Bumblebees of the Azores (Apidae: Bombus)

Bombus terrestris and Bombus pratorum were found for the first time in the Azores in 2005. Until then Bombus ruderatus was the only bumblebee species known from this group of nine midAtlantic islands. The identity of B. terrestris was confirmed by DNA barcoding

Proteomic Insights into the Hidden World of Phloem Sap Feeding

The physical interface between a phloem-feeding insect and its host plant is a single cell buried deep within the plant tissue. As such, the molecular interactions between these notorious agricultural pests and the crop plants upon which they feed are diffi cult to study. ‘Omic’ technologies have proved crucial in revealing some of the fascinating detail of the molecular interplay between these partners. Here we review the role of proteomics in identifying putative components of the secreted saliva of phloem-feeding insects, particularly aphids, and discuss the limited knowledge concerning the function of these proteins

The effect of entomopathogenic fungal culture filtrate on the immune response and haemolymph proteome of the large pine weevil, Hylobius abietis

peer-reviewedThe large pine weevil Hylobius abietis L. is a major forestry pest in 15 European countries, where it is a threat to 3.4 million hectares of forest. A cellular and proteomic analysis of the effect of culture filtrate of three entomopathogenic fungi (EPF) species on the immune system of H. abietis was performed. Injection with Metarhizium brunneum or Beauvaria bassiana culture filtrate facilitated a significantly increased yeast cell proliferation in larvae. Larvae co-injected with either Beauvaria caledonica or B. bassiana culture filtrate and Candida albicans showed significantly increased mortality. Together these results suggest that EPF culture filtrate has the potential to modulate the insect immune system allowing a subsequent pathogen to proliferate. Injection with EPF culture filtrate was shown to alter the abundance of protease inhibitors, detoxifing enzymes, antimicrobial peptides and proteins involved in reception/detection and development in H. abietis larvae. Larvae injected with B. caledonica culture filtrate displayed significant alterations in abundance of proteins involved in cellulolytic and other metabolic processes in their haemolymph proteome. Screening EPF for their ability to modulate the insect immune response represents a means of assessing EPF for use as biocontrol agents, particularly if the goal is to use them in combination with other control agents.This research was funded by the Irish Government (Department of Agriculture, Food and the Marine) (10/RD/MCOP/NUIM/720) under the National Development Plan 2007–2013 and through the MU Department of Biology Contingency Fund. The Q-Exactive quantitative mass spectrometer was funded under the SFI Research Infrastructure Call 2012; Grant Number: 12/RI/2346 (3) to Prof. S. Doyle

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 β-lactam antimicrobials but not ciprofloxacin, indicating a β-lactam stress response to exposure from this class of drugs, irrespective of β-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 β-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 β-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

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

Integrated Metabonomic-Proteomic Analysis of an Insect-Bacterial Symbiotic System

The health of animals, including humans, is dependent on their resident microbiota, but the complexity of the microbial communities makes these associations difficult to study in most animals. Exceptionally, the microbiology of the pea aphid Acyrthosiphon pisum is dominated by a single bacterium Buchnera aphidicola (B. aphidicola). A 1H NMR-based metabonomic strategy was applied to investigate metabolic profiles of aphids fed on a low essential amino acid diet and treated by antibiotic to eliminate B. aphidicola. In addition, differential gel electrophoresis (DIGE) with mass spectrometry was utilized to determine the alterations of proteins induced by these treatments. We found that these perturbations resulted in significant changes to the abundance of 15 metabolites and 238 proteins. Ten (67%) of the metabolites with altered abundance were amino acids, with nonessential amino acids increased and essential amino acids decreased by both perturbations. Over-represented proteins in the perturbed treatments included catabolic enzymes with roles in amino acid degradation and glycolysis, various cuticular proteins, and a C-type lectin and regucalcin with candidate defensive roles. This analysis demonstrates the central role of essential amino acid production in the relationship and identifies candidate proteins and processes underpinning the function and persistence of the association

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

Polyphenism in social insects: insights from a transcriptome-wide analysis of gene expression in the life stages of the key pollinator, Bombus terrestris

Background: Understanding polyphenism, the ability of a single genome to express multiple morphologically and behaviourally distinct phenotypes, is an important goal for evolutionary and developmental biology. Polyphenism has been key to the evolution of the Hymenoptera, and particularly the social Hymenoptera where the genome of a single species regulates distinct larval stages, sexual dimorphism and physical castes within the female sex. Transcriptomic analyses of social Hymenoptera will therefore provide unique insights into how changes in gene expression underlie such complexity. Here we describe gene expression in individual specimens of the pre-adult stages, sexes and castes of the key pollinator, the buff-tailed bumblebee Bombus terrestris. Results: cDNA was prepared from mRNA from five life cycle stages (one larva, one pupa, one male, one gyne and two workers) and a total of 1,610,742 expressed sequence tags (ESTs) were generated using Roche 454 technology, substantially increasing the sequence data available for this important species. Overlapping ESTs were assembled into 36,354 B. terrestris putative transcripts, and functionally annotated. A preliminary assessment of differences in gene expression across non-replicated specimens from the pre-adult stages, castes and sexes was performed using R-STAT analysis. Individual samples from the life cycle stages of the bumblebee differed in the expression of a wide array of genes, including genes involved in amino acid storage, metabolism, immunity and olfaction. Conclusions: Detailed analyses of immune and olfaction gene expression across phenotypes demonstrated how transcriptomic analyses can inform our understanding of processes central to the biology of B. terrestris and the social Hymenoptera in general. For example, examination of immunity-related genes identified high conservation of important immunity pathway components across individual specimens from the life cycle stages while olfactory-related genes exhibited differential expression with a wider repertoire of gene expression within adults, especially sexuals, in comparison to immature stages. As there is an absence of replication across the samples, the results of this study are preliminary but provide a number of candidate genes which may be related to distinct phenotypic stage expression. This comprehensive transcriptome catalogue will provide an important gene discovery resource for directed programmes in ecology, evolution and conservation of a key pollinator

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