RNA Viruses Are Prevalent and Active Tenants of the Predatory Mite Phytoseiulus persimilis (Acari: Phytoseiidae).

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.

Genomic characterization of viruses associated with the parasitoid Anagyrus vladimiri (Hymenoptera: Encyrtidae).

Knowledge on symbiotic microorganisms of insects has increased dramatically in recent years, yet relatively little data are available regarding non-pathogenic viruses. Here we studied the virome of the parasitoid wasp Anagyrus vladimiri Triapitsyn (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 AnvRV in the genus Idnoreovirus, and AnvDV in the genus Triatovirus. The genome of AnvRV comprises 10 distinct genomic segments ranging in length from 1.5 to 4.2 kb, but only two out of the 10 ORFs have a known function. AnvIFV and AnvDV each have one polypeptide ORF, which is typical of iflaviruses but very un-common among dicistroviruses. Five conserved domains were found along both the ORFs of those two viruses. AnvRV was found to be fixed in an A. vladimiri population that was obtained from a mass rearing facility, whereas its prevalence in field-collected A. vladimiri was ~15 %. Similarly, the prevalence of AnvIFV and AnvDV was much higher in the mass rearing population than in the field population. The presence of AnvDV was positively correlated with the presence of Wolbachia in the same individuals. Transmission electron micrographs of females' ovaries revealed clusters and viroplasms of reovirus-like particles in follicle cells, suggesting that AnvRV is vertically transmitted from mother to offspring. AnvRV was not detected in the mealybugs, supporting the assumption 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 potentially involved in the multitrophic system of mealybugs, their parasitoids and other members of the holobiont.

Frateuria defendens sp. nov., bacterium isolated from the yellows grapevine's disease vector Hyalesthes obsoletus.

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).

Dynamics of the endosymbiont Rickettsia in an insect pest.

A new heritable bacterial association can bring a fresh set of molecular capabilities, providing an insect host with an almost instantaneous genome extension. Increasingly acknowledged as agents of rapid evolution, inherited microbes remain underappreciated players in pest management programs. A Rickettsia bacterium was tracked sweeping through populations of an invasive whitefly provisionally described as the "B" or "MEAM1" of the Bemisia tabaci species complex, in the southwestern USA. In this population, Rickettsia provides strong fitness benefits and distorts whitefly sex ratios under laboratory conditions. In contrast, whiteflies in Israel show few apparent fitness benefits from Rickettsia under laboratory conditions, only slightly decreasing development time. A survey of B. tabaci B samples revealed the distribution of Rickettsia across the cotton-growing regions of Israel and the USA. Thirteen sites from Israel and 22 sites from the USA were sampled. Across the USA, Rickettsia frequencies were heterogeneous among regions, but were generally very high, whereas in Israel, the infection rates were lower and declining. The distinct outcomes of Rickettsia infection in these two countries conform to previously reported phenotypic differences. Intermediate frequencies in some areas in both countries may indicate a cost to infection in certain environments or that the frequencies are in flux. This suggests underlying geographic differences in the interactions between bacterial symbionts and this serious agricultural pest.

Novel Rickettsiella bacterium in the leafhopper Orosius albicinctus (Hemiptera: Cicadellidae).

Bacteria in the genus Rickettsiella (Coxiellaceae), which are mainly known as arthropod pathogens, are emerging as excellent models to study transitions between mutualism and pathogenicity. The current report characterizes a novel Rickettsiella found in the leafhopper Orosius albicinctus (Hemiptera: Cicadellidae), a major vector of phytoplasma diseases in Europe and Asia. Denaturing gradient gel electrophoresis (DGGE) and pyrosequencing were used to survey the main symbionts of O. albicinctus, revealing the obligate symbionts Sulcia and Nasuia, and the facultative symbionts Arsenophonus and Wolbachia, in addition to Rickettsiella. The leafhopper Rickettsiella is allied with bacteria found in ticks. Screening O. albicinctus from the field showed that Rickettsiella is highly prevalent, with over 60% of individuals infected. A stable Rickettsiella infection was maintained in a leafhopper laboratory colony for at least 10 generations, and fluorescence microscopy localized bacteria to accessory glands of the female reproductive tract, suggesting that the bacterium is vertically transmitted. Future studies will be needed to examine how Rickettsiella affects host fitess and its ability to vector phytopathogens.

Horizontal transmission of the insect symbiont Rickettsia is plant-mediated.

Bacteria in the genus Rickettsia, best known as vertebrate pathogens vectored by blood-feeding arthropods, can also be found in phytophagous insects. The presence of closely related bacterial symbionts in evolutionarily distant arthropod hosts presupposes a means of horizontal transmission, but no mechanism for this transmission has been described. Using a combination of experiments with live insects, molecular analyses and microscopy, we found that Rickettsia were transferred from an insect host (the whitefly Bemisia tabaci) to a plant, moved inside the phloem, and could be acquired by other whiteflies. In one experiment, Rickettsia was transferred from the whitefly host to leaves of cotton, basil and black nightshade, where the bacteria were restricted to the phloem cells of the plant. In another experiment, Rickettsia-free adult whiteflies, physically segregated but sharing a cotton leaf with Rickettsia-plus individuals, acquired the Rickettsia at a high rate. Plants can serve as a reservoir for horizontal transmission of Rickettsia, a mechanism which may explain the occurrence of phylogenetically similar symbionts among unrelated phytophagous insect species. This plant-mediated transmission route may also exist in other insect-symbiont systems and, since symbionts may play a critical role in the ecology and evolution of their hosts, serve as an immediate and powerful tool for accelerated evolution.

Rickettsia association with two Macrolophus (Heteroptera: Miridae) species: A comparative study of phylogenies and within-host localization patterns.

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.

The transmission efficiency of tomato yellow leaf curl virus by the whitefly Bemisia tabaci is correlated with the presence of a specific symbiotic bacterium species.

Tomato yellow leaf curl virus (TYLCV) (Geminiviridae: Begomovirus) is exclusively vectored by the whitefly Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae). TYLCV transmission depends upon a 63-kDa GroEL protein produced by the vector's endosymbiotic bacteria. B. tabaci is a species complex comprising several genetically distinct biotypes that show different secondary-symbiont fauna. In Israel, the B biotype harbors Hamiltonella, and the Q biotype harbors Wolbachia and Arsenophonus. Both biotypes harbor Rickettsia and Portiera (the obligatory primary symbionts). The aim of this study was to determine which B. tabaci symbionts are involved in TYLCV transmission using B. tabaci populations collected in Israel. Virus transmission assays by B. tabaci showed that the B biotype efficiently transmits the virus, while the Q biotype scarcely transmits it. Yeast two-hybrid and protein pulldown assays showed that while the GroEL protein produced by Hamiltonella interacts with TYLCV coat protein, GroEL produced by Rickettsia and Portiera does not. To assess the role of Wolbachia and Arsenophonus GroEL proteins (GroELs), we used an immune capture PCR (IC-PCR) assay, employing in vivo- and in vitro-synthesized GroEL proteins from all symbionts and whitefly artificial feeding through membranes. Interaction between GroEL and TYLCV was found to occur in the B biotype, but not in the Q biotype. This assay further showed that release of virions protected by GroEL occurs adjacent to the primary salivary glands. Taken together, the GroEL protein produced by Hamiltonella (present in the B biotype, but absent in the Q biotype) facilitates TYLCV transmission. The other symbionts from both biotypes do not seem to be involved in transmission of this virus.

An eco-systems biology approach for modeling tritrophic networks reveals the influence of dietary amino acids on symbiont dynamics of Bemisia tabaci.

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.

Wolbachia influence on the fitness of Anagyrus vladimiri (Hymenoptera: Encyrtidae), a bio-control agent of mealybugs.

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.

Introduction of a putative biocontrol agent into a range of phytoplasma- and liberibacter-susceptible crop plants.

BACKGROUND: Phytoplasma, the causative agent of Bois Noir disease of grapevines, are vectored by the planthopper Hyalesthes obsoletus (Hemiptera: Cixiidae). A Dyella-like bacterium (DLB) isolated from H. obsoletus inhibits the growth of Spiroplasma melliferum, a cultivable relative of phytoplasma. Additional evidence suggests that DLB can reduce the symptoms of yellows disease in grapevine plantlets. The aim of this study was to test whether DLB could colonize a range of phytoplasma- and liberibacter-sensitive crop plants, and thus assess its potential agricultural use. RESULTS: Vitex agnus-castus, the preferred host plant of H. obsoletus was found to be a natural host of DLB, which was successfully introduced into a range of crop plants belonging to seven families. The most effective DLB application method was foliar spraying. Microscopy observation revealed that DLB aggregated on the leaf surface and around the stomata, suggesting that this is its route of entry. DLB was also present in the vascular tissues of plants, indicating that it moved systemically through the plant. CONCLUSIONS: DLB is a potential biocontrol agent and its broad spectrum of host plants indicates the possibility of its future use against a range of diseases caused by phloem-limited bacteria. © 2017 Society of Chemical Industry.

Bacterial endosymbionts in field-collected samples of Trialeurodes sp. nr. abutiloneus (Hemiptera: Aleyrodidae).

Facultative bacterial endosymbionts are common, influential associates of arthropods, yet their movement among host species has not been well documented. Plant-mediated transmission of Rickettsia has been shown for the whitefly Bemisia tabaci. Bemisia tabaci in USA cotton fields harbors the secondary symbionts Rickettsia and Hamiltonella, and co-occurs with Trialeurodes sp. nr. abutiloneus whiteflies. To determine whether symbionts may be shared, the microbial diversity of these whiteflies on cotton across the USA was analyzed. Trialeurodes sp. nr. abutiloneus bore Portiera, Pseudomonas, Serratia, Arsenophonus and Wolbachia. No Rickettsia or Hamiltonella were detected. These results provide no evidence for horizontal transmission of symbionts between these whitefly genera.

Rickettsia 'in' and 'out': two different localization patterns of a bacterial symbiont in the same insect species.

Intracellular symbionts of arthropods have diverse influences on their hosts, and their functions generally appear to be associated with their localization within the host. The effect of localization pattern on the role of a particular symbiont cannot normally be tested since the localization pattern within hosts is generally invariant. However, in Israel, the secondary symbiont Rickettsia is unusual in that it presents two distinct localization patterns throughout development and adulthood in its whitefly host, Bemisia tabaci (B biotype). In the "scattered" pattern, Rickettsia is localized throughout the whitefly hemocoel, excluding the bacteriocytes, where the obligate symbiont Portiera aleyrodidarum and some other secondary symbionts are housed. In the "confined" pattern, Rickettsia is restricted to the bacteriocytes. We examined the effects of these patterns on Rickettsia densities, association with other symbionts (Portiera and Hamiltonella defensa inside the bacteriocytes) and on the potential for horizontal transmission to the parasitoid wasp, Eretmocerus mundus, while the wasp larvae are developing within the whitefly nymph. Sequences of four Rickettsia genes were found to be identical for both localization patterns, suggesting that they are closely related strains. However, real-time PCR analysis showed very different dynamics for the two localization types. On the first day post-adult emergence, Rickettsia densities were 21 times higher in the "confined" pattern vs. "scattered" pattern whiteflies. During adulthood, Rickettsia increased in density in the "scattered" pattern whiteflies until it reached the "confined" pattern Rickettsia density on day 21. No correlation between Rickettsia densities and Hamiltonella or Portiera densities were found for either localization pattern. Using FISH technique, we found Rickettsia in the gut of the parasitoid wasps only when they developed on whiteflies with the "scattered" pattern. The results suggest that the localization pattern of a symbiont may influence its dynamics within the host.