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Social wasps are invasive in many regions around the world. In their new communities, introduced predators such as these wasps may be beneficial as consumers of exotic pests, but they will also consume native species. Here, we examined the diet of the exotic European paper wasp (Polistes dominula) and the closely related congener, Polistes chinensis, in a region of New Zealand where they co-occur. DNA barcoding was used to analyse their diet. The diet of both wasp species was largely Lepidopteran but other orders such as Hemiptera, Diptera and Coleoptera were also represented. Our analysis showed substantial site-to-site variation in diet. The two wasps differed significantly in their prey, although these differences appear to be driven by taxa identified from a small number of DNA reads in a small number of samples. Native and introduced fauna were represented in the diets of both wasps and included important agricultural pests. Of the 92 prey taxa able to be identified to species level, 81 were identified as exotic or introduced to New Zealand. The remaining 11 were species native to New Zealand. However, our estimates suggest over 50% of the prey DNA in the wasp diet is derived from native species. These wasps are abundant in some coastal and urban habitats, where they are likely to consume pest species as well as native species of conservation importance. The ecosystem services or costs and benefits provided by these invasive species are likely to be contingent on the prey communities and habitats they occupy.

The most devastating pest to honey bees (Apis mellifera) worldwide is the parasitic miteVarroa destructor. The development of miticide-resistant mite populations has been a major driver of colony loss in many countries. We investigated the threat Varroa poses to honey bee populations in New Zealand and tested the effectiveness of the two most popular chemical treatments used by beekeepers. Colony losses reported by New Zealand beekeepers have risen over five consecutive years from 2017 to 2021, as have the proportion of losses attributed to Varroa, with this parasite found to be the main driver of colony loss in 2021. Varroa resistance to miticide treatments flumethrin and amitraz was tested. The concentration of flumethrin required to kill 50% of the mites (LC50) was 156 μg/g, 13 times greater than the adjusted LC50value of 12 μg/g observed in a trial also conducted in New Zealand in 2003, thus indicating evidence of developing mite resistance to flumethrin in New Zealand. Molecular analyses searching for mutations in the Varroa genome known to be associated with flumethrin resistance found no evidence of such mutations, suggesting that any extant resistance to flumethrin has evolved independently in New Zealand. No evidence of resistance to amitraz was found, as the LC50value of 12 μg/g was lower than what was observed in the 2003 trial (110 μg/g). Further development of integrated pest management, such as gene-silencing RNA interference (RNAi) and selective breeding of Varroa-resistant bees, is needed to effectively manage a parasite that threatens global agriculture.

Pest control methods that can target pest species with limited environmental impacts are a conservation and economic priority. Species-specific pest control using RNA interference is a challenging but promising avenue in developing the next generation of pest management. We investigate the feasibility of manipulating a biological invader's immune system using double-stranded RNA (dsRNA) in order to increase susceptibility to naturally occurring pathogens. We used the invasive Argentine ant as a model, targeting the immunity-associated genes Spaetzle and Dicer-1 with dsRNA. We show that feeding of Spaetzle dsRNA can result in partial target gene silencing for up to 28 days in the laboratory and 5 days in the field. Dicer-1 dsRNA only resulted in partial gene knockdown after 2 days in the laboratory. Double-stranded RNA treatments were associated with significant gene expression disruptions across immune pathways in the laboratory and to a lower extent in the field. Twelve viruses and four bacteria were found in these ant populations. Some changes in viral loads in dsRNA-treated groups were observed. For example, Linepithema humile Polycipivirus 2 (LhuPCV2) loads increased after 2 days of treatment with Spaetzle and Dicer-1 dsRNA treatments in the laboratory. After treatment with the dsRNA in the field, after 5 days the virus Linepithema humile toti-like virus 1 (LhuTLV1) was significantly more abundant. However, immune pathways disruption did not result in consistent increase in microbial infections, nor did they alter ant abundance in the field. Some viruses even declined in abundance after dsRNA treatment. Our study explores the feasibility of lowering a pest's immunity as a control tool. We demonstrate that it is possible to alter immune gene expression of pest species and pathogen loads, though in our specific system the affected pathogens did not appear to influence pest abundance. We provide suggestions on future directions for dsRNA-mediated immune disruption in pest species, including potential avenues to improve dsRNA delivery as well as the importance of the pest and pathogen biology. Double-stranded RNA targeting immune function might be especially useful for pest control in systems where viruses or other microorganisms are prevalent and have the potential to be pathogenic. This article is protected by copyright. All rights reserved.

The parasitic mite Varroa destructor is a leading cause of mortality for Western honey bee (Apis mellifera) colonies around the globe. We sought to confirm the presence and likely introduction of only one V. destructor haplotype in New Zealand, and describe the viral community within both V. destructor mites and the bees that they parasitise. A 1232 bp fragment from mitochondrial gene regions suggests the likely introduction of only one V. destructor haplotype to New Zealand. Seventeen viruses were found in bees. The most prevalent and abundant was the Deformed wing virus A (DWV-A) strain, which explained 95.0% of the variation in the viral community of bees. Black queen cell virus, Sacbrood virus, and Varroa destructor virus 2 (VDV-2) played secondary roles. DWV-B and the Israeli acute paralysis virus appeared absent from New Zealand. Ten viruses were observed in V. destructor, with > 99.9% of viral reads from DWV-A and VDV-2. Substantially more variation in viral loads was observed in bees compared to mites. Where high levels of VDV-2 occurred in mites, reduced DWV-A occurred in both the mites and the bees co-occurring within the same hive. Where there were high loads of DWV-A in mites, there were typically high viral loads in bees.

Invasive species populations periodically collapse from high to low abundance, sometimes even to extinction. Pathogens and the burden they place on invader immune systems have been hypothesised as a mechanism for these collapses. We examined the association of the bacterial pathogen (Pseudomonas spp.) and the viral community with immune gene expression in the globally invasive Argentine ant (Linepithema humile (Mayr)). RNA-seq analysis found evidence for 17 different viruses in Argentine ants from New Zealand, including three bacteriophages with one (Pseudomonas phage PS-1) likely to be attacking the bacterial host. Pathogen loads and prevalence varied immensely. Transcriptomic data showed that immune gene expression was consistent with respect to the viral classification of negative-sense, positive-sense and double-stranded RNA viruses. Genes that were the most strongly associated with the positive-sense RNA viruses such as the Linepithema humile virus 1 (LHUV-1) and the Deformed wing virus (DWV) were peptide recognition proteins assigned to the Toll and Imd pathways. We then used principal components analysis and regression modelling to determine how RT-qPCR derived immune gene expression levels were associated with viral and bacterial loads. Argentine ants mounted a substantial immune response to both Pseudomonas and LHUV-1 infections, involving almost all immune pathways. Other viruses including DWV and the Kashmir bee virus appeared to have much less immunological influence. Different pathogens were associated with varying immunological responses, which we hypothesize to interact with and influence the invasion dynamics of this species.

1. Variation in microbial communities between populations is increasingly hypothesised to affect animal fitness and performance, including for invasive species. Pathogenic species may be lost during the introduction process, enhancing invader fitness and abundance. 2. This study assessed fitness, immune gene expression, and microbial network complexity of invasive common wasps, Vespula vulgaris. Microbial networks were assayed using 16S and 18S sequencing and gene expression arrays in the native (Belgium) and introduced range (New Zealand). The immune gene expression of the wasp Down syndrome cell adhesion molecule (Dscam) gene homologue was examined. Dscam expression can be induced by viruses, Gram‐positive and Gram‐negative bacteria, and parasites. 3. Individual nest fitness was higher in the native range of Belgium than in the introduced New Zealand range. Microbial communities of wasps in the introduced range were more diverse with more complex networks, although some microorganisms were range‐specific. Microbial networks in the introduced range showed higher clustering coefficients, number of connected paths, network centralisation, number of neighbours and network density. 4. Larvae, workers, virgin and foundress queens had higher expression of Dscam in the New Zealand samples. These immune gene expression patterns were associated with higher pathogen pressure and lower relative fitness. 5. Epidemiological theory predicts that a high density of pathogen and microbial hosts should result in a high rate of disease infection, prevalence, and highly connected microbial networks. The results of this study support these predictions. Wasps displayed lower relative fitness and more highly connected microbial networks in New Zealand than in Belgium.

Wasps of the genus Vespula are social insects that have become major pests and predators in their introduced range. Viruses present in these wasps have been studied in the context of spillover from honey bees, yet we lack an understanding of the endogenous virome of wasps as potential reservoirs of novel emerging infectious diseases. We describe the characterization of 68 novel and nine previously identified virus sequences found in transcriptomes of Vespula vulgaris in colonies sampled from their native range (Belgium) and an invasive range (New Zealand). Many viruses present in the samples were from the Picorna-like virus family (38%). We identified one Luteo-like virus, Vespula vulgaris Luteo-like virus 1, present in the three life stages examined in all colonies from both locations, suggesting this virus is a highly prevalent and persistent infection in wasp colonies. Additionally, we identified a novel Iflavirus with similarity to a recently identified Moku virus, a known wasp and honey bee pathogen. Experimental infection of honey bees with this novel Vespula vulgaris Moku-like virus resulted in an active infection. The high viral diversity present in these invasive wasps is a likely indication that their polyphagous diet is a rich source of viral infections., publishedVersion

�� 2019, International Union for the Study of Social Insects (IUSSI). Introduced social wasps (Vespula spp.) are a pest in many parts of the world. Recently, a mite species (Pneumolaelaps niutirani) was described and associated with disease symptoms in wasps. The mite does not appear to directly parasitise the wasps, but has been observed in high abundance, feeding on exudates from the mouths of larvae. We investigated the viral and fungal pathogens community in these mites and wasps. We found known viruses including Moku virus in both wasps and mites. Moku virus replicated in mites, likely indicating parasitism. Deformed wing virus, commonly found in wasps, was also detected in mite samples. Furthermore, the presence of putative viral transcripts related to 38 distinct viruses, including seven viruses previously isolated from arthropods, indicated that there may be many more viruses associated with the mite that are potentially shared with Vespula wasps. We also found generalist entomopathogenic fungus Aspergillus to infect both mites and wasps. Twelve distinct Aspergillus species were observed, all of which were found in wasp larvae from nests displaying symptoms of disease, with only one species in larvae from apparently healthy nests. Aspergillus novofumigatus was the most common of these species observed in wasps. Six Aspergillus species, including A. novofumigatus were detected in mites. Aspergillus loads were significantly higher in larvae from diseased nests. Our exploratory study indicates that mites can harbour both viruses and fungi that infect wasps, providing avenues of research into biological control using mites as infection vectors.

CRISPR gene drives have potential for widespread and cost-efficient pest control, but are highly controversial. We examined a potential gene drive targeting spermatogenesis to control the invasive common wasp (Vespula vulgaris) in New Zealand. Vespula wasps are haplodiploid. Their life cycle makes gene drive production challenging, as nests are initiated by single fertilized queens in spring followed by several cohorts of sterile female workers and the production of reproductives in autumn. We show that different spermatogenesis genes have different levels of variation between introduced and native ranges, enabling a potential ‘precision drive’ that could target the reduced genetic diversity and genotypes within the invaded range. In vitro testing showed guide-RNA target specificity and efficacy that was dependent on the gene target within Vespula, but no cross-reactivity in other Hymenoptera. Mathematical modelling incorporating the genetic and life history traits of Vespula wasps identified characteristics for a male sterility drive to achieve population control. There was a trade-off between drive infiltration and impact: a drive causing complete male sterility would not spread, while partial sterility could be effective in limiting population size if the homing rate is high. Our results indicate that gene drives may offer viable suppression for wasps and other haplodiploid pests.

Despite the mitochondrion's long-recognized role in energy production, mitochondrial DNA (mtDNA) variation commonly found in natural populations was assumed to be effectively neutral. However, variation in mtDNA has now been increasingly linked to phenotypic variation in life history traits and fitness. We examined whether the relative fitness in native and invasive common wasp (Vespula vulgaris) populations in Belgium and New Zealand (NZ), respectively, can be linked to mtDNA variation. Social wasp colonies in NZ were smaller with comparatively fewer queen cells, indicating a reduced relative fitness in the invaded range. Interestingly, queen cells in this population were significantly larger leading to larger queen offspring. By sequencing 1,872 bp of the mitochondrial genome, we determined mitochondrial haplotypes and detected reduced genetic diversity in NZ. Three common haplotypes in NZ frequently produced many queens, whereas the four rare haplotypes produced significantly fewer or no queens. The entire mitochondrial genome for each of these haplotypes was sequenced to identify polymorphisms associated with fitness reduction. We found 16 variable sites; however, no nonsynonymous mutation that was clearly causing impaired mitochondrial function was detected. We discuss how detected variants may alter secondary structures, gene expression or mito-nuclear interactions, or could be associated with nuclear-encoded variation. Whatever the ultimate mechanism, we show reduced fitness and mtDNA variation in an invasive wasp population as well as specific mtDNA variants associated with fitness variation within this population. Ours is one of only a few studies that confirm fitness impacts of mtDNA variation in wild nonmodel populations.

A successful control or eradication programme using biological control or genetically-mediated methods requires knowledge of the origin and the extent of wasp genetic diversity. Mitochondrial DNA variation in the native and invaded range of the social wasp Vespula germanica was used to examine intraspecific genetic variation and invasive source populations.We also examined wasps for the presence of four viruses found in honey bees: Acute bee paralysis virus, Deformed wing virus, Israeli acute paralysis virus and Kashmir bee virus. German wasps showed reduced genetic diversity in the invaded range compared to that of their native range. Populations in the introduced range are likely to have arrived from different source populations. All four viral honey bee pathogens were found in V. germanica, although they varied in their distribution and strain. Multiple introductions of German wasps have occurred for most invaded regions, though some populations are genetically homogenous. The differing locations of origin will guide researchers searching for biocontrol agents andthe reduced genetic diversity may make these wasps a potentially viable target for control via gene drives. Fil: Brenton Rule, Evan C.. Centre For Biodiversity And Restoration Ecology; Nueva Zelanda Fil: Dobelmann, Jana. Centre For Biodiversity And Restoration Ecology; Nueva Zelanda Fil: Baty, James W.. Malaghan Institute Of Medical Research; Nueva Zelanda Fil: Brown, Robert L.. Crown Research Institutes. Landcare Research; Nueva Zelanda Fil: Dvorak, Libor. Mestske Museum Marianske Lazne; República Checa Fil: Grangier, Julien. Universite Lyon 2; Francia Fil: Masciocchi, Maité. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Patagonia Norte. Estación Experimental Agropecuaria San Carlos de Bariloche. Laboratorio de Ecología de Insectos; Argentina Fil: McGrannachan, Chris. Monash University; Australia Fil: Shortall, Chris R.. Rothamsted Insect Survey; Reino Unido Fil: Schmack, Julia. Centre For Biodiversity And Restoration Ecology; Nueva Zelanda Fil: van Zyl, Carolien. Centre For Biodiversity And Restoration Ecology; Nueva Zelanda Fil: Veldtman, Ruan. Centre For Biodiversity And Restoration Ecology; Nueva Zelanda Fil: Lester, Philip J.. Centre For Biodiversity And Restoration Ecology; Nueva Zelanda

Social insects host a diversity of viruses. We examined New Zealand populations of the globally widely distributed invasive Argentine ant (Linepithema humile) for RNA viruses. We used metatranscriptomic analysis, which identified six potential novel viruses in the Dicistroviridae family. Of these, three contigs were confirmed by Sanger sequencing as Linepithema humile virus-1 (LHUV-1), a novel strain of Kashmir bee virus (KBV) and Black queen cell virus (BQCV), while the others were chimeric or misassembled sequences. We extended the known sequence of LHUV-1 to confirm its placement in the Dicistroviridae and categorised its relationship to closest relatives, which were all viruses infecting Hymenoptera. We examined further for known viruses by mapping our metatranscriptomic sequences to all viral genomes, and confirmed KBV, BQCV, LHUV-1 and Deformed wing virus (DWV) presence using qRT-PCR. Viral replication was confirmed for DWV, KBV and LHUV-1. Viral titers in ants were higher in the presence of honey bee hives. Argentine ants appear to host a range of’ honey bee’ pathogens in addition to a virus currently described only from this invasive ant. The role of these viruses in the population dynamics of the ant remain to be determined, but offer potential targets for biocontrol approaches.

Within any one habitat, the relative fitness of organisms in a population can vary substantially. Social insects like the common wasp are among the most successful invasive animals, but show enormous variation in nest size and other fitness-related traits. Some of this variation may be caused by pathogens such as viruses that can have serious consequences in social insects, which range from reduced productivity to colony death. Both individual immune responses and colony-level traits such as genetic diversity are likely to influence effects of pathogen infections on colony fitness. Here we investigate how infections with Kashmir Bee Virus (KBV), immune response and intracolony genetic diversity (due to queen polyandry) affect nest size in the invasive common wasp Vespula vulgaris. We show that KBV is highly prevalent in wasps and expression of antiviral immune genes is significantly increased with higher viral loads across individuals. Patriline membership within a nest did not influence KBV susceptibility or immune response. A permutational MANCOVA revealed that polyandry, viral load and expression of the immune gene Dicer were significant predictors of variation in nest size. High intracolony genetic diversity due to polyandry has previously been hypothesized to improve colony-level resistance to parasites and pathogens. Consistent with this hypothesis, we observed genetically diverse colonies to be significantly larger and to produce more queens, although this effect was not driven by the pathogen we investigated. Invasive wasps clearly suffer from pathogens and expend resources, as indicated here by elevated immune gene expression, toward reducing pathogen-impact on colony fitness.

A series of N,N-bis(glycityl)amines with promising anti-cancer activity were prepared via the reductive amination of pentoses and hexoses, and subsequently screened for their ability to selectively inhibit the growth of cancerous versus non-cancerous cells. For the first time, we show that this class of compounds possesses anti-proliferative activity, and, while the selective killing of brain cancer (LN18) cells versus matched (SVG-P12) cells was modest, several of the amines, including d-arabinitylamine 1a and d-fucitylamine 1g, exhibited low micromolar IC50 values for HL60 cells. Moreover, these two amines showed good selectivity towards HL60 cells when compared to non-cancerous HEK-293 cells. The compounds also showed low micromolar inhibition of the leukaemic cell line, THP-1. The modes of action of amines 1a and 1g were then determined using yeast chemical genetics, whereby it was established that both compounds affect similar but distinct sets of biochemical pathways. Notably purine nucleoside monophosphate biosynthesis was identified as an enriched mechanism. The rapid synthesis of the amines and their unique mode of action thus make them attractive targets for further development as anti-cancer drugs.

Social wasps are a major pest in many countries around the world. Pathogens may influence wasp populations and could provide an option for population management via biological control. We investigated the pathology of nests of apparently healthy common wasps, Vespula vulgaris, with nests apparently suffering disease. First, next-generation sequencing and metatranscriptomic analysis were used to examine pathogen presence. The transcriptome of healthy and diseased V. vulgaris showed 27 known microbial phylotypes. Four of these were observed in diseased larvae alone (Aspergillus fumigatus, Moellerella wisconsensis, Moku virus, and the microsporidian Vavraia culicis). Kashmir Bee Virus (KBV) was found to be present in both healthy and diseased larvae. Moellerella wisconsensis is a human pathogen that was potentially misidentified in our wasps by the MEGAN analysis: it is more likely to be the related bacteria Hafnia alvei that is known to infect social insects. The closest identification to the putative pathogen identified as Vavraia culicis was likely to be another microsporidian Nosema vulgaris. PCR and subsequent Sanger sequencing using published or our own designed primers, confirmed the identity of Moellerella sp. (which may be Hafnia alvei), Aspergillus sp., KBV, Moku virus and Nosema. Secondly, we used an infection study by homogenising diseased wasp larvae and feeding them to entire nests of larvae in the laboratory. Three nests transinfected with diseased larvae all died within 19 days. No pathogen that we monitored, however, had a significantly higher prevalence in diseased than in healthy larvae. RT-qPCR analysis indicated that pathogen infections were significantly correlated, such as between KBV and Aspergillus sp. Social wasps clearly suffer from an array of pathogens, which may lead to the collapse of nests and larval death.

Emerging infectious diseases (EIDs) are a global threat to honeybees, and spillover from managed bees threaten wider insect populations. Deformed wing virus (DWV), a widespread virus that has become emergent in conjunction with the spread of the mite Varroa destructor , is thought to be partly responsible for global colony losses. The arrival of Varroa in honeybee populations causes a dramatic loss of viral genotypic diversity, favouring a few virulent strains. Here, we investigate DWV spillover in an invasive Hawaiian population of the wasp, Vespula pensylvanica , a honeybee predator and honey-raider. We show that Vespula underwent a parallel loss in DWV variant diversity upon the arrival of Varroa , despite the mite being a honeybee specialist. The observed shift in Vespula DWV and the variant-sharing between Vespula and Apis suggest that these wasps can acquire DWV directly or indirectly from honeybees. Apis prey items collected from Vespula foragers were positive for DWV, indicating predation is a possible route of transmission. We also sought cascading effects of DWV shifts in a broader Vespula pathogen community. We identified concurrent changes in a suite of additional pathogens, as well as shifts in the associations between these pathogens in Vespula . These findings reveal how hidden effects of the Varroa mite can, via spillover, transform the composition of pathogens in interacting species, with potential knock-on effects for entire pathogen communities.

A variety of 6,7-substituted-5,8-quinolinequinones were synthesised and assessed for their anti-tumour and anti-inflammatory activities, and their ability to inhibit the growth of Mycobacterium bovis BCG. In particular, the introduction of a sulfur group at the 7-position of the quinolinequinone led to the discovery of two compounds, 6-methylamino-7-methylsulfanyl-5,8-quinolinequinone ( 10a ) and 6-amino-7-methylsulfonyl-5,8-quinolinequinone ( 12 ), that exhibited selectivity for leukemic cells over T-cells, a highly desirable property for an anti-cancer drug. A number of anti-inflammatory (AI) compounds were also identified, with 6,7-bis-methylsulfanyl-5,8-quinolinequinone ( 18a ) exhibiting the highest AI activity (0.11 μM), while 6,7-dichloro-5,8-quinolinequinone ( 7a ), 6,7-dichloro-2-methyl-5,8-quinolinequinone ( 7b ), and 6,7-bis-phenylsulfanyl-quinoline-5,8-diol ( 19 ) also exhibited good AI activity and specificity. Several quinolinequinone TB-drug candidates were identified. Of these, 6-amino-7-chloro-5,8-quinolinequinone ( 11 ) and 6-amino-7-methanesulfinyl-5,8-quinolinequinone ( 14 ), exhibited low MICs (1.56–3.13 μg/mL) for the 100% growth inhibition of M. Bovis BCG. Some general trends pertaining to the functional group substitution of the quinolinequinone core and biological activity were also identified.

Although the redox-active synthetic isoflavene, phenoxodiol, is in Phase 3 clinical trials for drug-resistant ovarian cancer, and in early stage clinical trials for prostate and cervical cancer, its primary molecular target is unknown. Nevertheless, phenoxodiol inhibits proliferation of many cancer cell lines and induces apoptosis by disrupting FLICE-inhibitory protein, FLIP, expression and by caspase-dependent and -independent degradation of the X-linked inhibitor of apoptosis, XIAP. In addition, phenoxodiol sensitizes drug-resistant tumour cells to anticancer drugs including paclitaxel, carboplatin and gemcitabine. Here, we investigate the effects of phenoxodiol on plasma membrane electron transport (PMET) and cell proliferation in human leukemic HL60 cells and mitochondrial gene knockout HL60rho(o) cells that exhibit elevated PMET. Phenoxodiol inhibited PMET by both HL60 (IC(50) 32 microM) and HL60rho(o) (IC(50) 70 microM) cells, and this was associated with inhibition of cell proliferation (IC(50) of 2.8 and 6.7 microM, respectively), pan-caspase activation and apoptosis. Unexpectedly, phenoxodiol also inhibited PMET by activated murine splenic T cells (IC(50) of 29 microM) as well as T cell proliferation (IC(50) of 2.5 microM). In contrast, proliferation of WI-38 cells and HUVECs was only weakly affected by phenoxodiol. These results indicate that PMET may be a primary target for phenoxodiol in tumour cells and in activated T cells.

Oxidants can activate signaling pathways and modulate a variety of cellular activities. Their action at a molecular level involves the post-translational modification of protein thiols. We have developed a proteomic method to monitor the reduction and oxidation of protein thiols, and identify those thiol proteins most sensitive to oxidation. Cells were disrupted in the presence of N-ethylmaleimide to block the reduced thiol proteins and dithiothreitol was added to reduce the oxidized thiol proteins before labeling with 5-iodoacetamidofluorescein. Two-dimensional (2-D) electrophoresis was used to resolve the labeled samples. We applied the method to Jurkat T lymphocytes and examined the effect of diamide on the oxidized and reduced thiol protein profiles. A small percentage of protein thiols were already oxidized in untreated cells. Exposure of cells to 2 mM diamide for ten minutes led to a dramatic increase in thiol protein oxidation as seen in the oxidized thiol protein map. However, it was difficult to detect any change in the pattern of reduced thiol proteins. Separation of proteins by 2-D electrophoresis revealed approximately 200 thiol proteins that were oxidized by diamide treatment. This method will be valuable in elucidating redox signaling pathways.

The oxidation and reduction of cysteine residues is emerging as an important post-translational control of protein function. We describe a method for fluorescent labelling of either reduced or oxidized thiols in combination with two-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (2DE) to detect changes in the redox proteome of cultured cells. Reduced thiols are labelled with the fluorescent compound 5-iodoacetamidofluorescein. To monitor oxidized thiols, the reduced thiols are first blocked with N-ethyl-maleimide, then the oxidized thiols reduced with dithiothreitol and labelled with 5-iodoacetamidofluorescein. The method is illustrated by treating Jurkat T-lymphoma cells with hydrogen peroxide and monitoring increased labelling of oxidized thiol proteins. A decrease in labelling can also be detected, and this is attributed to the formation of higher oxidation states of cysteine that are not reduced by dithiothreitol.

The oxidation and reduction of cysteine residues is emerging as an important post-translational control of protein function. We describe a method for fluorescent labelling of either reduced or oxidized thiols in combination with two-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (2DE) to detect changes in the redox proteome of cultured cells. Reduced thiols are labelled with the fluorescent compound 5-iodoacetamidofluorescein. To monitor oxidized thiols, the reduced thiols are first blocked with N-ethyl-maleimide, then the oxidized thiols reduced with dithiothreitol and labelled with 5-iodoacetamidofluorescein. The method is illustrated by treating Jurkat T-lymphoma cells with hydrogen peroxide and monitoring increased labelling of oxidized thiol proteins. A decrease in labelling can also be detected, and this is attributed to the formation of higher oxidation states of cysteine that are not reduced by dithiothreitol.

Oxidants can activate signaling pathways and modulate a variety of cellular activities. Their action at a molecular level involves the post-translational modification of protein thiols. We have developed a proteomic method to monitor the reduction and oxidation of protein thiols, and identify those thiol proteins most sensitive to oxidation. Cells were disrupted in the presence of N-ethylmaleimide to block the reduced thiol proteins and dithiothreitol was added to reduce the oxidized thiol proteins before labeling with 5-iodoacetamidofluorescein. Two-dimensional (2-D) electrophoresis was used to resolve the labeled samples. We applied the method to Jurkat T lymphocytes and examined the effect of diamide on the oxidized and reduced thiol protein profiles. A small percentage of protein thiols were already oxidized in untreated cells. Exposure of cells to 2 mM diamide for ten minutes led to a dramatic increase in thiol protein oxidation as seen in the oxidized thiol protein map. However, it was difficult to detect any change in the pattern of reduced thiol proteins. Separation of proteins by 2-D electrophoresis revealed approximately 200 thiol proteins that were oxidized by diamide treatment. This method will be valuable in elucidating redox signaling pathways.

Thiol proteins are important in cellular antioxidant defenses and redox signalling. It is postulated that reactive oxidants cause selective thiol oxidation, but relative sensitivities of different cell proteins and critical targets are not well characterized. We exposed Jurkat cells to H2O2 for 10 min and measured changes in reversibly oxidized proteins by labelling with iodoacetamidofluorescein and two-dimensional electrophoresis. At 200 μM H2O2, which caused activation of the MAP (mitogen-activated protein) kinase ERK (extracellular-signal-regulated kinase), growth arrest and apoptosis, relatively few changes were seen. A total of 28 spots were reversibly oxidized (increased labelling intensity) and 24 decreased. The latter included isoforms of peroxiredoxins 1 and 2, which were irreversibly oxidized. Oxidation of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was striking, and other affected proteins included glutathione S-transferase P1-1, enolase, a regulatory subunit of protein kinase A, annexin VI, the mitotic checkpoint serine/threonine-protein kinase BUB1β, HSP90β (heat-shock protein 90β) and proteosome components. At 20 μM H2O2, changes were fewer, but GAPDH and peroxiredoxin 2 were still modified. Dinitrochlorobenzene treatment, which inhibited cellular thioredoxin reductase and partially depleted GSH, caused reversible oxidation of several proteins, including thioredoxin 1 and peroxiredoxins 1 and 2. Most changes were distinct from those with H2O2, and changes with H2O2 were scarcely enhanced by dinitrochlorobenzene. Relatively few proteins, including deoxycytidine kinase, nucleoside diphosphate kinase and a proteosome activator subunit, responded only to the combined treatment. Thus most of the effects of H2O2 were not linked to thioredoxin oxidation. Our study has identified peroxiredoxin 2 and GAPDH as two of the most oxidant-sensitive cell proteins and has highlighted how readily peroxiredoxins undergo irreversible oxidation.