Despite growing awareness of their importance in soil ecology, the genetic and physiological traits of bacterial predators are still relatively poorly understood. In the course of a Myxococcus xanthus predator evolution experiment, we identified a class of genotypes leading to enhanced predation against diverse species. RNA- seq analysis demonstrated that this phenotype is linked to the constitutive activation of a predation- specific program. Functional analysis of the mutations accumulated across the evolutionary time in a two- component system and Acyl- CoA- manipulating enzymes revealed the critical roles of fatty acid metabolism and antioxidant gene induction. The former likely adapts the predator to metabolites derived from the prey while the latter protects predatory cells from reactive oxygen species generated by prey cells under stress and released upon lysis during predation. These findings reveal interesting parallels between bacterial predator-prey dynamics and pathogen-host cell interactions.

Time and memory are intimately linked: the capability to learn and recall varies over the day and humans and many animals can associate important events with the time of day. However, how the circadian clock and memory centers are connected is not well understood. We time-trained honey bee foragers and used RNA-sequencing and RNAscope imaging to analyze gene expression changes in focal populations of mushroom body neurons. Thus, we identified three candidate functional modules of time-memory: synchronized peak-level expression of memory-related genes during training time, anticipatory activation of transcription in pdfr -expressing neurons, and cry2 and per co-expressing neurons that might represent local clocks. The complex interactions between the clock and memory centers, which appear to be more similar to mammals than other insects, might have been facilitated to optimize social foraging in honey bees.### Competing Interest StatementThe authors have declared no competing interest.

Myxococcus xanthus, a soil bacterium, predates collectively using motility to invade prey colonies. Prey lysis is mostly thought to rely on secreted factors, cocktails of antibiotics and enzymes, and direct contact with Myxococcus cells. In this study, we show that on surfaces the coupling of A-motility and contact-dependent killing is the central predatory mechanism driving effective prey colony invasion and consumption. At the molecular level, contact-dependent killing involves a newly discovered type IV filament-like machinery (Kil) that both promotes motility arrest and prey cell plasmolysis. In this process, Kil proteins assemble at the predator-prey contact site, suggesting that they allow tight contact with prey cells for their intoxication. Kil-like systems form a new class of Tad-like machineries in predatory bacteria, suggesting a conserved function in predator-prey interactions. This study further reveals a novel cell-cell interaction function for bacterial pili-like assemblages.

Myxococcus xanthus is a Gram-negative social bacterium belonging to the order Myxococcales of the class Deltaproteobacteria. It is a facultative social predator found in soils across the globe and is thought to be crucial for the microbial ecosystem. Here, we report a complete high-quality reference genome of the M. xanthus strain DZ2.

ETHNOPHARMACOLOGICAL RELEVANCE:Imperata cylindrica (L.) Raeusch (Gramineae) is a medicinal spice traditionally used in the treatment of hypertension and cancer.AIM OF THE STUDY:To assess the anti-metastatic potential of the methanol extract of I. cylindrica roots and determined its mechanisms of action.MATERIAL AND METHODS:The growth inhibition activity of I. cylindrica root extract in vitro and in vivo in human cervical cancer. The scratch assay and Boyden Chamber assay were used to determine the anti-migrative and anti-invasion actions of the plant extract. The whole-genome gene expression profiling using RNA-Seq was performed to determine the differentially expressed genes in CaSki cells after exposure to I. cylindrica to identify its targeted genes related to metastasis. Using protein analysis (western blotting) and gene expression analysis (RTqPCR), the targeted pathways of the key genes that were initially identified with RNA-Seq, were evaluated.RESULTS:I. cylindrica extract showed dose-dependent cytotoxicity in vitro and in vivo in mice bearing tumors. Furthermore, I. cylindrica root extract significantly inhibited cell migration and cell invasion. After the genome-wide transcriptome analysis, we found that important genes involved in cancer progression and metastasis of cervical cancer, that is, CD24 and TIMP-4 were significantly downregulated and upregulated, respectively. Moreover, I. cylindrica root extract significantly inhibited the PI3/AKT/Snail signaling pathway and blocked the EMT of CaSki cells.CONCLUSION:These findings provide an anti-metastatic mechanism of action of I. cylindrica root extract toward the human cervical cancer suggesting that this plant maybe developed into selective chemotherapy.

We performed an RNA-seq based comparison of gene expression levels in the antennae of honey bee drones and time-trained foragers (workers) collected at different times of the day and different activity states. Interestingly, olfaction-related genes (i.e. odorant receptor (Ors), odorant binding proteins (Obps), carboxyl esterases (CEst) etc.) showed stable gene expression differences between drone and worker antennae. Drone antennae showed higher expression of 24 Ors, of which 21 belong to the clade X which comprises the receptor for the major queen pheromone compound 9-ODA. This high number of drone-biased Ors suggests that more Ors than previously expected play a role in sex-pheromone communication. In addition, we found higher expression levels for many non-olfaction related genes including nitric oxide synthase (NOS), and the potassium channel Shaw. In contrast, workers showed higher expression of 67 Ors, which belong to different Or clades that are involved in pheromone communication as well as the perception of cuticular hydrocarbons and floral scents. Further, drone antennae showed higher expression of genes involved in energy metabolism, whereas worker antennae showed higher expression of genes involved in neuronal communication consistent with earlier reports on peripheral olfactory plasticity. Finally, drones that perform mating flight in the afternoon (innate) and foragers that are trained to forage in the afternoon (adapted) showed similar daily changes in the expression of two major clock genes, period and cryptochrome2. Most of the other genes showing changes with time or onset of daily flight activity were specific to drones and foragers.

Eusocial insects, like honey bees, which show an elaborate division of labor involving morphologically and physiologically specialized phenotypes provide a unique toolkit to study molecular underpinnings of behavior as well as neural processing. In this study, we performed an extensive RNA-seq based comparison of gene expression levels in the antennae of honey bee drones and foragers collected at different time of days and activity states to identify molecules involved in peripheral olfactory processing and provide insights into distinct strategies in sensory processing. First, honey bee drone and worker antennae differ in the number of olfactory receptor genes (ORs) showing a biased expression pattern. Only 19 Ors were higher expressed in drone antennae, whereas 54 Ors were higher expressed in workers. Second, drone antennae showed predominant higher expression of genes involved in energy metabolism, and worker antennae showed a higher expression of genes involved in neuronal communication. Third, drones and afternoon-trained foragers showed similar daily changes in the expression of major clock genes, per and cry2 . Most of the other genes showing changes with the onset of daily activity were specific to drones and foragers suggesting sex-specific circadian changes in antennae. Drone antennae are specialized to detect small amounts of queen’s pheromone and quickly respond to changes in pheromone concentration involving energetically costly action potentials, whereas forager antennae are predominantly involved in behavioral context dependent detection and discrimination of complex odor mixtures which requires mechanisms of sensory filtering and neural plasticity.

Honey bees have a remarkable sense of time and individual honey bee foragers are capable to adjust their foraging activity with respect to the time of food availability. Although, there is plenty of experimental evidence that foraging behavior is guided by the circadian clock, nothing is known about the underlying cellular and molecular mechanisms. Here we present a first study exploring whether the time-restricted foraging under natural light-dark condition affects the molecular clock in honey bees. In an enclosed flight chamber (12m × 4m × 4m), food was presented either for 2 hours in the morning or 2 hours in the afternoon for several consecutive days and daily cycling of the two major clock genes, cryptochrome2 (cry2) and period (per) , were analyzed in three different tissues involved in feeding-related behaviors: brain, antennae and subesophageal ganglion (SEG). We found that morning and afternoon trained foragers showed significant phase-differences in the cycling of both clock genes in all three tissues. Furthermore, the phase-differences were more pronounced when the feeder was scented with the general plant odor linalool. Our results clearly demonstrate that foraging time functions as a strong circadian Zeitgeber in honey bees. More surprisingly our results suggest that foraging time might have the potential to override the entrainment effect of the light-dark cycle.

In honey bees, continuous foraging is accompanied by a sustained up-regulation of the immediate early gene Egr-1 (early growth response protein-1) and candidate downstream genes involved in learning and memory. Here, we present a series of feeder training experiments indicating that Egr-1 expression is highly correlated with the time and duration of training even in the absence of the food reward. Foragers that were trained to visit a feeder over the whole day and then collected on a day without food presentation showed Egr-1 up-regulation over the whole day with a peak expression around 14:00. When exposed to a time-restricted feeder presentation, either 2 h in the morning or 2 h in the evening, Egr-1 expression in the brain was up-regulated only during the hours of training. Foragers that visited a feeder in the morning as well as in the evening showed two peaks of Egr-1 expression. Finally, when we prevented time-trained foragers from leaving the colony using artificial rain, Egr-1 expression in the brains was still slightly but significantly up-regulated around the time of feeder training. In situ hybridization studies showed that active foraging and time-training induced Egr-1 up-regulation occurred in the same brain areas, preferentially the small Kenyon cells of the mushroom bodies and the antennal and optic lobes. Based on these findings we propose that foraging induced Egr-1 expression can get regulated by the circadian clock after time-training over several days and Egr-1 is a candidate transcription factor involved in molecular processes underlying time-memory.

We developed a computational pipeline for homology based identification of the complete repertoire of olfactory receptor (OR) genes in the Asian honey bee species, Apis florea. Apis florea is phylogenetically the most basal honey bee species and also the most distant sister species to the Western honey bee Apis mellifera, for which all OR genes had been identified before. Using our pipeline, we identified 180 OR genes in A. florea, which is very similar to the number of ORs identified in A. mellifera (177 ORs). Many characteristics of the ORs including gene structure, synteny of tandemly repeated ORs and basic phylogenetic clustering are highly conserved. The composite phylogenetic tree of A. florea and A. mellifera ORs could be divided into 21 clades which are in harmony with the existing Hymenopteran tree. However, we found a few nonorthologous OR relationships between both species as well as independent pseudogenization of ORs suggesting separate evolutionary changes. Particularly, a subgroup of the OR gene clade XI, which had been hypothesized to code cuticular hydrocarbon receptors showed a high number of species-specific ORs. RNAseq analysis detected a total number of 145 OR transcripts in male and 162 in female antennae. Most of the OR genes were highly expressed on the female antennae. However, we detected five distinct male-biased OR genes, out of which three genes (AfOr11, AfOr18, AfOr170P) were shown to be male-biased in A. mellifera, too, thus corroborating a behavioral function in sex-pheromone communication.