Impact of ecological doses of the most widespread phthalate on a terrestrial species, the ant Lasius niger.

Phthalates are synthetic contaminants released into the environment notably by plastic waste. Semi-volatile, they adsorb to atmospheric particles and get distributed in all ecosystems. Effects of this major anthropogenic pollution in economical species in aquatic habitats have attracted large interest. On the contrary, very few studies have focused on wild terrestrial species. Yet, these lipophilic molecules are easily trapped by insect cuticle; ants and other insects have been shown to permanently bear among their cuticular components a non-negligible proportion of phthalates, meaning that they suffer from chronic exposure to these pollutants. Oral route could also be an additional way of contamination, as phthalates tend to stick to any organic particle. We show here via a food choice experiment that Lasius niger workers can detect, and avoid feeding on, food contaminated with DEHP (DiEthyl Hexyl Phthalate), the most widespread phthalate found in nature. This suggests that the main source of contamination for ants is atmosphere and that doses measured on the cuticle correspond to the chronic exposure levels for these animals. Such an ecologically relevant dose of DEHP was used to contaminate ants in lab and to investigate their physiological impact. Over a chronic exposure (1 dose per week for 5 weeks), the egg-laying rate of queens was significantly reduced lending credence to endocrine disruptive properties of such a pollutant, as also described for aquatic invertebrates. On the contrary, short term exposure (24h) to a single dose of DEHP does not induce oxidative stress in ant workers as expected, but leads to activation of the immune system. Because of their very large distribution, their presence in virtually all terrestrial ecosystems and their representation at all trophic levels, ants could be useful indicators of contamination by phthalates, especially via monitoring the level of activation of their immune state.

Deciphering the immune function and regulation by a TLR of the cytokine EMAPII in the lesioned central nervous system using a leech model.

A highly conserved ortholog of the human complex p43/endothelial monocyte-activating polypeptide II (EMAPII) was characterized in the CNS of the leech Hirudo medicinalis. As observed in mammals, the leech complex is processed to release the cytokine HmEMAPII. Taking advantages of these similarities, we have attempted to elucidate the role of EMAPII in the CNS using the leech model. Although EMAPII is considered a modulator of inflammatory reactions within the peripheral innate immune response in humans, its function in CNS immunity has yet to be described. Chemotaxis assays were conducted, revealing the ability of EMAPII to exert a chemoattractant effect on both leech and human microglial cells, indicating a novel function of this cytokine in the human brain. Quantitative RT-PCR analysis together with in situ hybridization and immunohistochemistry approaches showed that bacterial challenge induced the expression of HmEMAPII at the lesion site where microglial cells accumulated. Moreover, gene silencing experiments have demonstrated that the gene expression of HmEMAPII is under the control of a signaling pathway associated with the TLR HmTLR1, newly characterized in the CNS of our model. To the best of our knowledge, this is the first report showing evidence for (1) the chemoattractant properties of EMAPII on leech and human microglial cells, (2) the regulation by a TLR of the expression of a gene encoding a cytokine in the CNS of an invertebrate, and (3) an immune function of a TLR in a lophotrochozoan model.

The receptor protein tyrosine phosphatase HmLAR1 is up-regulated in the CNS of the adult medicinal leech following injury and is required for neuronal sprouting and regeneration.

LAR-like receptor protein tyrosine phosphatases (RPTPs), which are abundantly expressed in the nervous systems of most if not all bilaterian animals thus far examined, have been implicated in regulating a variety of critical neuronal processes. These include neuronal pathfinding, adhesion and synaptogenesis during development and, in adult mammals, neuronal regeneration. Here we explored a possible role of a LAR-like RPTP (HmLAR1) in response to mechanical trauma in the adult nervous system of the medicinal leech. In situ hybridization and QPCR analyses of HmLAR1 expression in individual segmental ganglia revealed a significant up-regulation in receptor expression following CNS injury, both in situ and following a period in vitro. Furthermore, we observed up-regulation in the expression of the leech homologue of the Abelson tyrosine kinase, a putative signaling partner to LAR receptors, but not among other tyrosine kinases. The effects on neuronal regeneration were assayed by comparing growth across a nerve crush by projections of individual dorsal P neurons (P(D)) following single-cell injection of interfering RNAs against the receptor or control RNAs. Receptor RNAi led to a significant reduction in HmLAR1 expression by the injected cells and resulted in a significant decrease in sprouting and regenerative growth at the crush site relative to controls. These studies extend the role of the HmLARs from leech neuronal development to adult neuronal regeneration and provide a platform to investigate neuronal regeneration and gene regulation at the single cell level.

Microbial challenge promotes the regenerative process of the injured central nervous system of the medicinal leech by inducing the synthesis of antimicrobial peptides in neurons and microglia.

Following trauma, the CNS of the medicinal leech, unlike the mammalian CNS, has a strong capacity to regenerate neurites and synaptic connections that restore normal function. In this study, we show that this regenerative process is enhanced by a controlled bacterial infection, suggesting that induction of regeneration of normal CNS function may depend critically upon the coinitiation of an immune response. We explore the interaction between the activation of a neuroimmune response and the process of regeneration by assaying the potential roles of two newly characterized antimicrobial peptides. Our data provide evidence that microbial components differentially induce the transcription, by microglial cells, of both antimicrobial peptide genes, the products of which accumulate rapidly at sites in the CNS undergoing regeneration following axotomy. Using a preparation of leech CNS depleted of microglial cells, we also demonstrate the production of antimicrobial peptides by neurons. Interestingly, in addition to exerting antibacterial properties, both peptides act as promoters of the regenerative process of axotomized leech CNS. These data are the first to report the neuronal synthesis of antimicrobial peptides and their participation in the immune response and the regeneration of the CNS. Thus, the leech CNS appears as an excellent model for studying the implication of immune molecules in neural repair.

Invertebrates facing environmental contamination by endocrine disruptors: Novel evidences and recent insights.

The crisis of biodiversity we currently experience raises the question of the impact of anthropogenic chemicals on wild life health. Endocrine disruptors are notably incriminated because of their possible effects on development and reproduction, including at very low doses. As commonly recorded in the field, the burden they impose on wild species also concerns invertebrates, with possible specificities linked with the specific physiology of these animals. A better understanding of chemically-mediated endocrine disruption in these species has clearly gained from knowledge accumulated on vertebrate models. But the molecular pathways specific to invertebrates also need to be reckoned, which implies dedicated research efforts to decipher their basic functioning in order to be able to assess its possible disruption. The recent rising of omics technologies opens the way to an intensification of these efforts on both aspects, even in species almost uninvestigated so far.

Antimicrobial Peptides and Ectosymbiotic Relationships: Involvement of a Novel Type IIa Crustin in the Life Cycle of a Deep-Sea Vent Shrimp.

The symbiotic shrimp Rimicaris exoculata dominates the macrofauna inhabiting the active smokers of the deep-sea mid Atlantic ridge vent fields. We investigated the nature of the host mechanisms controlling the vital and highly specialized ectosymbiotic community confined into its cephalothoracic cavity. R. exoculata belongs to the Pleocyemata, crustacean brooding eggs, usually producing Type I crustins. Unexpectedly, a novel anti-Gram-positive type II crustin was molecularly identified in R. exoculata. Re-crustin is mainly produced by the appendages and the inner surfaces of the cephalothoracic cavity, embedding target epibionts. Symbiosis acquisition and regulating mechanisms are still poorly understood. Yet, symbiotic communities were identified at different steps of the life cycle such as brooding stage, juvenile recruitment and molt cycle, all of which may be crucial for symbiotic acquisition and control. Here, we show a spatio-temporal correlation between the production of Re-crustin and the main ectosymbiosis-related life-cycle events. Overall, our results highlight (i) a novel and unusual AMP sequence from an extremophile organism and (ii) the potential role of AMPs in the establishment of vital ectosymbiosis along the life cycle of deep-sea invertebrates.

Transgenerational Immune Priming in the Field: Maternal Environmental Experience Leads to Differential Immune Transfer to Oocytes in the Marine Annelid Hediste diversicolor.

Transgenerational immune priming (TGIP) is an intriguing form of parental care which leads to the plastic adjustment of the progeny's immunity according to parental immune experience. Such parental effect has been described in several vertebrate and invertebrate taxa. However, very few empirical studies have been conducted from the field, with natural host-parasite systems and real ecological settings, especially in invertebrates. We investigated TGIP in wild populations of the marine annelid Hediste diversicolor. Females laid eggs in a mud tube and thus shared the local microbial threats with the first developmental stages, thus meeting expectations for the evolution of TGIP. We evidenced that a maternal bacterial challenge led to the higher antibacterial defense of the produced oocytes, with higher efficiency in the case of Gram-positive bacterial challenge, pointing out a prevalent role of these bacteria in the evolutionary history of TGIP in this species. Underlying mechanisms might involve the antimicrobial peptide hedistin that was detected in the cytoplasm of oocytes and whose mRNAs were selectively stored in higher quantity in mature oocytes, after a maternal immune challenge. Finally, maternal immune transfer was significantly inhibited in females living in polluted areas, suggesting associated costs and the possible trade-off with female's protection.

Immune failure reveals vulnerability of populations exposed to pollution in the bioindicator species Hediste diversicolor.

Human activities on the shoreline generate a growing pollution, creating deleterious habitats in coastal zones. Some species nevertheless succeed in such harsh milieus, raising the question of their tolerance to environmental stress. The annelid Hediste diversicolor lives buried in the sediments, directly exposed to contaminants trapped in the mud. After verifying the similarity of their genetic contexts, we compared reproductive output and individual immune resistance measures of populations living in polluted vs. 'clean' sediments, and related these assessments with measures of phthalates and metal pollution, and associated toxicity indices. Chemical analyses predicted no toxicity to the local infauna, and phenological studies evidenced no direct cost of living in noxious habitats. However, populations exposed to pollutants showed a significantly reduced survival upon infection with a local pathogen. Surprisingly, physiological studies evidenced a basal overinflammatory state in the most exposed populations. This over-activated baseline immune phenotype likely generates self-damage leading to enhanced immune cell death rate and immune failure. Monitoring the immune status of individual worms living in anthropic areas could thus be used as a reliable source of information regarding the actual health of wild populations.

Phthalate pollution in an Amazonian rainforest.

Phthalates are ubiquitous contaminants and endocrine-disrupting chemicals that can become trapped in the cuticles of insects, including ants which were recognized as good bioindicators for such pollution. Because phthalates have been noted in developed countries and because they also have been found in the Arctic, a region isolated from direct anthropogenic influence, we hypothesized that they are widespread. So, we looked for their presence on the cuticle of ants gathered from isolated areas of the Amazonian rainforest and along an anthropogenic gradient of pollution (rainforest vs. road sides vs. cities in French Guiana). Phthalate pollution (mainly di(2-ethylhexyl) phthalate (DEHP)) was higher on ants gathered in cities and along road sides than on those collected in the pristine rainforest, indicating that it follows a human-mediated gradient of disturbance related to the use of plastics and many other products that contain phthalates in urban zones. Their presence varied with the ant species; the cuticle of Solenopsis saevissima traps higher amount of phthalates than that of compared species. However, the presence of phthalates in isolated areas of pristine rainforests suggests that they are associated both with atmospheric particles and in gaseous form and are transported over long distances by wind, resulting in a worldwide diffusion. These findings suggest that there is no such thing as a "pristine" zone.

Reciprocal immune benefit based on complementary production of antibiotics by the leech Hirudo verbana and its gut symbiont Aeromonas veronii.

The medicinal leech has established a long-term mutualistic association with Aeromonas veronii, a versatile bacterium which can also display free-living waterborne and fish- or human-pathogenic lifestyles. Here, we investigated the role of antibiotics in the dynamics of interaction between the leech and its gut symbiont Aeromonas. By combining biochemical and molecular approaches, we isolated and identified for the first time the antimicrobial peptides (AMPs) produced by the leech digestive tract and by its symbiont Aeromonas. Immunohistochemistry data and PCR analyses evidenced that leech AMP genes are induced in the gut epithelial cells when Aeromonas load is low (starved animals), while repressed when Aeromonas abundance is the highest (post blood feeding). The asynchronous production of AMPs by both partners suggests that these antibiotic substances (i) provide them with reciprocal protection against invasive bacteria and (ii) contribute to the unusual simplicity of the gut microflora of the leech. This immune benefit substantially reinforces the evidence of an evolutionarily stable association between H. verbana and A. veronii. Altogether these data may provide insights into the processes making the association with an Aeromonas species in the digestive tract either deleterious or beneficial.

Regulation of reproduction in a queenless ant: aggression, pheromones and reduction in conflict.

In the monogynous queenless ant Diacamma ceylonense, the future reproductive (future gamergate) is very aggressive towards infertile workers during the first days of her adult life. Overt aggression disappears at about three weeks, when the future gamergate begins to lay male-destined eggs and is ready to mate. Over the same period, her cuticular hydrocarbon profile alters, changing from a chemical signature similar to that of a sterile worker towards that of a gamergate. In nature, these behavioural and chemical changes will coincide with a reduction in conflict within the nest: faced with a virgin future gamergate, infertile workers have an interest in producing male-destined eggs; however, once the gamergate produces female eggs, they have an interest in rearing her offspring. This demonstration of a shift from physical inhibition to chemical signalling is interpreted in terms of sociogenetic theory, the role of cuticular hydrocarbons as an indicator of fertility in insects and the fact that the regulation of reproduction in Diacamma involves mechanisms redolent of both queenless and queenright ant species.

Honey bee (Apis mellifera) transferrin-gene structure and the role of ecdysteroids in the developmental regulation of its expression.

Social life is prone to invasion by microorganisms, and binding of ferric ions by transferrin is an efficient strategy to restrict their access to iron. In this study, we isolated cDNA and genomic clones encoding an Apis mellifera transferrin (AmTRF) gene. It has an open reading frame (ORF) of 2136 bp spread over nine exons. The deduced protein sequence comprises 686 amino acid residues plus a 26 residues signal sequence, giving a predicted molecular mass of 76 kDa. Comparison of the deduced AmTRF amino acid sequence with known insect transferrins revealed significant similarity extending over the entire sequence. It clusters with monoferric transferrins, with which it shares putative iron-binding residues in the N-terminal lobe. In a functional analysis of AmTRF expression in honey bee development, we monitored its expression profile in the larval and pupal stages. The negative regulation of AmTRF by ecdysteroids deduced from the developmental expression profile was confirmed by experimental treatment of spinning-stage honey bee larvae with 20-hydroxyecdysone, and of fourth instar-larvae with juvenile hormone. A juvenile hormone application to spinning-stage larvae, in contrast, had only a minor effect on AmTRF transcript levels. This is the first study implicating ecdysteroids in the developmental regulation of transferrin expression in an insect species.

Ant cuticular response to phthalate pollution.

Phthalates are common atmospheric contaminants used in the plastic industry. Ants have been shown to constitute good bioindicators of phthalate pollution. Hence, phthalates remain trapped on ant cuticles which are mostly coated with long-chain hydrocarbons. In this study, we artificially contaminated Lasius niger ants with four phthalates: dibutyl phthalate (DBP), diisobutyl phthalate (DiBP), di(2-ethylhexyl) phthalate (DEHP), and benzyl butyl phthalate (BBP). The first three have previously been found on ants in nature in Touraine (France), while the fourth has not. The four phthalates disappeared rapidly (less than 5 days) from the cuticles of live ants. In contrast, on the cuticles of dead ants, DEHP quantities remained unchanged over time. These results indicate that phthalates are actively absorbed by the cuticles of live ants. Cuticular absorption of phthalates is nonspecific because eicosane, a nonnatural hydrocarbon on L. niger cuticle, was similarly absorbed. Ants are important ecological engineers and may serve as bioindicators of ecosystem health. We also suggest that ants and more generally terrestrial arthropods may contribute to the removal of phthalates from the local environment.

Characterization and function of the first antibiotic isolated from a vent organism: the extremophile metazoan Alvinella pompejana.

The emblematic hydrothermal worm Alvinella pompejana is one of the most thermo tolerant animal known on Earth. It relies on a symbiotic association offering a unique opportunity to discover biochemical adaptations that allow animals to thrive in such a hostile habitat. Here, by studying the Pompeii worm, we report on the discovery of the first antibiotic peptide from a deep-sea organism, namely alvinellacin. After purification and peptide sequencing, both the gene and the peptide tertiary structures were elucidated. As epibionts are not cultivated so far and because of lethal decompression effects upon Alvinella sampling, we developed shipboard biological assays to demonstrate that in addition to act in the first line of defense against microbial invasion, alvinellacin shapes and controls the worm's epibiotic microflora. Our results provide insights into the nature of an abyssal antimicrobial peptide (AMP) and into the manner in which an extremophile eukaryote uses it to interact with the particular microbial community of the hydrothermal vent ecosystem. Unlike earlier studies done on hydrothermal vents that all focused on the microbial side of the symbiosis, our work gives a view of this interaction from the host side.

Ant cuticles: a trap for atmospheric phthalate contaminants.

Phthalates are universal contaminants. We show that they are trapped by the ant cuticles and maintained permanently at a low level, generally less than 1% of cuticular components. They are found throughout the interior of the insect, predominately in the fat body, which suggests that they are adsorbed by the cuticle. In open plastic boxes free of phthalates the ants became more contaminated with phthalates over a period of time, whereas in closed glass jars they did not. This finding suggests that the main source of pollutants is the atmosphere. Different ant species collected from multiple places showed similar levels of contamination. It appeared that in some pristine places the contamination was lower, but this needs to be confirmed. Ants can be considered as bio-indicators of phthalate pollution.

Characterization and immune function of two intracellular sensors, HmTLR1 and HmNLR, in the injured CNS of an invertebrate.

Unlike mammals, the CNS of the medicinal leech can regenerate damaged neurites, thus restoring neural functions. Our group recently demonstrated that the injured leech nerve cord is able to mount an immune response, which promotes the regenerative processes. This defense mechanism is microorganism-specific, suggesting that the leech CNS is able to discriminate among microbial components. We report here the characterization of two receptors potentially implicated in this detection: HmTLR1 and HmNLR. Interestingly, HmTLR1 presents an endosomal distribution in neurons and appears as a chimera combining the mammalian intraendosomal domain of TLR3 and the cytoplasmic section of TLR13, while HmNLR is cytosolic and has the highest homology to NLRC3 receptors. Both receptors show patterns of induction upon stimulation that suggest their involvement in the leech neuroimmune response. This work constitutes the first demonstration in an invertebrate of (i) an intracellular TLR and (ii) a cytosolic PRR related to the NLR family.

Biogenic amine levels, reproduction and social dominance in the queenless ant Streblognathus peetersi.

Social harmony often relies on ritualized dominance interactions between society members, particularly in queenless ant societies, where colony members do not have developmentally predetermined castes but have to fight for their status in the reproductive and work hierarchy. In this behavioural plasticity, their social organisation resembles more that of vertebrates than that of the "classic" social insects. The present study investigates the neurochemistry of the queenless ant species, Streblognathus peetersi, to better understand the neural basis of the high behavioural plasticity observed in queenless ants. We report measurements of brain biogenic amines [octopamine, dopamine, serotonin] of S. peetersi ants; they reveal a new set of biogenic amine influences on social organisation with no common features with other "primitively organised societies" (bumble bees) and some common features with "highly eusocial" species (honey bees). This similarity to honey bees may either confirm the heritage of queenless species from their probably highly eusocial ancestors or highlight independent patterns of biogenic amine influences on the social organisation of these highly derived species.

Rapid modification in the olfactory signal of ants following a change in reproductive status.

In insect societies, the presence and condition of egg-layers can be assessed with pheromones. Exocrine secretions are expected to vary in time in order to give up-to-date information on an individual's reproductive physiology. In the queenless monogynous ant Streblognathus peetersi, we allowed a previously infertile high-ranking worker to accede to the alpha rank, thus triggering the onset of her oogenesis (15 replicates). We then studied her interactions with an established egg-layer from the same colony after different durations, ranging from 20 h to several days. Even though her eggs are only ready to be laid after 30 days, the new alpha was recognised within 1-2 days. Detection occurred at a distance of a few millimetres, suggesting the involvement of a pheromone with low volatility, such as cuticular hydrocarbons. When the new alpha had differentiated for >48 h, she was attacked by the established egg-layer. In all cases, low-ranking workers eventually immobilised one of the two alphas: the new alpha was the target if she had differentiated only recently, suggesting that police workers select the dominant worker with the "less fertile" odour. Using the behaviour of ants as our measure, we demonstrate that a dominant's olfactory signal changes rapidly with a modification in her social status, and it occurs well before the onset of egg-laying.