Mechanisms of transcriptional regulation in Anopheles gambiae revealed by allele specific expression

Abstract

Malaria control relies on insecticides targeting the mosquito vector, but this is increasingly compromised by insecticide resistance, which can be achieved by elevated expression of detoxifying enzymes that metabolize the insecticide. In diploid organisms, gene expression is regulated both in cis, by regulatory sequences on the same chromosome, and by trans acting factors, affecting both alleles equally. Differing levels of transcription can be caused by mutations in cis-regulatory modules (CRM), but few of these have been identified in mosquitoes. We crossed bendiocarb resistant and susceptible Anopheles gambiae strains to identify cis-regulated genes that might be responsible for the resistant phenotype using RNAseq, and cis-regulatory module sequences controlling gene expression in insecticide resistance relevant tissues were predicted using machine learning. We found 115 genes showing allele specific expression in hybrids of insecticide susceptible and resistant strains, suggesting cis regulation is an important mechanism of gene expression regulation in Anopheles gambiae. The genes showing allele specific expression included a higher proportion of Anopheles specific genes on average younger than genes those with balanced allelic expression.

Keywords: Allele; cis-regulation; insecticide; resistance; transcript.

Figure 1:. Crossing, DNA and RNA extraction schema

a. 13 Kisumu females (blue) were crossed to 13 Nagongera males (red). Females mate only once. Following mating, genomic DNA from Nagongera males was extracted and sequenced. b. In the reciprocal cross, 13 Nagongera females (red) were crossed to 13 Kisumu males (blue). Following mating, genomic DNA was extracted and sequenced from Kisumu males. c. Individual mated Kisumu females were transferred to laying cups. Following egg laying, genomic DNA was extracted and sequenced from three of these d. Individual mated Nagongera females were transferred to laying cups. Following egg laying, genomic DNA was extracted and sequenced from three of these. e. The F1 progeny (purple) from the three Kisumu mothers sequenced at step c. were raised to adulthood. Genomic DNA was extracted and sequenced from individual males f. The F1 progeny (purple) from the three Nagongera mothers sequenced at step d. were raised to adulthood. Genomic DNA was extracted and sequenced from individual males g. and h. Female F1 from each of the six mothers were raised to adulthood. RNA was extracted and sequenced from a pool of ten F1 females three to five days after eclosion.

Figure 2:. Reciprocal crosses show similar overall gene expression

a. Volcano plot of log2 fold change against -log10 P value comparing gene expression in the F1 progeny of reciprocal crosses between Kisumu and Nagongera strains. Blue points indicate genes downregulated in progeny of Kisumu mothers compared to progeny of Nagongera mothers, orange points indicate genes upregulated in progeny of Kisumu mothers compared to progeny of Nagongera mothers. b. Volcano plot of log2 fold change against -log10 P value comparing gene expression between F1 progeny of Nagongera and Kisumu with the Kisumu parental strain. Blue points indicate genes downregulated in the Kisumu compared to cross progeny, orange points indicate genes upregulated in Kisumu compared to cross progeny.

Figure 3:. ASE in progeny of crosses between strains

Plot of total read count against ASE at each SNP. Cross and source for the SNPs used to map and count reads are indicated at the top of each plot. Cross K6 is shown with ASE inferred using SNPs from both the parents of cross K4 and for the initially assumed parents of cross K6.

Figure 4:. Intersection of genes showing ASE between crosses

UpSet plot with the number of genes showing ASE for each cross and the intersection of these genes between crosses. Only the first 28 sets of overlaps are shown.

Figure 5:. Ages of genes showing ASE or not showing ASE

bar plots comparing the age of genes which showed ASE or did not in the progeny of six crosses between Nagongera and Kisumu strains, using the Wagner parsimony method. The cross name is indicated at the top left of each plot, Fisher’s exact test P values for the difference in fraction of genes in each age between genes showing ASE (red bars) and genes not showing ASE (black bars) are displayed above the bars; *: 0.001

Figure 6:. proportion of genes showing ASE versus genes with sufficient SNPs to detect ASE along each chromosome arm

Bar plot of the proportion of genes showing allele specific expression/ total genes containing suitable SNPs to detect ASE per chromosome arm. Colours indicate the different crosses. X chromosomal SNPs were not available for crosses B1 and B3