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. 2025 Jun 5;26(1):565.
doi: 10.1186/s12864-025-11755-y.

Molecular markers of reduced behavioral sensitivity to transfluthrin in Anopheles gambiae s.s. from Western Kenya

Stephen Okeyo  1   2 , Dieunel Derilus  3 , Lucy Mackenzie Impoinvil  3 , Nsa Dada  4   5 , Diana Omoke  6 , Helga Saizonou  4 , Cynthia Awuor Odhiambo  6 , Nicola Mulder  7 , Gerald Juma  8 , Benard W Kulohoma  8   9 , John E Gimnig  3 , Luc S Djogbénou  4   10   11 , Audrey Lenhart  3 , Eric Ochomo  12   13
Affiliations

Molecular markers of reduced behavioral sensitivity to transfluthrin in Anopheles gambiae s.s. from Western Kenya

Stephen Okeyo et al. BMC Genomics. .

Abstract

Background: The emergence and spread of insecticide resistance in malaria vectors threatens vector control efforts. The use of spatial repellent products (SR) containing volatile insecticides such as transfluthrin offer a promising complementary strategy to current vector control tools. Here, we employed whole transcriptome analysis to investigate the molecular mechanisms underlying reduced behavioral sensitivity to transfluthrin in two pyrethroid-resistant populations of Anopheles gambiae s.s. Using a high-throughput screening system (HITSS), we evaluated 600 mosquitoes across three populations (Bungoma field population, the insecticide-resistant Pimperena lab strain, and the susceptible Kisumu lab strain), categorizing them as responders or non-responders based on their SR avoidance behavior. Non-responders exhibited significantly reduced repellency (spatial activity index < 0.1) at standard transfluthrin concentrations (0.0025 μg/ml).

Results: RNA sequencing of pooled samples (n = 10 mosquitoes per pool, three replicates per condition) revealed distinct transcriptional profiles between responders and non-responders. The cytochrome P450 gene CYP12F12 showed significant overexpression (FC = 36.6389, p < 0.001) in Bungoma non-responders, suggesting its potential role in transfluthrin metabolism. Additionally, we observed population-specific distributions of voltage-gated sodium channel mutations, with fixation of kdr L995F in Pimperena non-responders and elevated frequency (80-100%) of kdr L995S in Bungoma non-responders.

Conclusions: These findings provide the first molecular evidence linking both metabolic and target-site mechanisms to reduced behavioral sensitivity to transfluthrin in malaria vectors. The co-occurrence of CYP12F12 overexpression and kdr mutations suggests multiple resistance mechanisms may affect spatial repellent efficacy, highlighting the need for resistance monitoring in spatial repellent deployment strategies.

Keywords: An. gambiae s.s; kdr mutation; CYP12 F12; Insecticide resistance; RNA-seq; Spatial repellents; Transfluthrin.

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Conflict of interest statement

Declarations. Ethics approval and consent to participate: This study received ethical approval from the Kenya Medical Research Institute—Scientific and involve human subjects (SERU 3309). The study protocol was reviewed by the US Centers for Disease Control and Prevention (CDC). Informed verbal consent was obtained from all participating household heads prior to adult mosquito collection. No monetary compensation was provided to participants. However, as part of a larger ongoing study with existing administrative approvals, these households were prioritized for the distribution of long-lasting insecticidal nets (LLINs) by the National Malaria Control Program (NMCP). At the time of sampling, National Commission for Science, Technology & Innovation (NACOSTI) approval was not a mandatory requirement. Consent for publication: This manuscript has been published with the permission of the Kenya Medical Research Institute (KEMRI) Director General. Competing interests: SO is at the time of this submission employed at Vestergaard Sarl, a company that manufacture’s vector control products, LLINs, not evaluated in this study. All other the authors declared that they have no competing interests.

Figures

Fig. 1
Fig. 1
Normalization of RNA-Seq library data. A Distribution of raw reads from the sequencing; RNA-Seq libraries are colored based on technical replicates. B TMM normalization to resolve technical variation between samples in the experiment. Raw counts were log transformed to further reduce differences in dimensions of counts before the normalization step
Fig. 2
Fig. 2
Volcano plots showing gene expression profiles in An. gambiae s.s. for the comparisons: (A) Bungoma non-responders versus Bungoma responders (BN vs BR); (B) Bungoma non-responders versus Bungoma unexposed (BN vs BU); (C) Bungoma non-responders versus Kisumu responders (BN-KR); (D) Pimperena non-responders versus Pimperena responders (PN vs PR); (E) Pimperena non-responders versus Pimperena unexposed (PN vs PU); (F) Pimperena non-responders versus Kisumu responders (PN vs KR). Red, green, black, pink, and grey points on volcano plots indicate gene families with major role in metabolic resistance to insecticides: cytochrome P450 monooxygenases (CYP, blue); glutathione-S transferases (GST, black); carboxylesterases (COE, red); cuticular proteins (CP, green); Salivary gland proteins (SGP, pink); Odorant binding proteins (OBP, orange); Odorant receptors (OR, purple). In each plot, genes over-expressed in the population are > 0 on the x-axis while genes under-expressed in the population are < 0 on the x-axis. Vertical dotted line indicates twofold expression differences, and the horizontal dotted line indicates an adjusted p-value of 0.05
Fig. 3
Fig. 3
Venn diagrams summarizing the numbers of differentially expressed (DE) genes between non-responders (N), unexposed (U) and responders (R) with a corrected/adjusted p-value < 0.01. A DE genes in the Bungoma population non-responder samples; B DE genes in the Pimperena population non-responder samples
Fig. 4
Fig. 4
Venn diagram summarizing the numbers of differentially expressed (DE) genes between non-responders (N) and responders (R) with a corrected/adjusted p-value < 0.05 in the Bungoma and Pimperena populations
Fig. 5
Fig. 5
Heatmaps summarizing differentially expressed genes, showing log2 fold-change and p < 0.05 values on a red-blue scale (red = overexpressed; blue = down expressed). A Cytochrome P450 monooxygenases family. B Olfactory genes. C Cuticular proteins. D Salivary gland proteins. E Glutathione-S-transferases and carboxylesterases
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