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Rapid cryopreservation of biological specimens is the gold standard for visualizing cellular structures in their true structural context. However, current commercial cryo-fluorescence microscopes are limited to low resolutions. To fill this gap, we have developed cryoSIM, a microscope for 3D super-resolution fluorescence cryo-imaging for correlation with cryo-electron microscopy or cryo-soft X-ray tomography. We provide the full instructions for replicating the instrument mostly from off-the-shelf components and accessible, user-friendly, open-source Python control software. Therefore, cryoSIM democratizes the ability to detect molecules using super-resolution fluorescence imaging of cryopreserved specimens for correlation with their cellular ultrastructure. Funding: Wellcome Trust (091911/Z/11/Z, 096144/Z/11/Z, 105605/Z/14/Z, 107457/Z/15/Z, 203141/Z/16/Z, 209412/Z/17/Z); H2020Marie Skłodowska-Curie Actions (700184).

SummaryWhile post-transcriptional control is thought to be required at the periphery of neurons and glia, its extent is unclear. Here, we investigate systematically the spatial distribution and expression of mRNA at single molecule sensitivity and their corresponding proteins of 200 YFP trap protein trap lines across the intact Drosophila nervous system. 98% of the genes studied showed discordance between the distribution of mRNA and the proteins they encode in at least one region of the nervous system. These data suggest that post-transcriptional regulation is very common, helping to explain the complexity of the nervous system. We also discovered that 68.5% of these genes have transcripts present at the periphery of neurons, with 9.5% at the glial periphery. Peripheral transcripts include many potential new regulators of neurons, glia and their interactions. Our approach is applicable to most genes and tissues and includes powerful novel data annotation and visualisation tools for post-transcriptional regulation.Brief outlineA novel high resolution and sensitive approach to systematically co-visualise the distribution of mRNAs and proteins in the intact nervous system reveals that post-transcriptional regulation of gene expression is very common. The rich data landscape is provided as a browsable resource (link), using Zegami, a cloud-based data exploration platform (link). Our solution provides a paradigm for the characterisation of post-transcriptional regulation of most genes and model systems.Highlights196/200 (98%) Drosophila genes show discordant RNA and protein expression in at least one nervous system region137/200 (68.5%) mRNAs are present in at least one synaptic compartmentNovel localised mRNA and protein discovered in periphery of glial processesNew paradigm for analysis of post-transcriptional regulation and data exploration

The impact of the cell community on its individual components in the prokaryotic realm is often overlooked. However, in the case of intracellular pathogens, where each infected cell can be considered as a single community, understanding how a population adapts to its environment to evolve and successfully propagate is key. Chlamydia infections are characterised by a silent propagation of the bacteria within individual hosts and the wider population. Chlamydia are strict intracellular pathogens residing within a specialised membrane-bound compartment called the inclusion. The life cycle of Chlamydia involves altering between the invasive elementary bodies (EBs) and replicative reticulate bodies (RBs). We have used cryo-soft X-ray tomography to observe individual inclusions, combining excellent resolution (40 nm) and large volume imaging (up to 16 µm). Combined with a semi-automated segmentation pipeline, we were able to consider each inclusion as an individual bacterial niche. Within the inclusion, we identified and classified different forms of the bacteria and confirmed the recent finding that RBs have a variety of volumes (small, large and abnormal). Moreover, we demonstrate that the proportions of these different RB forms depend on the bacterial concentration in the cell demonstrating the impact of the group on its individual component. We conclude that each inclusion operates as an autonomous community which regulates the characteristics of individual bacteria within the inclusion

Three-dimensional (3D) structured illumination microscopy (SIM) allows imaging of fluorescently labelled cellular structures at higher resolution than conventional fluorescence microscopy. This super-resolution (SR) technique enables visualization of molecular processes in whole cells and has the potential to be used in conjunction with electron microscopy and X-ray tomography to correlate structural and functional information. A SIM microscope for cryogenically preserved samples (cryoSIM) has recently been commissioned at the correlative cryo-imaging beamline B24 at the UK synchrotron. It was designed specifically for 3D imaging of biological samples at cryogenic temperatures in a manner compatible with subsequent imaging of the same samples by X-ray microscopy methods such as cryo-soft X-ray tomography. This video article provides detailed methods and protocols for successful imaging using the cryoSIM. In addition to instructions on the operation of the cryoSIM microscope, recommendations have been included regarding the choice of samples, fluorophores, and parameter settings. The protocol is demonstrated in U2OS cell samples whose mitochondria and tubulin have been fluorescently labelled.

A 3D cellular imaging platform developed at beamline B24 at Diamond Light Source has been used to identify cellular features such as filamentous actin in mammalian cells. This approach enabled virtual same-sample imaging of structures captured on separate microscopes through rigid transformation of 3D data in silico, bypassing the need for additional sample processing and ensuring artefact-free data correlation., Imaging of actin filaments is crucial due to the integral role that they play in many cellular functions such as intracellular transport, membrane remodelling and cell motility. Visualizing actin filaments has so far relied on fluorescence microscopy and electron microscopy/tomography. The former lacks the capacity to capture the overall local ultrastructure, while the latter requires rigorous sample preparation that can lead to potential artefacts, and only delivers relatively small volumes of imaging data at the thinnest areas of a cell. In this work, a correlative approach utilizing in situ super-resolution fluorescence imaging and cryo X-ray tomography was used to image bundles of actin filaments deep inside cells under near-native conditions. In this case, fluorescence 3D imaging localized the actin bundles within the intracellular space, while X-ray tomograms of the same areas provided detailed views of the local ultrastructure. Using this new approach, actin trails connecting vesicles in the perinuclear area and hotspots of actin presence within and around multivesicular bodies were observed. The characteristic prevalence of filamentous actin in cytoplasmic extensions was also documented.

Imaging techniques are fundamental in order to understand cell organization and machinery in biological research and the related fields. Among these techniques, cryo soft X-ray tomography (SXT) allows imaging whole cryo-preserved cells in the water window X-ray energy range (284-543 eV), in which carbon structures have intrinsically higher absorption than water, allowing the 3D reconstruction of the linear absorption coefficient of the material contained in each voxel. Quantitative structural information at the level of whole cells up to 10 µm thick is then achievable this way, with high throughput and spatial resolution down to 25-30 nm half-pitch. Cryo-SXT has proven itself relevant to current biomedical research, providing 3D information on cellular infection processes (virus, bacteria, or parasites), morphological changes due to diseases (such as recessive genetic diseases) and helping us understand drug action at the cellular level, or locating specific structures in the 3D cellular environment. In addition, by taking advantage of the tunable wavelength at synchrotron facilities, spectro-microscopy or its 3D counterpart, spectro-tomography, can also be used to image and quantify specific elements in the cell, such as calcium in biomineralization processes. Cryo-SXT provides complementary information to other biological imaging techniques such as electron microscopy, X-ray fluorescence or visible light fluorescence, and is generally used as a partner method for 2D or 3D correlative imaging at cryogenic conditions in order to link function, location, and morphology.

3D correlative microscopy methods have revolutionized biomedical research, allowing the acquisition of multidimensional information to gain an in-depth understanding of biological systems. With the advent of relevant cryo-preservation methods, correlative imaging of cryogenically preserved samples has led to nanometer resolution imaging (2-50 nm) under harsh imaging regimes such as electron and soft X-ray tomography. These methods have now been combined with conventional and super-resolution fluorescence imaging at cryogenic temperatures to augment information content from a given sample, resulting in the immediate requirement for protocols that facilitate hassle-free, unambiguous cross-correlation between microscopes. We present here sample preparation strategies and a direct comparison of different working fiducialization regimes that facilitate 3D correlation of cryo-structured illumination microscopy and cryo-soft X-ray tomography. Our protocol has been tested at two synchrotron beamlines (B24 at Diamond Light Source in the UK and BL09 Mistral at ALBA in Spain) and has led to the development of a decision aid that facilitates experimental design with the strategic use of markers based on project requirements. This protocol takes between 1.5 h and 3.5 d to complete, depending on the cell populations used (adherent cells may require several days to grow on sample carriers).

SummarySince the outbreak of the SARS-CoV-2 pandemic, there have been intense structural studies on purified recombinant viral components and inactivated viruses. However, investigation of the SARS-CoV-2 infection in the native cellular context is scarce, and there is a lack of comprehensive knowledge on SARS-CoV-2 replicative cycle. Understanding the genome replication, assembly and egress of SARS-CoV-2, a multistage process that involves different cellular compartments and the activity of many viral and cellular proteins, is critically important as it bears the means of medical intervention to stop infection. Here, we investigated SARS-CoV-2 replication in Vero cells under the near-native frozen-hydrated condition using a unique correlative multi-modal, multi-scale cryo-imaging approach combining soft X-ray cryo-tomography and serial cryoFIB/SEM volume imaging of the entire SARS-CoV-2 infected cell with cryo-electron tomography (cryoET) of cellular lamellae and cell periphery, as well as structure determination of viral components by subtomogram averaging. Our results reveal at the whole cell level profound cytopathic effects of SARS-CoV-2 infection, exemplified by a large amount of heterogeneous vesicles in the cytoplasm for RNA synthesis and virus assembly, formation of membrane tunnels through which viruses exit, and drastic cytoplasm invasion into nucleus. Furthermore, cryoET of cell lamellae reveals how viral RNAs are transported from double-membrane vesicles where they are synthesized to viral assembly sites; how viral spikes and RNPs assist in virus assembly and budding; and how fully assembled virus particles exit the cell, thus stablishing a model of SARS-CoV-2 genome replication, virus assembly and egress pathways.

Introduction: Hypoxia inducible factors (HIF) and prolyl hydroxylase domain (PHD) enzymes play a central role in tumor progression in clear cell renal cell carcinoma (ccRCC). However, there are currently no data regarding the behavior of this pathway (HIF/PHD) in a large number of benign renal tumors, the oncocytomas. The aim of the present study was to compare the expression levels of these factors between ccRCC and oncocytoma tumors. Material and methods: A total of 56 fresh frozen specimens from patients with ccRCC and 14 oncocytoma specimens were analyzed via reverse transcription-quantitative polymerase chain reaction in order to assess the expression levels of HIF-1α, HIF-2α, PHD1, PHD2, and PHD3. The analysis involved both fresh frozen tumor samples as well as adjacent normal kidney tissues. Results: In ccRCC, HIF-1α and HIF-2α levels were upregulated in 65.5% and 71.4% of cases, respectively. PHD3 was downregulated only in 15.4% of the ccRCC cases, in contrast with oncocytoma cases, which exhibited low expression levels in the majority. The upregulation of PHD3 messenger RNA (mRNA) levels in ccRCC when compared with oncocytoma was statistically significant ( PConclusion: To the best of our knowledge, this is the first time that the HIF/PHD pathway was compared between ccRCC and a common benign tumor, identifying the upregulation of PHD3 as the possible underlying factor guiding the difference in the behavior of ccRCC.

3D correlative microscopy methods have revolutionized biomedical research, allowing the acquisition of multidimensional information to gain an in-depth understanding of biological systems. With the advent of relevant cryo-preservation methods, correlative imaging of cryogenically preserved samples has led to nanometer resolution imaging (2-50 nm) under harsh imaging regimes such as electron and soft X-ray tomography. These methods have now been combined with conventional and super-resolution fluorescence imaging at cryogenic temperatures to augment information content from a given sample, resulting in the immediate requirement for protocols that facilitate hassle-free, unambiguous cross-correlation between microscopes. We present here sample preparation strategies and a direct comparison of different working fiducialization regimes that facilitate 3D correlation of cryo-structured illumination microscopy and cryo-soft X-ray tomography. Our protocol has been tested at two synchrotron beamlines (B24 at Diamond Light Source in the UK and BL09 Mistral at ALBA in Spain) and has led to the development of a decision aid that facilitates experimental design with the strategic use of markers based on project requirements. This protocol takes between 1.5 h and 3.5 d to complete, depending on the cell populations used (adherent cells may require several days to grow on sample carriers).

The aim of the present study was to evaluate the relative mRNA expression levels of genes involved in the hypoxia inducible factor (HIF) signalling pathway in renal cell carcinoma (RCC) and to analyse their associations with clinicopathological parameters and survival outcomes. Reverse transcription-quantitative PCR was used to quantify the mRNA expression levels of HIF-1α, HIF-2α, prolyl hydroxylase (PHD)1, PHD2 and PHD3 in formalin-fixed paraffin-embedded (FFPE) tumour tissue samples from 41 patients with RCC, including 33 cases of clear cell RCC (ccRCC). FFPE samples of corresponding adjacent normal kidney tissues were used as a comparison. mRNA expression levels were analysed in regard to clinical parameters, histological type, stage, nuclear grade, cancer specific survival and overall survival. Compared with adjacent normal kidney tissue, HIF-1α levels were lower in 16/33 ccRCC samples (48.48%), while HIF-2α, PHD1 and PHD2 levels did not exhibit a specific expression pattern. By contrast, the PHD3 mRNA level was higher in 29/33 (87.87%) of the tumour samples. HIF-1α was positively associated with HIF-2α, PHD1 and PHD2. HIF-2α levels were associated with PHD1, PHD2 and PHD3, while PHD3 was strongly associated with PHD2. PHD3 mRNA levels were inversely associated with nuclear grade (P=0.015). However, in univariate analysis, PHD3 was not associated with cancer-specific or overall survival rates. The present findings suggest an important involvement of PHD3 in ccRCC, since PHD3 mRNA expression was inversely associated with nuclear grade. However, PHD3 mRNA levels did not have an independent prognostic value. Further studies are required to investigate whether PHD3 could be used as either a therapeutic target or prognostic marker.

Summary Imaging of biological matter across resolution scales entails the challenge of preserving the direct and unambiguous correlation of subject features from the macroscopic to the microscopic level. Here, we present a correlative imaging platform developed specifically for imaging cells in 3D under cryogenic conditions by using X-rays and visible light. Rapid cryo-preservation of biological specimens is the current gold standard in sample preparation for ultrastructural analysis in X-ray imaging. However, cryogenic fluorescence localization methods are, in their majority, diffraction-limited and fail to deliver matching resolution. We addressed this technological gap by developing an integrated, user-friendly platform for 3D correlative imaging of cells in vitreous ice by using super-resolution structured illumination microscopy in conjunction with soft X-ray tomography. The power of this approach is demonstrated by studying the process of reovirus release from intracellular vesicles during the early stages of infection and identifying intracellular virus-induced structures., Graphical Abstract, Highlights • Correlative cryo-imaging used to study biological processes in vitrified cells • Reovirus intracellular release mechanism is elucidated in 3D with combined cryo-imaging • Reovirus core particles start modifying carrier endosomes at 1 h after infection • Correlation of X-ray and fluorescence cryo-imaging requires no data deformation, Kounatidis et al. describe a correlative microscopy platform that combines cryo-structured illumination fluorescence imaging with soft X-ray absorption imaging on the same sample allowing direct data correlation in 3D. The methods are used to track the intracellular progress of reovirus in human cells in the early stages of infection.

The genusRhagoletis(Diptera: Tephritidae) comprises more than 65 species distributed throughout Europe, Asia and America, including many species of high economic importance. Currently, there are threeRhagoletisspecies that infest fruits and nuts in Europe. The European cherry fruit fly,Rhagoletis cerasi(may have invaded Europe a long time ago from the Caucasian area of West Asia), and two invasive species (recently introduced from North America): the eastern American cherry fruit fly,R. cingulata, and the walnut husk fly,R. completa. The presence of differentRhagoletisspecies may enhance population dynamics and establish an unpredictable economic risk for several fruit and nut crops in Europe. Despite their excessive economic importance, little is known on population dynamics, genetics and symbiotic associations for making sound pest control decisions in terms of species-specific, environmental friendly pest control methods. To this end, the current paper (a) summarizes recently accumulated genetic and population data for the EuropeanRhagoletisspecies and their association with the endosymbiontWolbachia pipientis, and (b) explores the possibility of using the current knowledge for implementing the innovative biological control methods of sterile insect technique and incompatible insect technique.

Imaging of biological matter across resolution scales presents the challenge of preserving the direct and unambiguous correlation of subject features from the macroscopic to the microscopic level. We present here a correlative imaging platform developed specifically for imaging cells in 3D, under cryogenic conditions. Rapid cryo-preservation of biological specimens is the current gold standard in sample preparation for ultrastructural analysis in X-ray imaging. However, cryogenic fluorescence localisation methods are by and large diffraction-limited and fail to deliver matching resolution. We addressed this technological gap by developing an integrated, user-friendly, platform for 3D correlative imaging of cells in cryo-preserved states using super-resolution structured illumination microscopy (SIM) in conjunction with soft X-ray tomography (SXT). The power of this new approach is demonstrated by studying the process of reovirus release from intracellular vesicles during the early stages of infection and identifying novel virus-induced structures.

Rapid cryo-preservation of biological specimens is the gold standard for visualising cellular structures in their true structural context. However, current commercial cryo-fluorescence microscopes are limited to low resolutions. To fill this gap, we have developed cryoSIM, a microscope for 3D super-resolution fluorescence cryo-imaging for correlation with cryo electron microscopy or cryo soft X-ray tomography. We provide the full instructions for replicating the instrument mostly from off-the-shelf components and accessible, user-friendly open source Python control software. Therefore, cryoSIM democratises the ability to detect molecules using super-resolution fluorescence imaging of cryo-preserved specimens for correlation with their cellular ultrastructure.

Candida glabrata (C. glabrata) forms part of the normal human gut microbiota but can cause life-threatening invasive infections in immune-compromised individuals.C. glabratadisplays high resistance to common azole antifungals, which necessitates new treatments. In this investigation, we identified fiveC. glabratadeletion mutants (Δada2, Δbas1, Δhir3,Δino2andΔmet31) from a library of 196 transcription factor mutants that were unable to grow and activate an immune response inDrosophilalarvae. This highlighted the importance of these transcription factors inC. glabratainfectivity. Furtherex vivoinvestigation into these mutants revealed the requirement ofC. glabrata ADA2for oxidative stress tolerance. We confirmed this observationin vivowhereby growth of theC. glabrata Δada2strain was permitted only in flies with suppressed production of reactive oxygen species (ROS). Conversely, overexpression ofADA2promotedC. glabratareplication in infected wild type larvae resulting in larval killing. We propose thatADA2orchestrates the response ofC. glabrataagainst ROS-mediated immune defences during infection. With the need to find alternative antifungal treatment forC. glabratainfections, genes required for survival in the host environment, such asADA2, provide promising potential targets.

Gastrointestinal infection can provoke substantial disturbance at both a local as well as at a systemic level and may evolve into a chronic disease state. Our growing knowledge of gut-pathogen interactions has been based to a large extent on the use of genetically tractable model hosts such as the fruit fly Drosophila melanogaster. In this review we will summarise the growing literature and critically address the advantages and disadvantages of using this model to extrapolate results from studying pathogen virulence and intestinal responses to humans.

Area-wide integrated pest management requires an understanding of insect population dynamics and definition of suitable techniques to quantify spatio-temporal variability to make better pest management decisions. However, the viability of area-wide integrated pest management has often been questioned because of the high monitoring costs. The present study aimed to: (i) analyse the spatial and temporal dynamics of the olive fruit fly over a large olive growing area (Ormylia, Greece), and (ii) define a methodology to determine monitoring zones to optimize the monitoring effort over space and time in area-wide integrated pest management programmes. Data from an olive fruit fly monitoring network based on McPhail traps were utilized. The multi-variate spatial (elevation) and temporal (6 periods) data of olive fruit fly population density were analysed by principal component analysis, co-kriging and factor kriging to produce thematic maps and to delineate monitoring zones. Olive fruit fly density was spatially correlated from 200 to 4 000 m. The spatial pattern changed over the monitoring season. Areas with high density of olive fruit flies shifted from high altitudes in summer to lower altitudes towards autumn. Three recommended levels of monitoring intensity were defined, thus delineating homogeneous monitoring zones for summer (July to September) and October. It was concluded that delineating monitoring zones through multi-variate geostatistics is a suitable approach for optimising the monitoring effort, because population density distribution is spatially structured over large areas and changes over time.

The American eastern cherry fruit fly, Rhagoletis cingulata, a pest of cherries in the western hemisphere, invaded Europe in 1983, and since then dispersed to several European countries. Information on the genetics and cytogenetics of this pest is very scarce. The mitotic karyotype and detailed photographic maps of the salivary gland polytene chromosomes of R. cingulata are presented here. The mitotic metaphase complement consists of six pairs of chromosomes with the sex chromosomes being very small and similar in size. The analysis of the salivary gland polytene complement shows a total number of five long chromosomes (10 polytene arms), which correspond to the five autosomes of the mitotic nuclei and an extrachromosomal heterochromatic mass, which corresponds to the sex chromosomes. The banding patterns and the most characteristic features and prominent landmarks of each polytene chromosome are presented and discussed. Chromosomal homologies between R. cingulata, R. completa and R. cerasi are also proposed, based on the comparison of chromosome banding patterns. Furthermore, the detection and characterization of Wolbachia pipientis in the R. cingulata population studied is presented and the potential correlation with the asynaptic phenomena found in its polytene complement is discussed. In addition, 10 out of 24 microsatellite markers developed for other Rhagoletis species are cross-amplified, evaluated and proposed as useful markers for population and genetic studies in R. cingulata.

The genotoxicity of 10 essential oil constituents was evaluated using the Drosophila melanogaster (Meigen) somatic mutation and recombination test, also known as the wing spot test, in the frame of a screening project aiming at evaluating the mutagenic activity of widely used substances, natural or not. Of the compounds that we tested here, l-carveol, dihydrocarveol, (+)-dihydrocarvone, (−)-fenchone and (−)-carvyl acetate did not exhibit any mutagenic or recombinogenic activity, whereas (±)-linalool, S-(+)-carvone and S-(−)-limonene gave inconclusive results. In contrast, α-phellandrene and R-(−)-carvone significantly increased the frequency of mutant spots when compared with the negative control, suggesting mutagenic activity even at the lowest concentration used (1.5 µl/ml). Moreover, these data clearly demonstrate differences in activity between stereoisomers such as S-(+)- and R-(−)-carvone. Given that α-phellandrene and R-(−)-carvone are widely used compounds, further research is needed in order to have a better understanding of their activity and a clearer picture of their genotoxicity in order to decide whether they should remain or not in the lists of compounds that are safe to use. Copyright © 2011 John Wiley & Sons, Ltd.

BACKGROUND: The insecticides spinosad and deltamethrin are being increasingly used in pest management programmes. In order to assess further their toxic effects to target and non-target insect species, an evaluation was made of their insecticidal profile on Bactrocera oleae (Rossi) and Drosophila melanogaster (Meig.). Moreover, possible genotoxic effects of the two pesticides were investigated using the somatic mutation and recombination test (SMART) in D.melanogaster. RESULTS: Both insecticides were highly effective against B. oleae, exhibiting similar LC50 values. Moreover, they were found to be more effective against Bactrocera than against Drosophila adults. However, spinosad was significantly more toxic than deltamethrin to D. melanogaster. The results showed a lack of genotoxic activity of both insecticides under the invivo experimental procedure employed, at least at applied doses. CONCLUSION: The present study provides information for lethal and sublethal effects of spinosad and deltamethrin against a target and a non-target species. Both insecticides can exert high toxicity to B. oleae when adults are exposed even to very low doses for long periods of time. The results contribute to the database on the genotoxic potential of spinosad and deltamethrin, suggesting a safety profile for both insecticides. c � 2011 Society of Chemical Industry

The Ethiopian fruit fly, Dacus ciliatus, is an important pest of cucurbits, which recently invaded the Middle East. The genetics and cytogenetics of D. ciliatus have been scarcely studied. Such information is, however, an essential basis for understanding the biology of insect pests, as well as for the design of modern control strategies. We report here the mitotic karyotype and detailed photographic maps of the salivary gland polytene chromosomes of this species. The mitotic metaphase complement consists of six pairs of chromosomes, including one pair of heteromorphic sex (XX/XY) chromosomes. The heterogametic sex is ascribed to the male. The analysis of the salivary gland polytene complement shows a total number of five long chromosomes (10 polytene arms), which correspond to the five autosomes of the mitotic nuclei, and a heterochromatic mass corresponding to the sex chromosomes. Banding patterns, as well as the most characteristic features and prominent landmarks of each polytene chromosome are presented and discussed. Chromosomal homologies between D. ciliatus and Bactrocera oleae are proposed by comparing chromosome banding patterns and by in situ hybridization of the hsp70 gene.

Genetic and cytogenetic information is an essential basis for understanding the biology of insect pests, as well as for designing modern control strategies. The walnut husk fly, Rhagoletis completa (Cresson) (Diptera: Tephritidae), is an important pest of walnuts (Juglans spp.) in North America and has invaded Europe in the early 1990s. Studies on the genetics and cytogenetics of R. completa are scarce. The mitotic karyotype and detailed photographic maps of the salivary gland polytene chromosomes of this pest species are presented here. The mitotic metaphase complement consists of six pairs of chromosomes, the sex chromosomes being very small and similar in size. The analysis of the salivary gland polytene complement shows a total number of five long chromosomes (10 polytene arms) that correspond to the five autosomes of the mitotic nuclei and a heterochromatic mass corresponding to the sex chromosomes. The banding pattern as well as the most characteristic features and prominent landmarks of each polytene chromosome are presented and discussed.

The spatio-temporal patterns of olive fly populations in a managed olive-growing region in the area of Chalkidiki in northern Greece were followed during 2005. The trapping grid consisted of 700 traps. Throughout the growing season (July‐October), McPhail traps loaded with ammonium sulphate were inspected at 5-day intervals. Trapping data were analysed using Getis‐Ord local spatial statistics. Clustering of trapped flies was significantly related to elevation, which ranged from sea level to 700 m above sea level. The effect of elevation upon clustering depended on seasonal climatic patterns. During the summer, ‘hotspots’ (i.e. significant aggregations of captured insects) were located at intermediate to high elevations and ‘cold-spots’ (i.e. areas with significantly low numbers of captured insects) were found in the valleys. In contrast, during the fall, ‘hot-spots’ were detected at lower elevations and ‘cold-spots’ at higher elevations. Population phenology seemed to affect spatial patterns. During periods of low population levels across the entire region, clusters of traps with relatively high amounts of captured insects were found at lower elevations. Pest management activities may have affected population levels throughout the region, but our data set does not allow quantifying their effectiveness. Our data suggest that differences in environmental temperature, as a result of altitudinal cline and season, may affect both the size of the olive fly population and its spatial patterns. The implementation of spatio-temporal analyses for management of the olive fly is discussed. J. Appl. Entomol.

The European cherry fruit fly, Rhagoletis cerasi , is a major agricultural pest for which biological, genetic, and cytogenetic information is limited. We report here a cytogenetic analysis of 4 natural Greek populations of R. cerasi, all of them infected with the endosymbiotic bacterium Wolbachia pipientis . The mitotic karyotype and detailed photographic maps of the salivary gland polytene chromosomes of this pest species are presented here. The mitotic metaphase complement consists of 6 pairs of chromosomes, including one pair of heteromorphic sex chromosomes, with the male being the heterogametic sex. The analysis of the salivary gland polytene complement has shown a total of 5 long chromosomes (10 polytene arms) that correspond to the 5 autosomes of the mitotic nuclei and a heterochromatic mass corresponding to the sex chromosomes. The most prominent landmarks of each polytene chromosome, the “weak points”, and the unusual asynapsis of homologous pairs of polytene chromosomes at certain regions of the polytene elements are also presented and discussed.

A relatively unexplored nexus in Drosophila Immune deficiency (IMD) pathway is TGF-beta Activating Kinase 1 (TAK1), which triggers both immunity and apoptosis. In a cell culture screen, we identified that Lysine at position 142 was a K63-linked Ubiquitin acceptor site for TAK1, required for signalling. Moreover, Lysine at position 156 functioned as a K48-linked Ubiquitin acceptor site, also necessary for TAK1 activity. The deubiquitinase Trabid interacted with TAK1, reducing immune signalling output and K63-linked ubiquitination. The three tandem Npl4 Zinc Fingers and the catalytic Cysteine at position 518 were required for Trabid activity. Flies deficient for Trabid had a reduced life span due to chronic activation of IMD both systemically as well as in their gut where homeostasis was disrupted. The TAK1-associated Binding Protein 2 (TAB2) was linked with the TAK1-Trabid interaction through its Zinc finger domain that pacified the TAK1 signal. These results indicate an elaborate and multi-tiered mechanism for regulating TAK1 activity and modulating its immune signal., Author Summary Chronic activation of immune responses results in health problems including gastrointestinal infections, metabolic imbalances and inflammatory bowel diseases that may lead to colorectal cancer. Central to this, is the balance of activation/restriction of nuclear factor-κB (NF-κB) during innate immune responses. To study signaling through NF-κB, we use the fruit fly Drosophila melanogaster as a genetically tractable model system that reflects human biology (due to the evolutionary conservation between innate immunity in flies and mammals), while reducing the complexity of the human disease of interest. We have found a new negative regulator of the Drosophila NF-κB pathway named Trabid. Its loss released the pathway and resulted in constitutive immune activation both in the gut as well as in the whole fly. This spontaneous immune activation reduced life span in the absence of infection, especially when it was combined with loss of another known negative regulator of the same pathway, a protein named Pirk. Stem cell activity in the gut in a pirk;trabid double mutant was found to be significantly increased, as the gut was trying to balance enterocyte loss. Trabid was acting at the level of TGF-beta Activating Kinase 1 (TAK1), which triggers both immunity and cell death.

Drosophila has a primitive yet effective blood system with three types of haemocytes which function throughout different developmental stages and environmental stimuli. Haemocytes play essential roles in tissue modeling during embryogenesis and morphogenesis, and also in innate immunity. The open circulatory system of Drosophila makes haemocytes ideal signal mediators to cells and tissues in response to events such as infection and wounding. The application of recently developed and sophisticated genetic tools to the relatively simple genome of Drosophila has made the fly a popular system for modeling human tumorigensis and metastasis. Drosophila is now used for screening and investigation of genes implicated in human leukemia and also in modeling development of solid tumors. This second line of research offers promising opportunities to determine the seemingly conflicting roles of blood cells in tumor progression and invasion. This review provides an overview of the signaling pathways conserved in Drosophila during haematopoiesis, haemostasis, innate immunity, wound healing and inflammation. We also review the most recent progress in the use of Drosophila as a cancer research model with an emphasis on the roles haemocytes can play in various cancer models and in the links between inflammation and cancer.

The insecticides spinosad and deltamethrin are being increasingly used in pest management programmes. In order to assess further their toxic effects to target and non-target insect species, an evaluation was made of their insecticidal profile on Bactrocera oleae (Rossi) and Drosophila melanogaster (Meig.). Moreover, possible genotoxic effects of the two pesticides were investigated using the somatic mutation and recombination test (SMART) in D. melanogaster.Both insecticides were highly effective against B. oleae, exhibiting similar LC(50) values. Moreover, they were found to be more effective against Bactrocera than against Drosophila adults. However, spinosad was significantly more toxic than deltamethrin to D. melanogaster. The results showed a lack of genotoxic activity of both insecticides under the in vivo experimental procedure employed, at least at applied doses.The present study provides information for lethal and sublethal effects of spinosad and deltamethrin against a target and a non-target species. Both insecticides can exert high toxicity to B. oleae when adults are exposed even to very low doses for long periods of time. The results contribute to the database on the genotoxic potential of spinosad and deltamethrin, suggesting a safety profile for both insecticides.

Edible and nonedible grades of virgin olive oil (VOO), differing in quality characteristics, were evaluated for potential genotoxicity in the Drosophila somatic mutation and recombination test (SMART) before and after heating at high temperatures. Drosophila larvae were fed on medium containing 6 and 12% v/v of each of the examined oils. Edible VOOs did not exhibit any mutagenic or recombination activity even after thermal treatment. Lower grade VOO gave negative results at the concentration of 6% and inconclusive ones at 12%. However, after its thermal treatment, a statistically significant increase of large single spots was observed, giving a positive result for this spot category at both concentrations. Evaluation of the possible contribution of olive phenolic compounds to the nongenotoxic effects observed was carried out using a polar olive leaf extract and pure oleuropein. No significant increase in the frequency of any category of mutant spots was recorded for leaf extract (0.8-12 mg of total polar phenols/dose) or pure oleuropein (0.8-8 mg/ dose). These results are expected to contribute to the ongoing interest in the inherent properties of VOO as part of the everyday diet.

Following cultivation-dependent and -independent techniques, we investigated the microbiota associated with Bactrocera oleae , one of the major agricultural pests in olive-producing countries. Bacterial 16S rRNA gene libraries and ultrastructural analyses revealed the presence of several bacterial taxa associated with this insect, among which Acetobacter tropicalis was predominant. The recent increased detection of acetic acid bacteria as symbionts of other insect model organisms, such as Anopheles stephensi (G. Favia et al., Proc. Natl. Acad. Sci. USA 104:9047-9051, 2007) or Drosophila melanogaster (C. R. Cox and M. S. Gilmore, Infect. Immun. 75:1565-1576, 2007), prompted us to investigate the association established between A. tropicalis and B. oleae . Using an A. tropicalis -specific PCR assay, the symbiont was detected in all insects tested originating from laboratory stocks or field-collected from different locations in Greece. This acetic acid bacterium was successfully established in cell-free medium, and typing analyses, carried out on a collection of isolates, revealed that different A. tropicalis strains are present in fly populations. The capability to colonize and lodge in the digestive system of both larvae and adults and in Malpighian tubules of adults was demonstrated by using a strain labeled with a green fluorescent protein.