The Hidden Secrets of Psylloidea: Biology, Behavior, Symbionts, and Ecology.

Psyllids constitute a diverse group of sap-feeding Sternorrhyncha that were relatively obscure until it was discovered that a handful of species transmit bacterial plant pathogens. Yet the superfamily Psylloidea is much richer than the sum of its crop-associated vectors, with over 4,000 described species exhibiting diverse life histories and host exploitation strategies. A growing body of research is uncovering fascinating insights into psyllid evolution, biology, behavior, and species interactions. This work has revealed commonalities and differences with better-studied Sternorrhyncha, as well as unique evolutionary patterns of lineage divergence and host use. We are also learning how psyllid evolution and foraging ecology underlie life history traits and the roles of psyllids in communities. At finer scales, we are untangling the web of symbionts across the psyllid family tree, linking symbiont and psyllid lineages, and revealing mechanisms underlying reciprocal exchange between symbiont and host. In this review, we synthesize and summarize key advances within these areas with a focus on free-living (nongalling) Psylloidea.

Hiding in plain sight: a widespread native perennial harbors diverse haplotypes of 'Candidatus Liberibacter solanacearum' and its potato psyllid vector.

The unculturable bacterium 'Candidatus Liberibacter solanacearum' (CLso) is responsible for a growing number of emerging crop diseases. However, we know little about the diversity and ecology of CLso and its psyllid vectors outside of agricultural systems, which limits our ability to manage crop disease and understand the impacts this pathogen may have on wild plants in natural ecosystems. In North America, CLso is transmitted to crops by the native potato psyllid (Bactericera cockerelli). But the geographic and host plant range of the potato psyllid and CLso beyond the borders of agriculture are not well understood. A recent study of historic herbarium specimens revealed that a unique haplotype of CLso was present infecting populations of the native perennial Solanum umbelliferum in California decades before CLso was first detected in crops. We hypothesized that this haplotype, and other potentially novel CLso variants, are still present in S. umbelliferum populations. To test this, we surveyed populations of S. umbelliferum in Southern California for CLso and potato psyllid vectors. We found multiple haplotypes of CLso and the potato psyllid associated with these populations, with none of these genetic variants having been previously reported in California crops. These results suggest that CLso and its psyllid vectors are much more widespread and diverse in North American natural plant communities than suggested by data collected solely from crops and weeds in agricultural fields. Further characterization of these apparently asymptomatic haplotypes will facilitate comparison with disease-causing variants and provide insights into the continued emergence and spread of CLso.

Transcriptome analysis of aphid-resistant and susceptible near isogenic lines reveals candidate resistance genes in cowpea (Vigna unguiculata).

BACKGROUND: Cowpea (Vigna unguiculata) is a crucial crop for regions of the world that are prone to both heat and drought; however, the phytotoxic cowpea aphid (Aphis craccivora) impairs plant physiology at low population levels. Both antibiotic and antixenotic forms of resistance to the aphid have been mapped to two quantitative trait loci (QTLs) and near isogenic lines (NILs). The molecular mechanism for this resistance response remains unknown. RESULTS: To understand the genes underlying susceptibility and resistance, two cowpea lines with shared heritage were infested along a time course and characterized for transcriptome variation. Aphids remodeled cowpea development and signaling relative to host plant resistance and the duration of feeding, with resource acquisition and mobilization determining, in part, susceptibility to aphid attack. Major differences between the susceptible and resistant cowpea were identified including two regions of interest housing the most genetic differences between the lines. Candidate genes enabling aphid resistance include both conventional resistance genes (e.g., leucine rich repeat protein kinases) as well as multiple novel genes with no known orthologues. CONCLUSIONS: Our results demonstrate that feeding by the cowpea aphid globally remodels the transcriptome of cowpea, but how this occurs depends on both the duration of feeding and host-plant resistance. Constitutive expression profiles of the resistant genotype link aphid resistance to a finely-tuned resource management strategy that ultimately reduces damage (e.g., chlorosis) and delays cell turnover, while impeding aphid performance. Thus, aphid resistance in cowpea is a complex, multigene response that involves crosstalk between primary and secondary metabolism.

Epidemiology and Management of Plant Viruses Under a Changing Climate.

Plant viruses are an ever-present threat to agricultural production and provide a wide array of symptoms resulting in economic losses throughout the world. Diseases can be transmitted by insect vectors, as well as through pollen, seed, and other means. With the increased globalization of agriculture, the introduction of new viruses from exotic locations and their establishment in new production regions and even new crops is a growing concern. Advancing knowledge of the epidemiology of plant viruses including development of new diagnostic methods, virus surveillance, and modeling, virus ecology and evolution, virus interactions with insect vectors, and other factors are important toward reducing the spread of plant viruses and managing virus diseases.

Combined effects of mutualistic rhizobacteria counteract virus-induced suppression of indirect plant defences in soya bean.

It is increasingly clear that microbial plant symbionts can influence interactions between their plant hosts and other organisms. However, such effects remain poorly understood, particularly under ecologically realistic conditions where plants simultaneously interact with diverse mutualists and antagonists. Here, we examine how the effects of a plant virus on indirect plant defences against its insect vector are influenced by co-occurrence of other microbial plant symbionts. Using a multi-factorial design, we manipulated colonization of soya bean using three different microbes: a pathogenic plant virus (bean pod mottle virus (BPMV)), a nodule-forming beneficial rhizobacterium ( Bradyrhizobium japonicum) and a plant growth-promoting rhizobacterium ( Delftia acidovorans). We then assessed recruitment of parasitoids ( Pediobious foveolatus (Eulophidae)) and parasitism rates following feeding by the BPMV vector Epilachna varivestis (Coccinellidae). BPMV infection suppressed parasitoid recruitment, prolonged parasitoid foraging time and reduced parasitism rates in semi-natural foraging assays. However, simultaneous colonization of BPMV-infected hosts by both rhizobacteria restored parasitoid recruitment and rates of parasitism to levels similar to uninfected controls. Co-colonization by the two rhizobacteria also enhanced parasitoid recruitment in the absence of BPMV infection. These results illustrate the potential of plant-associated microbes to influence indirect plant defences, with implications for disease transmission and herbivory, but also highlight the potential complexity of such interactions.

Biochemical and physiological mechanisms underlying effects of Cucumber mosaic virus on host-plant traits that mediate transmission by aphid vectors.

The transmission of insect-vectored diseases entails complex interactions among pathogens, hosts and vectors. Chemistry plays a key role in these interactions; yet, little work has addressed the chemical ecology of insect-vectored diseases, especially in plant pathosystems. Recently, we documented effects of Cucumber mosaic virus (CMV) on the phenotype of its host (Cucurbita pepo) that influence plant-aphid interactions and appear conducive to the non-persistent transmission of this virus. CMV reduces host-plant quality for aphids, causing rapid vector dispersal. Nevertheless, aphids are attracted to the elevated volatile emissions of CMV-infected plants. Here, we show that CMV infection (1) disrupts levels of carbohydrates and amino acids in leaf tissue (where aphids initially probe plants and acquire virions) and in the phloem (where long-term feeding occurs) in ways that reduce plant quality for aphids; (2) causes constitutive up-regulation of salicylic acid; (3) alters herbivore-induced jasmonic acid biosynthesis as well as the sensitivity of downstream defences to jasmonic acid; and (4) elevates ethylene emissions and free fatty acid precursors of volatiles. These findings are consistent with previously documented patterns of aphid performance and behaviour and provide a foundation for further exploration of the genetic mechanisms responsible for these effects and the evolutionary processes that shape them.

Evaluation of a low-cost staining method for improved visualization of sweet potato whitefly (Bemisia tabaci) eggs on multiple crop plant species.

BACKGROUND: The sweet potato whitefly (Bemisia tabaci) is a globally important insect pest that damages crops through direct feeding and by transmitting viruses. Current B. tabaci management revolves around the use of insecticides, which are economically and environmentally costly. Host plant resistance is a sustainable option to reduce the impact of whiteflies, but progress in deploying resistance in crops has been slow. A major obstacle is the high cost and low throughput of screening plants for B. tabaci resistance. Oviposition rate is a popular metric for host plant resistance to B. tabaci because it does not require tracking insect development through the entire life cycle, but accurate quantification is still limited by difficulties in observing B. tabaci eggs, which are microscopic and translucent. The goal of our study was to improve quantification of B. tabaci eggs on several important crop species: cassava, cowpea, melon, sweet potato and tomato. RESULTS: We tested a selective staining process originally developed for leafhopper eggs: submerging the leaves in McBryde's stain (acetic acid, ethanol, 0.2% aqueous acid Fuchsin, water; 20:19:2:1) for three days, followed by clearing under heat and pressure for 15 min in clearing solution (LGW; lactic acid, glycerol, water; 17:20:23). With a less experienced individual counting the eggs, B. tabaci egg counts increased after staining across all five crops. With a more experienced counter, egg counts increased after staining on melons, tomatoes, and cowpeas. For all five crops, there was significantly greater agreement on egg counts across the two counting individuals after the staining process. The staining method worked particularly well on melon, where egg counts universally increased after staining for both counting individuals. CONCLUSIONS: Selective staining aids visualization of B. tabaci eggs across multiple crop plants, particularly species where leaf morphological features obscure eggs, such as melons and tomatoes. This method is broadly applicable to research questions requiring accurate quantification of B. tabaci eggs, including phenotyping for B. tabaci resistance.

Inbreeding in horsenettle (Solanum carolinense) alters night-time volatile emissions that guide oviposition by Manduca sexta moths.

Plant volatiles serve as key foraging and oviposition cues for insect herbivores as well as their natural enemies, but little is known about how genetic variation within plant populations influences volatile-mediated interactions among plants and insects. Here, we explore how inbred and outbred plants from three maternal families of the native weed horsenettle (Solanum carolinense) vary in the emission of volatile organic compounds during the dark phase of the photoperiod, and the effects of this variation on the oviposition preferences of Manduca sexta moths, whose larvae are specialist herbivores of Solanaceae. Compared with inbred plants, outbred plants consistently released more total volatiles at night and more individual compounds-including some previously reported to repel moths and attract predators. Female moths overwhelmingly chose to lay eggs on inbred (versus outbred) plants, and this preference persisted when olfactory cues were presented in the absence of visual and contact cues. These results are consistent with our previous findings that inbred plants recruit more herbivores and suffer greater herbivory under field conditions. Furthermore, they suggest that constitutive volatiles released during the dark portion of the photoperiod can convey accurate information about plant defence status (and/or other aspects of host plant quality) to foraging herbivores.

Volatiles from intact and Lygus-damaged Erigeron annuus (L.) Pers. are highly attractive to ovipositing Lygus and its parasitoid Peristenus relictus Ruthe.

Trap cropping and biological control can provide a sustainable means of controlling insect pests. Insects in the genus Lygus (Hemiptera: Miridae) are major pests on cotton and horticultural crops throughout the United States, and pesticide resistance within Lygus populations necessitates more sustainable long-term management techniques. Here, we explore behavioral responses of Lygus bugs (L. rubrosignatus Knight) and an introduced parasitoid, Peristenus relictus (Hymenoptera: Braconidae), to a common field edge plant, Erigeron annuus, which has the potential to serve as a trap host. Erigeron annuus is attractive to Lygus in the field, with Lygus preferentially moving to Erigeron patches compared to more abundant cotton plants. To determine the role of odor cues in mediating this attraction, we collected volatiles from E. annuus with and without Lygus damage, and then tested the attractiveness of these volatiles vs. those of cotton to Lygus females and female P. relictus wasps using Y-tube and wind tunnel bioassays. We found that undamaged E. annuus emits high concentrations of a complex volatile blend (60+ compounds), with novel compounds induced and constitutive compounds up-regulated in response to damage. Additionally, both female Lygus bugs and female P. relictus wasps are highly attracted to E. annuus volatiles over those of cotton in almost every combination of damage treatments. Our results suggest that Erigeron annuus would be an effective trap plant to control Lygus in cotton, since it is highly attractive to both the pest and its natural enemy.

Deceptive chemical signals induced by a plant virus attract insect vectors to inferior hosts.

Previous studies have shown that vector-borne pathogens can alter the phenotypes of their hosts and vectors in ways that influence the frequency and nature of interactions between them, with significant implications for the transmission and spread of disease. For insect-borne pathogens, host odors are particularly likely targets for manipulation, because both plant- and animal-feeding insects use volatile compounds derived from their hosts as key foraging cues. Here, we document the effects of a widespread plant pathogen, Cucumber mosaic virus (CMV), on the quality and attractiveness of one of its host plants (Cucurbita pepo cv. Dixie) for two aphid vectors, Myzus persicae and Aphis gossypii. Our results indicate that CMV greatly reduces host-plant quality-aphids performed poorly on infected plants and rapidly emigrated from them-but increases the attractiveness of infected plants to aphids by inducing elevated emissions of a plant volatile blend otherwise similar to that emitted by healthy plants. Thus, CMV appears to attract vectors deceptively to infected plants from which they then disperse rapidly, a pattern highly conducive to the nonpersistent transmission mechanism employed by CMV and very different from the pattern previously reported for persistently transmitted viruses that require sustained aphid feeding for transmission. In addition to providing a documented example of a pathogen inducing a deceptive signal of host-plant quality to vectors, our results suggest that the transmission mechanism is a major factor shaping pathogen-induced changes in host-plant phenotypes. Furthermore, our findings yield a general hypothesis that, when vector-borne plant or animal pathogens reduce host quality for vectors, pathogen-induced changes in host phenotypes that enhance vector attraction frequently will involve the exaggeration of existing host-location cues.

The aphid Myzus persicae (Hemiptera: Aphididae) acquires chloroplast DNA during feeding on host plants.

Aphids (Hemiptera: Aphididae) extract nutrients from host plant phloem via stylets that facilitate salivation and sap uptake. When navigating to the phloem, aphids periodically puncture nonvascular cells and sample cell contents, but rarely cause significant cell damage. As a result, aphids are considered "stealthy" feeders. In contrast, insects that do cause damage, such as chewing herbivores, will take up host cell contents-including DNA-into their guts. Researchers can use molecular barcoding methods to identify recent host use patterns of chewing herbivores. This information is valuable for both pest management and basic ecological studies. Because of their stealthy feeding style, it was assumed that host plant DNA could not be recovered from aphids and other Sternorrhyncha. However, several recent studies document host plant DNA uptake by psyllids, which feed in a similar manner to aphids. Therefore, we hypothesized that aphids may also acquire DNA from host plants. Since aphids puncture and sample cytosol contents from cells, we predicted that aphids would be most likely to acquire DNA from chloroplasts. To test this, we performed host feeding and host transfer experiments with Myzus persicae (Sulzer), then used PCR to recover and sequence a region between the trnT and trnF genes from acquired chloroplast DNA. We found that M. persicae readily acquires chloroplast DNA, even prior to phloem contact, and that fragment sizes sufficient for host plant identification can be recovered. Our work suggests that molecular gut content analysis is a viable tool for studying aphid-host interactions.

Bokashi fermentation of brewery's spent grains positively affects larval performance of the black soldier fly Hermetia illucens while reducing gaseous nitrogen losses.

Digestion of waste feedstocks by larvae of the black soldier fly Hermetia illucens (Diptera: Stratiomyidae) (BSF) results in proteins for animal feed and organic fertilizer with a reduced environmental footprint, but it can still have negative environmental effects through greenhouse gas (GHG) and ammonia (NH3) emissions. Both biomass conversion by BSF larvae and associated GHG and NH3 emissions can depend on substrate properties that may be optimized through microbial inoculation pre-treatments, such as bokashi fermentation. Here, we quantified the effects of bokashi fermentation of brewery's spent grains on BSF rearing metrics and associated GHG and NH3 emissions at benchtop scale. We found that bokashi fermentation increased larval biomass by 40% and shortened development time by over two days on average, compared with unfermented spent grains. In line with increased larval growth, CO2 emissions in BSF larvae treatments were 31.0 and 79.0% higher in the bokashi fermented spent grains and Gainesville substrates, respectively, compared to the unfermented spent grains. Adding BSF larvae to the spent grains increased cumulative N2O emissions up to 64.0 mg N2O kg substratedry-1 but there were essentially no N2O emissions when larvae were added to fermented spent grains. Bokashi fermentation also reduced NH3 fluxes from the volatilization of substrate nitrogen in the BSF larvae treatment by 83.7-85.8% during days 7 and 9, possibly by increasing N assimilation by larvae or by reducing the transformation of substrate NH4+ to NH3. Therefore, bokashi fermentation may be applied to improve performance of BSF larvae on a common industrial waste stream and reduce associated emissions.

Inbreeding alters volatile signalling phenotypes and influences tri-trophic interactions in horsenettle (Solanum carolinense L.).

The ecological consequences of inter-individual variation in plant volatile emissions remain largely unexplored. We examined the effects of inbreeding on constitutive and herbivore-induced volatile emissions in horsenettle (Solanum carolinense L.) and on the composition of the insect community attracted to herbivore-damaged and undamaged plants in the field. Inbred plants exhibited higher constitutive emissions, but weaker induction of volatiles following herbivory. Moreover, many individual compounds previously implicated in the recruitment of predators and parasitoids (e.g. terpenes) were induced relatively weakly (or not at all) in inbred plants. In trapping experiments, undamaged inbred plants attracted greater numbers of generalist insect herbivores than undamaged outcrossed plants. But inbred plants recruited fewer herbivore natural enemies (predators and parasitoids) when damaged. Taken together, these findings suggest that inbreeding depression negatively impacts the overall pattern of volatile emissions - increasing the apparency of undamaged plants to herbivores, while reducing the recruitment of predatory insects to herbivore-damaged plants.

A reproducible and sensitive method for generating high-quality transcriptomes from single whitefly salivary glands and other low-input tissues.

Transcriptomic studies are an important tool for understanding the molecular pathways underlying host plant use by agricultural pests, including vectors of damaging plant pathogens. Thus far, bulk RNA-Seq has been the main approach for non-model insects. This method relies on pooling large numbers of whole organisms or hundreds of individually dissected organs. The latter approach is logistically challenging, may introduce artifacts of handling and storage, and is not compatible with biological replication. Here, we tested an approach to generate transcriptomes of individual salivary glands and other low-input body tissues from whiteflies (Bemisia tabaci MEAM1), which are major vectors of plant viruses. By comparing our outputs to published bulk RNA-Seq datasets for whole whitefly bodies and pools of salivary glands, we demonstrate that this approach recovers similar numbers of transcripts relative to bulk RNA-Seq in a tissue-specific manner, and for some metrics, exceeds performance of bulk tissue RNA-Seq. Libraries generated from individual salivary glands also yielded additional novel transcripts not identified in pooled salivary gland datasets, and had hundreds of enriched transcripts when compared with whole head tissues. Overall, our study demonstrates that it is feasible to produce high quality, replicated transcriptomes of whitefly salivary glands and other low-input tissues. We anticipate that our approach will expand hypothesis-driven research on salivary glands of whiteflies and other Hemiptera, thus enabling novel control strategies to disrupt feeding and virus transmission.

Uncovering Symbionts Across the Psyllid Tree of Life and the Discovery of a New Liberibacter Species, "Candidatus" Liberibacter capsica.

Sap-feeding insects in the order Hemiptera associate with obligate endosymbionts that are required for survival and facultative endosymbionts that can potentially modify resistance to stress, enemies, development, and reproduction. In the superfamily Psylloidea, the jumping plant lice (psyllids), less is known about the diversity and prevalence of their endosymbionts compared to other sap-feeding pests such as aphids (Aphididae). To address this knowledge gap, using 16S rRNA sequencing we identify symbionts across divergent psyllid host lineages from around the world. Taking advantage of a new comprehensive phylogenomic analyses of Psylloidea, we included psyllid samples from 44 species of 35 genera of five families, collected from 11 international locations for this study. Across psyllid lineages, a total of 91 OTUs were recovered, predominantly of the Enterobacteriaceae (68%). The diversity of endosymbionts harbored by each psyllid species was low with an average of approximately 3 OTUs. Two clades of endosymbionts (clade 1 and 2), belonging to Enterobacteriaceae, were identified that appear to be long term endosymbionts of the psyllid families Triozidae and Psyllidae, respectively. We also conducted high throughput metagenomic sequencing on three Ca. Liberibacter infected psyllid species (Russelliana capsici, Trichochermes walkeri, and Macrohomotoma gladiata), initially identified from 16S rRNA sequencing, to obtain more genomic information on these putative Liberibacter plant pathogens. The phylogenomic analyses from these data identified a new Ca. Liberibacter species, Candidatus Liberibacter capsica, that is a potential pathogen of solanaceous crops. This new species shares a distant ancestor with Ca. L. americanus, which occurs in the same range as R. capsici in South America. We also detected the first association between a psyllid specializing on woody hosts and the Liberibacter species Ca. L. psyllaurous, which is a globally distributed pathogen of herbaceous crop hosts in the Solanaceae. Finally, we detected a potential association between a psyllid pest of figs (M. gladiata) and a Ca. Liberibacter related to Ca. L. asiaticus, which causes severe disease in citrus. Our findings reveal a wider diversity of associations between facultative symbionts and psyllids than previously reported and suggest numerous avenues for future work to clarify novel associations of ecological, evolutionary, and pathogenic interest.

A synthesis of virus-vector associations reveals important deficiencies in studies on host and vector manipulation by plant viruses.

Plant viruses face many challenges in agricultural environments. Although crop fields appear to be abundant resources for these pathogens, it may be difficult for viruses to "escape" from crop environments prior to host senescence or harvesting. One way for viruses to increase the odds of persisting outside of agricultural fields across seasons is by evolving traits that increase transmission opportunities between crops and wild plant communities. There is accumulating evidence that some viruses can achieve this by manipulating crop plant phenotypes in ways that enhance transmission by vectors. Putative manipulations occur through alteration of plant cues (color, size, texture, foliar volatiles, in-leaf metabolites, defenses, and leaf cuticles) that mediate vector orientation, feeding, and dispersal behaviors. Virus effects on host phenotypes are not uniform but appear to exhibit convergence depending on virus traits underlying transmission, particularly the duration of probing and feeding required to acquire and inoculate distinct types of plant viruses. This shared congruence in manipulation strategies and mechanisms across divergent virus lineages suggests that such effects may be adaptive. To discern if this is the case, researchers must consider molecular and environmental constraints on virus evolution, including those imposed by insect vectors from organismal to landscape scales. In this review, we synthesize applied research on vector-borne virus transmission in laboratory and field settings to identify the main factors determining transmission opportunities for plant viruses, and thus, selection pressure to evolve manipulative traits. We then examine these outputs in the context of studies reporting putative instances of plant virus manipulation. Our synthesis reveals important disconnects between virus manipulation studies and actual selection pressures imposed by vectors in real-world contexts.

Priming Melon Defenses with Acibenzolar-S-methyl Attenuates Infections by Phylogenetically Distinct Viruses and Diminishes Vector Preferences for Infected Hosts.

Plant virus management is mostly achieved through control of insect vectors using insecticides. However, insecticides are only marginally effective for preventing virus transmission. Furthermore, it is well established that symptoms of virus infections often encourage vector visitation to infected hosts, which exacerbates secondary spread. Plant defense elicitors, phytohormone analogs that prime the plant immune system against attack, may be a viable approach for virus control that complements insecticide use by disrupting pathologies that attract vectors. To explore this, we tested the effect of a commercial plant elicitor, acibenzolar-S-methyl (ASM), on infection rates, virus titers, and symptom development in melon plants inoculated with one of two virus species, Cucumber mosaic virus (CMV) and Cucurbit yellow stunting disorder virus (CYSDV). We also conducted behavioral assays to assess the effect of ASM treatment and virus inoculation on vector behavior. For both pathogens, ASM treatment reduced symptom severity and delayed disease progression. For CYSDV, this resulted in the attenuation of symptoms that encourage vector visitation and virion uptake. We did observe slight trade-offs in growth vs. defense following ASM treatment, but these effects did not translate into reduced yields or plant performance in the field. Our results suggest that immunity priming may be a valuable tool for improving management of insect-transmitted plant viruses.

Progress and challenges in identifying molecular mechanisms underlying host and vector manipulation by plant viruses.

Plant virus infection fundamentally alters chemical and behavioral phenotypes of hosts and vectors. These alterations often enhance virus transmission, leading researchers to surmise that such effects are manipulations caused by virus adaptations and not just by-products of pathology. But identification of the virus components behind manipulation is missing from most studies performed to date. Here, we evaluate causative empirical evidence that virus components are the drivers of manipulated host and vector phenotypes. To do so, we link findings and methodologies on virus pathology with observational and functional genomics studies on virus manipulation. Our synthesis provides an overview of progress, areas of synergy, and new approaches that will lead to an improved mechanistic understanding of host and vector manipulation by plant viruses.

Evolutionary Determinants of Host and Vector Manipulation by Plant Viruses.

Plant viruses possess adaptations for facilitating acquisition, retention, and inoculation by vectors. Until recently, it was hypothesized that these adaptations are limited to virus proteins that enable virions to bind to vector mouthparts or invade their internal tissues. However, increasing evidence suggests that viruses can also manipulate host plant phenotypes and vector behaviors in ways that enhance their own transmission. Manipulation of vector-host interactions occurs through virus effects on host cues that mediate vector orientation, feeding, and dispersal behaviors, and thereby, the probability of virus transmission. Effects on host phenotypes vary by pathosystem but show a remarkable degree of convergence among unrelated viruses whose transmission is favored by the same vector behaviors. Convergence based on transmission mechanism, rather than phylogeny, supports the hypothesis that virus effects are adaptive and not just by-products of infection. Based on this, it has been proposed that viruses manipulate hosts through multifunctional proteins that facilitate exploitation of host resources and elicitation of specific changes in host phenotypes. But this proposition is rarely discussed in the context of the numerous constraints on virus evolution imposed by molecular and environmental factors, which figure prominently in research on virus-host interactions not dealing with host manipulation. To explore the implications of this oversight, we synthesized available literature to identify patterns in virus effects among pathogens with shared transmission mechanisms and discussed the results of this synthesis in the context of molecular and environmental constraints on virus evolution, limitations of existing studies, and prospects for future research.

Addressing Research Needs in the Field of Plant Virus Ecology by Defining Knowledge Gaps and Developing Wild Dicot Study Systems.

Viruses are ubiquitous within all habitats that support cellular life and represent the most important emerging infectious diseases of plants. Despite this, it is only recently that we have begun to describe the ecological roles of plant viruses in unmanaged systems and the influence of ecosystem properties on virus evolution. We now know that wild plants frequently harbor infections by diverse virus species, but much remains to be learned about how viruses influence host traits and how hosts influence virus evolution and vector interactions. To identify knowledge gaps and suggest avenues for alleviating research deficits, we performed a quantitative synthesis of a representative sample of virus ecology literature, developed criteria for expanding the suite of pathosystems serving as models, and applied these criteria through a case study. We found significant gaps in the types of ecological systems studied, which merit more attention. In particular, there is a strong need for a greater diversity of logistically tractable, wild dicot perennial study systems suitable for experimental manipulations of infection status. Based on criteria developed from our quantitative synthesis, we evaluated three California native dicot perennials typically found in Mediterranean-climate plant communities as candidate models: Cucurbita foetidissima (buffalo gourd), Cucurbita palmata (coyote gourd), and Datura wrightii (sacred thorn-apple). We used Illumina sequencing and network analyses to characterize viromes and viral links among species, using samples taken from multiple individuals at two different reserves. We also compared our Illumina workflow with targeted RT-PCR detection assays of varying costs. To make this process accessible to ecologists looking to incorporate virology into existing studies, we describe our approach in detail and discuss advantages and challenges of different protocols. We also provide a bioinformatics workflow based on open-access tools with graphical user interfaces. Our study provides evidence that dicot perennials in xeric habitats support multiple, asymptomatic infections by viruses known to be pathogenic in related crop hosts. Quantifying the impacts of these interactions on plant performance and virus epidemiology in our logistically tractable host systems will provide fundamental information about plant virus ecology outside of crop environments.