Corinne Rouland-Lefèvre

The evolution of fungus-growing termites and their mutualistic fungal symbionts

We have estimated phylogenies of fungus-growing termites and their associated mutualistic fungi of the genus Termitomyces using Bayesian analyses of DNA sequences. Our study shows that the symbiosis has a single African origin and that secondary domestication of other fungi or reversal of mutualistic fungi to a free-living state has not occurred. Host switching has been frequent, especially at the lower taxonomic levels, and nests of single termite species can have different symbionts. Data are consistent with horizontal transmission of fungal symbionts in both the ancestral state of the mutualism and most of the extant taxa. Clonal vertical transmission of fungi, previously shown to be common in the genus Microtermes (via females) and in the species Macrotermes bellicosus (via males) [Johnson, R. A., Thomas, R. J., Wood, T. G. & Swift, M. J. (1981) J. Nat. Hist. 15, 751–756], is derived with two independent origins. Despite repeated host switching, statistical tests taking phylogenetic uncertainty into account show a significant congruence between the termite and fungal phylogenies, because mutualistic interactions at higher taxonomic levels show considerable specificity. We identify common characteristics of fungus-farming evolution in termites and ants, which apply despite the major differences between these two insect agricultural systems. We hypothesize that biparental colony founding may have constrained the evolution of vertical symbiont transmission in termites but not in ants where males die after mating.

Increased lead availability and enzyme activities in root-adhering soil of Lantana camara during phytoextraction in the presence of earthworms.

Earthworms are known to increase availability of heavy metals in soils and also play an important role in maintaining the structure and quality of soil. The introduction of earthworms into soils contaminated with metals in the presence of a potential hyperaccumulator has been suggested as an aid for phytoremediation processes. The present study was conducted to evaluate: (i) the effects of earthworms on lead availability in artificially contaminated soil at 500 and 1000 mg kg(-1) Pb in the presence of Lantana camara, a hyperaccumulator, (ii) the effects of earthworms and lead on soil properties such as pH, cation exchange capacity (CEC), organic matter (OM), total and available N, P and K and (iii) soil enzyme activities. Earthworms increased the bioavailable Pb in root-adhering soil by a factor of 2 to 3 in the contaminated soils at concentrations of 500 to 1000 mg Pb kg(-1), respectively. In lead contaminated soils, the presence of earthworms led to a significant decrease in soil pH by about 0.2 but increased CEC by 17% and OM by more than 30%. Earthworm activities also increased the activities of N-acetylglucosamidase, β-glucosidase, cellulase, xylanase, alkaline and acid phosphatase, urease and fluorescein diacetate assay (FDA). These results indicate that the ecological context for phytoremediation should be broadened by considering plant-soil-earthworm interactions as they influence both plant health and absorption of heavy metals. They also showed that the enzyme activities monitored could serve as useful proxies for phytoremediation capability and, more generally, for soil quality as a whole.

Effect of earthworms on plant Lantana camara Pb-uptake and on bacterial communities in root-adhering soil.

The present study aimed to assess the potential abilities of Lantana camara, an invasive plant species for phytoremediation in the presence of earthworm Pontoscolex corethrurus. Effects of earthworm on growth and lead (Pb) uptake by L. camara plant were studied in soil artificially contaminated at 500 or 1000mg of Pb kg(-1) soil. This species has a promising value for phytoremediation because it can uptake as much as 10% of 1000mgkg(-1) of Pb per year. Moreover, the presence of earthworms enhanced plant biomass by about 1.5-2 times and increased the uptake of lead by about 2-3 times. In the presence of earthworm, L. camara was thus able to uptake up 20% of Pb presence in the soil, corresponding to remediation time of 5 years if all organs are removed. As soil microorganisms are known to mediate many interactions between earthworms and plants, we documented the effect of earthworms on the bacterial community of root-adhering soil of L. camara. Cultivable bacterial biomass of root-adhering soil increased in the presence of earthworms. Similar trend was observed on bacterial metabolic activities. The increase of lead concentrations from 500 to 1000mgkg(-1) did not have any significant effect either on plant growth or on bacterial biomass and global activities but affected the structure and functional diversity of the bacterial community. These results showed that we should broaden the ecological context of phytoremediation by considering plant/microbial community/earthworm interactions that influence the absorption of heavy metals.

Termites as Pests of Agriculture

While pest species of termites are thought to belong predominantly to four families comprising the lower termites, the species causing the most damage to tropical agriculture belong to three of the four subfamilies of higher termites, lacking protist symbionts. A wide range of crops are affected, including trees in plantations and orchards, coconuts, palms, sugar cane, rice, maize, wheat, sorghum, groundnuts, coffee, tea, cocoa, yam, cassava and cotton. Market gardens and pastures can also be damaged. Organochlorine and organophosphate insecticides, once very effective agents of chemical control, are now prohibited or restricted, owing to their several toxicities, but alternatives of comparable efficacy in cropping systems are yet to be found. Integrated control strategies, combining improved cultural practices, attempts to increase plant vigour, and the introduction of biological agents to reduce termite populations may hold some hope for the future. Among the biological agents, entomopathogenic fungi and nematodes seem the most suitable for research and development.

Comparative Biology of Fungus Cultivation in Termites and Ants

We review the two known mutualistic symbioses between basidiomycete fungi and social insects: the attine ants and macrotermitine termites, comparing their origin, history and patterns of co-evolution, and stability. It is argued that ants are “specialised farmers of unspecialised crops”, whereas termites are “specialised farmers of specialised crops”. Furthermore, despite differences in symmetry and symbiont transmission mode, in both relationships there is a moderate specificity between partners. The unresolved debate about the main role of the symbiotic fungus in the fungus-growing termites is summarised and contrasted with the role in the fungus in attine ants, which is little debated. We compare colony foundation and structure, and the modes of symbiotic interaction between the two groups of social insects, highlighting gaps in our understanding of both systems. Finally, we discuss how these symbioses can be evolutionarily stable and the mechanisms by which the ant and termite symbionts ensure monopolies of host care. We conclude by identifying some lines of future research within the fungus-growing termite symbiosis.

Gut-specific actinobacterial community structure and diversity associated with the wood-feeding termite species, Nasutitermes corniger (Motschulsky) described by nested PCR-DGGE analysis

This comprehensive survey studied the actinobacterial community structure and putative representative members associated with the gut of the wood-feeding termite, Nasutitermes corniger (Motschulsky), using nested PCR-DGGE and 16S rDNA sequences analyses. The closest relatives of the actinobacteria inhabiting the gut of Nasutitermes corniger were in five families, regardless of the geographical origin of the termite colony: Propionibacteriaceae, Streptomycetaceae, Cellulomonodaceae, Corynebacteriaceae and Rubrobacteraceae. Feeding termites on beech wood did not result in substantial changes in the actinobacterial community structure as revealed by DGGE banding patterns. Most of the 16S rDNA sequences obtained after excision and sequencing of DGGE bands clustered with those previously retrieved in termite guts. These results confirm the presence of gut-specific actinobacteria. Except for the 16S rDNA sequences affiliated to Streptomycetaceae and Cellulomonodaceae, no sequence had more than 97% similarity with the closest isolated strains, indicating the presence of microorganisms that have not yet been cultivated. These results suggest that members of the Actinomycetales order account for the largest proportion of the Actinobacteria phylum inhabiting the gut of the termite N. corniger.

Molecular diversity and host specificity of termite-associated Xylaria

Studies have revealed that some Xylaria species were closely associated with fungus-growing termite nests. However this relationship rarely had been investigated and the host specificity of termite-associated Xylaria was not yet clearly established. Eighteen Xylaria rDNA-ITS sequences were obtained from fungus combs belonging to 11 Macrotermitinae species from eight regions. Low diversity was found between isolates, and nine sequences were retrieved. Termite-associated Xylaria were shown to be monophyletic, with three main clades, all including strains from various termite hosts and geographical localities. This new molecular study shows no species specificity with respect to fungus-growing termites, which suggests that there might be substrate specialization.

Profiling the Succession of Bacterial Communities throughout the Life Stages of a Higher Termite Nasutitermes arborum (Termitidae, Nasutitermitinae) Using 16S rRNA Gene Pyrosequencing.

Previous surveys of the gut microbiota of termites have been limited to the worker caste. Termite gut microbiota has been well documented over the last decades and consists mainly of lineages specific to the gut microbiome which are maintained across generations. Despite this intimate relationship, little is known of how symbionts are transmitted to each generation of the host, especially in higher termites where proctodeal feeding has never been reported. The bacterial succession across life stages of the wood-feeding higher termite Nasutitermes arborum was characterized by 16S rRNA gene deep sequencing. The microbial community in the eggs, mainly affiliated to Proteobacteria and Actinobacteria, was markedly different from the communities in the following developmental stages. In the first instar and last instar larvae and worker caste termites, Proteobacteria and Actinobacteria were less abundant than Firmicutes, Bacteroidetes, Spirochaetes, Fibrobacteres and the candidate phylum TG3 from the last instar larvae. Most of the representatives of these phyla (except Firmicutes) were identified as termite-gut specific lineages, although their relative abundances differed. The most salient difference between last instar larvae and worker caste termites was the very high proportion of Spirochaetes, most of which were affiliated to the Treponema Ic, Ia and If subclusters, in workers. The results suggest that termite symbionts are not transmitted from mother to offspring but become established by a gradual process allowing the offspring to have access to the bulk of the microbiota prior to the emergence of workers, and, therefore, presumably through social exchanges with nursing workers.

Occurrence of fungi in combs of fungus-growing termites (Isoptera: Termitidae, Macrotermitinae).

Fungus-growing termites cultivate their mutualistic basidiomycete Termitomyces species on a substrate called a fungal comb. Here, the Suicide Polymerase Endonuclease Restriction (SuPER) method was adapted for the first time to a fungal study to determine the entire fungal community of fungal combs and to test whether fungi other than the symbiotic cultivar interact with termite hosts. Our molecular analyses show that although active combs are dominated by Termitomyces fungi isolated with direct Polymerase Endonuclease Restriction - Denaturing Gradient Gel Electrophoresis (PCR-DGGE), they can also harbor some filamentous fungi and yeasts only revealed by SuPER PCR-DGGE. This is the first molecular evidence of the presence of non-Termitomyces species in active combs. However, because there is no evidence for a species-specific relationship between these fungi and termites, they are mere transient guests with no specialization in the symbiosis. It is however surprising to notice that termite-associated Xylaria strains were not isolated from active combs even though they are frequently retrieved when nests are abandoned by termites. This finding highlights the implication of fungus-growing termites in the regulation of fungi occurring within the combs and also suggests that they might not have any particular evolutionary-based association with Xylaria species.

Inter-Population Differences and Seasonal Dynamic of the Bacterial Gut Community in the Endangered Land Snail Helix pomatia (Gastropoda: Helicidae)

ABSTRACT Among the multifaceted environmental factors, the bacterial community ingested with soil and food might play an important role in physiological processes of terrestrial gastropods, with consequences on population dynamics and species distribution patterns. Therefore, we investigated the bacterial gut community structure and seasonal dynamic in three populations of the protected endangered land snail Helix pomatia. The PCR-DGGE fingerprinting analysis followed by Nonmetric Multidimensional Scaling (NMDS) showed that Gamma-and Alphaproteobacteria were common to all populations, while Mollicutes and Betaproteobacteria were population specific. Allochthonous strains might be transiently abundant in the gut of foraging snails with respect to habitat conditions. Autochthonous strains were permanently present in the gut, even after expulsion of the gut content at hibernation beginning. Some of these permanent strains are known to have ice-nucleating activity, which is consistent with the limited cold hardiness in this species. Snails that did not enter hibernation despite being exposed to winter conditions had a poor bacterial community. In conclusion, the bacterial community structure differed between habitats and physiological states, and might be important for physiological processes and survival. Further studies should focus on the individual variation in bacterial community and investigate how it is affected by environmental changes.