Unveiling Wolbachia pipientis transcriptomic signature in Aedes aegypti: a comprehensive meta-analysis

The mosquito Aedes aegypti, main vector of arboviruses such as Dengue and Zika, causes major health burdens worldwide and is expanding its geographical range. A promising arboviral control strategy consists in replacing native A. aegypti populations with others artificially infected with the intracellular bacteria Wolbachia pipientis, which reduces the mosquito’s vectorial capacity. Despite the current success of this strategy, preserving its efficacy over time will depend on a solid mechanistic knowledge of the A. aegypti-Wolbachia symbiosis. Previous genome-wide transcriptomic studies have made progress in such understanding but no work has focused on systematically identifying effects of Wolbachia that are consistent across datasets. To identify A. aegypti genes and functions consistently affected by Wolbachia, we performed de novo differential expression and gene set enrichment analysis on published Wolbachia-infected A. aegypti transcriptomic datasets, followed by a meta-analysis of the obtained p-values using the maxP method. Individual de novo analysis revealed the downregulation of six opsins due to wMelPop infection in mosquito heads, which may contribute to the fitness loss that currently impairs the use of this strain for arboviral control. Meta-analysis revealed 31 consistently differentially expressed genes, some of which have escaped the focus of A. aegypti-Wolbachia research, such as a flavin-containing monooxygenase and farnesoic acid methyltransferases. We also found consistent upregulation of GO terms related to electron transport chain, carbohydrate transmembrane transport and serine-type peptidase activity/inhibition, as well as downregulation of DNA replication. These results motivate new hypotheses on the dominant A. aegypti factors involved in Wolbachia’s pathogen blocking, such as induction of oxidative stress depending on upregulation of host electron transport chain rather than dual- and NADPH-oxidases, which showed downregulation or no regulation across datasets. This work shows how added value can be obtained from previous experimental efforts, developing integrated insights on biological systems that are not reachable at the time of the original publications. Author summary Aedes aegypti is the primary vector for arthropod-borne viruses, including Dengue, Zika, and Chikungunya, which impose severe global health burdens. The geographical range of Aedes aegypti is expanding, further exacerbating these challenges. A promising strategy to combat these arboviruses involves artificially infecting native A. aegypti mosquitoes with the bacteria Wolbachia pipientis, which reduces their competence as vectors. Gene expression studies comparing Wolbachia-infected versus uninfected A. aegypti have developed mechanistic understanding on this biological system, which is crucial for enhancing Wolbachia-based vector control. However, the generality of the observed effects is unclear, as no study has systematically compared the available gene expression profiles from different studies. Here we jointly analyzed gene expression profiles from Wolbachia-infected A. aegypti in order to identify consistent effects of the bacteria in this host. Our results provided new gene targets and hypotheses relevant for Wolbachia-based arboviral control. For example, we found consistent upregulation of A. aegypti’s electron transport chain and hypothesized it to be a persistent mechanism of viral blocking, which is essential for the success of the control strategy. Our work illustrates how accumulation of related genome-wide omic datasets and evolution of bioinformatic resources can be leveraged to develop integrated insights into biological systems, maximizing the value of previous experimental efforts.


Introduction
To identify functional classes overrepresented among up-or downregulated genes from each dataset, each gene list was sorted in decreasing order according to its logarithmic fold-change and subjected to Gene Set Enrichment Analysis (GSEA) [37] against Gene Ontology functional terms (database version 2024-01) [38].
GSEA was preferred over other methods as it does not require applying arbitrary significance or size-effect cutoffs on differential expression results, which may hinder the identification of functional effects reflected by moderate but coordinated expression changes of several genes [37].GSEA overcomes this limitation by considering the complete results of a differential expression analysis, quantifying the extent to which the genes from each functional set are enriched towards the up-or downregulated genes via a Normalized Enrichment Score (positive/negative scores reflect up/downregulation, respectively) and its p-value [37].We find this approach particularly valuable as Wolbachia is restricted to some host tissues and its effects are to a great extent tissue-specific [30,39], so that local transcriptomic signals may be diluted in whole body RNA samples.P-values were Benjamini-Hochberg adjusted to control the False Discovery Rate [51], applying a threshold of 0.05.

RNA-Seq data analysis
Quality of raw Aedes aegypti reads was assessed with FastQC v0.11.9 [41].Trimmomatic v0.38 [42] was used to trim adapter and low quality sequences, using a sliding window approach.STAR v2.7.8a [43] was used to align trimmed reads to the AaegL5 reference genome, with splicing event information obtained from NCBI RefSeq Aedes aegypti Annotation Release 101 [44,45].Aligned reads were counted at gene level using featureCounts v2.0.1 [46] and differential expression analyses were performed using DESeq2 v1.34.0 [47] in R, contrasting each Wolbachia-infected condition to its equivalent uninfected condition.GSEA was performed as described previously.Principal component analysis and heatmaps were based on gene counts normalized via the Variance Stabilizing Transformation (VST) from DESeq2 [47].

Meta-analysis
To search for A. aegypti genes and functions consistently affected by Wolbachia infection, we used the maxP method [48] implemented in the metap R package [49], to combine p-values derived from individual differential expression and GSEA tests.maxP is appropriate for finding non-zero effects in all statistical tests, using the maximum p-value as the test statistic [50,51].Combined p-values were adjusted with the Benjamini-Hochberg method to control the False Discovery Rate [51], applying a cutoff of 0.05 for genes and 0.01 for GO terms.Functional gene annotations were retrieved from VectorBase [52], InterPro [53] and Gene Ontology [38].
All datasets were analyzed de novo, yielding reliable results according to intermediate step quality assessments.
In particular, >90% reads mapped to the A. aegypti reference genome in each RNA-Seq sample (S2 Table ) and two-channel microarray MA-plots show consistency between expected and observed average intensities and red/green intensity fold-changes for control spots (S1 Fig) .In the following we present our findings from these new analyses, contrasting them to their original analyses.Infection status was not the main source of variation in this dataset (Figs 1.A and 1.B).Indeed, PCA showed that the first principal component correlates with slides rather than infection status, suggesting a residual batch effect not removed in the between-array normalization step.Nevertheless, an overall effect of wMelPop on host gene expression was still distinguishable by the second principal component, which comprised 14% of the total variance, allowing the identification of 13 up-and 30 downregulated GO terms (S3 Table ).

Kambris et al. (2009)
Results were widely consistent with the immune activation previously reported by  Results showed a strong transcriptomic effect of both infection status and Wolbachia strain (Fig

Ye et al. (2013)
Ye et al. ( 2013) characterized the methylome of female wMelPop-infected and uninfected A. aegypti (lines PGYP1 and PGYP1.tet)heads and muscles, examining its relationship with gene expression changes.12 twochannel Agilent microarrays were competitively hybridized to PGYP1 and PGYP1.tetcDNA samples (6 for heads and 6 for muscles), each sample derived from a pool of 20 heads or muscle tissues.Intensity data was normalized with the lowess method and differential expression analyses were conducted using a cross-gene error model and FDR q-value to correct for multiple testing [28].Differential expression analysis was used to search for correlations between gene methylation and expression across conditions, however, no functional characterization of the genome-wide expression patterns was performed [28].We analyzed this dataset to identify the main functional effects of wMelPop in A. aegypti's heads and tissues (Fig 3).aegypti gene sets that were curated from previous genomic and transcriptomic studies [30].In the following we present the results of our own analysis of this dataset (Fig 5).

Consistent transcriptomic effects of Wolbachia in Aedes aegypti
In order to find functions consistently affected by Wolbachia on its host, p-values derived from individual differential expression and GSEA tests were combined using the maxP approach (see Methods), finding 1,483 and 57 significant genes and GO terms, respectively (S4 Table ).This set was further constrained to focus on genes that: (i) had a consistent direction of differential expression across tests (i.e., logarithmic fold-change always positive or negative), and (ii) were individually significant (p<0.05) in at least 8 of the 12 differential expression tests.Genes fulfilling these conditions were called consistently up-or downregulated genes (Tables 2 and 3, respectively).We applied a similar criterion for finding consistently up-or downregulated functions but, given that no significant GO term was always up-or downregulated, we required at least 10 tests with the same regulation direction (Table 4).We found 21 consistently upregulated genes related to metabolism, innate immunity, transmembrane transport, proteolysis and other functions (Table 2).Metabolism-related genes included two putative farnesoic acid Omethyltransferases, involved in the synthesis of juvenile hormones [57], a flavin-dependent monooxygenase, involved in detoxification [58] and a putative dopachrome conversion enzyme, involved in melanin biosynthesis [53].Innate immunity-related genes comprised two C-type lectins and a PGRP, which are pathogen pattern recognition genes, as well as the extracellular iron transporter transferrin which serves immune functions by limiting pathogen access to iron [59], and the antimicrobial peptide defensin.
Transmembrane transport genes comprised three subunits of vacuolar ATP synthase, which is a proton pump responsible for the acidification of intracellular organelles [60].Additional genes included a putative mitochondrial ATPase inhibitor, a putative RNA-methyltransferase and myofilin, which is a component of the thick filaments of insect striated muscles [61].
We also found 10 consistently downregulated genes (Table 2) mainly related to nucleic acid interaction, including a Sds3-like gene (which are implicated in repression of gene expression [53]), Med21 (a component of the Mediator of transcriptional regulation, which mediates the interaction of RNA polymerase II and transcription activators or repressors [62]), and a putative RNA-exonuclease.Additional genes included a nuclear pore protein, a proteasome subunit and two genes with uncharacterized function.
Consistently upregulated GO terms were associated to the electron transport chain, carbohydrate transmembrane transport, serine-type peptidase activity and serine-type peptidase inhibition, while the GO term DNA replication was consistently downregulated (Table 3).

Discussion
Although Wolbachia pipientis is currently being used for arboviral disease control, a robust mechanistic understanding of the A. aegypti-Wolbachia system is still lacking.Our analysis provides new insights on previously published transcriptomic datasets and identifies host genes and functions commonly affected by Wolbachia transinfection, transcending differences such as tissue, age and bacterial strain, thus providing a crucial resource that could impact strategies to enhance and preserve the efficacy of this arboviral disease control approach.

New functional insights on the effects of Wolbachia in A. aegypti
De novo analyses performed in this work revealed functional impacts of Wolbachia transfection in A. aegypti that were concealed within published datasets.These included upregulation of transport processes in data from [25], downregulation of cell cycle and DNA metabolism in [25] and [27], and upregulation of respirasome in [30].The analysis of the dataset from Ye et al. ( 2013) is of particular interest as it comprises the only genomewide transcriptomic data of wMelPop-infected A. aegypti heads published so far, posing it as a unique source of tissue-specific mechanistic insight.We found that wMelPop downregulated six long and short wavelength opsins, light sensitive receptors that initiate phototransduction and mediate visual perception in mosquitoes from larval to adult stages [63,64].A coordinated downregulation of opsins has been previously identified for the native Wolbachia infection of Aedes fluviatilis [14], but not for A. aegypti transinfection.
It has been shown that wMelPop attenuates photofobicity (i.e., light avoidance) in A. aegypti larvae [65] and that inhibition of opsins impairs visual recognition of feeding targets in adult A. aegypti [55].Considering these reports in the light of our findings, we propose downregulation of opsins as a feasible mechanism for photofobicity attenuation by wMelPop and predict that this strain impairs A. aegypti's feeding target seeking.
As photophobicity and feed seeking ability are relevant for mosquito fitness at multiple developmental stages -the first one reducing predation and desiccation risk for larvae [56] and the second one allowing adult females to obtain blood meals [55]-testing these hypotheses will help clarify mechanistic bases of the dramatic fitness costs induced by wMelPop, which hinder its use for population replacement [66].

Wolbachia consistently upregulates electron transport chain genes in A. aegypti
Several GO terms associated with host electron transport chain (ETC) and carbohydrate transmembrane transport were found to be upregulated by Wolbachia in 10 out of the 12 analyzed datasets.ETC is a central step in mitochondrial aerobic respiration, responsible for most of adenosine triphosphate (ATP) production in the cell and is fueled by glycolysis of carbohydrates.Upregulation of host ETC and carbohydrate transmembrane transport can be interpreted as a tendency to increase ATP production along with the availability of sugars to fuel this process.Consistent upregulation of the putative mitochondrial ATPase inhibitor AAEL003417 also identified in this work may be a compensatory mechanism to control ATP production.
While it has been suggested that Wolbachia provides hosts with ATP from its own bacterial aerobic respiration [67,68], to the best of our knowledge our study is the first to show the persistence of host ETC upregulation by Wolbachia in A. aegypti.Interestingly, Wolbachia has a complete biosynthetic pathway for heme synthesis and may provide it to its hosts [67].As ETC heavily depends on heme, it could be hypothesized that Wolbachia further supports host ETC upregulation by providing this cofactor.
Electron leakage, which is an incomplete reduction of oxygen to form superoxide (O2 -) due to ETC activity, is a major source of cellular ROS [73].We propose that a generalized upregulation of ETC genes is a potential persistent host mechanism behind ROS induction by Wolbachia in A. aegypti.Additionally, as the extent of electron leakage is modulated by mitochondrial ATPase activity [73,74], the consistent upregulation of putative mitochondrial ATPase inhibitor AAEL003417 found here might be further modulating ROS production.
It has been previously proposed that the main A. aegypti factors responsible for Wolbachia-induced ROS are dual oxidases and NADPH-oxidases [26,75], which drive an immune oxidative burst in Drosophila under microbial infection [76].Surprisingly, we found that NADPH-oxidase AAEL002039 was insignificant (p>0.05) in 11 of 12 tests, while all six significant results for dual oxidase AAEL007563 and its maturation factor AAEL007562 corresponded to downregulation (S4 Table ).Typical downregulation of AAEL007562 and AAEL007563 could constitute a host mechanism to counterbalance oxidative stress associated with other mechanisms such as the bacterial aerobic respiration and, as previously discussed, host ETC upregulation.
Our results contrast with the 28-fold increase in AAEL007562 transcripts reported by Pan et al. (2012) for wAlbB-infected carcasses of non-blood fed A. aegypti [26], a discordance that may be attributed to the difference of Wolbachia strains or specific biological conditions.On the other hand, our results are consistent

A. aegypti
We found three consistently upregulated vacuolar ATP synthase (v-ATPase) subunits: AAEL006516, AAEL010819 and AAEL000291.v-ATPases are proton pumps that acidify intracellular membranous organelles, such as lysosomes, endosomes and parasitophorous vacuoles [60].As Wolbachia lives inside host-derived vacuoles [77,78], consistent upregulation of v-ATPases may have direct impacts in the immediate environment of the bacteria.

Wolbachia consistently upregulates two putative farnesoic acid methyl transferases in A. aegypti
We found the consistent upregulation of two putative farnesoic acid methyl transferases (FAMeTs), AAEL001508 and AAEL004667, which catalyze the conversion of farnesoic acid to methyl farnesoate, a late step in the biosynthesis of insect juvenile hormones [89,90].Interactions of Wolbachia with the juvenile hormone biosynthetic pathway have been previously reported in Diptera [83,84], but to the best of our knowledge, this is the first study showing Wolbachia's consistent upregulation of putative A. aegypti's FAMeTs.
Juvenile hormone III (JHIII) is a key regulator of development and reproduction in A. aegypti, and its titer determines the expression of thousands of genes [83,85,86].Also, the direct product of FAMeT -methyl farnesoate-can partially substitute the functions of JHIII in A. aegypti [87] and possibly serve additional uncharacterized functions [88].In light of these considerations, either by altering juvenile hormone or methyl farnesoate production, upregulation of FAMeTs could have deep consequences for A. aegypti physiology.
Further experiments are needed to confirm the enzymatic activity of AAEL001508 and AAEL004667, as well as to unveil their specific roles in the A. aegypti-Wolbachia symbiosis at different life stages.Checking if this phenomena has a parallel in male mosquitoes would be of particular interest, as alterations of JHIII have been proposed as a contributory mechanism for Wolbachia-induced CI in Drosophila [83].

Wolbachia consistently upregulates a flavin-containing monooxygenase implicated in xenobiotic metabolism in A. aegypti
We found the consistent upregulation of flavin-containing monooxygenase (FMO) AAEL000829, which catalyzes the incorporation of an oxygen atom from O2 to a compound, using flavin adenine dinucleotide as a prosthetic group [89,90].Another FMO, AAEL000834, shows a similar profile of expression being upregulated in 8 of the 9 significant tests (p<0.05)(S4 Table ).FMOs catalyze the oxygenation of nucleophilic nitrogen, sulphur, phosphorus and selenium atoms [53], and are implicated in the neutralization of xenobiotics (i.e. foreign substances) [58], particularly insecticides [89,91,92].Although direct effects of Wolbachia on A.
aegypti's resistance to typical insecticides is not clear [93], the consistent upregulation of FMOs found here opens the question if there are additional substances to which Wolbachia-infected A. aegypti is more resistant than its uninfected counterpart.
Coordinating Wolbachia-and insecticide-based vector control methods is important for the overall success of arboviral control [21,94].Particularly, coupling Wolbachia-introduction with that of insecticide resistance genotypes has been proposed as a way to facilitate spread of the bacteria in native mosquito populations, a paramount task for the use of strains that cause high fitness costs such as wMelPop [21,94].If the consistent upregulation of FMOs confers resistance to specific compounds in Wolbachia-infected A. aegypti, their controlled use might be considered to enhance Wolbachia introgression in native A. aegypti populations.By leveraging an effect already present in Wolbachia-infected A. aegypti, such a measure could avoid the introduction of new insecticide-resistant alleles in native populations, which is a major concern regarding the use of insecticides for the enhancement of Wolbachia-based arboviral control [21,94].

Wolbachia consistently downregulates genes with nucleic acid-related functions in A. aegypti
We found the GO term DNA replication downregulated by Wolbachia in 10 out of 12 tests.Together with the consistent upregulation of ETC and carbohydrate transmembrane transport, our results suggest a coordinated host metabolic shift towards enhanced aerobic energy metabolism and reduced cellular proliferation.This scenario may be induced by Wolbachia to favor its own preservation in A. aegypti adult stages such as those considered in this study, however, it is foreseeable that a similar effect in earlier developmental stages would critically impact development.It would be therefore interesting to study if this downregulation also stands in earlier stages of A. aegypti development, pinpointing the need for genome-wide transcriptomic analyses of Wolbachia-infected A. aegypti at new biological conditions.
Our results suggest that downregulation of genes with nucleic acid interaction is not restricted to DNA but includes RNA-interacting genes.For instance, the GO term mRNA metabolic process had a negative Normalized Enrichment Score in 10 out of 12 tests, 7 of which were significant (p<0.05)(S4 Table ).RNA viruses such as DENV, ZIKV and CHIKV have small genomes and heavily depend on host factors for the success of their life cycle, including various host RNA-interacting genes [80,95,96].Under this consideration, downregulation of RNA-interacting genes by Wolbachia may underlie the broad-range of RNA viruses to which Wolbachia offers protection.Particularly, the consistently downregulated gene AAEL009650 is homologous with Drosophila exonuclease-like CG6744, which contributes to DENV infection [80].This poses AAEL009650 as a concrete target of study for clarifying the link between downregulation of functions related to nucleic acid interaction and pathogen blocking.

A consistent signature of immune priming by Wolbachia in A. aegypti
The consistent upregulation of defensin DEFC, transferrin Tf1, peptidoglycan recognition protein PGRPS1 and GO terms related to serine-type peptidase activity and inhibition, are consistent with Wolbachia's priming of the Immunodeficiency (IMD) and Toll pathways in A. aegypti [27].Indeed, expression of the antimicrobial peptide defensin and the extracellular iron binding protein transferrin (the immunity function of which consists in reducing iron availability for pathogens [59]) is inducible by the Toll-and Immunodeficiency (IMD) pathways [59,97].These pathways in turn can be activated extracellularly by the action of pattern recognition receptors such as PGRPS1, which elicit protease signaling cascades mediated by serine-type peptidases and serine-type peptidase inhibitors [98].The expression pattern of Rel2 observed here is also consistent with the activation of the IMD pathway, as it has a positive expression change in 10 out of 12 tests, 9 of which are significant (p<0.05)(S4 Table ).
However, some genes of these pathways are downregulated.Particularly, for the Toll pathway, TUBE has a negative expression change in 9 out of 12 tests, 6 of which are significant (p<0.05),while Rel1A has a negative expression change in 7 out of 12 tests, 6 of which are significant (p<0.05)(S4 Table ).IKK1, part of the IMD pathway, has a negative expression change in 10 out of 12 tests, 3 of which are significant (p<0.05)(S4 Table ).
This suggests that Wolbachia elicit transcriptomic mechanisms tending to decrease activation of Toll and IMD pathways, which however are overcome by other effects that promote such activation.
A role of the Janus Kinase/Signal Transducer Activators of Transcription (JAK/STAT) pathway in Wolbachia's pathogen blocking has also been hypothesized [99], as components of this pathway were upregulated by Wolbachia in Drosophila [100], and this pathway restricts DENV in A. aegypti [101] and West Nile Virus in Culex mosquitoes [102].We found that JAK/STAT activators (DOME, HOP, STAT) and inhibitors (SOCS, PIAS1) are mainly downregulated, as from the 26 significant tests that they accumulate (p<0.05),23 correspond to downregulation (S4 Table ).Again, this suggests that there are conflicting transcriptomic mechanisms regarding immune priming by Wolbachia.
Knowing the specific mechanisms underlying Wolbachia's immune priming is crucial for understanding pathogen blocking by Wolbachia in A. aegypti, as it drives the expression of key antiviral factors such as DEFC [26] and transferrins [103].Our work contributes in identifying consistent transcriptional regulations associated with immune priming by Wolbachia in A. aegypti.We also highlight that there are transcriptomic mechanisms opposing immune activation.These require careful consideration and monitoring, as they may constitute a basis for attenuation of pathogen blocking in the future, a main concern regarding Wolbachia-based arboviral control.

Conclusion
Genome-wide omic datasets from related conditions accumulate in time and bioinformatic resources enabling their interpretation evolve.This offers the opportunity to extract new value from previously published datasets by both analyzing them with up-to-date resources, and comparing older to newer datasets by meta-analysis methods, offering global insights that no study by its own can provide.
In this work, by performing a de novo meta-analysis of transcriptomic data we were able to identify novel effects and a transcriptomic signature of Wolbachia in A. aegypti, which can explain previously observed phenotypes and provide new hypotheses and gene targets.Particularly, wMelPop infected mosquitos display a coordinated downregulation of opsins in A. aegypti heads, which can be related to the loss of light sensitivity in mosquito larval stages that has been previously reported.We revealed Wolbachia's consistent regulation of host genes that have escaped the focus of previous research, including a flavin-dependent monooxygenase and two farnesoic acid methyltransferases.We also highlight the common upregulation of functions associated with the electron transport chain, hypothesizing a role of this phenomena in pathogen blocking via induction of reactive oxygen species.
For this application, we note the relevance of availability of raw data, as identification of commonalities at the biological function level was mediated by gene set enrichment analyses that were not performed in the original publications, and that would not have been possible from pre-processed data.Follow-up work on our observations holds promise in producing valuable information towards the goals of preventing pathogen enhancement and loss of pathogen blocking, coupling Wolbachia-based control with other strategies, and enabling the use of specific Wolbachia strains, thus contributing towards the sustainability of Wolbachia-based arboviral control.

Fig 1 .
Fig 1. Transcriptomic effects of wMelPop on female A. aegypti whole bodies assessed from the analysis of

Fig 2 .
Fig 2. Transcriptomic effects of wMelPop and wMel on female A. aegypti whole bodies assessed from the

Fig 3 .
Fig 3. Transcriptomic effects of wMelPop on female A. aegypti muscles and heads assessed from the

Fig 4 .
Fig 4. Temporal progression of wMel transcriptomic effects on native A. aegypti populations assessed

Fig 5 .
Fig 5. Transcriptomic effects of wMel on female A. aegypti carcasses and midguts at different times post with the study ofCaragata et al. (2016) on wMel-infected A. aegypti whole bodies, where ROS induction was verified without upregulation of AAEL007562[70] and further supports an alternative mechanism for typical ROS production such as enhanced host ETC activity.This does not exclude the possibility that dual-and NADPH-oxidases plays a role in pathogen blocking by producing ROS early after a blood meal, which may have been missed in the transcriptomic datasets from Boehm et al. (2023) at 4 and 7 days post blood feeding analyzed in this work.

v-
ATPases are also responsible for the acidification of DENV-containing endosomes, which is a requisite for the release of viral RNA into the cytoplasm for further translation and replication.Consequently, inhibition of v-ATPases has proven antagonistic to DENV infection in Aedes mosquitoes, Aedes cells and HeLa cells[79-   81].Therefore, v-ATPase upregulation may be a pro-viral effect of Wolbachia, obscured however by its repertoire of antiviral effects.Existence of pro-viral Wolbachia effects should be carefully considered, as overall enhancement of arbovirus infection by Wolbachia is possible[82].Specifically, disruption of v-ATPase activity by RNAi or v-ATPase inhibitors hold potential to clarify the consequences of Wolbachia's upregulation of host v-ATPases for viral infection in A. aegypti.

Table 1 . Summary of selected transcriptomic studies.
a E-MEXP prefix corresponds to ArrayExpress accession numbers, while PRJNA prefix corresponds to SRA and GEO accession numbers.

Table). Rancès et al. (2012)
Kambris et al. (2009), as top 10 upregulated GO terms Serine-type endopeptidase activity, Extracellular region and Carbohydrate binding (Fig 1.C) were driven by the overexpression of CLIPs, pattern recognition receptors, prophenoloxidases and defensins (S3 Table).Our analysis further revealed effects of wMelPop in host transport processes (Fig 1.C)not reported in the original publication, suggesting an impact of wMelPop on amino acid homeostasis (including four amino acid transporters), transition metal homeostasis (including transporters of iron, zinc and copper), intercompartmental pH balance (comprising four vacuolar ATP synthase subunits) and neuronal signaling (including neurotransmitter receptors and ion channels) (S3 Table).

2.A and 2.B),
Our analysis of downregulated genes suggested disruption of DNA replication and cell cycle by both wMel and wMelPop, which was not identified in the original study.Top 10 downregulated terms Cell cycle and Chromosome (Fig 2.C, left and right) reflected underexpression of DNA polymerase subunits, DNA repair proteins, DNA helicases, anaphase-promoting complex subunits, M-phase inducer phosphatase and a putative cyclin (S3Table).For wMelPop, downregulation of these terms was further driven by additional cyclins, CDK, CDK activating kinase and a CDK regulatory subunit (S3 Table).wMelPop also downregulated RNA processing (Fig 2.C, left), with underexpression of ribonucleases, ribonucleoproteins, RNA methyltransferases, splicing factors and cap-binding protein subunits (S3 Table); as well as microtubule-mediated transport (Fig 2.C, left), with underexpression of tubulin chains, katanin P80 subunit, gamma-tubulin complex component, kinesin and dynein chains (S3 Table).

Table ) .
The downregulation of Extracellular region and Serine hydrolase activity was consistent with the original results of Boehm et al. (2023) for midguts at 7 dpf, however, it contrasted with our analysis of all previous datasets, where these GO terms were always upregulated (S3 Table).

Table 2 . Genes consistently upregulated by Wolbachia in Aedes aegypti
a Mean Fold-changes ± Standard Deviation.The reference condition for Fold-change calculation is Wolbachia-uninfected.

Table 3 . Genes consistently downregulated by Wolbachia in Aedes aegypti
a Mean Fold-changes ± Standard Deviation.The reference condition for Fold-change calculation is Wolbachia-infected.