Malaria remains a major cause of death and illness in Africa, with the greatest burden among children under 5 years of age. The most effective means of preventing the disease are insecticidal vector control agents that target adult Anopheles mosquitoes. As a result of the widespread use of these interventions, resistance to the most commonly used classes of insecticides is now widespread across Africa. Understanding the underlying mechanisms that lead to this phenotype is essential both to track the spread of resistance and to develop new tools to overcome it.
In this study, we compared the microbiome composition of insecticide-resistant Anopheles gambiae, Anopheles cruzi, and Anopheles arabiensis populations from Burkina Faso with insecticide-sensitive populations, also from Ethiopia.
We found no differences in the microbiota composition between insecticide-resistant and insecticide-susceptible populations in Burkina Faso. This result was confirmed by laboratory studies of colonies from two Burkina Faso countries. In contrast, in Anopheles arabiensis mosquitoes from Ethiopia, clear differences in the microbiota composition were observed between those that died and those that survived insecticide exposure. To further investigate the resistance of this Anopheles arabiensis population, we performed RNA sequencing and found differential expression of detoxification genes associated with insecticide resistance, as well as changes in respiratory, metabolic, and synaptic ion channels.
Our results suggest that in some cases the microbiota may contribute to the development of insecticide resistance, in addition to transcriptome changes.
Although resistance is often described as a genetic component of the Anopheles vector, recent studies have shown that the microbiome changes in response to insecticide exposure, suggesting a role for these organisms in resistance. Indeed, studies of Anopheles gambiae mosquito vectors in South and Central America have shown significant changes in the epidermal microbiome following exposure to pyrethroids, as well as changes in the overall microbiome following exposure to organophosphates . In Africa, pyrethroid resistance has been associated with shifts in the composition of the microbiota in Cameroon, Kenya, and Côte d’Ivoire, while laboratory-adapted Anopheles gambiae have shown shifts in their microbiota following selection for pyrethroid resistance . Furthermore, experimental treatment with antibiotics and the addition of known bacteria in laboratory-colonized Anopheles arabiensis mosquitoes showed increased tolerance to pyrethroids . Together, these data suggest that insecticide resistance may be linked to the mosquito microbiome and that this aspect of insecticide resistance could be exploited for disease vector control.
In this study, we used 16S sequencing to determine whether the microbiota of laboratory-colonized and field-collected mosquitoes in West and East Africa differed between those that survived and those that died after exposure to the pyrethroid deltamethrin. In the context of insecticide resistance, comparing microbiota from different regions of Africa with different species and levels of resistance can help understand regional influences on microbial communities. Laboratory colonies were from Burkina Faso and reared in two different European laboratories (An. coluzzii in Germany and An. arabiensis in the United Kingdom), mosquitoes from Burkina Faso represented all three species of the An. gambiae species complex, and mosquitoes from Ethiopia represented An. arabiensis. Here, we show that Anopheles arabiensis from Ethiopia had distinct microbiota signatures in live and dead mosquitoes, while Anopheles arabiensis from Burkina Faso and two laboratories did not. The aim of this study is to further investigate insecticide resistance. We performed RNA sequencing on the Anopheles arabiensis populations and found that genes associated with insecticide resistance were upregulated, while respiration-related genes were generally altered. Integration of these data with a second population from Ethiopia identified key detoxification genes in the region. Further comparison with Anopheles arabiensis from Burkina Faso revealed significant differences in transcriptome profiles, but still identified four key detoxification genes that were overexpressed across Africa.
Live and dead mosquitoes of each species from each region were then sequenced using 16S sequencing and relative abundances were calculated. No differences in alpha diversity were observed, indicating no differences in operational taxonomic unit (OTU) richness; however, beta diversity varied significantly between countries, and interaction terms for country and live/dead status (PANOVA = 0.001 and 0.008, respectively) indicated that diversity existed between these factors. No differences in beta variance were observed between countries, indicating similar variances between groups. The Bray-Curtis multivariate scaling plot (Figure 2A) showed that samples were largely segregated by location, but there were some notable exceptions. Several samples from the An. arabiensis community and one sample from the An. coluzzii community overlapped with a sample from Burkina Faso, whereas one sample from the An. arabiensis samples from Burkina Faso overlapped with the An. arabiensis community sample, which may indicate that the original microbiota was randomly maintained over many generations and across multiple regions. The Burkina Faso samples were not clearly segregated by species; this lack of segregation was expected since individuals were subsequently pooled despite originating from different larval environments. Indeed, studies have shown that sharing an ecological niche during the aquatic stage can significantly influence the composition of the microbiota [50]. Interestingly, while the Burkina Faso mosquito samples and communities showed no differences in mosquito survival or mortality after insecticide exposure, the Ethiopian samples were clearly segregated, suggesting that the microbiota composition in these Anopheles samples is associated with insecticide resistance. The samples were collected from the same location, which may explain the stronger association.
Resistance to pyrethroid insecticides is a complex phenotype, and while changes in metabolism and targets are relatively well studied, changes in the microbiota are only beginning to be explored. In this study, we show that changes in the microbiota may be more important in certain populations; we further characterize insecticide resistance in Anopheles arabiensis from Bahir Dar and show changes in known resistance-associated transcripts, as well as significant changes in respiration-related genes that were also evident in a previous RNA-seq study of Anopheles arabiensis populations from Ethiopia . Together, these results suggest that insecticide resistance in these mosquitoes may depend on a combination of genetic and non-genetic factors, likely because symbiotic relationships with indigenous bacteria may complement insecticide degradation in populations with lower levels of resistance.
Recent studies have linked increased respiration to insecticide resistance , consistent with the enriched ontology terms in Bahir Dar RNAseq and the integrated Ethiopian data obtained here; again suggesting that resistance results in increased respiration, either as a cause or consequence of this phenotype. If these changes lead to differences in reactive oxygen and nitrogen species potential, as previously suggested , this could impact vector competence and microbial colonization through differential bacterial resistance to ROS scavenging by long-term commensal bacteria .
The data presented here provide evidence that the microbiota can influence insecticide resistance in certain environments. We also demonstrated that An. arabiensis mosquitoes in Ethiopia display similar transcriptome alterations conferring insecticide resistance; however, the number of genes corresponding to those in Burkina Faso is small. Several caveats remain regarding the conclusions reached here and in other studies. First, a causal relationship between pyrethroid survival and the microbiota needs to be demonstrated using metabolomic studies or microbiota transplantation. In addition, validation of key candidates in multiple populations from different regions needs to be demonstrated. Finally, combining transcriptome data with microbiota data through targeted post-transplantation studies will provide more detailed information on whether the microbiota directly influences the mosquito transcriptome with respect to pyrethroid resistance. However, taken together, our data suggest that resistance is both local and transnational, highlighting the need to test new insecticide products in multiple regions.
Post time: Mar-24-2025