Spiroplasma shows a Wolbachia-like effect in hampering virus replication in spider mite

doi:10.21203/rs.3.rs-4868315/v1

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Spiroplasma shows a Wolbachia-like effect in hampering virus replication in spider mite

https://doi.org/10.21203/rs.3.rs-4868315/v1

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Background

Members of the Acari order, commonly known as mites, play a significant role as agricultural pests. Among these, the Tetranychidae family stands out due to its remarkable diversity, surrounding approximately 1200 species capable of infesting over 4000 plant species. By feeding on plant tissues, these mites directly harm crops and can also serve as vectors for viral pathogens, posing a substantial threat to global food security. In this study, we investigated the unexplored virome of Tetranychus truncatus exploring how biotic factors (Spiroplasma and Wolbachia endosymbionts), and abiotic stresses (abamectin and temperature) affect virus dynamics.

Results

Our metatranscriptomics analyses revealed sequences related to important arthropod- and crop-infecting viral families, including the known plant-pathogenic Potato Y virus and Cherry A virus strains and fourteen new species. Notably, abamectin treatment correlated with the absence of Potato virus Y and TtDV-2 virus, suggesting this pesticide impacts viral diversity. Interestingly, single infections of Wolbachia or Spiroplasma significantly decreased both the diversity and the abundance of viruses, with the greatest effect on dicistroviruses, indicating for the first time the potential of Spiroplasma to restrict viral infections. Surprisingly, Wolbachia-Spiroplasma co-infection leads to the loss of the virus restriction effect. Wolbachia-exclusive and Spiroplasma-exclusive responsive genes showed enrichment for similar pathways, with piRNA and autophagy enriched in up-regulated genes. In contrast, lipid metabolic processes were enriched in down-regulated elements.

Conclusions

Overall, our study describes the T. truncatus virome, unveiling the considerable influence of its microbiome, including Wolbachia and Spiroplasma, and Abamectin pesticide on both virus diversity and abundance.

Wolbachia

Spiroplasma

Virome

Tetranychidae

Transcriptomics

Bioinformatics

Mites (Arachnida: Acari) are an important group of arthropods known for their role as agricultural pests. They cause direct damage to plants by feeding on tissues or indirect damage by acting as vectors for viruses, which can lead to reduced plant vigor or, in severe cases, plant death [1, 2]. The Tetranychidae family, comprising spider mites, infests more than 4,000 plant species and include members known for virus transmission [3]. For example, Tetranychus telarius can transmit Potato virus Y [4], while Tetranychus urticae is associated with the transmission of tobacco ring spot virus (TRSV), tobacco mosaic virus (TMV), Southern bean mosaic virus (SBMV), and cotton leaf curl virus (CLCuV) [5–7]. Despite this, to this date the important agricultural pest Tetranychus truncatus has not been directly linked to the transmission of pathogens. Mite control is a current practice to prevent crop damage and the indiscriminate use of acaricides has led to the appearance of diversified resistance mechanisms in various mite species, including T. truncatus [8–10]. To overcome this issue, new biological alternatives for pest control are being developed. For example, the endosymbiotic bacteria Wolbachia and Spiroplasma have, in different occasions, shown potential in controlling arthropod populations [11–13]. Wolbachia is widely recognized as one of the most prevalent endosymbionts among arthropods with infection rates reaching as high as 76% in certain insect species [14]. Infection with Wolbachia has been described, in some occasions, to positively impact host fitness. However, some strains cause loss of cell replication synchronicity in the host, thus negatively affecting host lifespan, fecundity, and development [15]. Also, Wolbachia can disrupt host reproductive processes through cytoplasmic incompatibility (CI) and impair pathogen replication by competing for resources [16]. In a similar fashion, the endosymbiont Spiroplasma is also known to induce CI, which cause a negative impact on the overall fitness parameters of infected hosts [12, 17]. Due to these characteristics, Wolbachia and Spiroplasma endosymbionts have substantial implications for pest control and virus management [18, 19]. On a different aspect, temperature plays a critical role in mite development and can significantly influence virus transmission and replication in arthropods [20–22]. Taken together, understanding how abiotic stresses such as temperature fluctuations and chemical exposure, influence of endosymbionts, and understanding how naturally infecting viruses may affect mites, is crucial to gain insights into their resilience and adaptability, with implications for areas like agriculture, pest management, and conservation [11, 20, 23, 24].

In this study, we focused on unveiling the virome associated to T. truncatus and study the impact of biotic and abiotic stresses on its virome diversity and abundance. Using 39 publicly available high-throughput RNA-sequencing (RNAseq) libraries collected across diverse locations and under different sampling conditions (abamectin exposure, variable temperature, Wolbachia and/or Spiroplasma infection), we identified and characterized known and novel virus species and evaluated the impact of each treatment on the mite´s virome.

Virome of spider mites

To characterize the virome of T. truncatus, we retrieved publicly available RNAseq libraries covering a wide range of biological and environmental conditions and performed de novo virus identification using an unbiased virus discovery pipeline [25]. Our analysis revealed at least 30 potential virus-derived assembled contigs belonging to known and new virus species (Fig. 1 and Supplementary File 2). Among these, three contigs showed high sequence similarity to the genome of known viruses and coded for similar protein domains as their BLAST best hits. Among them, one contig showed 97% nucleotide identity and 95% amino acid identity to Potato virus Y, another contig had 99% nucleotide identity and 95% amino acid identity with Cherry virus A, while the last contig had 99% nucleotide identity and 80% amino acid identity with Acyrthosiphon pisum virus (Figure S1). Ten additional contigs showed sequence similarity above 50% at the amino acid level and similar conserved domains to positive single-strand RNA ((+) ssRNA) viruses (Fig. 1 and Supplementary File 2). Phylogenetic analyses grouped three putative contigs as viruses belonging to the Dicistroviridae family. The remaining contigs showed sequence similarity to the Kitaviridae, Betaflexiviridae, Virgaviridae, Botourmiaviridae and Nodaviridae families (Fig. 1 and Supplementary File 2). Among these, seven contigs contained polyproteins with start and stop codons, two contigs encoded for RdRp and capsid segments similar to nodaviruses, and one contig resemble a fragment of a narnavirus RdRp (Fig. 1, Figure S2-7, Figure S8A-F and Figure S9A-C, respectively). Four contigs exhibited sequence similarity above 30% and their ORFs contained conserved domains commonly present in the negative single-stranded RNA viral family Phenuiviridae. These contigs encoded for three incomplete and one complete RdRp protein (Figure S9D-G and Figure S10). The remaining 13 contigs matched both in amino acid sequence similarity and conserved domains to core genes of the dsDNA family Nudiviridae (Figure S11 and S12).

One major issue in virus discovery works is the presence of endogenous viral elements (EVEs) that can mislead the identification of exogenous viruses in metagenomics data. To avoid this issue, we performed de novo identification of EVEs on the T. truncatus genome. We identified three endogenized sequences sharing similarities with viral proteins of the Chuviridae and Rhabdoviridae families (see Supplementary file 6 for details). Assembled contigs from RNAseq libraries matching any of these potential EVEs with sequence similarity above 70% and similar length were discarded from our analyses.

Prevalence and widespread of T. truncatus-associated viruses

To have an overview of virus prevalence among samples analyzed in this study, we cross-mapped each library to all 30 putative viral sequences (Fig. 2). Except for AcPV, reads matching all viruses were present in at least two distinct samples, and most of them belonged to the same sequencing project. However, the dicistroviruses TtDV-1, TtDV-2 and AVT, together with the phenuiviruses GVT-1 and GVT-2, and the botourmiavirus TtOV were detected in libraries originated from independent projects (Fig. 1). Of note, AVT and TtDV-1 were present in approximately 82% and 52% of the libraries, respectively (Fig. 2). Interestingly, the dicistroviruses TtDV-1 and AVT, and the kitaviruses BVT-1 and BVT-2 showed normalized transcript abundance 10 to 40 times higher than constitutive genes (Fig. 2). On the other hand, the Goukoviruses (GVT-1–4) showed the lowest abundance.

We next examined the read coverage depth of each viral contig identified in our study. For the different datasets, we observed reads matching the sense and antisense strands of all viral contigs, which represents the viral genome and antigenome as depicted in Figure S13 and S14.

Effect of temperature and abamectin treatment on the T. truncatus virome

We took advantage of the public RNA libraries constructed from different treatments to assess the influence of biotic and abiotic stresses on the composition and dynamics of the T. truncatus virome. We first analyzed the impact of abamectin exposure and different temperatures on virome composition and abundance. Within the study investigating the impact of abamectin on mite fitness, Potato virus Y (PVY) and TtDV-2 were exclusively detected in T. truncatus samples collected from plants not exposed to abamectin (ABM–). Their abundance was approximately 10 and 100 TPM respectively, suggesting a notable association between the absence of the pesticide and virus abundance. Conversely, TtNoV was exclusively identified in libraries containing T. truncatus exposed to abamectin (ABM+), indicating a distinct viral dynamic of mites exposed to pesticide (Fig. 3A).

On the other hand, temperature did not cause any significant change in virus abundance comparing ordinary and high temperature conditions (Figure S15). However, virus diversity analyses showed that mites exposed to regular temperatures exhibited lower alpha diversity compared to mites subjected to high-temperature stress (Figure S16A). In contrast, samples subjected to abamectin exposure showed higher differences in alpha diversity, although not significant, comparing to samples derived from individuals not exposed to the abamectin pesticide (Figure S16B).

Wolbachia and Spiroplasma presence impact virus dynamics

To assess the impact of Wolbachia and/or Spiroplasma on the abundance and diversity of the mite virome we analyzed the virus-derived transcripts levels on samples infected solely with Wolbachia pipientis (W + S-) and Spiroplasma ixodetis (W-S+), as well as in those in coinfection or uninfected. In the presence of both Wolbachia and Spiroplasma, or in their absence, the dicistrovirus (+) ssRNA TtDV-1 virus showed consistently high abundance, reaching approximately 10³ TPM (Fig. 3B). However, in samples infected with Wolbachia (W + S-) or Spiroplasma (W-S+) alone, the abundance of TtDV-1 was significantly reduced in comparison to the groups with coinfection (W + S+) or not infected (W-S-) (Fig. 3B). This suggests that the presence of these endosymbionts may inhibit the replication of the dicistrovirus TtDV-1. A similar trend was also observed for the botourmiavirus TtOV that, despite having lower TPM than TtDV-1, showed to be significantly affected by the individual presence of each endosymbiont (Fig. 3B). The other dicistrovirus AVT also presented lower abundance, although not significantly, in mites carrying either Wolbachia or Spiroplasma alone (Fig. 3B). In all cases, the presence of both bacteria in the same individuals restored virus abundance to non-infected levels, suggesting a mutually antagonizing effect. We observed significant higher abundance of the phenuivirus ((-)ssRNA) GVT-2 in individuals co-infected with Wolbachia or Spiroplasma, indicating that the viruses are potentially impacted in a different manner by these endosymbionts (Fig. 3B).

Infections with Wolbachia and Spiroplasma in mites also influenced significantly virus diversity. Samples infected solely with Wolbachia or Spiroplasma displayed a notably lower alpha diversity compared to mites uninfected or coinfected with both endosymbionts. The reduction in alpha diversity suggests that Wolbachia and Spiroplasma interfere with the virome of T. truncatus (Fig. 4). Importantly, the combination of Wolbachia/Spiroplasma cancels the effect observed in T. truncatus carrying a single endosymbiont. Consistent with previous observations, uniform manifold approximation and projection (UMAP) analysis using the transcriptome and virome transcripts abundance of T. truncatus, revealed a clustering pattern where samples infected either with Wolbachia or Spiroplasma alone segregated from co-infected and uninfected samples (Figure S17).

Mite transcriptional responses to Wolbachia and/or Spiroplasma infection

Infection with Wolbachia and/or Spiroplasma significantly altered the virome composition and abundance of T. truncatus. Therefore, we investigated the transcriptional responses and pathways involved in the dynamics between endosymbiont bacteria and the host, focusing on the potential impact to virus infection (Fig. 5A).

Gene Set Enrichment Analysis (GSEA) revealed several pathways enriched in during Wolbachia (W + S-) and Spiroplasma (W-S+) infection, many overlapping between conditions. Upregulated enriched pathways included piRNA processing, adaptation of rhodopsin-mediated signaling and membrane bending (Fig. 5A-B, Figure S18, Figure S19 and Supplementary file 7). Notably, five unique pathways were significantly upregulated during Wolbachia or Spiroplasma infection alone. However, to our knowledge, none of these were yet described to have an association with antiviral activity (Fig. 5A and B, Figure S18 and S19). Conversely, GSEA also identified significantly downregulated pathways that were shared between W + S- and W-S + infected samples. These pathways included positive regulation of G protein-coupled receptor signaling, carbohydrate transport, octopamine regulation pathways and lipid metabolic processes. (Fig. 5A-B, Figure S18, Figure S19 and Supplementary file 7).

Notably, GSEA analysis revealed a minimal overlap of enriched pathways between solely infected and co-infected samples with endosymbionts. These pathways were related to host modulation of viral processes including viral genome replication and symbiosis-related interactions, translation readthrough and ribosomal large subunit export from the nucleus. Inversely, pathways involved regulation of oxidative stress response, cardiolipin metabolism and biosynthesis, sequestering of proteins and transcription factors were downregulated (Fig. 5A-B and Figure S20). An schematic illustration explaining the Wolbachia- and Spiroplasma-induced phenotypes can be visualized in the Fig. 5C.

Metatranscriptomics is a powerful approach to understanding complex ecosystems and has illuminated the interplay between microorganisms and their hosts [26, 27]. Our metatranscriptomic analysis of the important agricultural pest T. truncatus using data obtained from 39 libraries publicly available offered a glimpse into its virome landscape. Overall, most of the viral families identified have elements known to infect mites such as Dicistroviridae [28] and Nodaviridae [27]. Furthermore, we have also identified viruses from the families Phenuiviridae [29] and Nudiviridae [30], both known to infect arthropods. In addition, we have identified previously known and several new viruses associated with families that are traditionally associated with plants including Kitaviridae, Botourmiaviridae, Virgaviridae, Betaflexiviridae, and Potyviridae. Of note, transmission by mites of viruses belonging to these families has been described, as exemplified by the transmission of viruses from the Kitaviridae family by RAMOS-GONZÁLEZ et al.(2022) and TASSI et al.(2021), while virgaviruses transmission has been described by PLESHAKOVA et al. (2018). Furthermore, the mite-mediated transmission of Betaflexiviridae has been described by BERTAZZON et al. (2021) and Potyviridae transmission by CHOI et al. (1999). We could speculate that viruses identified in our study can potentially be transmitted to plants by T. truncatus. This hypothesis was supported by the identification of Potato virus Y and Cherry virus A, both known to infect economically important crops [4, 36, 37]. However, it is important to emphasize that this possibility cannot be substantiated solely through in silico approaches and would require confirmation through other methods.

Temperature is a critical factor influencing the development of mites and plays a critical role in both virus replication and transmission by these organisms [20, 38, 39]. Surprisingly, our study revealed higher viral diversity in T. truncatus specimens under heat-stressed conditions but we observed no statistically significant differences regarding the impact of temperature on abundance and diversity of viruses. Nevertheless, our study started shedding some light on the complex interplay between temperature and viral dynamics in mite populations.

Pesticides play a vital role in modern agriculture. They are currently used to protect crops from pests including insects, fungi, and mites, and are instrumental in ensuring food security and increasing agricultural productivity [40, 41]. Resistance to pesticides is of pressing concern, imposing limitations on effective pest control. Often, resistance arises from adaptations to environmental conditions and pose a significant challenge to agriculture [41, 42]. Interestingly, we observed distinct patterns in virus prevalence and abundance when comparing exposed and non-exposed individuals, with PVY and TtDV-2 exclusively found in specimens not exposed to abamectin. These results suggest a potential suppressive effect of the pesticide against some viruses. Conversely, TtNoV was predominantly detected in abamectin-exposed specimens, hinting at a complex interplay between the pesticide and viral dynamics. Our analysis further revealed differences in quantities of viral RNA and alpha diversity between exposed and unexposed mites, underscoring the multifaceted impact of abamectin on the virome composition and abundance. However, it is important to highlight that these differences could result from an impact of abamectin on mite’s fitness and, consequently, altered susceptibility to infections by viruses. Deeper exploration is necessary to better understand the mechanisms underlying this phenomenon and possible implications for pest management and ecological balance in agricultural settings.

Wolbachia is widely recognized as the most prevalent endosymbiotic microbe among arthropods, infecting up to 76% of known insect species [14]. Its presence has been found to have a positive impact on host fitness, contributing to improved nutrition, development, immune system function, and fecundity [13, 24]. On the other hand, certain strains of Wolbachia have undergone evolutionary changes that result in the loss of ability to synchronize their replication with the host cell that has negative consequences for the host, affecting its lifespan, fecundity, and development [15]. Such effects have been observed in various species, including Drosophila and several mosquito species [43]. Additionally, the presence of Wolbachia can interfere with the reproductive processes of the host and impede the replication of pathogens through several mechanisms including cytoplasmic incompatibility and resource scarcity. [12, 13, 44, 45]. The widespread effects of Wolbachia across different arthropods, including spider mites, have raised considerable interest as a tool for controlling virus transmission. Successful implementation in controlling viruses in mosquitoes paved the way for further use to manage viruses transmitted by mites and other arthropod vectors [12, 14, 24, 44]. Endosymbionts are known to play a key role in viral dynamics such as Rickettsia spp. [46], Arsenophonus [47] facilitating viral transmission in Bemisia tabaci and Rosenbergiella in reducing viral transmission in Aedes mosquitoes [43]. In this context, the symbiotic relationship between Wolbachia and Spiroplasma has been investigated in spider mites, particularly in T. truncatus [11]. Research shows that co-infection by these bacteria confers significant benefits to the host. For example, co-infected spider mites exhibit enhanced fitness, a higher fertilization rate, and a higher survival rate during the juvenile stage than uninfected individuals or individuals infected with only one of the bacteria. In addition, the presence of endosymbionts can induce competition for resources, such as space and nutrients, which can limit infection by other pathogens and hinder their transmission [16, 48, 49].

Our study revealed a substantial impact in the abundance of specific viral sequences in T. truncatus when infected alone with either Wolbachia or Spiroplasma. Notably, the discistrovirus TtDV-1, a virus in our study, is the most impacted by endosymbiont’s influence, with a considerable impact on virus abundance. Nevertheless, a distinct scenario emerges when considering populations co-infected with both Wolbachia and Spiroplasma (W + S+) or those uninfected (W-S-). Our analyses pointed out to a reduction in virus abundance of (+)ssRNA viruses in Wolbachia-infected samples, which is aligned with the Wolbachia's established role in regulating viral replication in other species [12, 18, 50–54]. A similar effect has also been reported in the dicistrovirus Cricket paralysis virus (CrPV) and Drosophila C virus (DCV) in D. melanogaster [55]. Moreover, we were not able to detect TtDV-1 in Spiroplasma infected samples, indicating a potentially stronger inhibition of virus replication in comparison to Wolbachia. Both TtOV and AVT exhibited comparable outcomes, revealing a consistent pattern of diminished viral abundance. This finding prompts intriguing questions about the interplay between endosymbionts and viral dynamics in mites, mirroring effects documented in Aedes spp. and other arthropods [12, 18, 45, 56, 57].

It's important to note that Spiroplasma has been demonstrated to function as a protective endosymbiont against bacterial infection in Drosophila melanogaster [58], parasitism of parasitoid wasps, nematodes, and fungi infection in Drosophila spp. and aphid species [59, 60]. However, no data regarding its protective role against viruses is currently available. In the context of nematode protection, certain strains of Spiroplasma produce the ribosome-inactivating protein (RIP), which depurinates the 28S rRNA of the invader. Notably, plants derived RIP’s are demonstrated to have antiviral activity in several viruses including HIV [61], DENV and CHIKV [62]. This mechanism could be related to the protection against viruses, as indicated by our findings. Certain Spiroplasma strains are also known to induce Cytoplasmic Incompatibility (CI) and other Wolbachia-like resource competition mechanisms, which can result in reduced viral loads [12, 13, 19, 45, 63].

Mites infected with either Wolbachia or Spiroplasma alone showed transcriptional regulation towards an induced expression of genes involved in piRNA processing pathways. This finding is particularly noteworthy as Wolbachia has been described to modulate host piRNAs, a class of small RNAs belonging to the RNA interference (RNAi) machinery [64–67]. Overall, RNAi pathways including the small interfering RNAs (siRNAs) are well described to control arbovirus infections in mosquitoes and other arthropods, and recent research are providing evidence that the piRNA pathway might also contribute to antiviral activity [68–72]. These results provide evidence that multifactorial events resulting of Wolbachia or Spiroplasma infections could explain the changes in viral abundance and diversity observed. Supporting this hypothesis, we also observed that autophagy was enriched by both endosymbionts’ infection. This important pathway is involved in cellular degradation previously shown to be activated by Wolbachia and Spiroplasma infection and can function as an antiviral host response [73–77].

Analysis of downregulated gene sets and pathways in Wolbachia- and Spiroplasma-exclusivelly infected mites revealed a link to lipid metabolism, particularly sterol and steroid metabolic processes. Cholesterol, the end-product of this pathway, is a vital molecule targeted by various viruses, including Dengue virus. Modulation of cholesterol dynamics and metabolism is a strategy employed by the host's innate immunity to combat viral infections [78, 79]. Interestingly, Wolbachia lacks the ability to synthesize cholesterol itself [80, 81]. This dependency leads to competition with the host for lipid molecules, a mechanism proposed to contribute to Wolbachia-mediated viral blocking [82, 83]. Wolbachia and Spiroplasma are known to deplete lipid availability, creating an unfavorable environment for viral replication [83] and larval development [84]. Furthermore, lipid metabolic processes are linked to changes in the membrane lipid composition of host cells, which can be critical for the formation of replication complexes for (+)ssRNA viruses [52]. Further research is needed to explore the potential antiviral capabilities of Spiroplasma and the specific mechanisms involved in providing protection against viral infections [59, 60].

Notably, W + S + populations exhibited similar diversity and abundance patterns to those of W-S- populations, suggesting that the virus-blocking effect of endosymbionts may be compromised in co-infection scenarios. Interestingly, the co-infected mite population displayed minimal overlap in enriched pathways compared to mites solely infected with Wolbachia or Spiroplasma. This distinction highlights the unique enrichment of host defense-specific pathways in the co-infected group. These enriched pathways encompass processes that modulate viral processes, such as viral genome replication, alongside pathways involved in symbiosis-related interactions. This implies that, despite the presence of endosymbionts, the host is actively responding to viral infections. While oxidative stress is a typical host response to viral infection, it appears to be connected to Wolbachia's presence [85]. Studies have shown a link between Wolbachia's antiviral function and increased oxidative stress levels [85, 86]. Interestingly, Spiroplasma acts in opposition. It promotes the production of antioxidants [87] which could potentially counteract the oxidative stress and weaken Wolbachia's antiviral effects in coinfected samples, where oxidative stress pathway is found enriched in downregulated genes.

Interestingly, potential viral-replication related pathways were also enriched, such as translation readthrough, sequestering of proteins and transcription factors, and ribosomal large subunit export from the nucleus. Viruses have evolved sophisticated mechanisms to hijack host cellular machinery for their replication [88]. During infection, viruses exploit host ribosomes to translate viral mRNA into proteins essential for viral replication and assembly [89]. Additionally, viruses sequester host transcription factors and proteins, redirecting them to facilitate viral gene expression while suppressing host defenses[90, 91]. This sequestration often involves the manipulation of RNA-binding proteins, which play crucial roles in RNA metabolism and gene regulation [92]. Furthermore, some viruses employ translation readthrough strategies, allowing ribosomes to bypass stop codons and produce extended viral proteins that enhance viral replication and pathogenicity[93, 94]. Such findings raise questions about the interplay between endosymbionts and host immune responses, which reinforces the need for further investigation into the mechanisms that are basis for these interactions.

The virome of Tetranychus truncatus, a pivotal agricultural pest within the Acari order, remains largely unexplored. In this study, we unravel the virome intricacies, investigating the interplay between biotic factors (Spiroplasma and Wolbachia endosymbionts) and abiotic stresses (abamectin exposure and temperature variations). Our metatranscriptomics analyses reveal novel viral species alongside known pathogens suggesting an emerging vector role. Notably, abamectin treatment disrupts specific viruses, while single infections of Wolbachia or Spiroplasma impact both diversity and abundance. Strikingly, Wolbachia-Spiroplasma co-infection reveals mutually exclusive mechanisms. Overall, our work sheds light on the T. truncatus virome, emphasizing the microbiome’s influence on virus dynamics and the role of pesticides in shaping agroecosystems.

RNA Libraries

Publicly available T. truncatus RNAseq libraries were obtained from the Sequence Read Archive (SRA) database at NCBI. A total of 39 libraries covering a wide range of sampling conditions and geographical origins were selected for this study. Among these, 25 libraries were used as input for virus discovery while 14 were used as biological replicates to access independent RNA levels. Overall, these libraries were constructed from total RNA of T. truncatus individuals reared on different host plants (soybean, eggplant, and tomato), samples from heat-stressed environments, samples infected or co-infected with Wolbachia pipientis and/or Spiroplasma ixodetis, and samples of mites exposed or unexposed to the acaricide abamectin. Individual library descriptions are available in Supplementary file 1.

Identification of Endogenous Viral Elements (EVEs)

To provide disambiguation between possible Endogenous Viral Elements (EVEs) and exogenous virus species circulating in T. truncatus, we screened the reference genome GCA_028476895.1 [95] using the pipeline previously described in Aguiar et al.(2020).

Metaviromic Analyses

De novo virus discovery in RNAseq libraries was performed as described in ESPINAL et al. (2023). Briefly, quality control of sequenced reads was assessed using FastQC (version 0.74 + galaxy0) [97] and low-quality reads (Phred < 20) and adaptor sequences were removed using Trimmomatic (Galaxy Version 0.38.1) [98]. Trimmed reads were aligned against the host genome using Bowtie2 (version 2.5.0 + galaxy0) [99] with default settings to identify host-related reads. Unaligned reads were assembled in parallel using different assembly tools including Trinity (version 2.15.1 + galaxy0) [100], SPAdes (version 3.15.4 + galaxy1) [101], rnaviralSPAdes (version 3.15.4 + galaxy2) [102], metaviralSPAdes [103], Megahit [104] and OASIS [105]. To identify potential virus-derived contigs, sequence similarity search was performed using Diamond (2.0.15 + galaxy0) [106] with BlastX mode, utilizing the NCBI Viral Refseq database (release 218) as reference. These analyses were conducted using the Galaxy Australia platform [107].

Manual curation of contigs annotated as viral genomes

Non-retroviral sequences were filtered based on a minimum size threshold of 500 nucleotides. Filtered sequences were manually examined through online version of BLAST against Nucleotide (NT) and Protein (NR) databases (release 04/2024). The ORFfinder [108] tool was used to predict open reading frames (ORFs) within contigs and InterPro [109], Hmmer [110–112] and CDblast [113] to identify conserved protein domains. An overview of BLAST best hits at nucleotide and amino acid levels for each virus-derived contig can be visualized in the Supplementary file 2.

Phylogenetic analysis

Sequences showing similarity to genes encoding for polymerases or polyproteins were used to build phylogenetic trees. MAFFT [114] was used for global alignment using standard parameters. ModelTest [115] was used to identify the best evolutionary model for the dataset and a maximum likelihood phylogenetic tree was inferred using 1000 bootstrap replicates. The CIPRES Science Gateway [116] was used for phylogenetic tree construction.

RNA abundance of viral segments

The tool Salmon (version 1.5.1 + galaxy0) [117] was used to assess the abundance of virus-derived sequences. The host mitochondrial ribosomal protein S11 (rps) protein and nuclear calmodulin-1 (cal) genes were selected as constitutive endogenous genes for comparison with virus abundance. An overview of the abundance of the viral sequences can be visualized in Supplementary file 3.

Diversity and Batch effect analysis

Assessment of alpha diversity was performed using the package VEGAN on R [118] utilizing the richness [119] to unravel the virus diversity within the conditions. In order to address potential confounding factors and ensure the robustness of our dataset, we performed batch effect analysis using RUVSeq[120].

Improving the annotation of T. truncatus genome

To improve the current T. truncatus gene annotation, we performed a comprehensive gene reannotation utilizing the Blast2GO [121] pipeline (November/2023). The process began with sequence alignment via Blast, followed by domain search using InterProScan. Orthologous groups were identified through eggNOG-mapper[122], after which GO mapping was performed. Finally, a functional annotation algorithm was employed to complete the reannotation. This pipeline ensured the inclusion of genes from the Arthropoda phylum, resulting in the annotation of over 400 additional genes compared to the previously available annotation. The novel annotation is available in the Supplementary file 4.

Ortholog inference, differential gene expression, and Gene Set Enrichment Analysis

In order to have a functional annotation for the T. truncatus genome (GCA_028476895.1), we deployed the tool OrthoFinder[123] v2.5.5.2 with default parameters to perform ortholog inference, using as reference the well described fly model Drosophila melanogaster (genome release FB2024_01) - available in Supplementary file 5. Subsequently, to unveil potential variations in gene expression across different conditions, we performed differential gene expression analysis utilizing DESeq2 [124]. Genes were considered significantly differentially expressed when having a fold change > 2 log₂ and p-value < 0.05. Gene Set Enrichment Analysis (GSEA – ClusterProfiler [125] (version 3.8) [126] was used to elucidate enriched pathways using D. melanogaster gene sets (version 3.13.0) as model obtained from Bioconductor annotation packages[127].

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Availability of data and materials

The scripts used to perform differential expression and diversity analysis are available at https://github.com/LymF/Truncatus-paper. The accession codes of public data analyzed in our work are provided in the Supplementary file 1. The viral sequences assembled in this work were deposited at NCBI Third Party Annotation database under the accession numbers (TPA: BK066973-BK066986, TPA: BK066963-BK066972, and TPA: BK067800-BK067802).

Competing Interests

The authors declare no conflict of interest. The funders had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results.

Funding

This study was funded by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior -CAPES- (Brazil), financial code 001, and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) which provided scholarships for students of the Programa de Pós-Graduação em Genética e Biologia Molecular (PPGGBM) at the Universidade Estadual de Santa Cruz. M.G.C.C. and E.R.G.R.A. are Research Fellows from CNPq.

Authors' contributions

Conceptualization, E.R.G.R.A.; methodology, L.Y.M.F., R.P.O. .; software, L.Y.M.F. S.F.S., A.G.S.S., J.P.N.S., V.C.S., R.P.O. and A.G.S.; validation, L.Y.M.F and E.R.G.R.A.; formal analysis, L.Y.M.F. and E.R.G.R.A.; investigation, L.Y.M.F. S.F.S., A.G.S.S., A.G.S., J.P.N.S., V.C.S., I.J.S.F, and P.L.C.F.; data curation, L.Y.M.F., S.F.S., A.G.S.S., J.P.N.S, P.L.C.F. and A.G.S.; writing—original draft preparation, L.Y.M.F., L.C.B.O. and E.R.G.R.A.; writing—review and editing, L.Y.M.F., L.C.B.O., I.J.S.F., A.R.O., M.G.C.C. and E.R.G.R.A. visualization, L.Y.M.F, J.P.N.S and E.R.G.R.A; supervision, E.R.G.R.A.; project administration, E.R.G.R.A.; funding acquisition, C.P.P and E.R.G.R.A. All authors have read and agreed to the published version of the manuscript.

Acknowledgments

We would like to express our gratitude to all members of the Virus Bioinformatics Laboratory – UESC for their valuable contributions and insightful discussions, especially Lucas Barbosa de Amorim Conceição for the exceptional help on Figure 5 design. We also extend our thanks to the Center of Biotechnology and Genetics for providing the necessary infrastructure to facilitate the development of this project. The authors would like to acknowledge the assistance of ChatGPT, a language model developed by OpenAI, in the text editing and grammar reviewing of this manuscript.

SarwarM.Mite (Acari Acarina) vectors involved in transmission of plant viruses.In:Applied Plant Virology.Elsevier;2020.p.257–73.
HoyMA.Agricultural acarology: introduction to integrated mite management.CRC press;2011.
MigeonA,DorkeldF.2006–2022—SpiderMitesWeb:acomprehensivedatabaseforTetranychidae[Internet]—Availablefrom:http://www.montpellier.inra.fr/CBGP/spmweb.LastaccessedonAugust.2023.
SchulzJT.Tetranychus telarius(L.),NewvectorofvirusY.In:Plantdiseasereporter.1963.p.594–6.
GranilloC.TobaccoandtomatoringspotvirusesandtheirrelationshipswithTetranychusurticae.1973;:494–9.
NaingH.Knowmoreaboutspidermites(Acari:Tetranychidae)inMyanmar.2015.p.257–75.
SarwarM.Mite (Acari Acarina) vectors involved in transmission of plant viruses.In:Applied Plant Virology.Elsevier;2020.p.257–73.
BachharA,BhaskarH,PathroseB,ShylajaMR.Resistance to acaricides in Tetranychus truncatus Ehara on vegetables.Indian Journal of Entomology.2019;81:130–3.
RimySJ,DasG,GotohT,UllahMS.Lethal and sublethal effects of bifenazate on the biological parameters of Tetranychus truncatus Ehara (Acari: Tetranychidae).Systematic and Applied Acarology.2021;26:2118–32.
UllahMS,GotohT.Laboratory-based toxicity of some acaricides to Tetranychus macfarlanei and Tetranychus truncatus (Acari: Tetranychidae).International Journal of Acarology.2013;39:244–51.
BingX,XiaC,YeQ,GongX,CuiJ,PengC,etal.Wolbachia manipulates reproduction of spider mites by influencing herbivore salivary proteins.Pest Management Science.2023;79:315–23.
HoffmannAA,MontgomeryBL,PopoviciJ,Iturbe-OrmaetxeI,JohnsonPH,MuzziF,etal.Successful establishment of Wolbachia in Aedes populations to suppress dengue transmission.Nature.2011;476:454–7.
Iturbe-OrmaetxeI,WalkerT,O’ NeillSL.Wolbachia and the biological control of mosquito‐borne disease.EMBO Reports.2011;12:508–18.
WeeksAR,Tracy ReynoldsK,HoffmannAA.Wolbachia dynamics and host effects: what has (and has not) been demonstrated?Trends in Ecology & Evolution.2002;17:257–62.
ZhangY,YangK,ZhuY,HongX.Symbiont-conferred reproduction and fitness benefits can favour their host occurrence.Ecology and Evolution.2018;8:1626–33.
XieK,LuY,YangK,HuoS,HongX.Co-infection of Wolbachia and Spiroplasma in spider mite Tetranychus truncatus increases male fitness.Insect Science.2020;27:921–37.
Mathé-HubertH,KaechH,GanesanandamoorthyP,VorburgerC.Evolutionary costs and benefits of infection with diverse strains of Spiroplasma in pea aphids*.Evolution.2019;73:1466–81.
MoreiraLA,Iturbe-OrmaetxeI,JefferyJA,LuG,PykeAT,HedgesLM,etal.A Wolbachia Symbiont in Aedes aegypti Limits Infection with Dengue, Chikungunya, and Plasmodium.Cell.2009;139:1268–78.
WerrenJH,BaldoL,ClarkME.Wolbachia: master manipulators of invertebrate biology.Nat Rev Microbiol.2008;6:741–51.
BounfourM,TanigoshiLK.Effect of Temperature on Development and Demographic Parameters of < I > Tetranychus urticae and < I > Eotetranychus carpini borealis (Acari: Tetranychidae).an.2001;94:400–4.
KilpatrickAM,MeolaMA,MoudyRM,KramerLD.Temperature,Viral Genetics, and the Transmission of West Nile Virus by Culex pipiens Mosquitoes.PLoS Pathog.2008;4:e1000092.
LowenAC,MubarekaS,SteelJ,PaleseP.Influenza Virus Transmission Is Dependent on Relative Humidity and Temperature.PLoS Pathog.2007;3:e151.
PuX,YangY,WuS,WuY.Characterisation of abamectin resistance in a field-evolved multiresistant population of Plutella xylostella: Abamectin resistance in Plutella xylostella.Pest Manag Sci.2010;66:371–8.
ZhuY-X,SongZ-R,ZhangY-Y,HoffmannAA,HongX-Y.Spider Mites Singly Infected With Either Wolbachia or Spiroplasma Have Reduced Thermal Tolerance.Front Microbiol.2021;12:706321.
EspinalRBA,DeSantanaSF,SantosVC,LizardoGNR,SilvaRJS,CorrêaRX,etal.Uncovering a Complex Virome Associated with the Cacao Pathogens Ceratocystis cacaofunesta and Ceratocystis fimbriata.Pathogens.2023;12:287.
BashiardesS,Zilberman-SchapiraG,ElinavE.Use of Metatranscriptomics in Microbiome Research.Bioinform Biol Insights.2016;10:BBI.S34610.
FrançoisS,MutuelD,DuncanA,RodriguesL,DanzelleC,LefevreS,etal.A New Prevalent Densovirus Discovered in Acari. Insight from Metagenomics in Viral Communities Associated with Two-Spotted Mite (Tetranychus urticae) Populations.Viruses.2019;11:233.
NiuJ,ZhangW,SunQ-Z,WangJ-J.Three novel RNA viruses in the spider mite Tetranychus urticae and their possible interactions with the host RNA interference response.Journal of Invertebrate Pathology.2019;166:107228.
YadavPD,NyayanitDA,SheteAM,JainS,MajumdarTP,ChaubalGY,etal.Complete genome sequencing of Kaisodi virus isolated from ticks in India belonging to Phlebovirus genus, family Phenuiviridae.Ticks and Tick-borne Diseases.2019;10:23–33.
HarrisonRL,HerniouEA,BézierA,JehleJA,BurandJP,TheilmannDA,etal.ICTV Virus Taxonomy Profile: Nudiviridae. Journal of General Virology.2020;101:3–4.
Ramos-GonzálezPL,Chabi-JesusC,TassiAD,CalegarioRF,HarakavaR,NomeCF,etal.A Novel Lineage of Cile-Like Viruses Discloses the Phylogenetic Continuum Across the Family Kitaviridae.Front Microbiol.2022;13:836076.
TassiAD,Ramos-GonzálezPL,SinicoTE,KitajimaEW,Freitas-AstúaJ.Circulative Transmission of Cileviruses in Brevipalpus Mites May Involve the Paracellular Movement of Virions.Front Microbiol.2022;13:836743.
PleshakovaTI,KakarekaNN,SapotskiyMV,LednevaVA,ShchelkanovMY.VARIETY OF VIRUSES AFFECTING CEREALS IN THE FAR EAST.
BertazzonN,RahaliM,AngeliniE,CrespanM,MigliaroD.First Report of Grapevine Pinot gris virus Infecting Grapevine in Algeria.Plant Disease.2021;105:234.
ChoiI-R,FrenchR,HeinGL,StengerDC.Fully Biologically Active In Vitro Transcripts of the Eriophyid Mite-Transmitted Wheat Streak Mosaic Tritimovirus.Phytopathology®.1999;89:1182–5.
SimkovichA,KohalmiS,WangA.First report of Cherry virus A infecting sweet cherry in Ontario, Canada.New Disease Reports.2021;44:e12022.
KarasevAV,GraySM.Continuous and Emerging Challenges of Potato virus Y in Potato.Annu Rev Phytopathol.2013;51:571–86.
MourierH,PoulsenKP.Control of insects and mites in grain using a high temperature/short time (HTST) technique.Journal of Stored Products Research.2000;36:309–18.
LiW,WuX,HuT,LiuL,WangS,SongL.The role of cytochrome P450 3A2 and 4V2 in response to high-temperature stress in Tetranychus truncatus (Acari: Tetranychidae).Exp Appl Acarol.2023;91:263–77.
KumarS,SharmaAK,RawatSS,JainDK,GhoshS.UseofpesticidesinagricultureandlivestockanimalsanditsimpactonenvironmentofIndia.
KoleRK,RoyK,PanjaBN,SankarganeshE,MandaT,WoredeRE.Use of pesticides in agriculture and emergence of resistant pests.ijah.2019;58:53.
HawkinsNJ,BassC,DixonA,NeveP.The evolutionary origins of pesticide resistance.Biological Reviews.2019;94:135–55.
ZhangL,WangD,ShiP,LiJ,NiuJ,ChenJ,etal.A naturally isolated symbiotic bacterium suppresses flavivirus transmission by Aedes mosquitoes.Science.2024;384:eadn9524.
MinK-T,BenzerS.Wolbachia, normally a symbiont of Drosophila, can be virulent, causing degeneration and early death.Proc Natl Acad Sci USA.1997;94:10792–6.
SinkinsSP.Wolbachia and cytoplasmic incompatibility in mosquitoes.Insect Biochemistry and Molecular Biology.2004;34:723–9.
KliotA,CiliaM,CzosnekH,GhanimM.Implication of the Bacterial Endosymbiont Rickettsia spp. in Interactions of the Whitefly Bemisia tabaci with Tomato yellow leaf curl virus.J Virol.2014;88:5652–60.
KaurR,SinghS,JoshiN.Pervasive Endosymbiont Arsenophonus Plays a Key Role in the Transmission of Cotton Leaf Curl Virus Vectored by Asia II-1 Genetic Group of Bemisia tabaci.Environmental Entomology.2022;51:564–77.
YangK,ChenH,BingX-L,XiaX,ZhuY-X,HongX-Y.Wolbachia and Spiroplasma could influence bacterial communities of the spider mite Tetranychus truncatus.Exp Appl Acarol.2021;83:197–210.
ZhuY-X,SongZ-R,HuoS-M,YangK,HongX-Y.Variation in the microbiome of the spider mite Tetranychus truncatus with sex, instar and endosymbiont infection.FEMS Microbiology Ecology.2020;96:fiaa004.
AliotaMT,WalkerEC,Uribe YepesA,Dario VelezI,ChristensenBM,OsorioJE.The wMel Strain of Wolbachia Reduces Transmission of Chikungunya Virus in Aedes aegypti.PLoS Negl Trop Dis.2016;10:e0004677.
MoussonL,MartinE,ZouacheK,MadecY,MavinguiP,FaillouxAB.Wolbachia modulates Chikungunya replication in Aedes albopictus.Molecular Ecology.2010;19:1953–64.
LoterioRK,MonsonEA,TemplinR,DeBruyneJT,FloresHA,MackenzieJM,etal.Antiviral Wolbachia strains associate with Aedes aegypti endoplasmic reticulum membranes and induce lipid droplet formation to restrict dengue virus replication.mBio.2024;15:e02495-23.
EdenboroughKM,FloresHA,SimmonsCP,FraserJE.Using Wolbachia to Eliminate Dengue: Will the Virus Fight Back?J Virol.2021;95:e02203-20.
CaragataE,DutraH,MoreiraL.Inhibition of Zika virus by Wolbachia in Aedes aegypti.Microbial Cell.2016;3:293–5.
BonningBC.The Dicistroviridae: An emerging family of invertebrate viruses.Virol Sin.2009;24:415–27.
AntTH,HerdC,LouisF,FaillouxAB,SinkinsSP.Wolbachia transinfections in Culex quinquefasciatus generate cytoplasmic incompatibility.Insect Molecular Biology.2020;29:1–8.
DeOliveiraCD,GonçalvesDS,BatonLA,ShimabukuroPHF,CarvalhoFD,MoreiraLA.Broader prevalence of Wolbachia in insects including potential human disease vectors.Bull Entomol Res.2015;105:305–15.
HrdinaA,Serra CanalesM,Arias-RojasA,FrahmD,IatsenkoI.The endosymbiont Spiroplasma poulsonii increases Drosophila melanogaster resistance to pathogens by enhancing iron sequestration and melanization.mBio.2024;:e00936-24.
BallingerMJ,PerlmanSJ.The defensive Spiroplasma.Current Opinion in Insect Science.2019;32:36–41.
HamiltonPT,PengF,BoulangerMJ,PerlmanSJ.A ribosome-inactivating protein in a Drosophila defensive symbiont.Proc Natl Acad Sci USA.2016;113:350–5.
KaurI,GuptaRC,PuriM.Ribosome inactivating proteins from plants inhibiting viruses.Virol Sin.2011;26:357–65.
CitoresL,IglesiasR,FerrerasJM.Antiviral Activity of Ribosome-Inactivating Proteins.Toxins.2021;13:80.
JupatanakulN,SimS,DimopoulosG.The Insect Microbiome Modulates Vector Competence for Arboviruses.Viruses.2014;6:4294–313.
AguiarERGR,OlmoRP,MarquesJT.Virus-derived small RNAs: molecular footprints of host–pathogen interactions.WIREs RNA.2016;7:824–37.
HessAM,PrasadAN,PtitsynA,EbelGD,OlsonKE,BarbacioruC,etal.Small RNA profiling of Dengue virus-mosquito interactions implicates the PIWI RNA pathway in anti-viral defense.BMC Microbiol.2011;11:45.
HussainM,EtebariK,AsgariS.Functions of Small RNAs in Mosquitoes.In:Advances in Insect Physiology.Elsevier;2016.p.189–222.
EtebariK,AsadS,ZhangG,AsgariS.Identification of Aedes aegypti Long Intergenic Non-coding RNAs and Their Association with Wolbachia and Dengue Virus Infection.PLoS Negl Trop Dis.2016;10:e0005069.
SchnettlerE,DonaldCL,HumanS,WatsonM,SiuRWC,McFarlaneM,etal.Knockdown of piRNA pathway proteins results in enhanced Semliki Forest virus production in mosquito cells.Journal of General Virology.2013;94:1680–9.
WuQ,LuoY,LuR,LauN,LaiEC,LiW-X,etal.Virus discovery by deep sequencing and assembly of virus-derived small silencing RNAs.Proc Natl Acad Sci USA.2010;107:1606–11.
MorazzaniEM,WileyMR,MurredduMG,AdelmanZN,MylesKM.Production of Virus-Derived Ping-Pong-Dependent piRNA-like Small RNAs in the Mosquito Soma.PLoS Pathog.2012;8:e1002470.
BlairCD.Mosquito RNAi is the major innate immune pathway controlling arbovirus infection and transmission.Future Microbiology.2011;6:265–77.
DonaldCL,KohlA,SchnettlerE.New Insights into Control of Arbovirus Replication and Spread by Insect RNA Interference Pathways.Insects.2012;3:511–31.
JhengJ-R,HoJ-Y,HorngJ-T.ER stress, autophagy, and RNA viruses.Front Microbiol.2014;5.
TallóczyZ,Virgin,IvH,LevineB.PKR-Dependent Xenophagic Degradation of Herpes Simplex Virus Type 1.Autophagy.2006;2:24–9.
VoroninD,CookDAN,StevenA,TaylorMJ.Autophagy regulates Wolbachia populations across diverse symbiotic associations.Proc Natl Acad Sci USA.2012;109.
SinkinsSP.Wolbachia and arbovirus inhibition in mosquitoes.Future Microbiology.2013;8:1249–56.
OuJ,LiuQ,BianY,LuanX,MengY,DongH,etal.Integrated analysis of mRNA and microRNA transcriptome related to immunity and autophagy in shrimp hemocytes infected with Spiroplasma eriocheiris.Fish & Shellfish Immunology.2022;130:436–52.
BlancM,HsiehWY,RobertsonKA,WattersonS,ShuiG,LacazeP,etal.Host Defense against Viral Infection Involves Interferon Mediated Down-Regulation of Sterol Biosynthesis.PLoS Biol.2011;9:e1000598.
HaasMJ,MooradianAD.Regulation of high-density lipoprotein by inflammatory cytokines: establishing links between immune dysfunction and cardiovascular disease.Diabetes Metabolism Res.2010;26:90–9.
MolloyJC,SommerU,ViantMR,SinkinsSP.Wolbachia Modulates Lipid Metabolism in Aedes albopictus Mosquito Cells.Appl Environ Microbiol.2016;82:3109–20.
WuM,SunLV,VamathevanJ,RieglerM,DeboyR,BrownlieJC,etal.Phylogenomics of the Reproductive Parasite Wolbachia pipientis wMel: A Streamlined Genome Overrun by Mobile Genetic Elements.PLoS Biol.2004;2:e69.
CaragataEP,RancèsE,HedgesLM,GoftonAW,JohnsonKN,O’NeillSL,etal.Dietary Cholesterol Modulates Pathogen Blocking by Wolbachia.PLoS Pathog.2013;9:e1003459.
KohC,IslamMN,YeYH,ChotiwanN,GrahamB,BelisleJT,etal.Dengue virus dominates lipid metabolism modulations in Wolbachia-coinfected Aedes aegypti.Commun Biol.2020;3:518.
ParedesJC,HerrenJK,SchüpferF,LemaitreB.The Role of Lipid Competition for Endosymbiont-Mediated Protection against Parasitoid Wasps in Drosophila.mBio.2016;7:e01006-16.
WongZS,BrownlieJC,JohnsonKN.Oxidative Stress Correlates with Wolbachia-Mediated Antiviral Protection in Wolbachia-Drosophila Associations.Appl Environ Microbiol.2015;81:3001–5.
ZugR,HammersteinP.Wolbachia and the insect immune system: what reactive oxygen species can tell us about the mechanisms of Wolbachia–host interactions.Front Microbiol.2015;6.
DingZ.Oxidative stress responses of the crayfish Procambrus clarkii to Spiroplasma eriocheiris challenge.Aquaculture Reports.2022;25:101219.
JaafarZA,KieftJS.Viral RNA structure-based strategies to manipulate translation.Nat Rev Microbiol.2019;17:110–23.
LiS.Regulation of Ribosomal Proteins on Viral Infection.Cells.2019;8:508.
DenBoonJA,DiazA,AhlquistP.Cytoplasmic Viral Replication Complexes. Cell Host & Microbe.2010;8:77–85.
AhlquistP,NoueiryAO,LeeW-M,KushnerDB,DyeBT.Host Factors in Positive-Strand RNA Virus Genome Replication.J Virol.2003;77:8181–6.
Serge AndigemaA.Viral Hijacking of Host RNA-binding Proteins: Implications for Viral Replication and Pathogenesis.JCLR.2024;7:01–6.
CiminoPA,NicholsonBL,WuB,XuW,WhiteKA.Multifaceted Regulation of Translational Readthrough by RNA Replication Elements in a Tombusvirus.PLoS Pathog.2011;7:e1002423.
PrasadA,SharmaS,PrasadM.Deeper look into viruses: replication intermediates do code!Plant Cell Rep.2024;43:52.
ChenL,YuX,XueX,ZhangF,GuoL,ZhangH,etal.The genome sequence of a spider mite, Tetranychus truncatus, provides insights into interspecific host range variation and the genetic basis of adaptation to a low-quality host plant.Insect Science.2023;30:1208–28.
AguiarERGR,DeAlmeidaJPP,QueirozLR,OliveiraLS,OlmoRP,DeFariaIJDS,etal.A single unidirectional piRNA cluster similar to the flamenco locus is the major source of EVE-derived transcription and small RNAs in Aedes aegypti mosquitoes.RNA.2020;26:581–94.
WingettSW,AndrewsS.FastQ Screen: A tool for multi-genome mapping and quality control.F1000Res.2018;7:1338.
BolgerAM,LohseM,UsadelB.Trimmomatic: a flexible trimmer for Illumina sequence data.Bioinformatics.2014;30:2114–20.
LangmeadB,SalzbergSL.Fast gapped-read alignment with Bowtie 2.Nat Methods.2012;9:357–9.
GrabherrMG,HaasBJ,YassourM,LevinJZ,ThompsonDA,AmitI,etal.Full-length transcriptome assembly from RNA-Seq data without a reference genome.Nat Biotechnol.2011;29:644–52.
PrjibelskiAD,VasilinetcI,BankevichA,GurevichA,KrivosheevaT,NurkS,etal.ExSPAnder: a universal repeat resolver for DNA fragment assembly.Bioinformatics.2014;30:i293–301.
AntipovD,KorobeynikovA,McLeanJS,PevznerPA.hybrid SPA des: an algorithm for hybrid assembly of short and long reads.Bioinformatics.2016;32:1009–15.
AntipovD,RaikoM,LapidusA,PevznerPA.Plasmid detection and assembly in genomic and metagenomic data sets.Genome Res.2019;29:961–8.
LiD,LiuC-M,LuoR,SadakaneK,LamT-W.MEGAHIT: an ultra-fast single-node solution for large and complex metagenomics assembly via succinct de Bruijn graph.Bioinformatics.2015;31:1674–6.
Oases. robustde novoRNA-seqassemblyacrossthedynamicrangeofexpressionlevels.Bioinformatics.2012;28:1086–92.
BuchfinkB,XieC,HusonDH.Fast and sensitive protein alignment using DIAMOND.Nat Methods.2015;12:59–60.
AfganE,BakerD,BatutB,van denBeekM,BouvierD,ČechM,etal.The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2018 update.Nucleic Acids Research.2018;46:W537–44.
RombelIT,SykesKF,RaynerS,JohnstonSA.ORF-FINDER: a vector for high-throughput gene identification.Gene.2002;282:33–41.
BlumM,ChangH-Y,ChuguranskyS,GregoT,KandasaamyS,MitchellA,etal.The InterPro protein families and domains database: 20 years on.Nucleic Acids Research.2021;49:D344–54.
FinnRD,ClementsJ,ArndtW,MillerBL,WheelerTJ,SchreiberF,etal.HMMER web server: 2015 update.Nucleic Acids Res.2015;43:W30–8.
FinnRD,ClementsJ,EddySR.HMMER web server: interactive sequence similarity searching.Nucleic Acids Research.2011;39suppl:W29–37.
PotterSC,LucianiA,EddySR,ParkY,LopezR,FinnRD.HMMER web server: 2018 update.Nucleic Acids Research.2018;46:W200–4.
Marchler-BauerA,BryantSH.CD-Search: protein domain annotations on the fly.Nucleic Acids Research.2004;32Web Server:W327–31.
KatohK,StandleyDM.MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability.Molecular Biology and Evolution.2013;30:772–80.
PosadaD,CrandallKA.MODELTEST: testing the model of DNA substitution.Bioinformatics.1998;14:817–8.
MillerMA,PfeifferW,SchwartzT.Creating the CIPRES Science Gateway for inference of large phylogenetic trees.In:2010 Gateway Computing Environments Workshop (GCE). New Orleans,LA, USA:IEEE;2010.p.1–8.
PatroR,DuggalG,LoveMI,IrizarryRA,KingsfordC.Salmon provides fast and bias-aware quantification of transcript expression.Nat Methods.2017;14:417–9.
OksanenJ,RoelandK,PierreL,BobO,GavinLS.TheveganPackage.2008.
FisherRA,CorbetAS,WilliamsCB.The Relation Between the Number of Species and the Number of Individuals in a Random Sample of an Animal Population.The Journal of Animal Ecology.1943;12:42.
RissoD,NgaiJ,SpeedTP,DudoitS.Normalization of RNA-seq data using factor analysis of control genes or samples.Nat Biotechnol.2014;32:896–902.
ConesaA,GötzS,García-GómezJM,TerolJ,TalónM,RoblesM.Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research.Bioinformatics.2005;21:3674–6.
CantalapiedraCP,Hernández-PlazaA,LetunicI,BorkP,Huerta-CepasJ.eggNOG-mapper v2: Functional Annotation, Orthology Assignments, and Domain Prediction at the Metagenomic Scale.Molecular Biology and Evolution.2021;38:5825–9.
EmmsDM,KellyS.OrthoFinder: phylogenetic orthology inference for comparative genomics.Genome Biol.2019;20:238.
LoveMI,HuberW,AndersS.Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2.Genome Biol.2014;15:550.
YuG,WangL-G,HanY,HeQ-Y.clusterProfiler: an R Package for Comparing Biological Themes Among Gene Clusters.OMICS: A Journal of Integrative Biology.2012;16:284–7.
ShiJ,WalkerM.Gene Set Enrichment Analysis (GSEA) for Interpreting Gene Expression Profiles.CBIO.2007;2:133–7.
GentlemanRC,CareyVJ,BatesDM,BolstadB,DettlingM,DudoitS,etal.Bioconductor: open software development for computational biology and bioinformatics.Genome Biol.2004;5:R80.

No competing interests reported.

FerreiraetalBMCBiologySupplementaryfigures.pdf
Supplementaryfile1.xlsx
Supplementary file 1. Table presenting key information for all utilized libraries.
Supplementaryfile2.xlsx
Supplementary file 2. Table summarizing blastN and blastX alignments for viral transcripts, including details such as alignment subjects, percentage identity, and E-values
Supplementaryfile3.xlsx
Supplementary file 3. Transcriptional activity of virus and host transcripts.
Supplementaryfile4.xlsx
Supplementary file 4. Tetranychus truncatustranscriptome re-annotation.
Supplementaryfile5.xlsx
Supplementary file 5. Analysis of T. truncatusgenes orthologues.
Supplementaryfile6.xlsx
Supplementary file 6. Sequence similarity results for Endogenous Viral Elements.
Supplementaryfile7.xlsx
Supplementary file 7. Gene Set Enrichment analysis for differentially expressed genes.

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Spiroplasma shows a Wolbachia-like effect in hampering virus replication in spider mite

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Background

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Virome of spider mites

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Conclusions

METHODS

RNA Libraries

Identification of Endogenous Viral Elements (EVEs)

Metaviromic Analyses

Manual curation of contigs annotated as viral genomes

Phylogenetic analysis

RNA abundance of viral segments

Diversity and Batch effect analysis

Ortholog inference, differential gene expression, and Gene Set Enrichment Analysis

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1