Investigating the role of glutathione and glutathione biosynthetic genes in the adaptation of Anopheles gambiae to insecticides

Malaria remains a serious public health challenge in the tropical world, with 584,000 deaths globally in 2013, of which 90% occurred in Africa, and mostly in pregnant women and children under the age of five. Anopheles gambiae (An. gambiae) is the principal malaria vector in Africa, where vector control measures involve the use of insecticides in the forms of long-lasting insecticide-treated nets (LLINs) and indoor residual spraying (IRS). The development of insecticides resistance mitigates these approaches. Glutathione (GSH) is widely distributed among all living organisms, and is associated with detoxification pathways, especially the Glutathione S-transferases (GSTs). Its direct involvement and relevance in insecticide resistance in An. gambiae has not been determined. Thus, this work examines the contribution of GSH, its biosynthetic genes (GCLM, GCLC) and their possible transcriptional regulator Nrf2 in insecticide resistance in An. gambiae sampled from agricultural setting (areas of intensive agriculture) and residential setting (domestic area). Bioinformatics analysis, W.H.O. adult susceptibility bioassays and molecular techniques were employed to investigate. Total RNA was first isolated from the adults An. gambiae mosquitoes raised from agricultural and residential field-caught larvae which had been either challenged or unchallenged with insecticides. Semi-quantitative RT-PCR using gel image densitometry was used to determine the expression levels of GCLM, GCLC genes and Nrf2. Bioinformatics’ results established the presence of putative AGAP010259 (AhR) and AGAP005300 (Nf2e1) transcription factor binding sites in An. gambiae GCLC and GCLM promoters in silico. An. gambiae s.l. studied here were highly resistant to DDT and permethrin but less resistant to bendiocarb. Both knockdown resistance (kdr) mutation variants L1014S and L1014F that confers resistance to pyrethroid insecticides were identified in both An. coluzzii and An. arabiensis sampled from northern Nigeria. The L1014F was much associated with An. coluzzii. A significant positive correlation (P=0.04) between the frequency of the L1014F point mutation and resistance to DDT and permethrin was observed. However, a weak or non-significant correlation (P=0.772) between the frequency of the L1014S point mutation and resistance was also found. L1014S and L1014F mutations co-occurred in both agricultural and residential settings with high frequencies. However, the frequencies of the two mutations were greater in the agricultural settings than in the residential settings. The levels of total, reduced and oxidized GSH were significantly higher in mosquitoes from agricultural sites than those from residential sites. Increased oxidized GSH levels appears to correlate with higher DDT resistance. The expression levels of GCLM, GCLC and Nrf2 were also significantly up-regulated in adults An. gambiae raised from agricultural and residential field-caught larvae when challenged with insecticide. However, there was higher constitutive expression of GCLM, GCLC and Nrf2 in mosquitoes from agricultural setting. The increased expression levels of these genes and also GSH levels in this population suggest their roles in the response and adaptation of An. gambiae to insecticide challenges. There exists the feasibility of using GSH status in An. gambiae to monitor adaptation and resistance to insecticides.

Development of gene expression-based biomarkers of exposure to metals and pesticides in the freshwater amphipod Hyalella azteca

Ecological risk assessment (ERA) is a framework for monitoring risks of exposure and adverse effects of environmental stressors to populations or communities of interest. One tool of ERA is the biomarker, which is a characteristic of an organism that reliably indicates exposure to or effects of a stressor like chemical pollution. Traditional biomarkers which rely on characteristics at the tissue level and higher often detect only acute exposures to stressors. Sensitive molecular biomarkers may detect lower stressor levels than traditional biomarkers, which helps inform risk mitigation and restoration efforts before populations and communities are irreversibly affected. In this study I developed gene expression-based molecular biomarkers of exposure to metals and insecticides in the model toxicological freshwater amphipod Hyalella azteca. My goals were to not only create sensitive molecular biomarkers for these chemicals, but also to show the utility and versatility of H. azteca in molecular studies for toxicology and risk assessment. I sequenced and assembled the H. azteca transcriptome to identify reference and stress-response gene transcripts suitable for expression monitoring. I exposed H. azteca to sub-lethal concentrations of metals (cadmium and copper) and insecticides (DDT, permethrin, and imidacloprid). Reference genes used to create normalization factors were determined for each exposure using the programs BestKeeper, GeNorm, and NormFinder. Both metals increased expression of a nuclear transcription factor (Cnc), an ABC transporter (Mrp4), and a heat shock protein (Hsp90), giving evidence of general metal exposure signature. Cadmium uniquely increased expression of a DNA repair protein (Rad51) and increased Mrp4 expression more than copper (7-fold increase compared to 2-fold increase). Together these may be unique biomarkers distinguishing cadmium and copper exposures. DDT increased expression of Hsp90, Mrp4, and the immune response gene Lgbp. Permethrin increased expression of a cytochrome P450 (Cyp2j2) and decreased expression of the immune response gene Lectin-1. Imidacloprid did not affect gene expression. Unique biomarkers were seen for DDT and permethrin, but the genes studied were not sensitive enough to detect imidacloprid at the levels used here. I demonstrated that gene expression in H. azteca detects specific chemical exposures at sub-lethal concentrations, making expression monitoring using this amphipod a useful and sensitive biomarker for risk assessment of chemical exposure.

The preparation of a certified reference material of polar pesticides in freeze-dried water (CRM 606)

The preparation of a certified reference material of polar pesticides in freeze-dried water is described. The pesticides selected were atrazine, simazine, carbaryl, propanil, linuron, fenamiphos and permethrin which were added to 6000 litres of tap water at 50–80 μg · L–1 (200–320 μg · L–1 for permethrin) level in presence of NaCl (2.5 g · L–1) prior lyophilization. After the freeze-drying process the residue was rehomogenized, filled into amber glass bottles and stored at –20 °C, +4 °C and +20 °C. All pesticides were determined by HPLC/diode array detector, except permethrin which was determined by GC/ECD. The results obtained for atrazine, simazine, carbaryl, propanil, linuron and fenamiphos showed no within- or between-bottle inhomogeneity, however the material was non-homogeneous for permethrin and therefore this was withdrawn from further studies. With respect to the stability for over one year, all pesticides were stable at –20 °C. At +4 °C all pesticides were stable for at least 9 months and at +20 °C the stability was demonstrated only during the first month of storage. The content (mass fractions) of atrazine, simazine, carbaryl, propanil and linuron in freeze-dried water (CRM 606) was certified by an interlaboratory testing and a certification campaign.