Ethics statement

No human participants, human data or human tissue were used in the present study. Adult mosquitoes were fed on pigeon blood once per week according to a protocol approved by the Institutional Animal Care and Use Committee of CIPEIN (IACUC/CICUAL 1531/13). Insects were housed, cared, fed and handled in accordance with resolution 1047/2005 (Consejo Nacional de Investigaciones Científicas y Técnicas, CONICET, Argentina) regarding the national reference ethical framework for biomedical research with laboratory, farm, and nature collected animals, which is in accordance with the standard procedures of the Office for Laboratory Animal Welfare, Department of Health and Human Services, NIH and the recommendations established by the 2010/63/EU Directive of the European Parliament, related to the protection of animals used for scientific purposes. Biosecurity considerations are in agreement with CONICET resolution 1619/2008, which is in accordance with the WHO Biosecurity Handbook (ISBN 92 4 354 6503).

Treatment with Eucalyptus camaldulensis EO and sample preparation

An insecticide-susceptible strain of Ae. aegypti (Rockefeller strain, Venezuela) was used in the bioassays. The colony has been kept in the laboratory since 1996, free of exposure to pathogens, insecticides, or repellents, at 25–30°C, 80–90% of relative humidity, and a L12/D12 photoperiod [25]. We selected this strain for the analysis, instead of a wild population, given that we are able to track its previous history of exposure to toxics and its status of susceptibility. All larval instars were fed on a mixture of rabbit pellets and yeast in a 3:1 proportion.

The larvicidal bioassay was performed with 1 mL of the E. camaldulensis EO diluted in pure acetone (Merck, Darmstadt, Germany), which was added to 224 mL of dechlorinated water in a 500-mL plastic jar, to a final concentration of 26.75 mg/L, which is the LC₅₀ for this EO (see extraction and analysis procedures of E. camaldulensis EO in [25]. The mixture was shaken slightly to ensure a homogeneous solution. Then, 20 late third or early fourth instar Ae. aegypti larvae were placed in 25 mL of dechlorinated water and transferred to that jar. For the control group, 20 larvae were introduced in a jar containing 1 mL pure acetone in 249 mL of dechlorinated water. No food was offered to the larvae during the exposure time given that, if the treatment affects the feeding behavior of the larvae, this would differentially affect the expression of genes in both experimental groups. Hence, we would not be able to differentiate those changes in gene expression due to the intoxication itself from those due to differences in feeding condition/nutritional state. The bioassays were conducted in a 27±2°C regulated chamber, with 80–90% relative humidity and a 12:12 hrs photoperiod [25]. After 14 hrs of exposure, surviving larvae from both experimental groups were collected in microtubes containing Trizol (Ambion, Sao Paulo, Brazil) (n = 8-10/group); this reagent was also used for total RNA extraction, according to the manufacturer’s instructions. We chose a 14 hrs period of exposure in order to allow differences in gene expression to reach a maximum, based on previous results on Ae. aegypti larvae intoxication [26]. Larvae were considered dead following the method previously reported [27]. The bioassay was repeated four independent times in order to obtain four independent biological replicates for each experimental group.

RNA sequencing and bioinformatic analysis

Library construction and high-throughput sequencing services were hired at Novogene Corporation Inc. (Sacramento, USA). A total of 8 cDNA libraries (4 per experimental condition) were constructed using the NEBNext Ultra RNA Library Prep Kit (New England Biolabs) with an insert length of 250–300 base pairs (bp). The libraries were sequenced using Illumina NovaSeq (paired-end reads with 150 bp length) with a sequencing depth of at least 26.9 million per library. The raw sequence dataset is available with the NCBI-SRA Bioproject number PRJNA671513.

The FASTQC tool [28] was used to analyze the presence of Illumina sequencing adapters and the read quality. After, Illumina adapters and those bases from 5’ and 3’ ends with Phred quality scores lower than 5 (TRAILING: 5 and LEADING: 5 parameters) were removed from the reads using Trimmomatic v0.32 in the paired-end mode [29]. Besides, the SLIDING-WINDOW parameter was set as 4:15 and only reads longer than 50 bp were maintained (MINLEN parameter = 50). The last version of the Ae. aegypti genome (Liverpool AGWG strain with the assembly AaegL5.0, uploaded on June 2017) was downloaded from VectorBase [30] with its corresponding General Feature Format (GFF) file, a tab-delimited text file that describes the genomic features (annotation AaegL5.2, uploaded 24th April 2019). STAR v.2.6.0 [31] was used to index the genome file and to map the trimmed reads with default parameters. The htseq-count command (with parameters -t exon -i Parent -r name and -s no) of HTSeq v.0.11.1 [32] was used to report the counts of the mapped paired-end reads from multiple index BAM files at specific intervals.

Gene expression analysis was carried out using edgeR package v3.6.8 [33] and the count data generated by HTSeq were used as input in R-studio. Read counts were initially normalized using calcNormFactors function, which implements the Trimmed Mean of M-values (TMM) method. Following, the estimateDisp function that calculates gene-specific biological variation (tagwise) based on the empirical Bayes method was applied. Afterwards, normalized counts were analyzed using the GLM approach and the quasi-likelihood F-test. First, the glmQLFit function was used to fit read counts to a quasi-likelihood negative binomial generalized log-linear model and, afterwards the glmTreat function was used to test for differential expression relative to a minimum required fold-change threshold = 2. Second, genes with low expression and high variation were filtered using HSTFilter package v. 1.32.0 [34]. Finally, we used the topTags function to rank and extract the differentially expressed genes according to their False Discovery Rate (FDR) values. Those genes with an FDR<0.05 were considered as differentially expressed genes (DEGs) between control and treated groups. We extracted the normalized Counts Per Million (CPM) values of the DEGs from the detoxification related protein families. A heatmap was generated using pheatmap v.1.0.12; this package calculates Z-scores (subtracting the mean and then dividing by the standard deviation) for each gene and plotted instead of the normalized CPMs.

The GO-terms of the Ae. aegypti predicted proteins were obtained from VectorBase [30] using the BioMart tool. The enrichment analysis was carried out with ermineR package using the Gene Score Resampling method (GSR) [35] and the absolute log fold change values for each gene to produce a score rank. In order to have a global analysis, complementary to that of the DEG set, all the genes in the database were considered for this analysis, along with their corresponding logFc as a continuous variable. A total of 200.000 interactions were performed. With this strategy, GO-terms that were enriched along the whole dataset were detected. For details on the strategy used see https://erminej.msl.ubc.ca/help/tutorials/running-an-analysis-resampling/.

In parallel, DEGs were mapped to the KEGG PATHWAY database and Fisher’s exact test followed by Benjamini and Hochberg FDR correction method was used to identify significantly enriched pathways (FDR<0.05) using KOBAS 3.0 server [36]. The pipelines used to perform the differential gene expression analysis (with EdgeR) and the GO-enrichment analysis (with ermineR) are available at: https://github.com/josmantorres/Differential-Gene-Expression-and-Enrichment-Analysis-pipeline.git

According to previous literature [37], we operationally defined a gene cluster when N genes belonging to the same gene family are arranged within a genomic region having fewer than N—1 genes that don’t belong to this family.

Identification, enrichment and clustering analysis of detoxificant-related gene families

The PFAM domains were used in the HMMER tool (Biosequence analysis using profile hidden Markov models) and as queries in tBLASTn searches [38] on the AaegL5.2 gene set (published on 24 Apr 2019). We used the following PFAM domain alignments as queries: PF00011 (HSP20); PF00012 (HSP70); PF00005 (ABC-transporters); PF00067 (CYP450); PF02798, PF00043 (GSTs); PF00201 (UGT) and PF03392 (CSP). The microsomal GSTs were identified using the orthologues from Drosophila melanogaster as queries. Those transcripts annotated as “pseudogenes” were not considered.

We used cpm (y, prior.count = 10, log = TRUE) function in e dgeR to generate a matrix of log2 CPM that was used to produce heatmaps, including all the members from each protein family, by means of gplot package v.3.1.1 in R-studio. Dendrograms were plotted with hierarchical clustering among genes based on Euclidean distances and complete linkage method for clustering. A g ene set enrichment analysis was conducted with each one of the detoxification-related gene families using the “Category” package (v.2.58.0) with the hyperg function, which performs a hypergeometric test for over- or under-representation of significant ‘genes’ amongst those assayed in a universe of genes. For each gene family, two lists were generated based on their PFAM domains: IDs of family members identified as differentially expressed (named significant); and IDs of family members identified in the entire genome (named assayed). Both lists were compared among them and with a third one containing the IDs of the entire genome with at least one PFAM domain (11.931 genes). The pipeline used for the hypergeometric test was also included in the GitHub link mentioned above.

Characterization of Chemosensory Proteins in Ae. aegypti

Analysis of CSPs was performed for the detection of a signal peptide (predicted by SignalP 5.0 [39]), the four characteristic cysteine domains and a secondary structure including six α-helices (predicted by PSIPRED [40]. For phylogenetic analysis, sequences were aligned in Multiple Alignment using Fast Fourier Transform (MAFFT) software version 7.0 (using G-INS-i strategy with—unalignlevel 0.1—leavegappyregion—ep 0.12—maxiterate 1000) and the resulting alignment was trimmed with trimAl v1.2 (using gap threshold = 0.3, [41]). Finally, maximum likelihood trees were built in IQ-TREE [42] and edited with iTol (https://itol.embl.de/). Branch support was determined using the approximate Likelihood Ratio Test (aLRT). Non-parametric branch support was based on the Shimodaira-Hasegawa-like (SH) procedure. CSP sequences from An. gambie [43] were included in the phylogenetic analysis in order to infer homologies.

Molecular modelling

CSP sequences were used to generate structural models using Modeller 9.25 [44]. A combination of multiple structures deposited in RSCB PDB [45] (1k19,1kx8,1n8u,1n8v,2gvs,2jnt) were used as templates, following the procedure indicated in modeller 9.25 manual for advanced modelling. A total of 100 models were built for each CSP and the one with the best (lower) Discrete Optimized Protein Energy (DOPE) score was selected.

Molecular docking

Each CSP structure was considered as a receptor in independent runs, using the modeled structure described in the previous section. p-cymene ligand structures were obtained from Drugbank 5.0 [46]. Previous to docking runs, receptor and ligand were prepared using the prepare_ligand.py and prepare_receptor.py scripts from MGLtools suite [47]. Docking runs were performed using Autodock VINA 1.1.2 [48], with a box that fully covers the receptor, under default parameters and producing 10 modes per complex. For each CSP, the lowest (best) and average binding energy is reported, together with the root-mean-square deviation (RMSD) for the different binding modes.

Sequencing and mapping metrics

RNA-Seq analysis generated more than 245 million reads with an average of 30.68 million reads per experimental replica. More than 97.5% of raw reads were retained after the trimming and filtering processes. High quality reads were further mapped to Ae. aegypti genome with an average of 24.65 million reads per replica (which represents 82.4% of the trimmed reads) (S1 Table). Principal component analysis showed that samples under the same treatment were grouped (control vs. EEO treated; S1 Fig).

Differential expression analysis after treatment with E. camaldulensis EO

Differential transcription analysis was performed on 11.151 transcripts (78% of the total predicted transcripts). A total of 239 genes (2.1% of the analyzed transcripts) were found differentially transcribed with an absolute fold-change >2 and an FDR<0.05 in the EEO treated group. These DEGs included 177 transcripts over-transcribed and 62 under-transcribed (see the complete list of DEGs in S2 Table and S2 Fig for a volcano plot).

Forty-two of the DEGs (17.6%) belong to gene families previously associated with detoxification in insects (13 HSPs, 9 CYPs, 6 UGTs, 5 CSPs, 5 GSTs and 4 ABC transporters).

From these, only 2 CYPs were underexpressed (AAEL003890 and AAEL014619/CYP9J22), the remaining detoxificative-related transcripts were overexpressed after EEO treatment (Fig 1). None of the members of CCEs, a superfamily related to xenobiotic detoxification in insects [49,50], was present in the DEG set (S2 Table). Conversely, previous transcriptomic studies demonstrated differential expression of CCEs in Ae. aegypti larvae exposed to the carbamate propoxur and the neonicotinoid imidacloprid, but not to the pyrethroid permethrin [4]. Besides, larvae from a population resistant to propoxur [51] and female adults resistant to the pyrethroid deltamethrin [52] presented differentially expressed CCEs members. On the other hand, Aedes albopictus larvae resistant to the organophospate larvicide temephos also presented an overexpression of CCEs when compared to a susceptible population [53]. Altogether, our results and previous data suggest that the transcriptional regulation of CCEs in response to an intoxication could be particular for different kinds of xenobiotics.

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Fig 1. Effect of EEO treatment on the transcription of differentially expressed genes related to xenobiotic detoxification families and major facilitator superfamily.

Heatmap was prepared using pheatmap that calculated z-score for each gene and plotted by means of a color scale, in which blue/red represent lowest/highest expression. Genes are identified by their VectorBase ID. Dendrogram was plotted with hierarchical clustering among samples and genes based on Euclidean distances and Ward method for clustering. C: control samples. T: treated samples.

https://doi.org/10.1371/journal.pntd.0009587.g001

Besides those genes directly involved in detoxification processes, we identified DEGs belonging to families that could have an important role in the defense against toxic xenobiotics, such as fatty acid synthesis related genes and cuticular proteins (S2 Table). The involvement of cuticular proteins and synthesis of cuticular lipids has been related to insecticide resistance as much in mosquitoes as in other species [14,15]. Overexpression of many genes encoding for cuticular proteins has been reported in Ae. aegypti larvae in response to propoxur, imidacloprid and permethrin, whereas the transcription of genes involved in lipid metabolism was detected in response to propoxur and imidacloprid [4]. Interestingly, we found that 19 (7%) of the DEGs encode proteins related to lipid metabolism (17) or cuticle (2) (S2 Table). From DEGs involved in lipid metabolism, 12 are overexpressed and 5 under-expressed in larvae exposed to EEO (S2 Table). Most of the overexpressed genes have a role in fatty acids biosynthesis (i.e. elongase, Acetyl-CoA synthase, AMP dependent ligases), whereas 3 out of 5 of the under-expressed are lipases, suggesting a net augment in fatty acid concentration. Besides, two cuticular-related genes were found differentially expressed; one cuticular protein was overexpressed, and a chitinase was under-expressed. Altogether, the results indicate that the cuticle reinforcement could be a mechanism of Ae. aegypti larvae to cope with a sustained exposure to EEO.

Hexamerins are abundant proteins in hemolymph, which have storage and transport roles, and were previously associated with resistance to insecticides in mosquitoes, in particular to pyrethroids [11,51], even though the exposure to synthetic insecticides or other xenobiotics did not modulate the expression of hexamerins [4]. In agreement, none of the transcripts modulated by the exposure to EEO belongs to the hexamerin family (S2 Table). These results suggest that, in spite of the probable role of hexamerins in insecticide resistance, their basal expression is not affected during the detoxificant response.

Major facilitator superfamily (MFS) members are membrane transports phylogenetically related [54]. They are involved in the transport of solutes through cell membranes. Opposite to ABC transporters that use ATP, MFS transport substrates in favor of an electrochemical gradient [54]. A role of MFS in the transport of toxic substances in bacteria and fungi has been demonstrated [55–57]. In our work, we found that 10 DEGs were MFS transporters. Besides, other 3 genes are solute transporters independent of ATP, but not presenting a MFS domain (AAEL005353, AAEL007458, and AAEL013109; S2 Table). Eight of these transporter genes were downregulated by the EEO treatment (Fig 1 and S2 Table; FDR<0.05). The tendency to downregulate the expression of genes involved in the transport of solutes in favor of their electrochemical gradient could help in the preservation of cells from intoxication. Interestingly, MFS expression modulation has been previously observed in arthropods exposed to toxins derived from plants [58,59], but not in Ae. aegypti larvae exposed to pollutants or synthetic insecticides [4].

Other DEGs encode mostly metabolic enzymes such as alcohol-dehydrogenases, hydrolases, peptidases, kinases/phosphatases, oxidases, reductases and transferases (S2 Table). Finally, the expression of one caspase, one odorant binding protein and two proteins induced by juvenile hormone were detected as upregulated (S2 Table).

Consistently with the DEG set, the GO-enrichment analysis revealed that biological processes related to biosynthesis and metabolism of alpha-amino acids, as well as sensory perception, were significantly enriched in the treated samples (Fig 2A). Considering the GO molecular function terms (Fig 2B), several activities were affected after the treatment: 1) detoxification, including iron, FAD and heme binding together with several metabolic enzymes (peptidase, reductases and monooxygenases); 2) odorant binding and sensory perception; 3) signaling and transduction and 4) structural constituents of the cuticle. The results of the KEGG enrichment analysis were similar to those generated by GO-term analysis, reinforcing the effect of the EEO at the metabolic level. For the complete list of KEGG enriched terms (FDR<0.05) see S3 Table).

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Fig 2. GO enrichment analysis of the RNA-Seq data.

A. Biological process. B. Molecular Function. Functional gene sets were defined using the Gene Ontology (GO) annotations of the Aedes aegypti genome (AaegL5.3 version) in VectorBase. Gene Score Resampling (GSR) method was applied and the reported IDs correspond to the significantly enriched GO-terms (FDR<0.05).

https://doi.org/10.1371/journal.pntd.0009587.g002

Families potentially involved in detoxification

A hypergeometric test was performed in order to evaluate the enrichment of HSP20, HSP70, CYP, UGT, GST, ABC-transporters and CSP gene families in the DEG set. All these gene families were over-represented and significantl enriched in this set (p<0.001; Fig 3).

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Fig 3. Hypergeometric test results for enrichment analysis of gene families potentially involved in detoxification.

Horizontal bars indicate total gene numbers. Black bars indicate the number of genes whose expression was unaltered after EEO treatment and gray bars indicate the number of genes differentially expressed in each gene family. P-values for each family are indicated. HSP20: Heat shock proteins 20; HSP70: Heat shock proteins 70; UDP: UDP glycosyltransferases; GST: Glutathione transferases; CSP: Chemosensory proteins; ABC: ABC transporters; CYP: Cytochromes P450.

https://doi.org/10.1371/journal.pntd.0009587.g003

Heat shock proteins

HSP expression is induced by a variety of stresses in insects, including heat and insecticide treatment [26,60]. We found that 12 DEGs are HSPs, all of them over-expressed in mosquito larvae treated with EEO. Nine of the HSP upregulated genes belong to the HSP20 (α-crystalline) class, whereas 3 belong to the HSP70 class. Also, one HSP20-like_chaperone (AAEL013314), which does not contain the HSP20 domain, was upregulated (FDR<0.05) (S2 Table).

The involvement of HSP20 in insecticide resistance in An. gambiae has been recently demonstrated [11]. Furthermore, the expression of HSP20 members is modulated in An. gambiae in response to treatment with a pyrethroid [11], but previous transcriptomic analyses did not report changes in the expression of HSPs in Ae. aegypti larvae in response to treatment with synthetic insecticides nor in resistant populations [4,51]. We detected 17 putative HSP20 sequences in Ae. aegyptitranscript dataset; all these transcripts are located in chromosome 2, conforming a gene cluster that spans <200,000 bp in the genome. Besides those 9 genes that reached FDR<0.05, most of the HSP20 transcripts tended to be upregulated in insects treated with EEO (see heat maps in S3 Fig), indicating that this family could be involved in the response to EEO in Ae. aegypti. Also, the disposition of HSP20 genes in a cluster and its expression pattern could indicate a transcriptional co-regulation. In agreement, transcriptional co-regulation of HSP20 members was suggested in An. gambiae [11].

The expression of most HSP70, which are overexpressed in response to heat stress in mosquitoes [61], tended to be upregulated during exposure to EEO, 3 of them reached statistical significance (FDR<0.05) (S3B Fig).

Cytochromes P450

From the 7 overexpressed CYPs, 3 belong to the CYP6 family, 2 to CYP9 family and 2 to CYP4 family (S2 Table). From these, AAEL001807/CYP9M9 was also observed overexpressed in response to both imidacloprid and propoxur [4], and in larvae from populations resistant to these two insecticides [51]. This could point to a role of CYP9M9 in the detoxificant response to a wide range of toxics.

The 3 CYP6 that were overexpressed with exposition to EEO (AAEL009124/CYP6N12, AAEL009127/CYP6M11 and AAEL009128/CYP6M6) were located in the same gene cluster in the genome, suggesting a co-regulation and/or a comparable role. Besides, one underexpressed CYP (AAEL014619/CYP9J22) belongs to CYP9 family, which was previously associated with detoxification in insects, particularly in Ae. aegypti [51,62]. Several CYP genes tended to be either underexpressed or overexpressed after exposure to EEO and most of these genes were classified in CYP9 or CYP6 families. Treatment with permethrin, which belongs to the pyrethroid family widely used as an adulticide for mosquitoes [2], did not modify the expression of CYPs in Ae. aegypti larvae [4], and the expression of three CYP6 and one CYP12 was underexpressed in larvae from a permethrin-resistant population [51]. Besides, in the larvae resistant to imidacloprid and in those resistant to propoxur, the expression of two genes encoding CYP6 (AAEL009131 and AAEL 014893) and one gene encoding a CYP9 (AAEL001807) were found upregulated [51]. Altogether, the results suggest that the members of CYP family involved in detoxification could differ with dependence of the toxic stimulus applied to Ae. aegypti larvae.

UDP-glycosyltransferases

Enzymes belonging to UDP-glycosyltransferase (UGT) superfamily catalyze glucosidation and transfer of glycosyl from UDP-glycosyl donor to a lipophilic molecule [13]. This superfamily has been involved in insect resistance to both plant allelochemicals and insecticides [63,64]. In our study, we found 6 overexpressed UGT encoding genes (S2 Table). Besides several other members of this superfamily tended to be overexpressed in the larvae treated with EEO (S3D Fig). From this gene family, AAEL003102 was overexpressed in untreated larvae from resistant populations to imidacloprid and propoxur [51], even though the transcription of this gene was not affected by treatment with EEO.

Chemosensory proteins

Forty-two transcripts encoding CSPs were detected in Ae. aegypti genome, 5 out of these genes were overexpressed in Ae. aegypti larvae treated with EEO (S3G Fig; FDR<0.05; AAEL001967, AAEL001999, AAEL002021, AAEL002026, and AAEL002028).

All of the CSP members found in Ae. aegypti genome presented the hallmarks of this protein family: the signal peptide, a pattern of 4 cysteines and 6 α-helical segments (Fig 4A). However, we found that the members of the CSP family have been annotated in the Ae. aegypti genome as “protein serine/threonine kinase” (www.vectobase.org). CSPs in Ae. aegypti genome are disposed in two clusters, one in chromosome 2 containing 34 genes, and a second one in chromosome 3 counting 7 genes (Fig 4A). The only CSP encoding gene outside of these clusters was AAEL012383, located in chromosome 3. The organization in clusters is a usual finding in CSP families [68], suggesting that large families are originated by gene duplications. The conservation of clusters in different species suggests an evolutive pressure to maintain this organization in the genome [68], and could point to a coordinated regulation of the gene expression. Interestingly, all the overexpressed CSPs are located in the chromosome 2 cluster. Other CSP encoded in this cluster (AAEL002024) also tended to be overexpressed (FDR = 0.077).

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Fig 4.

A. Multiple sequence alignment of chemosensory proteins identified in Ae. aegypti genome. Predicted signal peptide sequences are indicated underlined and with a light-gray shadow. Conserved cysteine residues are boxed. For clarity reasons, only the conserved region of the larger sequences (AAEL001985 and AAEL019813) are shown. In the last line of each alignment, an asterisk indicates a fully conserved residue, a colon indicates a conservative substitution with strongly similar properties, and a period indicates a semiconserved substitution with weakly similar properties. Black bar in the left indicates sequences located in chromosome 2 cluster; gray bar indicates sequences located in chromosome 3 cluster. B. Phylogenetic analysis of Ae. aegypti and An. gambiae chemosensory proteins constructed on IQ-Tree using the LG+I+G4 (Best-fit model according to Bayesian Information Criterion) as a model of amino-acid substitution and it is based on 1,000 replicates. The branch support values were estimated using the approximate Likelihood Ratio Test based on the Shimodaira-Hasegawa (aLRT-SH) procedure.

https://doi.org/10.1371/journal.pntd.0009587.g004

A bibliographic survey revealed that the expression of members of the CSP family (previously misinterpreted in the genome automatic annotation as “protein serine/threonine kinase”) has been also significantly modulated in Ae. aegypti populations resistant to different kinds of insecticides [50]. This kind of modulation also occurred in response to xenobiotics or synthetic insecticides[5]. We observed that the CSPs modulated both by xenobiotics and in resistant populations of Ae. aegypti were all located in the chromosome 2 cluster, reinforcing the evidence that suggests an involvement of this group of CSPs in detoxification. Besides, AAEL002028 was significantly upregulated both with EEO and with imidacloprid, whereas AAEL002021 was upregulated both with EEO and propoxur. Several m embers of the CSP family were found to be differentially regulated in Ae. aegypti larvae resistant to synthetic insecticides [51], but none of them overlapped with those CSPs overexpressed during the treatment with EEO. Furthermore, treatments with imidacloprid or propoxur modulated the expression of different sets of CSPs (6 transcripts were modulated only by imidacloprid; 6 transcripts were modulated only by propoxur and 3 transcripts overlapped in both responses) [4]. These results, in agreement to previous observations in An. gambiae [22], point to a specificity of different CSPs for dealing with different xenobiotics.

The number of CSP paralogues detected in Ae. aegypti (42) is much bigger than the reported for most mosquito genomes (8 in An. gambiae, 27 in Culex quinquefasciatus) [42,68,69], even though Ae. albopictus genome encodes 83 CSPs [69]. We performed a phylogenetic analysis to assign possible orthologues between Ae. aegypti and An. gambiae CSPs (Fig 4B). We observed orthologies between AAEL001963 and AgamCSP1, AAEL001985 and AgamCSP3, AAEL012383 and AgamCSP4, AAEL019813 and AgamCSP5. Besides, SAP1, SAP2 and SAP3 were grouped with most of the CSP proteins located in chromosome 2 cluster in the Ae. aegypti genome. Hence, the phylogenetic analysis indicates that CSPs clustered in chromosome 2 are orthologues of SAP proteins from An. gambiae, which were demonstrated to have a role in detoxification [22]. In parallel to the pattern of expression modulation with xenobiotics (see above), a detoxificant role of Ae. aegypti CSP clustered in chromosome 2 can be suggested as a suitable hypothesis for further physiological experiments.

The expansion observed in the CSP complement and the probable involvement of this gene family in detoxification suggest that Ae. aegypti could be especially versatile to deal with different toxic molecules and, as a consequence, to develop resistance to a wide spectrum of insecticides.

The main components of E. camaldulensis EO are 1,8-cineole, p-cymene and β-phellandrene [26]. According to previous results, p-cymene seems to be centrally implicated in toxicity against Ae. aegypti larvae [26]. Bioassays on larvae exposed to 40 parts per million of pure p-cymene exhibited a mortality rate of 100%, and there exists a significant correlation between larval mortality and the concentration of p-cymene in the EEO [26]. To further study the probable role of CSPs in EEO detoxification, we modeled the docking of p-cymene in all the members of the CSP family in Ae. aegypti. We found a favorable energy for docking in all the Ae. aegypti CSPs, going from -7.6 (AAEL001993) to -4.7 (AAEL002041) kcal/mol (Table 1). Two of the CSPs that are significantly overexpressed after EEO exposition (AAEL001999 and AAEL002021) also presented a highly favorable interaction energy (-7.3 kcal/mol for both). Fig 5 reports the best mode of AAEL001999 interaction with p-cymene found, where the aromatic/hydrophobic amino acids Phe42, Phe46, Leu49, Leu62, Leu66, Ile88, Ile89, Leu92, Trp100 and Leu103 are evidenced as defining the binding cavity. This cavity is bigger than the ligand and can hold it in two orientations (not shown). Given the size and hydrophobic nature of the cavity it could also bind other hydrophobic molecules of similar size. Furthermore, the nature of the residues surrounding the cavity in different CSPs could confer specificity/preference for different ligands. Altogether, binding analysis suggests a role of CSPs as detoxicants, with particular involvement of AAEL001999 and AAEL002021 for detoxification of p-cymene, a central component of EEOs. The overexpression of these genes in the presence of p-cymene could help to sequester the toxic molecules, reducing in this way its effects on physiology and survival of larvae.

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Fig 5. Docking of p-cymene inside a CSP.

A. Best binding mode of p-cymene (yellow) within AAEL001999 protein (ribbon representation). B. Zoom of the binding cavity, highlighting as solid colored sticks all amino acid sidechains within 5 Å of the ligand.

https://doi.org/10.1371/journal.pntd.0009587.g005

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Table 1. Docking of Ae. aegypti CSPs with p-cymene.

https://doi.org/10.1371/journal.pntd.0009587.t001