Symbiotic organs shaped by distinct modes of genome evolution in cephalopods.

Microbes have been critical drivers of evolutionary innovation in animals. To understand the processes that influence the origin of specialized symbiotic organs, we report the sequencing and analysis of the genome of Euprymna scolopes, a model cephalopod with richly characterized host-microbe interactions. We identified large-scale genomic reorganization shared between E. scolopes and Octopus bimaculoides and posit that this reorganization has contributed to the evolution of cephalopod complexity. To reveal genomic signatures of host-symbiont interactions, we focused on two specialized organs of E. scolopes: the light organ, which harbors a monoculture of Vibrio fischeri, and the accessory nidamental gland (ANG), a reproductive organ containing a bacterial consortium. Our findings suggest that the two symbiotic organs within E. scolopes originated by different evolutionary mechanisms. Transcripts expressed in these microbe-associated tissues displayed their own unique signatures in both coding sequences and the surrounding regulatory regions. Compared with other tissues, the light organ showed an abundance of genes associated with immunity and mediating light, whereas the ANG was enriched in orphan genes known only from E. scolopes Together, these analyses provide evidence for different patterns of genomic evolution of symbiotic organs within a single host.

SNP calling from RNA-seq data without a reference genome: identification, quantification, differential analysis and impact on the protein sequence.

SNPs (Single Nucleotide Polymorphisms) are genetic markers whose precise identification is a prerequisite for association studies. Methods to identify them are currently well developed for model species, but rely on the availability of a (good) reference genome, and therefore cannot be applied to non-model species. They are also mostly tailored for whole genome (re-)sequencing experiments, whereas in many cases, transcriptome sequencing can be used as a cheaper alternative which already enables to identify SNPs located in transcribed regions. In this paper, we propose a method that identifies, quantifies and annotates SNPs without any reference genome, using RNA-seq data only. Individuals can be pooled prior to sequencing, if not enough material is available from one individual. Using pooled human RNA-seq data, we clarify the precision and recall of our method and discuss them with respect to other methods which use a reference genome or an assembled transcriptome. We then validate experimentally the predictions of our method using RNA-seq data from two non-model species. The method can be used for any species to annotate SNPs and predict their impact on the protein sequence. We further enable to test for the association of the identified SNPs with a phenotype of interest.

The chemistry of negotiation: rhythmic, glycan-driven acidification in a symbiotic conversation.

Glycans have emerged as critical determinants of immune maturation, microbial nutrition, and host health in diverse symbioses. In this study, we asked how cyclic delivery of a single host-derived glycan contributes to the dynamic stability of the mutualism between the squid Euprymna scolopes and its specific, bioluminescent symbiont, Vibrio fischeri. V. fischeri colonizes the crypts of a host organ that is used for behavioral light production. E. scolopes synthesizes the polymeric glycan chitin in macrophage-like immune cells called hemocytes. We show here that, just before dusk, hemocytes migrate from the vasculature into the symbiotic crypts, where they lyse and release particulate chitin, a behavior that is established only in the mature symbiosis. Diel transcriptional rhythms in both partners further indicate that the chitin is provided and metabolized only at night. A V. fischeri mutant defective in chitin catabolism was able to maintain a normal symbiont population level, but only until the symbiotic organ reached maturity (∼ 4 wk after colonization); this result provided a direct link between chitin utilization and symbiont persistence. Finally, catabolism of chitin by the symbionts was also specifically required for a periodic acidification of the adult crypts each night. This acidification, which increases the level of oxygen available to the symbionts, enhances their capacity to produce bioluminescence at night. We propose that other animal hosts may similarly regulate the activities of epithelium-associated microbial communities through the strategic provision of specific nutrients, whose catabolism modulates conditions like pH or anoxia in their symbionts' habitat.

Animals in a bacterial world, a new imperative for the life sciences.

In the last two decades, the widespread application of genetic and genomic approaches has revealed a bacterial world astonishing in its ubiquity and diversity. This review examines how a growing knowledge of the vast range of animal-bacterial interactions, whether in shared ecosystems or intimate symbioses, is fundamentally altering our understanding of animal biology. Specifically, we highlight recent technological and intellectual advances that have changed our thinking about five questions: how have bacteria facilitated the origin and evolution of animals; how do animals and bacteria affect each other's genomes; how does normal animal development depend on bacterial partners; how is homeostasis maintained between animals and their symbionts; and how can ecological approaches deepen our understanding of the multiple levels of animal-bacterial interaction. As answers to these fundamental questions emerge, all biologists will be challenged to broaden their appreciation of these interactions and to include investigations of the relationships between and among bacteria and their animal partners as we seek a better understanding of the natural world.

The oxidative environment: a mediator of interspecies communication that drives symbiosis evolution.

Symbiotic interactions are ubiquitous in nature and play a major role in driving the evolution of life. Interactions between partners are often mediated by shared signalling pathways, which strongly influence both partners' biology and the evolution of the association in various environments. As an example of 'common language', the regulation of the oxidative environment plays an important role in driving the evolution of symbiotic associations. Such processes have been occurring for billions of years, including the increase in Earth's atmospheric oxygen and the subsequent evolution of mitochondria. The effect of reactive oxygen species and reactive nitrogen species (RONS) has been characterized functionally, but the molecular dialogue between partners has not been integrated within a broader evolutionary context yet. Given the pleiotropic role of RONS in cell-cell communication, development and immunity, but also their associated physiological costs, we discuss here how their regulation can influence the establishment, the maintenance and the breakdown of various symbiotic associations. By synthesizing recent developments in redox biology, we aim to provide an interdisciplinary understanding of the influence of such mediators of interspecies communication on the evolution and stability of symbioses, which in turn can shape ecosystems and play a role in health and disease.

The dual nature of haemocyanin in the establishment and persistence of the squid-vibrio symbiosis.

We identified and sequenced from the squid Euprymna scolopes two isoforms of haemocyanin that share the common structural/physiological characteristics of haemocyanin from a closely related cephalopod, Sepia officinalis, including a pronounced Bohr effect. We examined the potential roles for haemocyanin in the animal's symbiosis with the luminous bacterium Vibrio fischeri. Our data demonstrate that, as in other cephalopods, the haemocyanin is primarily synthesized in the gills. It transits through the general circulation into other tissues and is exported into crypt spaces that support the bacterial partner, which requires oxygen for its bioluminescence. We showed that the gradient of pH between the circulating haemolymph and the matrix of the crypt spaces in adult squid favours offloading of oxygen from the haemocyanin to the symbionts. Haemocyanin is also localized to the apical surfaces and associated mucus of a juvenile-specific epithelium on which the symbionts gather, and where their specificity is determined during the recruitment into the association. The haemocyanin has an antimicrobial activity, which may be involved in this enrichment of V. fischeri during symbiont initiation. Taken together, these data provide evidence that the haemocyanin plays a role in shaping two stages of the squid-vibrio partnership.

Development, feeding, and sex shape the relative quantity of the nutritional obligatory symbiont Wolbachia in bed bugs.

The common bed bug, Cimex lectularius, is a hemipteran insect that feeds only on blood, and whose bites cause public health issues. Due to globalization and resistance to insecticides, this pest has undergone a significant and global resurgence in recent decades. Blood is an unbalanced diet, lacking notably sufficient B vitamins. Like all strict hematophagous arthropods, bed bugs host a nutritional symbiont supplying B vitamins. In C. lectularius, this nutritional symbiont is the intracellular bacterium Wolbachia (wCle). It is located in specific symbiotic organs, the bacteriomes, as well as in ovaries. Experimental depletion of wCle has been shown to result in longer nymphal development and lower fecundity. These phenotypes were rescued by B vitamin supplementation. Understanding the interaction between wCle and the bed bug may help to develop new pest control methods targeting the disruption of this symbiotic interaction. The objective of this work was thus to quantify accurately the density of wCle over the life cycle of the host and to describe potential associated morphological changes in the bacteriome. We also sought to determine the impact of sex, feeding status, and aging on the bacterial population dynamics. We showed that the relative quantity of wCle continuously increases during bed bug development, while the relative size of the bacteriome remains stable. We also showed that adult females harbor more wCle than males and that wCle relative quantity decreases slightly in adults with age, except in weekly-fed males. These results are discussed in the context of bed bug ecology and will help to define critical points of the symbiotic interaction during the bed bug life cycle.

The first engagement of partners in the Euprymna scolopes-Vibrio fischeri symbiosis is a two-step process initiated by a few environmental symbiont cells.

We studied the Euprymna scolopes-Vibrio fischeri symbiosis to characterize, in vivo and in real time, the transition between the bacterial partner's free-living and symbiotic life styles. Previous studies using high inocula demonstrated that environmental V. fischeri cells aggregate during a 3 h period in host-shed mucus along the light organ's superficial ciliated epithelia. Under lower inoculum conditions, similar to the levels of symbiont cells in the environment, this interaction induces haemocyte trafficking into these tissues. Here, in experiments simulating natural conditions, microscopy revealed that at 3 h following first exposure, only ∼ 5 V. fischeri cells aggregated on the organ surface. These cells associated with host cilia and induced haemocyte trafficking. Symbiont viability was essential and mutants defective in symbiosis initiation and/or production of certain surface features, including the Mam7 protein, which is implicated in host cell attachment of V. cholerae, associated normally with host cilia. Studies with exopolysaccharide mutants, which are defective in aggregation, suggest a two-step process of V. fischeri cell engagement: association with host cilia followed by aggregation, i.e. host cell-symbiont interaction with subsequent symbiont-symbiont cell interaction. Taken together, these data provide a new model of early partner engagement, a complex model of host-symbiont interaction with exquisite sensitivity.

Influence of Wolbachia on host gene expression in an obligatory symbiosis.

BACKGROUND: Wolbachia are intracellular bacteria known to be facultative reproductive parasites of numerous arthropod hosts. Apart from these reproductive manipulations, recent findings indicate that Wolbachia may also modify the host's physiology, notably its immune function. In the parasitoid wasp, Asobara tabida, Wolbachia is necessary for oogenesis completion, and aposymbiotic females are unable to produce viable offspring. The absence of egg production is also associated with an increase in programmed cell death in the ovaries of aposymbiotic females, suggesting that a mechanism that ensures the maintenance of Wolbachia in the wasp could also be responsible for this dependence. In order to decipher the general mechanisms underlying host-Wolbachia interactions and the origin of the dependence, we developed transcriptomic approaches to compare gene expression in symbiotic and aposymbiotic individuals. RESULTS: As no genetic data were available on A. tabida, we constructed several Expressed Sequence Tags (EST) libraries, and obtained 12,551 unigenes from this species. Gene expression was compared between symbiotic and aposymbiotic ovaries through in silico analysis and in vitro subtraction (SSH). As pleiotropic functions involved in immunity and development could play a major role in the establishment of dependence, the expression of genes involved in oogenesis, programmed cell death (PCD) and immunity (broad sense) was analyzed by quantitative RT-PCR. We showed that Wolbachia might interfere with these numerous biological processes, in particular some related to oxidative stress regulation. We also showed that Wolbachia may interact with immune gene expression to ensure its persistence within the host. CONCLUSIONS: This study allowed us to constitute the first major dataset of the transcriptome of A. tabida, a species that is a model system for both host/Wolbachia and host/parasitoid interactions. More specifically, our results highlighted that symbiont infection may interfere with numerous pivotal processes at the individual level, suggesting that the impact of Wolbachia should also be investigated beyond reproductive manipulations.

Wolbachia interferes with ferritin expression and iron metabolism in insects.

Wolbachia is an intracellular bacterium generally described as being a facultative reproductive parasite. However, Wolbachia is necessary for oogenesis completion in the wasp Asobara tabida. This dependence has evolved recently as a result of interference with apoptosis during oogenesis. Through comparative transcriptomics between symbiotic and aposymbiotic individuals, we observed a differential expression of ferritin, which forms a complex involved in iron storage. Iron is an essential element that is in limited supply in the cell. However, it is also a highly toxic precursor of Reactive Oxygen Species (ROS). Ferritin has also been shown to play a key role in host-pathogen interactions. Measuring ferritin by quantitative RT-PCR, we confirmed that ferritin was upregulated in aposymbiotic compared to symbiotic individuals. Manipulating the iron content in the diet, we showed that iron overload markedly affected wasp development and induced apoptotic processes during oogenesis in A. tabida, suggesting that the regulation of iron homeostasis may also be related to the obligate dependence of the wasp. Finally, we demonstrated that iron metabolism is influenced by the presence of Wolbachia not only in the obligate mutualism with A. tabida, but also in facultative parasitism involving Drosophila simulans and in Aedes aegypti cells. In these latter cases, the expression of Wolbachia bacterioferritin was also increased in the presence of iron, showing that Wolbachia responds to the concentration of iron. Our results indicate that Wolbachia may generally interfere with iron metabolism. The high affinity of Wolbachia for iron might be due to physiological requirement of the bacterium, but it could also be what allows the symbiont to persist in the organism by reducing the labile iron concentration, thus protecting the cell from oxidative stress and apoptosis. These findings also reinforce the idea that pathogenic, parasitic and mutualistic intracellular bacteria all use the same molecular mechanisms to survive and replicate within host cells. By impacting the general physiology of the host, the presence of a symbiont may select for host compensatory mechanisms, which extends the possible consequences of persistent endosymbiont on the evolution of their hosts.

Vertical and horizontal transmission drive bacterial invasion.

A huge variety of Arthropod species is infected with endosymbiotic Wolbachia bacteria that manipulate their host's reproduction to invade populations. In addition to vertical transmission from mother to offspring through the egg cytoplasm, it has been demonstrated through phylogenetic analyses and natural transfer experiments that horizontal transmission of Wolbachia (i.e. contagion) can occur between Arthropod hosts. More recently, factors influencing horizontal transfer have also been explored. While it is clear that horizontal transmission between species plays a major role in the evolutionary history of Wolbachia infections among insects, its role in the spread of a new infection through a host population, notably through within-species transfers, remained unknown. In this issue of Molecular Ecology, Kraaijeveld et al. (2011) present the first evidence that horizontal transmission played a key role in the early spread of parthenogenesis-inducing Wolbachia through the parasitoid wasp Leptopilina clavipes. To support their finding, the authors studied genetic variation in three types of markers, including host nuclear DNA, mitochondrial DNA and Wolbachia DNA. Specifically, they examined potential associations between their diversity patterns. No diversity was detected in Wolbachia genes, indicating that a single Wolbachia strain must have infected and spread through L. clavipes. In addition, a correlation between substantial variation in mitochondrial and nuclear genotypes suggested that horizontal transmission played an important role in the current clonal genetic variation in this wasp. Such horizontal transmission could be facilitated by a specific host ecology (e.g. parasitoid wasps sharing the same host resource) and potentially impact co-evolution between host and symbiont.

Does a parthenogenesis-inducing Wolbachia induce vestigial cytoplasmic incompatibility?

Wolbachia is a maternally inherited bacterium that manipulates the reproduction of its host. Recent studies have shown that male-killing strains can induce cytoplasmic incompatibility (CI) when introgressed into a resistant host. Phylogenetic studies suggest that transitions between CI and other Wolbachia phenotypes have also occurred frequently, raising the possibility that latent CI may be widespread among Wolbachia. Here, we investigate whether a parthenogenesis-inducing Wolbachia strain can also induce CI. Parthenogenetic females of the parasitoid wasp Asobara japonica regularly produce a small number of males that may be either infected or not. Uninfected males were further obtained through removal of the Wolbachia using antibiotics and from a naturally uninfected strain. Uninfected females that had mated with infected males produced a slightly, but significantly more male-biased sex ratio than uninfected females that had mated with uninfected males. This effect was strongest in females that mated with males that had a relatively high Wolbachia titer. Quantitative PCR indicated that infected males did not show higher ratios of nuclear versus mitochondrial DNA content. Wolbachia therefore does not cause diploidization of cells in infected males. While these results are consistent with CI, other alternatives such as production of abnormal sperm by infected males cannot be completely ruled out. Overall, the effect was very small (9%), suggesting that if CI is involved it may have degenerated through the accumulation of mutations.

Chromosomal scale assembly of parasitic wasp genome reveals symbiotic virus colonization.

Endogenous viruses form an important proportion of eukaryote genomes and a source of novel functions. How large DNA viruses integrated into a genome evolve when they confer a benefit to their host, however, remains unknown. Bracoviruses are essential for the parasitism success of parasitoid wasps, into whose genomes they integrated ~103 million years ago. Here we show, from the assembly of a parasitoid wasp genome at a chromosomal scale, that bracovirus genes colonized all ten chromosomes of Cotesia congregata. Most form clusters of genes involved in particle production or parasitism success. Genomic comparison with another wasp, Microplitis demolitor, revealed that these clusters were already established ~53 mya and thus belong to remarkably stable genomic structures, the architectures of which are evolutionary constrained. Transcriptomic analyses highlight temporal synchronization of viral gene expression without resulting in immune gene induction, suggesting that no conflicts remain between ancient symbiotic partners when benefits to them converge.

Involvement of a host Cathepsin L in symbiont-induced cell death.

The cathepsin L gene of the host squid, Euprymna scolopes, is upregulated during the first hours of colonization by the symbiont Vibrio fischeri. At this time, the symbiotic organ begins cell death-mediated morphogenesis in tissues functional only at the onset of symbiosis. The goal of this study was to determine whether Cathepsin L, a cysteine protease associated with apoptosis in other animals, plays a critical role in symbiont-induced cell death in the host squid. Sequence analysis and biochemical characterization demonstrated that the protein has key residues and domains essential for Cathepsin L function and that it is active within the pH range typical of these proteases. With in situ hybridization and immunocytochemistry, we localized the transcript and protein, respectively, to cells interacting with V. fischeri. Activity of the protein occurred along the path of symbiont colonization. A specific Cathepsin L, nonspecific cysteine protease, and caspase inhibitor each independently attenuated activity and cell death to varying degrees. In addition, a specific antibody decreased cell death by ~50%. Together these data provide evidence that Cathepsin L is a critical component in the symbiont-induced cell death that transforms the host tissues from a colonization morphology to one that promotes the mature association.

Development of a PCR-RFLP assay to identify Drosophila melanogaster among field-collected larvae.

The fruit fly Drosophila melanogaster is a model organism to study several aspects of metazoan biology. Most of the work has been conducted in adult fruit flies, including laboratory and field-derived specimens, but Drosophila melanogaster larvae recently became a valuable model to better understand animal physiology, development, or host-microbe interactions. While adult flies can be easily assigned to a given Drosophila species based on morphological characteristics, such visual identification is more intricate at the larval stage. This could explain the limited number of studies focusing on larvae, especially field-derived samples. Here, we developed a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay that discriminates D. melanogaster from other ecologically relevant Drosophila species at the larval stage. The method, which targets the cytochrome oxidase I (COI) gene, was validated using laboratory-derived larvae from seven D. melanogaster populations originating from different geographic areas as well as six Drosophila species. We further validated this PCR-RFLP assay in a natural context, by identifying wild larvae collected in two locations in France. Notably, among all PCR-RFLP profiles that matched the D. melanogaster species, 100% were correctly identified, as confirmed by COI sequencing. In summary, our work provides a rapid, simple, and accurate molecular tool to identify D. melanogaster from field-collected larvae.

Persistent Interactions with Bacterial Symbionts Direct Mature-Host Cell Morphology and Gene Expression in the Squid-Vibrio Symbiosis.

In horizontally transmitted symbioses, structural, biochemical, and molecular features both facilitate host colonization by specific symbionts and mediate their persistent carriage. In the association between the squid Euprymna scolopes and its luminous bacterial partner Vibrio fischeri, the symbionts interact with two epithelial fields; they interact (i) transiently with the superficial ciliated field that potentiates colonization and regresses within days of colonization and (ii) persistently with the cells that line the internal crypts, whose ultrastructure changes in response to the symbionts. Development of the association creates conditions that promote the symbiotic partner over the lifetime of the host. To determine whether light organ maturation requires continuous interactions with V. fischeri or only the signaling that occurs during its initiation, we compared 4-week-old squid that were uncolonized with those colonized either persistently by wild-type V. fischeri or transiently by a V. fischeri mutant that triggers early events in morphogenesis but does not persist. Microscopic analysis of the light organs showed that, while morphogenesis of the superficial ciliated field is greatly accelerated by V. fischeri colonization, its eventual outcome is largely independent of colonization state. In contrast, the symbiont-induced changes in crypt cell shape require persistent host-symbiont interaction, reflected in the similarity between uncolonized and transiently colonized animals. Transcriptomic analyses reflected the microscopy results; host gene expression at 4 weeks was due primarily to the persistent interactions of host and symbiont cells. Further, the transcriptomic signature of specific pathways reflected the daily rhythm of symbiont release and regrowth and required the presence of the symbionts. IMPORTANCE A long-term relationship between symbiotic partners is often characterized by development and maturation of host structures that harbor the symbiont cells over the host's lifetime. To understand the mechanisms involved in symbiosis maintenance more fully, we studied the mature bobtail squid, whose light-emitting organ, under experimental conditions, can be transiently or persistently colonized by Vibrio fischeri or remain uncolonized. Superficial anatomical changes in the organ were largely independent of symbiosis. However, both the microanatomy of cells with which symbionts interact and the patterns of gene expression in the mature animal were due principally to the persistent interactions of host and symbiont cells rather than to a response to early colonization events. Further, the characteristic pronounced daily rhythm on the host transcriptome required persistent V. fischeri colonization of the organ. This experimental study provides a window into how persistent symbiotic colonization influences the form and function of host animal tissues.

Impact of Wolbachia on oxidative stress sensitivity in the parasitic wasp Asobara japonica.

The oxidative homeostasis is the balance between reactive oxygen species and antioxidant molecules. In addition to be considered as a key factor underlying life-history traits evolution, the oxidative homeostasis has been shown to be involved in many host-symbiont associations. Previous studies suggest an interaction between the bacterial endosymbiont Wolbachia and the oxidative homeostasis of some insect hosts. This interaction is likely to exert a strong influence on the host evolution, as it has been proposed in the wasp Asobara tabida, whose dependence upon Wolbachia is due to the evolutionary loss of its ability to regulate the oxidative homeostasis in the absence of the symbiont. Although such cases of complete dependence are rare, cases of insects having lost only a part of their autonomy over the control of the oxidative homeostasis might be more common. If so, one can expect that insects having coevolved with Wolbachia will be more sensitive to oxidative stress when cured of their symbionts. We tested this hypothesis by studying the effects of an experimentally-induced oxidative stress on various life-history traits of Asobara japonica, a species closely related to A. tabida. For most of the life-history traits studied, the sensitivity of the wasps to oxidative stress did not correlate with their infection status. The only exception was the parasitic success. However, contrarily to our expectation, the sensitivity to oxidative stress was increased, rather than decreased, when Wolbachia was present. This result suggests that Wolbachia does not participate to mitigate oxidative stress in A. japonica, and that on the contrary its presence might still be costly in stressful environments.

Adaptive male effects on female ageing in seed beetles.

Selection can favour the evolution of a high reproductive rate early in life even when this results in a subsequent increase in the rate of mortality, because selection is relatively weak late in life. However, the optimal reproductive schedule of a female may be suboptimal to any one of her mates, and males may thus be selected to modulate female reproductive rate. Owing to such sexual conflict, coevolution between males and females may contribute to the evolution of senescence. By using replicated beetle populations selected for reproduction at an early or late age, we show that males evolve to affect senescence in females in a manner consistent with the genetic interests of males. 'Late' males evolved to decelerate senescence and increase the lifespan of control females, relative to 'early' males. Our findings demonstrate that adaptive evolution in one sex may involve its effects on senescence in the other, showing that the evolution of optimal life histories in one sex may be either facilitated or constrained by genes expressed in the other.

Microbial impacts on insect evolutionary diversification: from patterns to mechanisms.

Symbiosis can favor rapid shifts in host phenotypic traits, particularly through the contribution of symbionts to the host's physiology. In addition, variations in the microbiota composition between individuals can be associated with pre-zygotic and post-zygotic barriers. All together, these phenomena may contribute to insect diversification and speciation. Recent advances have also shown that the host-microbiota molecular dialog, mediated notably by host immune and developmental pathways, is critical for the acquisition and control of the microbiota, and could also contribute to reproductive isolation. While still a controversial hypothesis, adaptation through symbiosis could thus trigger host-symbiont coevolution and accelerate differentiation.