Developmental and Caste-specific Expression Patterns of ATP-Binding Cassette (ABC) Transporters in Honey bees (Apis mellifera)

Developmental and Caste-specific Expression Patterns of ATP-Binding Cassette (ABC) Transporters in Honey bees (Apis mellifera) | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 21 January 2025 V1 Latest version Share on Developmental and Caste-specific Expression Patterns of ATP-Binding Cassette (ABC) Transporters in Honey bees (Apis mellifera) Authors : Angela Encerrado-Manriquez 0000-0002-9822-3549 , Zeke Spooner , Tina Truong , Julia Fine , and Sascha Nicklisch 0000-0003-3120-6485 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.173746687.74569861/v1 Published Environmental Toxicology and Pharmacology Version of record Peer review timeline 486 views 173 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract While honey bees play a vital role in global crop production, they face increasing exposure to xenobiotic chemicals during commercial pollination. Multidrug-resistance (MDR)-type ATP-binding cassette (ABC) transporters provide the first line of defense against xenobiotic chemicals and are upregulated in resistant pest organisms. While previous studies in bees have focused on the role of metabolic enzymes in insect detoxification, the presence and function of ABC transporters across the hive caste system remains largely unexplored. This study investigated the gene expression profiles of 12 ABC transporters known to be involved in chemical detoxification in arthropods across ten honey bee castes and life stages using quantitative real-time PCR. Protein homology to known MDR transporters in humans and Drosophila was inferred from BLAST and through phylogenetic analysis. Seven ABC genes that showed increased gene expression during worker bee development were identified as MDR-like transporters (AmeABCB1, AmeABCB6, AmeABCC1, AmeABCC4a-c, AmeABCG1); and their expression levels were further investigated in reproductive caste members (drone larvae, adult drones, queen ovaries, and queens). Significant variations were observed in defense gene expression among all castes suggesting reduced chemical defense capabilities in queens as evidenced by a dramatically reduced expression of five MDR-like transporter genes in queen bees relative to worker eggs: ABCB1 (4-fold), ABCC1 (2-fold), ABCC4a (2-fold), ABCC4b (3-fold), and ABCC4c (2-fold). Although our findings suggest that drones and queens are more vulnerable to direct xenobiotic exposure compared to workers, further research is required to better understand the different hive members’ responses to chemical threats. Developmental and Caste-specific Expression Patterns of ATP-Binding Cassette (ABC) Transporters in Honey bees ( Apis mellifera ) ABC Transporter Expression across Honey Bee Castes Angela M. Encerrado-Manriquez 1# , Zeke T. Spooner 1# , Tina T. Truong 1 , Julia D. Fine 2 , Sascha C.T. Nicklisch 1 * 1 Department of Environmental Toxicology, University of California-Davis, Davis, CA 95616 2 Invasive Species and Pollinator Health Research Unit, USDA-ARS, 3026 Bee Biology Rd., Davis, CA, 95616, USA # The authors Angela M. Encerrado-Manriquez and Zeke T. Spooner are co-first authors *Corresponding author: Sascha C.T. Nicklisch University of California, Davis College of Agricultural and Environmental Sciences, Department of Environmental Toxicology, 4117 Meyer Hall, Email: [email protected] Office: +1 (530) 752-1415 Conflict of Interest Disclosure The authors declare no conflict of interest. Data availability Statement All data and supplementary information supporting this study are available from Dryad at https://doi.org/10.5061/dryad.d2547d8c2. Funding Statement This work was supported by the NIFA-USDA (CA-D-ETX-2526-H) to S.C.T.N and by USDA NACA (58-2030-3-034) to J.D.F. and S.C.T.N. Abstract While honey bees play a vital role in global crop production, they face increasing exposure to xenobiotic chemicals during commercial pollination. Multidrug-resistance (MDR)-type ATP-binding cassette (ABC) transporters provide the first line of defense against xenobiotic chemicals and are upregulated in resistant pest organisms. While previous studies in bees have focused on the role of metabolic enzymes in insect detoxification, the presence and function of ABC transporters across the hive caste system remains largely unexplored. This study investigated the gene expression profiles of 12 ABC transporters known to be involved in chemical detoxification in arthropods across ten honey bee castes and life stages using quantitative real-time PCR. Protein homology to known MDR transporters in humans and Drosophila was inferred from BLAST and through phylogenetic analysis. Seven ABC genes that showed increased gene expression during worker bee development were identified as MDR-like transporters ( Ame ABCB1, Ame ABCB6, Ame ABCC1, Ame ABCC4a-c, Ame ABCG1); and their expression levels were further investigated in reproductive caste members (drone larvae, adult drones, queen ovaries, and queens). Significant variations were observed in defense gene expression among all castes suggesting reduced chemical defense capabilities in queens as evidenced by a dramatically reduced expression of five MDR-like transporter genes in queen bees relative to worker eggs: ABCB1 (4-fold), ABCC1 (2-fold), ABCC4a (2-fold), ABCC4b (3-fold), and ABCC4c (2-fold). Although our findings suggest that drones and queens are more vulnerable to direct xenobiotic exposure compared to workers, further research is required to better understand the different hive members’ responses to chemical threats. Keywords: Honey bees, castes, development, ABC transporters, multidrug resistance (MDR), xenobiotic defense, gene expression Introduction Historically, honey bees ( Apis mellifera ) have been recognized as the ideal pollinator for the commercial pollination industry due to their gentle disposition, high honey production, and lack of floral specificity. The demand for commercial pollination in tandem with large-scale agricultural chemical use has led to agrochemicals infiltrating their feeding habitats and hives 1 . Although worker bees are the caste that spend extensive periods of time outside the hive and are most often exposed to agrochemicals 2–4 , it has been suggested that interactions inside the hive provide the basis for chemical transport across multiple castes, potentially exposing new generations during development in addition to adults 5 . Furthermore, the use of in-hive medicines extends the presence of xenobiotic chemicals in the hive structure, with a high amount of residues often being found in the wax (e.g., fluvalinate 204 ppm, Amitraz 446 ppm) 5 . Moreover, genetic, physiological, and behavioral differences across castes could affect their exposures and responses to xenobiotic chemicals. Honey bee development and hive castes The honey bee superorganism is structured into three genetically, physiologically, and behaviorally distinct reproductive castes: queens, workers, and drones. Worker bees perform a wide variety of behaviors related to hive maintenance and protection, caretaking, and resource acquisition, with physiological characteristics that assist with these tasks (such as functioning stingers for protection). A drone bee’s primary function is to carry the genes of the hive; with a sole focus on reproduction, drones lack the physiological characteristics necessary to sustain and protect themselves. Queen bee physiology is similarly designed to prioritize reproductive success, though there is some evidence that queens may tolerate higher doses of certain topically applied miticides than workers 6 . Their reclusive nature may protect them from environmental dangers. Apart from mating flights and during swarming, they do not leave the colony 7 . Inside a hive, all three castes interact as a superorganism through intricate communication and food-sharing mechanisms 5 . Honey bee Chemical Defensome When exposed to potentially harmful chemicals, organisms can activate a sophisticated cellular defense system known as the cellular defensome 8 . A well-orchestrated defensome includes four metabolic stages defined as Phase 0 (uptake), Phase I (functionalization), Phase II (conjugation), and Phase III (export) 9 , that work in concert to protect cells from chemical toxicity. Multidrug resistance (MDR)-type ATP-binding cassette (ABC) transporters are ubiquitously expressed in cellular membranes and are a first line of defense against xenobiotic accumulation (Phase 0). Compounds that elude Phase 0 are typically chemically modified via functionalization (Phase I) and/or conjugation (Phase II) enzymes to enhance their inactivation and/or active elimination through Phase III efflux transporters 10 . Many chemical defensome studies in insects have focused on Phase I metabolic enzymes such as those in the cytochrome P450 family (CYPs). Although these enzymes are essential for the metabolism of xenobiotic chemicals, it is during Phase 0 where the members of the ABC family of transporter proteins have their first encounter with xenobiotics at the cell boundaries. Previous studies of Phase 0 focus on ABC transporters as the primary driver of insecticide resistance; these transporters were shown to provide protection against pesticides in multiple insect species, including Plutella xylostella (L.), Helicoverpa armigera, and Aedes aegypti 11–13 . This study explores the developmental and caste-specific expression of 12 different honey bee ABC transporters known to be involved in arthropod chemical defense 14–19 . Quantitative real-time PCR (qPCR) was used to establish baseline expression levels of these genes across ten developmental stages and three different castes (i.e., worker, queen, drone). In addition, the transporter gene expression patterns in Queen bee ovaries were explored since it is a key reproductive organ. The results show that different members of the hive superorganism may differ in their ability to defend themselves against a changing chemical environment, which is increasingly important given honey bees’ rising xenobiotic exposures 5,20 . Materials and Methods Sample Acquisition Honey bee samples were collected from hives maintained by the USDA-ARS Pollinator Health Research Unit in Davis, CA. Table S1 describes the sampling seasons, years, and locations. Qualitative monitoring of parasitic Varroa destructor mites was conducted using alcohol washes 21 , and levels were found to be low throughout the course of sample collection. Colonies were maintained according to standard best beekeeping practices in the United States 22 , and levels of Varroa destructor parasitic mites were maintained using treatments with Formic Pro TM (NOD Apiary Products Ltd., Trenton, Ontario). Ten different life stages were collected for this project: • Eggs (E) • Worker larvae instar 3 (WL3) • Worker larvae instar 5 (WL5) • Drone larvae instar 5 (DL5) • Worker pupae (P) • Newly emerged worker bees (NEB) • Nurses (N) • Foragers (F) • Drones (D) • Queens (Q) Five samples of each life stage were collected for biological replication purposes. Additional queens were collected for dissection of queen ovaries. Eggs (E) were collected by taking a frame containing E (from a hive or a queen monitoring cage 23 used in the laboratory) and tapping the E out onto a piece of dark-colored paper before pouring them into an RNase-free 1.5 mL tube. When E were sticking to the cells, they were removed using grafting tools. For each sample, 5-10 mg of E were collected. All larvae and pupae were collected by grafting directly from hive frames and were stored in the same way. Adult bees (NEBs, nurses, foragers, and drones) were identified on live frames, collected, and euthanized by flash-freezing. During an annual queen replacement event in April 2022 mated queens were collected in the field and immediately flash-frozen in liquid nitrogen. All bee samples collected in the field were stored on dry ice until they could be brought back to the lab and stored at -80°C. A subset of 5 queen samples from the previously collected mated queens had their ovaries isolated as an eleventh treatment group (QO) for the experiment. Dissections were performed using a Leica S9i stereo microscope (Leica Microsystems, Deerfield, IL). Frozen queens were dissected in a petri dish containing cool phosphate-buffered saline (PBS) (pH 7.4) solution on top of an ice block. Tissue Homogenization and RNA Extractions Honey bee tissues were homogenized using the BeadBug™ 6 Six-Position Homogenizer (Benchmark Scientific, Sayreville, NJ, USA). Steel beads of 2.8 mm (Omni International, Kennesaw, Georgia, USA) were selected over ceramic beads to effectively break through the insects’ exoskeletons and homogenize the samples. For each sample (except QO), the entire bee was used for RNA extraction. All samples were homogenized in DNA/RNA Protection Reagent (New England Biolabs, Ipswich, MA, USA) for three cycles of 30 seconds at a speed of 6 m/s with 45-second dwell periods. For samples with especially tough exoskeletons or wide bee samples (i.e., drones, queens) that impeded steel bead movement within the tube, up to three additional runs of the homogenization program were performed to ensure thorough soft tissue disruption. The sample was placed on ice between each run to prevent overheating that could potentially compromise RNA quality. RNA extraction was conducted using the Monarch® Total RNA Miniprep Kit (New England Biolabs, Ipswich, MA, USA). The quantities of reagents used in the initial steps varied based on sample weights. For instance, each WL3 sample weighing approximately 10 mg required minimal buffer and Proteinase K, whereas Q and D samples exceeding 200 mg necessitated maximal reagent usage. An optional on-column DNase 1 treatment was performed on every sample to ensure complete genomic DNA (gDNA) removal. RNA quality was validated using standard agarose gel electrophoresis and UV absorbance for determining A 260 /A 230 ratios. cDNA Conversion Total RNA was converted into cDNA for qPCR analysis using the SuperScript IV First-Strand Synthesis System (Invitrogen, Thermo Fisher Scientific, Waltham, MA). The manufacturer’s protocol was followed to prepare 1000 ng of cDNA per sample. If necessary, RNA samples were diluted with molecular biology-grade water before cDNA conversions to ensure accurate pipetting. For this reason, only 200 ng of cDNA was created for two of the E samples with particularly low RNA yield. Although the initial cDNA quantity differs between 1000 ng and 200 ng samples (while maintaining the same volume), the 1000 ng cDNA samples were diluted 1:4 to standardize the concentrations for all samples and to achieve the optimal concentration for qPCR. Primer Design and Quality Control The qPCR primers were designed using KEGG, CLC Genomic Workbench, Primer-BLAST (NCBI), and Primer3Plus 24 . The gene sequences for all genes of interest, including their respective isoforms, were downloaded into CLC. Within each gene, isoforms were aligned, and gaps were recorded. Primers were subsequently designed within regions of the alignments where no gaps were present to capture all isoforms of each gene. Target amplicons were set between 80 and 200 base pairs (bp), with an optimal size of 120 bp. The potential for primer dimerization was assessed using Beacon Designer (Premier Biosoft International, Palo Alto, CA), and all primers were screened for potential non-specific binding using Nucleotide BLAST (NCBI). A previously published primer was used for the housekeeping gene RAD1A 25 . For this project, three reference genes were selected: glyceraldehyde 3-phosphate dehydrogenase (GAPDH), ribosomal protein S5 (RS5), and Ras-related protein Rab-1A (RAD1A). The first two of these genes are commonly used as reference genes for honey bees, and the latter was recently identified as the most stable reference gene for qPCR experiments across honey bee life stages 25,26 . High-purity salt-free (HPSF) primers (Eurofins Genomics, Louisville, Kentucky, U.S.) were reconstituted and diluted using TE buffer (pH = 7.4). The working primer concentrations were 10 µM. Each primer pair specificity was assessed with nurse bee cDNA using standard PCR and agarose gel electrophoresis. Prior to qPCR analysis, the efficiency of the primers was also tested by creating a five-point cDNA serial dilution curve. The slope obtained from plotting concentrations against Ct values was used to calculate efficiency using the following equation 27 : [10^(-1/(slope))-1]*100 Efficiencies of the validated qPCR primers can be found in the supplemental table ( Table S2 ). Phylogeny The protein sequence for each honey bee transporter gene was blasted using NCBI Protein BLAST against human and Drosophila sequences. The results were sorted by Max score, selecting only the top annotated sequence from each organism. Sequences for each organism were downloaded and organized in CLC Main Workbench 21.0.5 to construct a phylogenetic tree using the following parameters: Neighbor-Joining (algorithm), Jukes-Cantor (distance measure), and 1000 replicates (bootstrap). The root (outgroup) was set to be human HRas GTPase (NP_001123914). Real-time – PCR (qPCR) All qPCR plates were designed with three technical replicates for each unique reaction. Each plate contained one biological replicate of three different life stages, with five genes of interest and three reference genes. The 20 µL reaction protocol using SsoAdvanced Universal SYBR® Green Supermix (Bio-Rad, Hercules, CA, USA) was applied to all samples and pipetted onto MicroAmp™ EnduraPlate™ optical 96-well fast clear reaction plates (Thermo Fisher Scientific, Waltham, MA). After loading the reactions, the plates were sealed with MicroAmp™ optical adhesive film (Thermo Fisher Scientific, Waltham, MA) and centrifuged for one minute to thoroughly mix all the reagents. All plates were run on a QuantStudio 3 Real-Time PCR system (Thermo Fisher Scientific, Waltham, MA) using the following protocol: a 30s hold at 98°C for polymerase activation and initial DNA denaturation (rate 4.3°C/s), followed by 40 cycles of 15s at 98°C for denaturation (rate 3°C/s), and 30s at 60°C for annealing/extension + plate read (rate 3°C/s). A melt-curve analysis was added after the 40 cycles: 15s at 95°C (rate 1.6°C/s), followed by 1min at 60°C (rate 1.6°C/s), and then 15s at 95°C (rate 0.05°C/s). Using the biological replicates (n=5) ΔC T values and technical plate replicates, an average value ΔC T was obtained for each of the eleven treatments (E, WL3, WL5, DL5, P, NEB, N, F, D, Q, QO) and twelve genes of interest ( Table 1 ). A geometric mean of all housekeeping genes was used to calculate the ΔC T values. The qPCR data was analyzed using the Livak method of calculating fold change with ΔΔC T 28 , comparing differences between treatments. Each tested life stage was considered a ”treatment” except for E, which was designated as the ”control” treatment. E was selected as the control group since all caste members pass through this stage. Gene expression was normalized to its corresponding expression in E (= value of 1). The fold change of gene expression was calculated using the equation: \begin{equation} 2\hat{}e-\ [\Delta\Delta CT\ =\ \Delta CT\ ("treatment")\ –\ \Delta CT\ ("control")]\nonumber \\ \end{equation} All qPCR results were assessed for quality criteria, including melting temperature, C T values greater than 35, and technical replicate C T s within 0.5 units of their mean. Quality parameters were applied using RStudio v.4.2.2 to ensure unbiased analysis. The filtered data were then used for statistical and interpretation. Table 1 . Metadata summary of the 12 honey bee ABC transporters investigated in this study . Gene ID and naming conventions in NCBI and BeeBase/HGD for honey bee ABC transporters are shown. The four different genes for honey bee ABCC4 (a-d) are all paralogous to human ABCC4. Two ABCG (a-b) genes were paralogous to human ABCG2. Ame -ABCB1 GB55378 551167 Mdr49 Multidrug resistance protein homolog 49 1343 Ame -ABCB6 GB54214 726867 Hmt-1 ABC transporter ATP-binding protein/permease Hmt-1 840 Ame -ABCC1 GB53134 412217 MRP Multidrug-Resistance like Protein 1 1536 Ame -ABCC10 GB44193 725993 LOC725993 Multidrug resistance-associated protein 7 1625 Ame -ABCC4a GB50195 413959 LOC413959 Multidrug resistance-associated protein 4 1356 Ame- ABCC4b GB45277 551061 LOC551061 Multidrug resistance-associated protein 4 1392 Ame -ABCC4c GB45278 725051 LOC725051 Multidrug resistance-associated protein 4 1418 Ame -ABCC4d GB17987; GB43189 410269 LOC410269 Probable multidrug resistance-associated protein lethal (2) 03659 1333 Ame -ABCG1 GB44770 412748 LOC412748 ATP-binding cassette sub-family G member 1 631 Ame -ABCG2a GB49098 410967 E23 Early gene at 23 657 Ame- ABCG2b GB43076 726508 LOC726508 Protein scarlet 623 Ame -ABCG5 GB41827 726513 LOC726513 ATP-binding cassette sub-family G member 5 625 Statistical analysis All the statistical analysis was performed using RStudio v.4.2.2. The filtered data were first checked for normality using a histogram, QQ-plot, and a Shapiro-Wilk normality test (W=0.97, p-value < 2.2 e -16 ). The normality test and the models created to analyze the data were applied to the housekeeping normalized C T values (ΔC T ). A one-way ANOVA test (“analysis of variance”) was conducted to determine if significant differences were present across the twelve treatments (E, WL3, WL5, DL5, P, NEB, N, F, D, Q, QO) for each gene of interest. Following the ANOVA, a Tukey test or Tukey’s Honest Significant Difference test was applied to the data to identify significant differences between combinations of treatments. The confidence interval for the Tukey model was set to 0.99. Comparisons were made across the same gene and across the same life stage, with values below 0.01 considered statistically significant. Results This study measured the life stage- and caste-dependent gene expression patterns of 12 different ABC transporters in honey bees ( Figure 1, Table 1 ). Homologs of these transporters in humans and other arthropod species are known to be involved in chemical defense and pesticide resistance mechanisms. All caste members expressed 2 ABCB, 6 ABCC, and 4 ABCG transcripts. The ABC transporters were expressed differently across life stages and caste members, with MDR-like ABC transporter homologs showing a characteristic temporal expression pattern that increased steadily throughout worker bee development. Figure 1. Phylogenetic analysis of full-length Apis mellifera suspected ABC transporters and their homologs in humans and Drosophila. The percentage concordance based on 1000 bootstrap iterations is shown at the nodes (blue numbers). Ame = Apis mellifera; Dme = Drosophila melanogaster; Has = Homo sapiens. The human Hras GTPase gene was used as a reference point to better determine evolutionary relationships between the ABC transporter genes and to properly root the tree. Identification of MDR-like ABC transporter homologs in honey bees NCBI BLAST and phylogeny analysis were used to identify MDR transporter homologs in humans ( Hsa ) and Drosophila ( Dme ) ( Figure 1, Table S3 ). Twelve genes for ABC-type transporter proteins in honey bees ( Ame ) that may be involved in chemical defense ( Table 1 ) were identified. The phylogenetic tree was rooted using a Hsa GTPase gene (NP_001123914) and the Neighbor-Joining model using CLC Main Workbench 21.0.5. From the tree, it was shown that Ame -Mdr49 & Ame -Hmt-1 are closely related to Dme -Mdr49 & Dme -Hmt-1, sharing the same node, with both being two nodes away from Hsa -ABCB1 & Hsa -ABCB6, respectively. Furthermore, in a separate cluster, Ame -MRP is found one node away from Dme -MRP1 and both two nodes away from Hsa -ABCC1. In a separate branch coming from the same node, Ame -LOC725993 clusters one node away from Dme -CG7806 and both two nodes away from Hsa -ABCC10. Also, in a separate branch from Hsa -ABCC1 and Hsa -ABCC10, Hsa -ABCC4 shares the same common ancestor with Ame -LOC551061, Ame -LOC725051, Ame -LOC413959, and Ame -LOC410269 as well as Dme -Mrp5. Ame -LOC410269 appears more closely related to Dme -Mrp5, one node apart, than Ame -LOC551061, Ame -LOC725051, and Ame -LOC413959, which are two and four nodes away from the previously mentioned Dme protein. One node away from the root, the branch with a bootstrap of 100% shows another set of clusters, with Ame -LOC412748 being in the same branch as Hsa -ABCG1. Dme -CG5853 seems more closely related to Hsa -ABCG1 than Ame -LOC412748. From a different branch, Ame -LOC726508 clusters one node away from Dme -st and two nodes away from Hsa -ABCG2. Ame -E23 is one node away from Dme -E23 and three nodes away from Hsa -ABCG2. Both Ame -E23 and Ame -LOC726508 seem to be related to the same human gene, Hsa -ABCG2. Finally, Ame -LOC726513 clusters one node away from Dme -CG11069 and two nodes away from Hsa -ABCG5, all belonging to the same clade and common ancestor. The proteins of interest were grouped in three main subfamily clusters (B, C, and G) based on the human ( Hsa ) proteins relations from the phylogenetic tree. Furthermore, based on our results of the phylogenetic analysis, protein sequence similarity BLAST (NCBI), and gene orthology (OrthoDB), we assigned new gene symbols to each honey bee ABC transporter that will be used throughout the manuscript ( Table 1 ) to describe our different proteins of interest. 3.2 Developmental expression patterns of ABC transporters in worker bees For our developmental ABC transporter gene expression analysis, seven life stages of worker bees were selected, from eggs to forager bees. Figure 2 shows the relative expression of these twelve transporters across these life stages. Both members of the ABCB family (B1, B6) showed a similar expression pattern across worker bee life stages, with the expression increasing as the worker bees age. After statistical analysis, the expression of ABCB1 was significantly different between pupa (1.67) and forager bee (3.83), with an apparent linear increase. ABCB1 was downregulated during WL3 and WL5. However, this downregulation was not significantly different from E’s. The same trend was observed for ABCB6, which significantly increased from WL3 (0.41) to F (2.16), with a significant downregulation compared to E during WL3 (0.41) and WL5 (0.61). Although ABCB6 was also downregulated in P, it was significantly different from E. Figure 2. Heat map of the relative gene expression (fold change) of twelve ABC-type transporters during worker bee development. The intensity of the color reflects the expression of each gene, with stronger colors representing relative upregulation (>1) and lighter colors representing relative downregulation (<1). The color scale reflects comparisons across all genes and all life stages. E (eggs), WL3 (Worker Larvae 3), WL5 (Worker Larvae 5), P (Pupa), NEB (Newly emerged bee), N (Nurse), F (Forager). Bolded ABC transporters are suspected MDR. The top images show the different life stages of the worker bees (pictures taken by A. Encerrado). Surprisingly, ABCC1 was downregulated throughout all life stages compared to E. However, only the downregulation in WL3 (0.35) and WL5 (0.43) were significantly different from E. Furthermore, ABCC1 followed the same pattern as the ABCB subfamily members, showing increasing expression as the bee ages. ABCC10 expression significantly decreased during WL3 (0.38) and increased afterward. However, only significantly increases compared to E were seen at F (1.75). ABCC4a was significantly upregulated in all life stages compared to E, with the expression decreasing from WL3 (3.12), WL5 (2.19) to P (1.94) and increasing linearly from there to forager (5.93). Significant differences were found between P and NEB to F. However, no significant differences were found between N and F, or NEB and N. ABCC4b differs considerably from the other two ABCC4 transporters with a significant decrease in expression during P (0.29) and a significant increase in N (3.83) and F (4.12). However, the expression between N and F was not considered significantly different. ABCC4c shows a similar trend to that of ABCC4a, with a decrease from WL5 to NEB and an increase from these to F. The expression in E is significantly different from N (2.21) and F (3.31), but these two are not significantly different from each other. ABCC4d expression was significantly different from E only at two points, in WL3 (0.47), which decreased, and P (2.46), which increased. The expression of ABCC4d seemed to decline after P linearly. The expression of ABCG1 was significantly higher than E from WL5 to F. Although it does not show a linear increase in expression, it is consistently high throughout development, with the highest level during foraging (5.27). The expression between F, N, and P were not significantly different from each other. ABCG2a had a unique expression pattern compared to the other eleven transporters, with the highest expression seen during P (15.66). The expression of ABCG2a was also significantly different from that of E during WL5 (2.57). However, this difference was not observed for the rest of the life stages. The expression of ABCG2b was significantly downregulated compared to E during WL3 (0.18) and WL5 (0.34), with WL3 being the only stage significantly different from WL5, NEB, N, and F. ABCG5 expression was mainly downregulated throughout the bee life stages, with the highest expression seen during P (2.40). ABCG5 was significantly downregulated compared to E during WL3 (0.25), NEB (0.44), N (0.34), and F (0.40). However, there were no significant differences in expression between these stages. ABCG2b expression is similar to that of ABCG5, mostly downregulated throughout the bee life stages, with the highest expression seen during P (4.16). Notably, an increased expression from larval to forager stage could be observed for seven distinct honey bee ABC transporters: Ame -ABCB1, Ame -ABCB6, Ame -ABCC1, Ame -ABCC4a, Ame -ABCC4b, Ame -ABCC4c, and Ame -ABCG1. Among these, the highest fold change was observed for Ame -ABCC4a with 5.93, followed by ABCG1 (5.27) and Ame -ABCC4b (4.12). The gene expression of these seven ABC transporters was subsequently investigated in queen and drone bees since they are the major reproductive castes in the hive. MDR transporter expression in reproductive caste members Figure 3 displays the gene expression patterns for seven selected MDR-like ABC transporters across worker bee life stages, reproductive caste members (DL5, D, Q), and queen ovaries (QO). Figure 3. Expression of MDR-like ABC transporter genes in reproductive hive members. Shown are fold changes in seven different ABC transporter gene expression patterns in drone larvae (L5), adult drones, queen bees, and queen ovaries relative to eggs. Statistical significance was validated by one-way ANOVA. The expression of Ame -ABCB1 in drones was significantly upregulated (2.38) compared to eggs (E). Conversely, Ame -ABCB1 expression in DL5 (0.46), Q (0.23), and QO (0.56) was downregulated compared to both E and D. Compared to NEB, N, and F, Ame -ABCB1 expression in D was not significantly different. The expression was the lowest in Q, significantly different from ovaries (QO) but not from Ame -ABCB1 in DL5. Ame -ABCB1 expression did not significantly differ in QO, WL3, WL5, and DL5. Ame -ABCB6 expression was upregulated in D (1.11), QO (1.33), and Q (1.24). There were no significant differences in Ame -ABCB6 expression among D, QO, and Q. However, Ame -ABCB6 expression was significantly downregulated in DL5 (0.49), with similar values to WL3 (0.41) and WL5 (0.61). Ame -ABCC1 expression was significantly downregulated compared to E in DL5 (0.31), D (0.65), and Q (0.56). While QO (0.89) was also downregulated, it was not significantly different from E. Ame -ABCC1 expression in Q and D did not significantly differ from each other but was significantly different from DL5, with the latter’s expression similar to WL3 (0.35) and WL5 (0.43). Ame -ABCC4a expression was significantly upregulated from E in both DL5 (1.94) and D (2.93) and downregulated in Q (0.95) and QO (0.66), though the latter two were not significant. Ame -ABCC4a expression in D and DL5 was similar to P (1.94) and NEB (2.88), with no significant differences between them. Ame -ABCC4b expression was downregulated in DL5 (0.64), D (0.96), QO (0.56), and Q (0.33), with expression only significantly different from E for Q. Ame -ABCC4b expression in P (0.29) was similar to that in the queen, with no significant difference. Ame -ABCC4c expression was upregulated in DL5 (1.42) and D (1.97), with the latter being significantly different from E but showing a similar expression to WL5 (1.99) and N (2.21), none of which were significantly different from each other. In QO (0.65) and Q (0.50), Ame - ABCC4c expression was downregulated compared to E, with Q being significantly different from E but not from WL3 (0.93). Finally , Ame -ABCG1 expression was upregulated in DL5 (2.35), D (2.30), QO (1.05), and Q (1.24). However, only Ame -ABCG1 expression in DL5 and D was significantly different from E, with levels similar to WL5 (2.35). Figure 3 shows that these seven proteins tend to be relatively lower expressed in the queen bee compared to the other caste members. Drones have Ame -ABCB1, Ame -ABCG1, Ame -ABCC4a, and Ame -ABCC4c expression similar to that of worker bees at different life stages. In almost all instances, QO showed downregulation of genes compared to E, or if gene expression was upregulated, it was never significantly different from E. In addition, when using DL5 as a control to examine the fold change of the twelve genes in D, only the expression of Ame -ABCB1, Ame -ABCB6, Ame -ABCC4b, and Ame -ABCG2b was significantly different between these two life stages (Figure S2). Discussion While ABC transporters have been extensively studied in pest insects in the context of insecticide resistance, their role in beneficial insects like honey bees remains poorly understood. This study examined the expression of twelve ABC transporters across multiple developmental stages of worker bees, providing insights into how chemical defense capabilities evolve as bees mature. Using a comprehensive transcriptomics analysis, Maiwald et al. ( 29 ) showed previously that the expression of major detoxification genes during worker bee development varies dramatically. The present work, focused on the expression patterns of 12 selected ABC-type transporters and expanded on Maiwald et al.’s ( 29 ) study by including the queen bee and drones, due to their importance in hive reproduction. Gaining a better understanding of how the roles and behaviors of all castes in the hive affect their exposure to chemicals, as well as their chemical defense capabilities, is vital for overall colony health and survival. Additionally, our analysis provides case-by-case discussions of the potential for these understudied proteins to confer multidrug resistance. Furthermore, we presented prospective MDR transporter proteins based on their expression trends in worker bees and their phylogenetic relationships to two well-studied model organisms, Homo sapiens (Hsa) and Drosophila melanogaster (Dme) . Orthology and Functional Insights into ABC Transporters in Honey Bee Workers Phylogenetic analysis shows that Ame -MDR49 is most closely related to Dme -MDR49 and Hsa -ABCB1 ( Figure 1 ). Hsa -ABCB1, also known as P-gp or P-glycoprotein, is a full transporter and the most extensively studied member of the ABC family 30 . Its presence in the human intestine, liver, and brain has been well-documented in relation to the pumping of xenobiotic compounds 15 . In Drosophila , there are three homologs of Hsa -ABCB1: MDR49, MDR50, and MDR65 15 . Dme -MRP49 is expressed in the adult head, endodermal anlage, head mesodermal anlage, larval brain, and mesoderm 31 . Furthermore, Dme -MRP49 is a transmembrane transporter contributing to insecticide resistance in Drosophila 32 . The expression pattern observed across developmental stages ( Figure 2 ) supports the potential role of Ame -MDR49 in conferring MDR resistance, as life stages with higher environmental exposure exhibit higher gene expression. Given its extensive presence in the literature and well-characterized function in model organisms, Ame -ABCB1 is a strong candidate for being an MDR in honey bees. Phylogenetic analysis shows Ame -ABCB6 is most closely related to Dme- ABCB6 and Hsa- ABCB6. Hsa- ABCB6 is a porphyrin transporter located in the outer membrane of mitochondria 30 . Hsa -ABCB6 is a half-transporter that functions as a homodimer and plays a role in protecting against oxidative stress and in the processing of iron and iron-containing biomolecules. It has been suggested that ABCB6 can also confer resistance against heavy metals in arthropods 33 . Dme -Hmt-1, or Heavy metal tolerance factor 1, is orthologous to Hsa -ABCB6, and it is involved in the transmembrane transport and binding of heme, as well as cellular detoxification of cadmium ions. Dme -Hmt-1 is expressed in the adult head, adult heart, embryonic/larval midgut, and gastric caecum 31 . These findings suggest that Ame -ABCB6 may play a significant role in chemical defense and cellular detoxification but not necessarily as an MDR transporter outside of chemicals that cause oxidative damage. The expression pattern determined with qPCR is similar to that of MDR49, further supporting the idea that Ame- ABCB6 is relevant to xenobiotic exposures ( Figure 2 ). Phylogenetic analysis shows Ame- MRP as homologous to Hsa- ABCC1 and Dme -MRP. In humans, ABCC1 was originally discovered as a cause of multidrug resistance in tumor cells. Further research has shown that it has a broad spectrum of drugs (xenobiotics) and physiological molecules as substrates 34 . Dme -MRP is involved in monoatomic anion transmembrane transport, renal tubular secretion, and transport of toxic substances. 31 In Drosophila , it is expressed in the adult head and heart 31 . Based on these pieces of evidence, one would expect the expression pattern of Ame- MRP to be similar to that of Ame- MDR49, given the likelihood of MRP conferring multidrug resistance. The expression pattern showed that adult workers did indeed have the highest expression of the gene although all the expressions were below the baseline (E). A potential explanation for this expression profile is that this gene is primarily called upon when there is significant exposure to an environmental chemical, and upon exposure the gene could be upregulated. Further research on this gene is necessary to determine if its expression changes with exposure to xenobiotics. Phylogenetic analysis shows that the paralogs of Ame -ABCC4 are most closely related to Dme- MRP5 and Hsa- ABCC4 ( Figure 1 ). In humans, Hsa -ABCC4 has been observed to transport nucleosides, 30 endogenous signaling molecules, and a wide range of drugs 35 . Furthermore, in lice ( Pediculus humanus humanus ), ABCC4 was also found to protect against the antiparasitic drug ivermectin 36 . In drosophila Dme -Mrp5 is involved in heme export and is orthologous to Hsa -ABCC4; it is expressed in the adult head and spermatozoon 31 . In honey bees, Ame -ABCC4b was reported to be highly expressed in the Malpighian tubules of the honey bee, an organ recognized for its major role in detoxification 29 . For Ame -ABCC4a, Ame -ABCC4b, and Ame -ABCC4c, the expression patterns bear similarities to other strong candidates for MDR function, specifically high levels in forager and nurse bees relative to other bees ( Figure 2 ). This trend is especially strong in Ame -ABCC4b; it is possible that Ame -ABCC4a and Ame -ABCC4c could have other functions besides those related to environmental exposure that become more relevant in earlier life stages. However, Ame -ABCC4d has strong expression in the pupa stage, suggesting that it may have a different function than its paralogous counterparts that is more relevant to the major physiological changes that occur at this life stage ( Figure 2 ). Based on the phylogenetic tree, Hsa -ABCC10 is orthologous to Ame -ABCC10 and Dme -CG7806. In humans ABCC10 also known as MRP7, confers drug resistance to cancer cells, and has a broad spectrum of substrates 30,37 . Hsa -ABCC10 expression is higher in the pancreas, followed by liver, placenta, lungs, kidneys, brain, ovaries, lymph nodes, spleen, heart, leukocytes and colon 37 . Dme -CG7806 is a protein predicted to be involved in transmembrane transport activity; it is highly expressed in the adult head and orthologous to Hsa -ABCC10 31 . Although it is shown to be an MRP, the expression pattern in honey bees does not reflect other MRP expressions. Suggesting that Ame- ABCC10 might not be an MRP-like protein. In insects, the ABCG subfamily G transporters are in the range of 10-25, with Drosophila having around 15 annotated G transporters similar to honey bee however in humans there are only five members of these subfamily 17 . The broad number of G transporters in insects and their presence in multiple detoxification tissues has raised the question if these transporters are used for detoxification and not only in sterol transport 14,38 . Phylogenetic analysis shows that Ame -ABCG1 is most closely related to Dme -CG5853 and Hsa -ABCG1. In humans Hsa -ABCG1 is present in the lungs, heart, spleen, brain, kidney and adrenal gland. Its function has been linked to cholesterol transport 30,38 . On the other hand in drosophila Dme -CG5853 is a predicted protein homolog to Hsa -ABCG1 and Hsa -ABCG4; its predicted function is to actively transport sterols outside the plasma membrane 31,39 . Dme -CG5853 is expressed in adult head and amnioserosa, embryonic/larval dorsal vessel, plasmatocyte primordium, and yolk nucleus 39 . The expression pattern has similarities to those of other MDR gene candidates, although the difference between adult workers and earlier life stages is not as stark as some other candidates. This suggests that Ame -ABCG1 may have some additional function besides cholesterol transport that relates to environmental exposure (such as the transport of environmental chemicals with similar properties to sterols). Ame -ABCG5, Ame -ABCG2a, and Ame -ABCG2b although suspected to have MDR capabilities, the results from the phylogenetic tree, and the expression profiles make them not suitable candidates. Hsa -ABCG5 is expressed in the liver and the intestine. Its function is to limit the absorption of sterols and promote its biliary excretion 30,38 . Dme -CG11069 is a predicted protein homolog to Hsa -ABCG5 predicted to actively transport sterols outside the plasma membrane 31 . Hsa -ABCG2 was seen to share the same ancestor with Ame -ABCG2a and Ame -ABCG2b. In humans, ABCG2 is present in the placenta, blood-brain barrier, liver, colon, intestine, mammary gland, and stem cells. Its function has been linked to xenobiotic defense and multidrug resistance for a broad range of substrates 30,38 . Hsa -ABCG2 also transports steroids, certain chlorophyll metabolites, and organic anions 30 . Orthologous to Ame -ABCG2b is Dme -st (Protein Scarlet), which is expressed in the Bolwig organ; Malpighian tubule main body primordium; Malpighian tubule primordium; adult head; and embryonic Malpighian tubule 31 . Dme -st transports bioamines, neurotransmitters, and metabolic intermediates 31 . And orthologous to Ame -ABCG2a is Dme -E23 which encodes an ABCG transporter of ecdysone (a steroid hormone) that regulates reproduction, molting, and metamorphosis 40 . The expression profile of both Ame -ABCG2a and Ame -ABCG2b contrasts with the expression profiles of the MDR candidates; notably, the expression is highest in the worker pupae and lower in other life stages and caste members ( Figure 2 ). This suggests that despite the well-characterized function of ABCG2 in drug resistance for humans, the most similar protein in honey bees is closer in function to that of Drosophila , given the high expression in a time of morphological change. Overall, worker bees exhibit changes in the expression of suspected multidrug resistance (MDR) transporters as they age, with notable differences observed in ABCB1 and ABCC4a expression levels from nurse bees to foragers. This pattern suggests an adaptation to the different environmental challenges encountered throughout their lifespan. Previous studies have highlighted differences in detoxification gene expression between nurses and foragers, particularly in tissues involved in nectar processing and social interactions 41 . This disparity may be attributed to the constant exposure of foragers to pesticide-laced nectar, pollen, and water from the field, necessitating greater expression of chemical defense genes to mitigate potential harm and maintain colony health. Honey bee larvae appear to require minimal MDR gene expression, likely due to protections by worker bees through food processing via trophallaxis. Workers probably cannot completely prevent xenobiotic exposure to the vulnerable larvae since the expression of ABCB1 in workers is only 3-5 times higher than the expression in larvae (i.e., WL3, WL5, and DL5). However, by using trophallaxis to transport and process their food, worker bees could act as a buffer to possible direct diet exposures 2 . Furthermore, genes such as ABCC4a/GB50195 have expression levels in larvae comparable to workers and drones, potentially serving a protective function (Figures 2 and 3) . Furthermore, some transporters showed vastly different expression profiles across life stages than ABCB1 and the other MDR genes. These included ABCC10, ABCG2a, ABCG2b, ABCG5 and ABCC4d. In general, the expression of these genes was more consistent across life stages than the MDR genes. However, these genes all had a significant spike in expression during the pupa stage. With the pupa stage being one of major development and physiological change, these results suggest that these transporters are involved in development rather than chemical defense. Caste-Specific Variations in Honey Bee MDR Transporters Expression Our results reveal significant variations in ABC transporter expression across different honey bee castes and life stages. Notably, mRNA expression of suspected MDR transporters is consistently lower in queens compared to other hive members. Although, this might not be true for all tissues since previous proteomics results had shown that queen Malpighian tubules (MT) relative to drone and worker tubules have a higher level of multidrug resistance (MDR) proteins 42 . Dietary influences emerge as critical regulators of gene expression dynamics during larval development. Huang et al. 2012 could show that cytochrome P450 (CYP) enzymes related to detoxification are particularly responsive to dietary compounds 43 . At 60 hours of larval development, when worker larvae are introduced to pollen grains, they encounter foreign compounds believed to stimulate the expression of defense-related genes. On the other hand, queen larvae stick to royal jelly (RJ), which contains only trace amounts of pollen 44 . Drones in contrast are exposed to pollen in their diet during development and throughout their adult life, which could explain why their gene expression looks more like that of the worker bees, although still lower. The difference in expression could explain why drones are more sensitive to imidacloprid, a reported ABCB1 possible substrate, than worker bees, showing a decrease in their survival 16–18 . Moreover, the genetic diversity and potential expression of defense genes in worker bees are influenced by the mating behavior of queens. During mating flights, a queen can mate with numerous drones, incorporating genetic material from multiple colonies into her spermatheca. As a result, worker bees inherit genetic material not only from the queen but also from the drones she mated with, enriching the genetic pool of the colony. While defense genes may be inherited from drones to worker bees, their expression and efficacy are likely subject to modulation by external factors, highlighting the complex interplay between genetics, environment, and caste-specific physiology in honey bee colonies. That said, the premature exposure of drones to toxic chemicals or diseases could affect their reproductive capacity by damaging sperm viability or preventing them from reaching sexual maturity 45,46 . This underscores the vulnerability of drones to environmental stressors and emphasizes the importance of safeguarding their health for colony sustainability. Our findings concerning the consistent downregulation of MDR-like genes in queen are potentially related to two interconnected factors: queen bees aren’t exposed to enough xenobiotics to necessitate high levels of transporters, or other components of their chemical defensome are robust enough to make up for a lack of transporters (i.e., metabolic enzymes). Previous studies suggest that secluded members of the hive have less need for chemical defense proteins since they are naturally exposed to fewer xenobiotics. Workers that collect and process the hive’s food are primarily exposed to contaminants, so when the highly refined food works its way to the queen, it is much cleaner than the raw materials 2 . Nevertheless, even with workers’ food processing, chemicals can still reach other members throughout the hive. A good example is the fungicide propiconazole (used in almonds, apples, blueberries, nectarines, oranges, peaches, raspberries, and strawberries), whose residues in the United States have been found at levels up to 227 ppb in wax and 361 ppb in pollen 5 . The results of this study are consistent with this idea; foragers that collect the food have the highest expression of MDR genes, followed by nurses, with queens having very low expression. This is also consistent with the idea that queens need to delegate as much of their energy budget as possible to reproduction (and not chemical defense) to maintain a healthy hive population; this could also explain why they exhibit lower expression of MDR genes. Should queens be seriously lacking in increasingly important chemical defense systems, then they could be particularly vulnerable to xenobiotics. With trophallaxis as a potential vector of pollutant transmission and increasing concentrations of pesticide use leading to accumulation in hive matrices, queens may be exposed to high doses of xenobiotics. Differential MDR-like Transporter Expression across Honey Bee Castes suggests Variation in Xenobiotic Defense Capabilities. The study of the chemical defense systems in honey bees against xenobiotic chemicals has mostly focused on metabolism (Phase I & II), while the input (Phase 0) and output (Phase III) stages are equally important in this process. Previous research has shown that as worker bees age, their chemical defense system becomes more robust, which is supported by the findings in this study that forager bees exhibit higher levels of xenobiotic efflux transporters than newly emerged bees. This study identifies seven MDR-like transporters based on changes in expression across worker bee life stages: ABCB1, ABCB6, ABCC1, ABCC4a, ABCC4b, ABCC4c, and ABCG1. Phylogenetic comparisons of human and Drosophila protein orthologs can help infer the function of these possible MDR transporters and their role in honey bee xenobiotic detoxification. The differences in relative expression of ABC transporter proteins across the three main castes suggests that castes are not equally protected against xenobiotics. Specifically, several major MDR transporters involved in xenobiotic efflux are consistently downregulated in queen bees compared to the other castes. Research by Cameron et al.( 47 ) and Corona et al.( 48 ) suggests that diet may play a role in regulating ABC transporter gene expression across castes, promoting the expression of oxidation-related genes in queen bees while decreasing that of chemical defense genes. Limited attention has been given to studying the defense genes of queen bees because trophallaxis and food processing are thought to create a dilution effect that minimizes queen exposure to xenobiotic chemicals from food sources. However, this hypothesis assumes an ideal scenario, raising questions about how long it would take for pollutants to accumulate in the hive under chronic exposure and reach all castes. In conclusion, ABC transporter gene expression in honey bee colonies varies by both life stage and caste. The reduced MDR gene expression in queens suggests that hive members may have different abilities to defend against chemical threats, which becomes increasingly important as honey bees encounter rising xenobiotic exposures during pollination season. Future research should focus on identifying additional molecular and/or biological components of the xenobiotic defense systems of the reproductive hive members while also applying field-relevant exposure scenarios. Acknowledgments The research reported in this publication was supported by a grant from the 2022-2025 PAm-Costco USA Scholarship, as well as by funds from the UC National Laboratory Fees Research Program of the University of California grant number L23GF6273, and the James and Rita Seiber Agricultural and Environmental Chemistry Support Fund. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer. Conflict of Interest The authors declare no conflict of interest. Data availability All data and supplementary information supporting this study are available from Dryad at https://doi.org/10.5061/dryad.d2547d8c2. Sample metadata: Related metadata can be found in the supplemental information of this article and is available from Dryad at https://doi.org/10.5061/dryad.d2547d8c2. Benefits Generated: A cross-disciplinary research collaboration between scientists from UC Davis and USDA ARS created this manuscript, with all collaborators included as co-authors. 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Supplementary Material File (image2.emf) Download 2.49 MB File (image3.emf) Download 11.55 MB Information & Authors Information Version history V1 Version 1 21 January 2025 Peer review timeline Published Environmental Toxicology and Pharmacology Version of Record 1 Sep 2025 Published Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords abc transporters castes gene expression honey bees multidrug resistance (mdr) xenobiotic defense Authors Affiliations Angela Encerrado-Manriquez 0000-0002-9822-3549 University of California Davis View all articles by this author Zeke Spooner University of California Davis View all articles by this author Tina Truong University of California Davis View all articles by this author Julia Fine USDA-ARS Pacific West Area View all articles by this author Sascha Nicklisch 0000-0003-3120-6485 [email protected] University of California Davis View all articles by this author Metrics & Citations Metrics Article Usage 486 views 173 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Angela Encerrado-Manriquez, Zeke Spooner, Tina Truong, et al. Developmental and Caste-specific Expression Patterns of ATP-Binding Cassette (ABC) Transporters in Honey bees (Apis mellifera). Authorea . 21 January 2025. 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