Glyphosate inhibits melanization and increases susceptibility to infection in insects.

Melanin, a black-brown pigment found throughout all kingdoms of life, has diverse biological functions including UV protection, thermoregulation, oxidant scavenging, arthropod immunity, and microbial virulence. Given melanin's broad roles in the biosphere, particularly in insect immune defenses, it is important to understand how exposure to ubiquitous environmental contaminants affects melanization. Glyphosate-the most widely used herbicide globally-inhibits melanin production, which could have wide-ranging implications in the health of many organisms, including insects. Here, we demonstrate that glyphosate has deleterious effects on insect health in 2 evolutionary distant species, Galleria mellonella (Lepidoptera: Pyralidae) and Anopheles gambiae (Diptera: Culicidae), suggesting a broad effect in insects. Glyphosate reduced survival of G. mellonella caterpillars following infection with the fungus Cryptococcus neoformans and decreased the size of melanized nodules formed in hemolymph, which normally help eliminate infection. Glyphosate also increased the burden of the malaria-causing parasite Plasmodium falciparum in A. gambiae mosquitoes, altered uninfected mosquito survival, and perturbed the microbial composition of adult mosquito midguts. Our results show that glyphosate's mechanism of melanin inhibition involves antioxidant synergy and disruption of the reaction oxidation-reduction balance. Overall, these findings suggest that glyphosate's environmental accumulation could render insects more susceptible to microbial pathogens due to melanin inhibition, immune impairment, and perturbations in microbiota composition, potentially contributing to declines in insect populations.

Gut Bacteria Mediate Nutrient Availability in Drosophila Diets.

Drosophila melanogaster gut microbes play important roles in host nutritional physiology. However, these associations are often indirect, and studies typically are in the context of specialized nutritional conditions, making it difficult to discern how microbiome-mediated impacts translate to physiologically relevant conditions, in the laboratory or nature. In this study, we quantified changes in dietary nutrients due to D. melanogaster gut bacteria on three artificial diets and a natural diet of grapes. We show that under all four diet conditions, bacteria altered the protein, carbohydrates, and moisture of the food substrate. An in-depth analysis of one diet revealed that bacteria also increased the levels of tryptophan, an essential amino acid encountered scarcely in nature. These nutrient changes result in an increased protein-to-carbohydrate (P:C) ratio in all diets, which we hypothesized to be a significant determinant of microbiome-mediated host nutritional physiology. To test this, we compared life history traits of axenic flies reared on the three artificial diets with increased P:C ratios or continuous bacterial inoculation. We found that while on some diets, an environment of nutritional plenitude had impacts on life history, it did not fully explain all microbiome-associated phenotypes. This suggests that other factors, such as micronutrients and feeding behavior, likely also contribute to life history traits in a diet-dependent manner. Thus, while some bacterial impacts on nutrition occur across diets, others are dictated by unique dietary environments, highlighting the importance of diet-microbiome interactions in D. melanogaster nutritional physiology.IMPORTANCE Both in the laboratory and in nature, D. melanogaster-associated microbes serve as nutritional effectors, either through the production of metabolites or as direct sources of protein biomass. The relationship between the microbiome and the resulting host nutritional physiology is significantly impacted by diet composition, yet studies involving D. melanogaster are performed using a wide range of artificial diets, making it difficult to discern which aspects of host-microbe interactions may be universal or diet dependent. In this study, we utilized three standard D. melanogaster diets and a natural grape diet to form a comprehensive understanding of the quantifiable nutritional changes mediated by the host microbial community. We then altered these artificial diets based on the observed microbe-mediated changes to demonstrate their potential to influence host physiology, allowing us to identify nutritional factors whose effects were either universal for the three artificial diets or dependent on host diet composition.

Bacterial Analogs of Plant Tetrahydropyridine Alkaloids Mediate Microbial Interactions in a Rhizosphere Model System.

Plants expend significant resources to select and maintain rhizosphere communities that benefit their growth and protect them from pathogens. A better understanding of assembly and function of rhizosphere microbial communities will provide new avenues for improving crop production. Secretion of antibiotics is one means by which bacteria interact with neighboring microbes and sometimes change community composition. In our analysis of a taxonomically diverse consortium from the soybean rhizosphere, we found that Pseudomonas koreensis selectively inhibits growth of Flavobacterium johnsoniae and other members of the Bacteroidetes grown in soybean root exudate. A genetic screen in P. koreensis identified a previously uncharacterized biosynthetic gene cluster responsible for the inhibitory activity. Metabolites were isolated based on biological activity and were characterized using tandem mass spectrometry, multidimensional nuclear magnetic resonance, and Mosher ester analysis, leading to the discovery of a new family of bacterial tetrahydropyridine alkaloids, koreenceine A to D (metabolites 1 to 4). Three of these metabolites are analogs of the plant alkaloid γ-coniceine. Comparative analysis of the koreenceine cluster with the γ-coniceine pathway revealed distinct polyketide synthase routes to the defining tetrahydropyridine scaffold, suggesting convergent evolution. Koreenceine-type pathways are widely distributed among Pseudomonas species, and koreenceine C was detected in another Pseudomonas species from a distantly related cluster. This work suggests that Pseudomonas and plants convergently evolved the ability to produce similar alkaloid metabolites that can mediate interbacterial competition in the rhizosphere.IMPORTANCE The microbiomes of plants are critical to host physiology and development. Microbes are attracted to the rhizosphere due to massive secretion of plant photosynthates from roots. Microorganisms that successfully join the rhizosphere community from bulk soil have access to more abundant and diverse molecules, producing a highly competitive and selective environment. In the rhizosphere, as in other microbiomes, little is known about the genetic basis for individual species' behaviors within the community. In this study, we characterized competition between Pseudomonas koreensis and Flavobacterium johnsoniae, two common rhizosphere inhabitants. We identified a widespread gene cluster in several Pseudomonas spp. that is necessary for the production of a novel family of tetrahydropyridine alkaloids that are structural analogs of plant alkaloids. We expand the known repertoire of antibiotics produced by Pseudomonas in the rhizosphere and demonstrate the role of the metabolites in interactions with other rhizosphere bacteria.

Reports from a Healthy Community: The 7th Conference on Beneficial Microbes.

The last two decades have seen an explosion in research about the beneficial microbial communities associated with plants and animals. Initially this explosion was driven by technological advances that enabled explorations of microbiomes on unprecedented scales. Increasingly, the drive is coming from conceptual advances that are the fruit of research investments into experimental systems to probe the functions of these beneficial microbes and their mechanisms of action. The Conference on Beneficial Microbes has been one of the premiere venues for this research. The 7th Conference on Beneficial Microbes was held July 8-11, 2018, at the University of Wisconsin-Madison Memorial Union. The 308 attendees-representing academia, industry, journals, and funding agencies-participated in an intense 4-day meeting encompassing research frontiers in beneficial microbiology and microbiome science.

Bloom of resident antibiotic-resistant bacteria in soil following manure fertilization.

The increasing prevalence of antibiotic-resistant bacteria is a global threat to public health. Agricultural use of antibiotics is believed to contribute to the spread of antibiotic resistance, but the mechanisms by which many agricultural practices influence resistance remain obscure. Although manure from dairy farms is a common soil amendment in crop production, its impact on the soil microbiome and resistome is not known. To gain insight into this impact, we cultured bacteria from soil before and at 10 time points after application of manure from cows that had not received antibiotic treatment. Soil treated with manure contained a higher abundance of ß-lactam-resistant bacteria than soil treated with inorganic fertilizer. Functional metagenomics identified ß-lactam-resistance genes in treated and untreated soil, and indicated that the higher frequency of resistant bacteria in manure-amended soil was attributable to enrichment of resident soil bacteria that harbor ß-lactamases. Quantitative PCR indicated that manure treatment enriched the blaCEP-04 gene, which is highly similar (96%) to a gene found previously in a Pseudomonas sp. Analysis of 16S rRNA genes indicated that the abundance of Pseudomonas spp. increased in manure-amended soil. Populations of other soil bacteria that commonly harbor ß-lactamases, including Janthinobacterium sp. and Psychrobacter pulmonis, also increased in response to manure treatment. These results indicate that manure amendment induced a bloom of certain antibiotic-resistant bacteria in soil that was independent of antibiotic exposure of the cows from which the manure was derived. Our data illustrate the unintended consequences that can result from agricultural practices, and demonstrate the need for empirical analysis of the agroecosystem.

Invasive and indigenous microbiota impact intestinal stem cell activity through multiple pathways in Drosophila.

Gut homeostasis is controlled by both immune and developmental mechanisms, and its disruption can lead to inflammatory disorders or cancerous lesions of the intestine. While the impact of bacteria on the mucosal immune system is beginning to be precisely understood, little is known about the effects of bacteria on gut epithelium renewal. Here, we addressed how both infectious and indigenous bacteria modulate stem cell activity in Drosophila. We show that the increased epithelium renewal observed upon some bacterial infections is a consequence of the oxidative burst, a major defense of the Drosophila gut. Additionally, we provide evidence that the JAK-STAT (Janus kinase-signal transducers and activators of transcription) and JNK (c-Jun NH(2) terminal kinase) pathways are both required for bacteria-induced stem cell proliferation. Similarly, we demonstrate that indigenous gut microbiota activate the same, albeit reduced, program at basal levels. Altered control of gut microbiota in immune-deficient or aged flies correlates with increased epithelium renewal. Finally, we show that epithelium renewal is an essential component of Drosophila defense against oral bacterial infection. Altogether, these results indicate that gut homeostasis is achieved by a complex interregulation of the immune response, gut microbiota, and stem cell activity.

Enterococcus faecalis 6-phosphogluconolactonase is required for both commensal and pathogenic interactions with Manduca sexta.

Enterococcus faecalis is a commensal and pathogen of humans and insects. In Manduca sexta, E. faecalis is an infrequent member of the commensal gut community, but its translocation to the hemocoel results in a commensal-to-pathogen switch. To investigate E. faecalis factors required for commensalism, we identified E. faecalis genes that are upregulated in the gut of M. sexta using recombinase-based in vivo expression technology (RIVET). The RIVET screen produced 113 clones, from which we identified 50 genes that are more highly expressed in the insect gut than in culture. The most frequently recovered gene was locus OG1RF_11582, which encodes a 6-phosphogluconolactonase that we designated pglA. A pglA deletion mutant was impaired in both pathogenesis and gut persistence in M. sexta and produced enhanced biofilms compared with the wild type in an in vitro polystyrene plate assay. Mutation of four other genes identified by RIVET did not affect persistence in caterpillar guts but led to impaired pathogenesis. This is the first identification of genetic determinants for E. faecalis commensal and pathogenic interactions with M. sexta. Bacterial factors identified in this model system may provide insight into colonization or persistence in other host-associated microbial communities and represent potential targets for interventions to prevent E. faecalis infections.

Whole-genome sequencing of Chromobacterium subtsugae strains exhibiting toxicity to Drosophila melanogaster.

Chromobacterium subtsugae exhibits toxicity to Drosophila melanogaster, providing a new infection model to study host homeostasis. Previous studies using pathogen models have proven to be a useful tool to understand host physiology. Here, we report on the whole-genome sequences of these microbes obtained from short and long reads.

Analysis of justification for author order and gender bias in author order among those contributing equally.

The practice of designating two or more authors as equal contributors (ECs) on a scientific publication is increasingly common as a form of sharing credit. However, EC authors are often unclearly attributed on curriculum vitae (CVs) or citation engines, and it is unclear how research teams determine author order within an EC listing. In response to studies showing that male authors were more likely to be placed first in an EC listing, the American Society for Microbiology (ASM) required that authors explain the reasons for author order beginning in 2020. In this study, we analyze data from over 2,500 ASM publications to see how this policy affected gender bias and how research teams are making decisions on author order. Data on publications from 2018 to 2021 show that gender bias was largely nonsignificant both before and after authors were asked by ASM to provide an EC statement. The most likely reasons for EC order included alphabetical order, seniority, and chance, although there were differences for publications from different geographic regions. However, many research teams used unique methods in order selection, highlighting the importance of EC statements to provide clarity for readers, funding agencies, and tenure committees. IMPORTANCE: First-author publications are important for early career scientists to secure funding and educational opportunities. However, an analysis published in eLife in 2019 noted that female authors are more likely to be placed second even when both authors report they have contributed equally. American Society for Microbiology announced in response that they would require submissions to include a written justification of author order. In this paper, we analyze the resultant data and show that laboratories are most likely to use some combination of alphabetical order, seniority, and chance to determine author order. However, the prevalence of these methods varies based on the research team's geographic location. These findings highlight the importance of equal contributor statements to provide clarity for readers, funding agencies, and tenure committees. Furthermore, this work is critically important for understanding how these decisions are made and provides a glimpse of the sociology of science.

Analysis of justification for and gender bias in author order among those contributing equally.

The practice of designating two or more authors as equal contributors (EC) on a scientific publication is increasingly common as a form of sharing credit. However, EC authors are often unclearly attributed on CVs or citation engines, and it is unclear how research teams determine author order within an EC listing. In response to studies showing that male authors were more likely to be placed first in an EC listing, the American Society of Microbiology (ASM) required that authors explain the reasons for author order beginning in 2020. In this study we analyze data from over 2500 ASM publications to see how this policy affected gender bias and how research teams are making decisions on author order. Data on publications from 2018-2021 show that gender bias was largely nonsignificant both before and after authors were asked by ASM to provide an EC statement. The most likely reasons for EC order included alphabetical order, seniority, and chance, although there were differences for publications from different geographic regions. However, many research teams used unique methods in order selection, highlighting the importance of EC statements to provide clarity for readers, funding agencies, and tenure committees.

Microbiome-derived acidity protects against microbial invasion in Drosophila.

Microbial invasions underlie host-microbe interactions resulting in pathogenesis and probiotic colonization. In this study, we explore the effects of the microbiome on microbial invasion in Drosophila melanogaster. We demonstrate that gut microbes Lactiplantibacillus plantarum and Acetobacter tropicalis improve survival and lead to a reduction in microbial burden during infection. Using a microbial interaction assay, we report that L. plantarum inhibits the growth of invasive bacteria, while A. tropicalis reduces this inhibition. We further show that inhibition by L. plantarum is linked to its ability to acidify its environment via lactic acid production by lactate dehydrogenase, while A. tropicalis diminishes the inhibition by quenching acids. We propose that acid from the microbiome is a gatekeeper to microbial invasions, as only microbes capable of tolerating acidic environments can colonize the host. The methods and findings described herein will add to the growing breadth of tools to study microbe-microbe interactions in broad contexts.

Massively parallel mutant selection identifies genetic determinants of Pseudomonas aeruginosa colonization of Drosophila melanogaster.

Pseudomonas aeruginosa is recognized for its ability to colonize diverse habitats and cause disease in a variety of hosts, including plants, invertebrates, and mammals. Understanding how this bacterium is able to occupy wide-ranging niches is important for deciphering its ecology. We used transposon sequencing [Tn-Seq, also known as insertion sequencing (INSeq)] to identify genes in P. aeruginosa that contribute to fitness during the colonization of Drosophila melanogaster. Our results reveal a suite of critical factors, including those that contribute to polysaccharide production, DNA repair, metabolism, and respiration. Comparison of candidate genes with fitness determinants discovered in previous studies on P. aeruginosa identified several genes required for colonization and virulence determinants that are conserved across hosts and tissues. This analysis provides evidence for both the conservation of function of several genes across systems, as well as host-specific functions. These findings, which represent the first use of transposon sequencing of a gut pathogen in Drosophila, demonstrate the power of Tn-Seq in the fly model system and advance the existing knowledge of intestinal pathogenesis by D. melanogaster, revealing bacterial colonization determinants that contribute to a comprehensive portrait of P. aeruginosa lifestyles across habitats.IMPORTANCEDrosophila melanogaster is a powerful model for understanding host-pathogen interactions. Research with this system has yielded notable insights into mechanisms of host immunity and defense, many of which emerged from the analysis of bacterial mutants defective for well-characterized virulence factors. These foundational studies-and advances in high-throughput sequencing of transposon mutants-support unbiased screens of bacterial mutants in the fly. To investigate mechanisms of host-pathogen interplay and exploit the tractability of this model host, we used a high-throughput, genome-wide mutant analysis to find genes that enable the pathogen P. aeruginosa to colonize the fly. Our analysis reveals critical mediators of P. aeruginosa establishment in its host, some of which are required across fly and mouse systems. These findings demonstrate the utility of massively parallel mutant analysis and provide a platform for aligning the fly toolkit with comprehensive bacterial genomics.

Preliminary evidence for chaotic signatures in host-microbe interactions.

Host-microbe interactions constitute dynamical systems that can be represented by mathematical formulations that determine their dynamic nature and are categorized as deterministic, stochastic, or chaotic. Knowing the type of dynamical interaction is essential for understanding the system under study. Very little experimental work has been done to determine the dynamical characteristics of host-microbe interactions, and its study poses significant challenges. The most straightforward experimental outcome involves an observation of time to death upon infection. However, in measuring this outcome, the internal parameters and the dynamics of each particular host-microbe interaction in a population of interactions are hidden from the experimentalist. To investigate whether a time-to-death (time-to-event) data set provides adequate information for searching for chaotic signatures, we first determined our ability to detect chaos in simulated data sets of time-to-event measurements and successfully distinguished the time-to-event distribution of a chaotic process from a comparable stochastic one. To do so, we introduced an inversion measure to test for a chaotic signature in time-to-event distributions. Next, we searched for chaos in the time-to-death of Caenorhabditis elegans and Drosophila melanogaster infected with Pseudomonas aeruginosa or Pseudomonas entomophila, respectively. We found suggestions of chaotic signatures in both systems but caution that our results are preliminary and highlight the need for more fine-grained and larger data sets in determining dynamical characteristics. If validated, chaos in host-microbe interactions would have important implications for the occurrence and outcome of infectious diseases, the reproducibility of experiments in the field of microbial pathogenesis, and the prediction of microbial threats.IMPORTANCEIs microbial pathogenesis a predictable scientific field? At a time when we are dealing with coronavirus disease 2019, there is intense interest in knowing about the epidemic potential of other microbial threats and new emerging infectious diseases. To know whether microbial pathogenesis will ever be a predictable scientific field requires knowing whether a host-microbe interaction follows deterministic, stochastic, or chaotic dynamics. If randomness and chaos are absent from virulence, there is hope for prediction in the future regarding the outcome of microbe-host interactions. Chaotic systems are inherently unpredictable, although it is possible to generate short-term probabilistic models, as is done in applications of stochastic processes and machine learning to weather forecasting. Information on the dynamics of a system is also essential for understanding the reproducibility of experiments, a topic of great concern in the biological sciences. Our study finds preliminary evidence for chaotic dynamics in infectious diseases.

Inside help from the microbiome.

Elucidating the role of one of the proteins produced by Lactiplantibacillus plantarum reveals a new molecule that allows this gut bacterium to support the development of fruit fly larvae.

Whole-genome sequences of two Drosophila melanogaster microbiome symbionts.

Lactiplantibacillus plantarum and Acetobacter tropicalis are bacterial symbionts commonly isolated from decaying fruits and from the microbiome of Drosophila melanogaster. Studies have shown that these organisms interact synergistically, imparting beneficial effects on the host. Here, we report whole-genome sequences of these microbes obtained from long and short reads.

Microbiome derived acidity protects against microbial invasion in Drosophila.

Microbial invasions underlie host-microbe interactions that result in microbial pathogenesis and probiotic colonization. While these processes are of broad interest, there are still gaps in our understanding of the barriers to entry and how some microbes overcome them. In this study, we explore the effects of the microbiome on invasions of foreign microbes in Drosophila melanogaster. We demonstrate that gut microbes Lactiplantibacillus plantarum and Acetobacter tropicalis improve survival during invasion of a lethal gut pathogen and lead to a reduction in microbial burden. Using a novel multi-organism interactions assay, we report that L. plantarum inhibits the growth of three invasive Gram-negative bacteria, while A. tropicalis prevents this inhibition. A series of in vitro and in vivo experiments revealed that inhibition by L. plantarum is linked to its ability to acidify both internal and external environments, including culture media, fly food, and the gut itself, while A. tropicalis diminishes the inhibition by quenching acids. We propose that acid produced by the microbiome serves as an important gatekeeper to microbial invasions, as only microbes capable of tolerating acidic environments can colonize the host. The methods described herein will add to the growing breadth of tools to study microbe-microbe interactions in broad contexts.

Massively parallel mutant selection identifies genetic determinants of Pseudomonas aeruginosa colonization of Drosophila melanogaster.

Pseudomonas aeruginosa is recognized for its ability to colonize diverse habitats and cause disease in a variety of hosts, including plants, invertebrates, and mammals. Understanding how this bacterium is able to occupy wide-ranging niches is important for deciphering its ecology. We used transposon sequencing (Tn-Seq, also known as INSeq) to identify genes in P. aeruginosa that contribute to fitness during colonization of Drosophila melanogaster. Our results reveal a suite of critical factors, including those that contribute to polysaccharide production, DNA repair, metabolism, and respiration. Comparison of candidate genes with fitness determinants discovered in previous studies of P. aeruginosa identified several genes required for colonization and virulence determinants that are conserved across hosts and tissues. This analysis provides evidence for both the conservation of function of several genes across systems, as well as host-specific functions. These findings, which represent the first use of transposon sequencing of a gut pathogen in Drosophila, demonstrate the power of Tn-Seq in the fly model system and advance existing knowledge of intestinal pathogenesis by D. melanogaster, revealing bacterial colonization determinants that contribute to a comprehensive portrait of P. aeruginosa lifestyles across habitats.

Chaotic signatures in host-microbe interactions.

Host-microbe interactions constitute dynamical systems that can be represented by mathematical formulations that determine their dynamic nature, and are categorized as deterministic, stochastic, or chaotic. Knowing the type of dynamical interaction is essential for understanding the system under study. Very little experimental work has been done to determine the dynamical characteristics of host-microbe interactions and its study poses significant challenges. The most straightforward experimental outcome involves an observation of time to death upon infection. However, in measuring this outcome, the internal parameters, and the dynamics of each particular host-microbe interaction in a population of interactions are hidden from the experimentalist. To investigate whether a time-to-death (time to event) dataset provides adequate information for searching for chaotic signatures, we first determined our ability to detect chaos in simulated data sets of time-to-event measurements and successfully distinguished the time-to-event distribution of a chaotic process from a comparable stochastic one. To do so, we introduced an inversion measure to test for a chaotic signature in time-to-event distributions. Next, we searched for chaos, in time-to-death of Caenorhabditis elegans and Drosophila melanogaster infected with Pseudomonas aeruginosa or Pseudomonas entomophila, respectively. We found suggestions of chaotic signatures in both systems, but caution that our results are preliminary and highlight the need for more fine-grained and larger data sets in determining dynamical characteristics. If validated, chaos in host-microbe interactions would have important implications for the occurrence and outcome of infectious diseases, the reproducibility of experiments in the field of microbial pathogenesis and the prediction of microbial threats.

Cuticular profiling of insecticide resistant Aedes aegypti.

Insecticides have made great strides in reducing the global burden of vector-borne disease. Nonetheless, serious public health concerns remain because insecticide-resistant vector populations continue to spread globally. To circumvent insecticide resistance, it is essential to understand all contributing mechanisms. Contact-based insecticides are absorbed through the insect cuticle, which is comprised mainly of chitin polysaccharides, cuticular proteins, hydrocarbons, and phenolic biopolymers sclerotin and melanin. Cuticle interface alterations can slow or prevent insecticide penetration in a phenomenon referred to as cuticular resistance. Cuticular resistance characterization of the yellow fever mosquito, Aedes aegypti, is lacking. In the current study, we utilized solid-state nuclear magnetic resonance spectroscopy, gas chromatography/mass spectrometry, and transmission electron microscopy to gain insights into the cuticle composition of congenic cytochrome P450 monooxygenase insecticide resistant and susceptible Ae. aegypti. No differences in cuticular hydrocarbon content or phenolic biopolymer deposition were found. In contrast, we observed cuticle thickness of insecticide resistant Ae. aegypti increased over time and exhibited higher polysaccharide abundance. Moreover, we found these local cuticular changes correlated with global metabolic differences in the whole mosquito, suggesting the existence of novel cuticular resistance mechanisms in this major disease vector.

Cuticular profiling of insecticide resistant Aedes aegypti.

Insecticides have made great strides in reducing the global burden of vector-borne disease. Nonetheless, serious public health concerns remain because insecticide-resistant vector populations continue to spread globally. To circumvent insecticide resistance, it is essential to understand all contributing mechanisms. Contact-based insecticides are absorbed through the insect cuticle, which is comprised mainly of chitin polysaccharides, cuticular proteins, hydrocarbons, and phenolic biopolymers sclerotin and melanin. Cuticle interface alterations can slow or prevent insecticide penetration in a phenomenon referred to as cuticular resistance. Cuticular resistance characterization of the yellow fever mosquito, Aedes aegypti , is lacking. In the current study, we utilized solid-state Nuclear Magnetic Resonance (ssNMR) spectroscopy, gas chromatography/mass spectrometry (GC-MS), and transmission electron microscopy (TEM) to gain insights into the cuticle composition of congenic cytochrome P450 monooxygenase insecticide resistant and susceptible Ae. aegypti . No differences in cuticular hydrocarbon content or phenolic biopolymer deposition were found. In contrast, we observed cuticle thickness of insecticide resistant Ae. aegypti increased over time and exhibited higher polysaccharide abundance. Moreover, we found these local cuticular changes correlated with global metabolic differences in the whole mosquito, suggesting the existence of novel cuticular resistance mechanisms in this major disease vector.