Immune challenge reduces gut microbial diversity and triggers fertility-dependent gene expression changes in a social insect.

BACKGROUND: The gut microbiome can influence life history traits associated with host fitness such as fecundity and longevity. In most organisms, these two life history traits are traded-off, while they are positively linked in social insects. In ants, highly fecund queens can live for decades, while their non-reproducing workers exhibit much shorter lifespans. Yet, when fertility is induced in workers by death or removal of the queen, worker lifespan can increase. It is unclear how this positive link between fecundity and longevity is achieved and what role the gut microbiome and the immune system play in this. To gain insights into the molecular regulation of lifespan in social insects, we investigated fat body gene expression and gut microbiome composition in workers of the ant Temnothorax rugatulus in response to an experimental induction of fertility and an immune challenge. RESULTS: Fertile workers upregulated several molecular repair mechanisms, which could explain their extended lifespan. The immune challenge altered the expression of several thousand genes in the fat body, including many immune genes, and, interestingly, this transcriptomic response depended on worker fertility. For example, only fertile, immune-challenged workers upregulated genes involved in the synthesis of alpha-ketoglutarate, an immune system regulator, which extends the lifespan in Caenorhabditis elegans by down-regulating the TOR pathway and reducing oxidant production. Additionally, we observed a dramatic loss in bacterial diversity in the guts of the ants within a day of the immune challenge. Yet, bacterial density did not change, so that the gut microbiomes of many immune challenged workers consisted of only a single or a few bacterial strains. Moreover, the expression of immune genes was linked to the gut microbiome composition, suggesting that the ant host can regulate the microbiome in its gut. CONCLUSIONS: Immune system flare-ups can have negative consequence on gut microbiome diversity, pointing to a previously underrated cost of immunity. Moreover, our results provide important insights into shifts in the molecular regulation of fertility and longevity associated with insect sociality.

What Is in Umbilicaria pustulata? A Metagenomic Approach to Reconstruct the Holo-Genome of a Lichen.

Lichens are valuable models in symbiosis research and promising sources of biosynthetic genes for biotechnological applications. Most lichenized fungi grow slowly, resist aposymbiotic cultivation, and are poor candidates for experimentation. Obtaining contiguous, high-quality genomes for such symbiotic communities is technically challenging. Here, we present the first assembly of a lichen holo-genome from metagenomic whole-genome shotgun data comprising both PacBio long reads and Illumina short reads. The nuclear genomes of the two primary components of the lichen symbiosis-the fungus Umbilicaria pustulata (33 Mb) and the green alga Trebouxia sp. (53 Mb)-were assembled at contiguities comparable to single-species assemblies. The analysis of the read coverage pattern revealed a relative abundance of fungal to algal nuclei of ∼20:1. Gap-free, circular sequences for all organellar genomes were obtained. The bacterial community is dominated by Acidobacteriaceae and encompasses strains closely related to bacteria isolated from other lichens. Gene set analyses showed no evidence of horizontal gene transfer from algae or bacteria into the fungal genome. Our data suggest a lineage-specific loss of a putative gibberellin-20-oxidase in the fungus, a gene fusion in the fungal mitochondrion, and a relocation of an algal chloroplast gene to the algal nucleus. Major technical obstacles during reconstruction of the holo-genome were coverage differences among individual genomes surpassing three orders of magnitude. Moreover, we show that GC-rich inverted repeats paired with nonrandom sequencing error in PacBio data can result in missing gene predictions. This likely poses a general problem for genome assemblies based on long reads.

Abdominal microbial communities in ants depend on colony membership rather than caste and are linked to colony productivity.

Gut bacteria aid their host in digestion and pathogen defense, and bacterial communities that differ in diversity or composition may vary in their ability to do so. Typically, the gut microbiomes of animals living in social groups converge as members share a nest environment and frequently interact. Social insect colonies, however, consist of individuals that differ in age, physiology, and behavior, traits that could affect gut communities or that expose the host to different bacteria, potentially leading to variation in the gut microbiome within colonies. Here we asked whether bacterial communities in the abdomen of Temnothorax nylanderi ants, composed largely of the gut microbiome, differ between different reproductive and behavioral castes. We compared microbiomes of queens, newly eclosed workers, brood carers, and foragers by high-throughput 16S rRNA sequencing. Additionally, we sampled individuals from the same colonies twice, in the field and after 2 months of laboratory housing. To disentangle the effects of laboratory environment and season on microbial communities, additional colonies were collected at the same location after 2 months. There were no large differences between ant castes, although queens harbored more diverse microbial communities than workers. Instead, we found effects of colony, environment, and season on the abdominal microbiome. Interestingly, colonies with more diverse communities had produced more brood. Moreover, the queens' microbiome composition was linked to egg production. Although long-term coevolution between social insects and gut bacteria has been repeatedly evidenced, our study is the first to find associations between abdominal microbiome characteristics and colony productivity in social insects.

Resource profitability, but not caffeine, affects individual and collective foraging in the stingless bee Plebeia droryana.

Plants and pollinators form beneficial relationships, with plants offering resources in return for pollination services. Some plants, however, add compounds to nectar to manipulate pollinators. Caffeine is a secondary plant metabolite found in some nectars that affects foraging in pollinators. In honeybees, caffeine increases foraging and recruitment to mediocre food sources, which might benefit the plant, but potentially harms the colonies. For the largest group of social bees, the stingless bees, the effect of caffeine on foraging behaviour has not been tested yet, despite their importance for tropical ecosystems. More generally, recruitment and foraging dynamics are not well understood in most species. We examined whether caffeine affects the foraging behaviour of the stingless bee Plebeia droryana, which frequently visits plants that produce caffeinated nectar and pollen. We trained bees to food sources containing field-realistic concentrations of sugar and caffeine. Caffeine did not cause P. droryana to increase foraging frequency and persistence. We observed P. droryana recruiting to food sources; however, this behaviour was also not affected by caffeine. Instead we found that higher sugar concentrations caused bees to increase foraging effort. Thus, unlike in other pollinators, foraging behaviour in this stingless bee is not affected by caffeine. As the Brazilian P. droryana population that we tested has been exposed to coffee over evolutionary time periods, our results raise the possibility that it may have evolved a tolerance towards this central nervous system stimulant. Alternatively, stingless bees may show physiological responses to caffeine that differ from those of other bee groups.

Enemy recognition is linked to soldier size in a polymorphic stingless bee.

Many ant and termite colonies are defended by soldiers with powerful mandibles or chemical weaponry. Recently, it was reported that several stingless bee species also have soldiers for colony defence. These soldiers are larger than foragers, but otherwise lack obvious morphological adaptations for defence. Thus, how these soldiers improve colony fitness is not well understood. Robbing is common in stingless bees and we hypothesized that increased body size improves the ability to recognize intruders based on chemosensory cues. We studied the Neotropical species Tetragonisca angustula and found that large soldiers were better than small soldiers at recognizing potential intruders. Larger soldiers also had more olfactory pore plates on their antennae, which is likely to increase their chemosensory sensitivity. Our results suggest that improved enemy recognition might select for increased guard size in stingless bees.

Evolutionary Dynamics of Pathoadaptation Revealed by Three Independent Acquisitions of the VirB/D4 Type IV Secretion System in Bartonella.

The α-proteobacterial genus Bartonella comprises a group of ubiquitous mammalian pathogens that are studied as a model for the evolution of bacterial pathogenesis. Vast abundance of two particular phylogenetic lineages of Bartonella had been linked to enhanced host adaptability enabled by lineage-specific acquisition of a VirB/D4 type IV secretion system (T4SS) and parallel evolution of complex effector repertoires. However, the limited availability of genome sequences from one of those lineages as well as other, remote branches of Bartonella has so far hampered comprehensive understanding of how the VirB/D4 T4SS and its effectors called Beps have shaped Bartonella evolution. Here, we report the discovery of a third repertoire of Beps associated with the VirB/D4 T4SS of B. ancashensis, a novel human pathogen that lacks any signs of host adaptability and is only distantly related to the two species-rich lineages encoding a VirB/D4 T4SS. Furthermore, sequencing of ten new Bartonella isolates from under-sampled lineages enabled combined in silico analyses and wet lab experiments that suggest several parallel layers of functional diversification during evolution of the three Bep repertoires from a single ancestral effector. Our analyses show that the Beps of B. ancashensis share many features with the two other repertoires, but may represent a more ancestral state that has not yet unleashed the adaptive potential of such an effector set. We anticipate that the effectors of B. ancashensis will enable future studies to dissect the evolutionary history of Bartonella effectors and help unraveling the evolutionary forces underlying bacterial host adaptation.

Repeated evolution of soldier sub-castes suggests parasitism drives social complexity in stingless bees.

The differentiation of workers into morphological castes represents an important evolutionary innovation that is thought to improve division of labor in insect societies. Given the potential benefits of task-related worker differentiation, it is puzzling that physical worker castes, such as soldiers, are extremely rare in social bees and absent in wasps. Following the recent discovery of soldiers in a stingless bee, we studied the occurrence of worker differentiation in 28 stingless bee species from Brazil and found that several species have specialized soldiers for colony defence. Our results reveal that worker differentiation evolved repeatedly during the last ~ 25 million years and coincided with the emergence of parasitic robber bees, a major threat to many stingless bee species. Furthermore, our data suggest that these robbers are a driving force behind the evolution of worker differentiation as targets of robber bees are four times more likely to have nest guards of increased size than non-targets. These findings reveal unexpected diversity in the social organization of stingless bees.Although common in ants and termites, worker differentiation into physical castes is rare in social bees and unknown in wasps. Here, Grüter and colleagues find a guard caste in ten species of stingless bees and show that the evolution of the guard caste is associated with parasitization by robber bees.

Local differences in parasitism and competition shape defensive investment in a polymorphic eusocial bee.

Many colonial animals rely for their defense on a soldier caste. Adaptive colony demography theory predicts that colonies should flexibly adjust the investment in different worker castes depending on the colony needs. For example, colonies should invest more in defensive workers (e.g., soldiers) in dangerous environments. However, evidence for this prediction has been mixed. We combined descriptive and experimental approaches to examine whether defensive investment and worker size are adjusted to local ecology in the only known bee with polymorphic workers, Tetragonisca angustula. Colonies of this species are defended by a morphologically specialized soldier caste. Our study included three populations that differed in the density of food competition and the occurrence of a parasitic robber bee. We found that colonies coexisting with robber bees had on average 43% more soldiers defending the nest entrance, while colonies facing stronger foraging competition had soldiers that were -6-7% smaller. We then experimentally relocated colonies to areas with different levels of competition. When released from intense food competition, body sizes of guards and foragers increased. After introducing chemical robber bee cues at nest entrances, we found both a short-term and a long-term up-regulation of the number of soldiers defending the colony. Active soldier numbers remained high after the experiment for a duration equivalent to 2-3 worker life spans. How information about past parasite threat is stored in the colony is currently unknown. In summary, T. angustula adjusts both the number and the body size of active soldiers to local ecological conditions. Competitor density also affects forager (or minor) size, an important colony trait with potential community ecological consequences. Our study supports adaptive colony demography theory in a eusocial bee and highlights the importance of colony threats and competition as selective forces shaping colony phenotype.

Context affects nestmate recognition errors in honey bees and stingless bees.

Nestmate recognition studies, where a discriminator first recognises and then behaviourally discriminates (accepts/rejects) another individual, have used a variety of methodologies and contexts. This is potentially problematic because recognition errors in discrimination behaviour are predicted to be context-dependent. Here we compare the recognition decisions (accept/reject) of discriminators in two eusocial bees, Apis mellifera and Tetragonisca angustula, under different contexts. These contexts include natural guards at the hive entrance (control); natural guards held in plastic test arenas away from the hive entrance that vary either in the presence or absence of colony odour or the presence or absence of an additional nestmate discriminator; and, for the honey bee, the inside of the nest. For both honey bee and stingless bee guards, total recognition errors of behavioural discrimination made by guards (% nestmates rejected + % non-nestmates accepted) are much lower at the colony entrance (honey bee: 30.9%; stingless bee: 33.3%) than in the test arenas (honey bee: 60-86%; stingless bee: 61-81%; P<0.001 for both). Within the test arenas, the presence of colony odour specifically reduced the total recognition errors in honey bees, although this reduction still fell short of bringing error levels down to what was found at the colony entrance. Lastly, in honey bees, the data show that the in-nest collective behavioural discrimination by ca. 30 workers that contact an intruder is insufficient to achieve error-free recognition and is not as effective as the discrimination by guards at the entrance. Overall, these data demonstrate that context is a significant factor in a discriminators' ability to make appropriate recognition decisions, and should be considered when designing recognition study methodologies.

Juvenile exposure to predator cues induces a larger egg size in fish.

When females anticipate a hazardous environment for their offspring, they can increase offspring survival by producing larger young. Early environmental experience determines egg size in different animal taxa. We predicted that a higher perceived predation risk by juveniles would cause an increase in the sizes of eggs that they produce as adults. To test this, we exposed juveniles of the mouthbrooding cichlid Eretmodus cyanostictus in a split-brood experiment either to cues of a natural predator or to a control situation. After maturation, females that had been confronted with predators produced heavier eggs, whereas clutch size itself was not affected by the treatment. This effect cannot be explained by a differential female body size because the predator treatment did not influence growth trajectories. The observed increase of egg mass is likely to be adaptive, as heavier eggs gave rise to larger young and in fish, juvenile predation risk drops sharply with increasing body size. This study provides the first evidence that predator cues perceived by females early in life positively affect egg mass, suggesting that these cues allow her to predict the predation risk for her offspring.