On the ecology and evolution of microorganisms associated with fungus-growing termites

Organisms living in symbiosis fascinate us with their adaptations to live in extreme proximity to, or even inside, a partner that may be from a completely different Class, Phylum or Kingdom. Combinations of organisms that live in mutualistic symbiosis seem very exceptional, but when studying any organism more closely one may find involvement in mutualistic symbiosis to be the rule rather than an exception. For example, most of the animals have microorganisms in their guts that help digestion, and many plants have fungi around their roots that aid in uptake of nutrients from the soil. Having complementary traits and reciprocally benefitting each other, cooperating organisms may evolve into extremely successful species. CHAPTER 1 introduces the topic of this thesis: fungus-growing termites. Fungus-growing termites play a dominant role as ecosystem engineers in sub-Saharan Africa and South Asia. They change soil properties by their building and foraging activities, and are major players in decomposition of wood and dead vegetation. Though they are often regarded as a pest, termites can be very useful for people. Besides eating the termites and mushrooms that emerge from the termite mound, people use termite soil-engineering to improve the fertility of agricultural fields. The termite and fungus live in obligate mutualistic symbiosis. Termites (Blattodea: Termitidae, subfamily Macrotermitinae) provide the fungus Termitomyces (Basidiomycota: Agaricales: Lyophyllaceae) with fragmented dead plant material and create a controlled environment perfect for the fungus, whereas Termitomyces decomposes the low-quality matter into a nutritious food source and produces mushroom primordia both of which are eaten by the termites. The symbiosis exists in a world where other organisms are awaiting their chance to exploit the richness of the termite nests. Hence, one could expect to find other organisms in the nest, next to termites and Termitomyces. There is at least one fungus associated with fungus-growing termites that emerges very prominently after termites are no longer active: species of Xylaria (Ascomycota: Xylariales: Xylariaceae, subgenus Pseudoxylaria) are frequently overgrowing the fungus gardens of dead termite nests. What is the status of Pseudoxylaria in the fungus-growing termite symbiosis, does it play a role? How are the fungus-growing termite gardens kept free of weeds, parasites and pathogens? These questions form the foundation of this thesis on the ecology and evolution of microorganisms associated with fungus-growing termites, with particular focus on the role and interactions with associated Pseudoxylaria. CHAPTER 2 investigates the specificity of Pseudoxylaria for fungus-growing termites. I hypothesize that specificity or selectivity for fungus-growing termites would mean that Pseudoxylaria is not present coincidentally as opportunist, but truly associated with fungus-growing termite symbiosis. Hundred and eight South-African fungus-growing termite nests were sampled for Pseudoxylaria, and it was found in most of the nests. Partial rDNA sequences of the obtained isolates were compared with those of Xylaria from the environment and isolates from other parts of the world. I found 16 different molecular types (‘species’) of Pseudoxylaria. They formed a separate group, showing that Pseudoxylaria specifically occurs in fungus-growing termite nests indeed. No specificity for the termite genus or species was found, implying that Pseudoxylaria may have specialised on the fungus garden substrate, rather than on the termite host or the mutualistic fungus Termitomyces. CHAPTER 3 focuses on the role of Pseudoxylaria in the fungus-growing termite nest. Pseudoxylaria is inconspicuous in healthy termite nests and usually only occurs when termites are no longer present in the nest, or when pieces of fungus garden are incubated without termites in the lab. Therefore, it seems to be suppressed and an unwelcome nest inhabitant. I postulate that Pseudoxylaria is a benign stowaway that practices a sit-and-wait strategy to survive in the termite nest. First, Pseudoxylaria and Termitomyces were grown independently on different carbon sources; to test if they have a complementary diet preference, degrading complementary substrate components as had been suggested previously. The carbon source use of both fungi overlapped, implying that Pseudoxylaria is not a beneficial or benign symbiont. Second, the role of Pseudoxylaria in termite nests was inferred from interactions between mycelia of Pseudoxylaria, Termitomyces, and their free-living relatives. Both fungi were grown on the same plate, and also combinations with each other’s free-living relatives were tested. This revealed that Pseudoxylaria is not parasitizing Termitomyces. Furthermore, Pseudoxylaria grew relatively less than its free-living relatives when combined with Termitomyces. This result suggests that the symbiotic lifestyle adopted by Pseudoxylaria went together with adaptations that changed the interaction between both fungi, consistent with Pseudoxylaria being a stowaway. CHAPTER 4 tests the hypothesis that termite workers play a crucial role in maintaining the fungus garden hygiene. The occurrence of microorganisms other than Termitomyces was monitored for pieces of fungus garden that were incubated with, without, or temporarily without termite workers. The effect that workers had on the fungus-comb hygiene, as well as observations on worker cleaning behaviour and their response to mycelium tissue of Pseudoxylaria and Termitomyces, show that termites play an important role in maintaining the fungus-garden hygiene indeed. CHAPTER 5 explores the potential of Actinobacteria for a mutualistic role as defensive symbiont against Pseudoxylaria in the fungus-growing termite nest. Actinobacteria play a mutualistic role as defensive symbionts in many biological systems. It was unclear by which mechanism the termites suppress Pseudoxylaria. Thirty fungus-growing termite colonies from two geographically distant sites were sampled for Actinobacteria. Resulting isolates were characterized based on morphology and 16S rRNA sequences. Next, the obtained Actinobacteria were tested for their antibiotic effect on both Pseudoxylaria and Termitomyces. This chapter describes the first discovery of an assembly of Actinobacteria occurring in fungus-growing termite nests. Actinobacteria were found throughout all sampled nests and materials, and in the phylogenetic tree their 16S rRNA sequences were interspersed with those of Actinobacteria from origins other than fungus-growing termites. The bioassays showed that many Actinobacteria inhibited both the substrate competitor Pseudoxylaria and the termite cultivar Termitomyces. The lack of specificity of the Actinobacteria for fungus-growing termites, and lack of specific defence against Pseudoxylaria, make it unlikely that Actinobacteria play a role as defensive symbionts in fungus-growing termites. Final CHAPTER 6 reflects on the previous chapters, focussing on underlying mechanisms. What caused fungus-growing termites to survive for thirty million years already, and what makes them so successful that they dominate semi-arid ecosystems in sub-Saharan Africa and South Asia? How are conflicts of interest between symbiotic partners resolved? How does cooperation between termites and Termitomyces remain stable over evolutionary time scales? The roles of termites, Termitomyces, Pseudoxylaria, and other organisms in the fungus-growing termite nest are discussed more elaborately. In addition, the question to what extent certain aspects determine whether an organism behaves parasitically or mutualistically, and the question whether symbiont role affects the level of specificity between symbiotic partners, are examined. An analogy is drawn with human agriculture and directions for future research are given. The chapter ends with main conclusions of this thesis. Fungus-growing termites are so successful in maintaining a Termitomyces monoculture that the means by which they accomplish this may be further studied for human agricultural interests. Pseudoxylaria species occur specifically in fungus-growing termite nests, where they are suppressed by termites while awaiting an opportunity to overgrow the fungus garden. </p

Muon localization site in U(Pt,Pd)3

The angular and temperature (10-250 K) variation of the Knight shift of single-crystalline U(Pt0.95Pd0.05)3 has been measured in transverse field (B=0.6 T) mSR experiments. By analysing the temperature variation of the Knight shift with a modified Curie-Weiss expression the muon localization site in this hexagonal material is determined at (0,0,0).Comment: 12 pages (including 4 figures); postscript file; Proc. 8th Int. Conf. on Muon Spin Rotation, Relaxation and Resonance (Aug.30-Sept.3, Les Diablerets); 2nd version with minor correction

On Scattering of Electromagnetic Waves by a Wormhole

We consider scattering of a plane electromagnetic wave by a wormhole. It is found that the scattered wave is partially depolarized and has a specific interference picture depending on parameters of the wormhole and the distance to the observer. It is proposed that such features can be important in the direct search of wormholes

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A real Lorentz-FitzGerald contraction

Many condensed matter systems are such that their collective excitations at low energies can be described by fields satisfying equations of motion formally indistinguishable from those of relativistic field theory. The finite speed of propagation of the disturbances in the effective fields (in the simplest models, the speed of sound) plays here the role of the speed of light in fundamental physics. However, these apparently relativistic fields are immersed in an external Newtonian world (the condensed matter system itself and the laboratory can be considered Newtonian, since all the velocities involved are much smaller than the velocity of light) which provides a privileged coordinate system and therefore seems to destroy the possibility of having a perfectly defined relativistic emergent world. In this essay we ask ourselves the following question: In a homogeneous condensed matter medium, is there a way for internal observers, dealing exclusively with the low-energy collective phenomena, to detect their state of uniform motion with respect to the medium? By proposing a thought experiment based on the construction of a Michelson-Morley interferometer made of quasi-particles, we show that a real Lorentz-FitzGerald contraction takes place, so that internal observers are unable to find out anything about their `absolute ' state of motion. Therefore, we also show that an effective but perfectly defined relativistic world can emerge in a fishbowl world situated inside a Newtonian (laboratory) system. This leads us to reflect on the various levels of description in physics, in particular regarding the quest towards a theory of quantum gravity.Comment: 6 pages, no figures. Minor changes reflect published versio

Cylindrical thin-shell wormholes

A general formalism for the dynamics of non rotating cylindrical thin-shell wormholes is developed. The time evolution of the throat is explicitly obtained for thin-shell wormholes whose metric has the form associated to local cosmic strings. It is found that the throat collapses to zero radius, remains static or expands forever, depending only on the sign of its initial velocity.Comment: 10 page

Exactly soluble model for self-gravitating D-particles with the wormhole

We consider D-particles coupled to the CGHS dilaton gravity and obtain the exact wormhole geometry and trajectories of D-particles by introducing the exotic matter. The initial static wormhole background is not stable after infalling D-particles due to the classical backreaction of the geometry so that the additional exotic matter source should be introduced for the stability. Then, the traversable wormhole geometry naturally appears and the D-particles can travel through it safely. Finally, we discuss the dynamical evolution of the wormhole throat and the massless limit of D-particles.Comment: 16 pages, 3 figures, revte

Serotoninergic, peptidergic and GABAergic innervation of the ventrolateral and dorsolateral motor nuclei in the cat S1/S2 segments: An immunofluorescence study

Indirect single- and double-staining immunofluorescence techniques were used to study the serotoninergic, peptidergic and GABAergic innervation of the ventrolateral (Onuf's nucleus) and dorsolateral (innervating intrinsic foot sole muscles) nuclei, located in the S1/S2 segments of the cat spinal cord. The relative density of 5-hydroxytryptamine-, thyrotropin-releasing hormone-, substance P- and γ-aminobuytric acid-immunoreactive axonal varicosities was similar in both nuclei. The highest relative density was recorded for varicosities immunoreactive to γ-aminobutyric acid, while those immunoreactive to 5-hydroxytryptamine or thyrotropin-releasing hormone yielded the lowest values. The density of enkephalin-immunoreactive varicosities was higher in the ventrolateral than in the dorsolateral nucleus. Calcitonin gene-related peptide-like immunoreactivity could be seen in neurons of the ventrolateral and dorsolateral nuclei. Occasionally, calcitonin gene-related peptide-immunoreactive axonal fibers were also encountered in these nuclei. Virtually all thyrotropin-releasing hormone-immunoreactive varicosities in the ventrolateral and dorsolateral nuclei also contained 5-hydroxytryptamine-like immunoreactivity, while a somewhat smaller number of them were co-localized with substance P. About 5–10% of the 5-hydroxytryptamine-immunoreactive varicosities were devoid of peptide-like immunoreactivity, and the number of 5-hydroxytryptamine-immunoreactive varicosities lacking thyrotropin-releasing hormone-like immunoreactivity was higher in the dorsolateral than in the ventrolateral nucleus. Finally, the free fraction of substance P-immunoreactive varicosities, i.e., those lacking both 5-hydroxytryptamine and thyrotropin-releasing hormone, was about 39% in the ventrolateral and 26% in the dorsolateral nucleus. Spinal cord transection at the lower thoracic level induced a depletion of 5-hydroxytryptamine and thyrotropin-releasing hormone-immunoreactive fibers from the ventrolateral and dorsolateral nuclei, indicating an exclusive supraspinal origin for these fibers. A reduction in substance P-like immunoreactivity following spinal cord transection alone or spinal cord transection combined with unilateral dorsal rhizotomy was also detected in both nuclei, suggesting a dual origin for substance P-immunoreactive fibers, i.e., both supra- and intraspinal. The decrease in number of substance P-immunoreactive fibers was however smaller than expected from the analysis of the fraction of substance P-immunoreactive fibers co-localized with 5-hydroxytryptamine, indicating thus that the experimental lesions may have triggered a sprouting of substance P-immunoreactive axons originating from spinal cord sources. The distribution of γ-aminobutyric acid in the ventrolateral and dorsolateral nuclei was not affected by the different lesion paradigms. It is therefore assumed that these inputs are intrinsic to the spinal cord. Finally, both in the ventrolateral and the dorsolateral nucleus a small but statistically significant increase of axonal fibers immunoreactive to enkephalin was seen in response to the experimental lesions

Quantum Collapse of a Small Dust Shell

The full quantum mechanical collapse of a small relativistic dust shell is studied analytically, asymptotically and numerically starting from the exact finite dimensional classical reduced Hamiltonian recently derived by H\'aj{\'\i}\v{c}ek and Kucha\v{r}. The formulation of the quantum mechanics encounters two problems. The first is the multivalued nature of the Hamiltonian and the second is the construction of an appropriate self adjoint momentum operator in the space of the shell motion which is confined to a half line. The first problem is solved by identifying and neglecting orbits of small action in order to obtain a single valued Hamiltonian. The second problem is solved by introducing an appropriate lapse function. The resulting quantum mechanics is then studied by means of analytical and numerical techniques. We find that the region of total collapse has very small probability. We also find that the solution concentrates around the classical Schwarzschild radius. The present work obtains from first principles a quantum mechanics for the shell and provides numerical solutions, whose behavior is explained by a detailed WKB analysis for a wide class of collapsing shells.Comment: 23 pages, 8 figures, Revtex4 fil