Caged Honey Bees Research Articles

Dietary dopamine supplementation has no effect on ovary activity in queen-less or queen-right honeybee (Apis mellifera) workers

Eusociality in honeybees (Apis mellifera) is characterised by a reproductive division of labour, where the queen monopolises reproduction while worker reproduction is constrained. This constraint is mediated by queen mandibular pheromone (QMP), which inhibits worker ovary development through Notch signalling and possibly oocyte apoptosis. Dopamine has been implicated in regulating reproduction in worker honeybees, with prior studies suggesting that dietary dopamine enhances ovary activity in queen-less workers. This study aimed to test whether dopamine supplementation could overcome QMP-mediated reproductive constraint in worker honeybees. Using caged honeybee experiments, we administered dopamine and its precursor L-dopa at a range of concentrations, both in the presence and absence of QMP. Results showed that neither dopamine nor L-dopa supplementation affected ovary development, survival rates, or food intake, which contrasts with a previous study. These findings suggest that dopamine may not be a major factor in QMP-mediated reproductive inhibition. Instead, we suggest that the multifaceted nature of QMP’s components together with the complexity of neuroendocrine signalling makes it likely that multiple redundant mechanisms regulate worker reproduction. Future research should investigate the interplay between nutrition, dopamine and QMP components to fully understand the regulation of ovary activation in honeybee workers.

Mechanistic exploration of royal jelly production in caged honey bees (Apis mellifera)

This study investigates the impact of bee pollen nutrition on the royal jelly production of honey bees (Apis mellifera). Results demonstrate that pollen diet significantly impacts hypopharyngeal gland (HPG) development and the expression of genes associated with royal jelly biosynthesis. Bees fed Brassica napus pollen exhibited superior HPG development, and increased mrjp1 expression (encoding a key royal jelly protein). While the cyp450 6AS8 gene expression (encoding a key enzyme in 10-HDA biosynthesis) was increased by pollen consumption, no distinct expression patterns were observed among the different pollen types tested. An in vitro bee cage platform for royal jelly production has been established to further understand the mechanisms behind royal jelly production in bees. The experiment demonstrated a positive correlation between the number of worker bees and the total yield of royal jelly per cage. However, when the number of worker bees is low, the amount of royal jelly each individual worker bee needs to produce increases. In conclusion, these findings enhance our understanding of the role of bee pollen nutrition in royal jelly production. Furthermore, the results from this in vitro bee cage platform suggest that the number of worker bees is a critical factor in royal jelly production, and that bees may possess a controllable mechanism for regulating royal jelly secretion.

Possible interactions between gut microbiome and division of labor in honey bees.

Recent studies have provided new insights into the role of the microbiome in shaping host behavior. However, the relationship between the temporal division of labor among honey bees (Apis mellifera) and their gut microbial community has not been widely studied. Therefore, we aimed to evaluate the link between the gut microbiome and division of labor in honey bees by examining the microbial absolute abundance and relative composition of 7-day-old nurse bees and 28-day-old forager bees from a natural hive, as well as those of worker bees of the same 14-day-old age showing different behaviors in a manipulated hive. We found that forager bees had fewer core bacteria, particularly gram-positive fermentative genera such as Lactobacillus and Bifidobacterium, with Bifidobacterium asteroides being the most sensitive to host behavioral tasks. Our results showed that forager bees have lower gut community stability compared to nurse bees, suggesting that their gut community is more susceptible to invasion by non-core members. Furthermore, a pollen limitation experiment using caged honey bees indicated that dietary changes during behavioral shifts may be a driving factor in honey bee microbial diversity. This study contributes to a greater understanding of the interaction between the gut microbiome and behavioral tasks and provides a foundation for future assays.

The effects of queen mandibular pheromone on Nosema (Vairimorpha) ceranae infections in caged honey bees

Honey bees utilize queen mandibular pheromone (QMP) for maintaining social hierarchy and colony development. In controlled cage studies, synthetic QMP is often introduced to mimic natural conditions. However, questions have arisen about the effects of QMP on nosema disease studies. This short report identifies significant early-stage suppression effects of QMP on Nosema (Vairimorpha) ceranae infections. QMP was found to significantly lower infection rates below the reported infectious dose for 50 % infectivity (ID50) and to slow disease development in a dose-independent manner. These effects diminished at doses exceeding ID100. We recommend that studies investigating treatment effects using caged bees avoid QMP to ensure unambiguous results. Additionally, employing multiple infectious doses with shorter incubation times would be useful for evaluating other treatments that may have subtle effects. Furthermore, our findings support previous field studies suggesting that queen replacement reduces nosema disease at levels similar to treatment with fumagillin.

Protective Role of Bergamot Polyphenolic Fraction (BPF) against Deltamethrin Toxicity in Honeybees (Apis mellifera).

Pesticide-induced poisoning phenomena are a serious problem for beekeeping and can cause large losses of honeybee populations due to acute and sub-acute poisoning. The reduced responsiveness of honeybees to the damage caused by pesticides used in agriculture can be traced back to a general qualitative and quantitative impoverishment of the nectar resources of terrestrial ecosystems. Malnutrition is associated with a decline in the functionality of the immune system and the systems that are delegated to the detoxification of the organism. This research aimed to verify whether bergamot polyphenolic extract (BPF) could have protective effects against poisoning by the pyrethroid pesticide deltamethrin. The studies were conducted with caged honeybees under controlled conditions. Sub-lethal doses of pesticides and related treatments for BPF were administered. At a dose of 21.6 mg/L, deltamethrin caused mortality in all treated subjects (20 caged honeybees) after one day of administration. The groups where BPF (1 mg/kg) was added to the toxic solution recorded the survival of honeybees by up to three days. Comparing the honeybees of the groups in which the BPF-deltamethrin association was added to the normal diet (sugar solution) with those in which deltamethrin alone was added to the normal diet, the BPF group had a statistically significant reduction in the honeybee mortality rate (p ≤ 0.05) and a greater consumption of food. Therefore, it can be argued that the inclusion of BPF and its constituent antioxidants in the honeybee diet reduces toxicity and oxidative stress caused by oral intake of deltamethrin. Furthermore, it can be argued that BPF administration could compensate for metabolic energy deficits often induced by the effects of malnutrition caused by environmental degradation and standard beekeeping practices.

Vital responses of caged honeybee (Apis mellifera) workers fed on new substitute diets in the laboratory

Vital responses of caged honeybee (Apis mellifera) workers fed on new substitute diets in the laboratory

Water provisioning increases caged worker bee lifespan and caged worker bees are living half as long as observed 50 years ago

The high loss rates of honey bee colonies drive research for solutions aimed to mitigate these losses. While honey bee colonies are superorganisms, experiments that measure the response to stressors often use caged individuals to allow for inference in a controlled setting. In an initial experiment, we showed that caged honey bees provisioned with various types of water (deionized, 1%NaCl in deionized, or tap) have greater median lifespans than those that did not. While researching the history of water provisioning in cage studies, we observed that the median lifespan of caged honey bees has been declining in the US since the 1970’s, from an average of 34.3 days to 17.7 days. In response to this, we again turned to historical record and found a relationship between this trend and a decline in the average amount of honey produced per colony per year in the US over the last 5 decades. To understand the relationship between individual bee lifespan and colony success we used an established honey bee population model (BEEHAVE) to simulate the predicted effects of decreased worker lifespans. Declines in downstream measures of colony population, overall honey production, and colony lifespan resulted from reduced worker bee lifespans. Modeled colony lifespans allowed us to estimate colony loss rates in a beekeeping operation where lost colonies are replaced annually. Resulting loss rates were reflective of what beekeepers’ experience today, which suggests the average lifespan of individual bees plays an important role in colony success.

A deeper understanding of system interactions can explain contradictory field results on pesticide impact on honey bees

While there is widespread concern regarding the impact of pesticides on honey bees, well-replicated field experiments, to date, have failed to provide clear insights on pesticide effects. Here, we adopt a systems biology approach to gain insights into the web of interactions amongst the factors influencing honey bee health. We put the focus on the properties of the system that depend upon its architecture and not on the strength, often unknown, of each single interaction. Then we test in vivo, on caged honey bees, the predictions derived from this modelling analysis. We show that the impact of toxic compounds on honey bee health can be shaped by the concurrent stressors affecting bees. We demonstrate that the immune-suppressive capacity of the widespread pathogen of bees, deformed wing virus, can introduce a critical positive feed-back loop in the system causing bistability, i.e., two stable equilibria. Therefore, honey bees under similar initial conditions can experience different consequences when exposed to the same stressor, including prolonged survival or premature death. The latter can generate an increased vulnerability of the hive to dwindling and collapse. Our conclusions reconcile contrasting field-testing outcomes and have important implications for the application of field studies to complex systems.

Increased Stress Levels in Caged Honeybee (Apis mellifera) (Hymenoptera: Apidae) Workers

Honeybees, Apis mellifera, usually live in large colonies consisting of thousands of individuals. Within the colony, workers interact with their social environment frequently. The large workforce, division of labour, and other features may promote the ecological success of honeybees. For decades, artificial mini colonies in cages within the laboratory have become the gold standard, especially in experiments related to toxicology, effects of pesticides and pathogens. Experiments using caged bees and full-sized colonies yielded contradictory results. Here, the effect of cage experiments on the stress level of individual bees is analysed. Two different stress response were targeted, the heat shock response and the mobilization of energetic resources. While no differences were found for varying group sizes of bees, very strong effects emerged by comparing caged workers with bees from natural colonies. Caged workers showed increased levels of hsp expression and reduced haemolymph titres for trehalose, the energy storage sugar. These results reveal that the lack of the social environment (e.g., lack of queen, lack of sufficient group size) induce stress in caged bees, which might act synergistically when bees are challenged by additional stressors (e.g., pesticides, pathogens) resulting in higher mortality than observed under field conditions.

Alterations in the Microbiota of Caged Honeybees in the Presence of Nosema ceranae Infection and Related Changes in Functionality

Several studies have outlined that changes in the honeybee gut microbial composition may impair important metabolic functions supporting the honeybees’ life. Gut dysbiosis may be caused by diseases like Nosema ceranae or by other anthropic, environmental or experimental stressors. The present work contributes to increasing knowledge on the dynamics of the gut microbiome acquisition in caged honeybees, an experimental condition frequently adopted by researchers, with or without infection with N. ceranae, and fed with a bacterial mixture to control N. ceranae development. Changes of the gut microbiota were elucidated comparing microbial profile of caged and open-field reared honeybees. The absolute abundance of the major gut microbial taxa was studied with both NGS and qPCR approaches, whereas changes in the functionality were based on RAST annotations and manually curated. In general, all caged honeybees showed important changes in the gut microbiota, with gamma-proteobacteria (Frischella, Gilliamella and Snodgrassella) lacking in all caged experimental groups. Caged honeybees infected with N. ceranae showed also a strong colonization of environmental taxa like Citrobacter, Cosenzaea and Morganella, as well as possibly pathogenic bacteria such as Serratia. The colonization of Serratia did not occur in presence of the bacterial mixture. The functionality prediction revealed that environmental bacteria or the supplemented bacterial mixture increased the metabolic potential of the honeybee gut microbiome compared to field and caged controls.

The effect of nutritional status on the synthesis ability, protein content and gene expression of mandibular glands in honey bee (Apis mellifera) workers

The mandibular glands (MGs) of honey bee workers are key exocrine glands responsible for the biosynthesis of multiple royal jelly acids, which have important functions within the colony. However, our knowledge about the nutrition requirement of MGs is quite limited. Here, we artificially manipulated the nutritional status of caged honey bee workers (pollen and sugar candy, sugar candy only, soybean meal and sugar candy, stearic acid supplemented sugar candy or citric acid supplemented sugar candy). Then we measured their impact on the 10-hydroxy-2-decanoic acid (10-HDA) and 10-hydroxydecanoic acid (10-HDAA) content in honey bee heads to evaluate the synthesis ability of workers’ MGs, and analyzed the protein content of MGs as well as gene expression in the MGs to reveal the possible physiological changes. Our result showed that nutrition strongly impacted the MGs acid synthesis, the 10-HDA and 10-HDAA contents were significantly higher in workers fed with pollen than with sugar candy only, while an additional supply of protein and organic acid showed no significant contribution. In addition, the 10-HDA/10-HDAA ratio was also significantly affected by the nutrition status. On the contrary, different diets showed no influence on the protein content or gene expressions of MGs, except that organic acid supplements could increase the expression of key genes in fatty acid β-oxidation pathways. Taken together, our study revealed that honey bee nutritional status is closely related to the function of worker MGs. The nutritional deficiency has a significant negative impact on the synthesis ability of worker MGs, but showed no effect on the physiology of MGs.

Nutritional efficacy of different diets supplemented with microalga Arthrospira platensis (spirulina) in honey bees (Apis mellifera)

In honey bees, food, gut microorganisms, and their nestmates may contribute to the health status of newly emerged worker bees. However, relatively little data are available regarding the extent to which supplemental protein feeding impacts bee colony health and mortality. The present research compared the efficacy of different diets supplements, i.e., bee pollen, ajeena (commercially available pollen substitute), and date paste either alone or admixed with varying percentages of microalga Arthrospira platensis (spirulina) for caged honey bees (Apis mellifera jemenitica). In addition, to investigate diet preference and mortality percentage in honey bees, the physicochemical analysis of these diets were also performed. Our results indicated that the honey bees consumed bee pollen (11.51 ± 2.22 mg/bee) and ajeena diet (10.68 ± 1.29 mg/bee) significantly higher than other diets.In contrast, the maximum consumption was 4.68 ± 2.82 mg/bee for date paste admixed with 2.5% spirulina than date paste diet only (2.41 ± 0.91 mg/bee). In addition, the mortality percentage was significantly lower when bees fed pollen (56.67% ± 3.88%) and ajeena diet (39.33% ± 2.08%) in comparison to combination with spirulina supplement. Overall, the mean maximum diet consumption for ajeena was 15.72 ± 3.65 mg/bee followed by bee pollen diet (11.97 ± 2.41 mg/bee) and date paste (2.08 ± 0.94 mg/bee), respectively, resulting mean percentage of mortality of 13.37% ± 3.77%. Physicochemical analysis revealed that glucose and fructose were higher in the date paste diet. In contrast, total sugar content, sucrose, and protein content were higher in the ajeena diet. Future research is needed to determine the impact of diet supplementation with spirulina on colony health and bee physiology.

Inert agricultural spray adjuvants may increase the adverse effects of selected insecticides on honey bees (Apis mellifera L.) under laboratory conditions

Currently, more than 360 spray adjuvants are registered in Germany (September 2021). Unlike plant protection products (PPPs), adjuvants are not subjected to regulatory risk assessment. In practice, numerous combinations of PPPs and adjuvants are therefore possible. Thus, tank mixtures containing insecticides that are classified as non-hazardous to bees up to the highest approved application rate or concentration may raise pollinator safety concerns when mixed with efficacy increasing adjuvants and applied in bee-attractive crops. This study analyzes whether selected “PPP–adjuvant” combinations result in increased contact mortality and pose an elevated risk to honey bees. To answer this question, we chose six common spray adjuvants of different classes for laboratory screening. These were then tested in a total of 30 tank mixtures, each with a neonicotinoid (acetamiprid), pyrethroid (lambda-cyhalothrin), diamide (chlorantraniliprole), carbamate (pirimicarb), and butenolide (flupyradifurone) formulation. We adapted an acute contact test (OECD Test Guideline 214) to our needs, e.g., by using a professional spray chamber for more realistic exposures. Our results showed that, in total, 50% of all combinations significantly increased the mortality of caged honey bees in comparison with individual application of insecticides. In contrast, none of the adjuvants alone affected bee mortality (Cox proportional hazard model, p > 0.05). With four of the five insecticide formulations, the organosilicone surfactant Break-Thru® S 301 significantly increased bee mortality within 72 h (for all insecticides except chlorantraniliprole). Furthermore, acetamiprid yielded the highest and second highest mortality increases from a tank mixture with the crop oil surfactant LI 700® (hazard ratio = 28.84, p < 0.05) and the organosilicone Break-Thru® S 301 (hazard ratio = 14.66, p < 0.05), respectively. To assess risk in a more field-realistic setting, field trials should be performed to provide a more realistic exposure scenario under colony conditions.

The active ingredients of a mitotoxic fungicide negatively affect pollen consumption and worker survival in laboratory-reared honey bees (Apis mellifera)

Recent observations of many sublethal effects of pesticides on pollinators have raised questions about whether standard short-term laboratory tests of pesticide effects on survival are sufficient for pollinator protection. The fungicide Pristine® and its active ingredients (25.2% boscalid, 12.8% pyraclostrobin) have been reported to have low acute toxicity to caged honey bee workers, but many sublethal effects at field-relevant doses have been reported and Pristine® was recently found to increase worker pollen consumption, reduce worker longevity and colony populations at field relevant concentrations (Fisher et al. 2021). To directly compare these whole-colony field results to more standard laboratory toxicology tests, the effects of Pristine®, at a range of field-relevant concentrations, were assessed on the survival and pollen consumption of honey bee workers 0–14 days of age. Also, to separate the effects of the inert and two active ingredients, bees were fed pollen containing boscalid, pyraclostrobin, or pyraclostrobin plus boscalid, at concentrations matching those in the Pristine® treatments. Pyraclostrobin significantly reduced pollen consumption across the duration of the experiment, and dose-dependently reduced pollen consumption on days 12–14. Pristine® and boscalid significantly reduced pollen feeding rate on days 12–14. Boscalid reduced survival in a dose-dependent manner. Consumption of Pristine® or pyraclostrobin plus boscalid did not affect survival, providing evidence against strong negative effects of the inert ingredients in Pristine® and against negative synergistic effects of boscalid and pyraclostrobin. The stronger toxic effects of Pristine® observed in field colonies compared to this laboratory test, and the opposite responses of pollen consumption in the laboratory and field to Pristine®, show that standard laboratory toxicology tests can fail to predict responses of pollinators to pesticides and to provide protection.

NUTRITIONAL EFFECTS OF SOME POLLEN TYPES ON HYPOPHARYNGEAL AND ACID GLANDS IN HONEYBEE WORKERS (APIS MELLIFERA L.)

Pollen had a wide range of effects on honey bee. Caged honey bee workers were used to determine the nutritional effects of four main types of pollen collected by honey bee colonies from the study area on the food consumption rates, bees age, the development of the hypopharyngeal (HPG) acini and acid gland (AG) sacs in honey bee workers. Additionally, chemical analysis of the selected pollen types was performed. Pollen diets were as follows: broad bean pollen, date palm pollen, clover pollen, maize pollen and mixture of the for mentioned typrs beside the control pollen diet. The highest mean consumption was observed between 4th and 6th of day aged bees in all diets (23.05 mg./bee/3days). The highest quantity of food consumption was recorded in the 18th day when the workers were fed on date palm pollen (75.42 mg), while the lowest recorded 59.17 mg from maize pollen treatment. The highest mean acinal surface area of HPG was 0.0451mm2, which measured at 9 days-old of bees in all diets. While the workers fed on mixture pollen diet had the highest significant mean recorded the acinal surface area of HPG 0.0450mm2 comparing of 0.0223mm2 in control. The considering increment in average volume of acid gland sac with had been obtained at 18 days-old (0.7276mm3) that resembles the highest significant degree, the greatest mean volume of AG sacs was observed when bees were fed on broad bean pollen (0.6419mm3), while the lowest mean was 0.3643mm3 in control.

A Potential Fungal Probiotic Aureobasidium melanogenum CK-CsC for the Western Honey Bee, Apis mellifera.

Aureobasidium melanogenum has been used as an animal feed additive for improving thehealth of pets, however, it has not yet been applied in honey bees. Here, a fungal strain CK-CsC isolated from bee bread pollen, was identified as A. melanogenum. Following characterizing CK-CsC fermentation broth, the 4-days fermentation broth (SYM medium or bee pollen) of the CK-CsC was used to feed newly emerged adult honey bees in cages under laboratory-controlled conditions for analysis of survival, gene expression of nutrient and antibacterial peptide, and gut microbiota of honey bees. It was found that the CK–CsC fermentation broth (SYM medium or bee pollen) is nontoxic to honey bees, and can regularly increase nutrient gene expression of honey bees. However, significant mortality of bees was observed after bees were fed on the supernatant liquid of the fermentation broth. Notably, this mortality can be lowered by the simultaneous consumption of bee pollen. The honey bees that were fed bee pollen exhibited more γ-Proteobacteria, Bacteriodetes, and Actinobacteria in their gut flora than did the honey bees fed only crude supernatant liquid extract. These findings indicate that A. melanogenum CK–CsC has high potential as a bee probiotic when it was fermented with bee pollen.

Chitosan-based gel application on model bees (Apis mellifera L.) for healing bite wounds caused by Varroa destructor

Chitin is basically responsible for protecting the body of honey bees against external attacks due to its hard cellulose structure. The chitin-structured body of honey bees is destroyed by Varroa destructor, the world’s most devastating pest of Western honey bees; causes honey bee decline. Varroa harms honey bees not only by feeding but also by the wounds it inflicts. Varroa bites cause a hole in the centre of the wound, which is a source of bacterial infection. In this study, the aim was to investigate the potential application of a chitosan-based gel to recover the chitin layer. The experiment was set up in eight cages. Varroa bites were artificially created on experimental bees called as model honey bees in cages. Different solutions of a chitosan-based gel at different doses were applied to the cages. Results revealed that Varroa bite wounds do not heal until day 9 in control cages. Healing of wounds by chitosan-based gel application on 1–3 and 3–6 days showed dose dependence. In addition, the gel prevented hair loss in honey bees, which was observed as a side effect of Varroa infestation. In addition, chitosan dissolved in organic acids is a key advantage for the treatment of V. destructor in parallel with wound healing.

A fermented Pollen Substitute Diet Affects Wax Construction by Honey Bee Workers, (Apies mellifera L.)

Pollen substitute diets have become increasingly important for maintaining strong and healthy honey bee colonies. Palatability and nutritional value are key attributes of a good diet. Since beebread, which is pollen fermented by the bees, is the main food of the worker-nurse bees that feed and care for the bee larvae, pollen substitutes should have similar attributes. In an attempt to simulate this natural food source, an inoculum prepared from beebread was used to ferment a pollen substitute diet. In this study, it was determined the differences in the wax building by caged workers fed with different diets (sucrose syrup only, bee bread, unfermented pollen substitute diet and fermented pollen substitute diet). Comb building in honeybees was analyzed with respect to the type of food. The effects on wax combs building were assessed of the presence of beeswax foundation, and of feeding honey bees in cages. Workers fed on unfermented diets produced significantly less wax than did those fed on the fermented diets; although variation in wax production among the treatments was not significant. We conclude that fermentation by beebread-derived microorganisms could improve the palatability, consumption and nutritional value of the dies and the behavioral activities such as wax building by honey bee workers.

Depletion kinetics and concentration- and time-dependent toxicity of a tertiary mixture of amitraz and its major hydrolysis products in honeybees

Although amitraz is one of the acaricides most commonly applied within beehives, to date, its time-dependent oral toxicity in honeybees has not been investigated, due to amitraz’s instability in aqueous media. In aqueous media such as honey, amitraz rapidly forms a continuously changing tertiary mixture with two of its major hydrolysis products, DMF and DMPF. The contribution of each hydrolysis product to the overall oral toxicity of this acaricide is not known. Therefore, we aimed to characterize the depletion and formation kinetics of amitraz and its hydrolysis products in 50% sucrose solution provided to caged honeybees, including the calculation of the 50% lethal oral concentration (LC50) of amitraz. We sought to determine the contribution of each component of the mixture to the overall observed toxicity. We also investigated the time- and concentration-dependent toxicity of the amitraz mixture and its hydrolysis products. A novel approach based on the analysis of the areas under the depletion and formation curves of amitraz and its hydrolysis products revealed that DMPF, amitraz and DMF accounted for 92%, 7% and 1% (respectively) of the overall toxicity of the mixture. The chronic oral LC50 of amitraz was 3300 μmol/L, of similar magnitude as that of the non-toxic hydrolysis product DMF. The toxicity of DMPF and the mixture decreased over time; whereas the toxicity of DMF increased over time. Amitraz’s instability in aqueous media and the highly toxic profile of DMPF, suggest that DMPF is the actual toxic entity responsible for amitraz’s toxicity toward honeybees.

Chronic exposure to imidacloprid or thiamethoxam neonicotinoid causes oxidative damages and alters carotenoid-retinoid levels in caged honey bees (Apis mellifera)

Over the last decade, the persistent dwindling of the populations of honey bees has become a growing concern. While this phenomenon is partly attributed to neonicotinoids (NEOCs), chronic exposures to these insecticides at environmentally-relevant concentrations are needed to fully estimate their implications. In this study, honey bees were orally exposed for 10 days to low field-realistic concentrations of NEOCs known for their effects on the cholinergic system (imidacloprid – IMI or thiamethoxam – THM). Selected biomarkers were measured such as acetylcholinesterase (AChE) activity, lipid peroxidation (LPO), α-tocopherol as well as several forms of vitamin A (retinoids) and carotenoids. Bees exposed to IMI showed lower levels of two carotenoids (α-carotene and α-cryptoxanthin) and α-tocopherol. The THM exposure increased the oxidized vitamin A metabolites in bees conjointly with the LPO. These results could be the consequence of a pro-oxidant effect of NEOCs and were observed at levels where no effects were recorded for AChE activity. This study reveals that exposure to low levels of NEOCs alters the carotenoid-retinoid system in honey bees. This would merit further investigation as these compounds are important in various aspects of bees’ health. Overall, this study contributes to the development of biomonitoring tools for the health of bees and other pollinators.