Gut Microbiota and Insecticide Resistance in the Mediterranean Fruit Fly (Ceratitis capitata)
Source: Charaabi, K., Hamdene, H., Djobbi, W.
et al. Assessing gut microbiota diversity and functional potential in resistant and susceptible strains of the mediterranean fruit fly.
Sci Rep 15, 33456 (2025).
https://doi.org/10.1038/s41598-025-01534-wDates: Received - 06 November 2024 | Accepted - 06 May 2025 | Published - 29 September 2025
Executive Summary
This briefing document synthesizes findings from a study investigating the link between gut microbiota and insecticide resistance in the Mediterranean fruit fly (Ceratitis capitata), a destructive agricultural pest. The research reveals a strong correlation between resistance to common insecticides (malathion, dimethoate, and spinosad) and significant alterations in the composition and functional potential of the fly's gut bacterial community.
Resistant strains of the medfly, developed over 36 generations of insecticide exposure, exhibit significantly lower microbial diversity compared to their susceptible counterparts. This reduction in diversity is accompanied by a profound shift in the gut's bacterial landscape. Specifically, the phylum Bacillota and the genera Enterococcus and Klebsiella are substantially enriched in resistant flies. Conversely, the dominant phylum Pseudomonadota and the genera Serratia and Buttiauxella are sharply reduced.
Functional analysis predicts that the gut microbiota of resistant flies possess enhanced metabolic capabilities for xenobiotic biodegradation. These enriched pathways are associated with the breakdown of various toxic environmental chemicals, suggesting a direct or indirect role in insecticide detoxification. The findings indicate that symbiont-mediated resistance is likely a key mechanism in the medfly, driven by the synergistic effect of multiple bacterial species rather than a single microbe. This research opens new avenues for pest management strategies that could target the gut microbiome to mitigate insecticide resistance.
Background and Research Objectives
The Mediterranean fruit fly (Ceratitis capitata), or medfly, is a highly polyphagous pest that infests over 300 plant species, causing billions of dollars in annual economic losses worldwide. These losses stem from reduced agricultural production, costly control measures, and restricted market access. While methods like the Sterile Insect Technique (SIT) are used, the predominant control practice remains the application of chemical insecticides.
The widespread and excessive use of insecticides has led to the development of significant resistance in medfly populations, undermining control efforts. While resistance is often linked to genetic traits in the insect, such as increased enzyme activity, recent evidence from other species suggests that symbiotic gut microorganisms can play a crucial role. These bacteria may contribute to resistance by directly metabolizing toxic substances or by modulating the host's detoxification gene expression.
Despite extensive research on the medfly's gut microbiota in relation to its fitness and SIT applications, the connection to insecticide resistance has remained largely unexplored. This study aimed to address this gap by investigating the potential association between the medfly gut microbiota and insecticide resistance. The primary objectives were to:
- Characterize and compare the gut microbiota community structure between insecticide-susceptible (IS) and insecticide-resistant (IR) strains of the medfly.
- Identify specific bacterial taxa that correlate with resistance phenotypes.
- Predict the functional differences between the microbiomes of susceptible and resistant strains.
Experimental Design and Methodology
To achieve its objectives, the study employed a controlled laboratory selection process and advanced sequencing techniques.
- Strain Development: Three insecticide-resistant (IR) strains were developed from a susceptible parent strain (IS) originally from Egypt (Egypt II). For 36 successive generations, populations were exposed to increasing concentrations of one of three insecticides: malathion (ML-SEL strain), dimethoate (Dm-SEL strain), or spinosad (Sp-SEL strain). The selection pressure was calibrated to achieve 50-70% mortality in each generation.
- Resistance Confirmation: Toxicological bioassays were conducted on the 36th generation of each IR strain and the IS strain. The lethal concentration required to kill 50% of the population (LC50) was calculated to quantify the level of resistance. The results confirmed a significant increase in tolerance in the selected strains.
| Strain | Insecticide | LC50 (ppm) | Resistance Ratio (RR) vs. IS Strain
| IS | Malathion | 18.8 | -
| ML-SEL (G36) | Malathion | 1872.2 | 99.23-fold
| IS | Dimethoate | 0.85 | -
| Dm-SEL (G36) | Dimethoate | 215.79 | 252.68-fold
| IS | Spinosad | 0.55 | -
| Sp-SEL (G36) | Spinosad | 133.79 | 241.49-fold
- Microbiota Analysis: Gut tissues were dissected from adult flies of all four strains. Genomic DNA was extracted, and the V3-V4 region of the 16S rRNA gene was amplified and sequenced. Bioinformatic analyses, including Principal Coordinate Analysis (PCoA), Non-metric Multidimensional Scaling (NMDS), and Linear discriminant analysis Effect Size (LEfSe), were used to analyze microbial diversity, structure, and to identify potential biomarkers. Functional potential was predicted using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database.
Key Findings: Shifts in Gut Microbiota Composition
The study revealed dramatic and statistically significant differences between the gut microbiomes of insecticide-susceptible and resistant medflies.
Reduced Microbial Diversity in Resistant Strains
A primary finding was that all three IR strains exhibited significantly lower bacterial richness and diversity compared to the IS parent strain (p < 0.05). This suggests that insecticide exposure acts as a strong selective pressure, favoring the growth of a specialized subset of bacteria that can tolerate or metabolize the toxic compounds. This "selection-cumulation effect" leads to an enrichment of resistance-associated bacteria at the expense of overall diversity.
Altered Bacterial Abundance at Phylum and Genus Levels
The composition of the gut microbiota was fundamentally altered in the resistant strains.
- Phylum-Level Shifts: While the phylum Pseudomonadota was dominant in all strains, its relative abundance decreased significantly in the IR strains (from 91.03% in IS to 70.85-75.27% in IR). Conversely, the abundance of the phylum Bacillota increased dramatically (from 8.94% in IS to 24.70-28.90% in IR).
- Genus-Level Shifts: The most pronounced changes occurred at the genus level, pointing to specific bacteria potentially involved in resistance.
| Bacterial Genus | Relative Abundance in IS Strain | Change in IR Strains | Specific Details
| Serratia | 22.28% | Sharp Decrease | Dropped to 0.5-0.9% in all IR strains.
| Buttiauxella | 10.29% | Sharp Decrease | Dropped to 0.2-0.89% in all IR strains.
| Enterococcus | 5.62% | Significant Increase | Rose to 24.43-28.37% in all IR strains.
| Klebsiella | 2.57% | Significant Increase | Rose to 8.46-40.92% across all IR strains.
| Cronobacter | 4.95% | Varied | Decreased in ML-SEL and Dm-SEL, but was identified as an enriched biomarker in the Sp-SEL strain.
| Providencia | Not specified | Enriched in Sp-SEL | Identified as an enriched biomarker in the Sp-SEL strain.
Distinct Microbial Community Structures
Statistical analyses confirmed that the gut microbial communities of the resistant strains were structurally distinct from the susceptible strain. Both PCoA and NMDS analyses showed a clear and significant separation between the IS and IR groups (p < 0.001). Furthermore, LEfSe analysis identified key bacterial taxa as statistically significant biomarkers associated with resistance to each specific insecticide. Enterococcus and Klebsiella were predominant biomarkers for malathion and dimethoate resistance, while Enterococcus and Cronobacter were key for spinosad resistance.
Key Findings: Predicted Functional Differences
Enhanced Xenobiotic Degradation Capabilities
To understand the functional implications of these compositional shifts, the study predicted the metabolic pathways present in each microbial community using the KEGG database. The results indicated that the microbiota of the IR strains were significantly enriched in functions related to xenobiotics biodegradation and metabolism.
- The ML-SEL (malathion) and Dm-SEL (dimethoate) strains showed enrichment in pathways for the degradation of various environmental chemicals, including toluene, xylene, fluorobenzoate, and polycyclic aromatic hydrocarbons.
- The Sp-SEL (spinosad) strain exhibited significant enrichment in pathways for nitrotoluene, naphthalene, and chloroalkane degradation, as well as drug metabolism.
While these pathways are not specific to the insecticides tested, their enrichment strongly suggests that the bacterial communities in resistant flies have an enhanced general capacity to break down and detoxify harmful exogenous compounds, which likely contributes to their insecticide tolerance.
Discussion and Conclusions
The Role of Symbiont-Mediated Resistance
This study provides the first report of the medfly gut microbiota's association with insecticide resistance. The findings strongly support the hypothesis that insecticide exposure reshapes the gut microbiome, favoring bacteria that contribute to the host's survival. The sharp decline in genera like Serratia and the proliferation of Enterococcus and Klebsiella is not random but rather a clear adaptive response to chemical stress. Previous research in other insects has implicated Enterococcus and Klebsiella in degrading various insecticides, corroborating their potential role in the medfly.
The evidence suggests that resistance is not conferred by a single bacterial species but by the synergistic metabolic activity of a reconfigured microbial community. This aligns with the hologenome concept of evolution, where the host and its microbiome evolve as a single unit, with the rapidly evolving bacteria bolstering the host's adaptability to new environmental pressures like insecticides.
Implications for Pest Management
The identification of a link between gut microbiota and insecticide resistance has significant implications for agricultural pest control.
- New Control Targets: The gut microbiome presents a novel target for managing medfly populations. Strategies could be developed to disrupt the beneficial, resistance-conferring bacteria or to introduce microbes that increase insecticide susceptibility.
- Mitigating Resistance: Understanding the microbial mechanisms of detoxification could lead to the development of synergistic compounds that inhibit bacterial degradation of insecticides, thereby restoring the efficacy of existing chemicals.
- Sustainable IPM: Microbiota-targeted approaches could reduce the reliance on conventional chemical insecticides, promoting more sustainable and environmentally friendly Integrated Pest Management (IPM) practices.
Study Limitations and Future Directions
The authors acknowledge that 16S rRNA sequencing, while excellent for identifying microbial composition, only allows for the prediction of function. The enriched KEGG pathways do not constitute direct proof of insecticide degradation by the identified bacteria.
Therefore, future research is necessary to validate these findings. Functional analyses using techniques such as metatranscriptomics (to study gene expression) and metametabolomics (to study metabolic products) are needed to definitively link specific bacterial species and their metabolic activities to the degradation of malathion, dimethoate, and spinosad. Additionally, culture-dependent manipulation—isolating and reintroducing key bacteria into flies—would provide direct evidence of their contributory role in insecticide resistance.