Correction: Proteomic profiling of cereal aphid saliva reveals both ubiquitous and adaptive secreted proteins.

[This corrects the article DOI: 10.1371/journal.pone.0057413.].

Establishing the extent of pesticide contamination in Irish agricultural soils.

To establish meaningful and sustainable policy directives for sustainable pesticide use in agriculture, baseline knowledge of pesticide levels in soils is required. To address this, five pesticides and one metabolite widely used in Irish agriculture and five neonicotinoid compounds pesticides were screened from soils from 25 fields. These sites represented a diversity of soil and land use types. Prothioconazole was detected in 16 of the 18 sites where it had been recently applied, with the highest maximum concentration quantified of 46 µg/kg. However, a week after application only four fields had prothioconazole concentrations above the limit of quantification (LOQ). Fluroxypyr was applied in 11 sites but was not detected above LOQ. Glyphosate and AMPA were not detected. Interestingly, neonicotinoids were detected in 96% of all sampling sites, even though they were not reported as recently applied. Excluding neonicotinoids, 60% of sites were found to contain pesticide residues of compounds that were not previously applied, with boscalid and azoxystrobin detected in 15 of the 25 sites sampled. The total number of pesticides detected in Irish soils were significantly negatively correlated with clay fraction, while average pesticide concentrations were significantly positively correlated with log Kow values. 17 fields were found to have total pesticide concentrations in excess of 0.5 µg/kg, even when recently applied pesticides were removed from calculations. Theoretical consideration of quantified pesticides determined that azoxystrobin has high leaching risk, while boscalid, which was detected but not applied, has an accumulation risk. This information provides insight into the current level of pesticide contamination in Irish agricultural soil and contributes to the European-level effort to understand potential impacts of pesticide contamination in soil.

The boundary of life and death: changes in mitochondrial and cytosolic proteomes associated with programmed cell death of Arabidopsis thaliana suspension culture cells.

Introduction: Despite the critical role of programmed cell death (PCD) in plant development and defense responses, its regulation is not fully understood. It has been proposed that mitochondria may be important in the control of the early stages of plant PCD, but the details of this regulation are currently unknown. Methods: We used Arabidopsis thaliana cell suspension culture, a model system that enables induction and precise monitoring of PCD rates, as well as chemical manipulation of this process to generate a quantitative profile of the alterations in mitochondrial and cytosolic proteomes associated with early stages of plant PCD induced by heat stress. The cells were subjected to PCD-inducing heat levels (10 min, 54°C), with/without the calcium channel inhibitor and PCD blocker LaCl3. The stress treatment was followed by separation of cytosolic and mitochondrial fractions and mass spectrometry-based proteome analysis. Results: Heat stress induced rapid and extensive changes in protein abundance in both fractions, with release of mitochondrial proteins into the cytosol upon PCD induction. In our system, LaCl3 appeared to act downstream of cell death initiation signal, as it did not affect the release of mitochondrial proteins, but instead partially inhibited changes occurring in the cytosolic fraction, including upregulation of proteins with hydrolytic activity. Discussion: We characterized changes in protein abundance and localization associated with the early stages of heat stress-induced PCD. Collectively, the generated data provide new insights into the regulation of cell death and survival decisions in plant cells.

Signatures of Adaptation, Constraints, and Potential Redundancy in the Canonical Immune Genes of a Key Pollinator.

All organisms require an immune system to recognize, differentiate, and defend against pathogens. From an evolutionary perspective, immune systems evolve under strong selective pressures exerted by fast-evolving pathogens. However, the functional diversity of the immune system means that different immune components and their associated genes may evolve under varying forms of selection. Insect pollinators, which provide essential ecosystem services, are an important system in which to understand how selection has shaped immune gene evolution as their populations are experiencing declines with pathogens highlighted as a potential contributing factor. To improve our understanding of the genetic variation found in the immune genes of an essential pollinator, we performed whole-genome resequencing of wild-caught Bombus terrestris males. We first assessed nucleotide diversity and extended haplotype homozygosity for canonical immune genes finding the strongest signatures of positive selection acting on genes involved in pathogen recognition and antiviral defense, possibly driven by growing pathogen spread in wild populations. We also identified immune genes evolving under strong purifying selection, highlighting potential constraints on the bumblebee immune system. Lastly, we highlight the potential loss of function alleles present in the immune genes of wild-caught haploid males, suggesting that such genes are potentially less essential for development and survival and represent redundancy in the gene repertoire of the bumblebee immune system. Collectively, our analysis provides novel insights into the recent evolutionary history of the immune system of a key pollinator, highlighting targets of selection, constraints to adaptation, and potential redundancy.

Investigating the effects of glyphosate on the bumblebee proteome and microbiota.

Glyphosate is one of the most widely used herbicides globally. It acts by inhibiting an enzyme in an aromatic amino acid synthesis pathway specific to plants and microbes, leading to the view that it poses no risk to other organisms. However, there is growing concern that glyphosate is associated with health effects in humans and an ever-increasing body of evidence that suggests potential deleterious effects on other animals including pollinating insects such as bees. Although pesticides have long been considered a factor in the decline of wild bee populations, most research on bees has focussed on demonstrating and understanding the effects of insecticides. To assess whether glyphosate poses a risk to bees, we characterised changes in survival, behaviour, sucrose solution consumption, the digestive tract proteome, and the microbiota in the bumblebee Bombus terrestris after chronic exposure to field relevant doses of technical grade glyphosate or the glyphosate-based formulation, RoundUp Optima+®. Regardless of source, there were changes in response to glyphosate exposure in important cellular and physiological processes in the digestive tract of B. terrestris, with proteins associated with oxidative stress regulation, metabolism, cellular adhesion, the extracellular matrix, and various signalling pathways altered. Interestingly, proteins associated with endocytosis, oxidative phosphorylation, the TCA cycle, and carbohydrate, lipid, and amino acid metabolism were differentially altered depending on whether the exposure source was glyphosate alone or RoundUp Optima+®. In addition, there were alterations to the digestive tract microbiota of bees depending on the glyphosate source No impacts on survival, behaviour, or food consumption were observed. Our research provides insights into the potential mode of action and consequences of glyphosate exposure at the molecular, cellular and organismal level in bumblebees and highlights issues with the current honeybee-centric risk assessment of pesticides and their formulations, where the impact of co-formulants on non-target organisms are generally overlooked.

Low-temperature exposure has immediate and lasting effects on the stress tolerance, chemotaxis and proteome of entomopathogenic nematodes.

Temperature is one of the most important factors affecting soil organisms, including the infective stages of parasites and entomopathogenic nematodes, which are important biological control agents. We investigated the response of 2 species of entomopathogenic nematodes to different storage regimes: cold (9°C), culture temperature (20°C) and temperature swapped from 9 to 20°C. For Steinernema carpocapsae, cold storage had profound effects on chemotaxis, stress tolerance and protein expression that were retained in temperature-swapped individuals. These effects included reversal of chemotactic response for 3 (prenol, methyl salicylate and hexanol) of the 4 chemicals tested, and enhanced tolerance to freezing (−10°C) and desiccation (75% RH). Label-free quantitative proteomics showed that cold storage induced widespread changes in S. carpocapsae, including an increase in heat-shock proteins and late embryogenesis abundant proteins. For Heterorhabditis megidis, cold storage had a less dramatic effect on chemotaxis (as previously shown for proteomic expression) and changes were not maintained on return to 20°C. Thus, cold temperature exposure has significant effects on entomopathogenic nematodes, but the nature of the change depends on the species. Steinernema carpocapsae, in particular, displays significant plasticity, and its behaviour and stress tolerance may be manipulated by brief exposure to low temperatures, with implications for its use as a biological control agent.

Assessing availability of European plant protection product data: an example evaluating basic area treated.

Besides the benefits of plant protection products (PPPs) for agricultural production, there is an increasing acknowledgement of the associated potential environmental risks. Here, we examine the feasibility of summarizing the extent of PPP usage at the country level, using Ireland as a case study, as well as at the European level. We used the area over which PPPs are applied (basic area) as an example variable that is relevant to initially assess the geographic extent of environmental risk. In Irish agricultural systems, which are primarily grass-based, herbicides fluroxypyr and glyphosate are the most widely applied active substances (ASs) in terms of basic area, followed by the fungicides chlorothalonil and prothioconazole that are closely associated with arable crops. Although all EU countries are subject to Regulation (EC) No 1185/2009, which sets the obligation of PPP usage data reporting at the national level, we only found usable data that met our criteria for Estonia, Germany, Finland, and Spain (4 of 30 countries reviewed). Overall, the most widely applied fungicide and herbicide in terms of basic area were prothioconazole (20%, 7% and 5% of national cultivated areas of Germany, Estonia and Ireland) and glyphosate (11%, 8% and 5% of national cultivated areas of Spain, Estonia and Ireland) respectively, although evaluations using application frequency may result in the observation of different trends. Several recommendations are proposed to tackle current data gaps and deficiencies in accessibility and usability of pesticide usage data across the EU in order to better inform environmental risk assessment and promote evidence-based policymaking.

The effect of temperature conditioning (9°C and 20°C) on the proteome of entomopathogenic nematode infective juveniles.

Entomopathogenic nematodes (EPN) of the genera Steinernema and Heterorhabditis are parasites which kill and reproduce within insects. While both have life cycles centred around their developmentally arrested, nonfeeding and stress tolerant infective juvenile (IJ) stage, they are relatively distantly related. These IJs are promising biocontrol agents, and their shelf life and stress tolerance may be enhanced by storage at low temperatures. The purpose of this study was to investigate how the proteome of the IJs of two distantly related EPN species is affected by storage at 9°C (for up to 9 weeks) and 20°C (for up to 6 weeks), using label-free quantitative proteomics. Overall, more proteins were detected in S. carpocapsae (2422) than in H. megidis (1582). The S. carpocapsae proteome was strongly affected by temperature, while the H. megidis proteome was affected by both time and temperature. The proteins which increased in abundance to the greatest extent in S. carpocapsae IJs after conditioning at 9°C were chaperone proteins, and proteins related to stress. The proteins which increased in abundance the most after storage at 20°C were proteins related to the cytoskeleton, cell signalling, proteases and their inhibitors, which may have roles in infection. The proteins which decreased in abundance to the greatest extent in S. carpocapsae after both 9°C and 20°C storage were those associated with metabolism, stress and the cytoskeleton. After storage at both temperatures, the proteins increased to the greatest extent in H. megidis IJs were those associated with the cytoskeleton, cell signalling and carbon metabolism, and the proteins decreased in abundance to the greatest extent were heat shock and ribosomal proteins, and those associated with metabolism. As the longest-lived stage of the EPN life cycle, IJs may be affected by proteostatic stress, caused by the accumulation of misfolded proteins and toxic aggregates. The substantial increase of chaperone proteins in S. carpocapsae, and to a greater extent at 9°C, and the general decrease in ribosomal and chaperone proteins in H. megidis may represent species-specific proteostasis mechanisms. Similarly, organisms accumulate reactive oxygen species (ROS) over time and both species exhibited a gradual increase in proteins which enhance ROS tolerance, such as catalase. The species-specific responses of the proteome in response to storage temperature, and over time, may reflect the phylogenetic distance and/or different ecological strategies.

Exposure to the Pseudomonas aeruginosa secretome alters the proteome and secondary metabolite production of Aspergillus fumigatus.

The fungal pathogen Aspergillus fumigatus is frequently cultured from the sputum of cystic fibrosis (CF) patients along with the bacterium Pseudomonas aeruginosa. A. fumigatus secretes a range of secondary metabolites, and one of these, gliotoxin, has inhibitory effects on the host immune response. The effect of P. aeruginosa culture filtrate (CuF) on fungal growth and gliotoxin production was investigated. Exposure of A. fumigatus hyphae to P. aeruginosa cells induced increased production of gliotoxin and a decrease in fungal growth. In contrast, exposure of A. fumigatus hyphae to P. aeruginosa CuF led to increased growth and decreased gliotoxin production. Quantitative proteomic analysis was used to characterize the proteomic response of A. fumigatus upon exposure to P. aeruginosa CuF. Changes in the profile of proteins involved in secondary metabolite biosynthesis (e.g. gliotoxin, fumagillin, pseurotin A), and changes to the abundance of proteins involved in oxidative stress (e.g. formate dehydrogenase) and detoxification (e.g. thioredoxin reductase) were observed, indicating that the bacterial secretome had a profound effect on the fungal proteome. Alterations in the abundance of proteins involved in detoxification and oxidative stress highlight the ability of A. fumigatus to differentially regulate protein synthesis in response to environmental stresses imposed by competitors such as P. aeruginosa. Such responses may ultimately have serious detrimental effects on the host.

Genome sequence of the English grain aphid, Sitobion avenae and its endosymbiont Buchnera aphidicola.

The English grain aphid, Sitobion avenae, is a major agricultural pest of wheat, barley and oats, and one of the principal vectors of barley yellow dwarf virus leading to significant reductions in grain yield, annually. Emerging resistance to and increasing regulation of insecticides has resulted in limited options for their control. Using PacBio HiFi data, we have produced a high-quality draft assembly of the S. avenae genome; generating a primary assembly with a total assembly size of 475.7 Mb, and an alternate assembly with a total assembly size of 430.8 Mb. Our primary assembly was highly contiguous with only 326 contigs and a contig N50 of 15.95 Mb. Assembly completeness was estimated at 97.7% using BUSCO analysis and 31,007 and 29,037 protein-coding genes were predicted from the primary and alternate assemblies, respectively. This assembly, which is to our knowledge the first for an insecticide resistant clonal lineage of English grain aphid, will provide novel insight into the molecular and mechanistic determinants of resistance and will facilitate future research into mechanisms of viral transmission and aphid behavior.

Global protein responses of multidrug resistance plasmid-containing Escherichia coli to ampicillin, cefotaxime, imipenem and ciprofloxacin.

OBJECTIVES: This study compared the proteomics of Escherichia coli containing the multidrug resistance plasmid pEK499 under antimicrobial stress and with no antimicrobial. METHODS: We utilised mass spectrometry-based proteomics to compare the proteomes of the bacteria and plasmid under antimicrobial stress and no antimicrobial. RESULTS: Our analysis identified statistically significant differentially abundant (SSDA) proteins common to groups exposed to the ß-lactam antimicrobials but not ciprofloxacin, indicating a ß-lactam stress response to exposure from this class of drugs, irrespective of ß-lactam resistance or susceptibility. Data arising from comparisons of the proteomes of ciprofloxacin-treated E. coli and controls detected an increase in the relative abundance of proteins associated with ribosomes, translation, the TCA cycle and several proteins associated with detoxification, and a decrease in the relative abundance of proteins associated with the stress response, including oxidative stress. We identified changes in proteins associated with persister formation in the presence of ciprofloxacin but not the ß-lactams. The plasmid proteome differed across each treatment and did not follow the pattern of antimicrobial-antimicrobial resistance (AMR) protein associations: a relative increase was observed in the amount of CTX-M-15 in the presence of cefotaxime and ciprofloxacin, but not the other ß-lactams, suggesting regulation of CTX-M-15 protein production. CONCLUSION: The proteomic data from this study provided novel insights into the proteins produced from the chromosome and plasmid under different antimicrobial stresses. These data also identified novel proteins not previously associated with AMR or antimicrobial responses in pathogens, which may well represent potential targets of AMR inhibition.

Simultaneous determination of pesticides from soils: a comparison between QuEChERS extraction and Dutch mini-Luke extraction methods.

The expanding nature of the agricultural sector has fuelled the intensification of plant protection products usage, including pesticides. These pesticides may persist in soils, necessitating their accurate determination in a variety of soil types. However, due to their complex nature, the effective extraction of pesticide residues from soil matrices can present challenges to pesticide detection and quantification. This research compared two well-known extraction methods, QuEChERS and Dutch mini-Luke, by assessing their specificity, sensitivity, accuracy, precision and reproducibility in extracting seven distinct pesticides with a range of chemico-physical characteristics from Irish soils. The HPLC-UV conditions were optimised to separate the seven pesticides, and it was shown that both extraction methods successfully extracted neonicotinoids with recovery values ranging between 85 and 115%. Fluroxypyr and prothioconazole could not be efficiently extracted using QuEChERS, however, the recovery values of both the analytes ranged between 59 and 117% using Dutch mini-Luke. Furthermore, with the exception of prothioconazole using Dutch mini-Luke, both extraction methods resulted in reproducibility and precision values below or equal to 20%. Lastly, Dutch mini-Luke is noted to have a lower matrix effect than QuEChERS, except for prothioconazole. The comparison results showed that Dutch mini-Luke resulted in superior method sensitivity, better recovery, and lower matrix effect towards most investigated analytes and was the only extraction technique that successfully extracted all pesticides analysed in soil matrices.

Bacterial Interactions with Aspergillus fumigatus in the Immunocompromised Lung.

The immunocompromised airways are susceptible to infections caused by a range of pathogens which increases the opportunity for polymicrobial interactions to occur. Pseudomonas aeruginosa and Staphylococcus aureus are the predominant causes of pulmonary infection for individuals with respiratory disorders such as cystic fibrosis (CF). The spore-forming fungus Aspergillus fumigatus, is most frequently isolated with P. aeruginosa, and co-infection results in poor outcomes for patients. It is therefore clinically important to understand how these pathogens interact with each other and how such interactions may contribute to disease progression so that appropriate therapeutic strategies may be developed. Despite its persistence in the airways throughout the life of a patient, A. fumigatus rarely becomes the dominant pathogen. In vitro interaction studies have revealed remarkable insights into the molecular mechanisms that drive agonistic and antagonistic interactions that occur between A. fumigatus and pulmonary bacterial pathogens such as P. aeruginosa. Crucially, these studies demonstrate that although bacteria may predominate in a competitive environment, A. fumigatus has the capacity to persist and contribute to disease.

The Aspergillus fumigatus Secretome Alters the Proteome of Pseudomonas aeruginosa to Stimulate Bacterial Growth: Implications for Co-infection.

Individuals with cystic fibrosis are susceptible to co-infection by Aspergillus fumigatus and Pseudomonas aeruginosa Despite the persistence of A. fumigatus in the cystic fibrosis lung P. aeruginosa eventually predominates as the primary pathogen. Several factors are likely to facilitate P. aeruginosa colonization in the airways, including alterations to the microbial environment. The cystic fibrosis airways are hypoxic, nitrate-rich environments, and the sputum has higher amino acid concentrations than normal. In this study, significant growth proliferation was observed in P. aeruginosa when the bacteria were exposed to A. fumigatus culture filtrates (CuF) containing a high nitrate content. Proteomic analysis of the A. fumigatus CuF identified a significant number of environment-altering proteases and peptidases. The molecular mechanisms promoting bacterial growth were investigated using label-free quantitative (LFQ) proteomics to compare the proteome of P. aeruginosa grown in the A. fumigatus CuF and in CuF produced by a P. aeruginosa-A. fumigatus co-culture, to that cultured in P. aeruginosa CuF. LFQ proteomics revealed distinct changes in the proteome of P. aeruginosa when cultured in the different CuFs, including increases in the levels of proteins involved in denitrification, stress response, replication, amino acid metabolism and efflux pumps, and a down-regulation of pathways involving ABC transporters. These findings offer novel insights into the complex dynamics that exist between P. aeruginosa and A. fumigatus Understanding the molecular strategies that enable P. aeruginosa to predominate in an environment where A. fumigatus exists is important in the context of therapeutic development to target this pathogen.

In silico Characterization of a Candidate Protein from Aphid Gelling Saliva with Potential for Aphid Control in Plants.

BACKGROUND: Sheath or gelling saliva, secreted during feeding by aphids, is a hard material that supports the piercing mouthparts and remains in the plant after feeding. Solidification or gelling of the saliva might be due to the composition of amino acids in the constituent proteins, many of which probably interact with plant defenses. OBJECTIVE: The complete complement of proteins in the gelling saliva are still unknown, although one sheath protein (SHP) has previously been identified as a potential candidate protein to control aphid feeding, but its structure and its physiochemical role remains obscure. The current study provides structural information and biochemical properties of the aphid sheath protein. METHODS: The Sheath protein encoding gene was amplified from cDNA of the pea aphid (Acyrthosiphon pisum) through PCR using specific gene primers. Sequence was in silico characterized by using EXPASY, Berkeley Drosophila Genome Project (BDGP) Neural Network Promoter Prediction, BioEdit, Mega7, ProtParam, Phyre server, 3D LigandSite SMART, MEME and GSDS programs, available online. RESULTS: BLASTp analysis revealed that the sequenced gene was identical (100%) to the sequence from Acyrthosiphon pisum, with 87% identity to Metpolophium dirhodum and 84% identity to Sitobion avenae. Phylogenetically monocot feeders such as M. dirhodum and S. avenae are in a sister taxa to dicot feeders. In silico analysis of the sequence revealed that sheath protein has a molecular weight of 144 kDa and 50% of the protein is composed of only six amino acids, i.e., threonine, serine, aspartic acid, glutamic acid, isoleucine and tyrosine. The computed IP value revealed that sheath protein is acidic in nature. Ligand binding sites for sheath protein were predicted on residues 1123 and 1125 (isoleucine and glutamine, respectively). Metallic heterogens are also present in sheath protein that are iron, zinc and magnesium, respectively. CONCLUSION: It is conceivable that variation in the salivary gene sequences may reveal important biological information of relevance to the insect-plant interaction. Further exploration of insect salivary proteins, their composition and structure will provide powerful information, especially when these proteins are interacting with plant proteins, and specific information about the sheath protein, which is interacting with plants at a molecular/cellular level, will be important to progress strategies aimed specifically against sucking pests such as aphids.

Characterization of the Proteomic Response of A549 Cells Following Sequential Exposure to Aspergillus fumigatus and Pseudomonas aeruginosa.

Aspergillus fumigatus and Pseudomonas aeruginosa are the most prevalent fungal and bacterial pathogens associated with cystic-fibrosis-related infections, respectively. P. aeruginosa eventually predominates as the primary pathogen, though it is unknown why this is the case. Label-free quantitative proteomics was employed to investigate the cellular response of the alveolar epithelial cell line, A549, to coexposure of A. fumigatus and P. aeruginosa. These studies revealed a significant increase in the rate of P. aeruginosa proliferation where A. fumigatus was present. Shotgun proteomics performed on A549 cells exposed to either A. fumigatus or P. aeruginosa or to A. fumigatus and P. aeruginosa sequentially revealed distinct changes to the host cell proteome in response to either or both pathogens. While key signatures of infection were retained among all pathogen-exposed groups, including changes in mitochondrial activity and energy output, the relative abundance of proteins associated with endocytosis, phagosomes, and lysosomes was decreased in sequentially exposed cells compared to cells exposed to either pathogen. Our findings indicate that A. fumigatus renders A549 cells unable to internalize bacteria, thus providing an environment in which P. aeruginosa can proliferate. This research provides novel insights into the whole-cell proteomic response of A549 cells to A. fumigatus and P. aeruginosa and highlights distinct differences in the proteome following sequential exposure to both pathogens, which may explain why P. aeruginosa can predominate.

Fungicides, herbicides and bees: A systematic review of existing research and methods.

Bees and the pollination services they deliver are beneficial to both food crop production, and for reproduction of many wild plant species. Bee decline has stimulated widespread interest in assessing hazards and risks to bees from the environment in which they live. While there is increasing knowledge on how the use of broad-spectrum insecticides in agricultural systems may impact bees, little is known about effects of other pesticides (or plant protection products; PPPs) such as herbicides and fungicides, which are used more widely than insecticides at a global scale. We adopted a systematic approach to review existing research on the potential impacts of fungicides and herbicides on bees, with the aim of identifying research approaches and determining knowledge gaps. While acknowledging that herbicide use can affect forage availability for bees, this review focussed on the potential impacts these compounds could have directly on bees themselves. We found that most studies have been carried out in Europe and the USA, and investigated effects on honeybees. Furthermore, certain effects, such as those on mortality, are well represented in the literature in comparison to others, such as sub-lethal effects. More studies have been carried out in the lab than in the field, and the impacts of oral exposure to herbicides and fungicides have been investigated more frequently than contact exposure. We suggest a number of areas for further research to improve the knowledge base on potential effects. This will allow better assessment of risks to bees from herbicides and fungicides, which is important to inform future management decisions around the sustainable use of PPPs.

Mating precedes selective immune priming which is maintained throughout bumblebee queen diapause.

BACKGROUND: Understanding the mechanisms by which organisms adapt to unfavourable conditions is a fundamental question in ecology and evolutionary biology. One such mechanism is diapause, a period of dormancy typically found in nematodes, fish, crustaceans and insects. This state is a key life-history event characterised by arrested development, suppressed metabolism and increased stress tolerance and allows an organism to avoid prolonged periods of harsh and inhospitable environmental conditions. For some species, diapause is preceded by mating which can have a profound effect on female behaviour, physiology and key biological processes, including immunity. However, our understanding of how mating impacts long-term immunity and whether these effects persist throughout diapause is currently limited. To address this, we explored molecular changes in the haemolymph of the ecologically important pollinator, the buff-tailed bumblebee Bombus terrestris. B. terrestris queens mate prior to entering diapause, a non-feeding period of arrested development that can last 6-9 months. Using mass-spectrometry-based proteomics, we quantified changes in the pre-diapause queen haemolymph after mating, as well as the subsequent protein expression of mated queens during and post-diapause. RESULTS: Our analysis identified distinct proteome profiles associated with diapause preparation, maintenance and termination. More specifically, mating pre-diapause was followed by an increase in the abundance of antimicrobial peptides, key effectors of the immune system. Furthermore, we identified the elevated abundance of these proteins to be maintained throughout diapause. This finding was in contrast to the general reduction observed in immune proteins during diapause suggestive of selective immune priming and expression during diapause. Diapause also affected the expression of proteins involved in cuticular maintenance, olfaction, as well as proteins of unknown function, which may have roles in diapause regulation. CONCLUSIONS: Our results provide clear molecular evidence for the consequences and benefits of mating at the immune level as it precedes the selective increased abundance of antimicrobial peptides that are sustained throughout diapause. In addition, our results provide novel insights into the molecular mechanisms by which bumblebees prepare for, survive, and recover from diapause, insights that may have implications for our general understanding of these processes in other insect groups.

The salivary gland proteome of root-galling grape phylloxera (Daktulosphaira vitifoliae Fitch) feeding on Vitis spp.

The successful parasitisation of a plant by a phytophagous insect is dependent on the delivery of effector molecules into the host. Sedentary gall forming insects, such as grape phylloxera (Daktulosphaira vitifoliae Fitch, Phylloxeridae), secrete multiple effectors into host plant tissues that alter or modulate the cellular and molecular environment to the benefit of the insect. The identification and characterisation of effector proteins will provide insight into the host-phylloxera interaction specifically the gall-induction processes and potential mechanisms of plant resistance. Using proteomic mass spectrometry and in-silico secretory prediction, 420 putative effectors were determined from the salivary glands or the root-feeding D. vitifoliae larvae reared on Teleki 5C (V. berlandieri x V. riparia). Among them, 170 conserved effectors were shared between D. vitifoliae and fourteen phytophagous insect species. Quantitative RT-PCR analysis of five conserved effector candidates (protein disulfide-isomerase, peroxidoredoxin, peroxidase and a carboxypeptidase) revealed that their gene expression decreased, when larvae were starved for 24 h, supporting their assignment as effector molecules. The D. vitifoliae effectors identified here represent a functionally diverse group, comprising both conserved and unique proteins that provide new insight into the D. vitifoliae-Vitis spp. interaction and the potential mechanisms by which D. vitifoliae establishes the feeding site, suppresses plant defences and modulates nutrient uptake.

The role of the liver in the migration of parasites of global significance.

Many parasites migrate through different tissues during their life-cycle, possibly with the aim to enhance their fitness. This is true for species of three parasite genera of global importance, Ascaris, Schistosoma and Plasmodium, which cause significant global morbidity and mortality. Interestingly, these parasites all incorporate the liver in their life-cycle. The liver has a special immune status being able to preferentially induce tolerance over immunity. This function may be exploited by parasites to evade host immunity, with Plasmodium spp. in particular using this organ for its multiplication. However, hepatic larval attrition occurs in both ascariasis and schistosomiasis. A better understanding of the molecular mechanisms involved in hepatic infection could be useful in developing novel vaccines and therapies for these parasites.