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Interplay between Selenium, selenoprotein genes, and oxidative stress in honey bee Apis mellifera L - PubMed

Interplay between Selenium, selenoprotein genes, and oxidative stress in honey bee Apis mellifera L

Mohamed Alburaki et al. J Insect Physiol. 2019 Aug-Sep.

Abstract

The honey bee, Apis mellifera L., is a major pollinator insect that lacks novel "selenoprotein genes", rendering it susceptible to elevated levels of Selenium (Se) occurring naturally in the environment. We investigated the effects of two inorganic forms of Se on biological traits, oxidative stress, and gene regulation. Using bioassay arenas in the laboratory, one-day old sister bees were fed ad libitum 4 different concentrations of selenate and selenite, two common inorganic forms of Se. The transcription levels of 4 honey bee antioxidant genes were evaluated, and three putative selenoprotein-like genes (SELENOT, SELENOK, SELENOF) were characterized as well as Sbp2, a Selenium binding protein required for the translation of selenoproteins mRNA. Oxidative stress and Se residues were subsequently quantified in honey bee bodies throughout the experiment. Se induced higher oxidative stress in treated honey bees leading to a significantly elevated protein carbonyl content, particularly at the highest studied concentrations. Early upregulations of Spb2 and MsrA were identified at day 2 of the treatment while all genes except SELENOT were upregulated substantially at day 8 to alleviate the Se-induced oxidative stress levels. We determined that doses between 60 and 600 mg.Se.L-1 were acutely toxic to bees (<48 h) while doses between 0.6 and 6 mg.Se.L-1 led to much lower mortality (7-16)%. Furthermore, when fed ad libitum, Se residue data indicated that bees tolerated accumulation up to 0.12 µg Se bee-1 for at least 8 days with a Se LC50 of ∼6 mg/L, a field realistic concentration found in pollen of certain plants in a high Se soil environment.

Keywords: Antioxidant gene; Honey bee; Oxidative stress; Selenium; Selenoprotein-like genes.

Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.

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Conflict of interest statement

Declaration of Competing Interest

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.

Shows the cage experimental design, concentrations of both selenate and selenite used in each treatment group as well as the sampling and acclimatization timelines. Each experiment lasted for 11 days and 50 sister bees were used per cage.

Fig. 2.
Fig. 2.

Syrup consumption throughout time exposed by treatment groups for both administrated selenate and selenite. Percentage of the daily bee mortality is also given for each treatment group. Day 1–3 is an acclimatization period where no treatment was applied. Treatments were applied at day 3 and lasted until day 11. Each data point represents (means ± SE) of 3 biological replicates.

Fig. 3.
Fig. 3.

Average of syrup consumption per bee. Average syrup consumption for each treatment group prior and after treatment for both selenate and selenite. Prior treatment represents the acclimatization period with no treatment applied while post treatment is the consumption from day 3 and onward. ANOVA levels of significance are *P < 0.05 and ***P < 0.001. Inter-group significant differences are indicated with different letters.

Fig. 4.
Fig. 4.

Average of bee survivorship. Percentage of bee survival for each treatment group for both selenate and selenite. Prior treatment stands for the acclimatization period in which all groups were only fed sugar syrup with no treatment. After treatment indicates the day 3 when treatments were administrated to bees and onward. ANOVA levels of significance are **P < 0.01 and ***P < 0.001. Inter-group significant differences are indicated with different letters.

Fig. 5.
Fig. 5.

Honey bee Oxidative Stress. Quantification of both hydrogen peroxide and protein carbonyl content in honey bees fed various concentrations of selenate and selenite at days 2. All treatment concentrations are in (μg/mL) and BSA is bovine serum albumin. Each boxplot represents an average of 6 biological replicates and BSA was not included in the statistical analysis. Error bars are the quartiles and outliers and ANOVA levels of significance are *P < 0.05, **P < 0.01 and ***P < 0.001.

Fig. 6.
Fig. 6.

Relative quantification conducted by RT-qPCR of three selenoprotein-like genes (SELENOT, SELENOK, SELENOF) exposed by individual biological replicates (cage) and total average (means ± SE) of 3 biological replicates for both selenate fed bees (6 μg/mL) and control bees. Gene expression is studied at two different time points (day 2–day 8) of the experiment. The 1.5% agarose gels show inter-tissue transcriptional verification of studied genes conducted by RT-PCR for representative set of samples (6 μg/mL selenate vs. control). Error bars represent the Standard Error SE and level of significance is *P < 0.05.

Fig. 7.
Fig. 7.

Relative quantification of the Selenium binding protein gene (Sbp2) at day 2 and 8 of the treatment conducted by RT-qPCR. The gene study describes the four target genes normalized against two housekeeping genes (GAPDH and RPS18). Sbp2 inter-tissue transcriptional verification run on 1.5% agarose gel is also shown. Error bars are the Standard Error SE and levels of significance are *P < 0.05, **P < 0.01. Housekeeping gene stability is given for each gene.

Fig. 8.
Fig. 8.

Relative quantification conducted by RT-qPCR of major honey bee antioxidant genes (Sod1, Trxr1, MsrA) exposed by individual biological replicates (cage) and total average (means ± SE) of 3 biological replicates for both selenate fed bees (6 μg/mL) and control bees. Gene expression is studied throughout time from day 2 to day 8 of the treatment and conducted on samples of the same cages (6 μg/mL vs. Control). Error bars represent the Standard Error SE and levels of significance are *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 9.
Fig. 9.

Relative quantification of the catalase (Cat) at day 2 and 8 of the treatment conducted by RT-qPCR. Gene study of the four target genes normalized against two housekeeping genes (GAPDH and RPS18). Error bars are the Standard Error SE and levels of significance are *P < 0.05, **P < 0.01, ***P < 0.001. Housekeeping gene stability is given for each gene.

Fig. 10.
Fig. 10.

Selenium residues (μg/g) identified in each of the four sampled sets of bees at day 2, 4, 6 and 8. Results are exposed by treatment groups and control for both selenate (A) and selenite (B) fed bees. Each boxplot represents an average of 3 biological replicates and error bars describe the quartiles and outliers. ANOVA level of significance among groups is ***P < 0.001.

Fig. 11.
Fig. 11.

Schema of the selenate case summarizing the major transcription and post-transcription events related to the Selenium residue levels identified per bee in each of the treatment groups. Overall percentage of survivorship is given for each treatment group as well as daily survivorship percentages for the (6 μg/mL) group.

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