Single-cell sequencing of tumor-associated macrophages in a Drosophila model.

Introduction: Tumor-associated macrophages may act to either limit or promote tumor growth, yet the molecular basis for either path is poorly characterized. Methods: We use a larval Drosophila model that expresses a dominant-active version of the Ras-oncogene (RasV12) to study dysplastic growth during early tumor progression. We performed single-cell RNA-sequencing of macrophage-like hemocytes to characterize these cells in tumor- compared to wild-type larvae. Hemocytes included manually extracted tumor-associated- and circulating cells. Results and discussion: We identified five distinct hemocyte clusters. In addition to RasV12 larvae, we included a tumor model where the activation of effector caspases was inhibited, mimicking an apoptosis-resistant setting. Circulating hemocytes from both tumor models differ qualitatively from control wild-type cells-they display an enrichment for genes involved in cell division, which was confirmed using proliferation assays. Split analysis of the tumor models further reveals that proliferation is strongest in the caspase-deficient setting. Similarly, depending on the tumor model, hemocytes that attach to tumors activate different sets of immune effectors-antimicrobial peptides dominate the response against the tumor alone, while caspase inhibition induces a shift toward members of proteolytic cascades. Finally, we provide evidence for transcript transfer between hemocytes and possibly other tissues. Taken together, our data support the usefulness of Drosophila to study the response against tumors at the organismic level.

The Influence of Selected Insecticides on the Oxidative Response of Atta sexdens (Myrmicinae, Attini) Workers.

Reactive oxygen species (ROS) are generated as products of normal cellular metabolic activities; however, the use of pesticides to control leafcutter ants leads to unbalanced ROS production. We evaluated the effects of two insecticides (fipronil, sulfluramid) and metallic insecticide complex (magnesium complex [Mg(hesp)2(phen)] (1)) on the superoxide dismutase (SOD), glutathione (GSH) and the overall antioxidant capacity using two different methodologies: total radical-trapping potential (TRAP) and oxygen radical absorbance capacity (ORAC). Media workers of Atta sexdens (C. Linnaeus) were exposed to the insecticides for 24 h, 48 h, 72 h and 96 h before their fat bodies were dissected for analysis. The results showed that although the sulfluramid may cause the production of ROS, its slow action in the organism does not lead to oxidative stress. There is a rise in oxidative stress in workers of leafcutter ants treated with fipronil because SOD significantly increased when compared to the control group. On the other hand, Mg1-complex suppressed both GSH and SOD, indicating that the immune system may be affected by Mg1-complex, which has a delayed activity ideal for its use in chemical pest control. Both TRAP and ORAC evaluated total antioxidant capacities; however, ORAC proved to be a more sensitive method. In conclusion, the Mg1-complex is a new compound that should be further investigated as a potential replacement for fipronil and sulfluramid in pest control.

Chitinase-like proteins promoting tumorigenesis through disruption of cell polarity via enlarged endosomal vesicles.

Introduction: Chitinase-like proteins (CLPs) are associated with tissue-remodeling and inflammation but also with several disorders, including fibrosis, atherosclerosis, allergies, and cancer. However, CLP's role in tumors is far from clear. Methods: Here, we utilize Drosophila melanogaster and molecular genetics to investigate the function of CLPs (imaginal disc growth factors; Idgf's) in RasV12 dysplastic salivary glands. Results and discussion: We find one of the Idgf's members, Idgf3, is transcriptionally induced in a JNK-dependent manner via a positive feedback loop mediated by reactive oxygen species (ROS). Moreover, Idgf3 accumulates in enlarged endosomal vesicles (EnVs) that promote tumor progression by disrupting cytoskeletal organization. The process is mediated via the downstream component, aSpectrin, which localizes to the EnVs. Our data provide new insight into CLP function in tumors and identifies specific targets for tumor control.

Omics-based analysis of honey bee (Apis mellifera) response to Varroa sp. parasitisation and associated factors reveals changes impairing winter bee generation.

The extensive annual loss of honey bees (Apis mellifera L.) represents a global problem affecting agriculture and biodiversity. The parasitic mite Varroa destructor, associated with viral co-infections, plays a key role in this loss. Despite years of intensive research, the complex mechanisms of Varroa - honey bee interaction are still not fully defined. Therefore, this study employed a unique combination of transcriptomic, proteomic, metabolomic, and functional analyses to reveal new details about the effect of Varroa mites and naturally associated factors, including viruses, on honey bees. We focused on the differences between Varroa parasitised and unparasitised ten-day-old worker bees collected before overwintering from the same set of colonies reared without anti-mite treatment. Supplementary comparison to honey bees collected from colonies with standard anti-Varroa treatment can provide further insights into the effect of a pyrethroid flumethrin. Analysis of the honey bees exposed to mite parasitisation revealed alterations in the transcriptome and proteome related to immunity, oxidative stress, olfactory recognition, metabolism of sphingolipids, and RNA regulatory mechanisms. The immune response and sphingolipid metabolism were strongly activated, whereas olfactory recognition and oxidative stress pathways were inhibited in Varroa parasitised honey bees compared to unparasitised ones. Moreover, metabolomic analysis confirmed the depletion of nutrients and energy stores, resulting in a generally disrupted metabolism in the parasitised workers. The combined omics-based analysis conducted on strictly parasitised bees revealed the key molecular components and mechanisms underlying the detrimental effects of Varroa sp. and its associated pathogens. This study provides the theoretical basis and interlinked datasets for further research on honey bee response to biological threats and the development of efficient control strategies against Varroa mites.

1H NMR Profiling of Honey Bee Bodies Revealed Metabolic Differences between Summer and Winter Bees.

In temperate climates, honey bee workers of the species Apis mellifera have different lifespans depending on the seasonal phenotype: summer bees (short lifespan) and winter bees (long lifespan). Many studies have revealed the biochemical parameters involved in the lifespan differentiation of summer and winter bees. However, comprehensive information regarding the metabolic changes occurring in their bodies between the two is limited. This study used proton nuclear magnetic resonance (1H NMR) spectroscopy to analyze the metabolic differences between summer and winter bees of the same age. The multivariate analysis showed that summer and winter bees could be distinguished based on their metabolic profiles. Among the 36 metabolites found, 28 metabolites have displayed significant changes from summer to winter bees. Compared to summer bees, trehalose in winter bees showed 1.9 times higher concentration, and all amino acids except for proline and alanine showed decreased patterns. We have also detected an unknown compound, with a CH3 singlet at 2.83 ppm, which is a potential biomarker that is about 13 times higher in summer bees. Our results show that the metabolites in summer and winter bees have distinctive characteristics; this information could provide new insights and support further studies on honey bee longevity and overwintering.

Winter honeybee (Apis mellifera) populations show greater potential to induce immune responses than summer populations after immune stimuli.

In the temperate climates of central Europe and North America, two distinct honeybee (Apis mellifera) populations are found in colonies: short-living summer bees emerge in spring and survive until summer, whereas long-living winter bees emerge in late August and overwinter. Besides the difference in their life spans, each of these populations fulfils a different role in the colonies and individual bees have distinct physiological and immunological adaptations depending on their roles. For instance, winter worker bees have higher vitellogenin levels and larger reserves of nutrients in the fat body than summer bees. The differences between the immune systems of both populations are well described at the constitutive level; however, our knowledge of its inducibility is still very limited. In this study, we focus on the response of 10-day-old honeybee workers to immune challenges triggered in vivo by injecting heat-killed bacteria, with particular focus on honeybees that emerge and live under hive conditions. Responses to bacterial injections differed between summer and winter bees. Winter bees exhibited a more intense response, including higher expression of antimicrobial genes and antimicrobial activity, as well as a significant decrease in vitellogenin gene expression and its concentration in the hemolymph. The intense immune response observed in winter honeybees may contribute to our understanding of the relationships between colony fitness and infection with pathogens, as well as its association with successful overwintering.

Bioactive Excreted/Secreted Products of Entomopathogenic Nematode Heterorhabditis bacteriophora Inhibit the Phenoloxidase Activity during the Infection.

Entomopathogenic nematodes (EPNs) are efficient insect parasites, that are known for their mutualistic relationship with entomopathogenic bacteria and their use in biocontrol. EPNs produce bioactive molecules referred to as excreted/secreted products (ESPs), which have come to the forefront in recent years because of their role in the process of host invasion and the modulation of its immune response. In the present study, we confirmed the production of ESPs in the EPN Heterorhabditis bacteriophora, and investigated their role in the modulation of the phenoloxidase cascade, one of the key components of the insect immune system. ESPs were isolated from 14- and 21-day-old infective juveniles of H. bacteriophora, which were found to be more virulent than newly emerged nematodes, as was confirmed by mortality assays using Galleria mellonella larvae. The isolated ESPs were further purified and screened for the phenoloxidase-inhibiting activity. In these products, a 38 kDa fraction of peptides was identified as the main candidate source of phenoloxidase-inhibiting compounds. This fraction was further analyzed by mass spectrometry and the de novo sequencing approach. Six peptide sequences were identified in this active ESP fraction, including proteins involved in ubiquitination and the regulation of a Toll pathway, for which a role in the regulation of insect immune response has been proposed in previous studies.

The Effect of Foraging on Bumble Bees, Bombus terrestris, Reared under Laboratory Conditions.

Bumble bees are important pollinators broadly used by farmers in greenhouses and under conditions in which honeybee pollination is limited. As such, bumble bees are increasingly being reared for commercial purposes, which brings into question whether individuals reared under laboratory conditions are fully capable of physiological adaptation to field conditions. To understand the changes in bumble bee organism caused by foraging, we compared the fundamental physiological and immunological parameters of Bombus terrestris workers reared under constant optimal laboratory conditions with workers from sister colonies that were allowed to forage for two weeks in the field. Nutritional status and immune response were further determined in wild foragers of B. terrestris that lived under the constant influence of natural stressors. Both wild and laboratory-reared workers subjected to the field conditions had a lower protein concentration in the hemolymph and increased antimicrobial activity, the detection of which was limited in the non-foragers. However, in most of the tested parameters, specifically the level of carbohydrates, antioxidants, total hemocyte concentration in the hemolymph and melanization response, we did not observe any significant differences between bumble bee workers produced in the laboratory and wild animals, nor between foragers and non-foragers. Our results show that bumble bees reared under laboratory conditions can mount a sufficient immune response to potential pathogens and cope with differential food availability in the field, similarly to the wild bumble bee workers.

The Year of the Honey Bee (Apis mellifera L.) with Respect to Its Physiology and Immunity: A Search for Biochemical Markers of Longevity.

It has been known for many years that in temperate climates the European honey bee, Apis mellifera, exists in the form of two distinct populations within the year, short-living summer bees and long-living winter bees. However, there is only limited knowledge about the basic biochemical markers of winter and summer populations as yet. Nevertheless, the distinction between these two kinds of bees is becoming increasingly important as it can help beekeepers to estimate proportion of long-living bees in hives and therefore in part predict success of overwintering. To identify markers of winter generations, we employed the continuous long-term monitoring of a single honey bee colony for almost two years, which included measurements of physiological and immunological parameters. The results showed that the total concentration of proteins, the level of vitellogenin, and the antibacterial activity of haemolymph are the best three of all followed parameters that are related to honey bee longevity and can therefore be used as its markers.

Adipokinetic hormone and adenosine interfere with nematobacterial infection and locomotion in Drosophila melanogaster.

This study examined how adipokinetic hormone (AKH) and adenosine affect defense responses in Drosophila melanogaster larvae infected with entomopathogenic nematodes (EPN, Steinernema carpocapsae and Heterorhabditis bacteriophora). Three loss-of-function mutant larvae were tested: Akh1, AdoR1 (adenosine receptor), and Akh1 AdoR1. Mortality decreased in all mutants post-EPN infection compared with the control (w1118). Additionally, co-application of external AKH with EPN significantly increased mortality beyond rates observed in EPN-only treatment, while also elevating carbon dioxide production, a measure of metabolism. Furthermore trehalose levels increased in both w1118 and Akh1 larvae post-EPN infection, but the latter group exhibited a lower increase and total trehalose levels. Interestingly, baseline trehalose was relatively high in untreated AdoR1 and Akh1 AdoR1 mutants, with levels remaining unaffected by infection. Infection also elevated haemolymph lipid content overall, but the different mutations did not substantially influence this change. In contrast, haemolymph protein content dropped after EPN infection in all tested groups, but this decline was more intense among Akh1. In uninfected larvae mutations decreased antioxidative capacity in Akh1 and increased in AdoR1, however, its post-infection increases were similar in all mutants, suggesting that antioxidant response in Drosophila involves mechanisms also beyond AKH and adenosine. Furthermore, AKH application in w1118 larvae significantly increased movement distance and percentage of larval activity, but reduced velocity. Mutations of Akh and AdoR did not strongly affect locomotion.

Caffeine administration alters the behaviour and development of Galleria mellonella larvae.

The effect of feeding caffeine on the behaviour and neural proteome of Galleria mellonella larvae was assessed. Caffeine was administered to larvae by force feeding and the metabolites theobromine and theophylline were subsequently detected by RP-HPLC analysis. Administration of caffeine to larvae resulted in reduced movement and a reduction in the formation of pupae. The production of the muscle relaxant theophylline may contribute to the reduction in larval movement. Analysis of the changes in proteome of the brain and surrounding tissues of caffeine fed larvae revealed an increase in the abundance of immune related proteins such as immune-related Hdd1 (6.28 fold increase) and hemolin (1.68 fold increase), ATPase associated proteins such as H+ transporting ATP synthase O subunit isoform 1 (1.87 fold increase) and H+ transporting ATP synthase delta subunit (1.53 fold increase) and proteins indicative of brain trauma such as troponin T transcript variant B, partial (1.55 fold increase). Proteins involved in development and protein degradation such as SUMO-activating enzyme subunit 1 (3.08 fold decrease) and chitin deacetylase, partial (3.67 fold decrease) were decreased in abundance. The results presented here indicate that caffeine is metabolised in a similar way in G. mellonella larvae to that in mammals and results in a variety of behavioural and developmental alterations. Utilisation of insects for studying the effects of caffeine and other neuroactive compounds may offer new insights into their mode of action and reduce the need to use mammals for this type of analysis.

Monitoring the effect of pathogenic nematodes on locomotion of Drosophila larvae.

One of the key factors that determine the interaction between hosts and their parasites is the frequency of their interactions, which depends on the locomotory behavior of both parts. To address host behavior we used natural infections involving insect pathogenic nematodes and Drosophila melanogaster larvae as hosts. Using a modified version of a recently described method (FIMTrack) to assess several parameters in larger sets of animals, we initially detected specific differences in larval food searching when comparing Drosophila strains. These differences were further influenced by the presence of nematodes. Given a choice, Drosophila larvae clearly avoided nematodes irrespective of their genetic background. Our newly developed methods will be useful to test candidate genes and pathways involved in host/pathogen interactions in general and to assess specific parameters of their interaction.

The Immune Phenotype of Three Drosophila Leukemia Models.

Many leukemia patients suffer from dysregulation of their immune system, making them more susceptible to infections and leading to general weakening (cachexia). Both adaptive and innate immunity are affected. The fruit fly Drosophila melanogaster has an innate immune system, including cells of the myeloid lineage (hemocytes). To study Drosophila immunity and physiology during leukemia, we established three models by driving expression of a dominant-active version of the Ras oncogene (RasV12 ) alone or combined with knockdowns of tumor suppressors in Drosophila hemocytes. Our results show that phagocytosis, hemocyte migration to wound sites, wound sealing, and survival upon bacterial infection of leukemic lines are similar to wild type. We find that in all leukemic models the two major immune pathways (Toll and Imd) are dysregulated. Toll-dependent signaling is activated to comparable extents as after wounding wild-type larvae, leading to a proinflammatory status. In contrast, Imd signaling is suppressed. Finally, we notice that adult tissue formation is blocked and degradation of cell masses during metamorphosis of leukemic lines, which is akin to the state of cancer-dependent cachexia. To further analyze the immune competence of leukemic lines, we used a natural infection model that involves insect-pathogenic nematodes. We identified two leukemic lines that were sensitive to nematode infections. Further characterization demonstrates that despite the absence of behavioral abnormalities at the larval stage, leukemic larvae show reduced locomotion in the presence of nematodes. Taken together, this work establishes new Drosophila models to study the physiological, immunological, and behavioral consequences of various forms of leukemia.

Analysis of the acute response of Galleria mellonella larvae to potassium nitrate.

Potassium nitrate (E252) is widely used as a food preservative and has applications in the treatment of high blood pressure however high doses are carcinogenic. Larvae of Galleria mellonella were administered potassium nitrate to establish whether the acute effects in larvae correlated with those evident in mammals. Intra-haemocoel injection of potassium nitrate resulted in a significant increase in the density of circulating haemocytes and a small change in the relative proportions of haemocytes but haemocytes showed a reduced fungicidal ability. Potassium nitrate administration resulted in increased superoxide dismutase activity and in the abundance of a range of proteins associated with mitochondrial function (e.g. mitochondrial aldehyde dehydrogenase, putative mitochondrial Mn superoxide dismutase), metabolism (e.g. triosephosphate isomerase, glyceraldehyde 3 phosphate dehydrogenase) and nitrate metabolism (e.g. aliphatic nitrilase, glutathione S-transferase). A strong correlation exists between the toxicity of a range of food preservatives when tested in G. mellonella larvae and rats. In this work a correlation between the effect of potassium nitrate in larvae and mammals is shown and opens the way to the utilization of insects for studying the in vivo acute and chronic toxicity of xenobiotics.