Non-Coding RNAs Potentially Involved in Pyrethroid Resistance of Anopheles funestus Population in Western Kenya

Abstract

Backgrounds: The resurgence of Anopheles funestus, a dominant vector of human malaria in western Kenya was partly attributed to insecticide resistance. However, evidence on the molecular basis of pyrethroid resistance in western Kenya is limited. Noncoding RNAs (ncRNAs) form a vast class of RNAs that do not code for proteins and are ubiquitous in the insect genome. Here, we demonstrated that multiple ncRNAs could play a potential role in An. funestusresistance to pyrethroid in western Kenya.

Materials and methods: Anopheles funestus mosquitoes were sampled by aspiration methods in Bungoma, Teso, Siaya, Port Victoria and Kombewa in western Kenya. The F1 progenies were exposed to deltamethrin (0.05%), permethrin (0.75%), DDT (4%) and pirimiphos-methyl (0.25%) following WHO test guidelines. A synergist assay using piperonyl butoxide (PBO) (4%) was conducted to determine cytochrome P450s' role in pyrethroid resistance. RNA-seq was conducted on a combined pool of specimens that were resistant and unexposed, and the results were compared with those of the FANG susceptible strain. This approach aimed to uncover the molecular mechanisms underlying pyrethroid resistance.

Results: Pyrethroid resistance was observed in all the sites with an average mortality rate of 57.6%. Port Victoria had the highest level of resistance to permethrin (MR=53%) and deltamethrin (MR=11%) pyrethroids. Teso had the lowest level of resistance to permethrin (MR=70%) and deltamethrin (MR=87%). Resistance to DDT was observed only in Kombewa (MR=89%) and Port Victoria (MR=85%). A full susceptibility to P-methyl (0.25%) was observed in all the sites. PBO synergist assay revealed high susceptibility (>98%) to the pyrethroids in all the sites except for Port Victoria (MR=96%, n=100). Whole transcriptomic analysis showed that most of the gene families associated with pyrethroid resistance comprised non-coding RNAs (67%), followed by imipenemase (10%), cytochrome P450s (6%), cuticular proteins (5%), olfactory proteins (4%), glutathione S-transferases (3%), UDP-glycosyltransferases (2%), ATP-binding cassettes (2%) and carboxylesterases(1%).

Conclusions: This study unveils the molecular basis of insecticide resistance in An. funestus in western Kenya, highlighting for the first time the potential role of non-coding RNAs in pyrethroid resistance. Targeting non-coding RNAs for intervention development could help in insecticide resistance management.

Keywords: Anopheles funestus; DDT; PBO; RNA-seq; insecticide resistance; non-coding RNAs; pyrethroid; western Kenya.

Figure 1. Map of study sites where mosquitoes were sampled in western Kenya.

The software ArcGIS Pro 2.6 was used to create the map. Map sources: USGS, ESRI, and CGIAR (www.esri.com)

Figure 2. Volcano plot indicating upregulation and downregulation for resistant vs susceptible (A), resistant vs unexposed (B) and unexposed (control) vs susceptible (C).

The X-axis indicates the log2 fold-change- positive and negative values are up and down-regulated respectively relative to the susceptible group in A and C. The Y-axis indicates −log₁₀ of the adjusted P-value (FDR) (−log₁₀FDR values >200 for A, > 9 for B and > 80 for C). In each volcano plot, genes that are overexpressed in the population are >0 on the x-axis. P-values of < 0.05 are indicated by the horizontal line, while 2-fold expression differences are indicated by vertical dotted lines.

Figure 3

A principal component analysis showing the gene expression pattern of the sample groups relative to the susceptible group.

Figure 4

Heatmap indicating the expression of genes in the sample groups relative to the susceptible group.

Figure 5. Venn diagram comparing upregulated and downregulated genes between-group comparisons.

A indicates upregulated genes between the groups and B indicates downregulated genes between groups. R-S: field-resistant population that survived pyrethroid exposure vs susceptible colony, R-C: field-resistant population that survived pyrethroid exposure vs unexposed (control) field population and C-S: unexposed (control) field population vs susceptible colony.

Figure 6. Pie chart showing the proportion of gene family involving pyrethroid resistance.

IMPs: Imipenemase, CYPs: Cytochrome P450s, CPs: cuticular proteins, OPs: olfactory proteins, GSTs: Glutathione S-transferases, UGTs: UDP-glycosyltransferases, ABCs:ATP-binding cassettes, COEs: carboxylesterases and ncRNA: non-coding RNA

Figure 7. Gene Ontology (GO) enrichment analysis of the differentially expressed genes.

The x-axis indicates the gene count/number of genes while the y-axis indicates the enriched terms. The colour is used to distinguish at different levels.