Identification of methylmercury tolerance gene candidates in Drosophila

Abstract

Methylmercury (MeHg) is a ubiquitous environmental contaminant that preferentially targets the developing nervous system. Variable outcomes of prenatal MeHg exposure within a population point to a genetic component that regulates MeHg toxicity. We therefore sought to identify fundamental MeHg tolerance genes using the Drosophila model for genetic and molecular dissection of a MeHg tolerance trait. We observe autosomal dominance in a MeHg tolerance trait (development on MeHg food) in both wild-derived and laboratory-selected MeHg-tolerant strains of flies. We performed whole-genome transcript profiling of larval brains of tolerant (laboratory selected) and nontolerant (control) strains in the presence and absence of MeHg stress. Pairwise transcriptome comparisons of four conditions (+/-selection and +/-MeHg) identified a "down-down-up" expression signature, whereby MeHg alone and selection alone resulted in a greater number of downregulated transcripts, and the combination of selection + MeHg resulted in a greater number of upregulated transcripts. Functional annotation cluster analyses showed enrichment for monooxygenases/oxidoreductases, which include cytochrome P450 (CYP) family members. Among the 10 CYPs upregulated with selection + MeHg in tolerant strains, CYP6g1, previously identified as the dichlorodiphenyl trichloroethane resistance allele in flies, was the most highly expressed and responsive to MeHg. Among all the genes, Turandot A (TotA), an immune pathway-regulated humoral response gene, showed the greatest upregulation with selection + MeHg. Neural-specific transgenic overexpression of TotA enhanced MeHg tolerance during pupal development. Identification of TotA and CYP genes as MeHg tolerance genes is an inroad to investigating the conserved function of immune signaling and phase I metabolism pathways in MeHg toxicity and tolerance in higher organisms.

FIG. 1.

Effect of MeHg exposure on eclosion of Canton S flies. Eclosion of Canton S flies was determined with first instar larvae placed on indicated concentration of MeHg in food (bars = SD of three trials, n = 150). Fifty percent eclosion occurred at ∼7.5μM MeHg. Assays performed with MeHg exposure during the larval to adult stages of the life cycle (inset).

FIG. 2.

MeHg tolerance of wild strains derived from various geographic regions. Forty-seven isolines from the indicated geographical region, plus Canton S and Oregon R strains, were assayed. Eclosion assays were performed with first instar larvae on 7.5μM MeHg food (bars = SD of three trials, n = 150).

FIG. 3.

Transmission of the tolerance trait in the progeny of wild strains. Eclosion assays were performed as in Fig. 1 using the indicated tolerant (RIH12) and non-tolerant (Sorell15 and Sor15) parental strains of wild flies. Larvae assayed were derived from the parent lines or the crosses indicated in the inset.

FIG. 4.

Transmission of the tolerance trait in the progeny of laboratory-selected strains. Eclosion assays were performed as in Fig. 1 using the indicated tolerant (H20) and nontolerant (H0) parental strains of laboratory-selected flies (see Fig. 5). Larvae assayed were derived from the parent lines or the crosses indicated in the inset.

FIG. 5.

Experimental design to study the transcript profiling of MeHg tolerance. Replicate selection and control strains were derived by rearing a starting population on either MeHg or control food (see “Materials and Methods” section) to generate six new lines (E0, F0, and H0 nontolerant; E20, F20, and H20 tolerant). Treatments for transcript profiling were done by feeding larvae of each of these lines (± 15μM MeHg, see “Materials and Methods” section). The nontolerant and tolerant groupings are referred to as S0 and S20, respectively.

FIG. 6.

Global changes in transcripts with selection and MeHg exposure. Scatter plots illustrating the pairwise comparisons of the entire probe set intensity data using the criteria of ≥ 1.5-fold change (black dots, 1.5-fold boundary marked by solid lines) and p ≤ 0.05. (Axes values are log₂ of probe intensity.) (A) MeHg exposure to S0 strain leads to differential expression of 362 transcripts with the majority (90%) downregulated. (B) Basal expression in the S20 versus S0 strains shows a change in 246 transcripts and majority of them (72%) are downregulated in the S20 because of selection. (C) MeHg exposure in S20 strains shows differential expression of 249 transcripts with 44% upregulated. (D) S20 versus S0 upon MeHg exposure shows differential expression of 233 transcripts with 74% showing upregulation. (Values in the parentheses are differential expression with ≥ twofold change and p value ≤ 0.05.).

FIG. 7.

Overlap of transcript changes in S20 and S0 strains in the presence and absence of MeHg. (A) Comparison of S20 treated with MeHg and S0 treated with MeHg. Of the 110 genes upregulated in S20 upon MeHg exposure, 52 are otherwise downregulated in S0 treated with MeHg. (B) Comparison of S20 treated with MeHg and S20 versus S0 (no MeHg). Of the 110 genes that are upregulated in S20 upon MeHg stress, 63 are otherwise downregulated by selection.

FIG. 8.

Overall changes in CYP transcripts. Scatter plots illustrating the pairwise comparisons of the CYP family probe set intensity data using the criteria of ≥ 1.5-fold change (boundary marked by solid lines) and a p value of ≤ 0.05. (Axes values are log₂ of probe intensity.) (A) MeHg exposure to S0 strains leads to differential expression of eight transcripts with all being downregulated. (B) Basal expression in the S20 versus S0 strains shows a change in four transcripts and majority of them are downregulated. (C) MeHg exposure in S20 strains shows differential expression of seven transcripts with a majority being upregulated. (D) S20 versus S0 both with MeHg exposure shows differential expression of 10 transcripts with all being upregulated. CYP6g1 (red circle) is the most highly expressed and the strongest responder to selection + MeHg exposure. (Values in the parentheses are total number of CYP probes with ≥ 1.5-fold change irrespective of p value.).

FIG. 9.

Validation of microarray data by qPCR. Microarray probe intensity (log₂) of Cyp6g1 (Ai–iii) and TotA (Bi–iii) with (+) and without (−) MeHg exposure (15μM). Relative transcript levels (fold change relative to −selection/−MeHg) of Cyp6g1 (Aiv–vi) and TotA (Aiv–vi) determined by qPCR using the same RNA samples used for the microarray.

FIG. 10.

Relative transcript levels of TotA and Cyp6g1 in wild-derived tolerant and nontolerant strains. The fold change of TotA and CYP6g1 expression determined by qPCR upon exposure to 15μM MeHg in larval brains is shown. Five tolerant (A) and nontolerant (B) strains were analyzed and presented. (No-change level indicated by dotted line.).

FIG. 11.

Transgenic expression of TotA induces MeHg tolerance. (A) Relative levels of TotA expression (by qPCR) under Gal4-driven expression in the nervous system (EG4 > TotA) or whole larvae (c754G4 > TotA) compared with controls (EG4 > 1118 and c754G4 > 1118, respectively). (B–C) Developmental tolerance to MeHg determined in TotA overexpressing larvae. Tolerance was determined by eclosion (dashed/dotted lines) or completion of development to the dark pupal stage (solid lines). TotA expression was driven in the nervous system (EG4 > TotA, B) or in the fat bodies (c754G4 > TotA, C) and development compared with controls (EG4 > 1118 and c754G4 > 1118, respectively) (bars = SD of three determinations, n = 150).