ABSTRACT Insect-virus associations have been studied extensively in the context of pathogenic viruses transmitted by insects, whereas research on nonpathogenic viruses remains relatively scarce. Recently, we discovered three nonpathogenic RNA viruses in the parasitoid wasp Anagyrus vladimiri: AnvRV, AnvDV, and AnvIfV. Here, using transmission electron microscopy, we detected AnvRV in the wasp oocytes and in a distinct group of cells in the ovaries, which we termed “satellite cells,” but not in the venom gland or venom reservoir, indicating that AnvRV is transmitted transovarially. Next, we analyzed gene expression in AnvRV-infected and uninfected wasps and found that AnvRV modulates the immune response and alters venom composition. Notably, the NF-κB inhibitor gene was upregulated in the wasp ovaries, where AnvRV is localized, suggesting that AnvRV locally suppresses the immune response of A. vladimiri to facilitate its transmission. Next, we studied the prevalence of the three viruses in field populations of A. vladimiri and its hosts, Planococcus citri and Planococcus ficus. AnvRV was absent from both mealybug species and detected at low prevalence in A. vladimiri, whereas AnvDV and AnvIfV were consistently present in P. citri. Lastly, by datamining of public RNAseq data sets, we investigated the prevalence of these viruses in other parasitoid species and revealed only a few related viruses. Taken together, we postulate that AnvRV is an active symbiont of A. vladimiri, influencing host gene regulation. These findings provide new insights into the diversity of insect–virus interactions and their potential roles in shaping parasitoid biology.IMPORTANCEViruses likely represent the majority of insect symbiotic microorganisms. Yet, viral symbionts and their interactions with insect hosts were less studied, mostly due to technical difficulties stemming from their small size and lack of universal markers. Although viral symbionts are usually perceived as pathogens, there are clear instances in which they are beneficial to their hosts, providing functions that are essential in some cases and conditionally beneficial in others, shaping insect ecology and evolution. This study provides several pieces of the puzzle on the road to understanding the complex interactions within the multi-trophic system consisting of a parasitoid wasp, its mealybug host, and a double-stranded RNA virus. This system may serve as a case study of viruses’ effect on insects and broaden our understanding of the possible effects of viruses on other arthropods.

Information on the impacts of RNA viruses inhabiting insect hosts is scarce. Here, we studied the effects of a recently described RNA virus, Anagyrus vladimiri Reovirus, on its host, the parasitoid wasp Anagyrus vladimiri (Hymenoptera: Encyrtidae), an important natural enemy of mealybug pest species. We found that Anagyrus vladimiri Reovirus is maternally transmitted with very high fidelity but not paternally. Additionally, Anagyrus vladimiri Reovirus is horizontally transferred at a frequency of 23% from infected to uninfected wasp larvae that develop together inside the same mealybug host (superparasitism). To test the effects of Anagyrus vladimiri Reovirus on A. vladimiri, the virus horizontal transmission was utilized to establish Anagyrus vladimiri Reovirus-infected (RV+) and uninfected (RV−) isogenic wasp lines, a method rarely applied and novel to RNA virus–parasitoid systems. Longevity, developmental time, sex ratio, and fecundity of RV+ and RV− A. vladimiri were very similar. Nonetheless, the egg hatching rate of RV+ wasps was markedly and significantly higher than that of RV- wasps, especially in hosts that were not superparasitized. Additionally, significantly fewer encapsulation marks (the main form of mealybug immunity) were found around RV+ eggs inside parasitized mealybug hosts. Taken together, the data shows that Anagyrus vladimiri Reovirus may benefit A. vladimiri by counteracting the encapsulation immune response of the mealybug. However, why the numbers of offspring in both wasp lines are similar remains unclear. These findings present a rare example of interaction between an RNA virus and a parasitoid and may provide a tool for the improvement of biological control efforts.

Information on the impacts of RNA viruses inhabiting insect hosts is scarce. Here, we studied the effects of a recently described RNA virus, termed AnvRV, on its host, the parasitoid wasp Anagyrus vladimiri (Hymenoptera: Encyrtidae), an important natural enemy of mealybug pests. AnvRV was found to be maternally transmitted with very high fidelity but not paternally. Additionally, AnvRV was horizontally transferred at an efficiency of 23% from infected to uninfected wasp larvae that develop together inside the same mealybug host (superparasitism). To test the effects of AnvRV on A. vladimiri , the virus horizontal transmission was utilized to establish AnvRV-infected (RV+) and uninfected (RV-) isogenic wasp lines, a method rarely applied and novel to RNA virus-parasitoid systems. Longevity, developmental time, sex ratio, and fecundity of RV+ and RV- A. vladimiri were very similar. Nonetheless, the egg hatching rate of RV+ wasps was markedly and significantly higher than that of RV- wasps, especially in hosts that were not superparasitized. Additionally, less encapsulation marks (the main form of mealybug immunity) were found around RV+ eggs inside parasitized mealybug hosts. Taken together, the data suggest that AnvRV is affecting the mealybugs' physiology in a way that improves first stages of wasps' development. These findings present a rare example of interaction between an RNA virus and a parasitoid and may provide a tool for the improvement of biological control efforts.  ### Competing Interest Statement The authors have declared no competing interest.

Many arthropods host bacterial symbionts, some of which are known to influence host nutrition and diet breadth. Omnivorous bugs of the genus Macrolophus (Heteroptera: Miridae) are mainly predatory, but may also feed on plants. The species M. pygmaeus and M. melanotoma (=M. caliginosus) are key natural enemies of various economically important agricultural pests, and are known to harbor two Rickettsia species, R. bellii and R. limoniae. To test for possible involvement of symbiotic bacteria in the nutritional ecology of these biocontrol agents, the abundance, phylogeny, and distribution patterns of the two Rickettsia species in M. pygmaeus and M. melanotoma were studied. Both of the Rickettsia species were found in 100 and 84% of all tested individuals of M. pygmaeus and M. melanotoma, respectively. Phylogenetic analysis showed that a co-evolutionary process between Macrolophus species and their Rickettsia is infrequent. Localization of R. bellii and R. limoniae has been detected in both female and male of M. pygmaeus and M. melanotoma. FISH analysis of female gonads revealed the presence of both Rickettsia species in the germarium of both bug species. Each of the two Rickettsia species displayed a unique distribution pattern along the digestive system of the bugs, mostly occupying separate epithelial cells, unknown caeca-like organs, the Malpighian tubules and the salivary glands. This pattern differed between the two Macrolophus species: in M. pygmaeus, R. limoniae was distributed more broadly along the host digestive system and R. bellii was located primarily in the foregut and midgut. In contrast, in M. melanotoma, R. bellii was more broadly distributed along the digestive system than the clustered R. limoniae. Taken together, these results suggest that Rickettsia may have a role in the nutritional ecology of their plant-and prey-consuming hosts.

Many arthropod species harbor a diverse range of viruses. While much is known about pathogenic viruses of some economically important insects and arthropods involved in disease transmission, viruses associated with mites have rarely been studied. The main objective of this study was to characterize the virome of Phytoseiulus persimilis (Phytoseiidae), a predatory mite commercially used worldwide for the biological control of the key pest Tetranychus urticae (Tetranichidae). A combination of de novo transcriptome assembly and virion sequencing, revealed that RNA viruses are highly prevalent and active tenants of commercial populations of P. persimilis, comprising on average 9% of the mite's total mRNA. Seventeen RNA viruses dominated the mite's virome (i.e., were highly transcribed) with over half (n = 10) belonging to the order Picornavirales, + ssRNA viruses that infect a large range of hosts, including arthropods. Screening of the 17 dominant virus sequences in P. persimilis and T. urticae revealed that three viruses (two Picornavirales of the families Iflaviridae and Dicistroviridae, and one unclassified Riboviria) are unique to P. persimilis and three others (two unclassified Picornavirales and one unclassified Riboviria) are present in both mite species. Most of the sequences were related to viruses previously documented in economically important arthropods, while others have rarely been documented before in arthropods. These findings demonstrate that P. persimilis, like many other arthropods, harbors a diverse RNA virome, which might affect the mite's physiology and consequently its efficiency as a biological control agent.

The knowledge on symbiotic microorganisms of insects has increased in recent years, yet relatively little data is available on non-pathogenic viruses. Here we studied the virome of the parasitoid wasp Anagyrus vladimiri (Hymenoptera: Encyrtidae), a biocontrol agent of mealybugs. By high-throughput sequencing of viral nucleic acids, we revealed three novel viruses, belonging to the families Reoviridae (provisionally termed AnvRV [Anagyrus vladimiri reovirus]), Iflaviridae (AnvIFV) and Dicistroviridae (AnvDV). Phylogenetic analysis further classified the AnvRV in the genus Idnoreovirus , and the AnvDV in the genus Triatovirus . The genome of AnvRV is comprised of 10 distinct genomic segments ranging in length from 1.5 to 4.2 Kbp, but only two out of the 10 open reading frames (ORFs) have a known function. AnvIFV and AnvDV each have one polypeptide ORF, which is typical to iflaviruses but very un-common among dicistroviruses. AnvRV was found to be fixed in a mass-reared population of A. vladimiri , whereas it’s prevalence in field-collected wasps was ~15%. Similarly, the prevalence of AnvIFV and AnvDV were much higher in the mass rearing population than in the field population. Transmission electron micrographs of females’ ovaries revealed clusters and viroplasms of Reovirus -like particles in follicle cells. AnvRV was not detected in the mealybugs, suggesting that this virus is truly associated with the wasps. The possible effects of these viruses on A. vladimiri ’s biology, and on biocontrol agents in general are discussed. Our findings identify RNA viruses as potential players involved in the multitrophic system of mealybugs, their parasitoids and other members of the holobiont. Importance Different biological control approaches use industrially mass-reared natural enemy insects to reduce damage of arthropod pests. Such mass-reared cultures may be positively and/or negatively affected by various microorganisms, including viruses. Yet, current knowledge on virus diversity, especially in arthropods, is limited. Here, we provide the first virome characterization of a member of the wasps family Encyrtidae - the member being the parasitoid Anagyrus vladimiri - a commercially important natural enemy of mealybug pests. We describe the genome of three previously unknown RNA viruses, co-inhabiting this parasitoid, and elaborate on the prevalence of those viruses in individual wasps of both mass-reared and environmental origins. Microscopy images suggest that at least one of the viruses is transmitted maternally via the ovaries. We discuss the genomic structure of the viruses and the possible relationship between those viruses and the A. vladimiri host, with implications on improvement of biocontrol of mealybug pests.

Yellows diseases in grapevine, associated with the presence of different phytoplasmas, are a major problem for growers, with no environmentally friendly means of control. Frateuria defendens (Frd), a bacterium with endophytic traits, has been shown to reduce yellows symptoms in grapevine plantlets under laboratory conditions. The objective of this study was to test whether similar effects could be achieved under field conditions. A trial was conducted in a heavily infected vineyard in northern Israel for two consecutive years. A suspension of Frd cells (108·mL-1) was applied bi-weekly by foliar spray on grapevines from bud burst to leaf senescence. Frd penetrated the leaves during the growing period but not during leaf senescence and could be detected in the leaves by PCR analysis up to 14 days post-spraying. The rate of yellows infection was lower in the treated grapevines compared to its increase in untreated grapevines and the yield of symptomatic plants was improved by 10 to 20 %. Taken together, the results suggest Frd acted as a biological control agent in vineyards under the experimental conditions tested.

Metabolic conversions allow organisms to produce essential metabolites from the available nutrients in an environment, frequently requiring metabolic exchanges among co-inhabiting organisms. Here, we applied genomic-based simulations for exploring tri-trophic interactions among the sap-feeding insect whitefly (Bemisia tabaci), its host-plants, and symbiotic bacteria. The simplicity of this ecosystem allows capturing the interacting organisms (based on genomic data) and the environmental content (based on metabolomics data). Simulations explored the metabolic capacities of insect-symbiont combinations under environments representing natural phloem. Predictions were correlated with experimental data on the dynamics of symbionts under different diets. Simulation outcomes depict a puzzle of three-layer origins (plant-insect-symbionts) for the source of essential metabolites across habitats and stratify interactions enabling the whitefly to feed on diverse hosts. In parallel to simulations, natural and artificial feeding experiments provide supporting evidence for an environment-based effect on symbiont dynamics. Based on simulations, a decrease in the relative abundance of a symbiont can be associated with a loss of fitness advantage due to an environmental excess in amino-acids whose production in a deprived environment used to depend on the symbiont. The study demonstrates that genomic-based predictions can bridge environment and community dynamics and guide the design of symbiont manipulation strategies.

The controlled application of olive mill wastewater (OMW) as a by-product of the olive oil extraction process is widespread in olive oil-producing countries. Therefore, a sustainable approach necessarily targets the positive effects of soil resilience between successive annual applications to exclude possible accumulations of negative consequences. To investigate this, we applied 50, 100, 100 with tillage and 150 m3 OMW ha−1 y−1 for five consecutive seasons to an olive orchard in a semi-arid region and monitored various soil physicochemical and biological properties. OMW increased soil water content with concentration of total phenols, cations, and anions as well as various biological and soil organic matter indices. Soil hydrophobicity, as measured by water drop penetration time (WDPT), was found to be predominantly in the uppermost layer (0–3 and 3–10 cm). OMW positively affected soil biology, increased the activity and abundance of soil arthropods, and served as a food source for bacteria and fungi. Subsequent shallow tillage reduced the extent of OMW-induced changes and could provide a simple means of OMW dilution and effect minimization. Despite potentially higher leaching risks, an OMW dose of 50–100 m3 ha−1 applied every two years followed by tillage could be a cost-effective and feasible strategy for OMW recycling.

Whiteflies (Hemiptera: Sternorrhyncha: Aleyrodidae) are a superfamily of small phloem-feeding insects. They rely on their primary endosymbionts "Candidatus Portiera aleyrodidarum" to produce essential amino acids not present in their diet. Portiera has been codiverging with whiteflies since their origin and therefore reflects its host's evolutionary history. Like in most primary endosymbionts, the genome of Portiera stays stable across the Aleyrodidae superfamily after millions of years of codivergence. However, Portiera of the whitefly Bemisia tabaci has lost the ancestral genome order, reflecting a rare event in the endosymbiont evolution: the appearance of genome instability. To gain a better understanding of Portiera genome evolution, identify the time point in which genome instability appeared and contribute to the reconstruction of whitefly phylogeny, we developed a new phylogenetic framework. It targeted five Portiera genes and determined the presence of the DNA polymerase proofreading subunit (dnaQ) gene, previously associated with genome instability, and two alternative gene rearrangements. Our results indicated that Portiera gene sequences provide a robust tool for studying intergenera phylogenetic relationships in whiteflies. Using these new framework, we found that whitefly species from the Singhiella, Aleurolobus, and Bemisia genera form a monophyletic tribe, the Aleurolobini, and that their Portiera exhibit genome instability. This instability likely arose once in the common ancestor of the Aleurolobini tribe (at least 70 Ma), drawing a link between the appearance of genome instability in Portiera and the switch from multibacteriocyte to a single-bacteriocyte mode of inheritance in this tribe.

Whiteflies (Hemiptera: Sternorrhyncha: Aleyrodidae) are a superfamily of small phloem-feeding insects. Their taxonomy is currently based on the morphology of nymphal stages that display phenotypic plasticity, which produces inconsistencies. To overcome this limitation, we developed a new phylogenetic framework that targets five genes of Portiera aleyrodidarum, the primary endosymbiont of whiteflies. lineages have been co-diverging with whiteflies since their origin and therefore reflect their host evolutionary history. We also studied the origin of stability and instability in genomes by testing for the presence of two alternative gene rearrangements and the loss of a functional polymerase proofreading subunit (), previously associated with genome instability. We present two phylogenetic reconstructions. One using the sequences of all five target genes from 22 whitefly species belonging to 17 genera. The second uses only two genes to include additional published sequences of 21 whitefly species, increasing our sampling size to 42 species from 25 genera. The developed framework showed low signal saturation, specificity to whitefly samples, and efficiency in solving inter-genera relationships and standing inconsistencies in the current taxonomy of the superfamily. Genome instability was found to be present only in the Aleurolobini tribe containing the and genera. This suggests that genome instability likely arose in the Aleurolobini tribe’s common ancestor, around 70 Mya. We propose a link between the switch from multi-bacteriocyte to a single-bacteriocyte mode of inheritance in the Aleurolobini tribe and the appearance of genome instability in .

While most insect herbivores are selective feeders, a small proportion of them feed on a wide range of plants. This polyphagous habit requires overcoming a remarkable array of defenses, which often necessitates an adaptation period. Efforts for understanding the mechanisms involved mostly focus on the insect’s phenotypic plasticity. Here, we hypothesized that the adaptation process might partially rely on transient associations with bacteria. To test this, we followed in a field-like experiment, the adaptation process of Bemisia tabaci, a generalist sap-feeder, to pepper (a less-suitable host), after switching from watermelon (a suitable host). Amplicon sequencing of 16S rRNA transcripts from hundreds of dissected guts revealed the presence of active “core” and “transient” bacterial communities, dominated by the phyla Proteobacteria, Actinobacteria, and Firmicutes, and increasing differences between populations grown on watermelon and pepper. Insects grown on pepper for over two generations, presented a significant increase in specific genera, mainly Mycobacterium, with a predicted enrichment in degradative pathways of xenobiotics and secondary metabolites. This result correlated with a significant increase in the insect’s survival on pepper. Taken together, our findings suggest that gut-associated bacteria can provide an additional flexible metabolic “tool-box” to generalist sap-feeders for facilitating a quick host switching process.

Bacterial symbionts in arthropods are common, vary in their effects, and can dramatically influence the outcome of biological control efforts. Macrolophus pygmaeus (Heteroptera: Miridae), a key component of biological control programs, is mainly predaceous but may also display phytophagy. M. pygmaeus hosts symbiotic Wolbachia, which induce cytoplasmic incompatibility, and two Rickettsia species, R. bellii and R. limoniae, which are found in all individuals tested. To test possible involvement of the two Rickettsia species in the feeding habits of M. pygmaeus, we first showed that the microbiome of the insect is dominated by these three symbionts, and later described the distribution pattern of the two Rickettsia species in its digestive system. Although both Rickettsia species were located in certain gut bacteriocyes, in caeca and in Malpighian tubules of both sexes, each species has a unique cellular occupancy pattern and specific distribution along digestive system compartments. Infrequently, both species were found in a cell. In females, both Rickettsia species were detected in the germarium, the apical end of the ovarioles within the ovaries, but not in oocytes. Although the cause for these Rickettsia distribution patterns is yet unknown, it is likely linked to host nutrition while feeding on prey or plants.

Candidatus Phytoplasma, the causative agent of yellows disease, inflicts substantial damage on several hundred plant species including perennials and annual plants. The endophytic bacterium Frateuria defendens reduces the symptoms of yellows disease in a number of agricultural crops. One possible mode of action is that the bacterium secretes antimicrobial metabolites. To test this hypothesis, the substances secreted by the endophyte during 10 days of growth in an artificial medium were identified by GC-MS (gas chromatography-mass spectrometry). Synthetic analogues to these substances were then used on periwinkle, a nurse culture plant infected by phytoplasma. Phytoplasma quantities were evaluated by quantitative PCR, and disease symptoms were monitored and recorded. It was found that specific compounds identified by the biochemical analysis caused a significant reduction in both the titer of phytoplasma and the disease symptoms in periwinkle when compared to untreated infected plants. Further research is required to examine the potential of these compounds as an effective treatment against yellows disease.

The compressed Supplementary_Data.tar.gz file contains all relevant scripts, R analysis, and files files generated during "Portiera gets wild" work. Sanger_sequencing.tar.gz includes Sanger raw files and Gap4 databases of the different whiteflies/Portiera sampled for the work.The scripts included are free software: you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation, either version 3 of the License, or (at your option) any later version. This program is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details. You should have received a copy of the GNU General Public License along with this program. If not, see .

BACKGROUND:Like numerous other animals, biocontrol agents (BCAs) of arthropod pests carry various microorganisms that may have diverse effects on the biology of their eukaryote hosts. We postulated that it is possible to improve the efficacy of BCAs by manipulating the composition of their associated microbiota. The parasitoid wasp Anagyrus vladimiri (Hymenoptera: Encyrtidae) from a mass-rearing facility was chosen for testing this hypothesis.RESULTS:High-throughput sequencing analysis indicated that fungal abundance in A. vladimiri was low and variable, whereas the bacterial community was dominated by the endosymbiont Wolbachia. Wolbachia was fixed in the mass-rearing population, whereas in field-collected A. vladimiri Wolbachia's prevalence was only approximately 20%. Identification of Wolbachia strains from the two populations by Multi Locus Sequence Typing, revealed two closely related but unique strains. A series of bioassays with the mass-rearing Wolbachia-fixed (W+ ) and a derived antibiotic-treated Wolbachia-free (W- ) lines revealed that: (i) Wolbachia does not induce reproductive manipulations; (ii) W- females have higher fecundity when reared individually, but not when reared with conspecifics; (iii) W+ females outcompete W- when they share hosts for oviposition; (iv) longevity and developmental time were similar in both lines.CONCLUSIONS:The findings suggest that W+ A. vladimiri have no clear fitness benefit under mass-rearing conditions and may be disadvantageous under lab-controlled conditions. In a broader view, the results suggest that augmentative biological control can benefit from manipulation of the microbiome of natural enemies.

Bois Noir is a major yellows disease in grapevine with no current means of control. The endophyte Frateuria defendens (Frd), isolated from the insect vector of Bois Noir, is a potential biocontrol agent for this disease. The aim of this study was to determine an efficient way to introduce Frd into mature vines under field conditions. Shoots of young and field-grown vines were sprayed with Frd suspension supplemented with either Tween 20, BB5, DX, or Triton X-100 as surfactants. The presence of Frd was confirmed by PCR in sprayed leaves, and in leaves below and above the sprayed site. The results showed that cells of Frd penetrate and move inside the vine shoots under field conditions. Highest penetration rate was achieved when leaves were sprayed with 108 or 109 colony forming units per mL (CFU mL-1) of Frd with addition of 0.1% Tween 20. The addition of a surfactant is essential to increase the proportion of shoots with Frd.

A Dyella-like bacterium was previously isolated from the planthopper Hyalesthes obsoletus (Hemiptera). Based on its 16S rRNA gene sequence, strain DHoT was assigned to the family Rhodanobacteraceae with Dyella and Frateuria as its closest relatives. The closest 16S rRNA gene sequences were Frateuria aurantia DSM 6220T (98.2 %), Dyella thiooxydans ATSB10T (98 %), Dyella terrae JS14-6T (97.8 %) and Dyella marensis CS5-B2T (97.8 %). Strain DHoT is a Gram-negative, aerobic, motile, yellow-pigmented, rod-shaped bacterium. Strain DHoT cells grew well at 28-30 °C and at pH 6.5-7.5 on a nutrient agar plate. DNA-DNA hybridization showed that the relatedness between strain DHoT and D. jiangningensis strain SBZ3-12T, and F. aurantia DSM 6220T was 42.7 and 42.6 %, respectively. Ubiquinone Q-8 was the predominant respiratory quinone, and the major fatty acids (>10 %) were iso-C15 : 0, iso-C16 : 0 and iso-C17 : 0. In silico analysis based on phylogenetics and sequence identity at the nucleotide and protein levels suggests that Frateuria is the closest known relative of strain DHoT. Based on the phenotypic, chemotaxonomic and phylogenetic data, strain DHoT was designated as a novel species of the genus Frateuria, for which the name Frateuria defendens sp. nov. is proposed. The type strain is DHoT (=NCCB 100648T; =DLBT=DSM 106169T).