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Entomopathogenic fungi (EPF) can display a plant-associated lifestyle as endophytes. Seed application of EPF can affect insect herbivory above ground, but the mechanisms behind this are not documented. Here we applied three EPF isolates, Beauveria bassiana, Metarhizium brunneum and M. robertsii, as seed inoculation of wheat and bean, and evaluated the effects on population growth of aphids, Rhopalosiphum padi and Aphis fabae, respectively. In wheat and bean leaves, we quantified benzoxazinoids and flavonoids, respectively, in response to EPF inoculation and aphid infestation to elucidate the role of specific plant secondary metabolites (PSMs) in plant–fungus–herbivore interactions. Inoculations of wheat and bean with M. robertsii and B. bassiana reduced aphid populations compared with control treatments, whereas M. brunneum unexpectedly increased the populations of both aphids. Concentrations of the majority of PSMs were differentially altered in EPF-treated plants infested with aphids. Changes in aphid numbers were associated with PSMs regulation rather than EPF endophytic colonisation capacity. This study links the effects of EPF seed inoculations against aphids with unique PSM accumulation patterns in planta. The understanding of PSM regulation in tri-trophic interactions is important for the future development of EPF for pest management.

The honey bee pollen/nectar diet is rich in bioactive phytochemicals and recent studies have demonstrated the potential of phytochemicals to influence honey bee disease resistance. To unravel the role of dietary phytochemicals in honey bee health it is essential to understand phytochemical uptake, bioavailability, and metabolism but presently limited knowledge exists. With this study we aim to build a knowledge foundation. For 5 days, we continuously fed honey bees on eight individual phytochemicals and measured the concentrations in whole and dissected bees by HPLC-MS/MS. Ample phytochemical metabolization was observed, and only 6–30% of the consumed quantities were recovered. Clear differences in metabolization rates were evident, with atropine, aucubin, and triptolide displaying significantly slower metabolism. Phytochemical gut uptake was also demonstrated, and oral bioavailability was 4–31%, with the highest percentages observed for amygdalin, triptolide, and aucubin. We conclude that differences in the chemical properties and structure impact phytochemical uptake and metabolism.

Honey bees are important pollinators and are subject to numerous stressors, such as changing floral resources, parasites, and agrochemical exposure. Pesticide exposure has been linked to the decline in the global honey bee population. We have limited knowledge of the metabolic pathways and synergistic effects of xenobiotics in bees. Quercetin is one of the most abundant phytochemicals in plants and is therefore abundant in the honey bee diet. Quercetin can upregulate the detoxification system in honey bees; however, it is still unknown to what extent quercetin ingestion can reduce the content of absorbed pesticides. In this study, we investigated the effect of dietary quercetin on the contents of three pesticides in honey bees: imidacloprid (insecticide), tebuconazole (fungicide), and tau-fluvalinate (insecticide and acaricide). Bees were divided into two main groups and fed either quercetin-sucrose paste or only sucrose for 72 h. Thereafter, they were orally exposed to ∼10 ng/bee imidacloprid or contact-exposed to ∼ 0.9 μg/bee tau-fluvalinate or ∼ 5.2 μg/bee tebuconazole. After 1 h of oral exposure or 24 h of contact exposure, the bees were anaesthetised with CO2, sacrificed by freezing, and extracted with a validated QuEChERS method. Subsequently, the concentrations of the three pesticides and quercetin in the bees were determined with a triple quadrupole tandem mass spectrometer coupled to an HPLC system. No significant effect on the concentration of tebuconazole or tau-fluvalinate was observed in bees fed quercetin. Intake of quercetin led to a reduction in the concentration of imidacloprid in honey bees. Quercetin-rich plants may be exploited in future beekeeping.

The honey bee pollen/nectar diet is rich in bioactive phytochemicals and recent studies have demonstrated the potential of phytochemicals to influence honey bee disease resistance. To unravel the role of dietary phytochemicals in honey bee health it is essential to understand phytochemical uptake, bioavailability, and metabolism but presently limited knowledge exists. With this study we aim to build a knowledge foundation. For 5 days, we continuously fed honey bees on eight individual phytochemicals and measured the concentrations in whole and dissected bees by HPLC-MS/MS. Ample phytochemical metabolization was observed, and only 6-30% of the consumed quantities were recovered. Clear differences in metabolization rates were evident, with atropine, aucubin, and triptolide displaying significantly slower metabolism. Phytochemical gut uptake was also demonstrated, and oral bioavailability was 4-31%, with the highest percentages observed for amygdalin, triptolide, and aucubin. We conclude that differences in the chemical properties and structure impact phytochemical uptake and metabolism.

The losses of honey bee colonies and declines of other insect pollinators have been associated with negative effects of pesticides. Honey bees as well as other pollinators are nectar and pollen foragers and thus are exposed to an extensive range of phytochemicals. Understanding the synergistic, additive, and antagonistic effects of plant secondary metabolites and pesticides in honey bees may help to protect honey bee colonies against agrochemicals. In this study, we used untargeted metabolomics to investigate the impact of dietary phytochemical composition on the residual concentration of three pesticides: imidacloprid, tau-fluvalinate and tebuconazole in exposed honey bees. Honey bees were given different diets based on pollen or nectar from four plants: Reseda odorata, Borago officinalis, Phacelia tanacetifolia, and Trifolium repens for two days. Thereafter, they were orally exposed to 10 ng/bee imidacloprid or contact-exposed to 0.9 μg/bee tau-fluvalinate or 5 μg/bee tebuconazole. After 1 h of oral exposure or 24 h of contact exposure, the honey bees were anaesthetised with CO2, sacrificed by freezing, extracted with a validated QuEChERS method, and residual pesticide concentrations were determined by LC-QTRAP-MS/MS. The phytochemical composition in the given diets were profiled with an UHPLC-Q Exactive-MS/MS. The results revealed that the dietary phytochemical composition has a noteworthy influence on the concentration of residual pesticides in honey bees. The correlation coefficient analysis demonstrated that flavonoids have a reducing effect on the residual concentration of imidacloprid and tau-fluvalinate in honey bees. The results also highlighted that exposure to imidacloprid impaired the metabolism of sugars in honey bees. Exploiting flavonoid-rich plants may protect honey bees against pesticides and hold promise as forage plants in future beekeeping.

Increasing demands are being placed on meat producers to verify more about their product with regards to safety, quality and authenticity. There are many methods that can detect aspects of these parameters in meat, yet most are too slow to keep up with the demands of modern meat processing plants and supply chains. A new technology, Rapid Evaporative Ionisation Mass Spectrometry (REIMS), has the potential to bridge the gap between advanced laboratory measurements and technology that can screen for quality, safety and authenticity parameters in a single measurement. Analysis with REIMS generates a detailed mass spectral fingerprint representative of a meat sample without the need for sample processing. REIMS has successfully been used to detect species fraud, detect use of hormones in meat animals, monitor meat processing and to detect off flavours such as boar taint. The aim of this review is to summarize these and other applications to highlight the potential of REIMS for meat analysis. Sampling methods and important considerations for data analysis are discussed as well as limitations of the technology and remaining challenges for practical adoption.

Honey bees regularly ingest quercetin, one of the most abundant phytochemicals in plants. Past research suggests that honey and pollen feeding upregulates the bees’ detoxification system and that dietary quercetin reduces the toxicity of imidacloprid and tau-fluvalinate [1,2].The aim of this study was to investigate the effect of dietary quercetin on the concentration of three pesticides present in honey bees. Honey bees (n = 600) were divided in five groups and fed either quercetin-sucrose paste or only sucrose for 72 h before being exposed to the neonicotinoid imidacloprid (oral exposure), the fungicide tebuconazole or the acaricide tau-fluvalinate (contact exposure). Bees were anesthetized with CO2, frozen, and extracted with a validated QuEChERS method. Finally, the concentrations of all three above-mentioned pesticides were determined by LC-MSMS. Quantification results of the three pesticides in honey bee samples [Fig. 1] displayed that there was a significant difference in concentration of imidacloprid (P = 2 ͯ 10-5) and tau-fluvalinate (P = 3 ͯ 10-2) between the bees that were fed quercetin-sucrose paste and bees that were fed only sucrose. However, there was no significant difference in the level of tebuconazole in the group of bees that were fed quercetin-sucrose paste and bees that were fed only sucrose (P = 4.2 ͯ 10-1). The results of this study demonstrate that honey bee intake of quercetin leads to a reduction in the concentration of imidacloprid and tau-fluvalinate by up regulating the detoxification systems in bees. Hence, quercetin may be a promising bioactive compound in chemo-ecological aspects to counteract the hazard of pesticides. Honey bees regularly ingest quercetin, one of the most abundant phytochemicals in plants. Past research suggests that honey and pollen feeding upregulates the bees’ detoxification system and that dietary quercetin reduces the toxicity of imidacloprid and tau-fluvalinate [1,2].The aim of this study was to investigate the effect of dietary quercetin on the concentration of three pesticides present in honey bees. Honey bees (n = 600) were divided in five groups and fed either quercetin-sucrose paste or only sucrose for 72 h before being exposed to the neonicotinoid imidacloprid (oral exposure), the fungicide tebuconazole or the acaricide tau-fluvalinate (contact exposure). Bees were anesthetized with CO2, frozen, and extracted with a validated QuEChERS method. Finally, the concentrations of all three above-mentioned pesticides were determined by LC-MSMS. Quantification results of the three pesticides in honey bee samples [Fig. 1] displayed that there was a significant difference in concentration of imidacloprid (P = 2 ͯ 10-5) and tau-fluvalinate (P = 3 ͯ 10-2) between the bees that were fed quercetin-sucrose paste and bees that were fed only sucrose. However, there was no significant difference in the level of tebuconazole in the group of bees that were fed quercetin-sucrose paste and bees that were fed only sucrose (P = 4.2 ͯ 10-1). The results of this study demonstrate that honey bee intake of quercetin leads to a reduction in the concentration of imidacloprid and tau-fluvalinate by up regulating the detoxification systems in bees. Hence, quercetin may be a promising bioactive compound in chemo-ecological aspects to counteract the hazard of pesticides.

Pheromones play an important role in the mating behavior of many insects, especially in bees, and it has recently been shown that female Osmia bicornis use the odor bouquet found on male antennae in female choice. Nevertheless, the glandular origin of the substances found on the male antennae is so far unknown. In this study, we investigated if antennal glands exist in males and if they are the glandular source of substances on the antennae’s cuticle, as has been suggested by previous studies. Since electron and light microscopy revealed no such glands, we investigated further possible places of origin for the antennal compounds by conducting chemical analyses of solvent extracts of the male abdominal surface and the antennae, as well as of two glands found in the bee’s head, the labial and mandibular gland. Our results clearly show that the substances found on the abdominal surface and in solvent extracts of the antennae differ significantly in relative amounts. Therefore, the compounds on the antennae must have additional sources. Since the relative amounts of compounds on the surface of antennae are different from the blends on the mandibular gland or the labial gland, neither one of them can be the sole source of the antennal compounds. Hence we believe another glandular source is also involved in the production of the bouquet on the antennae. Especially interesting were high amounts of sterols on the antennae, which also play a role in population differences. This sheds new light on the importance of the antennal compounds and also the importance sterols might play in communication.

Wheat grain nitrogen content displays large variations within different pearling fractions of grains because of radial gradients in the protein content. We identified how spatiotemporal mechanisms regulate this. The protein gradients emerged clearly at 19 days after anthesis, with the highest N content in aleurone and seed coat, followed by outer endosperm, whereas the lowest was in middle and inner endosperm. Laser microdissection, qRT-PCR and LC–MS were used to dissect tissue from aleurone, outer endosperm, middle endosperm, inner endosperm and transfer cells, measure gene expression and levels of free and protein-bound amino acids, respectively. The results showed that different FAA transportation pathways worked in parallel during grain filling stage while the grain protein gradient did not follow spatial expression of storage proteins. Additionally, two nitrogen (N) topdressing timings were conducted, either at the emergence of top third leaf (standard timing) or top first leaf (delayed timing), finding that delayed N topdressing enhanced both amino acids supply and protein synthesis capacity. The results provide insight into protein synthesis and amino acid transport pathways in endosperm and suggest targets for the enhancement of specialty pearled wheat with higher quality.

Secondary metabolites secreted by microbes and plants act as mediators in plant-microbe interactions including nutrient uptake. However, until now very little is known about their role in nutrient assimilation, particularly amino acids, which are important compounds due to their high N content. Here we show that the addition of flavonoid secondary metabolites, derived from clover, to soil changed the bacterial diversity, enhanced the flux of asparagine, and increased the pools of glutamine/glutamate in the soil. This indicates that flavonoids are functionally important qualitative and quantitative components of clover root exudates. Furthermore, the addition of microbial secondary metabolites negatively affected clover uptake of asparagine and plant performance, which demonstrates that microbial competition for nutrients may have multiple physiological targets in the plant. Finally, the detection of intact asparagine in clover roots confirms that amino acid uptake is significant to the plant in agricultural soil. In conclusion, amino-acid flow in the clover rhizosphere can be modified by the effects of clover-derived flavonoids on the bacterial community structure, which affects the flux and pools of amino acids; microbial secondary metabolites, which reduce clover uptake of asparagine; and direct recapture of amino acids by clover.

Weaver ants (genus Oecophylla) are tropical carnivorous ant species living in high numbers in the canopies of trees. The ants excrete copious amounts of fecal matter on leaf surfaces, and these feces may provide nutrients to host trees. This hypothesis is supported by studies of ant-plant interactions involving other ant species that have demonstrated the transfer of nutrients from ants to plants. In this 7-months study, a GC–MS-based metabolomics approach along with an analysis of total nitrogen and carbon levels was used to study metabolic changes in ant-hosting Coffea arabica plants compared with control plants. The results showed elevated levels of total nitrogen, amino acids, fatty acids, caffeine, and secondary metabolites of the phenylpropanoid pathway in leaves from ant-hosting plants. Minor effects were observed for sugars, whereas little or no effect was observed for organic acids, despite the fact that lower levels of total carbon were found in ant-hosting plants. The increased levels of total nitrogen, amino acids, fatty acids and caffeine and the decreased total carbon were consistent with changes observed in plants grown with an increased supply of nitrogen-containing nutrients. The up-regulation of the phenylpropanoid pathway could indicate biotic stress and/or nutrient deficiency. However, because nutrient deficiency was contradicted by the remaining results, this pathway up-regulation was ascribed to biotic stress caused by the physical presence of the weaver ants.

Weaver ants are tropical insects that nest in tree canopies, and for centuries these ants have been used for pest control in tropical orchards. Trees hosting weaver ants might benefit not only from the pest protective properties of these insects but also an additional supply of nutrients from ant feces deposited on the leaves. In a recent study, we demonstrated that Coffea arabica plants hosting Oecophylla smaragdina weaver ants under laboratory conditions experienced enhanced nitrogen availability compared with plants grown without ants. Therefore, the aim of the present study was to further investigate the interactions of weaver ants with the host plants with respect to plant nutrition. Here, we report the identification and quantification of urea, a highly effective foliar nutrient present in the fecal depositions of O. smaragdina. Feces samples obtained from six O. smaragdina colonies were analyzed, and urea concentrations ranging from 1.98 to 31.05 μg/mg ant feces were detected. Subsequently, we investigated the uptake and translocation of 15N2-urea in amounts corresponding to the estimated urea contribution via feces depositions on single host plant leaves under laboratory conditions. The results clearly demonstrated that fecal urea was not only assimilated but also translocated within the plant. This evidence strongly supports the hypothesis that the fecal urea of weaver ants is a source of nitrogen for the host trees. Thus, weaver ant feces likely contribute to an improved nutritional status of ant-hosting trees in tropical orchards, thereby adding value to the use of weaver ants for the biocontrol of insect pests. Weaver ants are tropical insects that nest in tree canopies, and for centuries these ants have been used for pest control in tropical orchards. Trees hosting weaver ants might benefit not only from the pest protective properties of these insects but also an additional supply of nutrients from ant feces deposited on the leaves. In a recent study, we demonstrated that Coffea arabica plants hosting Oecophylla smaragdina weaver ants under laboratory conditions experienced enhanced nitrogen availability compared with plants grown without ants. Therefore, the aim of the present study was to further investigate the interactions of weaver ants with the host plants with respect to plant nutrition. Here, we report the identification and quantification of urea, a highly effective foliar nutrient present in the fecal depositions of O. smaragdina. Feces samples obtained from six O. smaragdina colonies were analyzed, and urea concentrations ranging from 1.98 to 31.05 μg/mg ant feces were detected. Subsequently, we investigated the uptake and translocation of 15N2-urea in amounts corresponding to the estimated urea contribution via feces depositions on single host plant leaves under laboratory conditions. The results clearly demonstrated that fecal urea was not only assimilated but also translocated within the plant. This evidence strongly supports the hypothesis that the fecal urea of weaver ants is a source of nitrogen for the host trees. Thus, weaver ant feces likely contribute to an improved nutritional status of ant-hosting trees in tropical orchards, thereby adding value to the use of weaver ants for the biocontrol of insect pests.

Sterols are essential to insects because they are vital for many biochemical processes, nevertheless insects cannot synthesize sterols but have to acquire them through their diet. Studies of sterols in ants are sparse and here the sterols of the weaver ant genus Oecophylla are identified for the first time. The sterol profile and the dietary sterols provided to a laboratory Oecophylla longinoda colony were analyzed. Most sterols originated from the diet, except one, which was probably formed via dealkylation in the ants and two sterols of fungal origin, which likely originate from hitherto unidentified endosymbionts responsible for supplying these two compounds. The sterol profile of a wild Oecophylla smaragdina colony was also investigated. Remarkable qualitative similarities were established between the two species despite the differences in diet, species, and origin. This may reflect a common sterol need/aversion in the weaver ants. Additionally, each individual caste of both species displayed unique sterol profiles.

Relevance: Honey bees (Apis mellifera) are exposed to a range of agrochemicals when they pollinate our fields1,2. The neonicotinoid imidacloprid, the pyrethroid tau-fluvalinate and a triazole fungicide tebuconazole are common xenobiotics that induce stress in honey bees. Mao et al3 demonstrated that quercetin can up-regulate the detoxification systems and reduce the toxicity of tau-fluvalinate in bees. Here we investigate the effect of dietary quercetin, a flavonoid ubiquitous in pollen of many plants, on the concentration of three xenobiotics frequently found in honey bee colonies. Experimental: Honey bees were fed with either quercetin+sucrose or sucrose for 72 h and then exposed to the individual pesticides either by contact or oral exposure. A modified QuEChERS method was used for pesticide extraction with recovery percentages of 83.1 ±3.2, 86.3 ±3.3 and 100.3 ±1.5 for imidacloprid, tau-fluvalinate and tebuconazole, respectivelly. Subsequently, the samples were analyzed with a triple quadrupole linear ion trap mass spectrometer coupled to an HPLC system. Results: The results of this study demonstrates that intake of quercetin significantly reduces the concentration of imidacloprid (P = 2 ×10-5) and tau-fluvalinate (P = 3.5×10-3). Dietary quercetin did not affect the concentration of tebuconazole (P = 4.2×10-2) in honey bees.