Lethal, Sublethal, and Offspring Effects of Fluralaner and Dinotefuran on Three Species of Bactrocera Fruit Flies

Simple Summary Bactrocera dorsalis, Bactrocera cucurbitae, and Bactrocera tau are three invasive fruit fly pests that can cause significant economic damage to crops. Chemical agents have become the primary method for improving fruit fly management, leading to the development of insecticide resistance in these three species. Here, we assess the toxicity of a novel insecticide (fluralaner) and a commonly used insecticide (dinotefuran) against these three fruit fly species. Both insecticides exhibit strong lethal and sublethal effects on adult fruit flies and on the sex ratio of their offspring. Hence, fluralaner and dinotefuran can significantly control Bactrocera dorsalis, B. cucurbitae, and B. tau. These findings can help optimize insecticide application and ensure effective management of insecticide resistance. Abstract Fruit flies cause substantial economic damage, and their management relies primarily on chemical insecticides. However, pesticide resistance has been reported in several fruit fly species, the mitigation of which is crucial to enhancing fruit fly control. Here, we assess the toxicity of a novel insecticide (fluralaner) and a common insecticide (dinotefuran) against three fruit fly species, Bactrocera dorsalis (Hendel), Bactrocera cucurbitae (Coquillett), and Bactrocera tau (Walker). Both pesticides exhibit robust lethal and sublethal effects against all three fruit fly species, with fluralaner being more potent. Fluralaner and dinotefuran suppress the reproductive capacities and survival rates of fruit flies. However, at the 50% lethal concentration, fluralaner stimulates the reproductive capacity of B. dorsalis and the survival rate of B. tau. Fluralaner also causes significant transgenerational effects, impacting the offspring hatching rate of B. cucurbitae and B. tau and reducing the proportion of female offspring. Thus, both pesticides exhibit high potential for controlling fruit flies. However, their application should be tailored according to species variations and the diverse effects they may induce. Collectively, the findings of this study outline the sublethal effects of two insecticides against fruit flies, helping to optimize their application to ensure the effective management of insecticide resistance.


Introduction
Bactrocera dorsalis (Hendel), Bactrocera cucurbitae (Coquillett), and Bactrocera tau (Walker) are important invasive fruit fly pests of international concern that can cause significant economic crop damage [1][2][3][4][5].In China, these three species are widely distributed in regions like Taiwan, Fujian, and Guangdong, causing annual outbreaks and proving difficult to control.In fact, they have exhibited trends of a gradual spread northward [6][7][8].Ongoing research and scientific advancements have yielded diverse methods for the management of fruit flies, including RNA interference pesticides, insect sterilization techniques, and various trapping methods.However, chemicals remain the most commonly used modality of control [9,10].Fluralaner is a representative isoxazoline broad-spectrum insecticide that inhibits the γ-aminobutyric acid-gated chloride channels of pests, representing a novel mode of action with no significant cross-resistance to other insecticides [11][12][13].Fluralaner exhibits excellent insecticidal activity against over 30 insect species [14] while proving safe for non-target organisms (e.g., mammals, zebrafish, and poultry), suggesting broad application prospects [15][16][17].Meanwhile, dinotefuran is a third-generation neonicotinoid insecticide that exerts insecticidal effects primarily by acting on nicotinic acetylcholine receptors in insects [18][19][20].Although the use of neonicotinoid insecticides in agricultural and domestic applications is rapidly growing [21], their application in managing fruit flies has not been adequately investigated.
In practical applications, insecticides may directly induce lethal and sublethal effects.The latter can influence the behavior, physiological activities, or survival capabilities of insects over a particular period [22,23].For instance, flupyradifurone-a butanolide insecticide that targets the central nervous system of insects in a manner similar to neonicotinoidprolongs the development of Binodoxys communis Gahan while reducing its longevity [24,25].Additionally, the sublethal effects of the neonicotinoid insecticide thiacloprid may affect the learning and memory behaviors of Apis mellifera L. [26].Meanwhile, lethal and sublethal concentrations of cyantraniliprole (bisamide) affect the mating performance of B. dorsalis adults [27], whereas broflanilide-a meta-diamide insecticide-at its lethal concentration 50 (LC 50 ) affects the hatchability of B. cucurbitae and B. tau [3].
This study tests the hypothesis that fluralaner and dinotefuran exhibit insecticidal activity against B. dorsalis, B. cucurbitae, and B. tau.More specifically, their lethal and sublethal effects on fruit flies are evaluated, as well as their transgenerational effects.The results of this study provide a theoretical foundation for the future application of fluralaner and dinotefuran in controlling fruit flies in the field.

Determining the Lethal Concentrations of Fluralaner and Dinotefuran
The lethality of fluralaner and dinotefuran was determined using the residual contact method [28].The chemicals were prepared in acetone as 100 mg/L stock solutions.Fluralaner and dinotefuran stock solutions were diluted to 5-6 different concentrations with acetone; 5 mL of each diluted solution was added to a separate 250 mL clean conical flask, which was gently rotated so that the solution evenly coated the inside of the flask until the acetone evaporated.Twenty adult flies (1:1 sex ratio) (3-5 days after emergence) were placed in a prepared conical flask, which was sealed with gauze that had cotton dipped in 10% honey water placed on top (three replicates per concentration).The mortality rate was observed after 24 h.The adults were gently turned over with a brush and considered dead if they could not turn back over within 30 s.If the mortality rate in the control group (acetone only) was <10%, the experiments were considered valid, and the adjusted mortality was corrected using Abbott's formula [30]; if the mortality rate in the control group exceeded 10%, the experiment was considered invalid and repeated.

Bioassays
The lethal and sublethal effects of fluralaner and dinotefuran were studied under four separate conditions: treatment with either insecticide at the 15% lethal concentration (LC 15 ), LC 30 , LC 50 , and a vehicle control with acetone.After exposure to different treatments for 24 h, we selected 20 pairs of healthy adult flies (5 days after emergence; F0 generation) and transferred them to breeding cages for rearing (n = 3 replicates/treatment).Please refer to Section 2.2 for treatment details.Subsequently, adult mortality and egg laying were observed daily, starting on the second day (6 days after emergence).After the adults began laying eggs, eggs were collected every 3 days for 5 h until all the B. dorsalis flies died (eggs were collected 50 times each for B. cucurbitae and B. tau).Due to differences in the oviposition habits between the fruit fly species, oviposition cups with orange juice were used to collect B. dorsalis or eggs, and with pumpkin flesh to collect B. cucurbitae and B. tau eggs; simultaneously, the oviposition quantity of each species was recorded.Additionally, the egg-hatching rate, female fecundity, survival rate (male and female), and F1 sex ratio (proportion of females = Σ♀ ÷ Σ (♀ + ♂)) were observed after each treatment to assess the sublethal effects of both agents on all three species of fruit flies [3].Population parameters were calculated according to the protocol described by Zhang et al. [31].

Data Analysis
Probabilistic regression analyses were performed using SPSS version 22.0 (SPSS, Inc., IBM, Armonk, NY, USA) to calculate the LC 50 , LC 30 , and LC 15 values of both pesticides against fruit flies and the corresponding 95% confidence limits (CLs).The SAS software (version 9.4) was used for all other data analyses.Significance analyses of the experimental results were performed using Duncan's multiple-range test (DMRT) or the t-test (with validation of normal distribution before data analysis).We set p < 0.05 as the threshold for statistical significance.The data generated in this study were subjected to analysis of variance (ANOVA) followed by Tukey's or Friedman's post hoc tests.

Toxicity of Fluralaner and Dinotefuran in Laboratory Settings
The bioassays demonstrated high virulence levels for fluralaner and dinotefuran in the adults of all three fruit fly species.Notably, the LC 50 of fluralaner was consistently lower than that of dinotefuran in all the species (Table 1).The LC 50 , LC 30 , and LC 15 values of both pesticides against the various fruit fly species were calculated for the subsequent experiments.

Sublethal Effects on Survival and Reproduction
Bactrocera dorsalis survival decreased significantly after exposure to fluralaner or dinotefuran.However, the survival of the males was significantly lower than that of the females following fluralaner treatment (LC 30 and LC 50 ), whereas no difference was observed between the males and females treated with dinotefuran (Table 2).Almost half of both the male and female B. dorsalis flies died on day 4 (emergence at day 5 was considered the first day of treatment) of fluralaner treatment (9 days after emergence), whereas dinotefuran (LC 30 and LC 50 ) showed a sharp decline in survival after 16 days of treatment (21 days after emergence; Figures 1 and 2).

Sublethal Effects on Survival and Reproduction
Bactrocera dorsalis survival decreased significantly after exposure to fluralaner or di notefuran.However, the survival of the males was significantly lower than that of th females following fluralaner treatment (LC30 and LC50), whereas no difference was ob served between the males and females treated with dinotefuran (Table 2).Almost half o both the male and female B. dorsalis flies died on day 4 (emergence at day 5 was considered the first day of treatment) of fluralaner treatment (9 days after emergence), whereas di notefuran (LC30 and LC50) showed a sharp decline in survival after 16 days of treatmen (21 days after emergence; Figures 1 and 2).The treatment with fluralaner significantly reduced the survival rate of male and female B. cucurbitae, with a more pronounced effect observed in the females compared to the males (Table 2).With dinotefuran treatment, the survival rates of female and male B. cucurbitae flies did not differ from those of the control at LC30, although all other doses resulted in significantly lower survival than that of the control group.Unlike fluralaner treatment, the survival rates of both sexes under dinotefuran treatment were only The treatment with fluralaner significantly reduced the survival rate of male and female B. cucurbitae, with a more pronounced effect observed in the females compared to the males (Table 2).With dinotefuran treatment, the survival rates of female and male B. cucurbitae flies did not differ from those of the control at LC 30 , although all other doses resulted in significantly lower survival than that of the control group.Unlike fluralaner treatment, the survival rates of both sexes under dinotefuran treatment were only signifi-Insects 2024, 15, 440 6 of 12 cantly lower than that of the males at LC 15 (Table 2).Male and female B. cucurbitae survival rates declined sharply after 22 days (27 days after emergence) of fluralaner treatment but declined relatively slowly after dinotefuran treatment.Nevertheless, male survival fell below 50% by the following day (6 days post-emergence) after dinotefuran treatment at LC 50 (Figures 1 and 2).In contrast to B. dorsalis and B. cucurbitae, the male and female survival rates of B. tau were significantly higher in the fluralaner-treated groups than in the control group, with the females exhibiting significantly lower longevity than the males (LC 15 and LC 30 ).Meanwhile, dinotefuran treatment only reduced male and female survival significantly under LC 50 treatment; males and females did not exhibit survival differences under any of the treatment doses (Table 2).The survival of both male and female B. tau exhibited a sharp decline on the second day (6 days post-emergence) of fluralaner or dinotefuran treatment; subsequently, their survival rates declined slowly (Figures 1 and 2).
Neither pesticide altered the fecundity of B. cucurbitae or B. tau.However, both significantly altered the fecundity of B. dorsalis at their respective LC 30 and LC 50 values.Specifically, treating B. dorsalis with fluralaner at LC 50 resulted in higher fecundity than that observed in the control group, whereas the remaining treatments resulted in significantly lower fecundity levels (Table 2).

Sublethal Effects on Offspring Traits
The effects of both pesticides on the hatchability of B. dorsalis eggs were negligible.For B. cucurbitae, only the LC 50 dose of fluralaner significantly reduced egg hatchability, whereas for B. tau, both pesticides reduced egg hatchability (Table 2).
No differences were observed in the net reproductive rates (R 0 ) of B. cucurbitae or B. tau between either of the insecticides and the control treatment.However, treating B. dorsalis with fluralaner at LC 15 , LC 30 , and LC 50 reduced the R 0 value, which was two-fold higher than the control treatment.In contrast, R 0 exhibited a four-fold reduction after dinotefuran treatment at LC 30 (Table 3).No significant differences were observed among any of the fruit flies, except with the dinotefuran treatment at LC 50 , which slightly reduced the interval between each B. tau generation.Only B. dorsalis showed significantly lower intrinsic and finite rates of increase upon fluralaner treatment (at LC 30 ).In contrast, in B. cucurbitae and B. tau, these values did not differ significantly compared to the control group at any dose (Table 3).Abbreviations: λ, finite rate of increase; Din, dinotefuran; Flu, fluralaner; R 0 , net reproductive rate; r m , intrinsic rate of increase; SEM, standard error of the mean; T, interval between each generation.Data (means ± SEM) followed by the same lowercase letters (superscripts) are not significantly different (within each single species) according to Duncan's multiple-range test (DMRT) at the p = 0.05 level.
Fluralaner and dinotefuran also affected the sex ratio of the offspring (Figure 3).Both pesticides decreased the proportion of female B. dorsalis progeny.This was also induced by dinotefuran treatment in B. tau and fluralaner treatment at the LC 50 dose.In contrast, dinotefuran treatment did not alter the sex ratio of B. cucurbitae offspring, and only fluralaner at LC 15 significantly reduced the proportion of female progeny.

Discussion
The management of fruit flies depends predominantly on the use of chemical insecticides.However, some fruit fly species, including B. dorsalis [32,33], B. cucurbitae [34], and B. tau [35,36] have developed resistance to several chemical agents.Hence, it is advisable to rotate the use of insecticides for effective insecticide resistance management (IRM).Fluralaner has recently emerged as a new agent with considerable effectiveness in treating ectoparasites [37].Indeed, fluralaner exhibits high toxicity toward diverse agricultural insects [14].Meanwhile, although dinotefuran is highly effective against various insects, it is rarely used for fruit fly control.Therefore, our research provides essential theoretical groundwork for the future use of fluralaner and dinotefuran in controlling fruit flies, as well as for improving IRM.
Our findings revealed that both fluralaner and dinotefuran were highly toxic to all three fruit fly species, with fluralaner being more effective.This phenomenon was also observed in Plutella xylostella (L.) larvae, with the LC 50 of fluralaner being 0.02 mg/L, and that of dinotefuran exceeding 100 mg/L (feeding on a dipped leaf) [14,38].The high toxicity of fluralaner has also been reported in Sphodroxia maroccana Ley [39] and Spodoptera frugiperda (J.E.Smith) [14], suggesting that fluralaner holds promise for diverse applications.Dinotefuran is widely used on a global scale; however, the associated resistance is steadily increasing in various insects, including Nilaparvata lugens (Stål) [40] and Sogatella furcifera (Horváth) [41].Nevertheless, dinotefuran, which has not been extensively utilized for fruit fly control, may find potential application in the field through rotational strategies in the future.
The lethal or sublethal effects of insecticides [42] are achieved through their chemical components interfering with the insect's physiological processes [42][43][44].In this study, lethal and sublethal doses of both agents did not alter the fecundity of B. cucurbitae or B. tau.However, that of B. dorsalis significantly decreased under treatment with dinotefuran (LC 30 and LC 50 ) and fluralaner (LC 30 ).Notably, treatment with fluralaner at LC 50 significantly increased the reproductive capacity from 367.65 to 429.59 eggs/female.Indeed, the sublethal effects of insecticides can reportedly enhance or reduce the reproductive capacity of insects.For example, sublethal doses of sulfoxaflor decrease the reproductive capacity of Coccinella septempunctata L. [45], whereas B. dorsalis, Bactrocera correcta (Bezzi), and B. cucurbitae exhibit elevated fecundity after treatment with broflanilide [3].Moreover, the sublethal effects of fluralaner and dinotefuran positively and negatively impacted the survival rates of males and females across all three fruit fly species, respectively.
Most sublethal effects tend to negatively impact the survival rates of insects, such as Binodosys communis [24,25].In this study, we observed that the survival rates of the male and female B. tau insects increased after treatment with fluralaner (LC 15 and LC 30 ).Correspondingly, the hatching rate of the offspring decreased.Although the survival rate of the female B. tau insects (LC 30 ) was also higher than that of the control group under dinotefuran treatment, no difference was observed between the males and the controls, resulting in no difference in the hatching rates.Collectively, these observations indicate that insects incur a fitness cost at sublethal insecticide concentrations [46,47].This can cause insects to adopt complex survival strategies when facing chemical pressures in their environment, balancing survival, reproductive capacity, and the ability of offspring to hatch.
Insecticides can have transgenerational effects, affecting offspring hatchability and sex ratios.Thiodicarb significantly reduces the proportion of female Trichogramma pretiosum Riley offspring (F1) [48], while chlorantraniliprole impacts the hatchability of Agrotis ipsilon (Hufnagel) and A. segetum (Denis and Schiffermuller) [49].In this study, the proportion of females in the offspring (F1) of all three fruit fly species decreased to varying degrees, and the hatching rate of B. tau was reduced.This was likely due to a decrease in the quality of eggs after exposure to pesticides, which can affect hatching rates or elicit specific effects on the reproductive cells or developmental stages, influencing sex ratios.These transgenerational effects may involve genetic and non-genetic mechanisms [50,51].In some cases, pesticide exposure may induce genetic mutations that affect the offspring genome [52].In other instances, these effects may result from epigenetic changes induced by environmental pressures, which can be transmitted to subsequent generations [53,54].In our comparison, we found that the differences among the population parameters were not significant.Understanding the transgenerational effects of pesticides is crucial for comprehensively assessing their ecological risks and environmental impacts.

Conclusions
In this study, we investigated the sensitivity of three fruit fly species to a novel pesticide (fluralaner) and the widely used dinotefuran.Both pesticides exhibited strong toxicity against fruit flies, suggesting their potential for future rotational strategies in field applications.However, the study was confined to laboratory conditions, and specific field applications should consider sublethal effects, as well as the safety of the chemicals to non-target organisms and their environmental impact.In summary, this study on sublethal effects contributes to a more comprehensive understanding of the impact of insecticides on individual insects and populations, as well as their long-term effects on ecosystems.Our findings provide reliable data for the field application of fluralaner and dinotefuran in fruit fly control, as well as for improving IRM strategies.Funding: Yixiang Qi: the National Key R&D Project of China (2021YFC2600404), Yongyue Lu.: the Guangdong Modern Agriculture Innovation Team Project (2023KJ134), and the Guangzhou Key R&D Project (2023B04J0154).

Figure 1 .
Figure 1.Effects of fluralaner and dinotefuran treatments on the survival of female Bactrocera fru flies.

Figure 1 .
Figure 1.Effects of fluralaner and dinotefuran treatments on the survival of female Bactrocera fruit flies.

Figure 2 .
Figure 2. Effects of fluralaner and dinotefuran treatments on the survival of male Bactrocera fruit flies.

Figure 2 .
Figure 2. Effects of fluralaner and dinotefuran treatments on the survival of male Bactrocera fruit flies.
Author Contributions: Y.L. and X.L. designed the research; D.L. performed the research; X.L., D.L. and X.C. contributed reagents/materials/analyses; X.L. wrote the paper; Y.L. and Y.Q.provided funding support.All authors have read and agreed to the published version of the manuscript.

Table 1 .
Lethal activity of fluralaner and dinotefuran against three adult Bactrocera fruit fly species.

Table 3 .
Population parameters of Bactrocera fruit flies treated with sublethal concentrations of dinotefuran and fluralaner.