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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.

The 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.

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.

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.

Fluctuating asymmetry (FA) is hypothesized to be a useful predictor of population canalization, especially for organisms at risk from environmental change.Identification of traits that meet statistical criteria as FA measures remains a challenge.Here, a laboratory experiment subjected immature butterflies (Vanessa cardui) to diet and temperature conditions of varying stress levels. Variation in dietary macronutrient ratio (protein: carbohydrate) and rearing temperature (optimal: 25°C; elevated: 32°C) was introduced as stressors. Temperature and nutrition are key variables influencing ectotherm growth and fitness and so are likely to be important stressors that influence FA.Individuals subjected to stressful conditions were predicted to show elevated FA of three wing size traits, as well as increased mortality and decreased adult body size.Trait FA did not vary across treatments. Instead, treatment levels impacted viability: The combined incidence of pupal death and expression of significant wing malformations increased in treatment levels designated as stressful. Variation in adult dry mass also reflected predicted stress levels. Results suggest that individuals predicted to display increased FA either died or displayed gross developmental aberrations.This experiment illustrates important constraints on the investigation of FA, including selection of appropriate traits and identification of appropriate levels of stressors to avoid elevated mortality. The latter concern brings into question the utility of FA as an indicator of stress in vulnerable, natural populations, where stress levels cannot be controlled, and mortality and fitness effects are often not quantifiable.

The effects of rearing temperature and host stage parasitized on the development of three parasitoid species; Encarsia noyesi Hayat (Hymenoptera: Aphelinidae), Idioporus affinis LaSalle & Polaszek (Hymenoptera: Pteromalidae), and Entedononecremnus krauteri Zolnerowich & Rose (Hymenoptera: Eulophidae) were investigated. These parasitoids are part of the biological control program for the giant whitefly Aleurodicus dugesii Cockerell (Hemiptera: Aleyrodidae) in the United States. Temperature and host-dependent development was assessed for each species using the nonlinear Brière-1 model and simple linear regression to obtain critical thermal parameters. All three parasitoids successfully developed at constant temperatures ranging from 15°C to 30°C, which was narrower to their predicted thermal limits due to thermal constraints of A. dugesii. There were significant effects of both temperature and A. dugesii nymphal stage parasitized on immature parasitoid development times. All three parasitoid species' development time decreased as nymphal stage age increased. Thermal tolerance limits and development times varied by parasitoid species. The results of these findings in the context of biological control potential are discussed. [ABSTRACT FROM AUTHOR]

Sharpshooters (Hemiptera: Cicadellidae: Cicadellinae) and spittlebugs (Hemiptera: Aphrophoridae: Aphrophorinae) have a worldwide distribution and are often associated with many crops. Because the geographic range of sharpshooters and spittlebugs often overlaps with the range of the plant pathogenic bacterium Xylella fastidiosa, the importance of these groups of insects in spreading X. fastidiosa rises to the level of a major socioeconomic problem. Managing diseases caused by X. fastidiosa is challenging for several reasons. First, multiple sequence types of the pathogen have been identified, with sequence type affecting host plant specificity. Second, multiple vector species may be present in an agroecosystem, and the host range of vector species is often broad. Although multiple vector control options such as biological, chemical, physical and cultural controls have been described, disease control programs must take into account biological and ecological parameters of locally dominant X. fastidiosa sequence types and vector species to develop suitable control strategies. Our review demonstrates how fundamental principles of vector reproductive biology, behaviour, nutrition and population and community ecology have influenced research on insect vectors and provides insights into priority research areas. [ABSTRACT FROM AUTHOR]

Co-occurring plant species can influence the extent of damage to each other by altering the activity or abundance of a shared herbivore. One mechanism by which neighboring host plants exacerbate damage to a focal host is if the neighbor amplifies herbivore populations. We studied the performance of a shared herbivore on a native and an invasive plant, to estimate how strongly the presence of the invasive plant increases local herbivore abundance—in a system in which highly asymmetric spillover herbivory may occur. Specifically, we conducted a series of greenhouse experiments that measured reproduction, development, and survival of the invasive stink bug Bagrada hilaris Burmeister on an invasive annual forb, Sahara mustard (Brassica tournefortii), or a native perennial shrub, four-winged saltbush (Atriplex canescens). All measured aspects of stink bug performance revealed consistently greater performance on Br. tournefortii. Indeed, A. canescens appears to be insufficient for Ba. hilaris to complete its development. Nonetheless, preliminary damage assessments found that both plant species were used as feeding hosts, putative feeding lesions were a more reliable indicator of herbivory than was the degree of yellowing, and higher Ba. hilaris abundance was generally associated with greater sublethal damage to A. canescens. Thus, A. canescens appears to be susceptible to Ba. hilaris herbivory, though more research is needed to assess fitness impacts of this novel herbivore. Our results indicate that differential herbivore performance among host plants may be an important contributor to observed patterns of abundance of a shared herbivore and spillover herbivory between plants. [ABSTRACT FROM AUTHOR]

We examined dietary self-selection and rules of compromise for protein (P) and digestible carbohydrate (C) intake by fifth-instar Vanessa cardui L. ( Lepidoptera: Nymphalidae: Nymphalini). We presented six fat-free diet pairs to larvae in a choice trial to determine the 'intake target'. In addition, we fed larvae seven fat-free single diets differing in dietary nutrient ratio in no-choice trials to determine the rules of compromise they exhibit when constrained to a singular, sub-optimal dietary source. In choice trials, caterpillars regulated nutrient intake to a ratio of 1 protein to 1.09 carbohydrate (1P:1.09C), exhibiting tighter regulation of protein than of carbohydrate. Furthermore, larvae from different diet pair treatments did not differ in pupal mass or stadium duration. In no-choice experiments, larvae reduced consumption on increasingly protein-biased diets and increased consumption on increasingly carbohydrate-biased diets, relative to a 1P:1C ratio diet. Differences in carbohydrate consumption were much greater between no-choice treatments than differences in protein consumption. Dietary nutrient ratio affected pupal mass when accounting for initial larval mass. Pupal mass decreased as nutrient ratio was shifted off of 1P:1C, but to a greater extent when the ratio was skewed toward carbohydrate. Stadium duration increased as nutrient ratio diverged from 1P:1C, being more pronounced when shifted toward carbohydrate than toward protein. Regulation to near 1P:1C is consistent with results found for other Lepidoptera, and the rule of compromise exhibited by V. cardui is consistent with that expected for a generalist herbivore. [ABSTRACT FROM AUTHOR]

Acutaspis albopicta (Cockerell) is an exotic scale detected on imported 'Hass' avocados arriving in California from Mexico. An effort was made to understand how well this species might be biologically controlled by the parasitoid, Aphytis melinus DeBach. In no-choice trials, single life stages of A. albopicta and Aonidiella aurantii (Maskell) were exposed to one Ap. melinus female. Ap. melinus attacked and developed to adult on all A. albopicta life stages tested, which included early, middle, and late male and female second instars, and early, middle, and late female third instars. However, the proportion of scales attacked was relatively low (≤20%), as were the number of eggs laid on each scale stage. In six of seven comparisons of scale cover surface area between A. albopicta and Ao. aurantii that were similarly aged, Ao. aurantii was significantly larger. With intraspecific choice trials, females of Ap. melinus strongly preferred to parasitize the oldest and largest female scales in both single- and mixed-sex offerings. Younger, smaller scales were preferred for host feeding. Five comparisons of preference between A. albopicta and Ao. aurantii showed that Ap. melinus preferred 28- and 36-d-old Ao. aurantii females over similarly aged A. albopicta. However, Ap. melinus preferred 23-d-old males of A. albopicta. In three of five comparisons, Ao. aurantii was the preferred species to host feed on. More F1 females of Ap. melinus were recovered from both intraspecific choice trials compared with any stage in no-choice trials. Implications of incorporating Ap. melinus into biological control programs are discussed. [ABSTRACT FROM AUTHOR]