Sterile Insect Technique Research Articles

Dual suppression of Glossina pallidipes using entomopathogenic fungal-based biopesticides and sterile insect technique

Tsetse flies and trypanosomosis significantly impact bovine production and human health in sub-Saharan Africa, exacerbating underdevelopment, malnutrition, and poverty. Despite various control strategies, long-term success has been limited. This study evaluates the combined use of entomopathogenic fungi (EPF) and the sterile insect technique (SIT) to combat tsetse flies. Eleven EPF isolates were tested against teneral males of Glossina pallidipes, focusing on mortality rates, radial growth, and impacts on fly fitness. Temperature effects on conidial growth, sporulation, and spore yield of SIT-compatible/tolerant strains were also assessed. The fungal isolates significantly influenced mortality rates in both unirradiated and irradiated (SIT-treated) males (p < 0.0001). Metarhizium anisopliae strains ICIPE 20, ICIPE 32, ICIPE 41, ICIPE 62, ICIPE 78, and Beauveria bassiana ICIPE 603 showed higher SIT compatibility/tolerance with LT50 values of 11–30 days, compared to other more virulent isolates with LT50 values of 4–9 days. Temperature significantly affected the radial growth of SIT-compatible EPF strains (p < 0.0001), with M. anisopliae ICIPE 78 exhibiting the fastest conidia growth at 25°C. Spore yield varied significantly across temperatures (15–40°C), and the thermal range for conidia germination of SIT-compatible strains was 8.1–45.4°C, with an optimal range of 26.7–31.1°C. Moreover, infected unirradiated females and irradiated males (donors) successfully transmitted conidia to untreated flies (receivers) without significant differences in survival rates (p = 0.6438) and no observed sex dimorphism. Our findings highlight the potential of combining EPF and SIT as a novel dual approach that could effectively and synergistically suppress tsetse fly populations.

A mass rearing method to aliquot and hatch Aedes mosquito eggs using capsules

The sterile insect technique (SIT) is a sustainable genetic control method to effectively suppress mosquito vector populations below sanitary and nuisance thresholds. The SIT requires a cost-effective production and release of radio sterilized insects of high and consistent quality and quantity. In this study, we report a new procedure to hatch, aliquot and introduce Aedes albopictus and Aedes aegypti larvae into larval mass rearing units using dissolvable vegetable capsules containing brushed eggs and larval diet. The results demonstrated that the egg brushing procedures, the opening of the hatching container and the presence of diet in the hatching solution increased Aedes eggs hatching success. The diet added during the hatching process decreased the dissolved oxygen (DO) concentration, which constituted the main stimulus for egg hatching success in both species. However, a DO reduction below 3 mg/l can effectively stimulate hatching in both species regardless of the deoxygenation method adopted. The capsule method produced similar hatching success without affecting larval survival and pupation rates in comparison with standard hatching procedures. The use of capsules eliminates intermediate laborious procedures for hatching, counting and transferring larvae into mass rearing trays with an expected improvement in production times and costs for Aedes SIT operational programs.

Exploring the metabolic changes of Ceratitis capitata Vienna 8 strain across three developmental stages through probiotic larval diet supplementation.

The Sterile Insect Technique (SIT) is revolutionizing pest control through its environmentally friendly approach, which involves rearing and sterilizing male insect pests using ionizing radiation and subsequently releasing them into the field to mate with wild females without producing offspring. Recent research has unveiled a groundbreaking enhancement in the quality of released Ceratitis capitata (medfly) males through the addition of probiotics to their larval diet. To thoroughly examine the impact of probiotic supplementation on the medfly larval diet, we conducted an in-depth analysis with GC-MS from medfly extract supplemented with probiotic Enterobacter sp. strain. The supplementation revealed a notable difference in the metabolomic signature compared to non-supplemented extract across all medfly life stages. We identified 37 known metabolites for all the stages, 12 of them were identified as biomarkers for the larval stage, 12 for the pupal stage, and 13 for the adult stage exhibiting crucial activities such as anti-bacterial, anti-fungal, and sexual and aggregation pheromone. These findings underscore the considerable potential of SIT combined with probiotic supplementation for enhancing sustainable pest control strategies worldwide.

Exploring the impact of a chemical disinfectant and an antiviral drug for RNA virus management in the Mediterranean fruit fly mass-rearing.

The Mediterranean fruit fly is an agricultural pest of a wide variety of fruit crops. An effective method to counteract them in the field is through the application of the sterile insect technique, which requires the mass-production of sterile males. The presence of pathogens, and specifically viruses, threatens the well-being of mass-reared insects generating an interest on the development of strategies for viral elimination or containment. Thirteen RNA viruses have been described in the medfly although so far only one of them, Ceratitis capitata nora virus, has been associated with detrimental effects on medfly development. In this context, medfly larvae were supplied with a chemical compound (formaldehyde) and an antiviral compound (ribavirin) via oral feeding to (1) test the potential of these compounds for viral elimination and (2) analyze their effect on medfly development. Overall, formaldehyde treatment did not reduce the viral titer for any of the tested viruses, while ribavirin effectively reduced the levels of two widespread RNA viruses but not in a dose-response manner. However, the addition of both compounds correlated with detrimental effects on medfly fitness, arguing against their use in mass-rearing facilities.

The evaluation on control potential using X-ray to irradiate adult Spodoptera frugiperda (Lepidoptera: Noctuidae).

The sterile insect technique (SIT), involving the rearing of larvae for pupation followed by irradiating pupae, is employed for environmentally friendly invasive pest management. Despite its effectiveness, the cost of pupae production poses challenges for small farmers in developing countries. Alternatively, utilizing insect trapping techniques can capture abundant adult insects in their natural habitats, but application potential of adult SIT approach remains unclear. We used the invasive pest Spodoptera frugiperda (Lepidoptera: Noctuidae) to assess the effectiveness of X-ray irradiated moths on their sterility rates, mating competitiveness, flight ability, and larval control efficiency in the field. Our findings revealed that optimal X-ray doses for 1-, 3-, and 5-day-old adult S. frugiperda were 154, 173, and 180 Gy, respectively. These doses rendered males more than 80.0% infertile and females nearly completely infertile. Significantly, the flight ability of sterilized males remained unaffected by sub-sterilizing doses of irradiation. Furthermore, in a release ratio of 16:1:1 (irradiated males-unirradiated males-unirradiated females), irradiated males exhibited the highest mating competitiveness (0.79). In the field-cage experiments, the corrected leaf protection rate and the corrected population decline rate in the 16:1:1 release plot were 60.50% and 74.21%, respectively. These findings suggest that radiation-induced sterility in adult S. frugiperda holds promise for practical applications, offering a conceptual framework and novel approaches for advancing radiation-based pest control technology. © 2024 Society of Chemical Industry.

Investigating the Potential of X-Ray-Based Cancer Treatment Equipment for the Sterile Insect Technique in Aedes aegypti Control Programs.

The sterile insect technique (SIT) stands as an eco-friendly approach for mosquito control, but it is impeded by the limited availability of γ-ray radiation source. This research sought to investigate a different radiation source-the Varian Clinac 23EX linear accelerator, which is frequently used for X-ray therapy in cancer treatment. Evaluation parameters including emergence rate, average survival time, induced sterility (IS), male mating competitiveness of irradiated males and fecundity (the number of eggs per female per batch), and the egg hatch rate of females mated with irradiated males were assessed to gauge the application potential of this cancer treatment equipment in the realm of the SIT. The results indicated that X-rays from radiation therapy equipment can effectively suppress the hatch rate of offspring mosquitoes without adversely affecting the emergence rate of irradiated males or the fecundity of females. In addition, at an X-ray dose of 60 Gy, the induced sterility in Ae. aegypti was comparable to the sterility induced by 40 Gy of γ-rays with both treatments resulting in 99.6% sterility. Interestingly, when a release ratio of 7:1 (irradiated males:unirradiated males) was used to competitively mate with females, the IS results resulted by 60 Gy X-rays and 40 Gy γ-rays were still at 70.3% and 73.7%, respectively. In conclusion, the results underscored the potential of the Varian Clinac 23EX linear accelerator as an X-ray source in SIT research.

Linalool fumigation improves mating competitiveness of males for population suppression of the global fruit pest Cydia pomonella

AbstractBACKGROUNDThe implementation of sterile insect technique (SIT) has proven effective in the area‐wide suppression of several significant agricultural and sanitary pests by using traditional cobalt‐60 (60Co‐γ) as a radiation source. Recently, X‐ray has been validated as a feasible alternative to 60Co‐γ radiation sources. Nonetheless, higher doses of X‐ray irradiation led to insect sterility but diminish mating competitiveness, thereby impacting the effectiveness of SIT applications.RESULTSIn this study, we assessed the impact of various X‐ray irradiation doses (ranging from 0 to 366 Gy) on the fecundity, fertility, and mating competitiveness of Cydia pomonella, a globally invasive fruit pest. Results demonstrated that the sterility rate of irradiated males increased with dose up to 200 Gy, then stabilized. Exposure to 200 Gy reduced male mating competitiveness, with competitiveness index (CI) values of 0.17 in the laboratory and 0.096 in the orchard. This decline is likely linked to the decreased expression of genes associated with sex pheromones recognition, such as CpomOR3a, CpomOR3b, and CpomOR5, post‐irradiation. Fumigation of linalool at varying concentrations (70, 83, and 96 μL/m3) enhanced mating competitiveness of males, particularly at moderate levels, possibly by restoring pheromones recognition. Implementation of repeated releases of sterilized males on a pilot scale led to a notable reduction in the population of C. pomonella in the field.CONCLUSIONThese findings indicate that fumigation with plant volatiles has the potential to mitigate male sterility induced by X‐ray irradiation, offering a promising approach to enhance the efficacy of SIT applications for the control of C. pomonella. © 2024 Society of Chemical Industry.

Preparation for targeted sterile insect technique to control invasive Aedes aegypti (Diptera: Culicidae) in southern California: dose-dependent response, survivorship, and competitiveness.

Aedes aegypti is fast spreading across California, with over 300 cities within 22 central and southern counties being infested since its introduction in 2013. Due to its cryptic breeding habitats, control efforts have not been successful so far. This calls for innovative tools such as sterile insect technique (SIT) to reinforce the existing integrated pest management (IPM). Here, we assessed fitness, survivorship, and dose response of X-ray irradiated male Ae. aegypti in California. Locally acquired Ae. aegypti eggs were hatched and reared in temperature-controlled laboratory setting at the West Valley Mosquito and Vector Control District in Ontario, California. Freshly emerged adult male mosquitoes were manually separated using motor-operated aspirators and treated with X-ray radiation at different dosage (42-60 Gy). Dose response of irradiated males was analyzed and induced sterility determined. Survivorship of males treated with different X-ray doses was compared. Fecundity of females that mated with irradiated males at different X-ray doses was generally comparable. Overall, induced sterility increased with higher X-ray doses. Nulliparous females that mated with male Ae. aegypti treated with 55-60 Gy laid eggs with over 99% sterility. Non-irradiated male mosquitoes had higher survivorship (mean = 0.78; P = 0.0331) than irradiated mosquitoes (mean range = 0.50-0.65). The competitiveness index of irradiated males decreased with increasing X-ray treatment doses, 1.14 at 55 Gy and 0.49 at 60 Gy, and this difference was significant (P < 0.01). Irradiated males showed high survivorship and competitiveness-key for the anticipated SIT application for the control of invasive Ae. aegypti in California.

Effect of a Low Cost Diet on the Quality Control of the Greater Wax Moth, Galleria mellonella for Improving the Sterile Insect Technique

Effect of a Low Cost Diet on the Quality Control of the Greater Wax Moth, Galleria mellonella for Improving the Sterile Insect Technique

Effects of semiochemical pre-feeding, physiological state, and weather on the response of Bactrocera dorsalis to methyl eugenol baited traps

Plant-derived phenylpropanoids are semiochemicals that are often highly attractive to Bactrocera (Diptera: Tephritidae) males. One of these semiochemicals, methyl eugenol (ME), is used in the male annihilation technique (MAT) for the management of B. dorsalis (Hendel), a destructive horticultural pest. It is not normally viable to simultaneously implement MAT with the sterile insect technique (SIT), as released sterile males are attracted to MAT devices. However, prior semiochemical exposure can reduce the later response of Bactrocera males to the same or another semiochemical, which may allow the synchronous application of MAT and SIT. We determined how the interaction between semiochemical pre-feeding, weather, and fly physiology impacted the response of male B. dorsalis to ME baited traps. Response by a known number of males in field cages was determined in relation to temperature, relative humidity, semiochemical pre-feeding (ME, eugenol, or none), diet (protein supplemented and protein deprived) and age (4, 10 and 20 days old). Semiochemical pre-feeding of both ME and eugenol equally decreased the response of males that were 10 days old, or older, to ME baited traps. Adult diet had no effect on the response of males to ME baited traps. Response improved as temperature and relative humidity increased. These results highlight the feasibility of synchronous MAT-SIT programmes targeting B. dorsalis and the viability of continued protein supplementation of sterile males. We show that eugenol is an alternative to ME for suppression of male B. dorsalis response to MAT devices and that weather conditions significantly affect the variability and reliability of abundance estimates from trap captures.

Radiating South

Abstract From the 1960s to the 1990s, the US Department of Agriculture led a campaign across North America to eradicate a notorious livestock pest known as the screwworm. It did so by breeding flies in factories, sterilizing them with radiation, and scattering them from the air. Based on original multisite archival research, this article examines the overlooked role of Mexico in what was a flagship application of the “sterile male technique.” It argues that three main factors shaped this role: Mexico's long-term projects of agricultural and atomic modernization; diplomatic leverage provided by a shared border; and an earlier tradition of bilateral cooperation and conflict over animal disease that stretched back to the 1940s. The analysis deepens our understanding of how Mexico shaped science and agriculture at home and abroad. It also helps explain why this particular project of atomic agriculture succeeded on the scale it did.

Spatial and temporal characterization of Aedes albopictus oviposition activity in candidate urban settings for sterile insect technique testing in La Reunion Island

BackgroundUnderstanding of mosquito spatiotemporal dynamics is central to characterize candidate field sites for the sterile insect technique (SIT) testing, and is critical to the effective implementation and evaluation of pilot sterile male release programs. Here, we present a detailed description of Aedes albopictus (Skuse) egg-laying activity over a 6-year period in urban areas identified as potential SIT testing sites on Reunion Island.MethodWeekly entomological collections using ovitraps were carried out in residential and adjacent uninhabited habitats in two urban areas, Duparc and Bois Rouge, in the municipality of Sainte Marie, Reunion Island. Time-series data incorporating the frequency of positive ovitraps and the total number of eggs/ovitrap recorded each time at each locality during the study period from May 2013 to December 2018 were analyzed with multifaceted statistical approaches including descriptive statistics and spatiotemporal analyses incorporating the role of climatic factors on overall ovitrap productivity.ResultsDuring the ovitrap survey, the proportion of egg-positive ovitraps differed among study sites (χ2 = 50.21, df = 2, P < 0.001), being relatively lower in Duparc (89.5%) than in Bois-Rouges (95.3%) and the adjacent buffer zone (91.2%). Within each neighborhood, Ae. albopictus egg abundance varied by month in a roughly seasonal pattern marked by a single peak occurring more regularly February each year, a decline at the onset of the austral winter in July, followed by a period of lower ovitrap productivity in August and September. Fluctuation in both positivity rate and eggs densities per ovitraps were related to annual and seasonal variations in local temperature and rainfall (P < 0.001 in all cases). The spatial analysis also captured substantial between- and within-habitats heterogeneity, whereby the overall ovitrap productivity was higher in residential areas than in the buffer zone.ConclusionsCollectively, these results reveal that the distribution of Ae. albopictus oviposition activity is shaped by local habitat heterogeneity and seasonal climatic factors. Overall, this study provides baseline insights into the reproductive dynamics of Ae. albopictus, which would assist in planning locally tailored SIT interventions, while addressing concerns related to focal areas of high egg-laying intensity and potential immigration of females from natural areas.

The effects of diet and semiochemical exposure on male Bactrocera dorsalis (Hendel) (Diptera: Tephritidae) metabolic rate at a range of temperatures

The oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), is an invasive species that has rapidly spread across the African continent, endangering the security of agricultural industries. The sterile insect technique (SIT) is being investigated as a viable additional pest management tool to suppress B. dorsalis populations after its successful implementation in other parts of the world. There is evidence to suggest that pre-release nutritional and semiochemical treatments for sterilised males can enhance their competitive performance against wild type males in SIT programs. This study examined how sterilisation, a diet rich in protein (addition of yeast hydrolysate) or containing semiochemicals (methyl eugenol or eugenol) affected the resting metabolic (RMR) of male B. dorsalis at different temperatures (15 – 30 °C), measured using flow-through respirometry. Our results indicated that the negative effect of sterilisation on RMR decreased as temperature increased and that duration of exposure to semiochemicals for 1 to 4 days was not a significant influencing factor on male B. dorsalis RMR. Protein-rich diet increased average RMR, but the difference in RMR between dietary groups decreased as temperature increased. Semiochemical feeding reduced the average RMR in male B. dorsalis. The difference in RMR between males that consumed semiochemical and those that did not increased with as temperature increased.

Residual Longevity of Recaptured Sterile Mosquitoes as a Tool to Understand Field Performance and Reveal Quality.

Invasive mosquito species, such as Aedes albopictus, pose significant threats to both ecosystems and public health due to their role in transmitting diseases, such as dengue, Zika, and chikungunya. The Sterile Insect Technique (SIT) is a promising vector control strategy aimed at reducing mosquito populations by releasing sterile males to mate with wild females and reduce their reproduction rates. In this study, we employed the captive cohort method, which assesses the remaining longevity of randomly caught released individuals, to assess the longevity and frailty dynamics of sterile and non-sterile Ae. albopictus males. Using a mark-release-recapture approach (MRR), we compared the residual lifespan of sterile and non-sterile released males with that of wild, non-sterile males, aiming to understand the frailty dynamics of released males and, therefore, their quality and field performance. Contrary to expectations, our results revealed that released sterile males showed increased longevity compared to non-sterile males. Further, the marking process did not impact the longevity between lab-kept and marked males, suggesting that the marking process does not adversely affect survival under controlled conditions. These findings underscore the importance of optimizing pre-release and mass-rearing practices to enhance the effectiveness of SIT programs. Our study also demonstrates for the first time the use of the captive cohort method for understanding the biological dynamics of sterile mosquito populations in SIT programs, providing valuable insights for improving vector control strategies.

Loss-of-function in testis-specific serine/threonine protein kinase triggers male infertility in an invasive moth

Genetic biocontrol technologies present promising and eco-friendly strategies for the management of pest and insect-transmitted diseases. Although considerable advancements achieve in gene drive applications targeting mosquitoes, endeavors to combat agricultural pests have been somewhat restricted. Here, we identify that the testis-specific serine/threonine kinases (TSSKs) family is uniquely expressed in the testes of Cydia pomonella, a prominent global invasive species. We further generated male moths with disrupted the expression of TSSKs and those with TSSKs disrupted using RNA interference and CRISPR/Cas9 genetic editing techniques, resulting in significant disruptions in spermiogenesis, decreased sperm motility, and hindered development of eggs. Further explorations into the underlying post-transcriptional regulatory mechanisms reveales the involvement of lnc117962 as a competing endogenous RNA (ceRNA) for miR-3960, thereby regulating TSSKs. Notably, orchard trials demonstrates that the release of male strains can effectively suppress population growth. Our findings indicate that targeting TSSKs could serve as a feasible avenue for managing C. pomonella populations, offering significant insights and potential strategies for controlling invasive pests through genetic sterile insect technique (gSIT) technology.

Molecular Markers for Analyses of Genetic Diversity within the Anastrepha fraterculus Complex with Emphasis on Argentine Populations.

The South American fruit fly Anastrepha fraterculus (Wiedmann) has a vast range extending from northern Mexico, through Central America, to South America where it is an extremely polyphagous pest of wild and cultivated fruits. It is a complex of cryptic species currently composed of eight recognised morphotypes: "Mexican", "Venezuelan", "Andean", "Peruvian", "Ecuadorian", and the three Brazilian morphotypes "Brazilian-1", "Brazilian-2", and "Brazilian-3". Molecular markers that can identify the member species of the complex are crucial for the implementation of effective pest control measures, such as the sterile insect technique. The object of this study was to evaluate the use of the internal transcribed spacer 2 (ITS2) sequence for discriminating several members of the A. fraterculus cryptic species complex (Mexican, Peruvian, and Brazilian-1) and a related species, Anastrepha schultzi Blanchard. The analysis highlighted significant genetic differentiation between the evaluated morphotypes, allowed their discrimination within the A. fraterculus cryptic species complex, and provided new insights into their genetic relationships. The ITS2 marker provides a basis for the development of taxonomic keys for the discrimination of the cryptic taxa within the A. fraterculus cryptic species complex. ITS2 also represents an important marker for the poorly studied species A. schultzi.

Sperm Storage and Use Following Multiple Insemination in Aedes albopictus: Encouraging Insights for the Sterile Insect Technique.

The key to success in the application of the sterile insect technique (SIT) relies on the ability of released, sterile males to outcompete their fertile wild male counterparts to mate with wild females. However, many insect species exhibit multiple-mating behavior, which can be a way for females to select paternity for their progeny. This study aims to recognize the consequences of potential double-matings during an SIT program and to detect any evidence of sperm selection favoring sperm from fertile mates. This report provides a descriptive analysis of the storage and use of sperm by female Aedes albopictus. Stable isotopes were used to mark the sperm of fertile and sterile males. Mated females were allowed to oviposit before dissecting the spermathecae to link the presence of each type of sperm to the sterility of the eggs laid. It was found that sperm in females inseminated by both males was distributed in the three spermathecae with no obvious pattern, mostly mixed but also separately, and no evidence of any mechanism for sperm selection, sperm precedence, or sperm competition in Ae. albopictus females could be found. The fact that only a few double-mated females were double-inseminated and could also produce semi-sterile eggs, together with the finding that the sperm of sterile males appeared to be no less viable than that of fertile males, is an encouraging outcome for SIT approaches.

Mark-Release-Recapture Trial with Aedes albopictus (Diptera, Culicidae) Irradiated Males: Population Parameters and Climatic Factors.

Aedes albopictus is considered one of the major invasive species in the world and can transmit viruses such as dengue, Zika, or chikungunya. The Sterile Insect Technique (SIT) can be used to suppress the native populations of Ae. albopictus. Mark-release-recapture (MRR) studies are crucial to support the development of the release strategy during the SIT application. Meanwhile, weather conditions can affect the MRR trial's results and it is critical to understand the influence of climatic factors on the results. In October 2022, 84,000 irradiated sterile males were released for three consecutive weeks in Faro, Southern Portugal. Mosquitoes were recaptured by human landing collection (HLC) one, two, four, and six days after release. Generalized linear models with a negative binomial family and log function were used to estimate the factors associated with the number of recaptured mosquitoes, prevalence ratios, and the 95% confidence intervals (CIs). A total of 84,000 sterile male mosquitoes were released, with 528 recaptured (0.8%) by HLC. The prevalence of recaptured mosquitoes was 23% lower when the wind intensity was moderate. Marked sterile males had an average median distance travelled of 88.7 m. The median probability of daily survival and the average life expectancy were 61.6% and 2.1 days, respectively. The wild male population estimate was 443.33 males/ha. Despite no statistically significant association being found with humidity, temperature, and precipitation, it is important to consider weather conditions during MRR trial analyses to obtain the best determinant estimation and a more efficient application of the SIT in an integrated vector management program.

Population suppression with dominant female-lethal alleles is boosted by homing gene drive

BackgroundMethods to suppress pest insect populations using genetic constructs and repeated releases of male homozygotes have recently been shown to be an attractive alternative to older sterile insect techniques based on radiation. Female-specific lethal alleles have substantially increased power, but still require large, sustained transgenic insect releases. Gene drive alleles bias their own inheritance to spread throughout populations, potentially allowing population suppression with a single, small-size release. However, suppression drives often suffer from efficiency issues, and the most well-studied type, homing drives, tend to spread without limit.ResultsIn this study, we show that coupling female-specific lethal alleles with homing gene drive allowed substantial improvement in efficiency while still retaining the self-limiting nature (and thus confinement) of a lethal allele strategy. Using a mosquito model, we show the required release sizes for population elimination in a variety of scenarios, including different density growth curves, with comparisons to other systems. Resistance alleles reduced the power of this method, but these could be overcome by targeting an essential gene with the drive while also providing rescue. A proof-of-principle demonstration of this system in Drosophila melanogaster was effective in both biasing its inheritance and achieving high lethality among females that inherit the construct in the absence of antibiotic.ConclusionsOverall, our study shows that substantial improvements can be achieved in female-specific lethal systems for population suppression by combining them with various types of gene drive.

CRISPR/Cas12a ribonucleoprotein mediated editing of tryptophan 2,3-dioxygenase of Spodoptera frugiperda.

In insect genome editing CRISPR/Cas9 is predominantly employed, while the potential of several classes of Cas enzymes such as Cas12a largely remain untested. As opposed to Cas9 which requires a GC-rich protospacer adjacent motif (PAM), Cas12a requires a T-rich PAM and causes staggered cleavage in the target DNA, opening possibilities for multiplexing. In this regard, the utility of Cas12a has been shown in only a few insect species such as fruit flies and the silkworm, but not in non-model insects such as the fall armyworm, Spodoptera frugiperda, a globally important invasive pest that defies most of the current management methods. In this regard, a more recent genetic biocontrol method known as the precision-guided sterile insect technique (pgSIT) has shown successful implementation in Drosophila melanogaster, with certain thematic adaptations required for application in agricultural pests. However, before the development of a controllable gene drive for a non-model species, it is important to validate the activity of Cas12a in that species. In the current study we have, for the first time, demonstrated the potential of Cas12a by editing an eye color gene, tryptophan 2,3-dioxygenase (TO) of S. frugiperda by microinjecting ribonucleoprotein complex into pre-blastoderm (G0) eggs. Analysis of G0 mutants revealed that all five mutants (two male and three female) exhibited distinct edits consisting of both deletion and insertion events. All five edits were further validated through in silico modeling to understand the changes at the protein level and further corroborate with the range of eye-color phenotypes observed in the present study.