Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Open Access

Peer-reviewed

Research Article

Transcriptomic analysis to elucidate the response of honeybees (Hymenoptera: Apidae) to amitraz treatment

  • Liang Ye ,

    Roles Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing

    ‡ Co-first author

    Affiliation Anhui Agricultural University, Hefei, China

    ⨯
  • Peng Liu ,

    Roles Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Writing – review & editing

    ‡ Co-first author

    Affiliation Anhui Agricultural University, Hefei, China

    ⨯
  • Tengfei Shi ,

    Roles Methodology, Project administration, Resources, Software, Supervision, Validation, Writing – review & editing

    ‡ Co-first author

    Affiliation Anhui Agricultural University, Hefei, China

    ⨯
  • Anran Wang,

    Roles Funding acquisition, Investigation, Project administration, Resources

    Affiliation Anhui Agricultural University, Hefei, China

    ⨯
  • Yujie Zhu,

    Roles Conceptualization, Resources, Validation, Visualization

    Affiliation Anhui Agricultural University, Hefei, China

    ⨯
  • Lai Li,

    Roles Data curation, Investigation, Resources, Supervision

    Affiliation Anhui Agricultural University, Hefei, China

    ⨯
  • Linsheng Yu

    Roles Writing – review & editing

    yulinshengahau@163.com

    Affiliation Anhui Agricultural University, Hefei, China

    ⨯

Abstract

Amitraz is an acaricide that is widely used in apiculture. Several studies have reported that in honeybees (Apis mellifera Linnaeus; Hymenoptera: Apidae), amitraz affects learning, memory, behavior, immunity, and various other physiological processes. Despite this, few studies have explored the molecular mechanisms underlying the action of amitraz on honeybees. Here, we investigated the transcriptome of honeybees after exposure to 9.4 mg/L amitraz for 10 d, a subchronic dose. Overall, 279 differentially expressed genes (DEGs) were identified (237 upregulated, 42 downregulated). Several, including Pla2, LOC725381, LOC413324, LOC724386, LOC100577456, LOC551785, and P4504c3, were validated by quantitative PCR. According to gene ontology, DEGs were mainly involved in metabolism, biosynthesis, and translation. Kyoto Encyclopedia of Genes and Genomes pathway analyses revealed that amitraz treatment affected the relaxin signaling pathway, platelet activation, and protein digestion and absorption.

Citation: Ye L, Liu P, Shi T, Wang A, Zhu Y, Li L, et al. (2020) Transcriptomic analysis to elucidate the response of honeybees (Hymenoptera: Apidae) to amitraz treatment. PLoS ONE 15(3): e0228933. https://doi.org/10.1371/journal.pone.0228933

Editor: Yulin Gao, Chinese Academy of Agricultural Sciences Institute of Plant Protection, CHINA

Received: November 5, 2019; Accepted: January 27, 2020; Published: March 6, 2020

Copyright: © 2020 Ye et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper and SRA database(https://dataview.ncbi.nlm.nih.gov/object/PRJNA593612?reviewer=elfpv4vmb047ik7k9efhmla825).

Funding: This work was supported by the Earmarked Fund for China Agriculture Research System (No. CARS-45-KXJ10). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Approximately 80% of flowering plants, including many crops, require insects to pollinate [1–3]. Honeybees (Apis mellifera Linnaeus; Hymenoptera: Apidae) are the most important pollinators worldwide [4]. One-third of global food is linked to the pollination activity of honeybees [4]. In recent years, the substantial decline in apiculture has garnered much attention [5–8]; however, the underlying reasons for this remain poorly understood. Many factors affect the wellbeing of honeybees, including pathogens, pesticides, malnutrition and changing apicultural practices [9–11]. Among these, pesticide exposure has been widely accepted to be the major contributor to a decline in the honeybee population [10].

Honeybees are continuously exposed to agricultural pesticides, which are transported to hives by foraging bees [10]. Direct application of acaricides within beehives to control Varroa mites and other pests creates an extra pesticide burden on the bees [10]. Acaricides and pesticides contaminate apicultural products such as honey, beeswax, and pollen [8, 12, 13].

The midgut of honeybee is an absorptive organ and involved in degraded chemical compounds[14]. The epithelium, in particular, is responsible for detoxification of ingested xenobiotics[15]. Meanwhile, honey bee larvae exposed to sublethal concentrations of a broad range of pesticides resulted in midgut cell apoptosis[16]. As the same time, the midgut is the principal barrier to invasion of the honey bee for many pathogens [17].

Amitraz [1,5-di-(2,4-dimethylphenyl)-3-methyl-1,3,5-triaza-penta-1,4-diene] is a formamidine pesticide used globally to control pests on animals and crops [18]. It is an acaricide and mainly acts on the central nervous system of ectoparasites by interacting with octopamine receptors, causing lethal and sublethal effects [19]. In the apiary, beekeepers can control Varroa mites by fumigation of beehives with amitraz, but it results in contamination of honey stored in combs[20]. Amitraz does not persist in the hive environment [21], but its metabolite N-(2,4-dimethylphenyl)-N′-methylformamidine can accumulate and has been found in wax, pollen, and inside the bees themselves [13]. Moreover, acute exposure to amitraz also can kill honeybee larval midgut epithelial cells [22]. Amitraz also affects learning, memory [23, 24], immunity [25] and sensory organs [26] in honeybees. In addition, a recent study reported that amitraz affected the immune system of the queen [27]. Amitraz stress leads to increase glutathione S-transferases activity in the larval instars, pupae, newly emerged bees and nurse bees[28].

Despite the adverse effects of amitraz on honeybees, the relevant molecular mechanisms remain poorly explored. In this study, we conducted high-throughput RNA sequencing (RNA-Seq) analyses to investigate honeybee transcriptomes after exposure to 9.4 mg/L amitraz for 10 d, a subchronic level. Differentially expressed genes (DEGs) were identified and analyzed. Our aim was to help understand the molecular mechanisms underlying the action of amitraz to elucidate reasons for the decline in honeybee populations.

Materials and methods

Honeybee rearing

The honeybees were obtained as previously described [29, 30]. Two frames with sealed broods nearing adult emergence were collected from an apparently healthy colony at the Institute of Apiculture Research, Anhui Agriculture University, Hefei, China. The population had not been exposed to pesticides. The frames were held in darkness at 35 ± 1°C with relative humidity (RH) 50% ± 10%. Newly emerged honeybees were then placed into wooden cages (11 × 10 × 8 cm) in darkness for 2 d (28 ± 1°C, RH 60% ± 10%). Throughout the experimental period, bees were fed sufficient fresh pollen and 50% (w/v) sucrose-water solution. Dead bees were removed daily.

Amitraz treatment

We followed previously reported methods by Shi et al. with some minor modifications[31]. The median lethal concentration (LC50) of amitraz to honeybees is 94 mg/L [32]. Herein we used amitraz(99% purity) which was obtained from aladdin company(Shanghai, China) at a sublethal concentration (9.4 mg/L). A stock solution of amitraz (1000 mg/L) was prepared in acetone. Working solution (9.4 mg/L) was prepared by dissolving the stock solution in 50% sucrose-water solution. Sucrose–water solution added the equivalent acetone without amitraz as a negative controls.

Three-day-old bees were used for assays (45 bees/replicate, three replicates/treatment). After 10 d, all bees were collected and placed at 4°C for 5 min to anesthetized them; the bees were then dissected on ice, using liquid nitrogen to flash freeze the sample and the midgut was removed and stored at −80°C.

RNA extraction, library preparation and sequencing

Ten midguts from each replicate were pooled for RNA extraction using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA concentration was quantified and RNA integrity verified. Sequencing libraries were generated using a NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, CA, USA) following the recommended protocol; 3 μg RNA from each sample were used to prepare the library. Index codes were added to link sequences with the sample from which they originated. mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. mRNA was fragmented in 5× NEBNext First Strand Synthesis Reaction Buffer at elevated temperature. First strand cDNA was synthesized using random hexamer primers and M-MuLV Reverse Transcriptase (RNase H). Second strand cDNA was synthesized using DNA Polymerase I and RNase H. Overhangs were blunted using exonuclease/polymerase. 3′-ends of DNA fragments were adenylated and ligated with NEBNext Adaptors. DNA fragments (150–200 bp long) were selected by purification using an AMPure XP system (Beckman Coulter, Beverly, MA, USA). USER Enzyme (3 μL; NEB) was incubated with the size-selected, adaptor-ligated cDNA at 37°C for 15 min, then 5 min at 95°C. PCR was performed with Phusion High-Fidelity DNA polymerase, universal PCR primers. The amplicons obtained were purified using the AMPure XP system, and library quality was assessed on an Agilent Bioanalyzer 2100. Clustering of the index-coded samples was performed on a cBot Cluster Generation System using the TruSeq PE Cluster Kit v3-cBot-HS (Illumina). Then, the library preparations were sequenced (Illumina HiSeq 4000); 150-bp paired-end reads were generated.

Read processing

Raw reads (FASTQ) were initially processed using in-house Perl scripts. Clean reads were obtained by removing low-quality reads, and those containing adapter sequences or poly-N. All downstream analyses used high-quality clean reads. At the same time, the Q20, Q30, and GC contents were calculated. The index of the honeybee genome (NCBI: assembly Amel_HAv3.1) was built using Bowtie v2.2.3, and reads were aligned to the genome using TopHat v2.0.12. HTSeq v0.6.1 was used to count read numbers mapped to each gene.

Analysis of differential expression

Differential expression analysis used the DESeq R package v2.15.3. The resulting P-values were adjusted using the Benjamini and Hochberg approach for controlling false discovery rate; genes with adjusted P-value <0.05 were considered to be differentially expressed. Gene ontology (GO) enrichment analysis of DEGs was performed using the GOseq R package (gene length bias was corrected); GO terms with corrected P-values <0.05 were considered significantly enriched by DEGs. For Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, we used KOBAS to assess the statistical enrichment of DEGs.

Real-time quantitative PCR (qPCR) analysis

We selected seven DEGs identified following RNA-Seq (LOC725381, CYP4C3, LOC41332, Pla2, LOC724386, LOC100577456, and LOC551385) for verification by qPCR analysis. RpS5 and β-actin was used as the reference gene, and all the primers used were given in Table 1. We used 0.5 μg of total RNA (same as the total RNA for RNA-seq) for each sample. ReverTra Ace qPCR RT Master Mix Kit (Toyobo, Osaka, Japan) and a SuperReal PreMix Plus (SYBR Green) Kit (TIANGEN, Beijing, China) were used to obtain cDNA and perform qPCR, respectively. The relative expression levels of genes was calculated using the 2−ΔΔCt method [33]. Primer sets are listed in Table 1. Student’s t-test was used to assess differences in gene expression levels between amitraz-treated bees and controls. We used three replicates per group for qPCR validation.

thumbnail
Download:
Table 1. Primer sequences.

https://doi.org/10.1371/journal.pone.0228933.t001

Results

Survival

As shown in Fig 1, the average survival rate on day ten for the forager bees exposed to 9.4 mg/L amitraz and control of 50% sucrose-water solution were 77.8% and 88.9%, respectively. There were no significant differences among all treatments (Log-rank χ2 = 2.024, df = 1, P = 0.1548; Fig 1). The 9.4 mg/L amiteaz is a sublethal concentration.

thumbnail
Download:
Fig 1. Survival of forager bees subjected to chronic exposure to field-realistic concentrations of amitraz after 10 d.

https://doi.org/10.1371/journal.pone.0228933.g001

Raw read processing and quantitative gene expression

Six libraries were created from amitraz-treated bees and controls: amitraz-1, amitraz-2, amitraz-3, control-1, control-2, and control-3, which generated 46588584, 38742446, 39577344, 42787014, 47048748, and 44719946 usable reads, respectively. The Q20 were 99.97%, 99.97%, 99.97%, 99.97%, 99.97% and 99.97%, respectively, while the Q30 were 97.04%, 97.06%, 96.99%, 97.00%, 97.30% and 96.93%, respectively. Q20, Q30, and GC contents were listed in S2 Table. After mapping to the reference genome (NCBI: assembly Amel_HAv3.1) and junction database, 44944558, 37354986, 38094470, 41341014, 45468272 and 43453530 total mapped reads were acquired, respectively. The numbers of uniquely mapped reads were 27182392, 21407197, 24815509, 26929339, 28752629 and 27107676 respectively. Among these unique reads, 57.31%–65.14% were mapped to exons (S1 Table). The sequencing data are available in the SRA database (https://dataview.ncbi.nlm.nih.gov/object/PRJNA593612?reviewer=elfpv4vmb047ik7k9efhmla825) of the NCBI system.

The average number of genes expressed in the treatment and control groups was 11410 and 11303, respectively; 11034 genes were expressed in both groups (Fig 2).

thumbnail
Download:
Fig 2. Average number of genes specifically expressed in amitraz and control libraries.

Shown as the number of genes expressed in each class.

https://doi.org/10.1371/journal.pone.0228933.g002

In each library, 9.91%–11.74% of reads had reads per kilobase per million mapped reads (RPKM) values of <0.1; 9.76%–10.98% of reads had RPKM values of 0.1–0.3; 36.58%–37.69% of reads had RPKM values of 0.3–3.57; 19.12%–20.62% of reads had RPKM values of 3.57–15; 11.25%–12.29% of reads had RPKM values of 15–60; and 9.41%–10.07% of reads had RPKM values of >60 (S3 Table). Thus, a few genes were expressed at very high levels, but most were expressed at low levels, indicating that the distribution of our gene expression dataset was normal.

DEGs, GO enrichment analysis, and KEGG pathway analysis

Overall, 279 DEGs were detected in honeybees exposed to 9.4 mg/L amitraz for 10 d: 237 (84.9%) were upregulated and 42 (15.1%) were downregulated (Fig 3 and S4 Table). S5 Table lists the 23 most significantly differentially expressed genes; of these, one was downregulated and 22 were upregulated. Fig 4 shows the 30 most enriched GO terms. In GO classification “biological process”, most DEGs were involved in translation and metabolic and biosynthetic processes. In category “cellular components”, most DEGs were associated with the ribosome. Finally, considering classification “molecular function”, most DEGs were enriched in structural constituents of the ribosome, structural molecule activity, and oxidoreductase activity.

thumbnail
Download:
Fig 3. Volcano plot of differentially expressed genes in honeybees exposed to 9.4 mg/L of amitraz for 10 days.

Genes with an adjusted P value of <0.05 (FDR correction method) were considered to be differentially expressed. Red: upregulated genes in amitraz-treated bees; green: downregulated genes in amitraz-treated bees; blue: no significant difference.

https://doi.org/10.1371/journal.pone.0228933.g003

thumbnail
Download:
Fig 4. Gene ontology enrichment analysis of differentially expressed genes (DEGs) in honeybees exposed to 9.4mg/L amitraz for 10 days.

Green bars: DEGs enriched for biological process; orange bars: DEGs enriched for cellular components; purple bars: DEGs enriched for molecular function. * indicates that GO terms were significantly enriched by DEGs (corrected P values of <0.05, FDR correction method).

https://doi.org/10.1371/journal.pone.0228933.g004

In total, 135 DEGs (116 upregulated and 19 downregulated) were mapped to 67 KEGG pathways, 10 of which were significantly enriched (Table 2).

thumbnail
Download:
Table 2. The five significantly enriched pathways, corrected P-value < 0.05.

https://doi.org/10.1371/journal.pone.0228933.t002

qPCR analysis

To validate our RNA-Seq data, seven DEGs (LOC725381, CYP4C3, LOC41332, Pla2, LOC724386, LOC100577456, and LOC551385) were checked by qPCR. Consistent with our sequencing data, LOC724386 was downregulated in amitraz-treated bees, while LOC725381, CYP4C3, LOC41332, Pla2, LOC100577456, and LOC551385 were upregulated (Fig 5) (The reference gene is RPS5 and β-actin, LOC725381: t = 18.978, df = 2, P = 0.0276; CYP4C3: t = 3.165, df = 2, P = 0.0258; LOC413332: t = 15.623, df = 2, P = 0.0262; Pla2: t = 15.812, df = 2, P = 0.0063; LOC724386: t = −19.756, df = 2, P = 0.0095; LOC100577456: t = 8.232, df = 2, P = 0.0092; LOC551385: t = 4.551, df = 2, P = 0.0226).

thumbnail
Download:
Fig 5. Real-time quantitative PCR and RNA-seq analysis of LOC725381, CYP4C3, LOC41332, Pla2, LOC724386, LOC100577456, and LOC551385 genes expression multiple in honeybees exposed to 9.4 mg/L of amitraz for 10 days.

Reference gene were RPS5 and β-actin. Values represent means ± SEM. * indicates a significant difference in comparison with controls (P < 0.05) and ** indicates a statistically significant difference in comparison with controls (P < 0.01).

https://doi.org/10.1371/journal.pone.0228933.g005

Discussion

Herein, we exposed honeybees to 9.4 mg/L amitraz for 10 d, which led to the identification of 279 DEGs (237 upregulated and 42 downregulated genes) in the honeybee transcriptome. In order to further study the metabolic pathways influenced in honey bees after amitraz exposure, 91 detailed related pathways of the differential genes were constructed using KEGG pathway analysis. Among these, four pathways, relaxin signaling pathway, platelet activation, protein digestion and AGE-RAGE signaling pathway in diabetic complications were extremely significantly affected (P<0.01). Previous reviews have showed that a relaxin was a factor communicating abnormal growth status of Drosophila larval imaginal discs to the neuroendocrine centers that control the timing of the onset of metamorphosis.[36, 37]. Herein, we found that the relaxin signaling pathway were activated in honeybees after exposure to amitraz, which indicates that amitraz potentially influenced developmental processes of honeybees. To facilitate feeding, certain hematophagous invertebrates possess inhibitors of collagen-induced platelet aggregation in their saliva, inhibited platelet aggregation need inhibit signal transduction necessary for platelet activation by collagen[38]. The hemocytes phagocytosis may play an important role in the cellular immune responses in insects, and the platelet-activating factor can influence phagocytosis of cells[39]. In our study, up-regulation of two genes (LOC113219380 and LOC113219382) of Platelet activation pathway after exposure to amitraz (P <0.01), indicating that amitraz might influence the honeybees immunity. Previous study indicated that imidacloprid was involved in the intoxication of honeybees, it could compromise the viability of the midgut epithelium and affected protein digestion and absorption[40]. Comparison of transcriptome profiling between HearNPV-infected and control healthy Helicoverpa armigera larvae during an early stage post-inoculation, KEGG analysis indicated an enrichment of these differently expressed genes some pathways, including protein digestion and absorption, proved that the DEGs participated in nutritional digestion and exhibited specific expression patterns in a continuous time-course assessment[41]. In this study, we believe that in response to amitraz challenge, honeybees could repair the damages by inducing the expression levels of the protein digestion and absorption pathway. The study showed that six key pathways might be associated with longevity of Drosophila including the AGE-RAGE-signalling pathway in diabetic complications[42]. Comparison between control and Cr (VI)-treated samples of mantis shrimp, AGE-RAGE signaling pathway in diabetic complications were significantly enriched[43]. In honeybees, AGE-RAGE signaling pathway in diabetic complications governs the neural activity to drive the age-specific labor division[44]. In this study, expression levels of four genes (LOC113219380, LOC113219382, Plc and LOC724607) of AGE-RAGE signaling pathway in diabetic complications upregulated by exposure to amitraz (P <0.01), Chouquet et al. clarified the role of Plc in Spodoptera littoralis olfactory transduction[45]. Our study suggest that amitraz probably affected longevity, developmental processes and olfactory transduction of honeybees.

At the individual level, honeybees elicit both cellular and humoral innate immune responses against extraneous substances [19]. Antimicrobial peptides (AMPs) are a class of peptides with low molecular weight; they are encoded by specific genes and are important effectors of natural immunity [46]. Many studies have reported that pesticides affect immunocompetence by regulating the gene expression levels of AMPs in honeybees. For example, exposure to imidacloprid caused most immune related AMP genes (encoding apidaecin, hymenoptaecin and defensin-1) to be downregulated in white- and brown-eyed pupae, but in adults caused an increase in honey bee immune response[47]. Thiamethoxam treated honey bees were further exposed to either thiamethoxam or Nosema, which caused AMP genes abaecin, defensin-1 and defensin-2 to be upregulate[48]. In this study, AMP genes, like apidaecin (Apid1), were induced in amitraz-treated bees, indicating that amitraz also triggers the immune response in honeybees.

Among the identified DEGs, four serine/threonine-protein kinase (STK) genes were present: STK CG31145, STK A2, STK PAKm, and STK MKNK1II. STKs are enzymes involved in metabolism, cell differentiation, gene expression, disease resistance, and other processes [49, 50]. STKs participate in stress resistance in insects [51]. In honeybees, STKs are related to cold and heat stress [52, 53]. Herein, we found that the expression levels of STK CG31145, STK A2, STK PAKm, and STK MKNK1II were upregulated by exposure to amitraz, which indicates that they are potentially involved in conferring tolerance to amitraz.

Further, we found that two detoxification-related genes, CYP4C3 (encoding cytochrome P450 4c3) and CaE-I1 [encoding carboxylesterase (CarE) clade I], were differentially expressed. Cytochrome P450 monooxygenase (CYP) enzymes have been linked to insecticide resistance (i.e., detoxification) or environmental response [54]. Many xenobiotics and pesticides are metabolized by CYPs, such as pyrethroid lambda-cyhalothrin [55, 56], neonicotinoid insecticides [57, 58], aflatoxins [59], and the organophosphate coumaphos [60]. Honey bee are known to have the far fewer numbers of CYP family genes compared to other insects[61, 62], and some subfamilies that have been analyzed include CYP6 and CYP9. The CYP6 subfamily is insect-specific [54] and is involved in phytochemical metabolism [63]. The CYP9 subfamily is responsible for the degradation of various classes of plant protection products [56, 57], such as organophosphates, the pyrethroid cypermethrin, and chlorantraniliprole [64]. CYP9Q3 is known to metabolize tau-fluvalinate, and CYP9Q1 and CYP9Q2 are responsible for degrading bifenthrin [15]. However, the function of CYP4C3 has not yet been elucidated in honeybees. A previous study used transcriptome sequencing and bioinformatic analysis to compare transcription levels between a susceptible and resistant strain of Aedes aegypti; it was reported that the expression of genes such as CYP4C3 were significantly upregulated in resistant strain, suggesting the existence of a potential relationship between the expression of genes participating in metabolic processes and insecticide resistance [65]. CarEs include a group of enzymes involved in endocrine control, detoxification, and metabolism of nonpolar carboxyl hydrolases [66–68], such as malathion [66], methyl parathion [69], dichlorvos [70], and thiamethoxam [71, 72]. CarE has six isoforms in A. mellifera [73]; three isoforms (CarE1, CarE2, and CarE3) are involved in the metabolism of pesticides [74]. For example, CarE1 is directly involved in the detoxification of imidacloprid [71]. In the current study, the expression levels of CYP4c3 and CaE-I1 were upregulated by exposure to amitraz, indicating that these genes may be involved in amitraz degradation in honeybees.

Among the identified DEGs, genes encoding the protein Big Brother, fibrillin-2, Ral GTPase-activating protein, and Brachyury protein are vital for the growth and development of insects. An earlier study reported that Big Brother proteins are required during Drosophila development [75], and the fibrillin-like protein AD10 was found to affect wing morphogenesis in Bombyx mori [76]. The small GTP-binding protein Ral was reported to control the cytoskeletal structure required for cell shape changes during Drosophila development [77], and Brachyury is known to regulate gastrulation in Drosophila [78]. Our data show that expression of genes encoding these four proteins was upregulated in response to amitraz treatment. A previous study found that acute exposure to amitraz caused cell death in the honeybee larvae midgut [22]. Thus, we believe that in response to amitraz challenge, honeybees could repair the damage to the midgut by inducing the expression levels of the four aforementioned genes.

Supporting information

S1 Table. Details of read counts in each library.

https://doi.org/10.1371/journal.pone.0228933.s001

(XLSX)

S2 Table. Sequencing sequence statistics and quality control.

https://doi.org/10.1371/journal.pone.0228933.s002

(XLSX)

S3 Table. Abundance distribution of unigenes.

https://doi.org/10.1371/journal.pone.0228933.s003

(XLSX)

S4 Table. Differentially-expressed genes.

https://doi.org/10.1371/journal.pone.0228933.s004

(XLSX)

S5 Table. 23 genes with the most significant differential.

https://doi.org/10.1371/journal.pone.0228933.s005

(XLSX)

References

  1. 1. Andrikopoulos CJ, Cane JH. Comparative Pollination Efficacies of Five Bee Species on Raspberry. J Econ Entomol. 2018;111(6):2513–9. Epub 2018/08/24. pmid:30137356.
  2. 2. Fijen TPM, Scheper JA, Boom TM, Janssen N, Raemakers I, Kleijn D. Insect pollination is at least as important for marketable crop yield as plant quality in a seed crop. Ecol Lett. 2018;21(11):1704–13. Epub 2018/09/07. pmid:30189466.
  3. 3. Perrot T, Gaba S, Roncoroni M, Gautier J-L, Bretagnolle V. Bees increase oilseed rape yield under real field conditions. Agriculture, Ecosystems & Environment. 2018;266:39–48.
  4. 4. Aizen MA, Garibaldi LA, Cunningham SA, Klein AM. Long-term global trends in crop yield and production reveal no current pollination shortage but increasing pollinator dependency. Curr Biol. 2008;18(20):1572–5. Epub 2008/10/18. pmid:18926704.
  5. 5. Neumann P, Carreck NL. Honey bee colony losses. Journal of Apicultural Research. 2010;49(1):1–6.
  6. 6. Potts SG, Biesmeijer JC, Kremen C, Neumann P, Schweiger O, Kunin WE. Global pollinator declines: trends, impacts and drivers. Trends Ecol Evol. 2010;25(6):345–53. Epub 2010/03/02. pmid:20188434.
  7. 7. VanEngelsdorp D, Speybroeck N, Evans JD, Nguyen BK, Mullin C, Frazier M, et al. Weighing risk factors associated with bee colony collapse disorder by classification and regression tree analysis. J Econ Entomol. 2010;103(5):1517–23. Epub 2010/11/11. pmid:21061948.
  8. 8. Chauzat MP, Martel AC, Cougoule N, Porta P, Lachaize J, Zeggane S, et al. An assessment of honeybee colony matrices, Apis mellifera (Hymenoptera: Apidae) to monitor pesticide presence in continental France. Environ Toxicol Chem. 2011;30(1):103–11. Epub 2010/09/21. pmid:20853451.
  9. 9. Calatayud-Vernich P, Calatayud F, Simo E, Pico Y. Occurrence of pesticide residues in Spanish beeswax. Sci Total Environ. 2017;605–606:745–54. Epub 2017/07/06. pmid:28679118.
  10. 10. Goulson D, Nicholls E, Botias C, Rotheray EL. Bee declines driven by combined stress from parasites, pesticides, and lack of flowers. Science. 2015;347(6229):1255957. Epub 2015/02/28. pmid:25721506.
  11. 11. Ravoet J, Reybroeck W, de Graaf DC. Pesticides for apicultural and/or agricultural application found in Belgian honey bee wax combs. Bull Environ Contam Toxicol. 2015;94(5):543–8. Epub 2015/03/10. pmid:25749505; PubMed Central PMCID: PMC4391733.
  12. 12. Kasiotis KM, Anagnostopoulos C, Anastasiadou P, Machera K. Pesticide residues in honeybees, honey and bee pollen by LC-MS/MS screening: reported death incidents in honeybees. Sci Total Environ. 2014;485–486:633–42. Epub 2014/04/22. pmid:24747255.
  13. 13. Mullin CA, Frazier M, Frazier JL, Ashcraft S, Simonds R, Vanengelsdorp D, et al. High levels of miticides and agrochemicals in North American apiaries: implications for honey bee health. PLoS One. 2010;5(3):e9754. Epub 2010/03/25. pmid:20333298; PubMed Central PMCID: PMC2841636.
  14. 14. Gregorc A, Silva-Zacarin EC, Carvalho SM, Kramberger D, Teixeira EW, Malaspina O. Effects of Nosema ceranae and thiametoxam in Apis mellifera: A comparative study in Africanized and Carniolan honey bees. Chemosphere. 2016;147:328–36. Epub 2016/01/18. pmid:26774296.
  15. 15. Mao W, Schuler MA, Berenbaum MR. CYP9Q-mediated detoxification of acaricides in the honey bee (Apis mellifera). Proc Natl Acad Sci U S A. 2011;108(31):12657–62. Epub 2011/07/22. pmid:21775671; PubMed Central PMCID: PMC3150950.
  16. 16. Gregorc A, Ellis JD. Cell death localization in situ in laboratory reared honey bee (Apis mellifera L.) larvae treated with pesticides. Pesticide Biochemistry and Physiology. 2011;99(2):200–7.
  17. 17. Higes M, Meana A, Bartolome C, Botias C, Martin-Hernandez R. Nosema ceranae (Microsporidia), a controversial 21st century honey bee pathogen. Environ Microbiol Rep. 2013;5(1):17–29. Epub 2013/06/13. pmid:23757127.
  18. 18. Yilmaz HL, Yildizdas DR. Amitraz poisoning, an emerging problem: epidemiology, clinical features, management, and preventive strategies. Arch Dis Child. 2003;88(2):130–4. Epub 2003/01/23. pmid:12538314; PubMed Central PMCID: PMC1719437.
  19. 19. Evans JD, Aronstein K, Chen YP, Hetru C, Imler JL, Jiang H, et al. Immune pathways and defence mechanisms in honey bees Apis mellifera. Insect Mol Biol. 2006;15(5):645–56. Epub 2006/10/31. pmid:17069638; PubMed Central PMCID: PMC1847501.
  20. 20. Pohorecka K, Kiljanek T, Antczak M, Skubida P, Semkiw P, Posyniak A. Amitraz Marker Residues in Honey from Honeybee Colonies Treated with Apiwarol. J Vet Res. 2018;62(3):297–301. Epub 2018/12/26. pmid:30584608; PubMed Central PMCID: PMC6295988.
  21. 21. Martel A-C, Zeggane S, Aurières C, Drajnudel P, Faucon J-P, Aubert M. Acaricide residues in honey and wax after treatment of honey bee colonies with Apivar®or Asuntol®50. Apidologie. 2007;38(6):534–44.
  22. 22. Gregorc A, Bowen ID. Histochemical characterization of cell death in honeybee larvae midgut after treatment with Paenibacillus larvae, Amitraz and Oxytetracycline. Cell Biol Int. 2000;24(5):319–24. Epub 2000/05/12. pmid:10805966.
  23. 23. Hammer M, Menzel R. Multiple sites of associative odor learning as revealed by local brain microinjections of octopamine in honeybees. Learning & memory. 1998;5(1):146–56.
  24. 24. Schwaerzel M, Monastirioti M, Scholz H, Friggi-Grelin F, Birman S, Heisenberg M. Dopamine and octopamine differentiate between aversive and appetitive olfactory memories in Drosophila. Journal of Neuroscience. 2003;23(33):10495–502. pmid:14627633
  25. 25. O'Neal ST, Brewster CC, Bloomquist JR, Anderson TD. Amitraz and its metabolite modulate honey bee cardiac function and tolerance to viral infection. J Invertebr Pathol. 2017;149:119–26. Epub 2017/08/12. pmid:28797906.
  26. 26. Roeder T. Tyramine and octopamine: ruling behavior and metabolism. Annu Rev Entomol. 2005;50:447–77. Epub 2004/09/10. pmid:15355245.
  27. 27. Chaimanee V, Pettis JS. Gene expression, sperm viability, and queen (Apis mellifera) loss following pesticide exposure under laboratory and field conditions. Apidologie. 2019;50(3):304–16.
  28. 28. Loucif W, Noureddine S, Nadia A. Evaluation of Secondary Effects of some Acaricides on Apis Mellifera Intermissa (Hymenoptera, Apidae): Acetylcholinesterase and Glutathione STransferase Activities. European Journal of Scientific Research. 2008;21:642–9.
  29. 29. Naeger NL, Robinson GE. Transcriptomic analysis of instinctive and learned reward-related behaviors in honey bees. J Exp Biol. 2016;219(Pt 22):3554–61. Epub 2016/11/18. pmid:27852762; PubMed Central PMCID: PMC5117197.
  30. 30. Alburaki M, Karim S, Lamour K, Adamczyk J, Stewart SD. RNA-seq reveals disruption of gene regulation when honey bees are caged and deprived of hive conditions. The Journal of Experimental Biology. 2019;222(18). pmid:31413101
  31. 31. Shi TF, Wang YF, Liu F, Qi L, Yu LS. Sublethal Effects of the Neonicotinoid Insecticide Thiamethoxam on the Transcriptome of the Honey Bees (Hymenoptera: Apidae). J Econ Entomol. 2017;110(6):2283–9. Epub 2017/10/19. pmid:29040619.
  32. 32. Yang Y, Ma S, Yan Z, Liu F, Diao Q, Dai P. Effects of three common pesticides on survival, food consumption and midgut bacterial communities of adult workers Apis cerana and Apis mellifera. Environ Pollut. 2019;249:860–7. Epub 2019/04/08. pmid:30954834.
  33. 33. Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc. 2008;3(6):1101–8. Epub 2008/06/13. pmid:18546601.
  34. 34. Garrido PM, Porrini MP, Antunez K, Branchiccela B, Martinez-Noel GM, Zunino P, et al. Sublethal effects of acaricides and Nosema ceranae infection on immune related gene expression in honeybees. Vet Res. 2016;47(1):51. Epub 2016/04/28. pmid:27118545; PubMed Central PMCID: PMC4847213.
  35. 35. Lourenço AP, Mackert A, dos Santos Cristino A, Simões ZLP. Validation of reference genes for gene expression studies in the honey bee, Apis mellifera, by quantitative real-time RT-PCR. Apidologie. 2008;39(3):372–85.
  36. 36. Gontijo AM, Garelli A. The biology and evolution of the Dilp8-Lgr3 pathway: A relaxin-like pathway coupling tissue growth and developmental timing control. Mech Dev. 2018;154:44–50. Epub 2018/05/02. pmid:29715504.
  37. 37. Garelli A, Heredia F, Casimiro AP, Macedo A, Nunes C, Garcez M, et al. Dilp8 requires the neuronal relaxin receptor Lgr3 to couple growth to developmental timing. Nat Commun. 2015;6:8732. Epub 2015/10/30. pmid:26510564; PubMed Central PMCID: PMC4640092.
  38. 38. Morita A, Isawa H, Orito Y, Iwanaga S, Chinzei Y, Yuda M. Identification and characterization of a collagen‐induced platelet aggregation inhibitor, triplatin, from salivary glands of the assassin bug, Triatoma infestans. The FEBS journal. 2006;273(13):2955–62. pmid:16759235
  39. 39. Dubois AE, van der Heide S. Basophil-activation tests in Hymenoptera allergy. Current opinion in allergy and clinical immunology. 2007;7(4):346–9. pmid:17620828
  40. 40. Catae AF, Roat TC, Pratavieira M, da Silva Menegasso AR, Palma MS, Malaspina O. Exposure to a sublethal concentration of imidacloprid and the side effects on target and nontarget organs of Apis mellifera (Hymenoptera, Apidae). Ecotoxicology. 2018;27(2):109–21. pmid:29127660
  41. 41. Li Z, Lu Z, Wang X, Zhang S, Zhang Q, Liu X. Digital gene expression analysis of Helicoverpa armigera in the early stage of infection with Helicoverpa armigera nucleopolyhedrovirus. Journal of invertebrate pathology. 2015;132:66–76. pmid:26296928
  42. 42. Li J-q, Duan D-d, Zhang J-q, Zhou Y-z, Qin X-m, Du G-h, et al. Bioinformatic prediction of critical genes and pathways involved in longevity in Drosophila melanogaster. Molecular Genetics and Genomics. 2019;294(6):1463–75. pmid:31327054
  43. 43. Zhang D, Liu J, Qi T, Ge B, Wang Z, Jiang S, et al. Transcriptome Analysis of Hepatopancreas from the Cr (VI)-Stimulated Mantis Shrimp (Oratosquilla oratoria) by Illumina Paired-End Sequencing: Assembly, Annotation, and Expression Analysis. J Agric Food Chem. 2018;66(11):2598–606. Epub 2018/02/10. pmid:29425446.
  44. 44. Bezabih G, Cheng H, Han B, Feng M, Xue Y, Hu H, et al. Phosphoproteome Analysis Reveals Phosphorylation Underpinnings in the Brains of Nurse and Forager Honeybees (Apis mellifera). Sci Rep. 2017;7(1):1973. Epub 2017/05/18. pmid:28512345; PubMed Central PMCID: PMC5434016.
  45. 45. Chouquet B, Lucas P, Bozzolan F, Solvar M, Maïbèche-Coisné M, Durand N, et al. Molecular characterization of a phospholipase C β potentially involved in moth olfactory transduction. Chemical senses. 2010;35(5):363–73. pmid:20233741
  46. 46. Oren Z, Shai Y. Mode of action of linear amphipathic α‐helical antimicrobial peptides. Peptide Science. 1998;47(6):451–63. pmid:10333737
  47. 47. Tesovnik T, Zorc M, Gregorc A, Rinehart T, Adamczyk J, Narat M. Immune gene expression in developing honey bees (Apis mellifera L.) simultaneously exposed to imidacloprid and Varroa destructor in laboratory conditions. Journal of Apicultural Research. 2019;58(5):730–9.
  48. 48. Tesovnik T, Zorc M, Ristanic M, Glavinic U, Stevanovic J, Narat M, et al. Exposure of honey bee larvae to thiamethoxam and its interaction with Nosema ceranae infection in adult honey bees. Environ Pollut. 2019:113443. Epub 2019/11/18. pmid:31733951.
  49. 49. Krupa A, Preethi G, Srinivasan N. Structural modes of stabilization of permissive phosphorylation sites in protein kinases: distinct strategies in Ser/Thr and Tyr kinases. Journal of molecular biology. 2004;339(5):1025–39. pmid:15178245
  50. 50. Faucher SP, Viau C, Gros P-P, Daigle F, Le Moual H. The prpZ gene cluster encoding eukaryotic-type Ser/Thr protein kinases and phosphatases is repressed by oxidative stress and involved in Salmonella enterica serovar Typhi survival in human macrophages. FEMS microbiology letters. 2008;281(2):160–6. pmid:18312574
  51. 51. Giri J, Vij S, Dansana PK, Tyagi AK. Rice A20/AN1 zinc‐finger containing stress‐associated proteins (SAP1/11) and a receptor‐like cytoplasmic kinase (OsRLCK253) interact via A20 zinc‐finger and confer abiotic stress tolerance in transgenic Arabidopsis plants. New Phytologist. 2011;191(3):721–32. pmid:21534973
  52. 52. Xu K, Niu Q, Zhao H, Du Y, Jiang Y. Transcriptomic analysis to uncover genes affecting cold resistance in the Chinese honey bee (Apis cerana cerana). PLoS One. 2017;12(6):e0179922. Epub 2017/06/27. pmid:28650988; PubMed Central PMCID: PMC5484514.
  53. 53. Ma H, Huang Q, Lai X, Liu J, Zhu H, Zhou Y, et al. Pharmacological Properties of the Type 1 Tyramine Receptor in the Diamondback Moth, Plutella xylostella. Int J Mol Sci. 2019;20(12). Epub 2019/06/20. pmid:31212951; PubMed Central PMCID: PMC6627746.
  54. 54. Feyereisen R. Evolution of insect P450. Biochem Soc Tran. 2006;34:1252–5.
  55. 55. Pilling E, Bromleychallenor K, Walker C, Jepson P. Mechanism of synergism between the pyrethroid insecticide λ-cyhalothrin and the imidazole fungicide prochloraz, in the honeybee (Apis mellifera L.). Pesticide biochemistry and physiology. 1995;51(1):1–11.
  56. 56. Johnson RM, Wen Z, Schuler MA, Berenbaum MR. Mediation of pyrethroid insecticide toxicity to honey bees (Hymenoptera: Apidae) by cytochrome P450 monooxygenases. J Econ Entomol. 2006;99(4):1046–50. Epub 2006/08/30. pmid:16937654.
  57. 57. Iwasa T, Motoyama N, Ambrose JT, Roe RM. Mechanism for the differential toxicity of neonicotinoid insecticides in the honey bee, Apis mellifera. Crop Protection. 2004;23(5):371–8.
  58. 58. Manjon C, Troczka BJ, Zaworra M, Beadle K, Randall E, Hertlein G, et al. Unravelling the Molecular Determinants of Bee Sensitivity to Neonicotinoid Insecticides. Current Biology. 2018;28(7):1137–43.e5. pmid:29576476
  59. 59. Niu G, Johnson RM, Berenbaum MR. Toxicity of mycotoxins to honeybees and its amelioration by propolis. Apidologie. 2011;42(1):79–87.
  60. 60. Johnson RM, Pollock HS, Berenbaum MR. Synergistic interactions between in-hive miticides in Apis mellifera. J Econ Entomol. 2009;102(2):474–9. Epub 2009/05/20. pmid:19449624.
  61. 61. Claudianos C, Ranson H, Johnson R, Biswas S, Schuler MA, Berenbaum MR, et al. A deficit of detoxification enzymes: pesticide sensitivity and environmental response in the honeybee. Insect molecular biology. 2006;15(5):615–36. pmid:17069637
  62. 62. Johnson RM, Mao W, Pollock HS, Niu G, Schuler MA, Berenbaum MR. Ecologically appropriate xenobiotics induce cytochrome P450s in Apis mellifera. PLoS One. 2012;7(2):e31051. Epub 2012/02/10. pmid:22319603; PubMed Central PMCID: PMC3272026.
  63. 63. Johnson RM. Toxicogenomics of Apis Mellifera. Dissertations & Theses—Gradworks. 2008.
  64. 64. Christen V, Fent K. Exposure of honey bees (Apis mellifera) to different classes of insecticides exhibit distinct molecular effect patterns at concentrations that mimic environmental contamination. Environmental pollution. 2017;226:48–59. pmid:28402838
  65. 65. Nguyen Thi Kim L, Nguyen Thi Hong N, Nguyen Ngoc L, Nguyen Thu H, Nguyen Van T, Nguyen Thi Thanh N, et al. Transcriptome Sequencing and Analysis of Changes Associated with Insecticide Resistance in the Dengue Mosquito (Aedes aegypti) in Vietnam. American Journal of Tropical Medicine and Hygiene. 2019;100(5):1240–8. WOS:000476671900045. pmid:30834881
  66. 66. Yu S, Robinson F, Nation J. Detoxication capacity in the honey bee, Apis mellifera L. Pesticide Biochemistry and Physiology. 1984;22(3):360–8.
  67. 67. Stone D, Jepson P, Laskowski R. Trends in detoxification enzymes and heavy metal accumulation in ground beetles (Coleoptera: Carabidae) inhabiting a gradient of pollution. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology. 2002;132(1):105–12.
  68. 68. Dauterman W. Insect metabolism: extramicrosomal. Comprehensive insect physiology, biochemistry and pharmacology. 1985;12:713–30.
  69. 69. Attencia VM, Ruvolo-Takasusuki MCC, De Toledo VdAA. Esterase activity in Apis mellifera after exposure to organophosphate insecticides (Hymenoptera: Apidae). Sociobiology. 2005;45(3):587–95.
  70. 70. Figueiredo V, Bitondi M, Paulino-simõ, es Z. Esterase inhibition during several immature worker stages of Apis mellifera following topical application of DDVP insecticide. Journal of Apicultural Research. 1996;35(1):37–43.
  71. 71. Badiou-Bénéteau A, Carvalho SM, Brunet J-L, Carvalho GA, Buleté A, Giroud B, et al. Development of biomarkers of exposure to xenobiotics in the honey bee Apis mellifera: Application to the systemic insecticide thiamethoxam. Ecotoxicology and Environmental Safety. 2012;82:22–31. pmid:22683234
  72. 72. Hashimoto JH. Evaluation of the Use of the Inhibition Esterases Activity on Apis mellifera as Bioindicators of Insecticide. Sociobiology. 2003;42(3).
  73. 73. Bitondi MMG, Mestriner MA. Esterase isozymes of Apis mellifera: Substrate and inhibition characteristics, developmental ontogeny, and electrophoretic variability. Biochemical genetics. 1983;21(9–10):985–1002. pmid:6661179
  74. 74. Carvalho SM, Belzunces LP, Carvalho GA, Brunet JL, Badiou-Beneteau A. Enzymatic biomarkers as tools to assess environmental quality: a case study of exposure of the honeybee Apis mellifera to insecticides. Environ Toxicol Chem. 2013;32(9):2117–24. Epub 2013/05/29. pmid:23712883.
  75. 75. Karrenberg S, Kollmann J, Edwards PJ. Pollen vectors and inflorescence morphology in four species of Salix. Plant Systematics and Evolution. 2002;235(1):181–8.
  76. 76. Matsunaga TM, Fujiwara H. Identification and characterization of genes abnormally expressed in wing-deficient mutant (flu¨gellos) of the silkworm, Bombyx mori. Insect Biochemistry and Molecular Biology. 2002;32:691–9. pmid:12020843
  77. 77. Sawamoto K,., Yamada C,., Kishida S,., Hirota Y,., Taguchi A,., Kikuchi A,., et al. Ectopic expression of constitutively activated Ral GTPase inhibits cell shape changes during Drosophila eye development. Oncogene. 1999;18(11):1967. pmid:10208418
  78. 78. Brunet T, Bouclet A, Ahmadi P, Mitrossilis D, Driquez B, Brunet AC, et al. Evolutionary conservation of early mesoderm specification by mechanotransduction in Bilateria. Nat Commun. 2013;4:2821. Epub 2013/11/28. pmid:24281726; PubMed Central PMCID: PMC3868206.