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Development and validation of a larval bioassay and selection protocol for insecticide resistance in Drosophila suzukii.

ABSTRACT: The rapid invasion of Drosophila suzukii (Matsumura) throughout Europe and the Americas has led to an increased reliance on calendar-based broad-spectrum insecticide programs among berry and cherry growers. Relatively few active ingredients (AIs) are currently available for effective D. suzukii management, and studies from multiple growing regions indicate that susceptibility to at least some of these materials is declining. Greater effort is needed to understand the status of susceptibility across field populations and the potential for increased resistance to develop, as well as the possible fitness costs incurred by resistant individuals. However, current bioassay protocols used for resistance monitoring and selection studies (i.e. resistance risk assessments) are labor-intensive and costly, making large-scale studies difficult to conduct. Here, we first present a novel bioassay protocol using larvae that requires little effort or cost to implement beyond what is needed for basic D. suzukii laboratory colony maintenance. We then perform dose-response bioassays using this protocol to identify larval lethal concentrations for three commonly used insecticides (malathion, spinosad and zeta-cypermethrin) in a susceptible population. Finally, resistance risk assessments were conducted using a population of D. suzukii from commercial caneberry fields near Watsonville, CA. We find that five generations of larval selection with a discriminating dose is sufficient to significantly increase both larval (malathion and spinosad) and adult (spinosad) resistance to the target AIs. This approach provides a simple, cost-effective tool for assaying susceptibility of D. suzukii populations to insecticides and for selecting resistant insect lines for resistance management research.

SUBMITTER: Gress BE

PROVIDER: S-EPMC9242520 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Microorganisms play a central role in the biology of vinegar flies such as Drosophila suzukii and Drosophila melanogaster: serving as a food source to both adults and larvae, and influencing a range of traits including nutrition, behavior, and development. The niches utilized by the fly species partially overlap, as do the microbiota that sustain them, and interactions among these players may drive the development of crop diseases. To learn more about how the microbiota of one species may affect the other, we isolated and identified microbes from field-caught D. suzukii, and then characterized their effects on D. melanogaster larval development time in the laboratory. We found that the D. suzukii microbiota consistently included both yeasts and bacteria. It was dominated by yeasts of the genus Hanseniaspora, and bacteria from the families Acetobacteraceae and Enterobacteriaceae. Raising D. melanogaster under gnotobiotic conditions with each microbial isolate individually, we found that some bacteria promoted larval development relative to axenic conditions, but most did not have a significant effect. In contrast, nearly all the yeasts tested significantly accelerated larval development. The one exception was Starmerella bacillaris, which had the opposite effect: significantly slowing larval developmental rate. We investigated the basis for this effect by examining whether S. bacillaris cells could sustain larval growth, and measuring the survival of S. bacillaris and other yeasts in the larval gut. Our results suggest S. bacillaris is not digested by D. melanogaster and therefore cannot serve as a source of nutrition. These findings have interesting implications for possible interactions between the two Drosophilia species and their microbiota in nature. Overall, we found that microbes isolated from D. suzukii promote D. melanogaster larval development, which is consistent with the model that infestation of fruit by D. suzukii can open up habitat for D. melanogaster. We propose that the microbiome is an important dimension of the ecological interactions between Drosophila species.

Project description:The goal of this study is to proactively assess the risk of insecticide resistance development by determining susceptibility of field-collected D. suzukii and their response to insecticide treatment at the transcriptome level using next-generation sequencing technology. Methods:LC50 values were calculated for zeta-cypermethrin, spinosad, malathion treated D. suzukii field-collected and lab-reared populations. LC50 dosage survived and untreated (10 individuals per replicate) field-collected and lab reared D.suzukii transcript profiles were generated by RNA sequencing, in triplicate, using Illumina NextSeq 500. The 24 samples (paired-end reads, including replicates) were independently mapped onto the D. suzukii genome (SpottedWingFlybase v.1) by using TopHat followed by Cufflinks to estimate the expression values of the transcripts in FPKM (Fragments Per Kilobase per Million mapped reads) with the Cuffdiff 2 default geometric normalization. Differentially expressed genes (FDR < 0.05 after Benjamini-Hochberg correction for multiple-testing) were identified for insecticide-treated or untreated control for either (1) lab-reared populations or (2) field-collected populations. Results: As an approach to proactively assess the risk of insecticide resistance development, we determined the LC50 (lethal concentration, 50%) values of commonly used insecticides zeta-cypermethrin, spinosad, and malathion against laboratory-reared and field-collected D. suzukii populations. The LC50 values were significantly higher in field-collected populations when compared to lab-reared populations, indicating that field populations are less susceptible to these insecticides. Furthermore, we used RNA sequencing to analyze the response of D. suzukii at the transcriptome level upon treatment with the same three insecticide classes that are used in our bioassays. We identified differentially expressed genes (DEGs) in D. suzukii that survived LD50 doses of zeta-cypermethrin, spinosad, and malathion and gene classes that are overrepresented in DEGs. We observed that a high number of significantly DEGs are involved in detoxification and reduced cuticular penetration, especially in field population, thus providing potential molecular mechanisms for the higher LC50 values for field-collected population. Conclusion: Our study identified a high number of metabolic detoxification genes that are induced in field-collected D. suzukii upon insecticide treatment and a high degree of overlap when examining the lists of genes that are induced when D. suzukii is treated with three commonly used insecticides. Based on our results, we conclude that there is a substantial risk of insecticide resistance and cross-resistance development in D. suzukii in the field.

Project description:Drosophila suzukii Matsumura is an invasive species of vinegar fly that has become a prominent pest of berries and other soft-skinned fruits. Unlike most other Drosophila species, female D. suzukii flies lay their eggs in ripening and ripe fruits and larvae develop within the fruit. To understand how D. suzukii larvae utilize ripe and ripening fruits, which usually have low levels of protein, we investigated the microbiota of field-captured and laboratory-reared D. suzukii flies and further examined the combined influence of diet and microbes on host fitness. Field-captured flies were associated with diverse microbiota, which varied significantly with sampling location and season. In contrast, laboratory-reared flies possessed strikingly lower bacterial abundance and diversity. A comparison of conventionally reared (CR) and germ-free (GF) flies revealed that the microbiota of D. suzukii does not alter its development significantly but decreases its life span under conditions of a nutrient-sufficient diet. However, the microbiota is essential for D. suzukii development on strawberry-based or blueberry-based fruit diets. This developmental failure could be rescued by reassociation with single bacterial or fungal species or by the addition of a high quantity of heat-killed microbes. In addition, we found that proteins are limiting with respect to fly development on fruit-based diets and that GF flies show signs of protein starvation. Taken together, our study results demonstrate that the microbiota provides key proteins required for the development of D. suzukii reared on fresh fruit. Our work shows that the impact of microbes on fly fitness depends strongly on nutritional conditions.IMPORTANCE Animals are commonly associated with specific microbes, which play important roles in host development and fitness. However, little information about the function of microbes has been available for the important invasive pest Drosophila suzukii, also known as Spotted wing drosophila. Our study results demonstrate that the abundance and structure of microbiota in D. suzukii are strongly affected by the environment, where microbes have variable roles depending on the nutritional situation. For instance, we found that the presence of microbes is deleterious for flies growing on a protein-rich diet and yet is beneficial for flies growing on a diet of protein-poor fruits. Additionally, germ-free flies must feed on microbes to obtain the necessary protein for larval development on strawberries and blueberries. Our report validates the complexity seen in host-microbe interactions and may provide information useful for D. suzukii pest control.

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Development and validation of a larval bioassay and selection protocol for insecticide resistance in Drosophila suzukii.

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