Microsporidia MB mediated transcriptomics and gut microbiota shift in Plasmodium falciparum exposed Anopheles arabiensis mosquitoes

doi:10.21203/rs.3.rs-8191790/v1

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Microsporidia MB mediated transcriptomics and gut microbiota shift in Plasmodium falciparum exposed Anopheles arabiensis mosquitoes

https://doi.org/10.21203/rs.3.rs-8191790/v1

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Background

Microsporidia MB is a vertically and horizontally transmitted endosymbiont of Anopheles mosquitoes with the potential to impair Plasmodium falciparum transmission in malaria endemic regions. However, despite its ability to block Plasmodium transmission, the mechanistic basis underpinning this effect remains unknown.

Methods

We profiled gene expression and gut microbiota in Microsporidia MB-infected (positive) and -uninfected (negative) mosquitoes exposed to Plasmodium-infected and -uninfected blood from mosquito guts, across three time points 24-, 48-, and 72-hours post–blood meal.

Results

Microsporidia MB-negative mosquitoes exposed to P. falciparum infection exhibited suppressed immune signaling, followed by an upregulation of lipolytic and cellular stress related genes, suggesting parasite-driven immune evasion and host resource exploitation. In contrast, Microsporidia MB-positive mosquitoes exposed to Plasmodium-negative blood exhibited a sustained immune activation and lipid depletion, suggesting that Microsporidia MB infection contributes to a more sustained activation of immunity in mosquitoes exposed to Plasmodium-negative blood. In Microsporidia MB-positive mosquitoes exposed to P. falciparum-positive blood, gene expression indicated several possible routes by which parasite establishment could be disrupted. We observed indications of hormonal remodeling and nutrient restriction and suppression, which could potentially create an inhospitable environment for Plasmodium parasite development in Microsporidia MB-positive mosquitoes. Microbiota profiling further revealed the proliferation of taxa such as Serratia and Enterobacter, previously associated with parasite inhibition, in Microsporidia MB-positive mosquitoes.

Conclusion

Together, our data suggests that Microsporidia MB induces systemic changes in host metabolism, immunity, and gut microbial ecology to create an inhospitable environment for Plasmodium, supporting its potential as a sustainable transmission-blocking strategy.

Despite extensive global efforts to combat malaria, it remains one of the deadliest infectious diseases worldwide. Malaria is caused by protozoan parasites of the genus Plasmodium, which are primarily transmitted by female mosquitoes of the genus Anopheles. Of the approximately 460 identified Anopheles species, around 100 are recognized as malaria vectors, but only 30 to 40 species are known to effectively transmit Plasmodium parasites to humans (Sato, 2021; Sinka et al., 2012; Zekar & Sharman, 2023). Among the many Plasmodium species, only P. falciparum, P. vivax, P. malariae, P. ovale, and P. knowlesi are known to infect humans (Sato, 2021; Sinka et al., 2012; Zekar & Sharman, 2023).

The initial stages of Plasmodium development occur during a period that is also critical for mosquito reproduction. Female Anopheles mosquitoes may acquire Plasmodium parasites while feeding on the blood of an infected individual, a meal necessary for egg development (Meibalan & Marti, 2017). Following this blood meal, male and female Plasmodium gametocytes undergo gametogenesis in the mosquito midgut, where microgametes fertilize macrogametes to form zygotes. These zygotes develop into elongated, motile ookinetes within 22–24 hours post-blood meal, marking the establishment of infection (Meibalan & Marti, 2017).

The ookinetes then traverse the midgut epithelium and settle on the basal lamina, where they mature into oocysts, typically around 48 hours after the blood meal (Meibalan & Marti, 2017; Smith & Barillas-Mury, 2016). Over the next 7–14 days, the oocysts undergo sporogony, producing thousands of sporozoites. Upon maturation, the oocysts rupture and release sporozoites into the hemocoel. These sporozoites migrate through the hemolymph to the salivary glands, from where they can be transmitted to a vertebrate host during subsequent blood feeding, thus the completion of the Plasmodium developmental cycle in the mosquitoes (Meibalan & Marti, 2017; Smith & Barillas-Mury, 2016).

While the developmental cycle of Plasmodium within mosquitoes follows a relatively straightforward sequence, its success is modulated by a range of intrinsic and extrinsic factors (Bai et al., 2019; Barletta et al., 2025; Cirimotich et al., 2011; L. Gao et al., 2024; Shaw et al., 2022; Tripet et al., 2008; Y. O. Zhao et al., 2012). These include the physiological fitness of the mosquito, which influences its vectorial competence, as well as environmental conditions such as temperature and midgut pH that can affect parasite maturation and survival (Tripet et al., 2008). In addition, the ability of Plasmodium to evade the mosquito’s immune defenses and to withstand the hostile effects of the gut microbiota is critical for successful parasite development (Bai et al., 2019; Barletta et al., 2025; Cirimotich et al., 2011; Jaramillo-Gutierrez et al., 2009). Moreover, the mosquito’s metabolic state, particularly nutrient availability following a blood meal, can also impact the timing and efficiency of parasite maturation (Shaw et al., 2022; Y. O. Zhao et al., 2012). Additionally, the presence of microbial endosymbionts, such as Microsporidia MB, may impair Plasmodium transmission through immune priming or resource competition (Herren et al., 2020). Together, these factors shape the internal environment of the mosquito and ultimately determine whether transmission to a vertebrate host will occur.

In this study, we investigated the impact of mosquito infection with the endosymbiont Microsporidia MB on host biological processes and sought to elucidate the mechanistic basis of its Plasmodium transmission-blocking phenotype. Microsporidia MB is a naturally occurring endosymbiont of fungal ancestry that is predominant in major malaria vectors and has been shown to impair P. falciparum in An. arabiensis mosquitoes (Herren et al., 2020). The development of Microsporidia MB as a malaria transmission blocking strategy is feasible owing to its ability to be vertically and horizontally transmitted among mosquito populations (Herren et al., 2020; Nattoh et al., 2021). However, optimizing its transmission efficiency and enhancing its parasite-blocking capabilities are critical for its practical application in symbiont-based malaria control. Therefore, a deeper understanding of the tripartite interactions among Microsporidia MB, Plasmodium, and the mosquito host is essential to inform the development and deployment of this novel control strategy.

To achieve this, we analyzed transcriptomic data from the guts of An. arabiensis mosquitoes exposed to P. falciparum-infected and un-infected blood at multiple time points (24, 48 and 72 hours) post-blood meal. Since P. falciparum infections in mosquitoes rarely generate parasite burdens as high as those achieved in cultured P. falciparum gametocytes and rodent malaria laboratory models, owing to the ingestion of a blood meal with lower gametocytemia (Sangare et al., 2013), samples categorized as Plasmodium-infected mosquitoes may not have necessarily developed high-intensity infections. Instead, they were mainly included based on exposure to parasite-infected blood. Previously, it has been established that even low intensity Plasmodium infections are associated with significant transcriptional changes (Mendes et al., 2011). Differential gene expression analysis was conducted to identify host transcriptional changes associated with Microsporidia MB infection and to determine how these changes may contribute to the observed Plasmodium transmission-blocking phenotype. Our results highlight important immune and metabolic differences between Microsporidia MB–infected and uninfected mosquitoes exposed to either Plasmodium-infected or uninfected blood, shedding light on the mechanistic basis of the Microsporidia MB–Plasmodium transmission-blocking phenotype.

Principal component analysis of genes comprising the different samples constituting the different treatments in the study

We assessed the clustering patterns of Microsporidia MB-positive mosquitoes fed on Plasmodium-negative blood, Microsporidia MB-positive mosquitoes fed on Plasmodium-positive blood, Microsporidia MB-negative mosquitoes fed on Plasmodium-negative blood, and Microsporidia MB-negative mosquitoes fed on Plasmodium-positive blood across three time points including 24-, 48- and 72-hours post blood meal (Fig. 1A).

We observed that at the 24-hour time point, the samples from the various treatments clustered randomly (Fig. 1B), while 48 and 72 hours later they exhibited group specific clustering patterns (Fig. 1C and 1D).

DEGs distribution across treatments

In Microsporidia MB-negative mosquitoes exposed to Plasmodium-positive blood compared to Microsporidia MB-negative mosquitoes exposed to Plasmodium-negative blood, 20 genes were differentially expressed 24 hours post-blood meal, with 12 downregulated and 8 upregulated in mosquitoes exposed to Plasmodium positive blood (Supplementary table 1). At 48 hours, the number of DEGs increased to 54, with 29 upregulated and 25 downregulated (Supplementary table 2). None of the genes were differentially expressed 72 hours post blood meal (Supplementary table 3) (Fig. 2A).

In contrast, in Microsporidia MB-positive mosquitoes exposed to Plasmodium-negative blood compared to Microsporidia MB-negative mosquitoes exposed to Plasmodium-negative blood, 525 genes were differentially expressed 24 hours post-blood meal, including 30 upregulated and 495 downregulated (Supplementary table 4). At 48 hours, 230 genes were differentially expressed, with 132 upregulated and 98 downregulated (Supplementary table 5), while at 72 hours, 434 genes were differentially expressed, including 363 upregulated and 71 downregulated genes (Supplementary table 6) (Fig. 2B).

When comparing Microsporidia MB-positive mosquitoes exposed to Plasmodium-positive blood with Microsporidia MB-negative mosquitoes exposed to Plasmodium-positive blood, 68 genes were differentially expressed 24 hours post-blood meal, with 23 upregulated and 45 downregulated (Supplementary table 7). At 48 hours, 63 genes were differentially expressed, including 62 upregulated and 1 downregulated (Supplementary table 8), while at 72 hours only 1 gene was differentially expressed, which was upregulated (Supplementary table 9) (Fig. 2C).

In Microsporidia MB-positive mosquitoes exposed to Plasmodium-positive blood compared to Microsporidia MB-positive mosquitoes exposed to Plasmodium-negative blood, 2 genes were differentially expressed 24 hours post-blood meal (Supplementary table 10), with 1 upregulated and 1 downregulated. At 48 hours, 377 genes were differentially expressed, with 33 upregulated and 344 downregulated (Supplementary table 11), while at 72 hours, 6 genes were differentially expressed, including 5 upregulated and 1 downregulated (Supplementary table 12).

DEGs in Microsporidia MB-negative mosquitoes fed on P. falciparum-positive blood compared to -negative mosquitoes fed on P. falciparum-negative blood

24 hours post blood meal

The downregulated genes when comparing Microsporidia MB-negative mosquitoes fed on P. falciparum-positive blood to Microsporidia MB-negative mosquitoes fed on P. falciparum-negative blood at this time point included long-chain fatty acid transport protein 4-like, ketohexokinase-like, serine/threonine-protein kinase Smg1-like, aspartic peptidase, U1 & U2 spliceosomal RNA, and fatty acid synthase while the upregulated genes included zona pellucida domain and myofilin (Fig. 3A).

48 hours post blood meal

Histone-lysine N-methyltransferase Suv4-20, PHD finger protein 14, serine/threonine-protein kinase tousled-like 1, zinc finger SWIM domain-containing protein 8, mucin-19-like, glutamine-dependent NAD(+) synthetase, NPC intracellular cholesterol transporter 2-like, and glutathione S-transferase 1 included the downregulated genes while small heat shock protein, heat shock protein 70 A1, lipase 3-like, cytochrome P450 6g1-like, and D-beta-hydroxybutyrate dehydrogenase were among the upregulated genes when comparing Microsporidia MB-negative mosquitoes fed on P. falciparum-positive blood to -negative mosquitoes fed on P. falciparum-negative blood at this time point (Fig. 3B).

72 hours post blood meal

There were no significantly differentially expressed genes at this time point when comparing Microsporidia MB-negative mosquitoes fed on P. falciparum-positive blood to -negative mosquitoes fed on P. falciparum-negative blood (Fig. 3C).

Differentially expressed genes (DEGs) in Microsporidia MB-positive mosquitoes fed on P. falciparum-negative blood compared to -negative mosquitoes fed on P. falciparum-negative blood

24 hours post blood meal

Peptidoglycan-recognition protein, glutathione S-transferase 1, carboxypeptidase Q-like, phenoloxidase-activating factor, phenoloxidase 2-like, leucine-rich repeat, serine protease inhibitor, cecropin-A, toll-like receptor 7, chymotrypsin-2-like, cell death abnormality protein 1-like, low-density lipoprotein receptor, apolipophorin-3-like, lipid storage droplets surface-binding protein 1, lipid transport protein, fatty acid synthase, UDP-glycosyltransferase, NPC intracellular cholesterol transporter 2-like, glycine-rich cell wall structural protein 1.8-like and vitellogenin-A1 like were among the downregulated genes, while serine proteases, lipase 3-like, NADPH–cytochrome P450 reductase, adenylyltransferase and sulfurtransferase MOCS3 and neuropeptide F were included in the upregulated genes at this time point when comparing Microsporidia MB-positive mosquitoes fed on P. falciparum-negative blood to -negative mosquitoes fed on P. falciparum-negative blood (Fig. 4A).

48 hours post blood meal

The downregulated genes at this time point when comparing Microsporidia MB-positive mosquitoes fed on P. falciparum-negative blood to -negative mosquitoes fed on P. falciparum-negative blood included low-density lipoprotein receptor-related protein 2, alkaline phosphatase 4, proline dehydrogenase 1, dehydrogenase/reductase SDR family, aspartic peptidase domain superfamily, leucine-rich PPR motif-containing protein, serine protease inhibitor 88Ea-like, phenoloxidase-activating factor 2-like, serine/threonine-protein kinase Smg1-like, peptidase A2A, PDZ domain, zinc finger SWIM domain, V-type proton ATPase, major facilitator superfamily and tyrosine-protein kinase while serine proteases, lysozyme c-1-like, leucine-rich repeat neuronal protein 2, galectin-7-like, trypsin-5-like, antigen 5 like allergen, tumor necrosis factor alpha-induced protein (TNF), death-associated inhibitor of apoptosis 2-like, glutathione S-transferase 1-like, peptidase C45, cytochrome P450, UDP-glycosyltransferase, lipase 3-like, probable phospholipid-transporting ATPase IA, D-beta-hydroxybutyrate dehydrogenase, 17-beta-hydroxysteroid dehydrogenase, ecdysteroid kinase-like, estradiol 17-beta-dehydrogenase, and serine protease inhibitor were upregulated (Fig. 4B).

72 hours post blood meal

Among the downregulated genes at this time point when comparing Microsporidia MB-positive mosquitoes fed on P. falciparum-negative blood to -negative mosquitoes fed on P. falciparum-negative blood were SH3 domain, low-density lipoprotein receptor-like, glutamate dehydrogenase and isocitrate dehydrogenase, while leucine-rich repeat, L-dopachrome tautomerase yellow-f2-like, phenoloxidase-activating factor 2-like, phenoloxidase 2-like, serine proteases, CLIP domain-containing serine protease, antichymotrypsin-2-like, insulin-like growth factor-binding protein complex acid labile subunit, zinc finger protein, glycine-rich cell wall structural protein, fibrinogen-like protein A, apoptosis-inducing factor 3-like, carboxypeptidase N, serine protease inhibitor, apolipophorins, apolipoprotein D-like, lipid transport protein, lipid storage droplets surface-binding protein, lipase 3-like, fatty acid synthase, cytochrome P450, UDP-glucosyltransferase, glucose dehydrogenase, and mucin-5AC were among the upregulated genes (Fig. 4C).

DEGs in Microsporidia MB-positive mosquitoes fed on P. falciparum-positive blood compared to -negative mosquitoes fed on P. falciparum-positive blood

24 hours post blood meal

The toll-like receptor 7, peptidoglycan-recognition protein LB, NADH-quinone oxidoreductase subunit B-like, Aspartic peptidase domain, alkaline phosphatase 4, Ecdysteroid kinase-like, Haemolymph juvenile hormone binding, vitellogenin-A1-like, sodium-dependent nutrient amino acid transporter and glucosamine-6-phosphate isomerase were among the downregulated genes, while Acyltransferase 3 domain, lipase 3-like, homeobox protein PKNOX2-like, actin-binding Rho-activating protein-like, kinetochore protein NDC80 homolog and Ecdysteroid kinase-like genes were upregulated at this time point when comparing Microsporidia MB-positive mosquitoes fed on P. falciparum-positive blood to -negative mosquitoes fed on P. falciparum-positive blood (Fig. 5A).

48 hours post blood meal

Some of the downregulated genes included heat shock protein 70 A1-like, protein lethal(2)essential for life-like, Zinc finger, FAD-linked sulfhydryl oxidase ALR, fidgetin-like protein 1, complement component 1 Q subcomponent-binding protein, BTB/POZ domain-containing protein 9-like, sodium-coupled monocarboxylate transporter 1, aspartic peptidase domain, cytochrome P450 9b2-like and peroxiredoxin-6-like, while RNA pseudouridylate synthase domain was upregulated at this time point when comparing Microsporidia MB-positive mosquitoes fed on P. falciparum-positive blood to -negative mosquitoes fed on P. falciparum-positive blood (Fig. 5B).

72 hours post blood meal

At this point we only observed one differentially expressed gene, which was upregulated, namely serine proteases (Supplementary table 9, Fig. 5C).

DEGs in Microsporidia MB-positive mosquitoes fed on P. falciparum-positive blood compared to -positive mosquitoes fed on P. falciparum-negative blood

24 hours post blood meal

The D-amino-acid oxidase was upregulated when comparing Microsporidia MB-positive mosquitoes fed on Plasmodium-positive blood to those fed on -negative blood (Supplementary table 10, Fig. 6A).

48 hours post blood meal

Downregulated genes at this time point when comparing Microsporidia MB-positive mosquitoes fed on P. falciparum-positive blood to Microsporidia MB-positive mosquitoes fed on P. falciparum-negative blood comprised of peptidoglycan-recognition protein SC2-like, apoptosis-inducing factor 1 and 3, programmed cell death protein 7, glutaredoxin-C4, peroxiredoxin 1, thioredoxin-2, galectin-4-like, galectin-7-like, lipopolysaccharide-induced tumor necrosis factor-alpha factor homolog (LITAF), glutathione S-transferase 1-1-like, leucine-rich repeat neuronal protein 2, serine protease inhibitor, stress-activated protein kinase JNK-like, zinc finger protein 2 homolog, complement component 1 Q subcomponent-binding protein, probable cytochrome P450 6a14, FAD-linked sulfhydryl oxidase ALR, UDP-glycosyltransferase, NPC intracellular cholesterol transporter 2 homolog a, acyl-CoA desaturase, serine palmitoyltransferase, aspartic and glutamic acid-rich protein and fidgetin-like protein 1, while hexokinase type 2, protein HID1, neuronal calcium sensor 2 and mitochondrial uncoupling protein 4 comprised the upregulated genes (Fig. 6B).

72 hours post blood meal

The Cytochrome P450 4g15 was the only downregulated gene, while U1, U2, U4 spliceosomal RNA were upregulated when comparing Microsporidia MB-positive mosquitoes fed on P. falciparum-positive blood to Microsporidia MB -positive mosquitoes fed on P. falciparum-negative blood (Supplementary table 12, Fig. 6C).

Enriched pathways in Microsporidia MB-positive mosquitoes fed on P. falciparum-negative blood compared to -negative mosquitoes fed on P. falciparum-negative blood

24 hours post blood meal

When Microsporidia MB-positive mosquitoes were compared to Microsporidia MB-negative mosquitoes, 4 terms spanning biological, cellular and molecular pathways were enriched from the downregulated genes, including extracellular region, oxidoreductase activity, transmembrane transporter activity, and cellular amino acid metabolic process (Supplementary table 13, Fig. 7A).

48 hours post blood meal

None of the enriched terms from differentially expressed genes were significant when the guts from Microsporidia MB-positive mosquitoes were compared to those from -negative mosquitoes (Supplementary table 14).

72 hours post blood meal

At this time point, 7 terms spanning biological, cellular and molecular processes were significantly enriched from the upregulated genes when comparing guts from Microsporidia MB-positive mosquitoes to those from Microsporidia MB-negative mosquitoes, both fed on Plasmodium-negative blood. Significantly enriched terms included the cellular amino acid metabolic process, extracellular region and space, peptidase activity, oxidoreductase activity, and enzyme regulator activity (Supplementary table 15, Fig. 7B).

Gut microbiota profiling of the transcriptionally active microbes (dup: abstract ?)

QIIME2 analysis of bacteria sequences mined from the RNAseq data yielded 267,173 bacterial amplicon sequence variants (ASVs) cumulatively, from which Mitochondria, Chloroplast, Archaea, Cyanobacteria, Chloroflexi, Eukaryota, and those classified as unassigned were filtered leaving behind a total of 100,907 ASVs. Next, ASVs with low abundances were filtered leaving 18570 ASVs remaining that were used for quantitative analysis. Twenty-three microbes with relative abundances above 0.5% were classified as the most abundant in the whole dataset. Elizabethkingia, Serratia, Klebsiella, Pectobacterium and Enterobacter were the most abundant microbes cumulatively with relative abundances of 20.46%, 13.52%, 11.65%, 11.34% and 8.54% respectively (Supplementary table 16, Fig. 7). Other highly abundant bacterial members included Nissibacter, Izhakiella, Brenneria, Winslowiella, Asaia, Hafnia, Enterobacillus, Dickeya, Chryseobacteria, Acetobacter, Soonwooa, Kosakonia, Hydrocarboniclastica, Gluconobacter, Citrobacter, Erwinia, Gluconacetobacter, and Shewanella (Supplementary table 16, Fig. 8).

Interesting patterns were evident from the distribution of the relative abundances in some of the microbes across time points, which may inform about the physiology of the mosquitoes, across the various treatments. Case in point, Elizabethkingia and Cryseobacterium proliferated in relative abundances across all treatments from the 24th hour time point to the 48th. While Asaia also recorded proliferation across treatments through to the 48th hour time point, it recorded a drop in relative abundance from the 24th to the 48th hour in Microsporidia MB-positive mosquitoes fed on Plasmodium positive blood. Serratia and Enterobacter, also demonstrated interesting patterns as Serratia proliferated in Microsporidia MB-positive mosquitoes fed on Plasmodium-positive blood, 48 hours post blood meal, while Enterobacter sustained a relatively high abundance from the 24th up to the 48th hour time point in the same treatment, while it was dropping in the other treatment groups (Supplementary table 17, Fig. 8).

We report that Microsporidia MB infection in An. arabiensis is associated with distinct, time-dependent modulation of immune, metabolic, and physiological processes that differ with P. falciparum exposure. In Microsporidia MB-negative mosquitoes, P. falciparum infection induced structural and immune signaling changes in gut tissues, suggesting parasite-driven immune evasion. In Microsporidia MB-positive mosquitoes exposed to P. falciparum-negative blood on the other hand, responses ranged from early targeted immune activation, that was then sustained across the time points to metabolic remodelling that could be informative of existing Microsporidia MB-host interaction. Interestingly, Microsporidia MB-positive mosquitoes exposed to P. falciparum-positive blood, indicated an early disruption of parasite establishment through hormonal remodelling, and mid-phase nutrient restriction and immune suppression, suggestive of an inhospitable gut environment for the Plasmodium parasite’s development. Further, the gut microbiota profiling of the most abundant microbes from this study showed that the most abundant microbiota members encompassed both what has been considered the core mosquito gut microbiota members as well as transient gut microbiota members. Interestingly, some of the highly abundant microbes have associated roles in malaria transmission blocking and could be suggestive of a modulated gut microbiota structure in Microsporidia MB-positive mosquitoes to limit the proliferation of Plasmodium during an infection. Together, these patterns suggest that Microsporidia MB not only reprograms mosquito physiology in a phase-specific manner but also counteracts P. falciparum induced changes, potentially blocking transmission without major fitness costs to the host.

Additionally, the transcriptional responses revealed that Plasmodium infection alone induced only modest and transient changes in Microsporidia MB-negative mosquitoes, with few DEGs that disappeared by 72 hours post-blood meal, whereas Microsporidia MB infection triggered a much broader and more sustained modulation of mosquito gene expression. In Microsporidia MB-positive mosquitoes, early responses were dominated by widespread downregulation, followed by a shift toward strong upregulation at later time points, highlighting a dynamic regulatory pattern. When both infections were combined, transcriptomic differences peaked at 24–48 hours but largely reverted to baseline by 72 hours, suggesting that Microsporidia MB primarily alters the early host response to Plasmodium. Together, these patterns indicate that Microsporidia MB exerts a stronger and longer-lasting impact on mosquito gene expression than Plasmodium alone, potentially reshaping host physiology in ways that could influence parasite establishment and transmission.

DEGs in Microsporidia MB-negative mosquitoes fed on P. falciparum-positive blood compared to -negative mosquitoes fed on P. falciparum-negative blood

24 hours post-blood meal

We observed gut structural reinforcement, dampened immune signalling, suppressed spliceosomal activity, and metabolic remodelling in Microsporidia MB-negative mosquitoes exposed to Plasmodium. Specifically, myofilin, potentially linked to gut integrity (Qiu et al., 2005), was upregulated, while serine/threonine kinase, involved in immune signalling and hemocyte differentiation (Chiou et al., 1998), was downregulated. Such reduced immune signalling may create conditions more permissive for parasite development. Concurrently, downregulation of U1 and U2 spliceosomal RNAs (López et al., 2008), and fatty acid synthase (Chotiwan et al., 2022) suggests suppressed global gene expression and altered lipid metabolism respectively, consistent with parasite-driven modulation of host processes to favour infection.

48 hours post-blood meal

Microsporidia MB-negative mosquitoes exposed to Plasmodium infection showed sustained lipolysis and signs of nutrient depletion, accompanied by stress responses and chromatin modifications. In addition, immunity was suppressed, alongside reduced expression of structural and regulatory genes, suggesting ongoing parasite driven host reprogramming. Specifically, the lipase 3-like gene was upregulated, which is in contrast with previous studies that reported that lipolytic activity in mosquito midguts peaks ~ 15 hours after blood feeding and declines steadily thereafter, reaching low levels by 48 hours and the lowest levels by 72 hours (Geering & Freyvogel, 1975; Pirahanchi & Sharma, 2023). In addition, D-beta-hydroxybutyrate dehydrogenase, encoding a lipid-requiring enzyme involved in ketogenesis (Fleischer et al., 1983), was also upregulated. The upregulation of the two genes suggests sustained lipid catabolism while the downregulation of NPC intracellular cholesterol transporter involved in lipid transport suggests a potential state of nutrient depletion, possibly driven by metabolic demands of developing Plasmodium parasites. Such nutrient limitations may impose cellular stress, consistent with the upregulation of a small heat shock and heat shock proteins.

In addition, the downregulation of histone-lysine N-methyltransferase Suv4-20 and PHD finger protein 14 suggest chromatin modification in Microsporidia MB-positive mosquitoes exposed to Plasmodium infection. Chromatin reprogramming in response to Plasmodium infection was reported to be context dependent (Ruiz et al., 2019). In our data, the observed chromatin alterations may contribute to immune suppression, as further supported by the concurrent downregulation of glutathione S transferase that could function upstream in the melanization cascade. Genes associated with structural integrity (mucin-19) (Hodžić et al., 2025) and gene regulation (zinc finger SWIM domain-containing protein 8, serine/threonine-protein kinase tousled-like 1) (Darai et al., 2022; Gao et al., 2023) were downregulated, possibly reflecting near-completion of blood digestion and chromatin remodelling.

Cumulatively, our findings indicate that P. falciparum infection in Microsporidia MB-negative mosquitoes substantially alters metabolic, structural, and immune-related gene expression. The patterns suggest that parasite presence influences host nutrient allocation, stress responses, chromatin state, and immune readiness, potentially creating a more favourable environment for parasite development.

Differentially expressed genes (DEGs) in Microsporidia MB-positive mosquitoes fed on P. falciparum-negative blood compared to -negative mosquitoes fed on P. falciparum-negative blood

24 hours post-blood meal

The immune system in Microsporidia MB–positive mosquitoes appeared selectively modulated, with the upregulation of some effectors (serine proteases) (Dong & Dimopoulos, 2009; El Moussawi et al., 2019a), alongside the broad suppression of pattern-recognition receptors (PGRPs, toll like receptor 7, LRR proteins), melanization linked factors (including CLIP domain containing serine proteases, phenoloxidase 2-like, and phenoloxidase activators), cecropin A and cell-death abnormality protein 1-like (Gabrieli et al., 2021; Kumar et al., 2018). This pattern suggests immune remodeling, suppressing certain pathways to limit tissue damage or support microbial colonization, while priming others for parasite defense. Concurrently, genes involved in lipid storage and transport were downregulated (lipoprotein receptor-related protein 1, apolipophorin-3-like, lipid storage droplet surface-binding protein 1, lipid transport protein, apolipoprotein, NPC intracellular cholesterol transporter like and fatty acid synthase) (Gao et al., 2024), while lipase 3-like (lipid breakdown) was upregulated (Pirahanchi & Sharma, 2023). Given the acyltransferases in the Microsporidia MB genome (Ang’ang’o et al., 2024), this may reflect competition for host fatty acids that could indirectly reduce Plasmodium fitness.

48 hours post-blood meal

Lipid metabolism remained reprogrammed with the downregulation of lipid transport and metabolism-related genes, (e.g., low-density lipoprotein receptor-related protein 2, NPC cholesterol transporter 2-like), (Lei et al., 2023; Storch & Xu, 2009; Y. Zhao et al., 2024), and upregulation of catabolic enzymes including lipase 3-like, D-β-hydroxybutyrate dehydrogenase (ketone metabolism), and a phospholipid-transporting ATPase (FLEISCHER et al., 1983; Pirahanchi & Sharma, 2023). These changes suggest a continued metabolic remodeling in Microsporidia MB–positive mosquitoes, that may support host–symbiont interactions. Immunity on the other hand shifted towards activation, with increased expression of leucine-rich repeat proteins, zinc-finger proteins, serine proteases, lysozyme C1, galectin 7, GST1-like, apoptosis inhibitors, and TNF-α-induced protein 8-like (Kumar et al., 2018; Mitri & Vernick, 2012; Molina-Cruz et al., 2008; Rosales, 2017; Samantsidis et al., 2024), marking a shift from the primed immune state at 24 h towards stronger activation. Notably, TNF, linked to Plasmodium refractoriness and oenocytoid lysis in An. gambiae (Samantsidis et al., 2024), was strongly induced, together with suppression of serine protease inhibitor 88Ea-like, indicating activation of melanization pathways, despite downregulation of phenoloxidase-activating factor 2-like. TNF-driven oenocytoid lysis may release preformed prophenoloxidases, transiently sustaining phenoloxidase activity. Finally, upregulation of ecdysone kinase-like (Scanlan & Robin, 2024) suggests reduced active ecdysone levels, which could limit ovarian lipid accumulation and impair Plasmodium development (Gao et al., 2024).

72 hours post-blood meal

Immune activation persisted, with induction of pattern-recognition receptors (e.g., LRR proteins, fibrinogen-like protein A) (Kumar et al., 2018), melanization enzymes (e.g phenoloxidase-activating factor 2-like, phenoloxidase 2-like, L-dopachrome tautomerase yellow-f2-like) and apoptotic factors (apoptosis-inducing factor 3-like), consistent with a late-phase immune responses (De Gregorio et al., 2002; El Moussawi et al., 2019b). Lipid metabolism genes were broadly upregulated at this stage (apolipoprotein D-like, MD-2-related lipid-recognition protein-like, lipid storage droplet surface-binding protein 1, lipase 3-like, fatty acid synthase, lipid transport protein, and low-density lipoprotein receptor-related protein 2) (Gao et al., 2024), likely reflecting mobilization of lipids for oogenesis and storage.

Our data suggest that Microsporidia MB infection in An. arabiensis induces a time-dependent shift in immune and metabolic pathways, with early oenocytoid lysis–driven melanization and altered hormone and lipid regulation, followed later by classical phenoloxidase activation. These findings align with Waweru et al. (In Press), which also reported immune activation via factors such as LITAF, linked to hemocyte differentiation (Smith et al., 2012). The slight differences in timing of immune activation, occurring at 24 hours in the earlier study versus ~ 48 hours here, may reflect differences in blood meal source, environmental conditions, or gut microbiota composition. Despite these variations, both studies highlight the central role of cellular immunity in the Microsporidia MB–mosquito interaction.

DEGs in Microsporidia MB-positive mosquitoes fed on P. falciparum-positive blood compared to -negative mosquitoes fed on P. falciparum-positive blood

24 hours post-blood meal

In Microsporidia MB–positive mosquitoes exposed to P. falciparum–positive blood, most immune-related genes, including toll-like receptor 7 and peptidoglycan recognition protein LB (González-Santoyo & Córdoba‐Aguilar, 2012; Welte et al., 2009), were downregulated, suggesting immune remodeling not directly involving these pathways. Downregulation and upregulation of ecdysteroid kinase-like suggests tightly regulated levels of the ecdysone hormone (Gao et al., 2024; Scanlan & Robin, 2024; Werling et al., 2019). While elevated ecdysone hormone levels typically support lipid accumulation and Plasmodium growth, this effect may be offset by the possible tight regulation of the hormone levels, vitellogenin A1 downregulation and lipase upregulation, associated with depleted lipid reserves that impair parasite development (Gao et al., 2024). The additional downregulation of metabolic genes (e.g., alkaline phosphatase and amino acid transporters) likely further restricts the available nutrients for Plasmodium parasite development. Lastly, the reduced expression of juvenile hormone binding protein (JHBP) may indicate suppressed JH activity and a shift in immune strategy, favoring phagocytosis but limiting melanization, highlighting a possible trade-off in defense mechanisms (Ahmed et al., 1999; Kim et al., 2020).

48 hours post-blood meal

At this stage, several immune-related genes (e.g., FAD-linked sulfhydryl oxidase, zinc-finger proteins, complement-related factors, peroxiredoxin 6 like) (Andersen et al., 2024; Magalhaes et al., 2008; Zhang et al., 2025), and stress-response genes (heat shock protein and protein lethal(2) essential for life) (Singh et al., 2025), were downregulated, suggesting that sustained immune activation is not central to Microsporidia MB’s transmission-blocking effect at this time point. Instead, strong downregulation of nutrient acquisition and metabolic genes, including monocarboxylate transporters and aspartic peptidase domain, points to a resource-limiting environment unfavorable to Plasmodium (Halestrap, 2012; Rivera-Pérez et al., 2017).

Overall, the findings indicate that Microsporidia MB may impair Plasmodium development partly through cellular immunity, though no strong immune activation is evident in Plasmodium infected mosquitoes. In addition, Plasmodium appears to suppress immunity even in Microsporidia MB positive mosquitoes, but the consistency in the disruption of metabolic processes in Microsporidia MB positive mosquitoes even after exposure to Plasmodium infection likely underpins its Plasmodium transmission blocking phenotype. Lastly, hormonal pathways, particularly ecdysone and juvenille hormone, also emerge as potential regulators of Plasmodium transmission blocking, which warrants further investigations.

DEGs in Microsporidia MB–positive mosquitoes fed on P. falciparum–positive blood compared to Microsporidia MB–positive mosquitoes fed on P. falciparum–negative blood

24 hours post-bloodmeal

The most notable change was the upregulation of D-amino acid oxidase, encoding an enzyme that deaminates D-amino acids to produce ammonia and hydrogen peroxide (Corrigan et al., 1962), in positive mosquitoes exposed to Plasmodium infection. This activity could potentially disrupt the mosquito gut homeostasis through hydrogen peroxide production or microbiota modulation, thereby hindering early parasite establishment.

48 hours post-bloodmeal

A broad downregulation of immune (e.g. apoptosis-inducing factors, autophagy-regulators, melanization-associated, cellular-immunity related) (Ahn & Marygold, 2021; De Gregorio et al., 2002; El Moussawi et al., 2019b; Fisher, 2011; Gabrieli et al., 2021; González-Santoyo & Córdoba‐Aguilar, 2012; Kamhawi et al., 2004; Kumar et al., 2018; Lee et al., 2019; Raddi et al., 2020; Ranson & Hemingway, 2005; Sonu Koirala et al., 2022; Tzotzos, 2025), detoxification (cytochrome P450 6a14, GSTs) (Ahn & Marygold, 2021; Ranson & Hemingway, 2005; Sonu Koirala et al., 2022; Tzotzos, 2025) and lipid metabolism genes (cholesterol transporters, desaturases) was observed. These observations suggest parasite-mediated immune suppression, redox imbalance, and nutrient restriction that could limit parasite growth. Notably, genes associated with metabolism hexokinase type 2 and protein HID1 were upregulated.

72 hours post-bloodmeal

The expression patterns persisted from that at 48 h, with further downregulation of cytochrome P450 and sustained upregulation of spliceosomal RNAs, reinforcing the role of Microsporidia MB in altering transcriptional regulation and metabolic balance in infected mosquitoes exposed to Plasmodium-positive blood.

Collectively, these findings suggest a staged effect: early disruption of gut environment (24 h), followed by immune suppression and metabolic restriction (48–72 h), creating conditions that impair P. falciparum survival and transmission.

Gut microbiota profiling of the transcriptionally active microbes

Mosquito gut microbiota profiling revealed both core members (e.g. Elizabethkingia, Serratia) and transient taxa (e.g. Soonwooa, Kosakonia), with their relative abundances shifting across treatments. These findings suggest the dynamic nature of the mosquito gut microbiota and their responsiveness to both Microsporidia MB infection and P. falciparum exposure status. Notably, Elizabethkingia and Chryseobacterium proliferated from 24-48h post-blood meal across all treatments in this study. This may potentially be associated with digestion and provision of essential nutrients post blood meal, given the associated HemeS gene in both microbes which has a role in heme breakdown in blood fed mosquitoes (Ganley et al., 2020). The modulation of Asaia abundance, particularly its decline in Microsporidia MB-infected mosquitoes exposed to Plasmodium, is notable given its importance in paratransgenesis and capacity to interfere with Plasmodium development (Favia et al., n.d.; Ricci et al., 2012).

Likewise, the proliferation of Serratia and the sustained high levels of Enterobacter in Microsporidia MB–positive mosquitoes fed on Plasmodium-infected blood may suggest synergistic interactions between endosymbionts and specific gut bacteria that reinforce anti-Plasmodium responses, as both taxa have been associated with limiting parasite development via immune priming and release of reactive oxygen species, respectively (Bahia et al., 2014; Chiou et al., 1998; Cirimotich et al., 2011). The consistency in Serratia proliferation with that in the initial study by Waweru et al. (In Press), suggests that Serratia could be a key player in Microsporidia MB’s-Plasmodium transmission blocking phenotype. Although environmental variation may account for some differences in microbiota composition between this study and that of Waweru et al. (In press), together these findings strengthen the evidence that Microsporidia MB infection not only reshapes gut microbiota structure but also selectively favors taxa with functional traits that may enhance vector resistance to Plasmodium.

In conclusion, our findings indicate that Microsporidia MB infection reprograms the mosquito physiology in a manner that collectively limits Plasmodium development. The upregulation of phenoloxidases, CLIP-domain serine proteases, and dopachrome tautomerase in Microsporidia MB positive mosquitoes supports a reinforced melanization response, initiated by early oenocytoid lysis and sustained by enzymatic activation at later time points. These findings point to cellular immune responses being central to the Microsporidia MB – Plasmodium transmission blocking phenotype, which could also be associated with hormonal signalling in Microsporidia MB infected mosquitoes. Specifically, the differential expression of ecdysone kinase and juvenille hormone binding protein points to hormonal remodeling, which may influence reproductive investment, and immune homeostasis. In addition, enrichment of cellular amino acid metabolic process in Microsporidia MB positive mosquitoes alongside energy metabolism remodelling after Microsporidia MB mosquitoes are exposed to Plasmodium infections suggests nutrient reallocation, restricting resources available to the parasite while sustaining host defense. Finally, evidence from the gut microbiota profiling reinforces the Plasmodium transmission blocking phenotype exhibited by Microsporidia MB through the proliferation of microbes with anti-Plasmodium properties. Together, these changes—enhanced melanization, altered hormonal balance, redirected nutrient flow and gut microbiota profiling—create a hostile internal environment that helps explain the malaria transmission-blocking phenotype of Microsporidia MB.

Description of the study samples

Wild gravid female An. arabiensis mosquitoes were caught indoors from inside walls of houses neighboring the Ahero irrigation scheme (-34.9190W, -0.1661N) in Kenya. The gravid G₀ females were induced to oviposit inside perforated 1.5 ml microcentrifuge tubes containing 50 µl of distilled water and a soaked Whatman paper (1 cm × 1 cm in size). Upon egg laying, DNA was extracted from the G₀ females and screened for infection with Microsporidia MB using polymerase chain reaction (PCR), targeting the 18S rRNA gene. The eggs laid by each Microsporidia MB-positive and -negative gravid G₀ female were separately transferred to larval-rearing trays for larval development under ambient screenhouse conditions (26–28°C temperature, 70–80% humidity). Depending on infection status of the G₀ mother with Microsporidia MB, resultant pupae were pooled together and transferred to 30 X30 X30 cm netted cages for adult emergence and rearing. A maximum of 250 adult mosquitoes were placed in each cage and were maintained on 6% glucose solution ad libitum, at ambient laboratory conditions (26–28°C temperature, 70–80% humidity).

The experimental design included G₁ females from roughly 50 different Microsporidia MB-infected and -uninfected G₀ field collected mothers. To serve as controls, only G₁ females from Microsporidia MB-negative G₀ mothers were included. While G₁ females from Microsporidia MB-negative G₀ mothers may have different genetic backgrounds from G₁ females from Microsporidia MB-positive mothers, we controlled for genetic background differences in the experiment by including Microsporidia MB-positive G₁ females from different mothers and different field collections in the experiment across the different time points.

Plasmodium direct membrane feeding assays

Blood drawn from the study participants was first screened for malaria using the Rapid Diagnostic Test (RDT) kits (SD Bioline, UK). Microscopy was used to confirm the presence of Plasmodium gametocytes in RDT-positive samples. The blood samples positive for P. falciparum in this study had a gametocyte range count of between 2–3/200 WBC. A volume of 4 ml of blood was drawn from P. falciparum gametocyte positive patients, following consent from both patient and/or guardian. In addition, a similar volume of Plasmodium-free blood was obtained from study participants that were negative for P. falciparum infection, to serve as controls. Blood samples were preserved in vacutainers containing heparin. Prior to blood feeding, mosquitoes were starved for 4 hours after which they were allowed to blood feed for one hour, to allow them enough time to ingest the gametocytes. After blood feeding, the mosquitoes were sorted, and the fully engorged mosquitoes were separated and maintained for the duration of the experiment, while the non-blood-fed mosquitoes were discarded. The blood-fed mosquitoes from the various treatment conditions were dissected at 24, 48, and 72 hours post the blood meal, to obtain guts, fat body and the ovary tissues, which were processed for molecular detection of Microsporidia MB and P. falciparum parasites. The assay yielded four experimental groups: (i) those positive for Microsporidia MB but negative for P. falciparum (Microsporidia MB +|Plasmodium -), (ii) those positive for Microsporidia MB and exposed to P. falciparum (Microsporidia MB +|Plasmodium +) (iii) those negative for Microsporidia MB but exposed to P. falciparum (Microsporidia MB -|Plasmodium +), and iv) those negative for both Microsporidia MB and P. falciparum (Microsporidia MB -|Plasmodium -).

RNA and DNA extraction

The Trizol reagent (Invitrogen, Waltham, Massachusetts, United States) was used to extract RNA and DNA from the dissected tissue specimens according to the manufacturer’s instructions. Briefly, chloroform was used to isolate the RNA phase while isopropanol was used in precipitating RNA, followed by two washes with 75% ethanol, drying and elution using nuclease-free water. DNA on the other hand was extracted from the organic phase obtained after RNA extraction, using the back extraction buffer (BEB) [4 M Guanidine thiocyanate, 50 mM sodium citrate, 1 M Tris (free base)]. DNA was also precipitated using isopropanol followed by two 70% ethanol washes, drying and elution using nuclease-free water. RNA and DNA purity were determined using NanoDrop One/Oneᶜ Microvolume UV-Vis Spectrophotometer (ThermoFischer Scientific, Waltham, Massachusetts, United States), while their quantification was done using Qubit™ 4 Fluorometer (Invitrogen, Waltham, Massachusetts, United States). Of the total RNA extracted 50 ng per sample were used for cDNA synthesis while DNA from the corresponding samples was used for Microsporidia MB and P. falciparum screening. Samples were classified as Microsporidia MB-positive throughout the study based on detection of infection in the ovaries.

Complementary DNA (cDNA) synthesis and RNA sequencing

Complementary DNA synthesis was done on 80 gut tissues in this study. Six replicates were considered with the 24- and 48-hour time points, while 4 replicates were included with the 72 hour time point. The QuantiTect reverse transcriptase kit (Qiagen, Hilden, Germany)

was used for cDNA synthesis according to the manufacturer’s manual. Briefly, genomic DNA was first eliminated by incubating the samples together with the genomic DNA elimination reaction components for 2 minutes at 42°C. Next, first strand cDNA synthesis was done using the specified reagents in the kit, and incubation specifications for the step (30 minutes at 42°C), after which the reverse transcription was stopped by incubating for 3 minutes at 95°C. The generated cDNA for each sample was stored in -80°C until when shipped for sequencing at BGI Genomics (https://www.bgi.com/global) using the DNBSeq technology (second strand cDNA synthesis, agnostic cDNA library amplification and sequencing).

Bioinformatics analysis and differential gene expression analysis

The quality of the resulting sequencing data was determined using FastQC (Version 0.12.1) and MultiQC (Version 1.23). Eukaryotic ribosomal RNA was found to be overrepresented in this data as determined through BLAST (NCBI BLASTN). SORTMERNA (Version 4.3.4) was used to filter out ribosomal RNA contamination from the data by performing the filtering step twice. Next, the filtered reads were aligned against both the An. arabiensis (Anopheles arabiensis Dongola 2021, Species NCBI Taxon ID 7173) and P. falciparum (Plasmodium falciparum 3D7, Species NCBI Taxon ID 5833) transcriptome references from Vectorbase (release 68) using hisat2 (Version 2.1.0). The resulting SAM files were then converted to BAM files using samtools (Version 1.9). Next transcript counts were determined by using samtools (Version 1.9) and downstream statistical analysis done using edgeR (Version 4.2.1). To begin with, sample library sizes were normalized and the clustering of samples determined using PCA plots. Since none of the samples seemed to be an outlier on account of quality and library size, all variations observed were considered biological and hence all samples were included in the differential expression analysis. Genes were considered differentially expressed when their log fold change in expression was ≥ 1.5 and an adjusted P-value < 0.05. Lastly, gene enrichment analysis was done on the differentially expressed genes to identify their enriched Gene Ontology annotations using VectorBase (Release 68).

Bacterial reads were then mined from the RNAseq data by aligning them to the SORTMERNA (silva-bac-16s-id90.fasta reference database). The reads that mapped to the database were picked for downstream analysis using the QIIME2 software (Version 2024.5). Since the number of forward and reverse reads differed, downstream QIIME2 analysis was based on the forward reads as their counts were higher. The analysis steps in QIIME2 included denoising the data using the DADA2 pipeline in QIIME2, followed by taxonomic classification against the full-length Silva 138 database, using a pre-trained naïve Bayes classifier. The resulting feature, taxonomy and sequence tables from QIIME2 analysis were then read into R for further analysis. Amplicon sequence variants (ASVs) classified as Mitochondria, Chloroplast, Archaea, Cyanobacteria, Chloroflexi, Eukaryota and classified as unassigned reads at the kingdom level were filtered out from the analysis as a first step in R.

Subsequently, ASVs not classified up to the genus level and those with ambiguous classification had their sequences filtered out and re-classified using the NCBI blast 16S rRNA database. After this classification, reads classified by SILVA were merged with those classified by BLAST, a Phyloseq object was created, ASVs with abundance below 5 were filtered and an abundance plot of the highly present microbes was generated.

Ethical clearance

Ethical approval was obtained from the KEMRI Scientific Ethics Review Unit (KEMRI/RES/7/3/1) and the University of Cape Town HREC (REF: 663/2023). Written parental/guardian consent was secured for participating school children (5–15 years) from Mbita town and Homa Bay County. The study was classified as low risk, involving only blood collection for membrane feeding assays, with no direct contact between participants and mosquitoes.

Consent for publication

Not applicable

Availability of data and materials

Data supporting the findings reported by this study are available within the article or its supplementary files and can also be made available from the authors upon request.RNAseq and gut microbiota raw data have been submitted to the NCBI SequenceRetrieve Archive (SRA) database under bioproject number PRJNA1367057. The analysis scripts to this work have been archived in GitHub (https://github.com/Wahura/Host-Symbiont-Interraction-in-Microsporidia-MB-infected-An.-arabiensis-mosquitoes/tree/main)

Competing interests

The authors declare that they have no competing interests

Funding

The authors gratefully acknowledge the financial support for this research by the following organizations and agencies: Bill & Melinda Gates Foundation (INV0225840); the Swedish International Development Cooperation Agency (Sida); the Swiss Agency for Development and Cooperation (SDC); the Australian Centre for International Agricultural Research (ACIAR); the Government of Norway; the German Federal Ministry for Economic Cooperation and Development (BMZ); and the Government of the Republic of Kenya. The views expressed herein do not necessarily reflect the official opinion of the donors

Authors' contributions

J.W.W: conceptualization, designed experiments, molecular assays, data analysis, writing the initial draft and revising

D.M: Conceptualization, experimental design, analysis validation, writing and reviewing and supervision

J.G: dissection of samples, data analysis, manuscript writing and review

C.N.K: molecular analysis, data analysis and figure editing

N.M: Conceptualization, experimental design, analysis validation, writing and reviewing and supervision

J.K.H: Conceptualization, experimental design, analysis validation, writing and reviewing, supervision and funding acquisition

Acknowledgements

The authors acknowledge Ms Aclain Sishia, Dr Silvain Pinaud, and Dr Lilian Mbaisi for their incredible support with mosquito dissections and insights while performing all the experiments. We sincerely appreciate Faith Kyengo, Rose Marubu and Dr Thomas Onchuru for their administrative support and help with procurement of all reagents used in this study. We also sincerely acknowledge VectorBase (75), which was used to obtain genomic and functional annotation for Anopheles arabiensis genes, including gene ontology mappings and ortholog IDs (release version 67).

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Microsporidia MB mediated transcriptomics and gut microbiota shift in Plasmodium falciparum exposed Anopheles arabiensis mosquitoes

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DEGs distribution across treatments

Gut microbiota profiling of the transcriptionally active microbes (dup: abstract ?)

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Gut microbiota profiling of the transcriptionally active microbes

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RNA and DNA extraction

Bioinformatics analysis and differential gene expression analysis

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