Differential proteomic analysis of midguts from Nosema ceranae-infected honeybees reveals manipulation of key host functions.

Many invasive pathogens effectively bypass the insect defenses to ensure the completion of their life cycle. Among those, an invasive microsporidian species, Nosema ceranae, can cause nosemosis in honeybees. N. ceranae was first described in the Asian honeybee Apis cerana and is suspected to be involved in Western honeybee (Apis mellifera) declines worldwide. The midgut of honeybees is the first barrier against N. ceranae attacks. To bring proteomics data on honeybee/N. ceranae crosstalk and more precisely to decipher the worker honeybee midgut response after an oral inoculation of N. ceranae (10days post-infection), we used 2D-DIGE (2-Dimensional Differential In-Gel Electrophoresis) combined with mass spectrometry. Forty-five protein spots produced by the infected worker honeybee group were shown to be differentially expressed when compared to the uninfected group; 14 were subsequently identified by mass spectrometry. N. ceranae mainly caused a modulation of proteins involved in three key host biological functions: (i) energy production, (ii) innate immunity (reactive oxygen stress) and (iii) protein regulation. The modulation of these host biological functions suggests that N. ceranae creates a zone of "metabolic habitat modification" in the honeybee midgut favoring its development by enhancing availability of nutrients and reducing the worker honeybee defense.

Host-parasite molecular cross-talk during the manipulative process of a host by its parasite.

Many parasite taxa are able to alter a wide range of phenotypic traits of their hosts in ways that seem to improve the parasite's chance of completing its life cycle. Host behavioural alterations are classically seen as compelling illustrations of the 'extended phenotype' concept, which suggests that parasite genes have phenotype effects on the host. The molecular mechanisms and the host-parasite cross-talk involved during the manipulative process of a host by its parasite are still poorly understood. In this Review, the current knowledge on proximate mechanisms related to the 'parasite manipulation hypothesis' is presented. Parasite genome sequences do not themselves provide a full explanation of parasite biology nor of the molecular cross-talk involved in host-parasite associations. Recently, first-generation proteomics tools have been employed to unravel some aspects of the parasite manipulation process (i.e. proximate mechanisms and evolutionary convergence) using certain model arthropod-host-parasite associations. The pioneer proteomics results obtained on the manipulative process are here highlighted, along with the many gaps in our knowledge. Candidate genes and biochemical pathways potentially involved in the parasite manipulation are presented. Finally, taking into account the environmental factors, we suggest new avenues and approaches to further explore and understand the proximate mechanisms used by parasite species to alter phenotypic traits of their hosts.

Phylogenetic character mapping of proteomic diversity shows high correlation with subspecific phylogenetic diversity in Trypanosoma cruzi.

We performed a phylogenetic character mapping on 26 stocks of Trypanosoma cruzi, the parasite responsible for Chagas disease, and 2 stocks of the sister taxon T. cruzi marinkellei to test for possible associations between T. cruzi-subspecific phylogenetic diversity and levels of protein expression, as examined by proteomic analysis and mass spectrometry. We observed a high level of correlation (P < 10(-4)) between genetic distance, as established by multilocus enzyme electrophoresis, and proteomic dissimilarities estimated by proteomic Euclidian distances. Several proteins were found to be specifically associated to T. cruzi phylogenetic subdivisions (discrete typing units). This study explores the previously uncharacterized links between infraspecific phylogenetic diversity and gene expression in a human pathogen. It opens the way to searching for new vaccine and drug targets and for identification of specific biomarkers at the subspecific level of pathogens.

Diatom DNA metabarcoding to assess the effect of natural radioactivity in mineral springs on ASV of benthic diatom communities.

Little is still known about the low dose effects of radiation on the microbial communities in the environment. Mineral springs are ecosystems than can be affected by natural radioactivity. These extreme environments are, therefore, observatories for studying the influence of chronic radioactivity on the natural biota. In these ecosystems we find diatoms, unicellular microalgae, playing an essential role in the food chain. The present study aimed to investigate, using DNA metabarcoding, the effect of natural radioactivity in two environmental compartments (i.e. spring sediments and water) on the genetic richness, diversity and structure of diatom communities in 16 mineral springs in the Massif Central, France. Diatom biofilms were collected during October 2019, and a 312 bp region of the chloroplast gene rbcL (coding for the Ribulose Bisphosphate Carboxylase) used as a barcode for taxonomic assignation. A total of 565 amplicon sequence variants (ASV) were found. The dominant ASV were associated with Navicula sanctamargaritae, Gedaniella sp., Planothidium frequentissimum, Navicula veneta, Diploneis vacillans, Amphora copulata, Pinnularia brebissonii, Halamphora coffeaeformis, Gomphonema saprophilum, and Nitzschia vitrea, but some of the ASVs could not be assigned at the species level. Pearson correlation failed to show a correlation between ASV' richness and radioactivity parameters. Non-parametric MANOVA analysis based on ASVs occurrence or abundances revealed that geographical location was the main factor influencing ASVs distribution. Interestingly, 238U was the second factor that explained diatom ASV structure. Among the ASVs in the mineral springs monitored, ASV associated with one of the genetic variants of Planothidium frequentissimum was well represented in the springs and with higher levels of 238U, suggesting its high tolerance to this particular radionuclide. This diatom species may therefore represent a bio-indicator of high natural levels of uranium.

Assessing radiation dosimetry for microorganisms in naturally radioactive mineral springs using GATE and Geant4-DNA Monte Carlo simulations.

Mineral springs in Massif Central, France can be characterized by higher levels of natural radioactivity in comparison to the background. The biota in these waters is constantly under radiation exposure mainly from the α-emitters of the natural decay chains, with 226Ra in sediments ranging from 21 Bq/g to 43 Bq/g and 222Rn activity concentrations in water up to 4600 Bq/L. This study couples for the first time micro- and nanodosimetric approaches to radioecology by combining GATE and Geant4-DNA to assess the dose rates and DNA damages to microorganisms living in these naturally radioactive ecosystems. It focuses on unicellular eukaryotic microalgae (diatoms) which display an exceptional abundance of teratological forms in the most radioactive mineral springs in Auvergne. Using spherical geometries for the microorganisms and based on γ-spectrometric analyses, we evaluate the impact of the external exposure to 1000 Bq/L 222Rn dissolved in the water and 30 Bq/g 226Ra in the sediments. Our results show that the external dose rates for diatoms are significant (9.7 µGy/h) and comparable to the threshold (10 µGy/h) for the protection of the ecosystems suggested by the literature. In a first attempt of simulating the radiation induced DNA damage on this species, the rate of DNA Double Strand Breaks per day is estimated to 1.11E-04. Our study confirms the significant mutational pressure from natural radioactivity to which microbial biodiversity has been exposed since Earth origin in hydrothermal springs.

Update on the proteomics of major arthropod vectors of human and animal pathogens.

Vector-borne diseases (VBDs) are defined as infectious diseases of humans and animals caused by pathogenic agents such as viruses, protists, bacteria, and helminths transmitted by the bite of blood-feeding arthropod (BFA) vectors. VBDs represent a major public health threat in endemic areas, generally subtropical zones, and many are considered to be neglected diseases. Genome sequencing of some arthropod vectors as well as modern proteomic and genomic technologies are expanding our knowledge of arthropod-pathogen interactions. This review describes the proteomic approaches that have been used to investigate diverse biological questions about arthropod vectors, including the interplay between vectors and pathogens. Proteomic studies have identified proteins and biochemical pathways that may be involved in molecular crosstalk in BFA-pathogen associations. Future work can build upon this promising start and functional analyses coupled with interactome bioassays will be carried out to investigate the role of candidate peptides and proteins in BFA-human pathogen associations. Dissection of the host-pathogen interactome will be key to understanding the strategies and biochemical pathways used by BFAs to cope with pathogens.

Parasitology: parasite survives predation on its host.

As prisoners in their living habitat, parasites should be vulnerable to destruction by the predators of their hosts. But we show here that the parasitic gordian worm Paragordius tricuspidatus is able to escape not only from its insect host after ingestion by a fish or frog but also from the digestive tract of the predator. This remarkable tactic enables the worm to continue its life cycle.

Blood-feeding and immunogenic Aedes aegypti saliva proteins.

Mosquito-transmitted pathogens pass through the insect's midgut (MG) and salivary gland (SG). What occurs in these organs in response to a blood meal is poorly understood, but identifying the physiological differences between sugar-fed and blood-fed (BF) mosquitoes could shed light on factors important in pathogens transmission. We compared differential protein expression in the MGs and SGs of female Aedes aegypti mosquitoes after a sugar- or blood-based diet. No difference was observed in the MG protein expression levels but certain SG proteins were highly expressed only in BF mosquitoes. In sugar-fed mosquitoes, housekeeping proteins were highly expressed (especially those related to energy metabolism) and actin was up-regulated. The immunofluorescence assay shows that there is no disruption of the SG cytoskeletal after the blood meal. We have generated for the first time the 2-DE profiles of immunogenic Ae. aegypti SG BF-related proteins. These new data could contribute to the understanding of the physiological processes that appear during the blood meal.

Invasion of the body snatchers: the diversity and evolution of manipulative strategies in host-parasite interactions.

Parasite-induced alteration of host behaviour is a widespread transmission strategy among pathogens. Understanding how it works is an exciting challenge from both a mechanistic and an evolutionary perspective. In this review, we use key examples to examine the proximate mechanisms by which parasites are known to control the behaviour of their hosts. Special attention is given to the recent developments of post-genomic tools, such as proteomics, for determining the genetic basis of parasitic manipulation. We then discuss two novel perspectives on host manipulation (mafia-like strategy and exploitation of host compensatory responses), arguing that parasite-manipulated behaviours could be the result of compromises between host and parasite strategies. Such compromises may occur when collaborating with the parasite is less costly for the host in terms of fitness than is resisting parasite-induced changes. Therefore, even when changes in host behaviour benefit the parasite, the host may still play some role in the switch in host behaviour. In other words, the host does not always become part of the parasite's extended phenotype. For example, parasites that alter host behaviour appear to induce widely disseminated changes in the hosts' central nervous system, as opposed to targeted attacks on specific neural circuits. In some host-parasite systems, the change in host behaviour appears to require the active participation of the host (e.g., via host immune-neural connections). Even when the change in host behaviour results in clear fitness benefits for the parasite, these behavioural changes may sometimes be produced by the host. Changes in host behaviour that decrease the fitness costs of infection could be selected for, even if these changes also benefit the parasite.

Neurological and physiological disorders in Artemia harboring manipulative cestodes.

There are many impressive examples of host manipulation by parasites, but mechanisms underlying these ethological changes, as well as their physiological consequences, are not well characterized. Here, we analyzed part of the cerebral proteome of brine shrimp Artemia infected by manipulative cestodes, using for the first time the ProteinChip Surface-Enhanced Laser Desorption Ionization and Time of Fly Mass Spectrometry (SELDI-TOF MS) system, which has been proposed as an excellent way to analyze the host genome during the host-parasite interaction processes. We found 2 peptides downregulated in individuals infected by the dilepidid, Anomotaenia tringae (4.5 kDa), and by the 2 hymenolepidids, Flamingolepis liguloides and Confluaria podicipina (3.9 kDa), which are potential candidates for involvement with the manipulation process. The identification of 2 head peptides (4.1 and 4.2 kDa) overexpressed in all the categories in brine shrimp living at the surface (both infected individuals and uninfected controls) suggests its association with the different environmental conditions experienced at the water surface. In parallel, brine shrimp infected by C. podicipina showed significant values of triglycerides, potentially augmenting their profitability and attractiveness for the predaceous definitive host (grebes). We discuss our findings in relationship with current ideas on the complexity of parasitically modified organisms.

Reducing the ionizing radiation background does not significantly affect the evolution of Escherichia coli populations over 500 generations.

Over millennia, life has been exposed to ionizing radiation from cosmic rays and natural radioisotopes. Biological experiments in underground laboratories have recently demonstrated that the contemporary terrestrial radiation background impacts the physiology of living organisms, yet the evolutionary consequences of this biological stress have not been investigated. Explaining the mechanisms that give rise to the results of underground biological experiments remains difficult, and it has been speculated that hereditary mechanisms may be involved. Here, we have used evolution experiments in standard and very low-radiation backgrounds to demonstrate that environmental ionizing radiation does not significantly impact the evolutionary trajectories of E. coli bacterial populations in a 500 generations evolution experiment.

Hygroregulation, a key ability for eusocial insects: Native Western European honeybees as a case study.

Sociality has brought many advantages to various hymenoptera species, including their ability of regulating physical factors in their nest (e.g., temperature). Although less studied, humidity is known to be important for egg, larval and pupal development, and also for nectar concentration. Two subspecies of Apis mellifera of the M evolutionary lineage were used as models to test the ability of a superorganism (i.e. honeybee colony) to regulate the humidity in its nest (i.e. "hygroregulation hypothesis") in four conservation centers: two in France (A. m. mellifera) and two in Portugal (A. m. iberiensis). We investigated the ability of both subspecies to regulate the humidity in hives daily, but also during the seasons for one complete year. Our data and statistical analysis demonstrated the capacity of the bees to regulate humidity in their hive, regardless of the day, season or subspecies. Furthermore, the study showed that humidity in beehives is stable even during winter, when brood is absent, and when temperature is known to be less stable in the beehives. These results suggest that humidity is important for honeybees at every life stage, maybe because of the 'imprint' of the evolutionary history of this hymenopteran lineage.

Virulence and pathogenicity patterns of Trypanosoma brucei gambiense field isolates in experimentally infected mouse: differences in host immune response modulation by secretome and proteomics.

Human African trypanosomiasis is characterised by an important clinical diversity. Although Trypanosoma brucei gambiense field stocks isolated from patients in the same focus did not exhibit apparent genetic variability, they showed marked differences in terms of virulence (capacity to multiply inside a host) and pathogenicity (ability of producing mortality) in experimental murine infections. Two strains exhibiting opposite pathogenic and virulence properties in mouse were further investigated through their host-parasite interactions. In vitro, parasite bloodstream forms or soluble factors (or secretome) from both strains induced macrophage arginase as a function of their virulence. Arginase expression, a hallmark of macrophage alternative activation pathway, favours trypanosome bloodstream forms development. Moreover, a comparative proteomic study of the trypanosome stocks' secretomes evidenced both a differential expression of common molecules and the existence of stock specific molecules. This highlighted the potential involvement of the differential expression of the same genome in the diverse infectious properties of trypanosomes.

Understanding low radiation background biology through controlled evolution experiments.

Biological experiments conducted in underground laboratories over the last decade have shown that life can respond to relatively small changes in the radiation background in unconventional ways. Rapid changes in cell growth, indicative of hormetic behaviour and long-term inheritable changes in antioxidant regulation have been observed in response to changes in the radiation background that should be almost undetectable to cells. Here, we summarize the recent body of underground experiments conducted to date, and outline potential mechanisms (such as cell signalling, DNA repair and antioxidant regulation) that could mediate the response of cells to low radiation backgrounds. We highlight how multigenerational studies drawing on methods well established in studying evolutionary biology are well suited for elucidating these mechanisms, especially given these changes may be mediated by epigenetic pathways. Controlled evolution experiments with model organisms, conducted in underground laboratories, can highlight the short- and long-term differences in how extremely low-dose radiation environments affect living systems, shining light on the extent to which epimutations caused by the radiation background propagate through the population. Such studies can provide a baseline for understanding the evolutionary responses of microorganisms to ionizing radiation, and provide clues for understanding the higher radiation environments around uranium mines and nuclear disaster zones, as well as those inside nuclear reactors.

Nosema ceranae, Fipronil and their combination compromise honey bee reproduction via changes in male physiology.

The honey bee is threatened by biological agents and pesticides that can act in combination to induce synergistic effects on its physiology and lifespan. The synergistic effects of a parasite/pesticide combination have been demonstrated on workers and queens, but no studies have been performed on drones despite their essential contribution to colony sustainability by providing semen diversity and quality. The effects of the Nosema ceranae/fipronil combination on the life traits and physiology of mature drones were examined following exposure under semi-field conditions. The results showed that the microsporidia alone induced moderate and localized effects in the midgut, whereas fipronil alone induced moderate and generalized effects. The parasite/insecticide combination drastically affected both physiology and survival, exhibiting an important and significant generalized action that could jeopardize mating success. In terms of fertility, semen was strongly impacted regardless of stressor, suggesting that drone reproductive functions are very sensitive to stress factors. These findings suggest that drone health and fertility impairment might contribute to poorly mated queens, leading to the storage of poor quality semen and poor spermathecae diversity. Thus, the queens failures observed in recent years might result from the continuous exposure of drones to multiple environmental stressors.

Facultative virulence: a strategy to manipulate host behaviour?

Examples of behavioural manipulation by parasites are numerous, but the processes underlying these changes are not well characterized. From an evolutionary point of view, behavioural changes in infected hosts have often been interpreted as illustrations of the extended phenotype concept, in which genes in one organism (the parasite) have phenotypic effects on another organism (the host). Here, we approach the problem differently, suggesting that hosts, by cooperating with manipulative parasites rather than resisting them, might mitigate fitness costs associated with manipulation. By imposing extra fitness costs on their hosts in the absence of compliance, parasites theoretically have the potential to select for cooperative behaviour by their hosts. Although this 'mafia-like' strategy remains poorly documented, we believe that it has substantial potential to resolve issues specific to the evolution of behavioural alterations induced by parasites.

Simulating the Impact of the Natural Radiation Background on Bacterial Systems: Implications for Very Low Radiation Biological Experiments.

At very low radiation dose rates, the effects of energy depositions in cells by ionizing radiation is best understood stochastically, as ionizing particles deposit energy along tracks separated by distances often much larger than the size of cells. We present a thorough analysis of the stochastic impact of the natural radiative background on cells, focusing our attention on E. coli grown as part of a long term evolution experiment in both underground and surface laboratories. The chance per day that a particle track interacts with a cell in the surface laboratory was found to be 6 × 10-5 day-1, 100 times less than the expected daily mutation rate for E. coli under our experimental conditions. In order for the chance cells are hit to approach the mutation rate, a gamma background dose rate of 20 µGy hr-1 is predicted to be required.

Towards a new conceptual approach to "parasitoproteomics".

Many parasitologists are betting heavily on proteomic studies to explain biochemical host-parasite interactions and, thus, to contribute to disease control. However, many "parasitoproteomic" studies are performed with powerful techniques but without a conceptual approach to determine whether the host genomic responses during a parasite infection represent a nonspecific response that might be induced by any parasite or any other stress. In this article, a new conceptual approach, based on evolutionary concepts of immune responses of a host to a parasite, is suggested for parasitologists to study the host proteome reaction after parasite invasion. Also, this new conceptual approach can be used to study other host-parasite interactions such as behavioral manipulation.

First analysis of the proteome in two nematomorph species, Paragordius tricuspidatus (Chordodidae) and Spinochordodes tellinii (Spinochordodidae).

The proteome of most parasite species is currently unknown. Hairworms (Nematomorpha), 300 species distributed around the world, are parasitic in arthropods (mainly terrestrial species) when juveniles, but they are free-living in aquatic environments when adult. Most aspects of their systematics and biology are currently unknown. The aim of this paper was (i) to report a novel and reproducible protocol for the analysis of the proteome of hairworms using two-dimensional gel electrophoresis (2-DGE) and mass spectrometry (matrix laser desorption ionization-time of flight mass spectrometry (MALDI-TOF)) and (ii) to determine the level of proteomic divergence between two sympatric but taxonomically unrelated nematomorph species in the adult stage, Paragordius tricuspidatus Dufour (Nematomorpha, Gordiidae) and Spinochordodes tellinii Camerano (Nematomorpha, Gordiidae). In total, 689 protein spots were observed for P. tricuspidatus, 575 for S. tellinii. Only 36.2% spots were shared between the two species. Quantitative analysis of the proteins which are common to both parasite species reveals substantial differences in the pattern of protein expression. These results suggest a rapid evolutionary divergence between these two nematomorph families. Also, to test the value of our MALDI-TOF protocol, we used Actin-2 (Act-2), a protein highly conserved in the course of evolution. Peptide mass fingerprint (PMF) data obtained for Act-2 of P. tricuspidatus and S. tellinii suggest a very high homology with Act-2 of different worms species belonging to the Bilateria phylum (Annelida and Nematoda) and more specifically to Lumbricus terrestris (Annelida, Lumbricidae) and Caenorhabditis elegans (Nematoda, Rhabditidae). We discuss our results in relationship with current ideas concerning the use of proteomics in systematics.

Cross-talk in host-parasite associations: What do past and recent proteomics approaches tell us?

A cross-talk in host-parasite associations begins when a host encounters a parasite. For many host-parasite relationships, this cross-talk has been taking place for hundreds of millions of years. The co-evolution of hosts and parasites, the familiar 'arms race' results in fascinating adaptations. Over the years, host-parasite interactions have been studied extensively from both the host and parasitic point of view. Proteomics studies have led to new insights into host-parasite cross-talk and suggest that the molecular strategies used by parasites attacking animals and plants share many similarities. Likewise, animals and plants use several common molecular tactics to counter parasite attacks. Based on proteomics surveys undertaken since the post-genomic era, a synthesis is presented on the molecular strategies used by intra- and extracellular parasites to invade and create the needed habitat for growth inside the host, as well as strategies used by hosts to counter these parasite attacks. Pitfalls in deciphering host-parasite cross-talk are also discussed. To conclude, helpful advice is given with regard to new directions that are needed to discover the generic and specific molecular strategies used by the host against parasite invasion as well as by the parasite to invade, survive, and grow inside their hosts, and to finally discover parasitic molecular signatures associated with their development.