California’s $57.6 billion grape and wine industry faces an existential threat from an unlikely source: the glassy-winged sharpshooter (GWSS), Homalodisca vitripennis. This invasive leafhopper acts as the primary vector for Xylella fastidiosa, the bacterium causing Pierce’s Disease in grapes – a disease with no known cure.
Consequently, infected vines wither and die within years. The problem extends far beyond California; GWSS is native to the southeastern U.S. and northeastern Mexico and feeds on the xylem sap of over 100 host plants.
Moreover, it transmits Xylella strains responsible for other devastating plant diseases like Citrus Variegated Chlorosis and Olive Quick Decline Syndrome globally.
Alarmingly, traditional control methods are failing. Resistance to widely used pyrethroid and neonicotinoid insecticides (like bifenthrin and imidacloprid) is now confirmed in California GWSS populations, rendering these chemicals less effective and highlighting the urgent need for novel, sustainable solutions.
CRISPR Overcomes Bug Gene Barrier
Fortunately, a groundbreaking study published in Scientific Reports (April 2022) provides a revolutionary weapon: the first highly efficient CRISPR/Cas9 gene-editing platform specifically designed for the GWSS.
While CRISPR has transformed genetics in insects like flies and beetles, applying it to hemipteran pests (true bugs) like the GWSS proved significantly more challenging. Previously, CRISPR-mediated gene knockout had only been achieved in seven other hemipteran species across six families, with highly variable and often low success rates:
- Whitefly: 0.2–2.5% mutagenesis
- Brown planthopper: 0–27.3% mutagenesis
- Milkweed bug: 14–92.5% mutagenesis
The UC Riverside research team overcame this barrier through a remarkably simple yet ingenious innovation: injecting CRISPR components directly into GWSS embryos while they were still embedded in their natural environment – under the epidermis of sorghum leaves where females lay them in masses of 20-30 eggs.
Leaf-Injection Enables Rapid Gene Editing
The team’s innovative in situ approach cleverly leveraged the GWSS’s natural biology to achieve unprecedented efficiency. First, they carefully timed injections to occur just 1–2 hours after egg deposition, capitalizing on a critical early developmental window when embryos are most receptive.
Using finely beveled quartz needles, researchers then precisely pierced both the leaf tissue and embryonic chorion at optimal angles between 35° and 50°, while maintaining controlled pressures of 107–348 hPa. This meticulous approach delivered remarkable advantages:
Notably, even novice operators could process 20 embryos in just 10 minutes, dramatically accelerating experimental throughput. Equally important, the method achieved exceptional egg-to-nymph survival rates of 64.3%, far surpassing results from techniques requiring embryo isolation.
For real-time monitoring, a distinct melanized scar at injection sites became visible within just 48 hours, serving as a reliable viability indicator. Most crucially for efficiency, the developing eye pigment of embryos became clearly discernible by days 4-5, providing an exceptionally early visual confirmation of CRISPR success.
Ultimately, this integrated approach enabled unprecedented mutagenesis rates when targeting specific genes, with results significantly exceeding previous hemipteran CRISPR studies:
- Cinnabar (cn) Gene: Using sgRNAcn4-1 and Cas9 protein (300 ng/μl), they achieved 58.9% mutagenesis (33 mutants from 56 hatched embryos).
- White (w) Gene: Using two sgRNAs (sgRNAw6-1 and sgRNAw6-2) and a lower Cas9 concentration (150 ng/μl), they reached an astounding 80.0% mutagenesis (72 mutants from 90 hatched embryos).
Eye Color Genes Reveal Fundamental Biology
The researchers strategically targeted two classic insect “eye-color” genes, cinnabar (cn) and white (w), known components of conserved pigment pathways (ommochromes for brown/red and pteridines for red/yellow).
This allowed them to easily visualize the effects of CRISPR editing. First, targeting cinnabar (cn), which encodes the enzyme kynurenine 3-monooxygenase crucial for ommochrome production, resulted in dramatic changes.
Instead of the normal red-brown eyes, a high proportion (58.9%) of edited embryos, nymphs, and adults displayed a spectrum of orange and red eye colors, often in a mosaic pattern. Sequencing confirmed small deletions (2-16 base pairs) in the cn gene in these mutants, such as the cn² allele with a 5-bp deletion.
Importantly, they successfully bred these mosaic adults, establishing stable lines where the brilliant red-orange eye phenotype (caused by unmasked pteridine pigments) was inherited by 100% of the offspring through three generations (G3).
Second, targeting the white (w) gene, which codes for an ABC transporter protein needed to import pteridine precursors into cells, proved even more efficient. Using two guide RNAs simultaneously and optimizing Cas9 concentration, they achieved mutagenesis frequencies between 61.2% and 80.0%.
Mutant embryos were identifiable by their lack of normal eye pigmentation within days. Adult G0 insects showed varying degrees of eye mosaicism, and critically, the team established a stable white mutant line (WhA) maintained for four generations (G4).
All individuals in this line exhibited completely white eyes and ocelli. Deep genetic sequencing identified complex mutations (like the w¹ allele with an 83-bp insertion) that would completely disrupt the White protein’s function, explaining the total loss of pigmentation.
CRISPR Reveals Wing Color Secrets
While the eye color changes confirmed successful gene editing, the study yielded a truly surprising discovery upon closer examination of the white (w) mutant adults: their wings looked drastically different.
Wild-type GWSS possess distinctive bright red pigmentation within specific veins and interveinal spaces (like the brachial space and outer discal space) on their forewings, superimposed over darker brown pigments.
Strikingly, this characteristic red pigmentation was entirely absent in the *w* mutants! The red veins and patches were replaced by clear, unpigmented (white) areas. This was a major clue that the white gene’s function extended far beyond the eyes.
To definitively prove this, the researchers performed biochemical analyses, extracting and quantifying pigments from different body parts. They identified two primary types of pteridines based on their light absorption peaks: a 334-nm form (similar to common 6-biopterin, found more in heads) and a 467-nm form (likely an erythropterin-like compound, dominant in forewings). Crucially, the results were unambiguous:
- Wild-type Forewings: Showed high levels of the 467-nm pteridine, explaining the red pigment.
- *w* Mutant Forewings: Exhibited near-zero levels of both the 334-nm and 467-nm pteridines.
- Conclusion: The red pigments in GWSS wings are pteridines, and their presence absolutely depends on a functional White protein transporter. This overturned previous hypotheses suggesting the red color came from pheomelanins.
Precise Pest Gene Control
Establishing stable mutant lines through multiple generations—reaching the G3 for cinnabar and G4 for white—demonstrated that CRISPR-induced mutations were not only viable but also reliably inherited.
Critically, this generational stability enabled essential genetic crosses to pinpoint the chromosomal location of these genes. Through reciprocal pairings—mutant males with wild-type females and vice versa—a clear pattern emerged: all first-generation (F1) offspring exhibited wild-type eye color.
This outcome conclusively proved two key points: First, the cn and *w* mutations are recessive, requiring two mutant copies for the trait to manifest visibly.
Second, both genes reside on autosomes rather than sex chromosomes—a finding underscored by the absence of mutant phenotypes in male progeny from mutant female crosses, which would have occurred if the genes were X-linked.
Furthermore, addressing the vital concern of off-target effects—unintended DNA cuts at similar genomic sites—the team employed sophisticated amplicon sequencing. They scrutinized 4–5 potential off-target locations per guide RNA in edited G0 adults.
Reassuringly, results revealed mutations were either entirely absent or detectable only at minimal frequencies, with 10 out of 11 sites showing rates below 1%. A single site (for sgRNAw6-1) recorded a slightly higher but functionally insignificant 5.04% mutation rate.
Importantly, this precision aligns with gold-standard CRISPR systems in insects like Anopheles gambiae, validating the technique’s reliability for future applications.
Conclusion
This breakthrough transforms the glassy-winged sharpshooter into a tractable genetic model for Hemiptera—an order teeming with agricultural pests.
By combining high-efficiency editing (>55% success) and stable inheritance, researchers can now dissect the genetic drivers behind GWSS’s devastating impact, including its host-plant preferences, insecticide resistance, reproductive strategies, and role in transmitting Xylella fastidiosa.
While field deployment of gene-edited insects requires careful ethical and regulatory review, this work lays the essential groundwork for revolutionary control strategies. Potential long-term applications include:
- Gene drives to spread sterility or block Xylella transmission in wild populations,
- Releasing refractory strains genetically incapable of carrying the bacterium,
- Disrupting survival genes using this validated CRISPR platform.
Equally significant, the unexpected discovery of pteridine-based wing pigmentation—dependent on the white gene—opens new research pathways into insect coloration and evolution.
In summary, this precision gene-editing platform delivers more than academic insights: it offers tangible hope for sustainable, targeted solutions to safeguard California’s vineyards and global agriculture from a $57 billion threat.
Key Terms and Concepts
What is CRISPR/Cas9: A powerful tool scientists use to precisely edit an organism’s DNA, like genetic scissors. It’s important because it allows targeted changes to genes to study their function or fix problems. It’s used here to disrupt genes in the glassy-winged sharpshooter (GWSS). For example, researchers used it to mutate eye color genes (cinnabar and white) in GWSS. It involves the Cas9 protein guided by RNA.
What is Microinjection: A technique where tiny needles are used to inject substances (like CRISPR components) directly into cells or embryos. It’s crucial for delivering CRISPR tools into very small insect eggs. Researchers used it to inject Cas9 protein and guide RNAs into GWSS embryos while they were still on the leaf. This allowed them to create genetic changes efficiently.
What is Mutagenesis: The process of creating changes (mutations) in an organism’s DNA sequence. It’s important for studying what genes do by seeing what happens when they are broken. In this study, CRISPR/Cas9 caused mutagenesis in the cinnabar and white genes of GWSS. For example, mutations led to insects with white or bright orange eyes instead of red-brown.
What is a sgRNA (single-guide RNA): A specially designed RNA molecule that guides the Cas9 protein to a specific, matching sequence in the DNA to cut it. It’s essential for the precision of CRISPR/Cas9 gene editing. Researchers designed different sgRNAs targeting the cinnabar and white genes in GWSS. For instance, sgRNAw6-1 and sgRNAw6-2 targeted the white gene.
What is an Allele: A specific version of a gene. Different alleles can cause variations in traits, like eye color. Tracking alleles shows how mutations are inherited. Mutant alleles of the white (w) and cinnabar (cn) genes were created and passed down to offspring. For example, the w^1 allele had an 83-bp insertion disrupting the gene.
What is a Phenotype: The observable physical characteristics or traits of an organism, resulting from its genes and environment. Studying phenotypes reveals what a gene does. Mutations in white caused a white eye phenotype, while cinnabar mutations caused bright orange eyes and revealed hidden wing pigments.
What is a Genotype: The specific genetic makeup of an organism, particularly the alleles it carries for a gene. Knowing the genotype explains the phenotype and inheritance. Researchers sequenced DNA to find the genotypes (e.g., w^1 w^2) of mutant GWSS, confirming the mutations caused the observed eye and wing colors.
What is an Autosome: Any chromosome that is not a sex chromosome (X or Y). Genes on autosomes are inherited the same way regardless of sex. Researchers proved white and cinnabar genes are on autosomes in GWSS because mutant traits appeared equally in males and females after crosses. This is different from genes on sex chromosomes.
What is Xylem: The tissue in plants that transports water and dissolved minerals from the roots upwards. It’s important because GWSS feeds on xylem sap. GWSS is a xylem-feeder, meaning it pierces plants and sucks fluid from these vessels. This feeding habit makes it a vector for the xylem-dwelling bacterium Xylella fastidiosa.
What is Xylella fastidiosa: A bacterium that lives in the xylem of plants and causes serious diseases. It’s economically devastating because GWSS spreads it to crops. This bacterium causes Pierce’s Disease in grapes. GWSS transmits Xylella fastidiosa when feeding, threatening California’s multi-billion dollar grape industry.
What is Pierce’s Disease: A deadly disease of grapevines caused by the bacterium Xylella fastidiosa, blocking the xylem. It’s a major agricultural threat vectored by insects like GWSS. Pierce’s Disease destroys grapevines, costing the industry billions. Controlling GWSS is crucial to stop its spread.
What is a Vector: An organism (like an insect) that transmits a disease-causing pathogen (like a bacterium or virus) from one host to another. Vectors spread devastating plant and animal diseases. GWSS is a vector for Xylella fastidiosa, spreading Pierce’s Disease between grapevines and other plants as it feeds.
What is Off-target Mutagenesis: When CRISPR/Cas9 accidentally cuts DNA at sites other than the exact intended target. It’s important to minimize as it can cause unwanted, potentially harmful mutations. Researchers checked for off-target effects in GWSS but found them negligible or absent, showing their sgRNAs were specific.
What is Pteridine: A class of nitrogen-containing compounds that often act as pigments (colors) in insects. They are important for understanding insect coloration beyond just eye color. The study found pteridines give red color to GWSS wings and are reduced in white mutants, revealing a new role beyond eyes.
What is Ommochrome: A class of pigments, often brown or red, derived from the amino acid tryptophan, commonly found in insect eyes. They are key components of insect eye color. Mutations in the cinnabar gene disrupt ommochrome production in GWSS eyes, leading to orange eyes instead of red-brown.
What is Germline: The cells in an organism that give rise to eggs or sperm, passing genetic information to the next generation. Editing the germline ensures mutations can be inherited. CRISPR edits in GWSS germline cells allowed mutant white and cinnabar alleles to be passed stably to offspring for generations.
What is Mosaicism: When an organism (especially a G0 animal directly from an injected embryo) has a mixture of cells with different genotypes, some edited and some not. It shows the edit worked but wasn’t in all cells early on. Injected GWSS embryos hatched into adults with mosaic eyes (patches of mutant color).
What is Frameshift Mutation: A DNA mutation (insertion or deletion) that changes the grouping of nucleotides into codons, usually leading to a premature stop signal and a nonfunctional protein. It’s a common result of CRISPR cuts. Many CRISPR-induced mutations in GWSS cinnabar and white genes were frameshifts, disrupting protein function.
What is Amplicon Sequencing: A method where a specific DNA region is amplified (copied many times) by PCR and then sequenced in depth to find all variations (mutations) present. It detects the range of edits, especially in mosaic individuals. Researchers used it to analyze CRISPR edits in the white gene target site and off-target sites in GWSS.
What is Recessive Allele: An allele whose effect is masked or not seen if a dominant version of the gene is present. You need two copies (homozygous) to see the trait. The mutant white and cinnabar alleles are recessive; GWSS needed two mutant copies to show white or orange eyes fully.
What is Reciprocal Cross: Breeding experiments where the traits are crossed in both directions (e.g., Mutant Female x Wild Male AND Wild Female x Mutant Male). It tests if a gene is on a sex chromosome. Researchers did reciprocal crosses for white and cinnabar mutants; all F1 offspring were wild-type, proving the genes are autosomal, not sex-linked.
What is a Genetic Model System: A species studied intensively to understand fundamental biological processes, often because it’s easy to manipulate genetically. Establishing GWSS as one helps study Hemiptera traits. The ease of CRISPR in GWSS makes it a potential model for studying hemipteran pests and developing control strategies.
What is the white (w) Gene: A gene coding for an ABC transporter protein crucial for moving pigment precursors (for both ommochromes and pteridines) into cells in insects. Mutating it disrupts pigment accumulation. CRISPR disruption of white in GWSS caused white eyes and loss of red wing pigments (pteridines).
What is the cinnabar (cn) Gene: A gene coding for an enzyme (kynurenine 3-monooxygenase) in the ommochrome pigment biosynthesis pathway. Mutating it blocks brown/red eye pigment production. Mutating cn in GWSS led to bright orange eyes (revealing pteridines) but normal wing pigments, and uncovered eye cell patterns.
What is NHEJ (Non-Homologous End Joining): The cell’s primary way to repair broken DNA ends. It often results in small insertions or deletions (indels) at the break site. CRISPR cuts are repaired by NHEJ in GWSS, leading to the small indels that caused frameshift mutations in white and cinnabar.
Reference:
de Souza Pacheco, I., Doss, AL.A., Vindiola, B.G. et al. Efficient CRISPR/Cas9-mediated genome modification of the glassy-winged sharpshooter Homalodisca vitripennis (Germar). Sci Rep 12, 6428 (2022). https://doi.org/10.1038/s41598-022-09990-4
