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.

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.

Numerical and Experimental Analysis of the Load-Carrying Capacity of a Timber Semi-Rigid Dowel-Type Connection.

The paper deals with the analysis of the load-carrying capacity of a timber semi-rigid connection created from a system of two stands and a rung. The connection was made from glued laminated timber with metal mechanical dowel-type fasteners. Not only a common combination of bolts and dowels, but also fully threaded screws were used for the connection. The aim of the research and its motivation was to replace these commonly used fasteners with more modern ones, to shorten and simplify the assembly time, and to improve the load-carrying capacity of this type of connection. Each of these two types of connections was loaded statically, with a slow increase in force until failure. The paper presents results of the experimental testing. Three specimens were made and tested for each type of the connection. Experimental results were subsequently compared with numerical models. The achieved results were also compared with the assumption according to the currently valid standard. The results indicate that a connection using fully threaded screws provides a better load-carrying capacity.

Numerical and Experimental Analysis of the Rotational Stiffness of a Timber Semi-Rigid Dowel-Type Connection.

The paper deals with the analysis of the rotational stiffness of a semirigid connection created from a system of two stands and a rung. The connection was made from glued laminated timber with metal mechanical dowel-type fasteners. Not only a common combination of bolts and dowels but also fully threaded screws were used for the connection. The aim of the research and its motivation was to replace commonly used fasteners with more modern ones, to shorten and simplify the assembly time, and to improve the load-carrying capacity of this type of connection. Each of these two types of connection was loaded to the level of 60%, 80%, and 100% of the ultimate limit state value. Subsequently, the rotational stiffness was determined for each load level after five loading and unloading cycles. This paper presents the results and comparison of the experimental testing and the numerical modeling. The obtained results were also compared with the assumption according to the currently valid standard.

Load-Carrying Capacity of Double-Shear Bolted Connections with Slotted-In Steel Plates in Squared and Round Timber Based on the Experimental Testing, European Yield Model, and Linear Elastic Fracture Mechanics.

Nowadays, the use of timber as a building material is gaining more prominence. When designing timber structures, it is necessary to pay increased attention to the design of their connections. The commonly used connections are dowel-type connections, which are often used in combination with steel plates slotted into cut-outs in timber members. The presented paper deals with the behavior of double-shear bolted connections of squared timber and round timber with slotted-in steel plates. Several variants of connections with different distances between the fastener and the loaded end were selected for the experimental testing. A total of six types of test specimens were made from spruce timber, for which their selected physical properties were determined and evaluated before the experimental testing. Test specimens of bolted connections were first tested in tension parallel to the grain until failure under quasi-static loading. The connections were broken by splitting. Ductile failure preceded brittle failure. The actual load-carrying capacities were lowest for the lowest end distance. The load-carrying capacities for the middle and the longest end distances were comparable. The results of the experiments were then used for comparison with calculation procedures according to the standard for the design of timber structures and with calculations according to the theory of linear elastic fracture mechanics. The experiments and the analytical models were supported by a simple numerical analysis based on the finite element method.

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.

Changes in haemolymph parameters and insect ability to respond to immune challenge during overwintering.

Overwintering is a challenging period in the life of temperate insects. A limited energy budget characteristic of this period can result in reduced investment in immune system. Here, we investigated selected physiological and immunological parameters in laboratory-reared and field-collected harlequin ladybirds (Harmonia axyridis). For laboratory-reared beetles, we focused on the effects of winter temperature regime (cold, average, or warm winter) on total haemocyte concentration aiming to investigate potential effects of ongoing climate change on immune system in overwintering insects. We recorded strong reduction in haemocyte concentration during winter; however, there were only limited effects of winter temperature regime on changes in haemocyte concentration in the course of overwintering. For field-collected beetles, we measured additional parameters, specifically: total protein concentration, antimicrobial activity against Escherichia coli, and haemocyte concentration before and after overwintering. The field experiment did not investigate effects of winter temperature, but focused on changes in inducibility of insect immune system during overwintering, that is, measured parameters were compared between naïve beetles and those challenged by Escherichia coli. Haemocyte concentration decreased during overwintering, but only in individuals challenged by Escherichia coli. Prior to overwintering, the challenged beetles had a significantly higher haemocyte concentration compared to naïve beetles, whereas no difference was observed after overwintering. A similar pattern was observed also for antimicrobial activity against Escherichia coli as challenged beetles outperformed naïve beetles before overwintering, but not after winter. In both sexes, total protein concentration increased in the course of overwintering, but females had a significantly higher total protein concentration in their hemolymph compared to males. In general, our results revealed that insect's ability to respond to an immune challenge is significantly reduced in the course of overwintering.

Heptabladed ß-propeller lectins PLL2 and PHL from Photorhabdus spp. recognize O-methylated sugars and influence the host immune system.

O-methylation is an unusual sugar modification with a function that is not fully understood. Given its occurrence and recognition by lectins involved in the immune response, methylated sugars were proposed to represent a conserved pathogen-associated molecular pattern. We describe the interaction of O-methylated saccharides with two ß-propeller lectins, the newly described PLL2 from the entomopathogenic bacterium Photorhabdus laumondii, and its homologue PHL from the related human pathogen Photorhabdus asymbiotica. The crystal structures of PLL2 and PHL revealed up to 10 out of 14 potential binding sites per protein subunit to be occupied with O-methylated structures. The avidity effect strengthens the interaction by 4 orders of magnitude. PLL2 and PHL also interfere with the early immune response by modulating the production of reactive oxygen species and phenoloxidase activity. Since bacteria from Photorhabdus spp. have a complex life cycle involving pathogenicity towards different hosts, the involvement of PLL2 and PHL might contribute to the pathogen overcoming insect and human immune system defences in the early stages of infection. DATABASES: Structural data are available in PDB database under the accession numbers 6RG2, 6RGG, 6RFZ, 6RG1, 6RGU, 6RGW, 6RGJ, and 6RGR.

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.

Effects of Loaded End Distance and Moisture Content on the Behavior of Bolted Connections in Squared and Round Timber Subjected to Tension Parallel to the Grain.

This article presents the results of static tests on bolted connections in squared and round timber with inserted steel plates. The experiment evaluates structural timber connections with different distances between the fastener and the loaded end at different moisture contents. Specimens were loaded by tension parallel to the grain and load-deformation diagrams were recorded. Fifty-six specimens with three different distances between the fastener and the loaded end, at different moisture contents, were tested. The results were statistically evaluated using regression analysis, complemented with load-deformation curves, and compared with calculations according to the valid standard for design of timber structures. A decrease in the evaluated load-carrying capacity with increasing moisture content was confirmed experimentally. A slight increase in the evaluated load-carrying capacity with increasing fastener distance from the loaded end was found.

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.

Characterization of novel bangle lectin from Photorhabdus asymbiotica with dual sugar-binding specificity and its effect on host immunity.

Photorhabdus asymbiotica is one of the three recognized species of the Photorhabdus genus, which consists of gram-negative bioluminescent bacteria belonging to the family Morganellaceae. These bacteria live in a symbiotic relationship with nematodes from the genus Heterorhabditis, together forming a complex that is highly pathogenic for insects. Unlike other Photorhabdus species, which are strictly entomopathogenic, P. asymbiotica is unique in its ability to act as an emerging human pathogen. Analysis of the P. asymbiotica genome identified a novel fucose-binding lectin designated PHL with a strong sequence similarity to the recently described P. luminescens lectin PLL. Recombinant PHL exhibited high affinity for fucosylated carbohydrates and the unusual disaccharide 3,6-O-Me2-Glcß1-4(2,3-O-Me2)Rhaα-O-(p-C6H4)-OCH2CH2NH2 from Mycobacterium leprae. Based on its crystal structure, PHL forms a seven-bladed ß-propeller assembling into a homo-dimer with an inter-subunit disulfide bridge. Investigating complexes with different ligands revealed the existence of two sets of binding sites per monomer-the first type prefers l-fucose and its derivatives, whereas the second type can bind d-galactose. Based on the sequence analysis, PHL could contain up to twelve binding sites per monomer. PHL was shown to interact with all types of red blood cells and insect haemocytes. Interestingly, PHL inhibited the production of reactive oxygen species induced by zymosan A in human blood and antimicrobial activity both in human blood, serum and insect haemolymph. Concurrently, PHL increased the constitutive level of oxidants in the blood and induced melanisation in haemolymph. Our results suggest that PHL might play a crucial role in the interaction of P. asymbiotica with both human and insect hosts.

A Novel Fucose-binding Lectin from Photorhabdus luminescens (PLL) with an Unusual Heptabladed ß-Propeller Tetrameric Structure.

Photorhabdus luminescens is known for its symbiosis with the entomopathogenic nematode Heterorhabditis bacteriophora and its pathogenicity toward insect larvae. A hypothetical protein from P. luminescens was identified, purified from the native source, and characterized as an l-fucose-binding lectin, named P. luminescens lectin (PLL). Glycan array and biochemical characterization data revealed PLL to be specific toward l-fucose and the disaccharide glycan 3,6-O-Me2-Glcß1-4(2,3-O-Me2)Rhaα-O-(p-C6H4)-OCH2CH2NH2 PLL was discovered to be a homotetramer with an intersubunit disulfide bridge. The crystal structures of native and recombinant PLL revealed a seven-bladed ß-propeller fold creating seven putative fucose-binding sites per monomer. The crystal structure of the recombinant PLL·l-fucose complex confirmed that at least three sites were fucose-binding. Moreover, the crystal structures indicated that some of the other sites are masked either by the tetrameric nature of the lectin or by incorporation of the C terminus of the lectin into one of these sites. PLL exhibited an ability to bind to insect hemocytes and the cuticular surface of a nematode, H. bacteriophora.

Genome-wide transcriptional analysis of Drosophila larvae infected by entomopathogenic nematodes shows involvement of complement, recognition and extracellular matrix proteins.

Heterorhabditis bacteriophora is an entomopathogenic nematode (EPN) which infects its host by accessing the hemolymph where it releases endosymbiotic bacteria of the species Photorhabdus luminescens. We performed a genome-wide transcriptional analysis of the Drosophila response to EPN infection at the time point at which the nematodes reached the hemolymph either via the cuticle or the gut and the bacteria had started to multiply. Many of the most strongly induced genes have been implicated in immune responses in other infection models. Mapping of the complete set of differentially regulated genes showed the hallmarks of a wound response, but also identified a large fraction of EPN-specific transcripts. Several genes identified by transcriptome profiling or their homologues play protective roles during nematode infections. Genes that positively contribute to controlling nematobacterial infections encode: a homolog of thioester-containing complement protein 3, a basement membrane component (glutactin), a recognition protein (GNBP-like 3) and possibly several small peptides. Of note is that several of these genes have not previously been implicated in immune responses.

An improved method for nematode infection assays in Drosophila larvae.

The infective juveniles (IJs) of entomopathogenic nematodes (EPNs) seek out host insects and release their symbiotic bacteria into their body cavity causing septicaemia, which eventually leads to host death. The interaction between EPNs and their hosts are only partially understood, in particular the host immune responses appears to involve pathways other than phagocytosis and the canonical transcriptional induction pathways. These pathways are genetically tractable and include for example clotting factors and lipid mediators. The aim of this study was to optimize the nematode infections in Drosophila melanogaster larvae, a well-studied and genetically tractable model organism. Here we show that two nematode species namely Steinernema feltiae and Heterorhabditis bacteriophora display different infectivity toward Drosophila larvae with the latter being less pathogenic. The effects of supporting media and IJ dosage on the mortality of the hosts were assessed and optimized. Using optimum conditions, a faster and efficient setup for nematode infections was developed. This newly established infection model in Drosophila larvae will be applicable in large scale screens aimed at identifying novel genes/pathways involved in innate immune responses.

Clotting factors and eicosanoids protect against nematode infections.

We show that hemolymph clotting protects Drosophila melanogaster against infections with an entomopathogenic nematode and its symbiotic bacterium. We also provide biochemical and genetic evidence for an involvement of eicosanoids in the same infection model. Taken together, our results confirm the conserved nature of the immune function of clot formation.