A potential trade-off between offense and defense in honeybee innate immunity: Reduced phagocytosis in honeybee hemocytes correlates with a protective response after exposure to imidacloprid and amitraz.

Phagocytosis "offense" is a crucial process to protect the organism from diseases and the effects of foreign particles. Insects rely on the innate immune system and thus any hindrance to phagocytosis may greatly affect their resistance to diseases and response to pathogens. The European honeybee, a valuable species due to its economic and environmental contribution, is being challenged by colony collapse disorder leading to its decline. Exposure to multiple factors including pesticides like imidacloprid and amitraz may negatively alter their immune response and ultimately make them more susceptible to diseases. In this study, we compare the effect of different concentrations and mixtures of imidacloprid and amitraz with different concentrations of the immune stimulant, zymosan A. Results show that imidacloprid and amitraz have a synergistic negative effect on phagocytosis. The lowered phagocytosis induces significantly higher hemocyte viability suggesting a negatively correlated protective mechanism "defense" from pesticide-associated damage but may not be protective from pathogens.

Co-culture between Miscanthus x giganteus and Trifolium repens L. to enhance microbial activity, biomass and density in a PAH contaminated technosol.

Phytoremediation is a biological soil remediation technique using plants and their associated microorganisms to clean-up contaminated soils and improve soils' quality. We tested whether a co-culture between Miscanthus x giganteus (MxG) and Trifolium repens L. would enhance the soil biological quality. The objective was to determine the influence of MxG in mono- and in co-culture with white clover on the soil microbial activity, biomass and density. MxG was tested in mono- and in co-culture with white clover in a mesocosm over 148 days. The microbial respiration (CO2 production), the microbial biomass and the microbial density of the technosol were measured. Results showed that MxG induced an increase in microbial activity in the technosol compared to the non-planted condition with the co-culture having a greater impact. Regarding the bacterial density, MxG in mono- and in co-culture significantly increased the 16S rDNA gene copy number. The co-culture increased the microbial biomass, the fungal density and stimulated the degrading bacterial population, contrary to the monoculture and the non-planted condition. We can conclude the co-culture between MxG and white clover was more interesting than MxG monoculture in regards to the technosol biological quality and its potential for PAH remediation improvement.

Our precedent results have shown the benefits of using Miscanthus x giganteus in association with Trifolium repens L. to improve polycyclic aromatic hydrocarbons dissipation and decrease soil ecotoxicity compared to monocultures. In this study we focused on the plant species' influence on the soil's biological quality to improve MxG biomass productivity in the long term and phytoremediation. Many bioindicators were used such as microbial activity, microbial biomass as well as bacteria, fungi and PAH-degrading bacteria density.We showed it was more beneficial to use co-culture instead of MxG monoculture to improve biological technosol quality and in particular microbial activity and biomass as well as fungi and PAH-degrading bacteria density.

Imidacloprid and acetamiprid synergistically downregulate spaetzle and myD88 of the Toll pathway in haemocytes of the European honeybee (Apis mellifera).

Pollinator health has been of critical concern over the last few decades. The prevalence of the honeybee Colony Collapse Disorder (CCD), changing climate, and the rise of vector-borne honeybee diseases by Varroa destructor, have played a major role in the rapid decline of global honeybee populations. Honeybees are environmentally and economically significant actors in biodiversity. The impact of agricultural practices, such as pesticide use, has exacerbated the negative effects on honeybees. We demonstrate the synergistic effect of cocktails of the neonicotinoids imidacloprid and acetamiprid on honeybee haemocytes. Two genes responsible for critical immune responses, spaetzle and myD88, are consistently dysregulated following exposure to either neonicotinoid alone or as a mixture with or without an immune challenge. The 2018 ban of neonicotinoids in Europe, followed by the 2020 reauthorisation of imidacloprid in France and the current consideration to reinstate acetamiprid underscores the need to evaluate their cumulative impact on honeybee health.

Corrigendum: A clash on the Toll pathway: competitive action between pesticides and zymosan A on components of innate immunity in Apis mellifera.

[This corrects the article DOI: 10.3389/fimmu.2023.1247582.].

A clash on the Toll pathway: competitive action between pesticides and zymosan A on components of innate immunity in Apis mellifera.

Background: The immune system of honeybees includes multiple pathways that may be affected by pesticide exposure decreasing the immune competencies of bees and increasing their susceptibility to diseases like the fungal Nosema spp. infection, which is detected in collapsed colonies. Methods: To better understand the effect of the co-presence of multiple pesticides that interact with bees like imidacloprid and amitraz, we evaluated the expression of immune-related genes in honeybee hemocytes. Results: Imidacloprid, amitraz, and the immune activator, zymosan A, mainly affect the gene expression in the Toll pathway. Discussion: Imidacloprid, amitraz, and zymosan A have a synergistic or an antagonistic relationship on gene expression depending on the level of immune signaling. The presence of multiple risk factors like pesticides and pathogens requires the assessment of their complex interaction, which has differential effects on the innate immunity of honeybees as seen in this study.

Differential Production of Nitric Oxide and Hydrogen Peroxide among Drosophila melanogaster, Apis mellifera, and Mamestra brassicae Immune-Activated Hemocytes after Exposure to Imidacloprid and Amitraz.

Invertebrates have a diverse immune system that responds differently to stressors such as pesticides and pathogens, which leads to different degrees of susceptibility. Honeybees are facing a phenomenon called colony collapse disorder which is attributed to several factors including pesticides and pathogens. We applied an in vitro approach to assess the response of immune-activated hemocytes from Apis mellifera, Drosophila melanogaster and Mamestra brassicae after exposure to imidacloprid and amitraz. Hemocytes were exposed to the pesticides in single and co-exposures using zymosan A for immune activation. We measured the effect of these exposures on cell viability, nitric oxide (NO) production from 15 to 120 min and on extracellular hydrogen peroxide (H2O2) production after 3 h to assess potential alterations in the oxidative response. Our results indicate that NO and H2O2 production is more altered in honeybee hemocytes compared to D. melanogaster and M. brassicae cell lines. There is also a differential production at different time points after pesticide exposure between these insect species as contrasting effects were evident with the oxidative responses in hemocytes. The results imply that imidacloprid and amitraz act differently on the immune response among insect orders and may render honeybee colonies more susceptible to infection and pests.

Trace metal uptake by native plants growing on a brownfield in France: zinc accumulation by Tussilago farfara L.

Several human activities such as mining, smelting, or transportations lead to trace metal pollution in soil. The presence of these pollutants can represent environmental and organism health risks. Phytoextraction can be used to remediate trace metal-contaminated soils. It uses the plants' ability to remove trace metals from soil and to accumulate them in their shoots, which can then be harvested. We studied the spontaneous vegetation growing on a brownfield located in France. The use of native plants is interesting since spontaneous vegetation is already well adapted to the site's environmental conditions leading to a better survival and growth than non-native plants. Ten native plant species were sampled, and the Cr, Cu, Cd, Ni, Pb, and Zn concentrations present in their shoots were measured. In order to determine the plant's capacity to extract trace metals from the soil, the bioconcentration factor (BCF) was calculated for each plant and trace metal. Plants with a BCF greater than 1 are able to accumulate trace metals in their shoots and could be a good candidate to be used in phytoextraction. Results underscored one new accumulator plant for Zn, Tussilago farfara L., with a BCF value of 3.069. No hyperaccumulator was found among the other sampled plants. Our preliminary study showed that T. farfara is able to accumulate zinc in its shoots. Moreover, this native plant is a pioneer species able to quickly colonize various habitats by vegetative multiplication. That is why T. farfara  L. could be interesting for zinc phytoextraction and could be worth further studies.

Phagocytic activity of human macrophages and Drosophila hemocytes after exposure to the neonicotinoid imidacloprid.

Neonicotinoid insecticides are increasingly used in modern pest control and in conventional agriculture. Their residues are frequently found in our environment and in our food leading to chronic exposure of pollinating insects and humans. Indeed, evidence has become stronger that chronic exposure to neonicotinoids might have a direct impact on the immune response of invertebrates and vertebrates. Therefore, we compared the cellular immune response of human macrophages (THP-1) and Drosophila melanogaster hemocytes (Schneider 2 cells) after exposure to four different concentrations of the neonicotinoid imidacloprid. Cells were immune activated with LPS (lipopolysaccharide) of Escherichia coli to compare the phagocytic activity of immune activated and non-activated cells during pesticide exposure. Drosophila cells were more strongly affected by the insecticide than human macrophages. Even though imidacloprid showed an adverse effect on phagocytosis on both cells while immune activated, it decreased phagocytosis in Drosophila cells at shorter exposure time and without immune activation.

Imidacloprid intensifies its impact on honeybee and bumblebee cellular immune response when challenged with LPS (lippopolysacharide) of Escherichia coli.

Insect hemocytes play an important role in insects' defense against environmental stressors as they are entirely dependent on their innate immune system for pathogen defense. In recent years a dramatic decline of pollinators has been reported in many countries. The drivers of this declines appear to be associated with pathogen infections like viruses, bacteria or fungi in combination with pesticide exposure. The aim of this study was thus to investigate the impact of imidacloprid, a neonicotinoid insecticide, on the cellular immune response of two pollinators (Apis mellifera and Bombus terrestris) during simultaneous immune activation with LPS (lipopolysaccharide) of Escherichia coli. For this purpose the phagocytosis capacity as well as the production of H2O2 and NO of larval hemocytes, exposed to five different imidacloprid concentrations in vitro, was measured. All used pesticide concentrations showed a weakening effect on phagocytosis with but also without LPS activation. Imidacloprid decreased H2O2 and increased NO production in honeybees. Immune activation by LPS clearly reinforced the effect of imidacloprid on the immune response of hemocytes in all three immune parameters tested. Bumblebee hemocytes appeared more sensitive to imidacloprid during phagocytosis assays while imidacloprid showed a greater impact on honeybee hemocytes during H2O2 and NO production.

Comparison of two bacterial DNA extraction methods from non-polluted and polluted soils.

DNA extraction from soil samples is a critical step for molecular biology analyses. The present study compared the efficiency of two DNA isolation methods from non-polluted and polluted soils with or without the presence of a plant. Both applied methods used chemical and physical lyses, but method 1 had an additional physical disruption. The main difference between these two methods was the humic acid purification technique as it was carried out during cell lysis for method 1 and after cell lysis for method 2. Samples were assessed on the basis of their yield and DNA purity as well as their bacterial quantity and diversity. Based on our results, method 1 proved to be more effective at removing protein and RNA, whereas method 2 proved to be more effective at removing humic acids. Although no differences were obtained in terms of the DNA yield, both the bacterial quantity and community structure were affected by the method used. Method 1 allowed for the recovery of more information than method 2, and polluted soil was more sensitive to the DNA extraction procedure. We recommend carefully selecting the DNA extraction method, especially when soil is disturbed.

Hemocyte responses of Dreissena polymorpha following a short-term in vivo exposure to titanium dioxide nanoparticles: preliminary investigations.

The widespread use of titanium-based nanoparticles and their environmental release may pose a significant risk to aquatic organisms within freshwater ecosystems. Suspension-feeder invertebrates like bivalve molluscs represent a unique target group for nanoparticle toxicology. The aim of this work was to investigate the short-term responses of Dreissena polymorpha hemocytes after in vivo exposure to titanium dioxide nanoparticles (TiO(2) NP). For this purpose, freshwater mussels were exposed to P25 TiO(2) NP at the concentrations of 0.1, 1, 5 and 25mg/L during 24h. Viability, phagocytosis activity and mitogen activated protein kinase (MAPK) phosphorylation level of ERK 1/2 and p38 in hemocytes extracted from exposed mussels were compared to those from control specimens. Results demonstrated an inhibition of the phagocytosis activity after exposure to TiO(2) NP at 0.1 and 1mg/L. Similar trends, albeit less pronounced, were reported for higher concentrations of NP. Transmission electron microscopy showed for the first time the internalization of TiO(2) NP into Dreissena polymorpha hemocytes. Besides, exposure to NP increased the ERK 1/2 phosphorylation levels in all treatments. Concerning the phosphorylation level of p38, only exposures to 5 and 25mg/L of NP induced significant p38 activation in comparison to that of the control. Finally, these short-term effects observed at environmentally relevant concentrations highlighted the need for further studies concerning ecotoxicological evaluation of nanoparticle release into an aquatic environment.

Contribution of Miscanthus x giganteus root exudates to the biostimulation of PAH degradation: an in vitro study.

Phytoremediation is considered as a promising and cost-effective method to enhance bioremediation of polluted soils. Exudation of plant root secondary metabolites similar to organic pollutants may induce the expression of microbial degradative enzymes and favour cometabolism of xenobiotics. We investigated the contribution of Miscanthus x giganteus root exudates in the biostimulation of PAH-degradation. This perennial grass was chosen because of its capability to grow on polluted soils and its high biomass production for non-food purposes. First, the impact of cometabolism phenomena was evaluated on the selective enrichment of pyrene-degrading bacterial consortia. The identification of each isolated strains following incubation with pyrene only, "pyrene+phenanthrene", "pyrene+salycilate" or "pyrene+diesel fuel" showed a varying bacterial diversity and pyrene-degrading ability, depending on the co-substrate used. Then, a microplate assay was designed, based on the simultaneous measurement of bacterial consortia growth and degradation activity, in the presence of PAH and total root exudates. Results showed that i) the addition of root exudates was efficient for promoting bacterial growth, ii) but a selective enrichment of PAH-degraders compared to aliphatic ones could be clearly demonstrated, thereby conducing to an enhanced PAH catabolism. The identification of plant secondary metabolites showed the presence of a broad range of flavonoid-derived compounds that could play a role in cometabolic processes. Microplate assays with the two major molecules, quercetin and rutin, suggested a partial involvement of these compounds in biostimulation processes. Further investigations with the other identified secondary metabolites (apigenin, isovitexin, catechin, gallic and caffeic acid) should provide more information on the exudate-PAH cometabolic degradation phenomenon.

Influences of ozone exposure upon macrophage responsivity to N-formyl-methionyl-leucyl-phenylalanine: mobility and metabolic changes.

Alveolar macrophages represent one of the first lines of cell defence in the lungs. They employ several mechanisms, including phagocytosis and secretion of reactive oxygen and nitrogen species. fMLP, a formylated peptide of bacterial origin, is a potent inducer of phagocyte chemotaxis and is also involved in generating antimicrobial agents such as nitric oxide (NO) and hydrogen peroxide (H2O2). In this study we analysed the in vitro effects of fMLP on the mobility of the THP-1 cell line, which served as a model for alveolar macrophages. Cell mobility and cytotoxicity were also analysed after pre-exposures to an atmosphere polluted with ozone (0.03-0.5 ppm) followed by a fMLP treatment. Finally, the secreted molecules (H2O2 and NO) were measured after ozone exposures ranging from 5 to 30 min and fMLP action. Activation by fMLP alone induced cell movement, whereas pre-exposure to the ozone concentrations decreased it. Addition of fMLP had different effects on cytotoxicity, mobility and metabolite secretion by the cells: (1) cytotoxicity increased depending on ozone concentrations and exposure times; (2) during the first 5 min and for all ozone concentrations, an average decrease of 50% of activated cell mobility was observed; (3) H2O2 was increased, even in combination with ozone; (4) NO was detected at 731 nM, a result that was not affected by ozone pre-exposure.

Precipitation of silver-thiosulfate complex and immobilization of silver by Cupriavidus metallidurans CH34.

Cupriavidus metallidurans CH34 is a facultative chemolithotrophic bacterium that possesses two megaplasmids (pMOL28 and pMOL30) that confer resistance to eleven metals. The ability of Cupriavidus metallidurans CH34 to resist silver is described here. Electronic microscopy, energy-dispersive X-ray (EDX) and X-ray diffractometry (DRX) observations revealed that C. metallidurans CH34 strongly associated silver with the outer membrane, under chloride chemical form. Using derivate strains of C. metallidurans CH34, which carried only one or no megaplasmid, we show that this resistance seems to be carried by pMOL30.