Glassy-winged Sharpshooter Research Articles

Experimental Infection of California Ripe Olive Cultivars with Xylella fastidiosa Subspecies pauca De Donno and Acquisition by Glassy-Winged Sharpshooter

Strains of Xylella fastidiosa subspecies pauca can infect olive trees, and an introduction of this pathogen has caused significant damage to the olive industry in Europe. Although this subspecies is not known to be present in North America, the environment, plant hosts, and insect vectors in the United States olive-growing regions (mainly California) are conducive to X. fastidiosa spread and disease damage. This study evaluated several risk factors for X. fastidiosa disease in the California olive industry, including infection of the common cultivars and acquisition by insect vectors. Olive cultivars Manzanillo, Sevillano, and Mission all exhibited disease symptoms after inoculation with X. fastidiosa strain De Donno obtained from the olive disease outbreak in southern Italy. Glassy-winged sharpshooters were able to acquire X. fastidiosa De Donno from infected olive plants as soon as 30 days after plant inoculation. This information is valuable for risk assessment and disease management planning in the event of a future introduction of olive-pathogenic strains of X. fastidiosa to the United States. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 “No Rights Reserved” license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2024.

Chromosome-level genome assembly of the aster leafhopper (Macrosteles quadrilineatus) reveals the role of environment and microbial symbiosis in shaping pest insect genome evolution.

Leafhoppers comprise over 20,000 plant-sap feeding species, many of which are important agricultural pests. Most species rely on two ancestral bacterial symbionts, Sulcia and Nasuia, for essential nutrition lacking in their phloem and xylem plant sap diets. To understand how pest leafhopper genomes evolve and are shaped by microbial symbioses, we completed a chromosomal-level assembly of the aster leafhopper's genome (ALF; Macrosteles quadrilineatus). We compared ALF's genome to three other pest leafhoppers, Nephotettix cincticeps, Homalodisca vitripennis, and Empoasca onukii, which have distinct ecologies and symbiotic relationships. Despite diverging ~155 million years ago, leafhoppers have high levels of chromosomal synteny and gene family conservation. Conserved genes include those involved in plant chemical detoxification, resistance to various insecticides, and defence against environmental stress. Positive selection acting upon these genes further points to ongoing adaptive evolution in response to agricultural environments. In relation to leafhoppers' general dependence on symbionts, species that retain the ancestral symbiont, Sulcia, displayed gene enrichment of metabolic processes in their genomes. Leafhoppers with both Sulcia and its ancient partner, Nasuia, showed genomic enrichment in genes related to microbial population regulation and immune responses. Finally, horizontally transferred genes (HTGs) associated with symbiont support of Sulcia and Nasuia are only observed in leafhoppers that maintain symbionts. In contrast, HTGs involved in non-symbiotic functions are conserved across all species. The high-quality ALF genome provides deep insights into how host ecology and symbioses shape genome evolution and a wealth of genetic resources for pest control targets.

Geographical survey of the mycobiome and microbiome of Southern California glassy-winged sharpshooters.

The glassy-winged sharpshooter, Homalodisca vitripennis Germar, is an invasive xylem-feeding leafhopper with a devastating economic impact on California agriculture through transmission of the plant pathogen, Xylella fastidiosa. While studies have focused on X. fastidiosa or known symbionts of H. vitripennis, little work has been done at the scale of the microbiome (the bacterial community) or mycobiome (the fungal community). Here, we characterize the mycobiome and the microbiome of H. vitripennis across Southern California and explore correlations with captivity and host insecticide resistance status. Using high-throughput sequencing of the ribosomal internal transcribed spacer 1 region and the 16S rRNA gene to profile the mycobiome and microbiome, respectively, we found that while the H. vitripennis mycobiome significantly varied across Southern California, the microbiome did not. We also observed a significant difference in both the mycobiome and microbiome between captive and wild H. vitripennis. Finally, we found that the mycobiome, but not the microbiome, was correlated with insecticide resistance status in wild H. vitripennis. This study serves as a foundational look at the H. vitripennis mycobiome and microbiome across Southern California. Future work should explore the putative link between microbes and insecticide resistance status and investigate whether microbial communities should be considered in H. vitripennis management practices. IMPORTANCE The glassy-winged sharpshooter is an invasive leafhopper that feeds on the xylem of plants and transmits the devastating pathogen, Xylella fastidiosa, resulting in significant economic damage to California's agricultural system. While studies have focused on this pathogen or obligate symbionts of the glassy-winged sharpshooter, there is limited knowledge of the bacterial and fungal communities that make up its microbiome and mycobiome. To address this knowledge gap, we explored the composition of the mycobiome and the microbiome of the glassy-winged sharpshooter across Southern California and identified differences associated with geography, captivity, and host insecticide resistance status. Understanding sources of variation in the microbial communities associated with the glassy-winged sharpshooter is an important consideration for developing management strategies to control this invasive insect. This study is a first step toward understanding the role microbes may play in the glassy-winged sharpshooter's resistance to insecticides.

Anatomy of an agricultural antagonist: Feeding complex structure and function of three xylem sap-feeding insects illuminated with synchrotron-based 3D imaging.

Many insects feed on xylem or phloem sap of vascular plants. Although physical damage to the plant is minimal, the process of insect feeding can transmit lethal viruses and bacterial pathogens. Disparities between insect-mediated pathogen transmission efficiency have been identified among xylem sap-feeding insects; however, the mechanistic drivers of these trends are unclear. Identifying and understanding the structural factors and associated integrated functional components that may ultimately determine these disparities are critical for managing plant diseases. Here, we applied synchrotron-based X-ray microcomputed tomography to digitally reconstruct the morphology of three xylem sap-feeding insect vectors of plant pathogens: Graphocephala atropunctata (blue-green sharpshooter; Hemiptera, Cicadellidae) and Homalodisca vitripennis (glassy-winged sharpshooter; Hemiptera, Cicadellidae), and the spittlebug Philaenus spumarius (meadow spittlebug; Hemiptera, Aphrophoridae). The application of this technique revealed previously undescribed anatomical features of these organisms, such as key components of the salivary complex. The visualization of the 3D structure of the precibarial valve led to new insights into the mechanism of how this structure functions. Morphological disparities with functional implications between taxa were highlighted as well, including the morphology and volume of the cibarial dilator musculature responsible for extracting xylem sap, which has implications for force application capabilities. These morphological insights will be used to target analyses illuminating functional differences in feeding behavior.

Fitness Costs Associated With Insecticide Resistance in Populations of Homalodisca vitripennis Germar (Hemiptera: Cicadellidae).

The glassy-winged sharpshooter, Homalodisca vitripennis (Germar) (Hemiptera: Cicadellidae), is an important vector of bacterial Xyllela diseases throughout the southern and southwestern portions of the United States. Strong insect control measures, such that population densities of the insect vector are significantly reduced, are often necessary to limit the spread of Xylella fastidiosa. Glassy-winged sharpshooter populations within the Central Valley of California have developed a high resistance to imidacloprid (resistance ratio greater than 3,200) and tolerance to pyrethroids (ratio of less than 10) due to frequent applications of these materials. The purpose of this study was to determine the potential effects of insecticide resistance upon a variety of sharpshooter life history parameters associated with reproductive fitness. Our results indicate that individuals from susceptible populations of glassy-winged sharpshooters exhibited significantly higher fecundity and longer adult lifespans than those from the resistant populations. Additionally, resistant individuals were on average slightly larger than susceptible individuals. These results provide a strong indication that resistance to neonicotinoids imposes a reproductive fitness cost in an insecticide-free environment.

Glassy-Winged Sharpshooters Cease Feeding and Avoid Plants Treated With Sub-Lethal Doses of the Neonicotinoid Insecticide Imidacloprid.

Insecticides are a primary means for suppressing populations of insects that transmit plant pathogens. Application of insecticides for limiting the spread of insect-transmitted plant pathogens is often most effective when applied on an area-wide scale. The glassy-winged sharpshooter is a vector of the bacterial pathogen Xylella fastidiosa, which causes numerous plant diseases including Pierce's disease of grapevine. The glassy-winged sharpshooter has been the subject of an area-wide suppression program in California for nearly two decades. Overreliance on a limited number of active ingredients including the neonicotinoid imidacloprid has resulted in increased levels of resistance to commonly applied products. In California, glassy-winged sharpshooters move between citrus, an important overwintering host, and vineyards. Accordingly, imidacloprid is routinely applied via the irrigation system in vineyards and citrus orchards. For soil applied applications, it may take days to weeks for concentrations in plants to increase to lethal doses. Further, as the dose of imidacloprid required to kill sharpshooters increases due to resistance, so too does the period that sharpshooters are exposed to sub-lethal doses. Response of glassy-winged sharpshooter to cowpea plants treated with sub-lethal doses of imidacloprid was evaluated by conducting no-choice and choice tests. In no-choice feeding assays, glassy-winged sharpshooters caged on plants treated with sub-lethal doses of imidacloprid ceased feeding and produced little excreta. Further, sub-lethal exposure to a range of doses over a 4-d period did not affect viability over a 9-wk post-exposure holding period on untreated plants. In choice-tests, glassy-winged sharpshooters avoided treated plants and were observed predominately on untreated plants. Results suggest that application of imidacloprid to vineyards and citrus orchards may push glassy-winged sharpshooters out of treated habitats rather than kill them.

Climate change and the potential distribution of the glassy-winged sharpshooter (Homalodisca vitripennis), an insect vector of Xylella fastidiosa

Biological invasions represent a major threat for biodiversity and agriculture. Despite efforts to restrict the spread of alien species, preventing their introduction remains the best strategy for an efficient control. In that context preparedness of phytosanitary authorities is very important and estimating the geographical range of alien species becomes a key information. The present study investigates the potential geographical range of the glassy-winged sharpshooter (Homalodisca vitripennis), a very efficient insect vector of Xylella fastidiosa, one of the most dangerous plant-pathogenic bacteria worldwide. We use species distribution modeling (SDM) to analyse the climate factors driving the insect distribution and we evaluate its potential distribution in its native range (USA) and in Europe according to current climate and different scenarios of climate change: 6 General Circulation Models (GCM), 4 shared socioeconomic pathways of gas emission and 4 time periods (2030, 2050, 2070, 2090). The first result is that the climate conditions of the European continent are suitable to the glassy-winged sharpshooter, in particular around the Mediterranean basin where X. fastidiosa is present. Projections according to future climate conditions indicate displacement of climatically suitable areas towards the north in both North America and Europe. Globally, suitable areas will decrease in North America and increase in Europe in the coming decades. SDM outputs vary according to the GCM considered and this variability indicated areas of uncertainty in the species potential range. Both potential distribution and its uncertainty associated to future climate projections are important information for improved preparedness of phytosanitary authorities.

Transcriptome and population structure of glassy-winged sharpshooters (Homalodisca vitripennis) with varying insecticide resistance in southern California

BackgroundHomalodisca vitripennis Germar, the glassy-winged sharpshooter, is an invasive insect in California and a critical threat to agriculture through its transmission of the plant pathogen, Xylella fastidiosa. Quarantine, broad-spectrum insecticides, and biological control have been used for population management of H. vitripennis since its invasion and subsequent proliferation throughout California. Recently wide-spread neonicotinoid resistance has been detected in populations of H. vitripennis in the southern portions of California’s Central Valley. In order to better understand potential mechanisms of H. vitripennis neonicotinoid resistance, we performed RNA sequencing on wild-caught insecticide-resistant and relatively susceptible sharpshooters to profile their transcriptome and population structure.ResultsWe identified 81 differentially expressed genes with higher expression in resistant individuals. The significant largest differentially expressed candidate gene linked to resistance status was a cytochrome P450 gene with similarity to CYP6A9. Furthermore, we observed an over-enrichment of GO terms representing functions supportive of roles in resistance mechanisms (cytochrome P450s, M13 peptidases, and cuticle structural proteins). Finally, we saw no evidence of broad-scale population structure, perhaps due to H. vitripennis' relatively recent introduction to California or due to the relatively small geographic scale investigated here.ConclusionsIn this work, we characterized the transcriptome of insecticide-resistant and susceptible H. vitripennis and identified candidate genes that may be involved in resistance mechanisms for this species. Future work should seek to build on the transcriptome profiling performed here to confirm the role of the identified genes, particularly the cytochrome P450, in resistance in H. vitripennis. We hope this work helps aid future population management strategies for this and other species with growing insecticide resistance.

The Genomic Basis of Evolutionary Novelties in a Leafhopper.

Evolutionary innovations generate phenotypic and species diversity. Elucidating the genomic processes underlying such innovations is central to understanding biodiversity. In this study, we addressed the genomic basis of evolutionary novelties in the glassy-winged sharpshooter (Homalodisca vitripennis, GWSS), an agricultural pest. Prominent evolutionary innovations in leafhoppers include brochosomes, proteinaceous structures that are excreted and used to coat the body, and obligate symbiotic associations with two bacterial types that reside within cytoplasm of distinctive cell types. Using PacBio long-read sequencing and Dovetail Omni-C technology, we generated a chromosome-level genome assembly for the GWSS and then validated the assembly using flow cytometry and karyotyping. Additional transcriptomic and proteomic data were used to identify novel genes that underlie brochosome production. We found that brochosome-associated genes include novel gene families that have diversified through tandem duplications. We also identified the locations of genes involved in interactions with bacterial symbionts. Ancestors of the GWSS acquired bacterial genes through horizontal gene transfer (HGT), and these genes appear to contribute to symbiont support. Using a phylogenomics approach, we inferred HGT sources and timing. We found that some HGT events date to the common ancestor of the hemipteran suborder Auchenorrhyncha, representing some of the oldest known examples of HGT in animals. Overall, we show that evolutionary novelties in leafhoppers are generated by the combination of acquiring novel genes, produced both de novo and through tandem duplication, acquiring new symbiotic associations that enable use of novel diets and niches, and recruiting foreign genes to support symbionts and enhance herbivory.

Using X-ray Micro-Computed Tomography to Three-Dimensionally Visualize the Foregut of the Glassy-Winged Sharpshooter (Homalodisca vitripennis).

Simple SummaryThe foregut of the glassy-winged sharpshooter is difficult to dissect and observe in three dimensions with SEM or TEM. We describe an X-ray micro-computed tomography (microCT)-based method for studying the GWSS foregut including the precibarium and the cibarium, with high resolution. MicroCT imaging showed great promise for the study of the tissues and structures of small insects.Dissecting the heads of small insects belonging to the order Hemiptera for detailed anatomical investigation with light or scanning electron microscopy is difficult, time-consuming, and destructive, often resulting in sample preparation artifacts. Nevertheless, the structural details of these insects often hold critical information regarding their interactions with bacterial pathogens. For example, the glassy-winged sharpshooter (GWSS) is an efficient vector of the bacterium Xylella fastidiosa, the causal agent of Pierce’s disease in grape vines, but the foregut of this species is exceptionally difficult to dissect. Here, we describe a simple, non-destructive method to investigate the structure of the anterior gut of GWSS using high-resolution micro-computed X-ray tomography (microCT). The use of microCT eliminates the need for destructive dissection and reveals the morphology of small insects in three dimensions, allowing the user to virtually dissect the sample. The use of microCT imaging is a promising and powerful tool in the entomological sciences for studying the structures of vector insects, especially for difficult-to-dissect regions such as the foregut.

Metagenome-Assembled Genomes of Bacterial Symbionts Associated with Insecticide-Resistant and -Susceptible Individuals of the Glassy-Winged Sharpshooter (Homalodisca vitripennis).

ABSTRACTThe role of microbes in insecticide resistance is an emerging question. Here, we describe six metagenome-assembled genomes (MAGs) associated with the glassy-winged sharpshooter (Homalodisca vitripennis [Germar, 1821]) (Hemiptera, Cicadellidae). MAGs representing the obligate symbionts Candidatus Sulcia muelleri and Candidatus Baumannia cicadellinicola and the facultative symbiont Wolbachia were obtained from imidacloprid-resistant and imidacloprid-susceptible sharpshooters.

Efficient CRISPR/Cas9-mediated genome modification of the glassy-winged sharpshooter Homalodisca vitripennis (Germar)

CRISPR/Cas9 technology enables the extension of genetic techniques into insect pests previously refractory to genetic analysis. We report the establishment of genetic analysis in the glassy-winged sharpshooter (GWSS), Homalodisca vitripennis, which is a significant leafhopper pest of agriculture in California. We use a novel and simple approach of embryo microinjection in situ on the host plant and obtain high frequency mutagenesis, in excess of 55%, of the cinnabar and white eye pigmentation loci. Through pair matings, we obtained 100% transmission of w and cn alleles to the G3 generation and also established that both genes are located on autosomes. Our analysis of wing phenotype revealed an unexpected discovery of the participation of pteridine pigments in wing and wing-vein coloration, indicating a role for these pigments beyond eye color. We used amplicon sequencing to examine the extent of off-target mutagenesis in adults arising from injected eggs, which was found to be negligible or non-existent. Our data show that GWSS can be easily developed as a genetic model system for the Hemiptera, enabling the study of traits that contribute to the success of invasive pests and vectors of plant pathogens. This will facilitate novel genetic control strategies.

Nutritional and Physiological Regulation of Glassy-Winged Sharpshooter Oogenesis.

The glassy-winged sharpshooter (Homalodisca vitripennis (Germar); Hemiptera: Cicadellidae: Cicadellinae) is an invasive insect that transmits the plant pathogenic bacterium Xylella fastidiosa Wells et al. (Xanthomonadales: Xanthomonadacae). While adult glassy-winged sharpshooter must feed to produce eggs, the role of nutritional status on initiating oogenesis is poorly understood. To determine the effects of glassy-winged sharpshooter nutrition on nymphal development, oogenesis, and fecundity, glassy-winged sharpshooter were reared on cowpea, sunflower, sorghum, and a mixture of the three plant species. Adults emerging from cowpea, sunflower, or plant mixture treatments had shorter development times, attained larger size, and had greater estimated lipid reserves than females reared on sorghum. In choice tests, nymphs avoided sorghum and preferentially fed on cowpea and sunflower. Adult females provisioned with a single plant species during the nymphal stage were provided with either the same host plant species or a mixture of host plant species (cowpea, sunflower, sorghum) for a 9-wk oviposition period, with 37% of females initiating oogenesis. Ovipositing females had greater juvenile hormone and octopamine levels than reproductively inactive females, although topical application of the juvenile hormone analog Methoprene did not promote oogenesis. Across nymphal diets, reproductively active females produced more eggs when held on plant mixtures than on single plant species. In choice tests, adult females were observed most frequently on cowpea, although most eggs were deposited on sorghum, the host least preferred by nymphs. Results suggest that fecundity is largely determined by the quality of the adult diet, although the stimulus that initiates oogenesis does not appear to be related to nutrition.

Survey of Florida Olive Groves during Olive Fruit Development: Monitoring for Stink Bugs and Olive Fruit Flies

Olives, Olea europaea L. (Oleaceae), are an emerging commercial crop in Florida; however, potential arthropod threats during olive tree establishment and fruit development remain uncharacterized. Two potential pests that may threaten olive fruit production directly are native and invasive pentatomid stink bugs, which are important pest species of many crops in the southeast, and the invasive olive fruit fly, Bactrocera oleae (Gmelin) (Diptera: Tephritidae),which is not known to be established in Florida. Monitoring for stink bugs during fruit maturation was done using dual funnel tube traps baited with stink bug lures. Yellow sticky card traps baited with food and pheromone lures were used to monitor for the olive fruit fly. Both trap types were placed in tree canopies in 4 North Central Florida olive groves during the anticipated fruit development period for 2 growing seasons. Whereas neither of the invasive species targeted (Halyomorpha halys Stål [Hemiptera: Pentatomidae] or B. oleae) were detected, several other potential pests were identified including brown stink bugs (Euschistus spp.; Hemiptera: Pentatomidae), glassy winged sharpshooters, Homalodisca vitripennis Germar (Hemiptera: Cicadellidae), and grasshoppers. No fruit damage attributable to arthropod pests was detected although fruit production was very low with limited samples. These results contribute to awareness of potential pests that may jeopardize olive fruit production and aid in the future studies to develop effective monitoring activities for Florida growers.

Field-Collected Glassy-Winged Sharpshooters (Hemiptera: Cicadellidae) Perform More Xylella fastidiosa-Inoculating Behaviors on Susceptible Vitis vinifera cv. 'Chardonnay' Than on Resistant Vitis champinii Grapevines.

The glassy-winged sharpshooter, Homalodisca vitripennis (Germar) (Hemiptera: Cicadellidae: Cicadellinae), is an introduced vector of the xylem-dwelling bacterium Xylella fastidiosa Wells et al. (Xanthomonadales: Xanthomonadaceae) in California. Once acquired, X. fastidiosa colonizes the functional foregut of the vector. Bacteria can be inoculated directly into grapevine xylem during the xylem cell acceptance process in sharpshooter stylet probing, represented by the X wave using electropenetrography (EPG). Since 2001, an effort has been underway to develop PD-resistant grapevines, Vitis vinifera L., through classical breeding of various species of resistant wild grapevines with more susceptible V. vinifera. The present study used EPG to compare H. vitripennis stylet probing behaviors in a factorial experiment between V. champinii (a V. candicans/V. rupestris natural hybrid with moderate trichomes) and V. vinifera cv. 'Chardonnay' (which lacks trichomes) that had been gently scraped to remove trichomes or was not scraped. Results showed that sharpshooters performed significantly more X waves/X. fastidiosa inoculation behaviors of overall longer duration on Chardonnay than on V. champinii, regardless of shaving or not-shaving to remove trichomes. In addition, trichomes caused more frequent standing/walking/test-probing behaviors on V. champinii, whose xylem was rapidly accepted for sharpshooter ingestion once probing began. Thus, EPG can detect a novel type of grapevine resistance to X. fastidiosa-to the vector's probing process and inoculation of bacteria-in addition to the bacterial infection and symptom development processes that are the basis for most resistance breeding today. Future research could use EPG to screen grapevines for this novel type of resistance.

Optimizing Efficient RNAi-Mediated Control of Hemipteran Pests (Psyllids, Leafhoppers, Whitefly): Modified Pyrimidines in dsRNA Triggers.

The advantages from exogenously applied RNAi biopesticides have yet to be realized in through commercialization due to inconsistent activity of the dsRNA trigger, and the activity level of RNAi suppression. This has prompted research on improving delivery methods for applying exogenous dsRNA into plants and insects for the management of pests and pathogens. Another aspect to improve RNAi activity is the incorporation of modified 2′-F pyrimidine nucleotides into the dsRNA trigger. Modified dsRNA incorporating 32–55% of the 2′-F- nucleotides produced improved RNAi activity that increased insect mortality by 12–35% greater than non-modified dsRNA triggers of the same sequence. These results were repeatable across multiple Hemiptera: the Asian citrus psyllid (Diaphorina citri, Liviidae); whitefly (Bemisia tabaci, Aleyroididae); and the glassy-winged sharpshooter (Homalodisca vitripennis, Cicadellidae). Studies using siRNA with modified 2′-F- pyrimidines in mammalian cells show they improved resistance to degradation from nucleases, plus result in greater RNAi activity, due to increase concentrations and improved binding affinity to the mRNA target. Successful RNAi biopesticides of the future will be able to increase RNAi repeatability in the field, by incorporating modifications of the dsRNA, such as 2′-F- pyrimidines, that will improve delivery after applied to fruit trees or crop plants, with increased activity after ingestion by insects. Costs of RNA modification have decreased significantly over the past few years such that biopesticides can now compete on pricing with commercial chemical products.

Improved draft reference genome for the Glassy-winged Sharpshooter (Homalodisca vitripennis), a vector for Pierce's disease.

Homalodisca vitripennis (Hemiptera: Cicadellidae), known as the glassy-winged sharpshooter, is a xylem feeding leafhopper and an important agricultural pest as a vector of Xylella fastidiosa, which causes Pierce’s disease in grapes and a variety of other scorch diseases. The current H. vitripennis reference genome from the Baylor College of Medicine's i5k pilot project is a 1.4-Gb assembly with 110,000 scaffolds, which still has significant gaps making identification of genes difficult. To improve on this effort, we used a combination of Oxford Nanopore long-read sequencing technology combined with Illumina sequencing reads to generate a better assembly and first-pass annotation of the whole genome sequence of a wild-caught Californian (Tulare County) individual of H. vitripennis. The improved reference genome assembly for H. vitripennis is 1.93-Gb in length (21,254 scaffolds, N50 = 650 Mb, BUSCO completeness = 94.3%), with 33.06% of the genome masked as repetitive. In total, 108,762 gene models were predicted including 98,296 protein-coding genes and 10,466 tRNA genes. As an additional community resource, we identified 27 orthologous candidate genes of interest for future experimental work including phenotypic marker genes like white. Furthermore, as part of the assembly process, we generated four endosymbiont metagenome-assembled genomes, including a high-quality near complete 1.7-Mb Wolbachia sp. genome (1 scaffold, CheckM completeness = 99.4%). The improved genome assembly and annotation for H. vitripennis, curated set of candidate genes, and endosymbiont MAGs will be invaluable resources for future research of H. vitripennis.

Insect-to-insect horizontal transmission of a phytoreovirus in the absence of an infected plant host

Vertical transmission of Homalodisca vitripennis reovirus (HoVRV) from glassy-winged sharpshooter (GWSS, Homalodisca vitripennis (Germar)) females to progeny occurred in laboratory assays at frequencies too low (2.6%–15.4%) to account for HoVRV incidence (90–100%) in field populations resident in citrus. Because citrus is immune to HoVRV and no plant host is known, horizontal transmission of HoVRV from insect-to-insect was evaluated. Exposure of colony-reared, virus-free test nymphs to HoVRV-infected source adults held in the same cage for 10 days on virus-immune cowpea resulted in HoVRV transmission (13.3%–30.7%) to test nymphs. HoVRV was not transmitted when exposure was indirect and required passive movement of virions through the xylem of immune citrus seedlings. Collectively, these results demonstrate direct insect-to-insect horizontal transmission of HoVRV, providing a plausible explanation for high incidence of HoVRV in GWSS field populations in the absence of efficient vertical transmission or a plant host.

Mating Communication of the Variegated Leafhopper, Erasmoneura variabilis, With Notes on Vibrational Signaling of Other Grapevine Cicadellids in California

Abstract Leafhoppers in the tribe Erythroneurini are a concern for grape growers in California due to direct feeding damage by piercing the leaves. Management of leafhopper populations in vineyards may be accomplished by insecticide applications, the release of natural enemies, conservation biological control, exploitation of controlled deficit irrigation, or a combination of the above. Based on research on other leafhopper species, a behavioral mating disruption is a viable option, but nothing is known about the mating communication and circadian signaling of these species in vineyards. The objectives of this study were to identify and describe vibrational signals associated with mate selection behavior of Erasmoneura variabilis and Erythroneura ziczac, and evaluate the occurrence of vibrational signals within sympatric populations of E. variabilis, E. ziczac, and Homalodisca vitripennis on grapevines. Analysis of vibrational communication signals revealed that 1) one female signal and two distinct male signals are used in E. variabilis pair formation, 2) the pair formation process in E. variabilis is divided into three communication phases, 3) E. variabilis pre-copulatory communication is longer in the presence than in the absence of male-male rivalry, 4) two distinct signals are used in E. ziczac pair formation, and 5) E. variabilis, E. ziczac, and H. vitripennis sing at night and during the day. Results include detailed descriptions of leafhopper communication signals that are relevant for future development of vibrational disruption as a novel method to suppress populations under field conditions.

Copulatory Signaling and Polygamy of Glassy-Winged Sharpshooters (Hemiptera: Cicadellidae)

AbstractFinding a partner to mate with may be only part of ensuring successful siring of offspring. Females often exhibit cryptic female choice (CFC) during or after copulation, which can influence whose sperm from her multiple partners is chosen for egg fertilization. Known behavioral mechanisms for CFC include assessment of males by their nuptial gifts, duration of copulation, and seminal fluid contents. In this study, the glassy-winged sharpshooter, Homalodisca vitripennis (Germar) (Hemiptera: Cicadellidae), behaviors during the course of copulation were investigated. Glassy-winged sharpshooter (GWSS) use vibrational communication before copulation occurs. However, little is known about behaviors that occur during and after copulation. Results from this study determined that vibrational signaling also occurs during copulation. Vibrational signals similar to those emitted during precopulatory communication were identified during copulation alongside a new, ‘hum-like’ signal that typically occurred within 10 s after the pair joined in copulation. In addition, results determined the duration of copulation was on average of 15 h, though with a 10-h range (8.5–18.5 h) among observed male–female pairs. Finally, both males and females mated more than once. Collectively, results identified key reproductive parameters required for CFC to occur in GWSS. The study expands on the known animals that use CFC and emphasizes the role that copulatory vibrational communication may play setting the foundations for future more in-depth studies. Understanding of insect behaviors necessary for successful production of offspring is important from an ecological perspective and for development of pest control methods.