Age- and sex-related ABC transporter expression in pyrethroid-susceptible and –resistant Aedes aegypti

Resistance mechanisms to synthetic insecticides often include point mutations and increased expression of genes encoding detoxification enzymes. Since pyrethroids are the main adulticides used against Aedes aegypti, which vectors pathogens such as Zika virus, understanding resistance to this insecticide class is of significant relevance. We focused on adenosine triphosphate (ATP)-binding cassette (ABC) transporters in the pyrethroid-resistant Puerto Rico (PR) strain of Ae. aegypti. We investigated the expression patterns of six ABC transporters previously characterized as differentially expressed in insecticide-challenged mosquitoes, or increased mRNA expression in pyrethroid-resistant Ae. aegypti, by comparing PR to the Rockefeller (Rock) susceptible strain. No constitutive differential expression between strains was detected, but expression differences for these genes was influenced by sex and age, suggesting that their role is independent from resistance in PR. Instead, ABC transporters may be induced after insecticide exposure. Challenging mosquitoes with deltamethrin, with or without ABC transporter modulators, showed that Rock and PR responded differently, but a contribution of ABC transporters to deltamethrin toxicity is suspected. Moreover, the effect of dexamethasone, which enhanced the inhibition of nerve firing by deltamethrin, was observed using a Drosophila central nervous system preparation, showing synergy of these two compounds through the potential inhibition of ABC transporters.

Dexamethasone increases rate of inhibition to CNS firing after deltamethrin exposure. In addition to increased potency, we speculated that the inhibition of Pgps would reduce deltamethrin efflux from the CNS and, thus, increase the bioavailability of deltamethrin in the CNS. To test this, we measured the rate of inhibition of CNS firing after exposure to 80 nM deltamethrin, which is the approximate IC 50 value. Data show complete inhibition of CNS firing after exposure to 80 nM deltamethrin at 30 ± 5 min post-exposure. Importantly, the rate of nerve inhibition was increased by 2-fold after co-exposure to 30 µM dexamethasone and 80 nM deltamethrin, which is a statistically significant (P < 0.01) increase (Fig. 5C). Representative traces showing the rate of nerve inhibition for deltamethrin alone and after co-exposure to deltamethrin and dexamethasone are provided in Fig. 5E,F.

Discussion
This study primarily focused on determining if ABC transporters could be involved in pyrethroid resistance in PR mosquitoes by comparing various parameters to a susceptible counterpart, including constitutive gene expression, and expression after deltamethrin exposure. Additionally, the study aimed to determine if ABC transporters are involved in the response to deltamethrin exposure via toxicity in Ae. aegypti mosquitoes and electrophysiology bioassays via ABC transporter pharmacology with a prepared Drosophila CNS.
ABC transporters of the families ABCB and ABCG were considered good candidates for the export of pyrethroids out of neural tissue because they were differentially expressed in mosquitoes in response to insecticide exposure 35,36,38,39 or as potential resistance genes 19 . In particular, ABCB transporters are known to be specific for hydrophobic substrates 53 , such as the pyrethroid deltamethrin. Overall, there were no differences in expression of the ABC transporter transcripts of interest between PR and Rock in the experiment comparing sexes, ages and strains, but there were significant expression differences between males and females, and between age groups. Age dependent regulation of ABC transporter genes, as well as an involvement in embryo development, were previously shown in rats 54 . Sex was also a predominant factor of expression level variability, with a higher expression in female rats compared to males, resulting in quicker drug efflux by hepatic Pgps in females 55 . It was also observed that sex influenced the expression of the homologous genes in An. stephensi 38 .
A follow-up exposure using 5 to 7 d old female mosquitoes showed that all the ABC transporter transcripts investigated, except the transcript Pgp, exhibited significantly different expression levels between strains ( Fig. 2 and Supplementary Table 2). A particularly interesting result was the expression of ABCB4, which was lower in PR mosquitoes than in Rock, contradicting previously published results 19 . This experiment also illustrates the importance of accounting for the appropriate parameters in a gene expression experiment. There were many aspects to consider in the model for the analysis of the constitutive difference that did not detect more fine-tuned strain differences in the oldest age group, highlighted in the second experiment. These results may show that the higher expression of some of the ABC transporters tested is part of the resistance profile of PR mosquitoes, but it would have to be observed in males as well since they are also pyrethroid-resistant, and at all age groups 42 , which was not the case in the previous experiment.
This follow-up transcript expression assay also revealed that the exposure to deltamethrin did not have a significant effect on the expression of the genes of interest. This could be the result of using the LD 25 , respective to each strain, which may be too low at the 2 h timepoint to elicit a strong stress response and significantly alter (C) Average (n = 5-10) time to cessation of CNS firing after exposure to deltamethrin and a combination of deltamethrin + dexamethasone. Asterisk represents statistical significance at P < 0.05 as determined by an unpaired students t-test. (D-F) Representative CNS firing frequencies before and after exposure to dexamethasone, deltamethrin, and dexamethasone + deltamethrin, respectfully. Initial firing frequencies in spikes/second (Hz) for each experiment are given to the left of each representative trace. (2019) 9:19551 | https://doi.org/10.1038/s41598-019-56134-2 www.nature.com/scientificreports www.nature.com/scientificreports/ transcript expression. De Marco et al. 37 studied the expression dynamics of detoxification genes and ABC transporter genes following exposure to permethrin in An. stephensi. It was demonstrated that the response involved upregulation of certain genes while others were downregulated, and that this pattern would change over time between 6 and 48 h. Consequently, time is an important factor to account for with gene expression experiments and the timepoint we investigated may not have been the most representative of the expression changes related to ABC transporter gene expression. A time course experiment may provide more information about our genes of interest in a future study. Alternatively, our results could also indicate that the genes of interest are not involved in the response to deltamethrin exposure, which does not eliminate a role of ABC transporters in defense against pyrethroids, following the results of the toxicity assays.
The exposure to deltamethrin in combination with ABC transporter modulators highlighted differences between the susceptible and resistant strains, which could imply a different dynamic and function of ABC transporters in the two strains. The effect of the two main drugs used in the toxicity assays can give an idea of the differences between strains, but also a better understanding of the function of dexamethasone compared to verapamil. Verapamil is a competitive inhibitor of ABC transporter substrates, since it is itself a substrate of ABC transporters 49 , as well as a calcium channel blocker 43 . It stimulates ATPase activity while preventing efflux at the transporter level, which increases insecticide toxicity 56 . Verapamil was extensively used to determine the involvement of ABC transporters in the export of drugs and resistance to chemotherapy for instance, and to other drugs in mammals [57][58][59][60] and in insects 34,35,[39][40][41]56,61,62 . Verapamil had a synergistic effect on deltamethrin-induced mortality in Rock mosquitoes, regardless of pretreatment time, but it did not significantly affect mortality in PR mosquitoes. The increasing mortality trend after 60 minutes of exposure to deltamethrin may however indicate that verapamil is slow to act in PR mosquitoes.
Dexamethasone has been described in mammals as a stimulant of ABC transporter expression, as well as a substrate of ABCB and ABCG transporters, especially those involved in cancer resistance 44,45 , and is readily transported by MDR1 in mouse brain and human cells 46,48 . Thus, verapamil and dexamethasone share the ability to be transported by ABC transporters, hence, potentially sharing a similar role. In arthropods, however, it was thought to be an inducer of cytochrome P450 expression 63 . In Pediculus humanus for instance, dexamethasone induced tolerance to ivermectin, whereas verapamil increased ivermectin toxicity 63 , which led the authors to hypothesize that decreasing ABC transporter export with verapamil would increase toxicity, and that dexamethasone ultimately increased P450 activity. However, based on the results obtained in the present study using PR and Rock mosquitoes, as well as previous work 42 , increased P450 activity induces tolerance to deltamethrin and is likely part of the resistance mechanism in PR mosquitoes, whereas the toxicity assay exposing PR mosquitoes to deltamethrin did not increase survival in presence of dexamethasone. Survival was decreased when mosquitoes were exposed to these drugs, suggesting that increased P450 is either not the major modification induced by the exposure to dexamethasone, or is not the effect of this drug in Ae. aegypti. The effect of dexamethasone suggests that in Ae. aegypti, its role is more likely similar to the role of verapamil in Rock mosquitoes and can act as an ABC transporter competitive inhibitor. Dexamethasone and verapamil may differ in certain functional aspects, which would explain the divergence of results in PR after a 4 h pretreatment, as well as the temporal dimension of the effect of dexamethasone. Additionally, the results of the CNS preparation with exposure to dexamethasone show that it decreased the IC 50 of deltamethrin by increasing firing rate at the nerve, which suggests a direct role of dexamethasone at the blood brain barrier. Dexamethasone also significantly decreased the time needed for deltamethrin to induce nerve death.
The results highlight that ABC transporters may not play an important role in the efflux of deltamethrin in the PR strain, and their involvement would be delayed compared to Rock. It can be hypothesized that since PR mosquitoes carry mutations that confer target-site insensitivity 22 and exhibit increased P450 activity 42 , this strain may not rely as heavily on ABC transporter export compared to the susceptible Rock strain. The resistance mechanism would be relying on P450 activity instead 42 , which can be observed through the effect of PBO, used here as a positive control, which significantly increases mortality in both PR and Rock mosquitoes. Regarding the resistance mechanisms in PR, the expression analysis did not show a consistent higher expression of the select genes in PR or in case of exposure to deltamethrin, but the constitutive gene expression analysis did not show a decreased overall expression of the ABC transporter transcripts of interest either. The expression patterns were particularly interesting for the gene ABCB4, because it was previously found to be more highly expressed in other pyrethroid-resistant strains of mosquitoes when compared to the New Orleans susceptible strain of Ae. aegypti 19 , but does not follow the same pattern of expression here. Two hypotheses can be offered to explain the discrepancies between studies in terms of gene expression: (1) comparing a resistant strain to a separate susceptible strain, rather than a susceptible isogenic strain, can pose a challenge since there are likely other genetic differences between the two that could confound the results of such an experiment; (2) susceptible New Orleans and Rock strains can also present genetic differences resulting in diverging expression patterns when a resistant strain is compared. Interestingly, the expression pattern of ABCB4 may be part of a compensatory mechanism between P450s and ABC transporters, which could be selected to avoid fitness costs in resistant strains, due to a higher P450 activity that is energetically costly. However, if such a mechanism exists, then more experiments would need to be conducted to gain an understanding of this phenomenon and verify our hypothesis.
Overall, the competitive inhibition of ABC transporter export function by verapamil, and potentially dexamethasone, increased mortality, although no expression level change after exposure to deltamethrin was detected. These can be interpreted as discrepancies between mosquito species or insecticide classes, since the genes chosen for this study were either involved in response to other modes of action for Aedes species 39,40 , or extracted from work done with An. stephensi exposed to permethrin 35,36,38 , and may not be involved in the response to deltamethrin exposure. ABC transporter genes involved in the response to deltamethrin may be different from those genes examined in this study and their differential expression could explain the survival curves observed for Rock, and PR to a lesser extent. Consequently, this study contributes to the large body of work demonstrating www.nature.com/scientificreports www.nature.com/scientificreports/ the heterogeneity of the role of ABC transporters in insecticidal challenge 64 , especially across species. Overall, we observed differences between strains due to treatment with ABC transporter modulators, illustrated by the effect of verapamil in particular. There is also a temporal aspect to their contribution towards deltamethrin challenge, especially in PR, and in the expression of the tested ABC transporters, which was variable between age groups. Additionally, a different role in females and males was suggested by the differences between sexes, as previously observed 36,38 .
Although the genes involved in deltamethrin export were not clearly delineated, the toxicity assays in Ae. aegypti, complemented by the CNS preparation study in D. melanogaster, highlighted the participation of ABC transporters in deltamethrin cellular export, with an increased efficacy when using competitive inhibitors in Rock, and potentially in PR. This work also provided insight into the function of dexamethasone when applied in conjunction with deltamethrin, presumably allowing increased penetration, and eventually, increased nerve inhibition and death. These findings highlight the complicated nature of insecticide resistance by demonstrating the likely role of other proteins involved in insecticide detoxification or transport, emphasizing the need to continue investigating the mechanisms that confer resistance in different species.  42 . Mosquitoes were aspirated and transferred to a new cage within 24 h of emergence in order to maintain age-matched cohorts of each strain. Mosquitoes were anesthetized on ice and females and males were separated for experiments. Females and males of three age groups, 1 to 3 d old, 3 to 5 d old, and 5 to 7 d old, were collected for RNA extraction and subsequent gene expression analysis, whereas female mosquitoes of the two strains were collected 5 to 7 d after emergence for toxicity assays and gene expression confirmation.

Methods
Drosophila melanogaster. The wildtype Oregon-R (OR) strain of Drosophila melanogaster was used for CNS recordings. The OR strain was provided by Dr. Jeffrey Bloomquist at the University of Florida and was originally donated by Doug Knipple, Cornell University, Ithaca NY, USA. All fly strains were maintained in culture at Louisiana State University Agricultural Center (Department of Entomology) and were reared on standard medium in Drosophila tubes at 25 °C, 12:12 (light:dark) photoperiod and 55% relative humidity. For dissection, flies were anaesthetized by chilling on ice and decapitated before dissecting out CNS in Schneider's medium (Invitrogen, Paisley, Scotland, UK).

Gene expression. Deltamethrin exposure.
Two experiments were performed in order to 1) identify constitutive differences between susceptible and resistant strains, as well as sex and age differences, in the expression of ABC transporter transcripts, and 2) identify deltamethrin-induced changes in the expression of the same transcripts. The specimens used were either 1) untreated, in triplicates of 10 mosquitoes of the 2 strains, of 3 age groups for both sexes, or 2) 5 to 7 d old females that received their respective LD 25 (at 24 h) calculated based on weight differences observed by strain (resistant or susceptible). Deltamethrin was applied topically on the pronotum, then mosquitoes were placed in Petri dishes in groups of 10. Each condition was repeated in 3 replicates. Rock females received 0.2 µL of a stock solution of 22.5 nM in acetone and PR females received 0.2 µL of a stock solution of 1.5 µM of deltamethrin in acetone. The specimens were placed on ice before recovery after a 2 h exposure, and subsequently flash-frozen in liquid nitrogen and stored at −80 °C.
RNA extraction and cDNA synthesis. The RNA extraction area and materials were treated with RNase AWAY ™ (Molecular BioProducts ™ , San Diego, CA). RNA extraction was performed using Tri Reagent RT (Molecular Research Center, Inc., Cincinnati, OH) and 1 µg of RNA per 20 µL reaction volume was reverse transcribed using the iScript cDNA reverse transcription kit following manufacturer indications (BioRad, Hercules, CA). The resulting cDNA was diluted 2-fold (constitutive experiment) or 3-fold (deltamethrin exposure) to accommodate the volumes of reaction.
Quantitative reverse transcriptase polymerase chain reaction. The primers for ABCB2, ABCB4, ABCB6, and ABCG4 genes were originally designed for An. stephensi 35,36,38 , thus, the sequences were matched in the Basic Local Alignment Search Tool "BLAST" 65 to find the homologous sequence in Ae. aegypti and design custom primers with Primer 3 66 . Additionally, Porretta et al. 39 produced degenerate primers for Ae. caspius that also work in Ae. aegypti due to the conserved Walker A-Walker B region across species. Other primer sequences were gathered from Bariami et al. 19 (Supplementary Table 3 The statistical analysis of the qRT-PCR results was performed using the R 67 package MCMC.qpcr 68 . This method uses a generalized linear mixed model (GLMM). GLMM permits the analysis of complex designs by an ANOVA-type analysis. The model also considers primer efficiencies and allows the calculation of molecule counts from Ct values for all conditions. Prior to analysis, outliers of technical replicates were removed after visualization of the melt curve and amplification curve. All transcripts of interest and two housekeeping genes were analyzed. The housekeeping genes selected for the analysis were β-actin and ribosomal protein S17 (RPS17) 69 , however, these genes were not specified as "control" genes since no assumption was made about their stability, which is one of the features of the package used. The effect of (1) age, sex, and strain, or (2) strain and treatment, on the expression of the selected genes was measured. P-values were corrected for multiple tests by applying the method of Benjamini and Hochberg 70 with a false discovery rate of 5%. toxicity bioassays. The reagents used were acetone (Sigma Aldrich, Saint Louis, MO), verapamil hydrochloride, 99% (ThermoFisher Scientific, NJ), dexamethasone (Sigma Aldrich, Saint Louis, MO), and technical grade deltamethrin (Chem Service, Inc., PA).
A toxicity bioassay was performed with two pretreatment times: (1) Rock and PR female mosquitoes between 5 and 7 d old received a topical application of 0.2 µL of either acetone as a control treatment, PBO as a control (10 mM), verapamil (10 mM), or dexamethasone (8 mM), diluted in acetone, on the pronotum, and were immediately placed in bottles coated with deltamethrin. A sub-lethal concentration of PBO, verapamil and dexamethasone was used for each bioassay. Rock were exposed to 0.1 mg/bottle (1 mL/bottle) and PR were exposed to 1 mg/ bottle (1 mL/bottle). (2) All conditions were identical, but the topical application of the four compounds tested was performed 4 h before the transfer into the coated bottles. Controls were running simultaneously to account for natural mortality. Six replications were used for all the conditions. Mortality was recorded every 15 min for 2 h. The resulting survival curves were compared between treatments, separately for each pretreatment time and strain, and analyzed with a log-rank Mantel-Cox test. All statistical tests were carried out at a significance level of P ≤ 0.05. Analyses were performed with GraphPad Prism version 8.00 for Windows (GraphPad Software, La Jolla California USA, www.graphpad.com). electrophysiological studies of Drosophila melanogaster neural systems. Neurophysiological recordings were performed on the CNS of 3 rd -instar D. melanogaster larvae and slightly modified from previously described methods 50,51,71,72 . One primary difference in this study is the recordings were performed on intact CNS to ensure the blood brain barrier was fully functional. The CNS was excised from the larvae and placed in a separate dish with physiological saline (200 µL) containing: 157 mM NaCl, 3 mM KCl, 2 mM CaCl 2 , and 4 mM HEPES, pH = 7.25. Peripheral nerve trunks were drawn into a recording suction electrode and electrical activity was monitored from descending nerves originating from the ventral ganglia, with amplification by an AC/DC amplifier (Model 1700, A-M Systems, Inc., Carlsborg, WA, USA). Descending electrical activity was subjected to window amplitude discrimination and converted on-line into a rate plot, expressed in Hertz (Hz), using LabChart7 Pro (ADInstruments, Colorado Springs, CO, USA). Noise (60 Hz) was eliminated using Hum Bug (A-M Systems, Sequim, WA, USA). Activity was monitored for a 5 min time period to establish a constant baseline firing rate, as the spike frequency typically increased from 0 to 10 min before stabilization. After a baseline was established, the CNS preparation was directly exposed to test compounds by adding 200 µL of solution to the bath containing 200 µL of saline. The final concentration of solvent in the bath was 0.1% DMSO. Frequencies were measured for 3-5 min for each concentration prior to the addition of the next drug concentration. Mean spike frequencies over the 3-5 min recording period for each concentration were used to construct concentration-response curves in order to determine IC 50 values, which were calculated by nonlinear regression (variable slope) using GraphPad Prism TM (GraphPad Software, San Diego, CA, USA). Each drug concentration was replicated 5-10 times.

Data availability
The datasets generated during and/or analyzed during the current study are contained within the paper and its Supplementary Information Files, or can be obtained from the corresponding author on reasonable request.