Glassy-winged Sharpshooter Research Articles

A paratransgenic strategy to block transmission of Xylella fastidiosa from the glassy-winged sharpshooter Homalodisca vitripennis

BackgroundArthropod-borne diseases remain a leading cause of human morbidity and mortality and exact an enormous toll on global agriculture. The practice of insecticide-based control is fraught with issues of excessive cost, human and environmental toxicity, unwanted impact on beneficial insects and selection of resistant insects. Efforts to modulate insects to eliminate pathogen transmission have gained some traction and remain future options for disease control.ResultsHere, we report a paratransgenic strategy that targets transmission of Xylella fastidiosa, a leading bacterial pathogen of agriculture, by the Glassy-Winged Sharpshooter (GWSS), Homalodisca vitripennis. Earlier, we identified Pantoea agglomerans, a bacterial symbiont of the GWSS as the paratransgenic control agent. We genetically engineered P. agglomerans to express two antimicrobial peptides (AMP)-melittin and scorpine-like molecule (SLM). Melittin and SLM were chosen as the effector molecules based on in vitro studies, which showed that both molecules have anti-Xylella activity at concentrations that did not kill P. agglomerans. Using these AMP-expressing strains of P. agglomerans, we demonstrated disruption of pathogen transmission from insects to grape plants below detectable levels.ConclusionThis is the first report of halting pathogen transmission from paratransgenically modified insects. It is also the first demonstration of paratransgenic control in an agriculturally important insect vector.

Reduced Susceptibility of Homalodisca vitripennis (Hemiptera: Cicadellidae) to Commonly Applied Insecticides.

Pest management for the glassy-winged sharpshooter, Homalodisca vitripennis Germar (Hemiptera: Cicadellidae), in Kern County, California relies on the application of insecticides. These treatments have contributed to low H. vitripennis field counts since applications were initiated in 2001. However, densities have been high in recent years despite continued management, prompting efforts to evaluate the susceptibility of current populations to insecticides. H. vitripennis adults were subjected to bioassays with five commonly applied insecticides, and the results were compared to baseline toxicities determined in 2002. Two neonicotinoids, imidacloprid and thiamethoxam, were evaluated using systemic uptake bioassays. Contact toxicities of the neonicotinoid acetamiprid and pyrethroids bifenthrin and fenpropathrin were estimated using leaf dip bioassays. Dose-mortality responses were analyzed by probit analysis. For each compound, there was no significant difference in annual LC50 values determined over 2 yr. Compared to baseline toxicities, acetamiprid and bifenthrin were found to be significantly less toxic to H. vitripennis. The LC50 values of these two compounds increased sevenfold and 152-fold, respectively. Tests with the neonicotinoids revealed a trend of decreasing susceptibility levels within each season followed by reversion back to early season LC50 estimates in the following year. In addition, data showed seasonal and site variation in susceptibility to imidacloprid, possibly due to differential applications in nearby fields.

Effects of Nymphal Diet and Adult Feeding on Allocation of Resources to Glassy-Winged Sharpshooter Egg Production.

The glassy-winged sharpshooter is an invasive insect capable of transmitting the bacterial pathogen Xylella fastidiosa. Pre-oviposition periods of laboratory-reared glassy-winged sharpshooters are variable. Here, two questions were addressed: does nymphal diet affect pre-oviposition period and how do allocation patterns of resources differ for females that produce eggs versus females that do not? Nymphs were reared on one of three host plant species: cowpea, sunflower, or sorghum. Half of the females were sacrificed at emergence. The remaining adult females were held on cowpea, a host plant species known to support egg maturation via adult feeding. Females were sacrificed on the day of first oviposition or after 9 wk if no eggs were deposited. Females reared as nymphs on sorghum had longer development times and were smaller (head capsule width and hind tibia length) than females reared as nymphs on cowpea and sunflower. However, nymphal diet did not affect percentage of dry weight that was lipid at emergence. Further, nymphal diet did not affect time to deposition of the first egg mass or total number of eggs matured at the time of first oviposition. Egg production reduced the allocation of resources to insect bodies, with body lipid content decreasing with increasing egg production. In general, females increased wet weight 1.4-fold during the first week after adult emergence, with wet weights plateauing over the remaining 9 wk that adults were monitored. Thus, it seems reasonable to hypothesize that resources required for egg production were acquired via adult feeding during the first week after adult emergence.

Mating disruption of Homalodisca vitripennis (Germar) (Hemiptera: Cicadellidae) by playback of vibrational signals in vineyard trellis.

Glassy-winged sharpshooter (GWSS), Homalodisca vitripennis (Germar) (Hemiptera: Cicadellidae) is an important vector of the bacterium Xylella fastidiosa, the causal agent of Pierce's disease of grapevine. Area-wide insecticide applications have suppressed GWSS populations for ∼25 years, but reduced levels of insecticide susceptibility have been reported. Therefore, alternative methods of control are needed. Objectives of this study were to evaluate the efficacy of playback of vibrational mating communication signals for disrupting mating of GWSS in a natural vineyard setting and to evaluate spectral properties of signal transmission through vineyard trellis. Playback reduced mating of GWSS on grapevines. A total of 28 (of 134) male-female pairs mated in the control treatment (silence) and only one (of 134) pair mated when treated with the vibrational signal playback. Playback of vibrational signals through vineyard trellis was affected by distance from the signal source, with frequency composition being the highest at the source and lowest on vines positioned away from the source. Frequency composition in canes housing test insects decreased exponentially as distance from the source increased, whereas the relative amplitude of analyzed frequencies decreased linearly. Although further studies are needed prior to method implementation, data from this study continue to support integration of vibrational mating disruption with current methods to suppress GWSS populations. Published 2018. This article is a U.S. Government work and is in the public domain in the USA.

Mating vibrational signal transmission through and between plants of an agricultural pest, the Glassy-Winged Sharpshooter

The agricultural pest, glassy-winged sharpshooter (GWSS), Homalodisca vitripennis, relies primarily on successful vibrational communication across its home plant. Males and females engage in a vibrational duet to identify correct species, attractiveness of mate, and location on the plant. The signal produced by these animals has a dominant frequency component between 80 and 120 Hz, with harmonics spaced approximately 100 Hz apart. However, our analysis revealed that not all harmonics are present in every recorded signal. Therefore, we sought to understand how the GWSS vibrational communication signal changes over distance on the plant. We have confirmed that first, with increasing distance fewer high frequency harmonics are present. Second, at distances of only 50 cm, there is a difference in the latency of signal arrival based on the frequency, with higher frequencies arriving sooner. Finally, the animal appears to generate no airborne signal component, yet, the low frequencies are clearly detectable in neighboring plants by the signal “jumping” from leaf-to-air-to-leaf. Together, these results highlight the complexity of vibration transmission in plants and the possibility of alteration and disruption of the GWSS signal.

Effects of Energy Reserves and Diet on Glassy-Winged Sharpshooter Egg Maturation.

The glassy-winged sharpshooter is an invasive insect capable of transmitting the plant pathogen Xylella fastidiosa. As rates of pathogen spread are a function of vector abundance, identification of factors contributing to glassy-winged sharpshooter egg production will aid in predicting population growth. Here, effects of stored energy reserves and adult diet on glassy-winged sharpshooter egg maturation were evaluated. To estimate energy reserves available to adult females at the beginning of feeding assays, residuals from a regression of wet weight on size were used. Analysis of a subset of females sacrificed at the beginning of feeding assays, demonstrated that females with a positive residual wet weight had higher lipid content and carried more eggs than females with a negative residual wet weight. To evaluate effects of diet and energy reserves on egg maturation, energy reserves available to females entering feeding assays on cowpea and grapevine were estimated. For females held on cowpea, residual wet weight and quantity of excreta produced over a 6-d feeding period affected egg production. In contrast, for females held on grapevine, only residual wet weight affected egg production. Comparison of cowpea and grapevine xylem sap determined that eight amino acids were more concentrated in xylem sap from cowpea than from grapevine. Collectively, the results suggest that glassy-winged sharpshooter population growth within crop monocultures will not depend solely on the nutritional quality of the specific crop for producing mature eggs but also on the quantity of energy reserves accumulated by females prior to entering that crop habitat.

Genome-Wide Transcriptional Dynamics in the Companion Bacterial Symbionts of the Glassy-Winged Sharpshooter (Cicadellidae: Homalodisca vitripennis) Reveal Differential Gene Expression in Bacteria Occupying Multiple Host Organs.

The agricultural pest known as the glassy-winged sharpshooter (GWSS) or Homalodisca vitripennis (Hemiptera: Cicadellidae) harbors two bacterial symbionts, “Candidatus Sulcia muelleri” and “Ca. Baumannia cicadellinicola,” which provide the 10 essential amino acids (EAAs) that are limited in the host plant-sap diet. Although they differ in origin and symbiotic age, both bacteria have experienced extensive genome degradation resulting from their ancient restriction to specialized host organs (bacteriomes) that provide cellular support and ensure vertical transmission. GWSS bacteriomes are of different origins and distinctly colored red and yellow. While Sulcia occupies the yellow bacteriome, Baumannia inhabits both. Aside from genomic predictions, little is currently known about the cellular functions of these bacterial symbionts, particularly whether Baumannia in different bacteriomes perform different roles in the symbiosis. To address these questions, we conducted a replicated, strand-specific RNA-seq experiment to assay global gene expression patterns in Sulcia and Baumannia. Despite differences in genomic capabilities, the symbionts exhibit similar profiles of their most highly expressed genes, including those involved in nutrition synthesis and protein stability (chaperonins dnaK and groESL) that likely aid impaired proteins. Baumannia populations in separate bacteriomes differentially express genes enriched in essential nutrient synthesis, including EAAs (histidine and methionine) and B vitamins (biotin and thiamine). Patterns of differential gene expression further reveal complexity in methionine synthesis. Baumannia’s capability to differentially express genes is unusual, as ancient symbionts lose the capability to independently regulate transcription. Combined with previous microscopy, our results suggest that the GWSS may rely on distinct Baumannia populations for essential nutrition and vertical transmission.

Do spittlebugs feed on grape? Assessing transmission potential for <i>Xylella fastidiosa</i>

The bacterium Xylella fastidiosa is a causal agent of Pierce’s disease in grapevines and is considered to be vectored by several xylem-feeding insects. Xylella fastidiosa and its primary insect vector Homalodisca vitripennis are not currently present in New Zealand, but considered a biosecurity threat to the wine industry. Should it be introduced, infection and dispersal of X. fastidiosa within New Zealand vineyards could occur through extant vectors, especially spittlebugs. Electrical Penetration Graph technology was used to compare the feeding behaviour of three spittlebug species (Philaenus spumarius, Carystoterpa fingens and Carystoterpa minor) on grape (Vitis vinifera Sauvignon blanc) and their original host plants. Results suggest that Philaenus spumarius feeds on grape more than the other two spittlebug species. As Philaenus spumarius has been reported as a vector of X. fastidiosa, their potential to transmit the bacterium into grapevines is discussed based on the real feeding times recorded by EPG.

Design of a candidate vibrational signal for mating disruption against the glassy-winged sharpshooter, Homalodisca vitripennis.

The glassy-winged sharpshooter (GWSS), Homalodisca vitripennis, is an important pest of grapevines due to its ability to transmit Xylella fastidiosa, the causal agent of Pierce's disease. GWSS mating communication is based on vibrational signals; therefore, vibrational mating disruption could be an alternative to insecticides for suppression of the GWSS population. Our objectives were to identify spectral features of the female signal that elicit male signaling, design disruptive signals able to alter male perception and acceptance of a female, and determine the signal intensity required for future field applications. Male responses to playback of modified female signals were significantly reduced by 60-75% when part of the female signal spectral components above or below 400 Hz were deleted. Playback bioassays showed that transmission of an 80 Hz pure frequency tone to plants completely suppressed male signaling to female signal playback, even if the disruptive signal amplitude was 10 dB lower than the female signal playback. Although the mechanism underlying cessation of male signaling activity in the presence of disruption is not yet understood, results suggest that an 80 Hz vibrational signal should be tested in laboratory and field experiments to assess its efficacy in disrupting mating of GWSS. © 2017 Society of Chemical Industry.

Mating interference of glassy‐winged sharpshooters, Homalodisca vitripennis

AbstractAnimal communication is a complex behavior that is influenced by abiotic and biotic factors of the environment. Glassy‐winged sharpshooters (GWSS), Homalodisca vitripennis (Germar) (Hemiptera: Cicadellidae), primarily use vibrational signaling for courtship communication. Because GWSS is a major pest, transmitting the plant pathogenic bacterium Xylella fastidiosa Wells et al., interruption of communication is a possible avenue for control. Playback of white noise, pre‐recorded female signals, and artificial female noise (continuously overlapping female signals) significantly reduced mating of GWSS when compared to silent control mating trials. Furthermore, to begin to determine the mechanism underlying playback control, female signaling activity was recorded in the presence of stimuli. In response to playback of female signals, females signaled (duet‐like) more often than females tested in the absence of playback. After the first playback, almost two‐thirds of females signaled a response within 3 s. Additionally, one‐third of the females signaled within 1 s after cessation of white noise, and significantly more in the time periods following noise termination. Results highlight how GWSS responds to differing competitive disturbances in the environment and lays important ground work that possibly could be used to develop pesticide‐free control methods.

Effects of Xylem-Sap Composition on Glassy-Winged Sharpshooter (Hemiptera: Cicadellidae) Egg Maturation on High- and Low-Quality Host Plants.

Glassy-winged sharpshooters must feed as adults to produce mature eggs. Cowpea and sunflower are both readily accepted by the glassy-winged sharpshooter for feeding, but egg production on sunflower was reported to be lower than egg production on cowpea. To better understand the role of adult diet in egg production, effects of xylem-sap chemistry on glassy-winged sharpshooter egg maturation was compared for females confined to cowpea and sunflower. Females confined to cowpea consumed more xylem-sap than females held on sunflower. In response, females held on cowpea produced more eggs, had heavier bodies, and greater lipid content than females held on sunflower. Analysis of cowpea and sunflower xylem-sap found that 17 of 19 amino acids were more concentrated in cowpea xylem-sap than in sunflower xylem-sap. Thus, decreased consumption of sunflower xylem-sap was likely owing to perceived lower quality, with decreased egg production owing to a combination of decreased feeding and lower return per unit volume of xylem-sap consumed. Examination of pairwise correlation coefficients among amino acids indicated that concentrations of several amino acids within a plant species were correlated. Principal component analyses identified latent variables describing amino acid composition of xylem-sap. For females held on cowpea, egg maturation was affected by test date, volume of excreta produced, and principal components describing amino acid composition of xylem-sap. Principal component analyses aided in identifying amino acids that were positively or negatively associated with egg production, although determining causality with respect to key nutritional requirements for glassy-winged sharpshooter egg production will require additional testing.

Mating behavior and vibrational mimicry in the glassy-winged sharpshooter, Homalodisca vitripennis

The glassy-winged sharpshooter (GWSS), Homalodisca vitripennis, is an important vector of Xylella fastidiosa, the causal agent of Pierce’s disease of grapevine. GWSS control relies mainly on insecticides; therefore, an alternative method, such as vibrational mating disruption, is required. However, knowledge of GWSS intraspecific communication is necessary to evaluate applicability of such methods. Mating behavior and associated vibrational signals were described in different social contexts: individuals, pairs, and one female with two competing males. Behavioral analysis showed that GWSS mating communication involved the emission of three male and two female signals, with specific roles in two distinct phases of mating behavior, identification and courtship. Mating success depended on vibrational duets between genders, which were temporarily interrupted in the presence of male rivalry. Male rivalry behavior involved the emission of three distinct rivalry signals. Two rivalry signals resemble female signals and were associated with replacement of the female in the duet by the rival male. The third rivalry signal was emitted by competing males. Data suggested that rival males used mimicry and hostile signals to interrupt the ongoing duet and gain access to a female. In the future, knowledge acquired from this study will be essential to develop a mechanical mating disruption method for GWSS control.

Development and validation of a real-time PCR assay for the glassy-winged sharpshooter Homalodisca vitripennis (Hemiptera: Cicadellidae).

The glassy-winged sharpshooter (Homalodisca vitripennis) is an invasive pest organism, which is found throughout Central America and has recently invaded a few countries in the Pacific Islands. As a carrier of the highly virulent plant pathogenic bacterium Xylella fastidiosa, it is of great economic significance to horticulture and is estimated to cost Californian vineyards over US$100 million per year in control and losses. New Zealand is currently free from this pest, but its recent spread through the Pacific has raised concerns of it establishing in New Zealand, potentially as a result of introduction through human travel. We report here a real-time polymerase chain reaction assay for the rapid identification of H. vitripennis. The assay was extensively validated in silico then optimized and tested against a range of Cicadellidae species, both internationally collected and local to New Zealand. This assay was able to correctly identify H. vitripennis samples, and distinguish between H. vitripennis and close relatives, such as the smoke-tree sharpshooter (Homalodisca liturata) and will be of great benefit to New Zealand biosecurity.

Sequence polymorphism in an insect RNA virus field population: A snapshot from a single point in space and time reveals stochastic differences among and within individual hosts

Population structure of Homalodisca coagulata Virus-1 (HoCV-1) among and within field-collected insects sampled from a single point in space and time was examined. Polymorphism in complete consensus sequences among single-insect isolates was dominated by synonymous substitutions. The mutant spectrum of the C2 helicase region within each single-insect isolate was unique and dominated by nonsynonymous singletons. Bootstrapping was used to correct the within-isolate nonsynonymous:synonymous arithmetic ratio (N:S) for RT-PCR error, yielding an N:S value ~one log-unit greater than that of consensus sequences. Probability of all possible single-base substitutions for the C2 region predicted N:S values within 95% confidence limits of the corrected within-isolate N:S when the only constraint imposed was viral polymerase error bias for transitions over transversions. These results indicate that bottlenecks coupled with strong negative/purifying selection drive consensus sequences toward neutral sequence space, and that most polymorphism within single-insect isolates is composed of newly-minted mutations sampled prior to selection.

Transmission of phage by glassy-winged sharpshooters, a vector of Xylella fastidiosa.

ABSTRACTXylella fastidiosa subsp. fastidiosa (Xff) is the causal agent of Pierce's Disease (PD) of grapevines and is vectored by the glassy-winged sharpshooter (GWSS, Homalodisca vitripennis). Previously we have reported the development of a bacteriophage (phage) based biocontrol system for PD, but no information on insect transmission of phages has been reported. Here we communicate that laboratory reared GWSSs fed on cowpea plants (Vigna unguiculata subsp. unguiculata) harboring the virulent phage Paz were able to uptake of phage efficiently when the phage was present in high concentration, but were inefficient in transfer to plants.

Deep 16S rRNA gene sequencing of anterior foregut microbiota from the blue‐green sharpshooter (Graphocephala atropunctata)

AbstractGraphocephala atropunctata or the blue‐green sharpshooter (BGSS) has been long recognized as the principal native vector of Xylella fastidiosa in coastal, wine‐grape‐growing areas of California. X. fastidiosa is the causative agent of Pierce's disease of grapevine and of numerous other leaf‐scorching diseases of agronomically important plants. X. fastidiosa has been shown to colonize the cibarium and precibarium (anterior foregut) of sharpshooters, where it may encounter other naturally occurring bacterial species. Here, deep 16S rRNA sequencing was used to survey the microbiota associated with the BGSS anterior foregut. DNA was extracted from dissected cibaria and precibaria; a portion of the 16S rRNA gene was amplified and sequenced using Illumina MiSeq technology. An average of approximately 32 000 sequence reads per insect was obtained. Agrobacterium was the most common genus detected; additional sequencing of the full‐length 16S rRNA gene further identified this as Agrobacterium tumefaciens or A. fabrum. A number of additional plant‐associated bacterial genera were also detected (Pseudomonas and Ensifer), along with genera known to be associated with insects (Baumannia), and soil (Stenotrophomonas, Caulobacter, Delftia, Achromobacter, Acinetobacter and Novosphingobium). Approximately half of the genera reported here have been previously reported to be prevalent in the cibarium and precibarium of glassy‐winged sharpshooter (GWSS; Homalodisca vitripennis). Many of these cibarium‐ and precibarium‐associated genera likely interact with X. fastidiosa.

The complete mitochondrial genome of Tambocerus sp. (Hemiptera: Cicadellidae)

The complete mitochondrial genome of Tambocerus sp. (Hemiptera: Cicadellidae) from Zhejiang and Anhui provinces of China was sequenced. The total length of the mitogenome is 15 955 bp (GenBank accession no. KT827824) and consists of 22 transfer RNAs, 13 protein-coding genes, 2 ribosomal RNAs and 1 control region. The base composition of the heavy strand for A, T, C, and G is 41.39, 35.02, 14.00, and 9.59%, respectively. All of the protein-coding genes (PCGs) start with ATN. Five protein-coding genes use TAA as stop codons, four use TAG as stop codons, and others use incomplete stop codons ‘‘T––’’ or ‘‘TA–’’. The control region has a length of 1581 bp which is between rrnS and trnI genes with the AT content high to 85.96%. Phylogenetic analysis indicated that Tambocerus sp. was clustered in a closely related subgroup with Homalodisca vitripennis and Empoasca vitis. This is consistent with the result of the traditional taxonomy.

Adult Age Structure and Trends inXylella fastidiosaIncidence inHomalodisca vitripennis(Hemiptera: Cicadellidae) from Texas Grape Vineyards

The glassy-winged sharpshooter, Homalodisca vitripennis (Germar) (Hemiptera: Cicadellidae), is the primary insect vector of the xylem-limited plant pathogen Xylella fastidiosa fastidiosa Wells (Xanthomonadales: Xanthomonadaceae), the bacterium that causes Pierce's disease of grape, Vitus sp. Because it is mobile and polyphagous, the glassy-winged sharpshooter is key to Pierce's disease outbreaks in California, Florida, and Texas. Adult age structure and X. fastidiosa foregut infectivity rates of glassy-winged sharpshooter from nine Texas vineyards in summer 2007 were assessed from archived samples. The ages of individuals were determined by measuring the red pigmentation of forewing intersections. Presence or absence of X. fastidiosa was determined in the foregut of pathogen-positive insects by quantitative PCR. Larger amounts of the associated pathogen in insects from two vineyards sampled in 2005 and 2006 were observed. No significant differences in average insect age among vineyards or collection dates were found in those years. However, significant differences in average pathogen load among multiple vineyards and collection dates were observed.

Evaluation of Insecticide Residues on the Mortality of Adult Glassy-winged Sharpshooter on Citrus, 2013: Table 1

During the fall of 2014, we evaluated the residues of insecticides for their effectiveness against glassy-winged sharpshooter (GWSS). On 24 September, 35 young potted citrus trees were organized in a completely randomized design with 5 replicates of 7 treatments. A squirt bottle was used to spray each tree with 160 ml (runoff) of insecticide solutions that were prepared according to the per acre use …

Evaluation of Insecticide Residues on the Mortality of Adult Glassy-winged Sharpshooter on Citrus, 2014: Table 1

Evaluation of Insecticide Residues on the Mortality of Adult Glassy-winged Sharpshooter on Citrus, 2014: Table 1