Introduction                                                                               

Induction refers to the process in which a chemical stimulus enhances the activity of a detoxification system by production of additional enzymes (Tieriere, 1984). Experiments on induction often involves exposing animals reared in the laboratory to xenobiotic inducers and using biochemical or molecular procedures to demonstrate evidence for involvement of a particular detoxification pathway (Ding et al., 2005; Lumjuan et al., 2011; Wen-Wu zhou et al., 2011; Bamigdele et al., 2017). Three large enzyme families, monooxygenases, carboxylesterases and glutathione s –transferases have been associated with detoxification of xenobiotic compounds in biological systems (Hemingway et al., 2004; Ranson et al., 2011; Mashimaya et al., 2014). In insects, these enzyme families are important in metabolism of insecticides and constitute the basis for metabolic resistance mechanisms (Ranson and Hemingway, 2000; Che-mendoza et al., 2009). The insect specific delta and epsilon glutathione s – transferases in particular have been implicated in detoxification of organophosphates (Chiang and Sun, 1993; Huang et al., 1998), pyrethroids (Vontas et al., 2001; Rakotondranaivo et al., 2018) and organochlorines (Prapanthadra et al., 1995; Ranson et al., 2001) insecticides. Evidence for the role of GSTe2 in resistance to permethrin and DDT has been shown in various species of Anopheline mosquitoes including, An. cracens (Wongtrakul et al., 2010), An. funestus (Gregory et al., 2011), An. gambiae (Ortelli et al., 2003; Patience et al., 2016) and An. arabiensis (Yayo et al., 2018a). Also, some GSTs from the Sigma, Delta and Epsilon classes including GSTe2 have been shown to possess peroxidase activity and protect against oxidative stress due to xenobiotic compounds (Singh et al., 2001; Vontas et al., 2002; Giardano et al., 2007; Lumjuan et al., 2011).

Exposure to xenobiotic compounds either in the environment or laboratory have caused overexpression of GST genes in various species of mosquitoes including Aedes aegypti, An. stephensi, and An. gambiae (Ding et al., 2005; David et al., 2010; Gregory et al., 2011). Aedes aegypti larvae exposed to the herbicides glyphosate, the carcinogene benzo(a)pyrene and the synthetic insecticides propoxur and permethrin were found to induce differentially four GSTs including GSTx (David et al., 2010). A two – fold change in expression of GSTe2 was reported in the pupae of pyrethroid resistant An. stephensi compared to the susceptible laboratory strains (Gregory et al., 2011). Ding et al. (2005) found significant increases in transcripts of GSTe1 and GSTe2 in larvae of An. gambiae exposed to H₂O_2.. Fang (2012), has reviewed in detail studies on inducible GSTs.

An. arabiensis is the most closely related ecologically and taxonomically to An. gambiae s.s but is genetically distinct and tends to be more adapted to human environment habitually containing xenobiotic contaminants (Muller et al., 2007; Matowo et al., 2014; Cassone et al., 2014). An. arabiensis occurs sympatrically with An. gambiae and is one of the dominant malaria vectors in tropical Africa (Coetzee et al., 2000; Coetzee et al., 2013). Previously, we isolated and characterized eight Epsilon GSTe genes from An. arabiensis MAT strain and demonstrated differential expression of three of GSTe genes in developmental stages. We reported overexpression of the GSTe1 and GSTe2 in DDT selected lines compared to susceptible strains (Yayo et al., 2018a;b). To investigate the inducible expression of GSTe2, we exposed the fourth instar larvae of two laboratory strains of An. arabiensis to sub-lethal concentrations of H₂O₂, Permethrin and DDT and monitored the expression of the gene by quantitative PCR.

1 Materials and Methods

1.1 Mosquito strains and chemicals

Mosquitoes from the parental lines of MAT and KGB laboratory strains of An. arabiensis (Patton) were used in the induction experiments. The parental lines of these strains were selected for the induction studies because both strains have been maintained in the insectary for a least 3 (MAT) and 2 (KGB) years without any insecticidal selection pressure.  When tested for susceptibility to DDT, the adult mosquitoes from the KGB parental line were more tolerant to DDT compared to the MAT strain (Yayo et al., 2018a). However, susceptibility of the larvae of these mosquitoes to permethrin and DDT was not known. The choice of the chemicals for the induction work was based on the need to simulate the type of xenobiotic contaminants in the mosquito’s natural breeding habitats (Muller et al., 2007; Cassone et al., 2014; Kamdem et al., 2014). Hence, H₂O₂, permethrin and DDT were chosen as representative xenobiotic compounds which An. arabiensis may typically encounter in the environment. Previously, sub-lethal doses of 3mM H₂O₂ (Ding et al., 2005) and 0.02 mg/L of permethrin (David JP, personal communication) were used to treat larvae from ZAN/U and RSP strains of An. gambiae, therefore these dosages were adopted in this experiment.  In order to determine the sub-lethal dose of DDT, 3 batches each of 25 larvae were exposed, each for one hour, to the following concentrations of DDT: 0.5 mg/L, 0.05 mg/L and 0.005 mg/L. Larval mortalities were determined after 24 hours holding.  The dose 0.05 mg/L, which gave a mortality of >20%, was adopted and used for subsequent induction.

1.2 Recruitment of larvae for induction

Rearing activities were harmonised to minimise any compounding effect, possibly due to excess food or overcrowding. Egg batches laid by the adult females were collected from filter paper placed in the egg-cup. The filter paper containing eggs was carefully cut to split the egg-batch into two halves. Each half paper was placed in an egg pot containing 10~20 mL of water to enable the eggs to hatch to 1^st instar larvae. About 25~30 1^st instar larvae were transferred to shallow trays measuring 28×20 cm with a depth of 6.5 cm, which were only half filled with distilled water. All larvae from the two strains were fed on Tetramin fish food flakes, using grinded flakes for the first instars. Sufficient food was added each time without causing the water to go scummy. Distilled water was squirted into the larval trays on a daily basis to aerate the water and excess food removed using a pipette. The water was changed if there were any signs of cloudiness, along with sluggish swimming behaviour of the larvae. Extreme precaution was taken to avoid contamination between the strains and lines of the An. arabiensis. Pipettes and egg pots were colour-coded for each strain. This scrupulous method of larval rearing was used to generate all the 4^th instar larvae which were used for the induction experiments.

1.3 Exposure of 4^th instar larvae to H₂O₂, Permethrin and DDT

A total of six hundred 4^th instar larvae each taken from MAT and KGB strains were exposed to H₂O₂, permethin and DDT in a series of separate experiments. In each experiment, a group of 40~50 larvae were immersed for one hour in either 3 mM H₂O₂, 0.02 mg/L permethrin or 0.05 mg/L DDT. The larvae were then transferred to sterile water to recover. Unexposed larvae of the same life-stage from both strains were used as control. Larval samples in batches of 10 were collected in triplicate at one, two and 24 hour time points post exposure.

1.4 Extraction of RNA and cDNA synthesis

RNA extraction and cDNA synthesis were performed as previously described (Yayo et al., 2018a). Total RNA was extracted from each batch of the larval samples obtained after exposure at each of the time points mentioned above. The samples were homogenized in 400 µL of TRI reagent (sigma) and centrifuged at 12,000 g for 10 min at 4°C. The supernatant was transferred to a DEPC- treated centrifuge tube, homogenized for 15 secs with chloroform and was then incubated at 4°C after which the upper aqueous phase containing RNA was transferred to a fresh tube. The RNA was precipitated by adding 200 µL of molecular grade isopropanol and incubated at room temperature for 10 min. After centrifugation at 12,000 g for 10 min at 4°C, the pellet was air- dried and re-suspended in 26 µL of nuclease-free water. The RNA was treated with RQI DNase (Promega) to remove contaminating genomic DNA. cDNA was synthesized from total RNA using superscript TM111RNase H- reverse transcriptase (Invitrogen) and an oligo (dT) adapter primer (5´-GACTCGAGTCGACATCGA (dT) 17 – 3´). One microgram of RNA was mixed with 1 µg of the primer and heated to 65°C for five minutes to dissociate the secondary conformation. The reaction mixture was first chilled on ice for 1 minute and then pre-warmed to 50°C for 2 minutes after addition of 8 µL of 5X first strand buffer (250 mM Tris-HC1 pH 8.3, 375 mM KCL, 15 mM MgCl₂), 2 µL of 10 mM of each dNTP and 2 µL of 0.1 M DTT. One half microlitre of Superscript TM111Rnase H- reverse transcriptase was added and the reaction was incubated at 50°C for 90 minutes. The reaction was then heated to 70^oc for 15 minutes to inactivate the enzyme. First strand cDNA samples were stored at -20°C.

1.5 Plasmid construction and qPCR

Primer sequences specific for An. arabiensis GSTe2 described previously (Yayo et al., 2018b) were used to amplify the full-length cDNA from the KGB and MAT strains. PCR reactions contained 1.5 mM MgCl₂, 0.2 mM of each dNTPs, and 0.5 µm of each primer, 1 X reaction buffer (Qiagen) and 1.25 units of Taq DNA polymerase (Hotstar) in a final volume of 25 µL. The cycling parameters were 95°C for 15 minutes, followed by 30 cycles of 95°C for 30 seconds, 50°C for 30 seconds and 72°C for 30 seconds and a final extension of 72°C for 10 minutes. The amplification was conducted using PCR machine PTC-200 (MJ Research). GSTe2 standard plasmid was constructed by insertion PCR products of cDNA from the MAT and KGB strains into the PGEM-T Easy Vector (Promega) according to manufacture’s instructions. The ribosomal protein gene SP7 (accession no. AY 380336) (Salazar et al., 1993), used as an internal control plasmid, was amplified using forward GCA CGT CGT GTT CAT TGC CG  and reverse GAA CAT TAA CGT CAC GGC CAG TCA primers and ligated into the PGEM-T Easy Vector. The concentrations of the plasmids were determined using an ND- 1000 spectrophotometer (Nanodrop Technologies).

1.6 Quantitative PCR

The GSTe2 and SP7 standard plasmids were diluted serially to concentrations ranging from 1 ng/µL to 1 fg/µL. To compare the abundance of GSTe2 transcript in the larvae exposed to each of the three xenobiotic inducers and control samples from the MAT and KGB strains, quantitative real time PCR was performed. In each experiment, duplicate reactions were set up for each of the seven standard plasmids and the cDNA samples. Quantitative PCR was performed in 15 µL reactions with a quantitative PCR Kit (Qiagen), which composed of 1 µL of DNA template, 1 × Master Mix, 0.2 mM of each primer. Details of the primer sequences are given in Table 1. The PCR conditions were 95°C for 15min, followed by 35 cycles of 94°C for 20 s, 54°C for 25 s and 72°C for 30 s with the fluorescence read at 82°C. Plasmid DNA standards and negative controls were included in the same plate, for each experiment. Three biological replicate samples from each strain and batches of samples of larvae exposed to each of the xenobiotic were used as templates. A sample was analysed in duplicate in each experiment and results were averaged from three separate experiments. The number of copies of GSTe2 and SP7 was calculated by measuring the incorporation of the SYBR Green 1 into the PCR products using the DNA Engine Opticon (MJ Research). The fluorescence was plotted in relation to the number of cycles and the crossing line was produced to obtain the standard concentration related to the cycle number. A straight line standard curve was obtained by plotting the cycle number against the logarithmic value of the standard concentration. The ribosomal gene SP7 was used to normalize for variation in total cDNA concentration.

Table 1 Oligo nucleotide Primer Sequences used in quantitative real time PCR

The normalised copy number of GSTe2 was thus determined for each batch of larval samples obtained at one, two and twenty-four hours post exposure to DDT, Permethrin and H₂O₂ in the separate experiments. One-way analysis of variance was used initially to test for differences between treatments and the mosquito strains. Significant results were in investigated in detail using the Turkey significant difference multiple comparison test (Winter, 1962). Statistical significance was set at the conventional 5% level for all tests.

2 Results

2.1 Inducible expression of Epsilon GSTe2 by DDT, permethrin and hydrogen peroxide.

2.1.1 DDT Induced expression

The geometric means levels of GSTe2 expressed at 1, 2 and 24 hours post-exposure to 0.05mg/l DDT for one hour and control are shown individually in Figure 1. At 1 hour post-exposure to DDT, the mean expression of GSTe2 was raised relative to the control larvae by just over one half (57%) MAT strain (p = 0.025) but was more than doubled (108% increase) in the KGB strain (p < 0.001). At 2-hours, the mean GSTe2 expression was now 60% lower than in the control larvae in the MAT strain (p < 0.001); in the KGB strain, however the mean gene expression remained numerically higher than control (by 29% i.e. by just over one quarter), this difference was not statistically significant (p = 0.146). At 24 hours , mean gene expression was 79% lower than in the control larvae in the MAT strain (p < 0.001); in the KGB strain, mean gene expression was lower than control by just 16% - this difference was not statistically significant (p = 0.445).

Figure 1 Epsilon GSTe2 expression in control and larvae exposed to 0.05mg/L DDT for one hour – geometric means and 95% confidence intervals. Statistical differences in geometric means of the observations were calculated using Turkey honestly significant multiple tests

2.1.2 Permethrin induced expression

The expression levels of GSTe2 in the control and larvae exposed to permethrin are shown in (Figure 2). The mean GSTe2 expression peaked at 2 hours post-exposure, increasing by 132% in the MAT strain and by 146% in the KGB strain (p < 0.001 for both strains).The gene expression was 17% and 15.6% lower in the MAT and the KGB strains respectively at 5-hours post-exposure; this difference relative to the control strain was statistically not significant for the KGB strain (p = 0.360) and for the MAT strain (p = 0.350). At 24 hours post-exposure, mean GSTe2 expression was 9% lower than control in the MAT strain but 16% higher in the KGB strain; however, neither difference was statistically significant (p = 0.846 for MAT and p = 0.700 for KGB).

Figure 2 Induction of Epsilon GSTe2 expression at 0, 2, 5 and 24 hours post-exposure to 0.02mg/L permethrin for one hour – geometric means and 95% confidence intervals

2.1.3 Hydrogen peroxide induced expression

The geometric means and their 95% confidence intervals for observed levels of GSTe2 expressed at 0 and 1 hour post-exposure to H₂O₂ are shown individually in (Figure 3).

Figure 3 Induction of Epsilon GSTe2 expression at 1 hours post-exposure to H2O2 – geometric means and 95% confidence intervals

Hydrogen peroxide produced increase in the expression of GSTe2 at 1 hour post-exposure of (24%) in the MAT strain (24%) and (28%) in the KGB strain (p = 0.029 and p = 0.005 respectively). The relative expression of GSTe2 induced by DDT, Permethrin and Hydrogen peroxide at different time points after exposure compared to control in larvae from the MAT and KGB strains to each of the xenobiotic is summarized on Table 2 and Table 3 respectively.

Table 2 (Geometric) Mean GSTe2 expression levels (with 95% confidence limits) in MAT strains

Table 3 (Geometric) Mean GSTe2 expression levels (with 95% confidence limits) in KGB strains

3 Discussion

We investigated patterns of expression of the epsilon GSTe2 gene induced by DDT, permethrin and hydrogen peroxide in the fourth instar larvae of the parental (non-selected) lines of KGB and MAT laboratory strains of An. arabiensis. The expression of GSTe2 increased significantly, 2.1 fold in the KGB and 1.6 fold in MAT strains one hour post-exposure to DDT. Similar patterns of GSTe2 expression in response to exposure to DDT have been reported in Aedes aegypti (Lumjuan et al., 2005), An. stephensi (Sanil et al., 2014), An. funestus Djouaka et al. (2011) and An. gambiae (Ding et al., 2005). Increase in levels of GSTe2 expression has been consistently associated to DDT resistance and AgGSTe2 have been shown to possess DDT dehydrochlorinase activity (Ranson et al., 2001; Pontes et al., 2016). Recent investigations on structure and function relationship and simulations based on conformational studies indicate higher efficiency and binding affinity for DDT of isoforms of GSTe2 in An. gambiae (Wang et al., 2008; Pontes et al., 2016; Kettman et al., 2011). It remains to be shown if the GSTe2 in An. arabiensis can metabolize DDT and posses the structural characteristics for the function. The expression of GSTe2 decreased after two hours by approximately 42% in the DDT-treated compared to control larvae in MAT strain, but in the KGB strain, the expression remained induced though not significantly (p = 0.146) (Figure 1). This observed difference in the induction pattern of GSTe2 suggests higher intrinsic levels of GST activity in the KGB compared to the MAT strain. Adults from the colony of the parental line of the KGB were more resistant to DDT, resistant ratio 1.5 compared to those from the MAT strain (Yayo et al., 2018a). DDT was a more potent inducer of GSTe2 than permethrin and H₂O₂ (Table 2; Table 3). The stronger induction of GSTe2 expression by DDT compared to permethrin and the H₂O₂ may be explained on the basis of substrate specificities (Aravindan et al., 2014). Permethrin and hydrogen peroxide consecutively increased the expression of GSTe2 by 40% and 30% in the KGB strain and in the MAT strain, permethrin increased the expression of the gene by 2.3 fold and hydrogen peroxide by 1.2 fold. Samra et al. (2012) have reported a two-fold increase in GSTe2 expression in Culex pipiense exposed to permethrin. In An. funestus permethrin has caused an increase of 2.55 in the expression of GSTe2 (Djuaka et al., 2011). We cannot explain the reason for the low expression recorded in the KGB strain. A border line significant induction of GSTe2 expression was observed (1.2-fold) in MAT and (1.4-fold) in KGB strains following exposure to 3 mM H₂O₂ (Table 2). This result is very similar to that reported for H₂O₂ induced expression of GSTe2 at one hour time point in An. gambiae (Ding et al., 2005). Exposure to DDT, permethrin and hydrogen peroxide can induce oxidative stress in the mosquitoes and the GSTs may contribute to antioxidant defence by direct peroxidase activity or conjugation of the toxic aldehyde 4-hydroxyl nonenol (Vontas et al., 2002; Sawicki et al., 2003; Abdullahi et al., 2004). The GSTe2 in Aedes aegypti and An. gambiae have been shown to possess peroxidase activity suggesting its potential role in defense against oxidative stress induced by xenoboitics (Ortelli et al., 2003; Lumjuan et al., 2005). Ding et al. (2005) attributed the induced expression of GSTe2 by H₂O₂ in An. gambiae to the presence of transcription factors responsive to oxidative stress in the promoter region of the gene this indicates the need for identifying the orthologous putative promoter elements in An arabiensis GSTe2.

The induction of GSTe2 expression by different chemical compounds may suggest its involvement in the detoxification of xenobiotic compounds found in the natural environment of the mosquito larvae. Elevated tolerance of mosquito larvae to chemical insecticides after their exposure to common herbicides has been demonstrated and this indicated the potential of pesticide residues in larval habitats to confer mosquito larvae pre-adaptive advantage to develop insecticide resistance (David et al. 2010). In this respect, a comparative study of molecular mechanisms controlling the induction of individual GST genes between An. arabiensis and An. gambiae may help to understand the ecological differences identified between these important malaria vectors (Coluzzi et al., 1979).

4 Conclusion

GSTe2 has been induced differentially by DDT, permethrin and hydrogen peroxide in laboratory strains of An. arabiensis. There is need to conduct further investigations on biochemical and molecular evidence for involvement of AaGSTe2 in metabolism of xenobiotics.

Authors’ contributions

Yayo A.M. and Hemingway J. draft the experimental design and conduct the Lab work; Yayo A.M., Hemingway J and Ado A. conduct the literature search; Yayo A.M., Hemingway J., Ado A. and Safiyanu M. review and edit the manuscript; Safiyanu M. Sambo F.I., Abubakar A. conduct the statistical analysis; All the respective authors have read the manuscript and approved its submission to Molecular Entomology Journal.

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