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4  Discussion

Successful infection by entomopathogenic fungi involves suppression of both the mosquito’s innate immune system and modulation of its resident microbiota (Butt et al., 2016; Wei et al., 2017; Ramirez et al., 2021). In this study, we characterized microbiome changes in Ae. aegypti following infection with four entomopathogenic fungi, revealing a dynamic and fungus-specific interaction with the mosquito microbiota. Our results highlight the profound impact of fungal infection on microbiome composition and structure.

One of the most important findings was the dissociation between fungal load colonization success and total bacterial load. All four entomopathogenic fungi achieved significant colonization of the mosquito body; however, only C. amoenerosea-infections led to a significant increase in total bacterial abundance. This suggests that bacteriome expansion is not a passive consequence of fungal infection intensity but rather reflects specific fungi- mosquito interactions. The genus most responsible for this increase was Pandoraea, a Betaproteobacterium that has been isolated from soil and insect guts, and some members are associated with opportunistic infection in immunocompromised hosts (Coenye et al., 2000; Yi et al., 2024). Pandoraea’s expansion under C. amoenerosea’s infection could potentially indicate that this genus is promoting a state of immune dysregulation or may be disrupting the gut homeostasis that benefits bacterial proliferation. Similar patterns have been observed in other insect-fungal systems where immune suppression by entomopathogenic fungi facilitates bacterial expansion (Wei et al., 2017; Wang et al., 2023; Li et al., 2024b), which suggests that C. amoenerosea may mediate mosquito mortality through a dual mechanism of direct mycosis and bacterially-driven systemic stress. However, further experiments are necessary to link this phenotype to mosquito mortality.

Our  findings  also  reveal  that  fungal  infection  affects  the alpha  diversity  of  the  mosquito  microbiome.  The  reductions  in  Shannon  and  Simpson  indices  without  any  corresponding changes in observed ASVs likely indicate that fungal infection induces community composition shifts driven by dominance  rather than by bacterial elimination. This type of dysbiosis, in which community structure  is  shifted  toward  a  small  number of dominant opportunistic bacteria while other core taxa are relatively suppressed, leads to impairment of several host metabolic functions including disruption in immune homeostasis and larval development as well as increased susceptibility to secondary infections (Coon et al., 2016; Dickson et al., 2017; Li et al., 2024b). Thus, the significant reductions in evenness observed under C. javanica and C. cateniannulata-infections may translate to significant physiological costs to the mosquito apart from the direct effects of fungal infection. These findings are similar to previous reports of alpha diversity reductions following entomopathogenic fungal infections in insects (Noskov et al., 2021; Liu et al., 2023b; Mesquita et al., 2023) and suggest that dysbiosis these community shifts may contribute itself may contribute to accelerated mosquito mortality (Wei et al., 2017).

Although entomopathogenic fungi infect through the cuticle rather  than  the  gut,  the  gut-associated  bacterial  community    is nonetheless affected through systemic processes. The mechanism for these shifts is likely two fold: (1) the  direct  impact  of  fungal-derived  enzymes,   secondary   metabolites   and proteases that proliferate in the hemocoel and circulate systemically (Humber, 2008; Liu et al., 2023a; Zhang et al., 2024), potentially altering gut homeostasis  indirectly;  and  (2)  the collateral damage from the mosquito immune responses mounted to combat the fungal invasion (including oxidative stress, melanization, and antimicrobial peptide production) that indirectly suppresses commensal populations while leaving more resilient opportunists to fill the void (Krams et al., 2017; Boucias et al., 2018; McMillan and Adamo, 2020; Negroni et al., 2020). Experimental evidence from Wei et al. (2017) demonstrates that gut microbiota modulates mosquito susceptibility to  fungal  infection  despite  the infection initiating at the  cuticle.  This  is  further supported by Ramirez et al. (2018), who showed temporospatial immune modulation, including gut-associated immune responses, during cuticular fungal challenge in Ae. aegypti.

Beta diversity analysis further indicated that each fungal species induced unique shifts in microbial community composition across all treatment groups. For instance, mosquitoes infected with C. cateniannulata exhibited the most pronounced shift in overall community composition. This result indicates that fungal entomopathogenic species vary  in  their  ability  to  restructure  the mosquito microbiome, likely due to differences in virulence, systemic  colonization,  and   metabolite   production   profiles (Bai et al., 2020; Peng et al., 2023). The magnitude of these differences has practical implications  for  how  we  conduct fungal biocontrol agent selection. Accounting for experimental replicate as a covariate in the PERMANOVA model reinforced these conclusions.

Heatmap clustering analysis indicated genus-level shifts in response to fungal infection. Specifically, it revealed a shared directional shift in community composition among  Cordyceps  spp. that is distinct from the B. bassiana-associated microbiome response. For instance, infections by C. cateniannulata resulted   in an increase in the genera Kluyvera and Pantoea and a relative decline in genera that have been previously characterized as core components of the mosquito bacteriome, including Elizabethkingia, Serratia, Nubsella, Delftia, Sphingobium, Burkholderia, and Rhizobium (Kim and Lee, 2015; Bar-Shmuel et al., 2020; Li et al., 2020; Miyamoto et al., 2022; Gong et al., 2023; Chen et al., 2024). The loss of these taxa likely has critical functional consequences. Elizabethkingia is a genus commonly found in the microbiome    of Anopheles and Aedes mosquitoes at all life  stages  and  has been found to be associated with mosquito’s physiology and has antiparasitic activity (Chen et al., 2024). Some Delftia species are known to produce the toxic alkaloid harmane which also has antiparasitic activity (Huang et al., 2023). Therefore, their decline could compromise the mosquito’s resistance to parasites and opportunistic microbes, a shift that has the potential to promote mortality in the mosquito by enriching other opportunistic microbes residing in the gut. Two other important genera,  Nubsella and Burkholderia, were also depleted. Some members   of Nubsella have been found exclusively in healthy insects, and are thought to contribute to insect fitness and survival (Li et al., 2020), while Burkholderia is a well-characterized genus with many strains acting as insect symbionts with protective roles against entomopathogenic fungi and a putative role in digestion (Santos et al., 2004; Olivier-Espejel et al., 2011; Boissière et al., 2012). Thus, these losses could potentially lead to an impairment of immune competence and further enhance susceptibility to fungal infection. The decline of Sphingobium and  Rhizobium, taxa involved in xenobiotic degradation and nitrogen fixation, respectively (Bar-Shmuel et al., 2020; Miyamoto et al., 2022), adds another layer of potential metabolic disruption. Given that uric acid production mitigates oxidative stress in blood-feeding insects (Weihrauch and O’Donnell, 2021),  it  is  plausible  that loss of nitrogen-fixing or xenobiotic-degrading bacteria may exacerbate ROS accumulation and accelerate mortality of fungus- infected mosquitoes. Alternatively, their decline may simply reflect sensitivity to the antimicrobial peptide production and oxidative defenses the mosquito mounts against the fungus. In any case, fungal entomopathogenic infections may not only perturb microbial diversity but may also displace functionally significant community members, potentially affecting metabolic processes such as detoxification and oxidative stress management.

The enrichment of Kluyvera and Pantoea specifically under C. cateniannulata, and not under the other fungal infections, further demonstrates C. cateniannulata’ s ability to influence the mosquito microbiome. Kluyvera is a gram-negative Enterobacteriaceae found in insect gut microbiomes with some species been associated with degradation of foreign particles including polystyrene (Li et al., 2024a;  Ndotono et al.,  2024)  and   others   are   known   to   act as opportunistic entomopathogens causing mortality in certain insects (Ogodo, 2020). Pantoea, a bacterial genus frequently found in the Aedes mosquito, has been shown to influence vector competence through the secretion of anti-Plasmodium effector proteins (Ranasinghe et al., 2021). Expansion of Pantoea under C. cateniannulata infection most likely reflects opportunistic colonization of niches vacated by suppressed core taxa, plausibly due to host immune modulation, fungal metabolite production, and tissue damage that this species induces. Our LEfSe results, which identified both genera as strongly associated with C. cateniannulata treatment, support their potential utility as microbiome-based indicators of this specific fungal infection.

Infection by C. amoenerosea produced a community composition broadly  similar  to  the  C.  cateniannulata  group  but with a distinct profile marked by relative increases in Bacillus, Pandoraea, and Achromobacter alongside a decrease of Serratia. The increase in Bacillus is of interest given that specific members of this genus have documented insecticidal activity (Toukabri et al., 2023; Deng et al., 2024) suggesting the possibility that its increase may contribute to mosquito mortality independently of the fungal infection. However, further research directly coupling fungal infection, microbiome profiling and survival is needed to link this microbiome disruption to mosquito mortality. Of  significance  was the statistically robust increase in bacterial load observed in C. amoenerosea-infected mosquitoes, which our analysis indicates Pandoraea as the primary driver of the bacterial load in this treatment group. While some Pandoraea species are opportunistic pathogens, others participate in the biodegradation of organic compounds and chemosynthetic processes (Kostygov et al., 2017); thus, its expansion may reflect a compensatory metabolic response to the nutrient and energetic demands of fighting off the fungal infection or may indicate an opportunistic proliferation in a disrupted microbiome. Some species of the genus Achromobacter are known to degrade many pesticides as a  nutritional  source (Pan et al., 2024), while others have insecticidal activity, suggesting that its increase could be either protective or harmful depending on which species is actually expanding. However, our genus-level data cannot determine which species are present. The reduction   in Serratia under C. amoenerosea is also of significance. Given that certain Serratia strains have immunosuppressive qualities, with high  cytotoxicity  to  granular  cells  and  plasmatocytes (Kim et al., 2009; Toukabri et al., 2023), its decline may reflect active suppression by an upregulated mosquito immune response fighting off the fungal infection. Further experiments are needed to confirm the functional role of these bacteria.

Infection by B. bassiana  was  characterized  by  an  increase in Pseudomonas, Nubsella, Burkholderia, and Rhizobium with a relative decrease in Chryseobacterium, Kluyvera, and Pantoea. The increase in Pseudomonas is particularly interesting  given  that certain members  of  this  genus  have  been  shown  to provide nutrients and protection  against  entomopathogenic  fungi, with in vitro studies identifying compounds produced by Pseudomonas that decrease the growth of entomopathogenic fungi (Saati-Santamaría et al., 2021). Its enrichment under B. bassiana infection could therefore represent a mosquito microbiome-level defense response with the recruitment or selective enrichment of bacteria capable of fighting the invading fungus. As previously mentioned, Nubsella, Burkholderia, and Rhizobium all play protective roles in insects such as increased fitness, protection against pathogens, and nitrogen fixation, respectively; thus, their retention and even enrichment under B. bassiana infection, in contrast to their suppression under Cordyceps species, further distinguishes this fungus from others. This differential retention of these taxa may also reflect differences in the secondary metabolite profiles these fungi deploy against the mosquito resident microbes. Mosquitoes infected with C. javanica most closely resembled uninfected controls in overall microbial community composition, but one consistent feature across all fungal treatments (including C. javanica) was  the  reduction  of  Chryseobacterium.  This genus was the most abundant taxon in the uninfected control  group and was consistently reduced across all fungus-infected treatments,  suggesting  that  it   is   playing   a   stabilizing   role in  the  healthy  mosquito  microbiome.  Its  importance  is   further supported by previous studies demonstrating its high prevalence in the microbiomes of healthy insects (Shelomi et al., 2023; Akintola and Hwang, 2024) and its decline  following fungal infections (Noskov et al., 2021). Members of the genus Chryseobacterium have been associated with immune homeostasis (Lee et al., 2016), and production of antimicrobial  compounds that protect against opportunistic infections (Dahal et al., 2021). Thus, its consistent suppression across all fungal treatments may represent a shared early consequence of infection, removal of a key stabilizing taxon which consequently opens the door to secondary pathogens and opportunistic expansions described above. This makes Chryseobacterium a potential indicator for host microbiome health in the context of fungal-based microbial control strategies regardless of species.

Several of these genus-level associations were independently confirmed using two complementary differential abundance methods, MaAsLin2 (batch-corrected) and EdgeR. For example, MaAsLin2 and EdgeR results agree with the LEfSe analysis that Pandoraea was enriched under C. amoenerosea infection, indicating this genus as the primary indicator of C. amoenerosea infection. However, in some cases, associations were not detected by all three methods. For instance, MaAsLin2 indicated the enrichment of Kluyvera and Pantoea during C. cateniannulata infection, which also had the highest LEfSe LDA scores, while enrichment of these two genera was not detected by EdgeR, likely due to these signals belonging to specific ASVs that are diluted upon genus aggregation. On the other hand, EdgeR provided a wider coverage of core microbiome depletion under C. cateniannulata infection, which provided expanded information on the microbiome loss in addition to what MaAsLin2 and LEfSe detected. The three methods are therefore complementary rather than contradictory. The combined use of these two additional methods strengthens the conclusions drawn from the LEfSe and heatmap analysis.

While   previous   studies   on   mosquito-microbe interactions have  focused  mostly  on  single  Beauveria   bassiana   strains, this approach overlooks the diverse pathogenic mechanisms of entomopathogenic fungi. Our study addresses  this  critical  gap  by investigating the effect of infection by four distinct Beauveria and Cordyceps strains on the microbiome of the yellow fever mosquito Ae. aegypti through infection bioassays and 16S rRNA metagenomic sequencing. The important changes in the core mosquito microbiome under the context of a fungal infections, described in the present study, likely have important consequences in mosquito physiology, immunity, and vector competence  (Hegde et al., 2015; Song et al., 2018), making mosquitoes more susceptible  to  additional  infections  and/or  accelerated mortality during fungal infections. However, further research is needed to elucidate interactions of observed taxa with entomopathogenic fungi and establish a causal link between the microbiome changes reported here and their potential contributions to mosquito mortality. From an applied point of view, these findings have direct relevance to the development and optimization of fungal biocontrol strategies. The fungal species-specific disruption of the mosquito microbiome documented in this study means that the choice of fungal agent should not just focus on killing efficiency, but also on its secondary microbiome-mediated effects on the mosquito host. The genera identified in this study, Kluyvera for C. cateniannulata, Burkholderia for B. bassiana, and Pandoraea for C. amoenerosea, could be useful as indicators of successful fungal colonization and microbiome disruption. This set of candidate bacterial discriminating taxa could aid in the development of novel strategies to enhance the efficacy of fungal entomopathogens in vector control programs.

Overall, our findings demonstrate that entomopathogenic fungal infections significantly reshape the mosquito microbiome. Infection by C. cateniannulata and C. amoenerosea showed the most pronounced disruptions. Although survival data were not collected in this study and it will be necessary to draw any correlation between microbiome disruption and  host  mortality, the findings reported in this study make a case for incorporating microbiome assessment as part of an entomopathogenic fungal biocontrol evaluation. Understanding how different fungal species reshape the mosquito microbiome adds a mechanistic dimension to biocontrol research and open new doors for refining the selection and application of fungal entomopathogens in integrated vector control programs.

Data availability statement

The original contributions presented in the study are included in the article/Supplementary material. Further inquiries can be directed to the corresponding author/s.

Author contributions

EE: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Software, Validation, Visualization, Writing – original draft, Writing – review & editing. HG: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Software, Validation, Visualization, Writing – original draft, Writing – review & editing. SK: Formal analysis, Investigation,  Methodology,  Writing  –  original  draft,  Writing – review & editing. KD: Conceptualization, Data curation,  Formal analysis, Investigation, Methodology, Software, Validation, Visualization, Writing – original draft, Writing – review & editing. LF-W: Formal analysis, Investigation, Methodology, Validation, Writing – original draft, Writing – review & editing. JM: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Supervision, Validation, Writing – original draft, Writing – review & editing. JR: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing.

Funding

The author(s) declared that financial support  was  received  for this work and/or its publication. This research was supported  in part by the U.S. Department of Agriculture, Agricultural Research Service Project Number 5010-10400-001-000D to JLR and a Summer Research and Artistry Fellowship to EE from the Bradley University College of Liberal Arts and Sciences. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer.

Conflict of interest

The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The author(s) JR declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.

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Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2026. 1879658/full#supplementary-material