Complex interplay between facilitation and competition in two invasive fruit pests

Globalization has contributed enormously to emergence and expansion of invasive pests. A recent example is the invasion in Europe and the USA by the cherry vinegar fly Drosophila suzukii. The pest has high potential to invade and establish itself in different climatic zones, the fact that is made clear by looking at the current global distribution map of the fly. The use of pesticide chemicals is so far the first line of defense against it due to lack of reliable alternatives. The use of pesticides against this fly has major problems including possible emergence of insecticide resistance due to the short generation time and number of generations per year. Secondly, the fly lays eggs inside the fruit which make the egg and larvae not exposed to insecticide. Thirdly and most importantly is the time of invasion with regard to ripening and harvest of the fruits. This makes the pesticide choice a very hard one. To be able to fight this onerous pest, the Sterile Insect Technique (SIT) offers an effective, environment friendly genetic pest management that does not interfere with the natural enemies and pollinators. Here, I propose the use of biotechnological approaches to develop transgenic strains suitable for SIT against D. suzukii. In the first part of this study, the use of a CRISPR/Cas9 genome editing system to engineer a sex conversion suppression gene drive in Drosophila melanogaster is presented. We found that current designs of homing-based gene drive induce development of resistance against them. We proposed possible solutions to avoid rapid development of resistance and the application of such drive in Tephritid flies where targeting the sex determination gene transformer leads to fertile XX males. Second, the hyperactive version of the piggyBac transposase was successfully used to improve insect transgenesis with demonstration in three species belonging to two different orders, the genetic model Drosophila melanogaster, the global fruit pest Ceratitis capitata and the new beetle model for development and evolution Tribolium castaneum. In the third chapter improvement in CRISPR/Cas9 genome editing of the invasive fruit pest Drosophila suzukii is presented. Different endogenous regulatory elements were isolated and used to drive the expression of Cas9 and the guide RNAs. In addition, the application of CRISPR/Cas9 to engineer the first sperm marking strain for D. suzukii is presented, which represents an important contribution to the establishment of the SIT for this pest. In the fourth part, the φC31 integrase based site-specific germline transformation of the fruit pest D. suzukii was established both by integration in one attP landing site and by recombinase mediated cassette exchange (RMCE). This provides a platform for testing several enhancers and/or complete system in the same genome context. We also present the development of embryonic and spermatogenesis specific driver lines that can be used for different biotechnological improvements of SIT against D. suzukii. The last three chapters present a perspective paper describing a new reproductive sterility system and the first steps toward its development. It is based on CRISPR/Cas9 chromosome shredding during spermatogenesis that should lead to the same results commonly achieved by ionizing radiation but with no deleterious effect on the males’ fitness. This should culminate in embryonic lethality due to chromosomal aberration causing aneuploidy. We first generated several spermatogenesis specific driver lines and Cas9 responder lines to test the suitability of the tet-off binary system to control gene expression during spermatogenesis. Suitable genomic targets for chromosome shredding were identified and gRNAs to target them were designed. Genome editing and insect transgenesis tools developed in this study will facilitate further biotechnological improvements of the SIT and its transfer to the invasive agricultural pest D. suzukii.

BackgroundThe invasive fruit pest Drosophila suzukii was reported for the first time in Europe and the USA in 2008 and has spread since then. The adoption of type II clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) as a tool for genome manipulation provides new ways to develop novel biotechnologically-based pest control approaches. Stage or tissue-specifically expressed genes are of particular importance in the field of insect biotechnology. The enhancer/promoter of the spermatogenesis-specific beta-2-tubulin (β2t) gene was used to drive the expression of fluorescent proteins or effector molecules in testes of agricultural pests and disease vectors for sexing, monitoring, and reproductive biology studies. Here, we demonstrate an improvement to CRISPR/Cas-based genome editing in D. suzukii and establish a sperm-marking system.ResultsTo improve genome editing, we isolated and tested the D. suzukii endogenous promoters of the small nuclear RNA gene U6 to drive the expression of a guide RNA and the Ds heat shock protein 70 promoter to express Cas9. For comparison, we used recombinant Cas9 protein and in vitro transcribed gRNA as a preformed ribonucleoprotein. We demonstrate the homology-dependent repair (HDR)-based genome editing efficiency by applying a previously established transgenic line that expresses DsRed ubiquitously as a target platform. In addition, we isolated the Ds_β2t gene and used its promoter to drive the expression of a red fluorescence protein in the sperm. A transgenic sperm-marking strain was then established by the improved HDR-based genome editing.ConclusionThe deployment of the endogenous promoters of the D. suzukii U6 and hsp70 genes to drive the expression of gRNA and Cas9, respectively, enabled the effective application of helper plasmid co-injections instead of preformed ribonucleoproteins used in previous reports for HDR-based genome editing. The sperm-marking system should help to monitor the success of pest control campaigns in the context of the Sterile Insect Technique and provides a tool for basic research in reproductive biology of this invasive pest. Furthermore, the promoter of the β2t gene can be used in developing novel transgenic pest control approaches and the CRISPR/Cas9 system as an additional tool for the modification of previously established transgenes.

Distance decay principles predict that species with larger geographic ranges would have greater intraspecific genetic diversity than more restricted species. However, invasive pest species may not follow this prediction, with confounding implications for tracking phenomena including original ranges, invasion pathways and source populations. We sequenced an 815 base-pair section of the COI gene for 441 specimens of Bactrocera correcta, 214 B. zonata and 372 Zeugodacus cucurbitae; three invasive pest fruit fly species with overlapping hostplants. For each species, we explored how many individuals would need to be included in a study to sample the majority of their haplotype diversity. We also tested for phylogeographic signal and used demographic estimators as a proxy for invasion potency. We find contrasting patterns of haplotype diversity amongst the species, where B. zonata has the highest diversity but most haplotypes were represented by singletons; B. correcta has ~7 dominant haplotypes more evenly distributed; Z. cucurbitae has a single dominant haplotype with closely related singletons in a ‘star-shape’ surrounding it. We discuss how these differing patterns relate to their invasion histories. None of the species showed meaningful phylogeographic patterns, possibly due to gene-flow between areas across their distributions, obscuring or eliminating substructure.

Drosophila suzukii, commonly known as the spotted-wing Drosophila, is an invasive polyphagous fruit pest, which has emerged as a global threat to agriculture in the Americas and in Europe. Due to the rapid spread, great economic losses and its pest behavior, D. suzukii represents a powerful model for invasion biology and pest management studies. However, its current European distribution, invasion routes and levels of genetic diversity in populations of D. suzukii are poorly understood. We present the first report of D. suzukii from Ukraine, with the invasion likely occurring close to 2014. The pattern of genetic variation at cytochrome oxidase I among D. suzukii populations from Europe, USA and Asia reveals comparatively high genetic diversity in the Ukrainian population of this pest species, suggesting a complex invasion scenario from multiple sources. Further monitoring patterns of genetic variation across space and time, to better understand the invasion routes of this invasive insect pest, will be an essential part for developing successful pest management strategies.

Simple SummaryThe codling moth, Cydia pomonella (L.), is an invasive agricultural pest species of pome fruits and walnuts that has developed resistance to many insecticides. Alternative eco-friendly approaches are warranted to reduce the dependence on insecticides for the sustainable management of C. pomonella. The sterile insect technique (SIT) is an autocidal strategy of control that is being trialed in experiments against various pests and that could reduce the risk of the development of insecticide resistance. In our previous study, we found that 366 Gy X-ray irradiation can effectively make the C. pomonella male sterile. In this study, we report on the investigation of the effect of X-ray irradiation on the fitness and adaptability of sterile insects, as well as the first pilot release of sterile male adults of C. pomonella in orchards in China. Results show that 366 Gy of X-ray irradiation significantly shortened the lifespan of the sterile male moths, reduced the mating competitiveness of males, and resulted in males being sterile in the orchards.The codling moth, Cydia pomonella (L.), is an invasive agricultural pest of pome fruits and walnuts in China that threatens the apple industry in the Loess Plateau and Bohai Bay; it has developed resistance to many insecticides. Sterile insect technique (SIT) combined with area-wide integrated pest management (AW-IPM) can reduce the risk of resistance to insecticides and effectively control some insect pest species. Our previous laboratory experiment found that irradiation with 366 Gy of X-ray caused the males of the codling moth to become sterile. However, the sterility and adaptability of males after being irradiated with 366 Gy X-ray in the field are still unclear. In this study, we investigated the effect of X-ray irradiation on the fitness of male adults that emerged from pupae irradiated with 366 Gy to explore their adaptability and mating competitiveness, and to examine the effect of releasing sterile male insects in orchards in northeast China on the fruit infestation rate of the Nanguo pear. The results showed that 366 Gy of X-ray irradiation significantly reduced the mating competitiveness of males and the hatching rate of the eggs laid by females pairing with sterile males. Meanwhile, the lifespan of the sterile male moths was significantly shorter than that of the normal ones in the field. A pilot test showed that the release twice of sterile male moths in the orchards had no significant effect on the fruit infestation rate. Our field experiments provide a scientific basis for the further optimization of the SIT technology program for controlling C. pomonella.

This fruit devastating pest of white mango scale (Aulacaspis tubercularis Newstead; Hempitera: Diaspididae) insect is an economically important pest and damages the mango fruit by sucking the sap through its piercing-sucking mouth part from mango leaves and fruits. Since the 2010 outbreak in Ethiopia due to a poor internal quarantine system this insect pest was distributed and invaded the whole mango-producing region in the country. Therefore, the aim of this study was to assess the infestation and severity status of WMS insects to get evidence for upcoming management investigations. The survey was carried out at Boloso Bombe and Boloso Sore districts in South Ethiopia Wolaita Zone in 2023. Among mango-producing farmers by using a semi-structural questionnaire 45 farmers were intervened through random selection. Geographic location and ten samples of mango leaf were collected from four cardinal directions per tree of each stop for examining the infestation and severity status of WMS. The occurrence of this pest at study locations was in 2016 as interviewed farmers respond. Since then, due to a lack of attention by farmers currently, it was distributed and covered the whole study area. As confirmed by this study the infestation ranged from 60 to 100% and damage or severity rated from moderate to very high. Among surveyed locations, 29% of mango farms were very highly, 52% highly and 20% moderately damaged. From the total study area, almost 31% of mango farms reduced 100% yield and the reduction of yield ranged from 50% to 100%. Before the outbreak of this invasive insect pest, the study area farmers were harvesting nearly 272 kg of mango fruit per tree and at normal times yield reduction was almost 43 kg per tree. After the prevalence of WMS insect per tree yield was almost 44 kg and the reduction of yield was alarmingly increased to 228 kg per tree as shown by this study data. Even though this insect pest is a devastating pest of mango fruit, however, it can be controlled through different management practices. Therefore, the management practice of this pest is categorized into three such as cultural, biological and biochemical. Culturally managed by Mango tree pruning, planting resistant materials, using proper spacing, cleaning or sanitation, burying infected residues and fruits, smoking different repellents of dry grass, animal dung, mango leaves and lemon bark or lemon mixed organic materials in one smoking can/pot and hanging inside mango tree. Foliar Spraying of various botanical extracts such as Neem seed extract (Azadirachta indica). Several concoctions made from ash, soap and goat urine were mixed and sprayed on infested mango fruit leaves and twigs. Various predators and parasitoids are used as Biological control. Soil drenching of systemic chemical insecticides also can control this insect pest. The way to control this serious damage and hinder the expansion of this invasive insect pest, integrated experimental investigation using the above-explained mechanisms will be advisable to enhance mango fruit yield. Int. J. Agril. Res. Innov. Tech. 14(2): 28-37, December 2024

Simple SummarySpotted-wing drosophila (SWD), Drosophila suzukii, a major pest of small fruits and cherries, is often managed with conventional insecticides. RNA interference (RNAi) technology has been investigated to develop an alternative control method for SWD. Our previous RNAi work showed that the RNAi efficacy on SWD was limited because double-stranded RNA (dsRNA) was degraded by the midgut nuclease (= dsRNA − degrading enzyme) before passing through the midgut membrane of the fly. To overcome this obstacle, RNAi directly targets the midgut genes, thus eliminating the need for dsRNA to pass through the midgut membrane. The primary focus of this study is to identify the fly adult midgut’s genes. We compared differential gene expressions between the midgut and the rest of the whole-body using transcriptomic analysis. We found that 1921 genes were upregulated, and 1834 genes were downregulated in the midgut. We chose ten midgut-specifically upregulated genes involved in various biological functions and confirmed their gene expressions. Particularly, the midgut membrane proteins found in this study would be potential targets for developing RNAi and other biological tools for controlling SWD in the future.Spotted-wing drosophila (SWD), Drosophila suzukii, is a destructive and invasive pest that attacks most small fruits and cherries. The current management for SWD involves the use of conventional insecticides. In an effort to develop a biologically based control option, the application of RNA interference (RNAi) has been investigated. To develop an RNAi approach, suitable targets must be identified, and an efficient delivery method must be developed for introducing the double-stranded RNA (dsRNA) in the midgut. In D. suzukii, we previously found that dsRNA nucleases actively degrade dsRNA molecules in the midgut. In this study, we focused on identifying biological targets focused on the midgut membrane. The profile of midgut-specific genes was analyzed and compared with the genes expressed in the whole-body using transcriptome analysis. Differential gene expression analysis revealed that 1921 contigs were upregulated and 1834 contigs were downregulated in the midgut when compared to genes from other body tissues. We chose ten midgut-specifically upregulated genes and empirically confirmed their expressions. We are particularly interested in the midgut membrane proteins, including G protein-coupled receptors (GPCRs) such as diuretic hormone 31 (DH31) receptor, neuropeptide F (NPF) recepror, toll-9, adhesion receptors, methuselah (mth), and gustatory receptor, because insect GPCRs have been offered great potential for next-generation pest management.

Insecticidal and oviposition deterrent effects of essential oils and their constituents against the invasive pest Drosophila suzukii (Matsumura) (Diptera: Drosophilidae)

Chilli thrips, (Scirtothrips dorsalis Hood; Thysanoptera: Thripidae), is an economically important pest of vegetable, fruit, and ornamental crops throughout Asia, Africa, Oceania, the Caribbean, and some parts of South America and is an invasive pest in several US states. Chilli thrips were first observed in Florida in 1991. It was first recorded in blueberries in Hernando, Pasco and Sumter counties in July of 2008. This 4-page fact sheet written by Oscar E. Liburd, Babu R. Panthi, and Douglas A. Phillips and published by the UF/IFAS Entomology and Nematology Department discusses the life cycle of the chilli thrips, plant damage it causes, and management recommendations for chilli thrips in blueberries in Florida.https://edis.ifas.ufl.edu/in1298

Simple SummaryThe spotted-wing Drosophila is an invasive pest of berry fruits in Florida. The pest is difficult to control with pesticides, and resistance is developing. The current study was carried out in north Florida to see if non-pesticidal pest control tactics using three mulching practices could reduce its population density to increase growers’ berries productivity. The experiments were conducted for two years while using two plant-based mulches (short pine needles and pine bark) and one fabric-based mulch in the open blueberry fields. In general, the fly population was reduced within the selected pine bark and black weed fabric mulches. This is the first report of such practices deployed to manage the spotted-wing Drosophila in Florida. We are confident that these selective cultural practices could be utilized in open blueberry fields before berries settings to reduce pest pressure for both conventional and organic blueberry growers in Florida. Indeed, this will increase growers’ berry production and profitability.The spotted-wing Drosophila (SWD), Drosophila suzukii, is native species in Southeast Asia. For over a decade, this invasive pest has been globally expanding. The economic losses to soft fruits and stoned fruits in the United States are increasing every year. Presently, the only viable tool to reduce the SWD population is the continued use of broad-spectrum insecticides. Pesticide resistance is appearing in the populations for the SWD. Organic farmers have limited options to control this pest in open fields. The major goal of this study was to develop cost-effective pest management strategies to manage the SWD using three types of mulches (two plant-based and one fabric-based) to reduce fly population and damage in open blueberry fields in north Florida. The study was conducted in two fruiting seasons (2017 and 2018). The study results demonstrated that the fly trap catches in 2017 shortleaf pine needle mulch had much higher populations (about 2.5-fold) of the SWD than all other treatments. In 2018, the numbers were about 1.7-fold more on shortleaf pine needle mulch than on other treatments. The fine texture of the mulch (pine needles) can easily facilitate the emergence of the SWD if the mulch is not thick enough. Although the pine needles covered the soil surface, it may have been too thin and thus allowed the SWD adults to emerge from the soil without much hindrance. In 2018, a higher population of the SWD was recorded from all the mulching practices. However, there were no significant differences in trap catches between all treatments. In general, the fly population is reduced with the use of pine bark and black weed fabric mulches. This is the first study that reports the effects of three mulches in controlling the SWD populations, which could benefit conventional and organic blueberry growers.

Drosophila suzukii (Matsumura) is an invasive pest of soft-skin fruits. Due to its recent introduction, there are currently no studies about its chemical control in Argentina. The aim of this research was to evaluate five formulated insecticides with different mode of action against eggs, larvae and adults of D. suzukii under laboratory conditions. The product formulated with spinosad was the most effective insecticide, since it showed high efficacy against all life stages. Furthermore, females exposed to spinosad and lambda-cyhalothrin laid fewer eggs. Further studies need to be conducted in order to corroborate these results under field conditions.

Quercetin stimulates an accelerated burst of oviposition-based reproductive strategy in codling moth controlled by juvenile hormone signaling pathway

This review focuses on the process of adapting the original concept of Integrated Pest Management (IPM) to the wider conception of the Integrated Fruit Production (IFP) implemented in Europe. Even though most of the pest management strategies still rely on the use of synthetic pesticides, a wide array of innovative and environmentally friendly tools are now available as possible alternative to the pesticides within the modern apple production system. We also highlight how recent pest management strategies and tools have created an opening for research towards IPM improvement, including the use of biorational pesticides, semiochemicals and biological control. Forecasting models, new tree training systems and innovative spray equipment have also been developed to improve treatment coverage, to mitigate pesticide drift and to reduce chemical residues on fruits. The possible threats that jeopardize the effective implementation of IPM and particularly the risks related to the development of the pesticide resistance and the introduction of new invasive pests are also reviewed. With the directive 128/09, the European legislation recognizes IPM as a strategic approach for the sustainable use of pesticides. Within this context, IPM and related guidelines is called to meet different areas of concern in relation to the worker and bystander safety. Beside the traditional economic criteria of the market-oriented agriculture, sustainable agriculture includes the assessment of the environmental impact of the agronomic practices within the societal context where they take place. As a consequence of the raising consumer concerns about environmental impacts generated by the fruit production, IFP certification over product standards, including process aspects, are frequently required by consumers and supermarket chains.

The implementation of sterile insect technique (SIT) has proven effective in the area-wide suppression of several significant agricultural and sanitary pests by employing traditional cobalt-60 (60 Co-γ) as a radiation source. Recently, X-ray has been validated as a feasible alternative to 60 Co-γ radiation sources. Nonetheless, higher doses of X-ray irradiation lead to insect sterility but diminish mating competitiveness, thereby impacting the effectiveness of SIT applications. Thus, it is crucial to ascertain the optimal irradiation dose and develop strategies to enhance the mating competitiveness of sterile insects to enhance SIT efficacy. In this study, we determined the effect 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 sterile males increased proportionally with irradiation dose up to 200 Gy, beyond which it plateaued. Notably, exposure to 200 Gy of irradiation notably decreased the mating competitiveness of male, as evidenced by a mating competitiveness index of 0.17 in laboratory and 0.096 in the orchard. This decline in mating competitiveness is likely linked to the down-regulation of genes associated with the recognition of sex pheromones, specifically CpomOR3a, CpomOR3b, and CpomOR5, following X-ray irradiation. Fumigation of the plant volatile, linalool at varying concentrations (70, 83, and 96 µL/m³) resulted in differential enhancements in male mating competitiveness, with the moderate concentration significantly improving the competitiveness of sterilized males, possibly by restoring their ability to recognize sex pheromones. 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. These 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.

During October and November 2014, a survey was conducted in order to establish the presence of the invasive pest Drosophila suzukii (Matsumura, 1931) on the territory of Serbia. Survey revealed the presence of this fly in four districts (Rasinski, Mačvanski, Raški and Pčinjski), in sampled raspberry and blackberry ripe fruits, as well as in common fig and grape. Collected fruits were used for the rearing of Drosophilid larvae to adults, which were subsequently subjected to morphological and molecular characterization. The presence of D. suzukii was confirmed in all surveyed districts, as well as in Zemun (City of Belgrade). This is the first report of the highly invasive fruit pest D. suzukii on the territory of Serbia.