Use of Farnesyl Transferase Inhibitors in an Ageing Model in Drosophila.

The presence of farnesylated proteins at the inner nuclear membrane (INM), such as the Lamins or Kugelkern in Drosophila, leads to specific changes in the nuclear morphology and accelerated ageing on the organismal level reminiscent of the Hutchinson-Gilford progeria syndrome (HGPS). Farnesyl transferase inhibitors (FTIs) can suppress the phenotypes of the nuclear morphology in cultured fibroblasts from HGPS patients and cultured cells overexpressing farnesylated INM proteins. Similarly, FTIs have been reported to suppress the shortened lifespan in model organisms. Here, we report an experimental system combining cell culture and Drosophila flies for testing the activity of substances on the HGPS-like nuclear morphology and lifespan, with FTIs as an experimental example. Consistent with previous reports, we show that FTIs were able to ameliorate the nuclear phenotypes induced by the farnesylated nuclear proteins Progerin, Kugelkern, or truncated Lamin B in cultured cells. The subsequent validation in Drosophila lifespan assays demonstrated the applicability of the experimental system: treating adult Drosophila with the FTI ABT-100 reversed the nuclear phenotypes and extended the lifespan of experimentally induced short-lived flies. Since kugelkern-expressing flies have a significantly shorter average lifespan, half the time is needed for testing substances in the lifespan assay.

Long-term field-realistic exposure to a next-generation pesticide, flupyradifurone, impairs honey bee behaviour and survival.

The assessment of pesticide risks to insect pollinators have typically focused on short-term, lethal impacts. The environmental ramifications of many of the world's most commonly employed pesticides, such as those exhibiting systemic properties that can result in long-lasting exposure to insects, may thus be severely underestimated. Here, seven laboratories from Europe and North America performed a standardised experiment (a ring-test) to study the long-term lethal and sublethal impacts of the relatively recently approved 'bee safe' butenolide pesticide flupyradifurone (FPF, active ingredient in Sivanto®) on honey bees. The emerging contaminant, FPF, impaired bee survival and behaviour at field-realistic doses (down to 11 ng/bee/day, corresponding to 400 µg/kg) that were up to 101-fold lower than those reported by risk assessments (1110 ng/bee/day), despite an absence of time-reinforced toxicity. Our findings raise concerns about the chronic impact of pesticides on pollinators at a global scale and support a novel methodology for a refined risk assessment.

Honeybee colonies compensate for pesticide-induced effects on royal jelly composition and brood survival with increased brood production.

Sublethal doses of pesticides affect individual honeybees, but colony-level effects are less well understood and it is unclear how the two levels integrate. We studied the effect of the neonicotinoid pesticide clothianidin at field realistic concentrations on small colonies. We found that exposure to clothianidin affected worker jelly production of individual workers and created a strong dose-dependent increase in mortality of individual larvae, but strikingly the population size of capped brood remained stable. Thus, hives exhibited short-term resilience. Using a demographic matrix model, we found that the basis of resilience in dosed colonies was a substantive increase in brood initiation rate to compensate for increased brood mortality. However, computer simulation of full size colonies revealed that the increase in brood initiation led to severe reductions in colony reproduction (swarming) and long-term survival. This experiment reveals social regulatory mechanisms on colony-level that enable honeybees to partly compensate for effects on individual level.

Geographical Distribution and Selection of European Honey Bees Resistant to Varroa destructor.

Developing resistance to the varroa mite in honey bees is a major goal for apicultural science and practice, the development of selection strategies and the availability of resistant stock. Here we present an extended literature review and survey of resistant populations and selection programs in the EU and elsewhere, including expert interviews. We illustrate the practical experiences of scientists, beekeepers, and breeders in search of resistant bees. We describe numerous resistant populations surviving without acaricide treatments, most of which developed under natural infestation pressure. Their common characteristics: reduced brood development; limited mite population growth; and low mite reproduction, may cause conflict with the interests of commercial beekeeping. Since environmental factors affect varroa mite resistance, particular honey bee strains must be evaluated under different local conditions and colony management. The resistance traits of grooming, hygienic behavior and mite reproduction, together with simple testing of mite population development and colony survival, are significant in recent selection programs. Advanced breeding techniques and genetic and physiological selection tools will be essential in the future. Despite huge demand, there is no well-established market for resistant stock in Europe. Moreover, reliable experience or experimental evidence regarding the resistance of stocks under different environmental and management conditions is still lacking.

Immunosuppression response to the neonicotinoid insecticide thiacloprid in females and males of the red mason bee Osmia bicornis L.

Solitary bees are frequently exposed to pesticides, which are considered as one of the main stress factors that may lead to population declines. A strong immune defence is vital for the fitness of bees. However, the immune system can be weakened by environmental factors that may render bees more vulnerable to parasites and pathogens. Here we demonstrate for the first time that field-realistic concentrations of the commonly used neonicotinoid insecticide thiacloprid can severely affect the immunocompetence of Osmia bicornis. In detail, males exposed to thiacloprid solutions of 200 and 555 µg/kg showed a reduction in hemocyte density. Moreover, functional aspects of the immune defence - the antimicrobial activity of the hemolymph - were impaired in males. In females, however, only a concentration of 555 µg/kg elicited similar immunosuppressive effects. Although males are smaller than females, they consumed more food solution. This leads to a 2.77 times higher exposure in males, probably explaining the different concentration thresholds observed between the sexes. In contrast to honeybees, dietary exposure to thiacloprid did not affect melanisation or wound healing in O. bicornis. Our results demonstrate that neonicotinoid insecticides can negatively affect the immunocompetence of O. bicornis, possibly leading to an impaired disease resistance capacity.

Tribolium castaneum as a whole-animal screening system for the detection and characterization of neuroprotective substances.

Parkinson's disease (PD) is a movement disorder caused by the progressive loss of dopaminergic neurons. Natural antioxidants and plant extracts with neuroprotective properties offer a promising new therapeutic approach for PD patients, but a suitable large-scale screening system is required for their discovery and preclinical analysis. Here we used the red flour beetle (Tribolium castaneum ) as a whole-animal screening system for the detection and characterization of neuroprotective substances. Paraquat was added to the diet of adult beetles to induce PD-like symptoms, which were quantified using a novel positive geotaxis behavioral assay. These paraquat-induced behavioral changes were reduced in beetles fed on diets supplemented with l-dihydroxyphenylalanine, ascorbic acid, curcumin, hempseed flour, or the Chinese herb gou-teng. T. castaneum is, therefore, a valuable model for the screening of neuroprotective substances in chemical libraries and plant extracts and could be developed as a model for the preclinical testing of therapeutic candidates for the treatment of neurodegenerative diseases, such as PD.

A Comparison of Wolbachia Infection Frequencies in Varroa With Prevalence of Deformed Wing Virus.

Wolbachia are widely distributed bacterial endosymbionts of arthropods and filarial nematodes. These bacteria can affect host fitness in a variety of ways, such as protecting hosts against viruses and other pathogens. Here, we investigate the possible role of Wolbachia in the prevalence of the deformed wing virus (DWV), a highly virulent pathogen of honey bees (Apis mellifera) that is transmitted by parasitic Varroa mites (Varroa destructor). About 180 Varroa mites from 18 beehives were tested for infection with Wolbachia and DWV. We first screened for Wolbachia using two standard primers (wsp and 16S rDNA), and found 26% of the mites to be positive for Wolbachia using the wsp primer and 64% of the mites to be positive using the 16S rDNA primer. Using these intermediate Wolbachia frequencies, we then tested for statistical correlations with virus infection frequencies. The analysis revealed a significant positive correlation between DWV and Wolbachia using the wsp primer, but no significant association between DWV and Wolbachia using the 16S rDNA primer. In conclusion, there is no evidence for an anti-pathogenic effect of Wolbachia in V. destructor, but weak evidence for a pro-pathogenic effect. These results encourage further examination of Wolbachia-virus interactions in Varroa mites since an increased vector competence of the mites may significantly impact disease outbreaks in honey bees.

Immunosuppression in Honeybee Queens by the Neonicotinoids Thiacloprid and Clothianidin.

Queen health is crucial to colony survival of honeybees, since reproduction and colony growth rely solely on the queen. Queen failure is considered a relevant cause of colony losses, yet few data exist concerning effects of environmental stressors on queens. Here we demonstrate for the first time that exposure to field-realistic concentrations of neonicotinoid pesticides can severely affect the immunocompetence of queens of western honeybees (Apis mellifera L.). In young queens exposed to thiacloprid (200 µg/l or 2000 µg/l) or clothianidin (10 µg/l or 50 µg/l), the total hemocyte number and the proportion of active, differentiated hemocytes was significantly reduced. Moreover, functional aspects of the immune defence namely the wound healing/melanisation response, as well as the antimicrobial activity of the hemolymph were impaired. Our results demonstrate that neonicotinoid insecticides can negatively affect the immunocompetence of queens, possibly leading to an impaired disease resistance capacity.

Temporal dynamics of whole body residues of the neonicotinoid insecticide imidacloprid in live or dead honeybees.

In cases of acute intoxication, honeybees often lay in front of their hives for several days, exposed to sunlight and weather, before a beekeeper can take a sample. Beekeepers send samples to analytical laboratories, but sometimes no residues can be detected. Temperature and sun light could influence the decrease of pesticides in bee samples and thereby residues left for analysis. Moreover, samples are usually sent via normal postal services without cooling. We investigated the temporal dynamics of whole-body residues of imidacloprid in live or dead honeybees following a single-meal dietary exposure of 41 ng/bee under various environmental conditions, such as freezing, exposure to UV light or transfer of individuals through the mail system. Immobile, "dead" looking honeybees recovered from paralysis after 48 hours. The decrease of residues in living but paralysed bees was stopped by freezing (= killing). UV light significantly reduced residues, but the mode of transport did not affect residue levels. Group feeding increased the variance of residues, which is relevant for acute oral toxicity tests. In conclusion, elapsed time after poisoning is key for detection of neonicotinoids. Freezing before mailing significantly reduced the decrease of imidacloprid residues and may increase the accuracy of laboratory analysis for pesticides.

The neonicotinoids thiacloprid, imidacloprid, and clothianidin affect the immunocompetence of honey bees (Apis mellifera L.).

A strong immune defense is vital for honey bee health and colony survival. This defense can be weakened by environmental factors that may render honey bees more vulnerable to parasites and pathogens. Honey bees are frequently exposed to neonicotinoid pesticides, which are being discussed as one of the stress factors that may lead to colony failure. We investigated the sublethal effects of the neonicotinoids thiacloprid, imidacloprid, and clothianidin on individual immunity, by studying three major aspects of immunocompetence in worker bees: total hemocyte number, encapsulation response, and antimicrobial activity of the hemolymph. In laboratory experiments, we found a strong impact of all three neonicotinoids. Thiacloprid (24h oral exposure, 200 µg/l or 2000 µg/l) and imidacloprid (1 µg/l or 10 µg/l) reduced hemocyte density, encapsulation response, and antimicrobial activity even at field realistic concentrations. Clothianidin had an effect on these immune parameters only at higher than field realistic concentrations (50-200 µg/l). These results suggest that neonicotinoids affect the individual immunocompetence of honey bees, possibly leading to an impaired disease resistance capacity.

The Fruit Fly Drosophila melanogaster as a Model for Aging Research.

: Average human life expectancy is increasing and so is the impact on society of aging and age-related diseases. Here we highlight recent advances in the diverse and multidisciplinary field of aging research, focusing on the fruit fly Drosophila melanogaster, an excellent model system in which to dissect the genetic and molecular basis of the aging processes. The conservation of human disease genes in D. melanogaster allows the functional analysis of orthologues implicated in human aging and age-related diseases. D. melanogaster models have been developed for a variety of age-related processes and disorders, including stem cell decline, Alzheimer's disease, and cardiovascular deterioration. Understanding the detailed molecular events involved in normal aging and age-related diseases could facilitate the development of strategies and treatments that reduce their impact, thus improving human health and increasing longevity.

The farnesylated nuclear proteins KUGELKERN and LAMIN B promote aging-like phenotypes in Drosophila flies.

The nuclear lamina consists of a meshwork of lamins and lamina-associated proteins, which provide mechanical support, control size and shape of the nucleus, and mediate the attachment of chromatin to the nuclear envelope. Abnormal nuclear shapes are observed in aging cells of humans and nematode worms. The expression of laminDelta50, a constitutively active lamin A splicing variant in Hutchinson-Gilford progeria syndrome patients, leads to the lobulation of the nuclear envelope accompanied by DNA damage, and loss of heterochromatin. So far, it has been unclear whether these age-related changes are laminDelta50 specific or whether proteins that affect nuclear shape such as KUGELKERN or LAMIN B in general play a causative role in senescence. Here we show that in adult Drosophila flies, the size of the nuclei increases with age and the nuclei assume an aberrant shape. Moreover, induced expression of the farnesylated lamina proteins Lamin B and Kugelkern cause aberrant nuclear shapes and reduce the lifespan of adult flies. The shorter lifespan correlates with an early decline in age-dependent locomotor behaviour. Expression of kugelkern or lamin B in mammalian cells induces a nuclear lobulation phenotype in conjunction with DNA damage, and changes in histone modification similar to that found in cells expressing laminDelta50 or in cells from aged individuals. We conclude that lobulation of the nuclear membrane induced by the insertion of farnesylated lamina-proteins can lead to aging-like phenotypes.

Developmental control of nuclear size and shape by Kugelkern and Kurzkern.

BACKGROUND: The shape of a nucleus depends on the nuclear lamina, which is tightly associated with the inner nuclear membrane and on the interaction with the cytoskeleton. However, the mechanism connecting the differentiation state of a cell to the shape changes of its nucleus are not well understood. We investigated this question in early Drosophila embryos, where the nuclear shape changes from spherical to ellipsoidal together with a 2.5-fold increase in nuclear length during cellularization. RESULTS: We identified two genes, kugelkern and kurzkern, required for nuclear elongation. In kugelkern- and kurzkern-depleted embryos, the nuclei reach only half the length of the wild-type nuclei at the end of cellularization. The reduced nuclear size affects chromocenter formation as marked by Heterochromatin protein 1 and expression of a specific set of genes, including early zygotic genes. kugelkern contains a putative coiled-coil domain in the N-terminal half of the protein, a nuclear localization signal (NLS), and a C-terminal CxxM-motif. The carboxyterminal CxxM motif is required for the targeting of Kugelkern to the inner nuclear membrane, where it colocalizes with lamins. Depending on the farnesylation motif, expression of kugelkern in Drosophila embryos or Xenopus cells induces overproliferation of nuclear membrane. CONCLUSIONS: Kugelkern is so far the first nuclear protein, except for lamins, that contains a farnesylation site. Our findings suggest that Kugelkern is a rate-determining factor for nuclear size increase. We propose that association of farnesylated Kugelkern with the inner nuclear membrane induces expansion of nuclear surface area, allowing nuclear growth.