Social isolation shortens lifespan through oxidative stress in ants.

Social isolation negatively affects health, induces detrimental behaviors, and shortens lifespan in social species. Little is known about the mechanisms underpinning these effects because model species are typically short-lived and non-social. Using colonies of the carpenter ant Camponotus fellah, we show that social isolation induces hyperactivity, alters space-use, and reduces lifespan via changes in the expression of genes with key roles in oxidation-reduction and an associated accumulation of reactive oxygen species. These physiological effects are localized to the fat body and oenocytes, which perform liver-like functions in insects. We use pharmacological manipulations to demonstrate that the oxidation-reduction pathway causally underpins the detrimental effects of social isolation on behavior and lifespan. These findings have important implications for our understanding of how social isolation affects behavior and lifespan in general.

Basic Structures of Gut Bacterial Communities in Eusocial Insects.

Gut bacterial communities assist host animals with numerous functions such as food digestion, nutritional provision, or immunity. Some social mammals and insects are unique in that their gut microbial communities are stable among individuals. In this review, we focus on the gut bacterial communities of eusocial insects, including bees, ants, and termites, to provide an overview of their community structures and to gain insights into any general aspects of their structural basis. Pseudomonadota and Bacillota are prevalent bacterial phyla commonly detected in those three insect groups, but their compositions are distinct at lower taxonomic levels. Eusocial insects harbor unique gut bacterial communities that are shared within host species, while their stability varies depending on host physiology and ecology. Species with narrow dietary habits, such as eusocial bees, harbor highly stable and intraspecific microbial communities, while generalists, such as most ant species, exhibit relatively diverse community structures. Caste differences could influence the relative abundance of community members without significantly altering the taxonomic composition.

Local Necrotic Cells Trigger Systemic Immune Activation via Gut Microbiome Dysbiosis in Drosophila.

Necrotic cells elicit an inflammatory response through their endogenous factors with damage-associated molecular patterns. Blocking apoptosis in Drosophila wings leads to the necrosis-driven systemic immune response by unknown mechanisms. Here, we demonstrate that immune activation in response to necrotic cells is mediated by commensal gut microbiota. Removing the microbiome attenuates hyperactivation of the innate immune signaling IMD pathway in necrosis-induced flies. Necrotic cells in wings trigger Gluconobacter expansion in the gut. An isolated Gluconobacter sp. strain is sufficient for pathological IMD activation in necrosis-induced flies, while it is not inflammatory for control animals. In addition, bacterial colonization shifts the host metabolome and shortens the lifespan of necrosis-induced flies. This study shows that local necrosis triggers a pathological systemic inflammatory response through interaction between the host and the dysbiotic gut microbiome.

Deep Sequencing Uncovers Caste-Associated Diversity of Symbionts in the Social Ant Camponotus japonicus.

Symbiotic microorganisms can have a profound impact on the host physiology and behavior, and novel relationships between symbionts and their hosts are continually discovered. A colony of social ants consists of various castes that exhibit distinct lifestyles and is, thus, a unique model for investigating how symbionts may be involved in host eusociality. Yet our knowledge of social ant-symbiont dynamics has remained rudimentary. Through 16S rRNA gene deep sequencing of the carpenter ant Camponotus japonicus symbiont community across various castes, we here report caste-dependent diversity of commensal gut microbiota and lineage divergence of "Candidatus Blochmannia," an obligate endosymbiont. While most prevalent gut-associated bacterial populations are found across all castes (Alphaproteobacteria, Gammaproteobacteria, Bacteroidetes, and Cyanobacteria), we also discovered uncultured populations that are found only in males (belonging to Corynebacteriales, Alkanindiges, and Burkholderia). Most of those populations are not detected in laboratory-maintained queens and workers, suggesting that they are facultative gut symbionts introduced via environmental acquisition. Further inspection of "Ca. Blochmannia" endosymbionts reveals that two populations are dominant in all individuals across all castes but that males preferentially contain two different sublineages that are diversified from others. Clearly, each caste has distinct symbiont communities, suggesting an overlooked biological aspect of host-symbiont interaction in social insects.IMPORTANCE Social animals, such as primates and some insects, have been shown to exchange symbiotic microbes among individuals through sharing diet or habitats, resulting in increased consistency of microbiota among social partners. The ant is a representative of social insects exhibiting various castes within a colony; queens, males, and nonreproductive females (so-called workers) show distinct morphologies, physiologies, and behaviors but tightly interact with each other in the nest. However, how this social context affects their gut microbiota has remained unclear. In this study, we deeply sequenced the gut symbiont community across various castes of the carpenter ant Camponotus japonicus We report caste-dependent diversity of commensal gut microbial community and lineage divergence of the mutualistic endosymbiont "Candidatus Blochmannia." This report sheds light on the hidden diversity in microbial populations and community structure associated with guts of males in social ants.

A novel system of bacterial cell division arrest implicated in horizontal transmission of an integrative and conjugative element.

Integrative and conjugative elements (ICEs) are widespread mobile DNA elements in the prokaryotic world. ICEs are usually retained within the bacterial chromosome, but can be excised and transferred from a donor to a new recipient cell, even of another species. Horizontal transmission of ICEclc, a prevalent ICE in proteobacteria, only occurs from developed specialized transfer competent (tc) cells in the donor population. tc cells become entirely dedicated to the ICE transmission at the cost of cell proliferation. The cell growth impairment is mediated by two ICEclc located genes, parA and shi, but the mechanistic and dynamic details of this process are unknown. To better understand the function of ParA and Shi, we followed their intracellular behavior from fluorescent protein fusions, and studied host cell division at single-cell level. Superresolution imaging revealed that ParA-mCherry colocalized with the host nucleoid while Shi-GFP was enriched at the membrane during the growth impairment. Despite being enriched at different cellular locations, the two proteins showed in vivo interactions, and mutations in the Walker A motif of ParA dislocalized both ParA and Shi. In addition, ParA mutations in the ATPase motif abolished the growth arrest on the host cell. Time-lapse microscopy revealed that ParA and Shi initially delay cell division, suggesting an extension of the S phase of cells, but eventually completely inhibit cell elongation. The parA-shi locus is highly conserved in other ICEclc-related elements, and expressing ParA-Shi from ICEclc in other proteobacterial species caused similar growth arrest, suggesting that the system functions similarly across hosts. The results of our study provide mechanistic insight into the novel and unique system on ICEs and help to understand such epistatic interaction between ICE genes and host physiology that entails efficient horizontal gene transfer.

Dronc-independent basal executioner caspase activity sustains Drosophila imaginal tissue growth.

Caspase is best known as an enzyme involved in programmed cell death, which is conserved among multicellular organisms. In addition to its role in cell death, caspase is emerging as an indispensable enzyme in a wide range of cellular functions, which have recently been termed caspase-dependent nonlethal cellular processes (CDPs). In this study, we examined the involvement of cell death signaling in tissue-size determination using Drosophila wing as a model. We found that the Drosophila executioner caspases Dcp-1 and Decay, but not Drice, promoted wing growth independently of apoptosis. Most of the reports on CDPs argue the importance of the spatiotemporal regulation of the initiator caspase, Dronc; however, this sublethal caspase function was independent of Dronc, suggesting a more diverse array of CDP regulatory mechanisms. Tagging of TurboID, an improved promiscuous biotin ligase that biotinylates neighboring proteins, to the C terminus of caspases revealed the differences among the neighbors of executioner caspases. Furthermore, we found that the cleavage of Acinus, a substrate of the executioner caspase, was important in promoting wing growth. These results demonstrate the importance of executioner caspase-mediated basal proteolytic cleavage of substrates in sustaining tissue growth. Given the existence of caspase-like DEVDase activity in a unicellular alga, our results likely highlight the original function of caspase-not cell death, but basal proteolytic cleavages for cell vigor.

Oxytocin/vasopressin-like peptide inotocin regulates cuticular hydrocarbon synthesis and water balancing in ants.

Oxytocin/vasopressin-like peptides are important regulators of physiology and social behavior in vertebrates. However, the function of inotocin, the homologous peptide in arthropods, remains largely unknown. Here, we show that the level of expression of inotocin and inotocin receptor are correlated with task allocation in the ant Camponotus fellah Both genes are up-regulated when workers age and switch tasks from nursing to foraging. in situ hybridization revealed that inotocin receptor is specifically expressed in oenocytes, which are specialized cells synthesizing cuticular hydrocarbons which function as desiccation barriers in insects and for social recognition in ants. dsRNA injection targeting inotocin receptor, together with pharmacological treatments using three identified antagonists blocking inotocin signaling, revealed that inotocin signaling regulates the expression of cytochrome P450 4G1 (CYP4G1) and the synthesis of cuticular hydrocarbons, which play an important role in desiccation resistance once workers initiate foraging.

Oral transfer of chemical cues, growth proteins and hormones in social insects.

Social insects frequently engage in oral fluid exchange - trophallaxis - between adults, and between adults and larvae. Although trophallaxis is widely considered a food-sharing mechanism, we hypothesized that endogenous components of this fluid might underlie a novel means of chemical communication between colony members. Through protein and small-molecule mass spectrometry and RNA sequencing, we found that trophallactic fluid in the ant Camponotus floridanus contains a set of specific digestion- and non-digestion related proteins, as well as hydrocarbons, microRNAs, and a key developmental regulator, juvenile hormone. When C. floridanus workers' food was supplemented with this hormone, the larvae they reared via trophallaxis were twice as likely to complete metamorphosis and became larger workers. Comparison of trophallactic fluid proteins across social insect species revealed that many are regulators of growth, development and behavioral maturation. These results suggest that trophallaxis plays previously unsuspected roles in communication and enables communal control of colony phenotypes.

In vivo monitoring of caspase activation using a fluorescence resonance energy transfer-based fluorescent probe.

Caspases, which constitute a family of cysteine proteases, are highly conserved in multicellular organisms and function as a central player in apoptosis. The detection of apoptosis is intrinsically difficult because dying cells are rapidly removed from tissues by phagocytosis. Thus, the development of a method for detecting caspase activation is critical for the in vivo study of apoptosis. In this chapter, we describe a genetically encoded fluorescent probe for live imaging of caspase activation.

Who lives and who dies: Role of apoptosis in quashing developmental errors.

Apoptosis is essential for normal development. Large numbers of cells are eliminated by apoptosis in early neural development and during the formation of neural connections. However, our understanding of this life-or-death decision is incomplete, because it is difficult to identify dying cells by conventional strategies. Live imaging is powerful for studying apoptosis, because it can trace a death-fated cell throughout its lifetime. The Drosophila sensory organ development is a convenient system for studying neural-cell selection via lateral inhibition. We recently showed that about 20% of the differentiating neuronal cells die during sensory organ development, which results in the characteristic spatial patterning of the sensory organs. The eliminated differentiating neurons expressed neurogenic genes and high levels of activated Notch. Thus, live imaging allowed us to document the role of apoptosis in neural progenitor selection, and revealed that Notch activation is the mechanism determining which cells die during sensory organ development.

Apoptosis controls the speed of looping morphogenesis in Drosophila male terminalia.

In metazoan development, the precise mechanisms that regulate the completion of morphogenesis according to a developmental timetable remain elusive. The Drosophila male terminalia is an asymmetric looping organ; the internal genitalia (spermiduct) loops dextrally around the hindgut. Mutants for apoptotic signaling have an orientation defect of their male terminalia, indicating that apoptosis contributes to the looping morphogenesis. However, the physiological roles of apoptosis in the looping morphogenesis of male terminalia have been unclear. Here, we show the role of apoptosis in the organogenesis of male terminalia using time-lapse imaging. In normal flies, genitalia rotation accelerated as development proceeded, and completed a full 360° rotation. This acceleration was impaired when the activity of caspases or JNK or PVF/PVR signaling was reduced. Acceleration was induced by two distinct subcompartments of the A8 segment that formed a ring shape and surrounded the male genitalia: the inner ring rotated with the genitalia and the outer ring rotated later, functioning as a 'moving walkway' to accelerate the inner ring rotation. A quantitative analysis combining the use of a FRET-based indicator for caspase activation with single-cell tracking showed that the timing and degree of apoptosis correlated with the movement of the outer ring, and upregulation of the apoptotic signal increased the speed of genital rotation. Therefore, apoptosis coordinates the outer ring movement that drives the acceleration of genitalia rotation, thereby enabling the complete morphogenesis of male genitalia within a limited developmental time frame.

Apoptosis ensures spacing pattern formation of Drosophila sensory organs.

BACKGROUND: In both vertebrates and invertebrates, developing organs and tissues must be precisely patterned. One patterning mechanism is Notch/Delta-mediated lateral inhibition. Through the process of lateral inhibition, Drosophila sensory organ precursors (SOPs) are selected and sensory bristles form into a regular pattern. SOP cell fate is determined by high Delta expression and following expression of neurogenic genes like neuralized. SOP selection is spatially and temporally regulated; however, the dynamic process of precise pattern formation is not clearly understood. RESULTS: In this study, using live-imaging analysis, we show that the appearance of neuralized-positive cells is random in both timing and position. Excess neuralized-positive cells are produced by developmental errors at several steps preceding and accompanying lateral inhibition. About 20% of the neuralized-positive cells show aberrant cell characteristics and high Notch activation, which not only suppress neural differentiation but also induce caspase-dependent cell death. These cells never develop into sensory organs, nor do they disturb bristle patterning. CONCLUSIONS: Our study reveals the incidence of developmental errors that produce excess neuralized-positive cells during sensory organ development. Notch activation in neuralized-positive cells determines aberrant cell fate and typically induces caspase-dependent cell death. Apoptosis is utilized as a mechanism to remove cells that start neural differentiation at aberrant positions and timing and to ensure robust spacing pattern formation.

Temporal regulation of Drosophila IAP1 determines caspase functions in sensory organ development.

The caspases comprise a family of cysteine proteases that function in various cellular processes, including apoptosis. However, how the balance is struck between the caspases' role in cell death and their nonapoptotic functions is unclear. To address this issue, we monitored the protein turnover of an endogenous caspase inhibitor, Drosophila IAP1 (DIAP1). DIAP1 is an E3 ubiquitin ligase that promotes the ubiquitination of caspases and thereby prevents caspase activation. For this study, we developed a fluorescent probe to monitor DIAP1 turnover in the external sensory organ precursor (SOP) lineage of living Drosophila. The SOP divides asymmetrically to make the shaft, socket, and sheath cells, and the neuron that comprise each sensory organ. We found that the quantity of DIAP1 changed dramatically depending on the cell type and maturity, and that the temporal regulation of DIAP1 turnover determines whether caspases function nonapoptotically in cellular morphogenesis or cause cell death.