Marie Diogon

Exposure to sublethal doses of fipronil and thiacloprid highly increases mortality of honeybees previously infected by Nosema ceranae.

Background The honeybee, Apis mellifera, is undergoing a worldwide decline whose origin is still in debate. Studies performed for twenty years suggest that this decline may involve both infectious diseases and exposure to pesticides. Joint action of pathogens and chemicals are known to threaten several organisms but the combined effects of these stressors were poorly investigated in honeybees. Our study was designed to explore the effect of Nosema ceranae infection on honeybee sensitivity to sublethal doses of the insecticides fipronil and thiacloprid. Methodology/Finding Five days after their emergence, honeybees were divided in 6 experimental groups: (i) uninfected controls, (ii) infected with N. ceranae, (iii) uninfected and exposed to fipronil, (iv) uninfected and exposed to thiacloprid, (v) infected with N. ceranae and exposed 10 days post-infection (p.i.) to fipronil, and (vi) infected with N. ceranae and exposed 10 days p.i. to thiacloprid. Honeybee mortality and insecticide consumption were analyzed daily and the intestinal spore content was evaluated 20 days after infection. A significant increase in honeybee mortality was observed when N. ceranae-infected honeybees were exposed to sublethal doses of insecticides. Surprisingly, exposures to fipronil and thiacloprid had opposite effects on microsporidian spore production. Analysis of the honeybee detoxification system 10 days p.i. showed that N. ceranae infection induced an increase in glutathione-S-transferase activity in midgut and fat body but not in 7-ethoxycoumarin-O-deethylase activity. Conclusions/Significance After exposure to sublethal doses of fipronil or thiacloprid a higher mortality was observed in N. ceranae-infected honeybees than in uninfected ones. The synergistic effect of N. ceranae and insecticide on honeybee mortality, however, did not appear strongly linked to a decrease of the insect detoxification system. These data support the hypothesis that the combination of the increasing prevalence of N. ceranae with high pesticide content in beehives may contribute to colony depopulation.

Parasite-insecticide interactions: a case study of Nosema ceranae and fipronil synergy on honeybee

In ecosystems, a variety of biological, chemical and physical stressors may act in combination to induce illness in populations of living organisms. While recent surveys reported that parasite-insecticide interactions can synergistically and negatively affect honeybee survival, the importance of sequence in exposure to stressors has hardly received any attention. In this work, Western honeybees (Apis mellifera) were sequentially or simultaneously infected by the microsporidian parasite Nosema ceranae and chronically exposed to a sublethal dose of the insecticide fipronil, respectively chosen as biological and chemical stressors. Interestingly, every combination tested led to a synergistic effect on honeybee survival, with the most significant impacts when stressors were applied at the emergence of honeybees. Our study presents significant outcomes on beekeeping management but also points out the potential risks incurred by any living organism frequently exposed to both pathogens and insecticides in their habitat.

Genome sequence of the stramenopile Blastocystis, a human anaerobic parasite

BackgroundBlastocystis is a highly prevalent anaerobic eukaryotic parasite of humans and animals that is associated with various gastrointestinal and extraintestinal disorders. Epidemiological studies have identified different subtypes but no one subtype has been definitively correlated with disease.ResultsHere we report the 18.8 Mb genome sequence of a Blastocystis subtype 7 isolate, which is the smallest stramenopile genome sequenced to date. The genome is highly compact and contains intriguing rearrangements. Comparisons with other available stramenopile genomes (plant pathogenic oomycete and diatom genomes) revealed effector proteins potentially involved in the adaptation to the intestinal environment, which were likely acquired via horizontal gene transfer. Moreover, Blastocystis living in anaerobic conditions harbors mitochondria-like organelles. An incomplete oxidative phosphorylation chain, a partial Krebs cycle, amino acid and fatty acid metabolisms and an iron-sulfur cluster assembly are all predicted to occur in these organelles. Predicted secretory proteins possess putative activities that may alter host physiology, such as proteases, protease-inhibitors, immunophilins and glycosyltransferases. This parasite also possesses the enzymatic machinery to tolerate oxidative bursts resulting from its own metabolism or induced by the host immune system.ConclusionsThis study provides insights into the genome architecture of this unusual stramenopile. It also proposes candidate genes with which to study the physiopathology of this parasite and thus may lead to further investigations into Blastocystis-host interactions.

The RhoGAP RGA-2 and LET-502/ROCK achieve a balance of actomyosin-dependent forces in C. elegans epidermis to control morphogenesis

Embryonic morphogenesis involves the coordinate behaviour of multiple cells and requires the accurate balance of forces acting within different cells through the application of appropriate brakes and throttles. In C. elegans, embryonic elongation is driven by Rho-binding kinase (ROCK) and actomyosin contraction in the epidermis. We identify an evolutionary conserved, actin microfilament-associated RhoGAP (RGA-2) that behaves as a negative regulator of LET-502/ROCK. The small GTPase RHO-1 is the preferred target of RGA-2 in vitro, and acts between RGA-2 and LET-502 in vivo. Two observations show that RGA-2 acts in dorsal and ventral epidermal cells to moderate actomyosin tension during the first half of elongation. First, time-lapse microscopy shows that loss of RGA-2 induces localised circumferentially oriented pulling on junctional complexes in dorsal and ventral epidermal cells. Second, specific expression of RGA-2 in dorsal/ventral, but not lateral, cells rescues the embryonic lethality of rga-2 mutants. We propose that actomyosin-generated tension must be moderated in two out of the three sets of epidermal cells surrounding the C. elegans embryo to achieve morphogenesis.

Standard methods for toxicology research in Apis mellifera

Summary Modern agriculture often involves the use of pesticides to protect crops. These substances are harmful to target organisms (pests and pathogens). Nevertheless, they can also damage non-target animals, such as pollinators and entomophagous arthropods. It is obvious that the undesirable side effects of pesticides on the environment should be reduced to a minimum. Western honey bees (Apis mellifera) are very important organisms from an agricultural perspective and are vulnerable to pesticide-induced impacts. They contribute actively to the pollination of cultivated crops and wild vegetation, making food production possible. Of course, since Apis mellifera occupies the same ecological niche as many other species of pollinators, the loss of honey bees caused by environmental pollutants suggests that other insects may experience a similar outcome. Because pesticides can harm honey bees and other pollinators, it is important to register pesticides that are as selective as possible. In this manuscript, we describe a selection of methods used for studying pesticide toxicity/selectiveness towards Apis mellifera. These methods may be used in risk assessment schemes and in scientific research aimed to explain acute and chronic effects of any target compound on Apis mellifera.

Expression and Immunogenicity of the Mycobacterial Ag85B/ESAT-6 Antigens Produced in Transgenic Plants by Elastin-Like Peptide Fusion Strategy

This study explored a novel system combining plant-based production and the elastin-like peptide (ELP) fusion strategy to produce vaccinal antigens against tuberculosis. Transgenic tobacco plants expressing the mycobacterial antigens Ag85B and ESAT-6 fused to ELP (TBAg-ELP) were generated. Purified TBAg-ELP was obtained by the highly efficient, cost-effective, inverse transition cycling (ICT) method and tested in mice. Furthermore, safety and immunogenicity of the crude tobacco leaf extracts were assessed in piglets. Antibodies recognizing mycobacterial antigens were produced in mice and piglets. A T-cell immune response able to recognize the native mycobacterial antigens was detected in mice. These findings showed that the native Ag85B and ESAT-6 mycobacterial B- and T-cell epitopes were conserved in the plant-expressed TBAg-ELP. This study presents the first results of an efficient plant-expression system, relying on the elastin-like peptide fusion strategy, to produce a safe and immunogenic mycobacterial Ag85B-ESAT-6 fusion protein as a potential vaccine candidate against tuberculosis.

Complete circular DNA in the mitochondria-like organelles of Blastocystis hominis

Blastocystis hominis is an anaerobic parasite of the human intestinal tract belonging to the Stramenopile group. Using genome sequencing project data, we describe here the complete sequence of a 29,270-bp circular DNA molecule that presents mitochondrial features (such as oxidative phosphorylation complex I subunits) but lacks complexes III, IV and V. Transmission electron microscopy analyses reveal that this molecule, as well as mitochondrial (NADH:ubiquinone oxidoreductase subunit 7 (NAD7), beta-succinyl-CoA synthetase (beta-SCS)) and hydrogenosomal (pyruvate ferredoxin oxido-reductase (PFOR), iron-hydrogenase) proteins, are located within double-membrane surrounded-compartments known as mitochondria-like organelles (MLOs). As there is no evidence for hydrogen production by this organism, we suggest that MLOs are more likely anaerobic mitochondria.

CRT-1/calreticulin and the E3 ligase EEL-1/HUWE1 control hemidesmosome maturation in C. elegans development.

Hemidesmosomes connect the extracellular matrix (ECM) to intermediate filaments through ECM receptors and plakins (plectin and BPAG1e). They affect tissue integrity, wound healing, and carcinoma invasion. Although biochemical and time-lapse studies indicate that alpha6beta4-integrin (ECM receptor) and plectin play a central role in modulating hemidesmosome disassembly, the mechanisms controlling hemidesmosome biogenesis in vivo remain poorly understood. The nematode C. elegans provides a powerful genetic model to address this issue. We performed a genome-wide RNA interference screen in C. elegans, searching for genes that decrease the viability of a weak VAB-10A/plakin mutant. We identified 14 genes that have human homologs with predicted roles in different cellular processes. We further characterized two genes encoding the chaperone CRT-1/calreticulin and the HECT domain E3 ubiquitin ligase EEL-1/HUWE1. CRT-1 controls by as little as 2-fold the abundance of UNC-52/perlecan, an essential hemidesmosome ECM ligand. Likewise, EEL-1 fine tunes by 2-fold the abundance of myotactin, the putative hemidesmosome ECM receptor. CRT-1 and EEL-1 activities, and by extension other genes identified in our screen, are essential during embryonic development to enable hemidesmosomes exposed to mechanical tension to mature into a tension-resistant form. Our findings should help understand how hemidesmosome dynamics are regulated in vertebrate systems.

Molecular structure of the N-terminal domain of the APC/C subunit Cdc27 reveals a homo-dimeric tetratricopeptide repeat architecture

The anaphase promoting complex/cyclosome (APC/C) is a large multi-subunit E3 ubiquitin ligase that targets specific cell cycle regulatory proteins for ubiquitin-dependent degradation, thereby controlling cell cycle events such as the metaphase to anaphase transition and the exit from mitosis. Biochemical and genetic studies are consistent with the notion that subunits of APC/C are organised into two distinct sub-complexes; a catalytic sub-complex including the cullin domain and RING finger subunits Apc2 and Apc11, respectively, and a tetratricopeptide repeat (TPR) sub-complex composed of the TPR subunits Cdc16, Cdc23 and Cdc27 (Apc3). Here, we describe the crystal structure of the N-terminal domain of Encephalitozoon cuniculi Cdc27 (Cdc27(Nterm)), revealing a homo-dimeric structure, composed predominantly of successive TPR motifs. Mutation of the Cdc27(Nterm) dimer interface destabilises the protein, disrupts dimerisation in solution, and abolishes the capacity of E. cuniculi Cdc27 to complement Saccharomyces cerevisiae Cdc27 in vivo. These results establish the existence of functional APC/C genes in E. cuniculi, the evolutionarily conserved dimeric properties of Cdc27, and provide a framework for understanding the architecture of full-length Cdc27.

Antimicrosporidian activity of sulphated polysaccharides from algae and their potential to control honeybee nosemosis

Nosemosis is one of the most common and widespread diseases of adult honeybees. The causative agents, Nosema apis and Nosema ceranae, belong to microsporidia some obligate intracellular eukaryotic parasites. In this study, 10 sulphated polysaccharides from algae were evaluated for their antimicrosporidian activity. They were first shown to inhibit the in vitro growth of the mammal microsporidian model, Encephalitozoon cuniculi. The most efficient polysaccharides were then tested for their ability to inhibit the growth of Nosema ceranae in experimentally-infected adult honeybees. Two polysaccharides extracted from Porphyridium spp. did not show any toxicity in honeybees and one of them allowed a decrease of both parasite load and mortality rate due to N. ceranae infection. A decrease in parasite abundance but not in mortality rate was also observed with an iota carrageenan. Our results are promising and suggest that algal sulphated polysaccharides could be used to prevent and/or control bee nosemosis.