Pollen contaminated with a triple-action fungicide induced oxidative stress and reduced bee longevity though with less impact on lifespan in bees from well fed colonies

doi:10.21203/rs.3.rs-4836495/v1

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Pollen contaminated with a triple-action fungicide induced oxidative stress and reduced bee longevity though with less impact on lifespan in bees from well fed colonies

https://doi.org/10.21203/rs.3.rs-4836495/v1

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There is increasing evidence that besides insecticides, fungicides also affect bee health. However, there has been little research on how bees are affected by recently developed modern fungicides that contain various active ingredients to help overcome fungal pathogen resistance. Experiments were conducted to determine how this type of fungicide affects bees and whether annutritional supplements can ameliorate eventual negative impacts for bees. Newly-emerged bees from well fed and from nutritionally restricted honey bee colonies were maintained in groups of 20 in plastic cages in an incubator and fed for five days with pollen from sunflower plants that had been sprayed or not during flowering with a three-component commercial fungicide containing bixafen, prothioconazole and trifloxystrobin. Bees from the well-fed colonies were significantly larger and consumed more uncontaminated pollen in the cage tests. They also had increased glutathione peroxidase activity and higher concentrations of pyridine nucleotides. Feeding on the fungicide-contaminated pollen resulted in decreased catalase activity of bees from well-fed colonies and damage to cell membranes of bees indepent of nutritional condition. Bee longevity was reduced by both fungicide contamination of the pollen diet and poor nutritional condition of the donor colony. In conclusion, the triple action commercial fungicide adversely affected bees fed with contaminated pollen, though nutritional supplementation of bee colonies that provided the bees partially compensated for these effects.

bixafen

nutrition

prothioconazole

redox

trifloxystrobin

Bees play a vital role in pollination, contributing to agricultural production and maintenance of biodiversity in natural ecosystems (Potts et al. 2018). Concerns about the health and longevity of bees have grown, with various factors being associated with the decline of these natural insect pollinator populations (Hernández et al. 2021). The honey bee (Apis mellifera) is a managed crucial pollinator of crops worldwide, frequently exposed to various pesticides, especially in crop fields (Poquet et al. 2016). When visiting sprayed crops or other areas affected by pesticide drift, bees can become contaminated. The damage may increase when two pesticides are applied together or when a product with more than one active ingredient is used, as is the case with some modern fungicides (Böhme et al. 2017; Prado et al. 2019; Rondeau and Raine, 2022).

Fungicides are generally considered to have low toxicity for bees based on acute toxicity tests; however, fungicides provoke behavioral (Tadei et al. 2019), cellular (Batista et al. 2020), microbial (Carneiro et al. 2020), and immunological changes (Cizelj et al. 2016) in bees. Additionally, they can reduce food consumption (Cizelj et al. 2016; Liao et al. 2017; Mao et al. 2017) and bee longevity (Fisher et al. 2017. Given the stress on bee health resulting from fungicide exposure, it is pertinent to consider the nutritional condition of colonies in agricultural landscapes, especially since a deficient diet negatively affects the ability of bees to deal with pathogens and toxins (Fisher et al. 2023). A deficiency in pollen availability can result in a reduced colony population and compromise the health of the bees, decreasing resistance to biotic and abiotic stresses at individual and colony levels (Corona et al. 2023; Fisher et al. 2023).

In a scenario of intensive pesticide use and landscapes with spatial and temporal limitations on food resources for bees, nutritional support for these beneficial insects should be considered with increased attention, as it is crucial for their development and survival and is intrinsically linked to their resilience against numerous stressors, including pesticides (Wickramasinghe et al. 2020; Quinlan and Grozinger, 2023). Moreover, a better understanding of the interaction between nutrition, pesticide exposure, and the functioning of the antioxidant system in bees is essential, considering impairments to bee health that impact their behavior, productive performance, oxidative metabolism, and longevity (Tong et al. 2019; Fisher et al. 2023).

A triple-action fungicide, containing the active ingredients bixafen, prothioconazole, and trifloxystrobin, is now commonly used in large-scale agricultural productions, including cotton, sunflower, and soybeans (Bayer, 2019). These crops are visited by bees for pollen and/or nectar, and crop yields are benefitted by entomophilous pollination (Klein et al. 2007). However, little is known about the isolated and combined effects of these active ingredients on bee health, especially the oxidative metabolism of nurse honey bees. Increases in intracellular reactive oxygen species (ROS) or failures in the antioxidant defense system lead to oxidative stress, a state detrimental to cellular homeostasis. Defenses against oxidative stress operate through enzymes such as superoxide dismutase, catalase, and glutathione peroxidase, as well as non-enzymatic components, which can be obtained endogenously or from natural or artificial food sources (Sies et al. 2017).

Nutritional stress has been identified as one of the factors that harm bee health and longevity, while non-enzymatic antioxidants present in high-quality diets can assist in the antioxidant defenses of bees (Mucci et al. 2021; Corona et al. 2023). In this study, we evaluated whether nutritional supplementation of colonies can mitigate the harmful effects on the oxidative system and longevity resulting from the exposure of adult bees to protein food contaminated with a modern triple-component fungicide.

Experiment location, sunflower cultivation, fungicide application, and pollen collection

The experiment was conducted in Dracena, state of São Paulo, at 21°27'33" South Latitude and 51°33'22" West Longitude, with an altitude of 392 meters. This study involved the use of two nutritional management practices for honey bee colonies to obtain bees that developed under different conditions regarding food availability during their brood stage. Bees were obtained from these colonies and maintained in groups of 20 in small cages in an incubator, where they were fed with protein paste based on sunflower pollen from plants that were treated or not treated with a commercial fungicide.

The CATI Multissol cultivar of sunflowers (Helianthus annuus) were cultivated in the field in an area of 3,000 m², with a spacing of 0.45 m between rows and 0.40 m between plants. Planting and topdressing fertilizations followed current recommendations for Brazil (Cantarella et al. 2022). No pesticides were applied to the crop.

To obtain pollen from the sunflower plants, approximately 500 inflorescences were covered with non-woven fabric bags before anthesis. Once the majority of the capitula were open, the sunflower stems were cut, and the inflorescences were taken to the laboratory.

The inflorescences were removed from the bags and laid out on the floor of a spray test facility with the same spacing used in the field. The area occupied by the inflorescences was calculated to determine the amount of fungicide to be applied. Fungicide application followed the instructions provided in the commercial product label of Fox Xpro fungicide (Bayer, 2019) for the control of Alternaria helianthi (Pleosporaceae), a major fungal pathogen of sunflowers, at a rate of 0.5L ha⁻¹ in 144 liters of spray volume per hectare, with spraying equipment set at a pressure of 200 kPa, 0.5 m above the flowers, under windless conditions. After 15 minutes, the time needed for the spray to dry, the inflorescences were collected.

The pollen was collected in a plastic tray, upon which the inverted inflorescences were manually shaken. For the control group pollen, no fungicide spray was applied, and the pollen was collected immediately after inflorescence harvesting. The pollen was stored in 50 mL plastic tubes, which were stored in an ultrafreezer (-80ºC), until it was used in the preparation of the protein pastes for the bees.

Dietary Management of Honey Bee Colonies

Eight Africanized honey bee (Apis mellifera) colonies maintained in standard Langstroth hives were subjected to one of two dietary management systems for 24 weeks. In addition to the food freely collected in the field, four colonies had their diet supplemented (S) with 100g of protein paste, prepared with three parts of multifloral bee pollen and one part of wildflower honey, provided once a week, along with 500 mL of sucrose syrup made from water and crystallized cane sugar in equal proportions (w/w), offered twice a week.

The dietary management for the second group involved restricted food availability (R) of the colonies by installing a pollen collector at the entrance of each hive. Colonies in this group did not receive any dietary supplementation. The colonies had naturally mated queens and at the beginning of the experiment were standardized with six frames of brood and four frames of food. The 24-week period was set to ensure that the bees used in the experiments experienced the nutritional management of the colonies during the brood stage, so that the data collected from the bees would not be influenced by the food stocks accumulated before the beggining of the colony treatments.

Pollen collection and efficiency of pollen collectors in colonies with restricted feeding

Bee pollen obtained from the pollen collectors installed in the hives of group R was collected daily during the 24 weeks of dietary management. After each collection, the pollen obtained from each hive was weighed on a semi-analytical balance. The weekly amount of pollen obtained from each hive was recorded.

To estimate the quantity of protein food available to the R colonies, the efficiency of the pollen collectors was determined. An observer stood near each pollen collector for 30 minutes, between 8 and 10 AM during three days, recording the number of bees returning from the field with pollen in their corbiculae. After this 30 minute period, the pollen pellets inside the pollen collector drawer were counted. Observations were conducted in triplicate for each hive. These data were used to determine the efficiency of the pollen collectors as described by Kato et al (2024).

Evaluation of the weight of newly emerged bees

After the dietary management period of the colonies, frames with bees about to emerge were obtained from all colonies to create a pool of bees originating from each dietary management (S and R). We used 100 individuals from each nutritional management group. The bees were placed in a freezer for one hour. Subsequently, samples of 10 bees each were placed in crucibles, with five replications, and dryed in an oven with air circulation at 105ºC for 16 hours (AOAC, 1990). Each group of 10 bees was weighed before and after the drying process.

Obtaining newly emerged bees, preparation of experimental diets and protein consumption

The brood combs from which bees were obtained for weight evaluation of newly emerged bees. Combs with bees about to emerge were obtained from all colonies to create a pool of bees originating from each dietary management (S and R). These frames were placed in an incubator for 24 hours at 33ºC and 70% relative humidity. Subsequently, 0.25L round clear plastic countainers were used as cages to house 20 bees from the same dietary management group; container lids were perforated to allow gas exchange. An Eppendorf tube used as a syrup feeder was installed in the lid with access to food through an opening made with a needle, and another tube partially cut open with a knife was installed through the side of the cage for providing protein food.

The protein foods were formulated from a mixture containing three parts of freshly harvested sunflower pollen from flowers treated (F+) or untreated (F-) with the fungicide under study and one part wildflower honey. Sugar syrup was prepared containing two parts cane sugar and one part water. Daily replenishment of the food in the cages was provided, and the amount consumed of the protein food measured with an analytical balance in order to determine protein consumption.

Analysis of oxidative stress in nurse honey bees

For the evaluation of oxidative metabolism, 320 bees were maintained in 16 cages for five days (2 dietary managements of colonies, 2 diets for the newly emerged bees, 4 repetitions, 20 bees per cage). The bees were kept in an incubator with controlled temperature (33°C) and humidity (70%). An extra cage with 20 bees for each treatment was used to replace any dead bees.

Obtaining and quantifying protein in the thorax homogenate

After the period in which the bees received the experimental diets, the cages were placed in a freezer at -20^oC for a few minutes to anesthetize the bees. Subsequently, the thorax of the bee was separated from other body parts, including legs and wings, with the aid of fine tipped scissors (Hoskins et al. 1956). The thoraxes of bees from the same cage were placed in a mortar containing the isolation medium (250 mM sucrose, 1 mM EGTA, and 10 mM HEPES-KOH, pH 7.2), kept at 4ºC, and macerated with a porcelain pestle. The resulting homogenate pool was filtered through gauze folded eight times. Total protein concentration was determined using the biuret reaction, according to Cain and Skilleter (1987), with bovine serum albumin (BSA) used as a standard.

Evaluation of antioxidant enzyme activity in the thorax homogenate

Glutathione peroxidase (GPx) enzyme activity

GPx enzyme activity was determined by an indirect method based on oxidation of reduced glutathione (GSH) to oxidized glutathione (GSSG), catalyzed by GPx, and the subsequent oxidation of NADPH by GSSG (FLOHÉ and GÜNZLER, 1984). The reaction volume was 1.5 mL containing: GSH 1.0 mM, NADPH 0.2 mM, H₂O₂ 0.25 mM, EDTA 0.5 mM, and sodium phosphate buffer 0.10 M (pH 7.6), along with the bee thorax homogenate (1 mg protein/mL). Enzymatic activity was evaluated at 30ºC in a Beckman-Coulter DU-800 spectrophotometer at a wavelength of 340 nm, and the oxidation of 1 µmol of NADPH/minute was considered one unit of glutathione peroxidase. Specific activity was expressed as units per mg of protein/minute.

Catalase enzyme (CAT) activity

Evaluation of catalase enzyme activity was measured in thorax homogenate (1 mg of protein) in 1.75 mL of 50 mM potassium phosphate buffer (pH 7). The reaction was initiated by adding 200 µL of 10 mM H₂O₂. Catalase activity was defined as the amount of enzyme required to decompose 1 µmol of H₂O₂per minute at 25°C and pH 7. Absorbance was read on a Beckman-Coulter model DU-800 spectrophotometer at 230 nm. Specific activity was expressed as units per mg of protein/minute (AEBI, 1974).

Determination of non-enzymatic antioxidants

Reduced glutathione (GSH) concentration

GSH concentration in the thorax homogenate was measured according to Hissin and Hilf (1976), using 2 mL microtubes. The homogenate (1 mg of protein) was added to medium containing 125 mM sucrose, 65 mM KCl, and 10 mM HEPES-KOH, pH 7.4, to complete 1 mL. After gentle homogenization, 500 µL of 13% trichloroacetic acid was added. The mixture was shaken and then centrifuged at 9000 g for 3 minutes. In 5 mL test tubes, 1800 µL of buffer containing 0.1 M NaH₂PO₄, pH 8.0, with 5 mM EDTA, 100 µL of the supernatant obtained from centrifugation, and 100 µL of OPT (o-phthalaldehyde) 1 mg/mL were added. The tubes were then shaken and kept in the dark at room temperature for 15 minutes. Reads were made in a Shimadzu RFPC 5301 spectrofluorometer, with a wavelength of 350 nm for emission and 420 nm for excitation, with a slit width of 3 mm in both cases.

Oxidized glutathione (GSSG) concentration

For the GSSG assay, 250 µL of the initial supernatant was treated with 250 µL of 0.04 M N-ethylmaleimide (NEM) and subjected to the same mixing procedure with OPT. The concentrations of GSH and GSSG were estimated using a standard curve.

Oxidative state of pyridine nucleotides (NAD(P)H)

Homogenate samples (1 mg of protein/mL) were added to a reaction medium containing 125 mM sucrose, 65 mM KCl, and 10 mM HEPES-KOH, pH 7.4, with a final volume of 2 mL. Reads were made on a Shimadzu RFPC 5301 spectrofluorometer, using 366 nm for excitation and 450 nm for emission. The results were expressed as Relative Fluorescence Units (RFU).

Lipoperoxidation assessment

Lipid peroxidation was determined using the TBARS (thiobarbituric acid reactive substances) method (Buege and Aust, 1978). The homogenate (5 mg of protein) was placed in test tubes, and 0.2 mL of sodium dodecyl sulfate (SDS) (8.1%), 1.5 mL of acetic acid (20%), and 1.5 mL of thiobarbituric acid (TBA) (0.67% aqueous solution) were added. The volume was completed to 4 mL with deionized water (milli-Q), and the mixture was placed in a water bath at 95°C for 60 minutes. After the incubation period, the tubes were removed and cooled in an ice bath. Then, 1 mL of milli-Q water was added, and the MDA-TBA complex was extracted with 5 mL of n-butanol. The tubes were centrifuged at 2000 g for 10 minutes, the organic phase was collected, and the absorbance was measured at 535 nm. The amount of lipid peroxidation was determined using the molar extinction coefficient of 1.56 x 10⁵ M^-1.

Longevity test

For the longevity test, 480 bees were used, with half from each nutritional management (S or R). Groups of 20 newly emerged individuals were placed in plastic cages and fed the same experimental diets used for evaluation of oxidative metabolism (2 colony nutritional managements, fed with pollen contaminated with fungicide (F+) or not (F-), with 6 replications). The number of dead bees was recorded daily until the death of the last bee. The cages were maintained in an incubator at 33ºC and 70% relative humidity.

Statistical Analysis

The weekly amount of honey bee pollen collected from the colonies of group R was recorded. The efficiency of the pollen collectors was determined based on three observations for each colony, and data dispersion was assessed through the standard error of the mean. The data collected for the evaluation of bee weight, not considering the factor related to fungicide contamination, as bees were weighed immediately after emergence, was analyzed with analysis of variance (ANOVA), and in cases where a significant effect was observed (P < 0.05), pairwise mean comparisons were made using the Tukey test.

For the analysis of each variable related to oxidative metabolism and protein consumption, a completely randomized design in a split-plot scheme with two factors was used: nutritional management of the colonies (supplemented – S or restricted – R) and adult bee protein diet (with fungicide – F + or without fungicide – F-), which were used to establish four treatments (SF+, SF-, RF+, RF-). The main factor, nutritional management, was applied first, followed by feeding the bees from the S or R colonies with protein (F + or F-). A mixed linear model was fitted considering the two factors as well as their interaction. The adjusted means for each treatment combination were compared using Tukey's test (5%).

For the assessment of bee longevity, we analyzed the results of the four treatments using the non-parametric Kaplan-Meier method and the Log-Rank test. Next, we used the hazard ratios for each factor to compare bee longevity across different treatments by fitting a Cox proportional hazards model. All analyses were conducted using SAS software (SAS Institute, 2024).

Dietary Management and Pollen Collection

During the 24 weeks of dietary management of the colonies, honey bee pollen was collected daily from the colonies in the restricted diet group R). On average, 65.23 ± 12.35g of bee pollen was obtained weekly per colony. Throughout the experimental period, 1,171.27 to 1,828.42g of pollen was collected per colony. In the group of colonies supplemented weekly (S), 2,400g of protein paste containing 1,800g of bee pollen was provided to each colony during the same period.

The mean efficiency of the pollen collectors was 61.2 ± 4.8%. Thus, considering the amount of pollen collected with the pollen traps, the estimated quantity of this proteinaceous food that was brought into the colonies over the 24 weeks ranged from 454.80 to 709.98g. Weekly, the available amount of pollen for each colony in the restricted diet group (R) was estimated to be 18.95 to 29.58g.

Weight of newly emerged bees

The live weight of bees from colonies with dietary supplementation did not differ significantly from that of bees from colonies with restricted feeding (Table 1). After sample drying, it was observed that the dry matter content and the dry matter: original matter ratio were higher in bees from well-fed colonies (S) compared to bees from nutritionally restricted colonies (R).

Table 1

Mean live weight, dry weight in mg, and dry weight/live weight ratio of newly emerged bees obtained from colonies with supplemented or restricted food availability.
Effects		Live weight (LW)	Dry weight (DW)	DW:LW
Feeding	Supplemented	82.72	18.06 a	21.83 a
Feeding	Restricted	79.37	15.53 b	19.57 b
SE		1.21	0.54	0.46
P value		0.178	0.007	0.004

Means followed by different letters in the same column differ significantly from each other (P < 0.05), according to the Tukey test. Means for 10 individuals from each colony.

Protein food consumption during five days and oxidative metabolism

The consumption of protein paste by the bees in the cages was influenced by the interaction between the nutritional management of the colonies from which the bees were obtained and the applicationi of fungicide to the sunflowers from which the pollen was obtained (Table 2). Bees from supplemented colonies that received protein paste containing sunflower pollen from inflorescences untreated with fungicide (SF-) consumed a significantly more protein diet compared to bees from restricted diet colonies that received the same food. These bees (RF) consumed more protein paste than the others (SF + and RF+), which did not differ from each other. Fungicide contamination of the food led to a reduction in food consumption, both in bees from colonies with nutritional supplementation and in those with less available protein food during the brood phase.

Glutathione peroxidase (GPx) activity:

The activity of the GPx enzyme was influenced by nutritional management and pollen treatment with fungicide (Table 2), being higher in bees that had more food available for their development during the brood phase (S) in comparison with R, and that ingested protein food without fungicide (F-), compared to bees that ingested the contaminated diet F+.

Catalase (CAT) enzyme activity:

The activity of the catalase enzyme was influenced by the interaction of nutritional condition of the colony of origen with fungicide contamination of the pollen that they consumed, with a reduction in its activity in bees from group SF + compared to bees from other treatments, which did not differ from each other (Table 2).

Reduced glutathione (GSH) and oxidized glutathione (GSSG) concentrations

The concentration of reduced glutathione and oxidized glutathione was higher in bees obtained from S compaed to those from R colonies. No effect of the fungicide was observed for the concentration of non-enzymatic antioxidants. No difference in the ratio between GSH and GSSG concentrations was observed for bees from the four treatments, indicating that there was no alteration in the oxidative state of glutathione, when considering the factors nutritional management of the colonies and ingestion of food contaminated with fungicide.

Oxidative State in Pyridine Nucleotides (NAD(P)H)

The concentration of NAD(P)H in the thorax homogenate was higher in bees from colonies that had their diet supplemented (S) (Table 2). No effect of the fungicide in pollen fed to the caged bees was observed.

Evaluation of Lipoperoxidation

In the analysis of lipid peroxidation through the quantification of malondialdehyde (MDA), it was observed that pollen contaminated with fungicide led to an increase in lipoperoxidation levels (Table 2). This oxidation was not influenced by the dietary management in the colonies from which the newly emerged bees were obtained.

Table 2

Individual consumption of protein paste over five days, activity of antioxidant enzymes, and parameters related to oxidative stress in the thorax homogenate of bees after the feeding period.
Effects		Consumption (mg/bee)	GPx (mU/mg of protein)	CAT (µmol/mg of protein/min)	GSH (nmol/mg of protein)	GSSG (nmol/mg of protein)	GSH/ GSSG	NAD(P)H (Relative Fluorescence Units)	MDA (nmol/mg of protein)
Feeding	Supplemented (S)	10.973	127.570 a	24.065	1.453 b	0.067 b	22.144	23.873 a	0.197
Feeding	Restricted (R)	7.968	92.725 b	30.785	2.477 a	0.098 a	26.471	7.630 b	0.188
Fungicide	With fungicide (F+)	6.716	104.400 b	23.705	1.928	0.077	25.289	15.750	0.200 a
Fungicide	Without fungicide (F-)	12.223	115.890 a	31.145	2.002	0.088	23.325	15.758	0.185 b
	SF+	6.823 c	126.800	18.523 b	1.423	0.070 b	20.973	23.870	0.210
Interaction	SF-	15.122 a	128.330	29.607 a	1.483	0.063 b	23.317	23.877	0.183
Interaction	RF+	6.608 c	82.009	28.887 a	2.433	0.083 b	29.608	7.630	0.190
	RF-	9.325 b	103.440	32.683 a	2.520	0.114 a	23.333	7.640	0.187
Standard error of the mean		0.533	3.089	1.064	0.187	0.010	2.470	0.047	0.006
Source of variation					Probability
Feeding (a)		< 0.001	< 0.001	< 0.001	< 0.001	0.022	> 0.200	< 0.001	>0.200
Fungicide (b)		0.001	0.030	< 0.001	> 0.200	> 0.200	> 0.200	> 0.200	0.043
Interaction (a x b)		0.001	0.052	0.009	> 0.200	0.140	0.095	> 0.200	0.098

Means followed by the same letter for the same effect (bee feeding or use of fungicide on sunflower plants or interaction between these factors) for each variable do not differ significantly from each other (P > 0.05), according to the Tukey test. Food consisting of three parts of sunflower pollen treated or not with the fungicide Fox Xpro and one part honey.

Longevity of workers

Bee survival was affected by the feeding management to which the colonies of origin were subjected and contamination of the diet fed to the caged bees with fungicide (𝑋² = 40.167, p < 0.0001; Fig. 1). The longevity of bees that developed during the larval stage in supplemented colonies and received uncontaminated food with fungicide in the adult stage (15.20 ± 0.34 days) was greater than that of bees from the same colonies that were fed fungicide-contaminated food in the adult stage (12.88 ± 0.33 days). Bees that developed in colonies with restricted feeding and received uncontaminated food in the adult stage had a mean survival of 13.04 ± 0.36 days. Longevity was lowest for bees from colonies with restricted feeding that received fungicide-contaminated food in the adult stage (12.08 ± 0.27 days).

When considering the hazard ratios for each factor evaluated, we observed that fungicide (F+) in the bees diet results in lower survival of bees from R colonies compared to bees from S colonies (Table 3). Even when bees were treated with pollen without fungicide (F-), the survival of bees from S colonies was significantly higher than bees from R colonies. Regarding the effect of fungicide within each nutritional management group, we observed that for both S and R bee groups, survival was lower due to exposure to food containing pollen from inflorescences treated with fungicide.

Table 3

Chi-square, P values and hazard ratios for dietary management of honey bee colonies (S for supplemented and R for restricted feeding) and presence of fungicide in protein paste provided to the bees (F + means with fungicide and F- without fungicide).
Effect	Chi-Square	Pr > ChiSq	SE
Feeding	4.0595	0.0439	0.1367
Fungicide	12.8742	0.0003	0.1361
Feeding x Fungicide	0.3744	0.5406	0.1928
Hazard Ratios
	Point estimate	Confidence Limits
S x R at F+	0.759	0.581	0.993
S x R at F-	0.675	0.517	0.881
F + x F- at S	1.630	1.248	2.128
F + x F- at R	1.448	1.105	1.900

According to Corona et al. (2023), nutritional stress significantly contributes to weakening and loss of colonies; it is intrinsically linked to colony health and vigor, habitat degradation, pollen diversity, and innate foraging differences among bees from different colonies. Pollen deprivation induces bees to forage early, which is associated with accelerated aging and reduced longevity of these insects (Perry et al. 2015; Corona et al. 2023). With food restriction, colonies undergo a population adjustment to balance pollen demand and supply. In our experiment, the estimated amount of food that bees were able to bring into the hives is compatible with the minimum amount needed to maintain about 4,000 individuals, considered a small colony of honey bees (Danner et al. 2017). The weight of bees can be influenced by increased food availability, resulting in positive effects on the health and longevity of adult bees, provided that the diet is nutritionally balanced (Ihle et al. 2014). In our study, the weight of bees upon emerging from the combs was favored by the more suitable nutritional conditions of colonies with food supplementation (Table 1).

During the brood phase, honey bees require 125 to 187 mg of pollen. As adults, about 3.9 mg of pollen is needed daily, with peaks during the nurse bee phase (Brodschneider and Crailsheim, 2010). In this phase, consumption is higher to ensure the proper development of the hypopharyngeal glands. In our study, newly emerged bees from the SF- treatment consumed about 2.27 mg of sunflower pollen per day during the first five days of life (Table 2).

Bees from colonies subjected to food restriction consumed less food than those that developed in an environment with greater food availability during the brood phase, considering sunflower pollen untreated with fungicide (Table 2). Thus, it is considered that food restriction during the brood phase hinders the development of the bee in the adult phase, with no compensatory gain in food consumption during adulthood. Larvae reared during deficiency of an essential nutrient may become adults with lower survival or impaired brood care and foraging skills (Brodschneider and Crailsheim, 2010).

Bee bread is useful as a source of antioxidants that protect cells against oxidative damage to the cytoplasmic structure within cellular organelles and in the extracellular fluid (Martinello and Mutinelli, 2021). Bee bread from different botanical sources has different antioxidant capacities, more related to specific profiles of flavones and phenolic acids (Borycka et al. 2015), highlighting the importance of protein nutrition for the physiological well-being of bees (Wright et al. 2018).

Food contamination with fungicide caused reduced food consumption, independent of their being from well fed or nutritionally deprived colonies. The sensitivity of bees to pesticides may be linked to the life stage of the bee (Zhu et al. 2020), as changes occur in endocrine and metabolic activity during behavioral maturation related to age, inherent to the transition from nursing tasks to foraging (Robinson, 2002). Forager bees show higher detoxifying enzymatic activity than bees working solely within the colony (Smirle and Robinson, 1989; Berenbaum and Johnson, 2015). However, it seems that susceptibility among castes depends on the type of pesticide being evaluated, as some active ingredients are more toxic to foragers in comparison with nurse bees (Barascou et al., 2022).

Sunflower pollen has been indicated in some studies as having lower nutritional quality compared to various other types of pollen (Schmidt et al. 1987), with low protein content (Radev, 2019), and low levels of two essential amino acids for honey bees, methionine and tryptophan, below the minimum required by bees (Nicolson and Human, 2013). Pollen has a complex chemical composition, including amino acids, lipids, sugars, secondary metabolites, and other elements (Palmer-Young et al. 2019). Indeed, plant secondary compounds have stood out for their potential positive impacts on pollinators, including protective action against pesticide exposure (Ardalani et al. 2021). Bee foraging on various plant species favors obtaining a more balanced and nutrient-rich diet (Vaudo et al. 2015). The hypothesis that adequate nutrition can protect animals against the adverse effects of stressors, such as pesticides, is a well-established perspective in biology (Wahl and Ulm, 1983).

Antioxidant defense systems in honey bees encompass both enzymatic and non-enzymatic mechanisms, both crucial for mitigating damage caused by reactive oxygen species (ROS) (Corona et al. 2023). Among the antioxidant enzymes, glutathione S-transferase (GST), glutathione peroxidase (GPx), catalase (CAT), and superoxide dismutase (SOD) are noteworthy. Simultaneously, non-enzymatic components such as glutathione, NAD(P)H, and vitamins C and E, actively participate in neutralizing ROS (Badiou-Bénéteau et al. 2012; Tawfik et al. 2020).

GPx activity was higher in the thoracic homogenate of bees that consumed more food. According to Wahl and Ulm (1983), sensitivity to oxidative stress in bees is reduced with improved pollen quality. Therefore, considering the low quality of sunflower pollen (Radev, 2019), we presumed that lower food consumption implies lower activity of this enzyme. A reduction in GPx enzyme activity observed in bees exposed to food contaminated with fungicide may be due to a reduction in energy availability in cells, given the inhibition of the respiratory chain inherent to the action of the active principles bixafen and trifloxystrobin (Bartlett et al. 2002; Oliver and Hewitt, 2014; Nicodemo et al. 2020).

CAT enzyme activity was only altered in the SF + group, which was lower than in the other groups. Even though there was no significant difference in food consumption between SF + and RF+, it is suggested that the slightly higher consumption in the former group may have resulted in a more effective action of the active principles bixafen and trifloxystrobin in reducing cellular work capacity. In this sense, the antioxidant defense would not neutralize the levels of ROS generated by exposure to the fungicide.

The GSH:GSSG ratio is recognized as a fundamental indicator of intracellular redox balance and antioxidant capacity. In our study, this ratio was not affected by the nutritional management of the colonies of origin of the bees or by feeding on food contaminated with fungicide. The concentration of NAD(P)H was higher in bees that consumed more food, with no effect detected of the pesticide, considering the exposure level, which was according to the fungicide application protocol. These results may differ from those obtained in other investigations (Kapoor et al. 2011; Bal et al. 2012; Chakrabarti et al. 2015) regarding the impact of pesticides on the concentration of these non-enzymatic oxidants. However, the differences between populations, doses, and exposure modes used in each of the works should be considered.

A significant increase in lipid peroxidation levels, evidenced by the marker MDA, was observed in samples of bees fed pollen from inflorescences exposed to the triple-action fungicide, regardless of the dietary management of the colony of origin of the bees. The active principle protioconazole blocks the sterol biosynthesis pathway, which is fundamental for the organization and structuring of cell membranes (Schuhmann et al. 2022). Lipoperoxidation is recognized as a sign of cellular damage and oxidative stress, which can affect bee cells exposed to xenobiotics (Balieira et al. 2018).

Regarding longevity, we observed that the fungicide affected bee longevity. Bees that developed in supplemented colonies during the brood phase and did not receive contaminated food during adulthood lived 16.6% longer than bees that had restricted feeding during the brood phase and received uncontaminated food. The greatest impacts on bee survival were observed when bees developed during the brood phase in colonies with food restriction and, during adulthood, received food contaminated with fungicide, with a 20.5% reduction in the average survival of workers compared to the longer-lived bees (SF-), highlighting the synergistic effect between food restriction and exposure to fungicide.

The longevity of honey bees is reduced when exposed to pesticides; however, the presence of some phytochemicals in food, even those without nutritional value, can mitigate the effects on bee survival (Liao et al. 2020). The availability of food in greater quantity (Mattos et al. 2017; Hýbl et al. 2021) and of higher quality (Castle et al. 2022; Costa et al. 2022) can favor the health and survival of bees exposed to pesticides. Despite the use of the pesticide according to application protocols, there was damage to the health of these insects. Therefore, it is suggested that the recommendation for the use of this fungicide during the flowering period of target crops be reconsidered.

Feeding pollen from sunflowers sparyed with a commercial fungicide containing bixafen, prothioconazole and trifloxystrobin caused oxidative stress in honey bees, even though the application was made in accordance with agronomic recommendations.

Bee survival was adversely affected by exposure to fungicide, with a synergistic effect on bees from colonies subjected to food restriction.

Funding

This research was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo - FAPESP (process n^o 2021/00702-1) to Daniel Nicodemo and by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior-Brasil (CAPES)-Finance Code 001 to Thais Regina Ramos Alves.

Competing interests

Daniel Nicodemo reports financial support was provided by FAPESP. All the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

The datasets generated during this study are available from the corresponding author on reasonable request.

Author Statement

TRRA, MFT, TALF, AYK, CRAG, YMMF, JAS and CAM: data collection. DN, DDJ, FEM, EPP and EFV: research design. DN and TGP: data analysis. DN, ROO, FEM and DDJ: manuscript writing.

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Pollen contaminated with a triple-action fungicide induced oxidative stress and reduced bee longevity though with less impact on lifespan in bees from well fed colonies

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Abstract

Figures

Introduction

Material and methods

Experiment location, sunflower cultivation, fungicide application, and pollen collection

Dietary Management of Honey Bee Colonies

Pollen collection and efficiency of pollen collectors in colonies with restricted feeding

Evaluation of the weight of newly emerged bees

Obtaining newly emerged bees, preparation of experimental diets and protein consumption

Analysis of oxidative stress in nurse honey bees

Obtaining and quantifying protein in the thorax homogenate

Evaluation of antioxidant enzyme activity in the thorax homogenate

Glutathione peroxidase (GPx) enzyme activity

Catalase enzyme (CAT) activity

Determination of non-enzymatic antioxidants

Reduced glutathione (GSH) concentration

Oxidized glutathione (GSSG) concentration

Oxidative state of pyridine nucleotides (NAD(P)H)

Lipoperoxidation assessment

Longevity test

Statistical Analysis

Results

Dietary Management and Pollen Collection

Weight of newly emerged bees

Protein food consumption during five days and oxidative metabolism

Glutathione peroxidase (GPx) activity:

Catalase (CAT) enzyme activity:

Reduced glutathione (GSH) and oxidized glutathione (GSSG) concentrations

Oxidative State in Pyridine Nucleotides (NAD(P)H)

Evaluation of Lipoperoxidation

Longevity of workers

Discussion

Conclusion

Declarations

References

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