Dietary‐driven variation effects on the symbiotic flagellate protist communities of the subterranean termite Reticulitermes grassei Clément

The ability of subterranean termites to digest lignocellulose relies not only on their digestive tract physiology, but also on the symbiotic relationships established with flagellate protists and bacteria. The objective of this work was to test the possible effect of different cellulose‐based diets on the community structure (species richness and other diversity metrics) of the flagellate protists of the subterranean termite Reticulitermes grassei. Termites belonging to the same colony were subjected to six different diets (natural diet, maritime pine wood, European beech, thermally modified European beech, cellulose powder and starvation), and their flagellate protist community was evaluated after the trials. All non‐treated sound woods produced similar flagellate protist communities that were more diverse and of high evenness (low dominance). On the contrary, flagellate protist communities from cellulose‐fed termites and starving termites were considered to be significantly different from all non‐treated woods; they were less diverse and some morphotypes became dominant as a consequence of flagellate protist communities having suffered major adaptations to these diets. The flagellate protist communities of untreated beech and thermally modified beech‐fed termites were considered to be significantly different in terms of abundance and morphotype diversity. This may be caused by a decrease in lignocellulose quality available for termites and from an interference of thermally treated wood with the chemical stability of the termite hindgut. Our study suggests that as a consequence of the strong division of labour among these protists to accomplish the intricate process of lignocellulose digestion, termite symbiotic flagellate protist communities are a dynamic assemblage able to adapt to different conditions and diets. This study is important for the community‐level alteration approach, and it is the first study to investigate the effects of thermally modified wood on the flagellate protist communities of subterranean termites.     

Efficient but occasionally imperfect vertical transmission of gut mutualistic protists in a wood‐feeding termite

Although mutualistic associations between animals and microbial symbionts are widespread in nature, the mechanisms that have promoted their evolutionary persistence remain poorly understood. A vertical mode of symbiont transmission (from parents to offspring) is thought to ensure partner fidelity and stabilization, although the efficiency of vertical transmission has rarely been investigated, especially in cases where hosts harbour a diverse microbial community. Here we evaluated vertical transmission rates of cellulolytic gut oxymonad and parabasalid protists in the wood‐feeding termite Reticulitermes grassei. We sequenced amplicons of the 18S rRNA gene of protists from 24 colonies of R. grassei collected in two populations. For each colony, the protist community was characterized from the gut of 14 swarming reproductives and from a pool of 10 worker guts. A total of 98 operational taxonomic units belonging to 13 species‐level taxa were found. The vertical transmission rate was estimated for each protist present in a colony based on its frequency among the reproductives. The results revealed that transmission rates were high, with an average of 0.897 (±0.164) per protist species. Overall, the protist community did not differ between reproductive sexes, suggesting that both the queen and the king could contribute to the gut microbiota of the offspring. A positive relationship between the transmission rate of protists and their prevalence within populations was also detected. However, transmission rates alone do not explain the prevalence of protists. In conclusion, these findings reveal key forces behind a conserved, multispecies mutualism, raising further questions on the roles of horizontal transfer and negative selection in shaping symbiont prevalence.     

Competition between invasive and indigenous species: an insular case study of subterranean termites

An important requirement for the management of invasive species is to identify the biological and ecological factors that influence the ability of such species to become established and spread within a new environment. Although competition is one of the key interactions determining the coexistence of species and exclusion, few studies directly examine the mechanism of competitive interactions within invasive communities. This study focused on putative competition in a social insect invader, R. flavipes, an American termite introduced into France, and an indigenous European termite, R. grassei. We first characterized and mapped a zone of sympatry between these two species. We then evaluated the degree of direct and indirect competition by comparing several life-history traits: behavioral aggression, chemical recognition and dispersion modes. Interspecific competition revealed that R. flavipes was dominant over R. grassei. Intraspecific competition was not found in R. flavipes while it appeared in varying degrees in R. grassei. These findings seemed to be correlated with the remarkable chemical homogeneity found in R. flavipes in comparison with R. grassei. Genetic analyses revealed that R. flavipes foraged over a greater distance than R. grassei colonies and might suggest a difference in the capacity to produce secondary reproductives. These findings suggest that R. flavipes has a significant advantage owing to competitive asymmetry that may enable the species to become dominant. The interspecific superiority, lack of intraspecific aggression and large extensive colonies, seem to be some of the reasons for its invasive success.     

SYMBIODINIUM (DINOPHYTA) DIVERSITY AND STABILITY IN AQUARIUM CORALS1

Indo‐Pacific reef corals growing for years in closed‐system aquaria provide an alternate means to investigate host–symbiont specificity and stability. The diversity of dinoflagellate endosymbionts (Symbiodinium spp.) from coral communities in private and public aquaria was investigated using molecular‐genetic analyses. Of the 29 symbiont types (i.e., species) identified, 90% belonged to the most prevalent group of Symbiodinium harbored by Indo‐Pacific reef corals, Clade C, while the rest belonged to Clade D. Sixty‐five percent of all types were known from field surveys conducted throughout the Pacific and Indian oceans. Because specific coral–dinoflagellate partnerships appear to have defined geographic distributions, correspondence of the same symbionts in aquarium and field‐collected specimens identifies regions where particular colonies must have been collected in the wild. Symbiodinium spp. in clade D, believed to be “stress‐tolerant” and/or “opportunistic,” occurred in a limited number of individual colonies. The absence of a prevalent, or “weedy,” symbiont suggests that conditions under which aquarium corals are grown do not favor competitive replacements of their native symbiont populations. The finding of typical and diverse assemblages of Symbiodinium spp. among aquarium corals living many years under variable chemical/physical conditions, artificial and natural light, while undergoing fragmentation periodically, indicates that individual colonies maintain stable, long‐term symbiotic associations.     

A GHF7 CELLULASE FROM THE PROTIST SYMBIONT COMMUNITY OF Reticulitermes flavipes ENABLES MORE EFFICIENT LIGNOCELLULOSE PROCESSING BY HOST ENZYMES

Termites and their gut microbial symbionts efficiently degrade lignocellulose into fermentable monosaccharides. This study examined three glycosyl hydrolase family 7 (GHF7) cellulases from protist symbionts of the termite Reticulitermes flavipes. We tested the hypotheses that three GHF7 cellulases (GHF7‐3, GHF7‐5, and GHF7‐6) can function synergistically with three host digestive enzymes and a fungal cellulase preparation. Full‐length cDNA sequences of the three GHF7s were assembled and their protist origins confirmed through a combination of quantitative PCR and cellobiohydrolase (CBH) activity assays. Recombinant versions of the three GHF7s were generated using a baculovirus‐insect expression system and their activity toward several model substrates compared with and without metallic cofactors. GHF7‐3 was the most active of the three cellulases; it exhibited a combination of CBH, endoglucanase (EGase), and β‐glucosidase activities that were optimal around pH 7 and 30°C, and enhanced by calcium chloride and zinc sulfate. Lignocellulose saccharification assays were then done using various combinations of the three GHF7s along with a host EGase (Cell‐1), beta‐glucosidase (β‐glu), and laccase (LacA). GHF7‐3 was the only GHF7 to enhance glucose release by Cell‐1 and β‐glu. Finally, GHF7‐3, Cell‐1, and β‐glu were individually tested with a commercial fungal cellulase preparation in lignocellulose saccharification assays, but only β‐glu appreciably enhanced glucose release. Our hypothesis that protist GHF7 cellulases are capable of synergistic interactions with host termite digestive enzymes is supported only in the case of GHF7‐3. These findings suggest that not all protist cellulases will enhance saccharification by cocktails of other termite or fungal lignocellulases.     

Genetic diversity and colony structure of Tapinoma melanocephalum on the islands and mainland of South China

Aim

Tapinoma melanocephalum is listed as one of the most important invasive pest species in China. Information regarding the patterns of invasion and effects of geographic isolation on the population genetics of this species is largely lacking.

Location

South China.

Methods

To address this problem, we genotyped 39 colonies (two colonies were collapsed due to genetic similarity) using microsatellite markers and mitochondrial DNA sequencing to compare colony genetic structure of T. melanocephalum on the mainland and islands of South China.

Results

An analysis of the colony genotypes showed that the genetic diversity of the mainland population was slightly higher than that of the island populations but not significantly so. However, the observed heterozygosity on Shangchuan Island (SCD) was significantly lower than that of the other colonies. We also found six haplotypes in 111 mitochondrial DNA COI sequences. The relatedness (r) value between colonies of SCD was 0.410, higher than that of the other populations. The genetic clusters among colonies were not related to geographic locations and exhibited admixture likely due to frequent human‐mediated dispersal associated with trade between the mainland population and the islands. Pairwise F_STs between populations showed differentiation among mainland populations, while SCD displayed high levels of divergence (F_ST > 0.15) from most mainland populations. There was no significant isolation by distance among colonies. Most populations showed signs of a bottleneck effect.

Main conclusions

Our study suggests that there was no significant difference in the genetic diversity among the islands and the mainland; however, the lower genetic diversity, the higher degree of genetic divergence from other colonies, and the higher relatedness among nestmates made the SCD population stand out from all the others.     

Microbiota associated with Mollitrichosiphum aphids (Hemiptera: Aphididae: Greenideinae): diversity,host species specificity and phylosymbiosis

Symbiotic association is universal in nature, and an array of symbionts play a crucial part in host life history. Aphids and their diverse symbionts have become a good model system to study insect-symbiont interactions. Previous symbiotic diversity surveys have mainly focused on a few aphid clades, and the relative importance of different factors regulating microbial community structure is not well understood. In this study, we collected 65 colonies representing eight species of the aphid genus Mollitrichosiphum from different regions and plants in southern China and Nepal and characterized their microbial compositions using Illumina sequencing of the V3 − V4 hypervariable region of the 16S rRNA gene. We evaluated how microbiota varied across aphid species, geography and host plants and the correlation between microbial community structure and host aphid phylogeny. Heritable symbionts dominated the microbiota associated with Mollitrichosiphum, and multiple infections of secondary symbionts were prevalent. Ordination analyses and statistical tests highlighted the contribution of aphid species in shaping the structures of bacterial, symbiont and secondary symbiont communities. Moreover, we observed a significant correlation between Mollitrichosiphum aphid phylogeny and microbial community composition, providing evidence for a pattern of phylosymbiosis between natural aphid populations and their microbial associates.     

Towards an integrated understanding of the consequences of fungus domestication on the fungus‐growing termite gut microbiota

Approximately 30 million years ago (MYA), the subfamily of higher termites Macrotermitinae domesticated a fungus, Termitomyces, as the main plant decomposer and food source for the termite host. The origin of fungiculture shifted the composition of the termite gut microbiota, and some of the functional implications of this shift have recently been established. I review reports on the composition of the Macrotermitinae gut microbiota, evidence for a subfamily core gut microbiota, and the first insight into functional complementarity between fungal and gut symbionts. In addition, I argue that we need to explore the capacities of all members of the symbiotic communities, including better solidifying Termitomyces role(s) in order to understand putative complementary gut bacterial contributions. Approaches that integrate natural history and sequencing data to elucidate symbiont functions will be powerful, particularly if executed in comparative analyses across the well‐established congruent termite–fungus phylogenies. This will allow for testing if gut communities have evolved in parallel with their hosts, with implications for our general understanding of the evolution of gut symbiont communities with hosts.     

Asymmetric interaction specificity between two sympatric termites and their fungal symbionts

Abstract.  1. Fungus-growing termites live in an obligate mutualistic symbiosis with Termitomyces fungi. The functions of the fungal symbiont have been hypothesised to differ between species and to range from highly specific roles of providing plant-degrading enzymes complementary to termite gut enzymes, to non-specific roles of providing protein-rich food to the termites.
2. Termite species with unspecialised fungal symbionts are predicted to be associated with a wider range of symbionts than species with specialised symbionts. Recent DNA data have confirmed this prediction, but evidence for differences in functional specificity has been sparse and indirect.
3. Here the consequences of symbiont interaction specificity are experimentally tested by reciprocally exchanging the fungal symbionts of sympatric colonies of Macrotermes natalensis and Odontotermes badius , which were inferred to have specialised and non-specialised symbionts respectively.
4. As expected, survival of O. badius termites on M. natalensis fungus was not significantly worse than on their own fungus, but survival of M. natalensis termites on O. badius fungus was significantly reduced.
5. This asymmetric result confirms that symbiont roles differ significantly between macrotermitine genera and indicates that symbiont transplantation experiments are a powerful tool for testing the functional details of mutualistic symbioses.     

Farming termites determine the genetic population structure of Termitomyces fungal symbionts

Symbiotic interactions between macrotermitine termites and their fungal symbionts have a moderate degree of specificity. Consistent with horizontal symbiont transmission, host switching has been frequent over evolutionary time so that single termite species can often be associated with several fungal symbionts. However, even in the few termite lineages that secondarily adopted vertical symbiont transmission, the fungal symbionts are not monophyletic. We addressed this paradox by studying differential transmission of fungal symbionts by alate male and female reproductives, and the genetic population structure of Termitomyces fungus gardens across 74 colonies of Macrotermes bellicosus in four west and central African countries. We confirm earlier, more limited, studies showing that the Termitomyces symbionts of M. bellicosus are normally transmitted vertically and clonally by dispersing males. We also document that the symbionts associated with this termite species belong to three main lineages that do not constitute a monophyletic group. The most common lineage occurs over the entire geographical region that we studied, including west, central and southern Africa, where it is also associated with the alternative termite hosts Macrotermes subhyalinus and Macrotermes natalensis. While Termitomyces associated with these alternative hosts are horizontally transmitted and recombine freely, the genetic population structure of the same Termitomyces associated with M. bellicosus is consistent with predominantly clonal reproduction and only occasional recombination. This implies that the genetic population structure of Termitomyces is controlled by the termite host and not by the Termitomyces symbiont.     

Parasitoids as drivers of symbiont diversity in an insect host

Immune systems have repeatedly diversified in response to parasite diversity. Many animals have outsourced part of their immune defence to defensive symbionts, which should be affected by similar evolutionary pressures as the host’s own immune system. Protective symbionts provide efficient and specific protection and respond to changing selection pressure by parasites. Here we use the aphid Aphis fabae, its protective symbiont Hamiltonella defensa, and its parasitoid Lysiphlebus fabarum to test whether parasite diversity can maintain diversity in protective symbionts. We exposed aphid populations with the same initial symbiont composition to parasitoid populations that differed in their diversity. As expected, single parasitoid genotypes mostly favoured a single symbiont that was most protective against that particular parasitoid, while multiple symbionts persisted in aphids exposed to more diverse parasitoid populations, which in turn affected aphid population density and rates of parasitism. Parasite diversity may be crucial to maintaining symbiont diversity in nature.     

A widespread plant-fungal-bacterial symbiosis promotes plant biodiversity,plant nutrition and seedling recruitment

Highly diverse microbial assemblages colonize plant roots. It is still poorly understood whether different members of this root microbiome act synergistically by supplying different services (for example, different limiting nutrients) to plants and plant communities. In order to test this, we manipulated the presence of two widespread plant root symbionts, arbuscular mycorrhizal fungi and nitrogen-fixing rhizobia bacteria in model grassland communities established in axenic microcosms. Here, we demonstrate that both symbionts complement each other resulting in increased plant diversity, enhanced seedling recruitment and improved nutrient acquisition compared with a single symbiont situation. Legume seedlings obtained up to 15-fold higher productivity if they formed an association with both symbionts, opposed to productivity they reached with only one symbiont. Our results reveal the importance of functional diversity of symbionts and demonstrate that different members of the root microbiome can complement each other in acquiring different limiting nutrients and in driving important ecosystem functions.     

Vertical transmission as the key to the colonization of Madagascar by fungus-growing termites?

The mutualism between fungus-growing termites (Macrotermitinae) and their mutualistic fungi (Termitomyces) began in Africa. The fungus-growing termites have secondarily colonized Madagascar and only a subset of the genera found in Africa is found on this isolated island. Successful long-distance colonization may have been severely constrained by the obligate interaction of the termites with fungal symbionts and the need to acquire these symbionts secondarily from the environment for most species (horizontal symbiont transmission). Consistent with this hypothesis, we show that all extant species of fungus-growing termites of Madagascar are the result of a single colonization event of termites belonging to one of the only two groups with vertical symbiont transmission, and we date this event at approximately 13 Mya (Middle/Upper Miocene). Vertical symbiont transmission may therefore have facilitated long-distance dispersal since both partners disperse together. In contrast to their termite hosts, the fungal symbionts have colonized Madagascar multiple times, suggesting that the presence of fungus-growing termites may have facilitated secondary colonizations of the symbiont. Our findings indicate that the absence of the right symbionts in a new environment can prevent long-distance dispersal of symbioses relying on horizontal symbiont acquisition.     

Microbial communities and interactions in the lone star tick,Amblyomma americanum

To quantify microbial composition and interactions, we identified prokaryotic communities in the lone star tick (Amblyomma americanum) based on 16S rRNA gene sequences and direct probing. The lone star tick is the vector of emerging diseases and host to additional symbionts of unknown activity, and is representative of other blood‐sucking arthropods. We evaluated the potential for vertical (transovarial) transmission by molecular analysis of microbial symbionts from egg and larval clutches. Direct probing of adults (N = 8 populations from the southeastern and midwestern USA, 900 ticks total) revealed three vertically transmitted symbionts: a Coxiella symbiont occurred at 100% frequency, Rickettsia species occurred in 45–61% of all ticks in every population and an Arsenophonus symbiont occurred in 0–90% of ticks per population. Arsenophonus and Rickettsia exhibited significant heterogeneity in frequency among populations. The human pathogens Ehrlichia chafeensis and Borrelia lonestari were rare in most populations. Additional microbes were detected sporadically. Most ticks (78%) were co‐infected by two or three microbes but statistical analysis indicated no significant deviation from random co‐occurrence. Our findings indicate that microbial communities within lone star ticks are diverse, and suggest that direct probing for a wider range of prokaryotes and application of quantitative polymerase chain reaction (PCR) may provide further insights into microbial interactions within disease vectors. Our results also emphasize the close phylogenetic relationship between tick symbionts and human pathogens, and consistent differences in their prevalence.     

Genetic Diversity in Remnant Mainland and “Pristine” Island Populations of Three Endemic Australian Macropodids (Marsupialia): Macropus Eugenii, Lagorchestes Hirsutus and Petrogale Lateralis

Since European settlement, mainland Australia has experienced a wave of mammal extinctions and population declines. However, some species have persisted on off-shore islands, which are now viewed as important wildlife refuges. In this study, we assessed the level of genetic diversity, at 7–11 microsatellite loci, in island and remnant mainland populations of three endemic species of macropodid marsupial; the tammar wallaby Macropus eugenii(n = 92); rufous hare-wallaby Lagorchestes hirsutus(n = 40) and black-footed rock-wallaby Petrogale lateralis(n = 164). There was a consistent pattern of significantly higher levels of microsatellite diversity in the remnant mainland population (A= 4.9–13.9; He= 0.61–0.86) of each species compared to conspecific pristine island populations (A= 1.2–3.7; He= 0.05–0.44). These marked differences are even apparent where island populations currently have a substantially larger census size. In addition, island populations were substantially inbred (Fe= 0.49–0.91). Although island populations have been insulated from the relatively recent threatening processes operating on the mainland, they have nevertheless been significantly impacted by increased inbreeding and the substantial erosion of genetic diversity. Despite the difficulties of ensuring the survival of remnant mainland populations, they appear to retain substantially more genetic diversity than their island counterparts and therefore are more likely to contribute to the long-term persistence of their species. These data also demonstrate that small remnant mainland populations (n < 10–20) are often capable of rapid recovery and are not necessarily genetically depauperate.     

Occurrence of the putatively heat-tolerant <Emphasis Type="Italic">Symbiodinium</Emphasis> phylotype D in high-latitudinal outlying coral communities

Biogeographic investigations have suggested that coral-symbiont associations can adapt to higher temperatures by hosting a heat-tolerant Symbiodinium, phylotype D. It is hypothesized that phylotype D is absent in high latitudes due to its heat-tolerant characteristics. In this study, this hypothesis was tested by examining the symbiont diversity in a scleractinian coral, Oulastrea crispata, throughout its entire latitudinal distribution range in the West Pacific. Molecular phylotyping of the 5′-end of the nuclear large subunit of ribosomal DNA (lsu rDNA) indicated that phylotype D was the dominant Symbiodinium in O. crispata from the tropical reefs to the marginal non-reefal coral communities. Several colonies of tropical populations were associated with phylotype C, either alone or simultaneously with phylotype D. Analysis of the polymerase chain reaction products using single-strand conformation polymorphism (SSCP) detected relatively low densities of phylotype C in most of the O. crispata colonies surveyed. These results provide evidence for the occurrence of phylotype D in cold-water outlying coral communities. The dominant occurrence of phylotype C in some O. crispata colonies on tropical reefs and the relatively low densities of phylotype C identified by SSCP in subtropical and temperate populations show that the dominant symbiont type can vary in this coral species and that multiple symbionts can co-occur in the same host.