination of FISH and 16S sequencing, that the Camponotus symbionts localized intracellularly to bacteriocytes lining the ants’ midgut, and that the 16S genes of the bacteria had a phylogenetic relationship that recapitulated that of their hosts. When an additional study using a larger number of taxa confirmed this result, the bacteria were proposed to form a new genus, Blochmannia, named after their 19th-century discoverer (Sauer et al., 2000). 
So in some respects, the ant-bacterial relationship looked a lot like the better-described partnership between aphids and their intracellular symbionts, in which vertical transmission of the symbionts through the aphid eggs over millions of years had led to symbionts with dramatically reduced genomes, and a phylogeny that essentially mirrors that of their host. (More on this system in a future post!) In the aphid system, it had been determined that relatively small bacterial population sizes and release from the vagaries of a fluctuating extracellular environment had permitted a dramatic acceleration of mutation in the genome, leading to extensive gene loss as elements of the genome that were no longer strictly necessary to a stable intracellular existence were deleted or rendered inoperable. The genes that are retained (primarily involved in synthesis of essential amino acids) are thought to be of significant benefit to the host. More studies on Camponotus confirmed a similar pattern in their symbionts, for which a small genome size was verified through pulse-field gel electrophoresis (PFGE; Wernegreen et al., 2002).
Functionally, though, the comparison to the aphid symbionts didn’t seem to hold much water. In aphids, a good dose of antibiotics kills off most of the symbionts, leaving you with messed up, infertile, unhappy aphids. Ants fed antibiotics, however, seemed at first glance to be just fine: they kept eating, living, and having babies, even when microscopy showed that most or all of their symbiotic bacteria had been cleared. Moreover, unlike aphids, the Camponotus species studied had omnivorous diets, not clearly lacking in sources of nitrogen or essential amino acids. If killing them with antibiotics didn’t seem to hurt the ants, were these really mutualists? And if they weren’t really mutualists, then why did every species of Camponotus people could find seem to have the bacteria?
The first hint of an answer came when researchers noticed that older queen ants, which in Camponotus can live for decades, often seemed to have lost the Blochmannia symbionts from their gut, but retained dense populations in their eggs (Sauer et al., 2002). Furthermore, these researchers noted an observation in a 1959 paper that this may be true for older workers, as well. Perhaps, rather than playing a crucial role throughout the lifespan of the individual ants, the symbionts were important for a specific, early life stage. 
Genome analysis leant some plausibility to this hypothesis. Using techniques developed by the Moran lab to sequence the aphid symbiont genome, Gil et al. (2003) reported a similarly reduced genome with strong AT bias that, in addition to coding the complete pathways for a number of essential amino acids and a possible pathway for nitrogen upgrading, coded for two elements (arginine catalysis and synthesis of the non-essential amino acid tyrosine) that have been shown to be important in insect metamorphosis. 
With an apparent target, plus a rich reserve of sequence information with which to develop molecular tools, researchers returned to the functional questions with gusto. A series of papers employing qPCR to track microbial density and gene expression, and creative experimental design with antibiotic treatments, suggested that Blochmannia may play a major role in facilitating the development of immature ants, particularly relating to successful eclosion from the terminal pupae stage (Wolschin et al., 2004; Zientz et al., 2006). In these studies, bacterial densities were shown to increase dramatically during pupation, corresponding to an increased expression of a tyrosine synthesis pathway (tyrosine is an aromatic side chain amino acid important in the sclerotization [hardening] of the emerging insect cuticle). Furthermore, nitrogen isotope labeling experiments demonstrated that labeled nitrogen from urea was being incorporated into host amino acids (Feldhaar et al., 2007). 
The effects of these microbial contributions on host fitness are a little harder to ascertain. A number of studies have demonstrated that antibiotic treatments decrease the success of the ants at raising brood (Zientz et al., 2006; Feldhaar et al., 2007; de Souza et al., 2009), though the effect sizes aren’t terribly large, the relationship between  abundance of essential amino acids and nitrogen sources in the host diet and the effects of antibiotic treatment are still poorly constrained, and the possible confounding effects of antibiotic toxicity remain fairly murky, at least in my humble opinion. Still, although the experimental results may be a little unsatisfying, the circumstantial evidence for the importance of Blochmannia to the Camponotini continues to grow. Every paper to examine additional members of the tribe (Degnan et al., 2004; Wernegreen et al., 2009) confirms the ubiquity and tight cospeciation of the bacteria within the ants, indicating that an endosymbiont acquired is not easily lost. 
If this is the case, though -- if the extraordinary phylogenetic conservation of these endosymbionts in fact reflects a crucial functional role -- then why are the experimental results so half-assed? Why, if this relationship with a bacterial mutualist were so key in establishing the worldwide Camponotine dominance of the ant world, do the hosts, upon application of antibiotic, not wither in dramatic, technicolor Wizard of Ozzian fashion?

C. rosariensis tending scale bugs; photo courtesy Alexander Wild
I wonder whether the problem here is one of perspective. Our natural inclination is to think of mutualisms in terms of an interaction between different species of organisms, each benefitting relative to their state if they were not interacting. With vertically transmitted obligate endosymbionts like Blochmannia, though, it’s unclear to what degree it is appropriate to think of the partners as separate organisms. Since the origin of the interaction 30 to 40 million years ago, Blochmannia may have represented a sort of default state for the Camponotines, something without which a fitness measurement would be nonsensical. 
Furthermore, the role played by the bacterium may have changed over the course of evolution. Today, the Camponotines occupy an incredibly diverse range of niches and ecologies, to which they have each adapted over the course of millions of years. Perhaps the ancestor to the group did depend more explicitly on nutrition from its endosymbiont, and like the aphids, used this relationship to diversify into a new habitat. There are still species of Camponotus which have the sort of arboreal, primarily liquid-feeding lifestyle in which you might expect a more dramatic functional role to be apparent for the symbiont; unfortunately, these species are often logistically difficult to acquire, and most of the laboratory studies to date have focused on the more abundant and accessible ground-dwelling omnivores -- perhaps, even now, my counterpart in Germany is hard at work to rectify that situation. With such diversity, the genus is certainly ripe for comparative study!
Next week, in honor of the snow peas I’ll be planting, we’re talking about Rhizobia! Get your shovels ready, and I’ll see you on (hopefully) Monday.

Some Questions:

  • How is the mutualism affecting the host? (Is it right to focus on just one attribute? With an ancient mutualism, is it possible that the underlying factors have shifted?)
  • Does the symbiosis affect different species of Camponotus differently? Ie, do the arboreal species predicted to have more restricted diets rely more on the nitrogen upgrading and amino acid synthesis capabilities of their endosymbionts? 
  • Are the symbionts of mealybugs and the Camponotini truly monophyletic (Wernegreen et al., 2009)? If so, at what point in their evolutionary history did they jump the shark and become vertically transmitted primary endosymbionts?
  • Could an endosymbiotic origin of the Blochmannia explain the unusually fast evolution of their 16S molecule, as compared to other bacteria and even Buchnera (in Degnan et al, 2004)?
  • What are the implications of the long, well-supported branches separating symbionts from different genera within the Camponotini; as well as the giant polytomy at the base of the Camponotus (Wernegreen et al., 2009)? Are the deeper divisions within the Camponotus uncertain because of a very fast diversification in the genus, or due to some degree of horizontal transfer at the beginning of the association?

An Annotated Bibliography:

Boursaux-Eude and Gross. New insights into symbiotic associations between ants and bacteria. Res Microbiol (2000) vol. 151 (7) pp. 513-519

Early review of microbe-ant associations, focusing heavily on Camponotus  and the Attini. Notes that Blochmannia codiversified with Camponotus (Saur et al 2000), but at time of publication functional role wasn't clear. Most information in this section is actually interpretation in light of the aphid symbiosis. 
Schroder et al. Intracellular endosymbiotic: Bacteria of Camponotus species (carpenter ants): Systematics, evolution and ultrastructural characterization. Mol Microbiol (1996) vol. 21 (3) pp. 479-489

First paper to describe Camponotus symbionts using molecular techniques, specifically 16S sequence and FISH.

Used EM to look at bacteriocytes in four Camponotus species, including old world C. herculeanus + ligniperdus and new world C. floridanus + rufipes. Bacteriocytes intercalated between epithelial cells i the midgut. C. rufipes had lower bacterial load. Apparently not in a membrane-bound bacteriosome within the bacteriocyte, however.

16S analysis showed congruent trees with host; 94.7% minimum similarity between strains from diff. Camponotus hosts, max 98.8% identity.

Use some shady molecular dating assumptions to suggest that the Camponotus-bacterial relationship predates the first fossil ant, and such associations might be ancestral in ants. 
Sauer et al. Systematic relationships and cospeciation of bacterial endosymbionts and their carpenter ant host species: proposal of the new taxon Candidatus Blochmannia gen. nov. Int J Syst Evol Micr (2000) vol. 50 pp. 1877-1886

This paper first established Blochmannia as a genus, based on FISH and molecular phylogenetic evidence. Primarily a taxonomic expansion of the 1996 Schroder et al paper. 
Wernegreen et al. Small genome of Candidatus Blochmannia, the bacterial endosymbiont of Camponotus, implies irreversible specialization to an intracellular lifestyle. Microbiol-Sgm (2002) vol. 148 pp. 2551-2556

Used pulse-field gel electrophoresis following percoll density-gradient purification of symbiont cells to estimate Blochmannia genome size at ~810kb, 5-fold smaller than in free-living enterobacteria and consistent with reduced genomes of other vertically-transmitted insect endosymbionts. 
Sauer et al. Tissue localization of the endosymbiotic bacterium "Candidatus Blochmannia floridanus" in adults and larvae of the carpenter ant Camponotus floridanus. Applied and Environmental Microbiology (2002) vol. 68 (9) pp. 4187-4193

Used oligonucleotide probes ad microscopy to more deeply describe the localization of Blochmannia in Camponotus floridanus. As previously described, symbionts were typically densely packed in the cytoplasm of intercalated cells in the midgut, as well as in oocytes.

Surprisingly, mature queens had very few bacteria or bacteriocytes in the midgut. Observations by Kolb in 1959 suggest this may be true to some degree for older workers, as well. There were also no bacteria detected in other ovarial cells, leaving open the question of how the bacteria get to the eggs themselves.

Association with the midgut is a little leaky in early development, with a few bacterial cells apparently present in other tissues until pupation. 
Gil et al. The genome sequence of Blochmannia floridanus: Comparative analysis of reduced genomes. P Natl Acad Sci Usa (2003) vol. 100 (16) pp. 9388-9393

Isolated Blochmannia cells from C. floridanus, prepared libraries, and Sanger sequenced to ~9x coverage. Revealed a 705kb genome with 27% GC, similar to the sequence Buchnera genomes. Genes are virtually a subset of the E. coli genome, but with extensive chromosomal rearrangement.

Of interest is B. floridanus’s lack of any DnaA boxes that might be associated with replication; the authors not that the only other bacterium for which this was true was another cytosolic endosymbiont, and posit that localization to the cytosol (as opposed to a vacuole) may posit greater danger to the host cell and thus require greater host control over replication. Also lacks flagellar genes (which had been suggested to play a role in transport and/or localization to oocytes), but maintains most cell wall structural proteins (like the other cytosolic endosymbiont Wigglesworthia), suggesting the cytoplasm may be a more difficult physical environment than the vacuole.

Gene content suggests a role in amino acid metabolism; symbiont contains glutamine synthetase, permitting ammonia recycling. Lacks part of the arginine synthesis pathway, suggesting a role in arginine catalysis, possibly to permit host amine storage during metamorphosis. Also maintains a complete sulfur reduction pathway, permitting incorporation of sulfate.

Note in conclusion that present function may not be equivalent to past function -- genome degradation may have eliminated some functions that were important to the initial symbiosis. How do these symbioses change over time? Did Camponotus used to have a more restricted diet?
Degnan et al. Host-symbiont stability and fast evolutionary rates in an ant-bacterium association: Cospeciation of Camponotus species and their endosymbionts, Candidatus Blochmannia. Systematic Biology (2004) vol. 53 (1) pp. 95-110

Despite the hallmarks of vertical transmission in previous papers, Degnan et al. suggest that the very limited analyses of codiversification in previous papers was insufficient, and note that the lack of immediately obvious experimental support for obligacy of the symbiont leaves long term transmission fidelity an open question.

Sequence 16 host EF-1a and COI/II, plus Blochmannia 16S, groEL, gidA, and rpsB to do a good codiversification analysis. Used Bayesian and ML tree searches on concatenated data sets for phylogenetic analysis, and compared branch lengths of congruent branches for further corroboration of codiversification using PAML. Used r8s, with fossil constraints on host nodes, to estimate rates of divergence.

Trees were congruent except for a few deeper banchings, which were likely due to homoplasies. The tree made with combined host/symbiont data had very high support at almost all nodes.

Rates were calculated at .0854-.1372 substitutions per synonymous site per MY for Blochmannia gidA and groEL coding genes, and 0.0011-0.0027 s/s/MY for 16S, quite a bit faster (14x!) than observed in Buchnera. This fast rate of evolution (constrained in this study by the internal Camponotus node) is what led earlier researchers to posit a 100mya origin, which would put it in the ancestor of ants themselves.
Zientz et al. Insights into the microbial world associated with ants. Arch Microbiol (2005) vol. 184 (4) pp. 199-206

A fairly short review focusing on the Camponotus association, but also touching on the Tetraponera gut flora, Wolbachia inefection, and the Atta-funagl-actinomycete association.
Wolschin et al. Replication of the endosymbiotic bacterium Blochmannia floridanus is correlated with the developmental and reproductive stages of its ant host. Applied and Environmental Microbiology (2004) vol. 70 (7) pp. 4096-4102

Used qPCR to track bacterial genome copy number in C. floridanus at various developmental stages; initial (egg) copies dropped by an order of magnitude during larval development, then increased by 10
1.5 upon pupation, and again by 101.5 by eclosion. In older workers, copy count had dropped back down by >2 orders of magnitude, to be about half what was present in the egg.

Males seemed particularly prone to losing bacteriocytes, apparently by active ejection into the gut lumen; this was enhanced by starvation. Perhaps an extra burst of energy for a suicidal caste?

Authors posit that primary role of symbiosis may be the production of aromatic amino acids (such as tyrosine) which are needed during eclosion for scleratinization of the cuticle.
Zientz et al. Relevance of the endosymbiosis of Blochmannia floridanus and carpenter ants at different stages of the life cycle of the host. Applied and Environmental Microbiology (2006) vol. 72 (9) pp. 6027-6033

Tried to elucidate the functional role of Blochmannia to Camponotus by 1) performing qPCR on a number of genes involved in amino acid biosynthesis and nitrogen metabolism over the host lifecycle, and 2) comparing larval rearing success in ant treated with antibiotics to those untreated.

Gene expression results suggested upregulation of nitrogen metabolism by the bacteria in the entire pupal stage, with tyrosine biosynthesis increased at the end-pupal stage. Overall gene expression appeared dramatically higher in workers than in immatures; authors take this as diagnostic of continued relevance the symbiosis to workers.

Brood rearing was depressed in colonies treated with antibiotics, although the difference was not significant after colonies were taken off the antibiotic (antibiotic toxicity?). Authors suggest that bacteria may have an important role in upgrading food shared among workers and between workers and larvae.
Feldhaar et al. Nutritional upgrading for omnivorous carpenter ants by the endosymbiont Blochmannia. Bmc Biol (2007) vol. 5 pp. 48

Note that many Camponotini contain Blochmannia, suggesting an origin of 30-40 MYA. Other background: hypothesis for nutritive function is 1) synthesis of essential amino acids, 2) extra synthesis of tyrosine for cuticle sclerotization, 3) reduction and bioincorporation of oxidized sulfur compounds, and 4) nitrogen upgrading via hydrolysis of urea to CO
2 + ammonia -> incorporation of ammonia via glutamine synthetase pathway.

To test this, authors followed host response when constrained to defined diets omitting essential amino acids, and tested N-upgrading with
15N-labeled urea. Found that worker groups fed antibiotic and no essential amino acids raised significantly fewer pupae than those fed either control, artificial diet + essential AA with or without antibiotic, or artificial diet - essential AA and without antibiotic, indicating bacteria play an important essential AA provisioning role. Worker mortality was also higher on artificial diets (but not sucrose). Ants fed antibiotics or only sucrose also appeared to eat more of the brood. Generally confusing results.

Labeling experiments were more clear, with 3-9 mol % increase relative to controls for six amino acids (Gly, Ala, Glu, Asp, Met, and Phe). Reports also that Camponotus compressus has been shown to attract to urea solutions, and others in the genus collect bird droppings.

Also of interest, were unable to detect much other bacterial presence via TGGE.
Wernegreen and Wheeler. Remaining Flexible in Old Alliances: Functional Plasticity in Constrained Mutualisms. Dna Cell Biol (2009) vol. 28 (8) pp. 371-382

An excellent review considering the various ways in which vertically transmitted endosymbioses (‘constrained mutualisms’) are able to react to changing environments (‘functional plasticity’). Authors focus on work done in Camponotus, while placing those results in context of other insect endosymbioses. Note that these microbes typically have limited regulatory ability (possibly due to genomic degradation), and constitutively express chaperones thought to assist in folding of proteins with deleterious mutations. Mention that endosymbionts of C. pennsylvanicus retains a number of regulatory genes lost in B. floridanus.

Authors note the potential import of the ant superorganism and caste structure, although exactly how this is relevant is somewhat unclear. They mention the lack of visible morbidity in workers after antibiotic treatment, and something about how nutrients can flow through the superorganism, but I don’t understand how precisely this connection is supposed to work.

Also suggest that transcriptional slippage on poly-A tracts in pseudogenes could be a bass-ackwards mechanism for regulation. This also seems somewhat dubious to me.

Finally, contains an excellent table of regulatory genes present in a Wigglesworthia, two Blochmannia, and three Buchnera genomes.
de Souza et al. Blochmannia endosymbionts improve colony growth and immune defence in the ant Camponotus fellah. Bmc Microbiol (2009) vol. 9 pp. 29

This group used another Camponotus species to investigate the role of symbionts in host colony founding and immunity. Found some correlation of antibiotic treatment with decreased production of workers and brood. Encapsulation response seemed to be correlated with endogenous Blochmannia density in control colonies, but was universally elevated in ants treated with antibiotics.
Wernegreen et al. One nutritional symbiosis begat another: Phylogenetic evidence that the ant tribe Camponotini acquired Blochmannia by tending sap-feeding insects. BMC Evol Biol (2009) vol. 9 pp. 292

Does Cephalotes have a metapleural gland? These authors suggest that endosymbiosis (and potential immune benefits derived therefrom) may be correlated with occasional loss of this immune-implicated organ in Camponotus.

Otherwise, this paper is primarily about a much broader taxon sampling of Blochmannia from a number of species (>50) from across the Camponotini. As predicted, bacterial lineages recapitulate host phylogeny, with an outgroup recently cut from the main tribe (Notostigma) posessing bacterial sequences that nest within a bunch of other insect endosymbionts but well outside the Blochmannia group.

One thing I find puzzling is the taxon sampling chosen by the authors -- why did they not include any aphid symbionts, and why so few free-living bug sequences? If they are meaning to suggest an endosymbiotic origin for Blochmannia, I would find it more convincing if they tried to break the endosymbiont clade with some closely related free-living sequences from GenBank. 

No comments: