Key Message

  • The effect of insect growth regulators on offspring production by D. suzukii was evaluated.

  • Lufenuron, cyromazine and pyriproxyfen completely suppressed the production of viable offspring.

  • The effects were not permanent and required a continuous exposure to be maintained.

  • A vertical transmission of lufenuron and cyromazine from females to their offspring was observed.

  • Lufenuron, cyromazine and pyriproxyfen could be useful for the control of D. suzukii.

Introduction

Drosophila suzukii (Matsumura) (Diptera: Drosophilidae) has led to an increasing concern among the producers of stone and small fruits due to the great damages that this insect can produce on these crops. This fly is endemic to Southeast Asia, but since 2008 this invasive species has experienced a rapid expansion to new areas so now it is widely present in Europe and North America (Asplen et al. 2015) and it has also been detected in South America (Deprá et al. 2014; Andreazza et al. 2017a) and Africa (Boughdad et al. 2021; Kwadha et al. 2021). This colonization has been mediated to a great extent by human activity, in particular the international movement of infested fruits (Cini et al. 2014; Haye et al. 2016). The wide variety of hosts in which D. suzukii is able to develop (Walsh et al. 2011; Poyet et al. 2015; Kenis et al. 2016), the short generation times and high reproductive potential of this fly (Sánchez-Ramos et al. 2019a, 2019b), its great plasticity and adaptability to the environmental conditions found in the new zones (Little et al. 2020) and the absence of its original natural enemies in the new colonization areas (Daane et al. 2016; Haye et al. 2016) has favoured this rapid expansion and the increase in its populations.

Drosophila suzukii females have a serrated ovipositor which enables them to lay eggs in healthy ripening stone and small fruits, such as strawberries, cherries, blueberries, raspberries, blackberries, apricots, figs, plums and grapes (Lee et al. 2011; Walsh et al. 2011). The larvae feed on the fruit pulp what results in reduced production and high economic losses (Walsh et al. 2011; Wiman et al. 2016). Moreover, larvae are able to feed on a great variety of fruits of wild plants (Lee et al. 2015; Poyet et al. 2015; Kenis et al. 2016). This allows the maintenance of the populations of this fly when no fruit crops are present in the fields and facilitates reinfestation.

Many strategies have been investigated and developed to be implemented in integrated pest management programmes for the control of this pest (Haye et al. 2016; García, 2020; Tait et al. 2021). However, despite this wide range of possible control methods, the main control strategies rely on insecticide applications. The most common products used against this pest are organophosphates, pyrethroids, spinosyns and carbamates (Haye et al. 2016; Van Timmeren et al. 2018). Concern about the health and environmental risks associated with the use of these pesticides is driving the need for products with better ecotoxicological profiles.

Insect growth regulators (IGRs) are compounds that adversely interfere with the normal growth and reproduction of insects (Smagghe et al. 2019). These substances have a range of modes of action, for example chitin-synthesis inhibitors, juvenile hormone mimics, ecdysone agonists or moulting disruptors (Pener and Dhadialla 2012; Smagghe et al. 2019; IRAC 2022). They are considered safer than conventional products for beneficial organisms and they have low toxicity to vertebrates (Dhadialla et al. 1998, 2005; Pener and Dhadialla 2012). Laboratory studies have shown that, when ingested, these products produce sterilizing effects on adult insects and negative effects on their fecundity, including several species of Diptera (Moya et al. 2010; Sánchez-Ramos et al. 2013; Bensebaa et al. 2015; Chang 2017). Although their efficacy against D. suzukii has not been widely tested, promising results have been reported for lufenuron, a chitin-synthesis inhibitor (Sampson et al. 2017a, 2017b). Accordingly, IGRs might be an interesting alternative for the control of D. suzukii applied as bait treatments, similar to other insecticides (Andreazza et al. 2017b; Noble et al. 2023).

The aim of this work was to evaluate the effect of several IGRs with different modes of action (the chitin-synthesis inhibitor lufenuron; the moulting disruptor cyromazine; the juvenile hormone mimic pyriproxyfen; azadirachtin, which can inhibit reproduction and produce sterility in insects; and the ecdysone agonist tebufenozide) on fertility, fecundity and adult offspring production of D. suzukii when fed on a treated artificial diet.

Materials and methods

Mass rearing of D. suzukii

A D. suzukii population was established from individuals collected in San Pol de Mar, Maresme, Barcelona (Spain). Rearing was conducted in an environmental chamber (IBERCEX, Spain) at 19 °C, 70% RH and a 16:08 h light/dark photoperiod. Artificial diet and rearing procedures were the same that those described in Sánchez-Ramos et al. (2019a).

Chemicals

The following commercial formulations were used: Match 5® [5% w:v (emulsifiable concentrate) lufenuron] (Syngenta Agro SA, Madrid, Spain); MFRC (maximum field recommended concentration): 200 mL hL−1 (100 μg AI mL−1). Trigard® [75% w:w (wettable powder) cyromazine] (Syngenta Agro SA, Madrid, Spain); MFRC: 40 g hL−1 (300 μg AI mL−1). Juvinal® [10% w:v (emulsifiable concentrate) pyriproxyfen] (Kenogard SA, Barcelona, Spain); MFRC: 75 mL hL−1 (75 μg AI mL−1). Align® [3.2% w:v (emulsifiable concentrate) azadirachtin] (Sipcam Inagra SA, Valencia, Spain); MFRC: 150 mL hL−1 (48 μg AI mL−1). Mimic 2F® [24% w:v (suspension concentrate) tebufenozide] (Certis Europe BV, Elche, Spain); MFRC: 75 mL hL−1 (180 μg AI mL−1). Insecticide preparations were made up in distilled water, which was used as control.

Effect of IGRs on the fertility and offspring production of D. suzukii

Assays were carried out in rearing units consisting of plastic tubes (15 cm high × 5.5 cm diameter) placed vertically in the upper half of a 5.8 cm diameter Petri dish with the upper opening covered by a piece of translucent nylon cloth held in place by a rubber band, which provided ventilation for the rearing unit (Sánchez-Ramos et al. 2019a). A round box (4 cm diameter × 2 cm height) filled with artificial diet (1.5 cm height) was introduced without cover within each rearing unit. The diet served as food source for both larvae and adults and as oviposition substrate.

Two experiments with different periods of exposure to the IGRs were performed. For the assays, pairs of < 16 h old adult flies were used. In the first assay, pairs were fed for 3 days after emergence with treated diet and then transferred to rearing units with untreated diet for 16 days. In the second assay, pairs were fed continuously after emergence with the treated diet for 19 days. The products were administered at the MFRC by treating the surface of the artificial diet employed as oviposition substrate by means of a computer controlled sprayer (Burkard Manufacturing Co Ltd, England). The amount of solution applied on the diet was 115 μl which was equivalent to the maximum volume of the product solution applicable in the field (1000 L/ha). Diets sprayed with the same amount of distilled water were used as control treatment. Ten replicates were used per product. Experiments were performed at 22 °C, 90% RH and a 16:08 h light:dark photoperiod in environmental test chambers (MLR-352H, Panasonic, Japan). A stock of males treated under the same conditions for each experiment was kept to replace any which died before the females.

After the first 3-day exposure, diets containing the eggs laid were replaced every 2 days by new fresh diets (treated or untreated, depending on the experiment). Also, in the continuous feeding experiment, the surviving pairs corresponding to those products that showed the highest effect were transferred to fresh untreated diet for 11 more days after the 19-day exposure period to test if they were able to recover the ability to produce viable offspring. In all cases, round boxes containing the diet and the eggs laid were transferred and fixed with double-sided sticky tape to the base of transparent disposable plastic boxes (250 mL capacity) where the development from egg to adult occurred at 25°C, 70% RH and a 16:08 h light:dark photoperiod in a walk-in environmental chamber (IBERCEX, Spain). After 12 days, the boxes with the diets were frozen to count the pupae and adults produced. The percentage of fertile mating and the total number of pupae and adults produced were established for each treatment.

Choice assays

Choice experiments were performed to establish the possible repellent effects on oviposition of those products that showed the greatest effect in the previous assays. For these assays, ten pairs < 16 h old per product were previously exposed for 6 days to treated diet in the same rearing units employed in the previous assays. Each product was administered at the MFRC by treating the surface of the artificial diet following the procedures described before. Daily, the diet was removed, the eggs counted under a stereomicroscope to estimate fecundity and a new fresh diet provided to each pair. The diets containing the eggs were transferred to the same transparent disposable plastic boxes described above to check for pupation and adult emergence. After the 6-day period, pairs were transferred to the same plastic cages used for the mass rearing (Sánchez-Ramos et al. 2019a), containing two round boxes as described above filled with artificial diet (1.5 cm height). One of the boxes was treated with the product to be evaluated and the other was treated with the same quantity of distilled water acting as a control. A double-control with both diets treated with distilled water was also established for comparative purposes. Pairs were maintained for two days in these cages and each day, new fresh diet was provided. The number of eggs laid daily in each substrate was counted. Then, the diets with the eggs were transferred individually to disposable plastic boxes to check for pupation and adult emergence. The number of eggs laid during the two-day oviposition period and the pupae and adults obtained from them were used to calculate an index of oviposition deterrence and indices of reduction in pupation and adult offspring production according to the following equation (Blaney et al. 1987):

$$I = 100 \times \frac{C - T}{{C + T}}$$

where I is the index, C is the value of the corresponding parameter in the control treatment and T is the value of the corresponding parameter in the treatment. The index for each parameter was also established for the control replicates for comparative purposes, since its value should not be statistically different from zero.

As above, oviposition experiments were performed at 22 °C, 90% RH and a 16:08 h light:dark photoperiod in environmental test chambers and development from egg to adult occurred at 25 °C, 70% RH and a 16:08 h light:dark photoperiod in a walk-in environmental chamber.

Egg-transfer assays

Two additional experiments were performed to determine if the observed effects were due to direct feeding by emerged larvae on the treated diet and interference on their development or to alterations of the reproduction because of the adult feeding on treated diet. In the first one, eggs laid by flies that had been fed on untreated diet were transferred to treated diet. In the second one, eggs laid by flies that had been fed on treated diet were transferred to untreated diet. In these experiments, eggs < 2 h laid at 22 °C, 90% RH and a 16:08 h light:dark photoperiod were used. To obtain the eggs, for the first type of experiments, flies on undetermined age were introduced in rearing cages with new artificial untreated diet and allowed to lay eggs for 2 h. For the second one, flies were fed for 3 days after emergence with treated diet and then they were transferred to rearing units with new treated diet for 2 h. Individual eggs laid in the cages were collected by scooping a small amount of diet around the egg with a laboratory spatula. Afterwards, the eggs were completely extracted from the diet with the help of a moistened fine camel hair brush and transferred to the surface of the diet contained in round boxes (4 cm diameter × 2 cm height) filled with artificial diet (1.5 cm height) that had been treated or untreated depending on the experiment. For both type of experiments, eggs laid on untreated diet were transferred to new fresh untreated diet and were employed as controls. Ten replicates per product were used and ten eggs were deposited in each round box, which was introduced into a transparent disposable plastic box. Development from egg to adult occurred within these boxes at 25°C, 70% RH and a 16:08 h light:dark photoperiod in a walk-in environmental chamber. During the first three days, the boxes were observed for egg hatching and after 12 days the boxes with the diets were frozen to count the pupae and adults produced in both types of experiments.

Statistical analysis

The effects of the products tested on the different parameters considered were compared with the control mainly by means of parametric tests (Student t-tests for two samples and one-way ANOVA followed by Dunnett two-tailed test for more than two samples). When necessary, data were previously transformed by ln(x + 1) to meet the assumptions of parametric statistics. However, if any of these assumptions were violated after appropriate transformations, the effect of the treatments was analysed by means of nonparametric tests (Mann–Whitney U-tests for two samples and Kruskal–Wallis test followed by Dunn test for more than two samples). In some experiments, some data did not meet the assumptions for performing parametric statistics while others did. In those cases, data were compared separately with the control using both nonparametric and parametric tests, since these latter tests have more statistical power to detect significant effects than their nonparametric counterparts. When offspring production was compared between treated and untreated diets within the same cages in the choice assays, paired samples t-tests for normal data and paired samples Wilcoxon signed rank tests for non-normal data were employed. The level of significance was P < 0.05 in all cases. Analyses were performed using Statgraphics® Centurion XV, except the Dunn test that was done according to the procedure described in Gibbons (1985).

Results

When the IGRs were administered to adults by feeding on treated diet for three days or continuously, all the pairs were able to lay eggs for all treatments.

In the 3-day treatment assay, no significant differences were observed in pupation or adult production for any of the compounds tested when the whole duration of the experiment was considered (F5, 54 = 0.70, P = 0.6281, ANOVA test for pupation; F5, 54 = 0.96, P = 0.4491, ANOVA test for adult emergence) (Table 1). However, no emergence of adults was observed in the diets corresponding to the 1st to 3rd oviposition days for lufenuron, cyromazine and pyriproxyfen (H = 33.25, P < 0.0001, Kruskal–Wallis test followed by Dunn test) whereas there was no significant effect of azadirachtin and tebufenozide (F2, 27 = 2.31, P = 0.1184, ANOVA test) (Table 2). In the 4th to 5th and 6th to 7th oviposition days, lufenuron was the only compound that conserved some effect in reducing adult production (F5, 54 = 23.21, P < 0.0001 for 4th to 5th oviposition days; F5, 54 = 3.44, P < 0.01 for 6th to 7th oviposition days, ANOVA test followed by Dunnett test). By the 8th to 9th oviposition days, none of the compounds maintained any effect (F5, 54 = 0.73, P = 0.6007, ANOVA test).

Table 1 Offspring production for 19 days by Drosophila suzukii pairs initially fed for three days or fed continuously on artificial diets treated on surface with different insect growth regulators
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Table 2 Offspring production for the first 9 oviposition days by Drosophila suzukii pairs initially fed for three days on artificial diets treated on surface with different insect growth regulators
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A continuous feeding on treated diet produced a significant reduction in the number of pupae and adult offspring produced in all cases (Pupation: H = 27.49, P < 0.0001, Kruskal–Wallis test followed by Dunn test for lufenuron and cyromazine; F3, 36 = 9.28, P < 0.0005, ANOVA test followed by Dunnett test for pyriproxyfen, azadirachtin and tebufenozide; Adult emergence: H = 37.96, P < 0.0001, Kruskal–Wallis test followed by Dunn test for lufenuron, cyromazine and pyriproxyfen; F2, 27 = 17.28, P < 0.0001, ANOVA test followed by Dunnett test for azadirachtin and tebufenozide) (Table 1). Moreover, lufenuron, cyromazine and pyriproxyfen completely inhibited viable offspring production since no adult emergence was observed. However, when the surviving pairs of these treatments were transferred to untreated diet for 11 more days after the period of continuous exposure to treated diet, they were able to produce viable offspring that reached the adult stage (Fig. 1).

Fig. 1
figure 1

Mean number of flies emerged from eggs laid on untreated diet for 11 days by females exposed previously to diet treated with different insect growth regulators for 19 days

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Hence, lufenuron, cyromazine and pyriproxyfen were selected for further evaluation of their effect on D. suzukii. In the six previous days to the choice tests, no significant differences were found in the total number of eggs laid for these products compared to the control (F3, 36 = 2.48, P = 0.0768, ANOVA test) (Table 3). However, pupation and adult emergence were significantly reduced by the three compounds. No pupation was observed for lufenuron and cyromazine, and the number of pupae observed for pyriproxyfen was significantly reduced compared with the control (H = 27.49, P < 0.00001, Kruskal–Wallis test followed by Dunn test for lufenuron and cyromazine; t = 3.40, P < 0.01, Student t-test for pyriproxyfen) (Table 3). In addition, no emergence was observed for lufenuron and cyromazine (since no pupation occurred), and in the case of pyriproxyfen only one fly was able to emerge from the pupae produced (H = 35.63, P < 0.0001, Kruskal–Wallis test followed by Dunn test) (Table 3).

Table 3 Offspring production for the six previous oviposition days to the choice assays by Drosophila suzukii pairs fed on artificial diets treated on surface with different insect growth regulators
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During the two-day period of the choice assay, no pupation occurred in the treated and non-treated diets within the replicates for lufenuron treated pairs, and consequently no adults were produced (W = 0.0, P = 1.0 in both cases, paired samples Wilcoxon signed rank test) (Table 4). For cyromazine-treated pairs, no pupation and adult emergence were registered in the treated diets, and only a much reduced number of pupae and adults were observed in their corresponding non-treated diets within the replicates (Table 4). Differences were detected in the case of the pupation (W = 2.11, P < 0.05, paired samples Wilcoxon signed rank test), but not in the case of the emergence (W = 1.90, P = 0.0579, paired samples Wilcoxon signed rank test). For pyriproxyfen treated pairs, pupation occurred at the same level both in the treated diets and in their corresponding non-treated diets within the replicates (t = 1.18, P = 0.2691, paired samples t-test). However, emergence of adults was only observed in their non-treated diets (W = 2.76, P < 0.01, paired samples Wilcoxon signed rank test). As expected, no differences in pupation and adult emergence were detected between the diets within the double-control reference treatment (t = 0.34, P = 0.7413 for pupation and t = 0.28, P = 0.7861 for adult emergence, paired samples t-tests). In addition, when pupation and adult emergence registered in the treated and non-treated diets within the replicates were compared with regard to their corresponding control diets of the double-control reference treatment, a significant reduction in the number of pupae and adults produced was observed in all cases (H = 27.51, P < 0.0001, Kruskal–Wallis test followed by Dunn test for the pupation in the diets treated with lufenuron and cyromazine, and t = 2.93, P < 0.01, Student t-test for the pupation in the diets treated with pyriproxyfen; H = 24.14, P < 0.0001, Kruskal–Wallis test followed by Dunn test for the pupation in the non-treated diets corresponding to lufenuron and cyromazine, and t = 4.04, P < 0.001, Student t-test for the pupation in the non-treated diets corresponding to pyriproxyfen; H = 37.99, P < 0.0001, Kruskal–Wallis test followed by Dunn test for the emergence in the diets treated with all compounds; H = 23.96, P < 0.0001, Kruskal–Wallis test followed by Dunn test for the emergence in the non-treated diets corresponding to lufenuron and cyromazine, and t = 4.43, P < 0.0005, Student t-test for the pupation in the non-treated diets corresponding to pyriproxyfen) (Table 4).

Table 4 Offspring production for two oviposition days by Drosophila suzukii pairs fed on artificial diets treated or not treated on surface with different insect growth regulators in choice assays
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None of the products had an oviposition repellent effect (F3, 36 = 1.59, P = 0.2086, ANOVA test) (Table 5). The indices of reduction in pupation and adult offspring production could not be calculated for lufenuron because no pupae were produced in the treated and non-treated diets (Table 4). In the case of cyromazine, both indices were significantly different to the control, and for pyriproxyfen, only the index of reduction in adult offspring production resulted in significant differences compared to the control (F2, 23 = 117.24, P < 0.0001, ANOVA test followed by Dunnett test for the index of reduction in pupation; H = 22.06, P < 0.0001, Kruskal–Wallis test followed by Dunn test for the index of reduction in adult offspring production) (Table 5).

Table 5 Effect of different insect growth regulators on offspring production of Drosophila suzukii adults tested in choice assays on artificial diets
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When eggs laid on non-treated diet were transferred to treated diet, no effect was observed on egg hatching for lufenuron, cyromazine and pyriproxyfen (F3, 36 = 1.89, P = 0.1493, ANOVA test) (Table 6). However, no pupation was registered on lufenuron and cyromazine-treated diets (H = 27.58, P < 0.0001, Kruskal–Wallis test followed by Dunn test). This effect was not observed for pyriproxyfen (t = 0.43, P = 0.6705, Student t-test), but this compound together with lufenuron and cyromazine completely inhibited adult emergence (H = 38.02, P < 0.0001, Kruskal–Wallis test followed by Dunn test) (Table 6). When eggs laid on treated diet were transferred to non-treated diet, no egg hatching was observed in the case of lufenuron (U = 50.0, P < 0.0001, Mann–Whitney U-test), whereas this effect was not observed for cyromazine and pyriproxyfen (F2, 27 = 1.11, P = 0.3428, ANOVA test) (Table 7). Also, no pupae or adults were obtained in the case of lufenuron and only one fly was able to emerge in the case of cyromazine (H = 26.18, P < 0.0001, Kruskal–Wallis test followed by Dunn test for pupation; H = 26.09, P < 0.0001, Kruskal–Wallis test followed by Dunn test for adult emergence) (Table 7). Pyriproxyfen did not have any effect on pupation and adult emergence (t = 0.57, P = 0.5724, Student t-test for pupation; t = 0.49, P = 0.6319, Student t-test for adult emergence).

Table 6 Percentages of egg hatching, pupation and adult emergence obtained from eggs laid by Drosophila suzukii females fed on non-treated artificial diet and transferred to diet treated on surface with different insect growth regulators
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Table 7 Percentages of egg hatching, pupation and adult emergence obtained from eggs laid by Drosophila suzukii females fed on artificial diet treated on surface with different insect growth regulators and transferred to non-treated diet
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Discussion

The activity of different IGRs was tested to evaluate their suitability for the control of D. suzukii. Lufenuron and azadirachtin have been previously tested on this pest and the results obtained here agree in general with the findings reported by other authors (Andreazza et al. 2017b; Pavlova et al. 2017; Sampson et al. 2017a, 2017b). Cyromazine, pyriproxyfen and tebufenozide have, to our knowledge, been assayed for the first time and the first two have shown negative effects on D. suzukii, making them promising candidates for further evaluation of their utility in the control of this pest.

Lufenuron belongs to the benzoylphenyl urea compounds, which act as chitin-synthesis inhibitors, and accordingly interferes with the deposition of new cuticle during moulting. Furthermore, these substances may have ovicidal effects and alter reproduction (Dhadialla et al. 2005). This compound had no effect on fecundity of D. suzukii females fed on treated diet, similar to what has been reported before (Sampson et al. 2017a), but, as shown in the choice and egg-transfer assays, it prevented the oviposition of viable eggs. Eggs failed to hatch because lufenuron was transmitted to them transovarially through the female feeding on the treated diet, a mechanism well known for benzoylphenyl ureas (Subramanian and Shankarganesh 2016; Sampson et al. 2017a). However, the effect was transitory and disappeared when D. suzukii flies were transferred to untreated diet, which is similar to the results obtained previously (Sampson et al. 2017a). Nevertheless, dose-dependent long-lasting sterilizing effects of lufenuron have been observed in fruit flies of family Tephritidae after transferring the adults to untreated diet (Moya et al. 2010; Sánchez-Ramos et al. 2013; Chang 2017). Also, chemosterilant bait stations containing lufenuron enhanced the population reduction achieved by the sterile insect technique against Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) (Navarro-Llopis et al. 2011). In any case, despite the transitory effects on treated females, the application of lufenuron in blueberry and blackberry fields reduced larval infestation of D. suzukii by 75% (Sampson et al. 2017b). This is in accordance with the egg and larval mortality registered in laboratory assays when blueberries and blackberries were dipped in lufenuron solutions before exposing them to D. suzukii adults (Sampson et al. 2017a). These authors concluded that eggs received the compound through the treated exocarp or from the contaminated ovipositor when they were laid.

Cyromazine acts as a moulting disruptor altering development of Diptera and this effect seems to be related to the hormonal control of larval development (Van de Wouw et al. 2006). This substance completely inhibited the production of viable offspring of D. suzukii when adults fed on treated diet. However, this effect was transitory and disappeared when they were transferred to an untreated diet, similar to that reported for other flies (Alam and Motoyama 2000). Since the eggs were able to hatch, the effect observed might be due to the feeding of the emerged larvae on the treated diet. However, in the choice assay, D. suzukii females fed previously with cyromazine-treated diet produced small numbers of adults in the control diets, in addition to non-viable offspring in the treated diets. Furthermore, in the egg-transfer assay, eggs laid on treated diet and transferred to non-treated diet were able to hatch, but almost all the larvae died without reaching the pupa stage (only one specimen was able to reach the adult stage). This indicates a vertical transmission of cyromazine from the females fed on treated diet to the eggs laid by these females, which impedes the subsequent larval development in the vast majority of larvae. Similar findings have been observed in other dipteran species (Budia and Viñuela 1996; Alam and Motoyama 2000; Alam et al. 2001).

Moreover, it has been observed a decrease of 58% in the number of eggs laid by Drosophila melanogaster Meigen (Diptera: Drosophilidae) when fed on a diet treated with cyromazine at 50 ppm, thus suggesting additional effects on the reproductive processes of females (Khalid et al. 2022). However, another study reported no effect on fecundity and egg hatch of treated females of this species, although the dose employed was much smaller (0.3–3 ppm) (Wilson 1997). The results obtained here in the six days previous to the choice assay did not show any effect of cyromazine on the fecundity of D. suzukii, so it seems that no alterations of the reproduction of this fly are occurring. In any case, variable results have been obtained for other dipteran species indicating that the response of reproduction of adults to cyromazine is species-specific (Wilson 1997).

Pyriproxyfen is a juvenile hormone mimic that is reported to interfere with the development of immature stages and the reproduction of adults (Dhadialla et al. 1998). This compound impeded the adult emergence of D. suzukii when females fed and laid eggs in treated diet. However, viable offspring was produced as soon as the flies were transferred to untreated diet. Many larvae where able to pupate in the continuous feeding experiment (about 60% compared with the control), although finally no adult emergence was recorded. This is similar to what has been reported for D. melanogaster (Bensebaa et al. 2015). In the choice assay, pyriproxyfen produced a decrease in the production of viable offspring by adults fed previously on treated diet for six days, since the number of pupae produced was significantly reduced both in the treated and non-treated diets compared with the double-control diets. This effect was not due to a reduction in fecundity, as this was not observed in the six previous days to the choice assay. However, in the egg-transfer experiment, eggs laid by females that fed on treated diet for three days and transferred to non-treated diet produced a number of adults not significantly different from the control, what is not in accordance to what was obtained in the choice assay. The different period of previous exposure to treated diet (6 vs. 3 days) might explain the differences observed. On the other hand, adult production was completely inhibited in the treated diets of the choice assay, whereas a great majority of the pupae obtained in their corresponding non-treated diets were able to become adults, suggesting an effect of pyriproxyfen through the feeding of larvae on treated diet. This was corroborated with the results obtained in the egg-transfer experiment because no adult emergence was obtained from eggs laid on untreated diet and transferred to treated diet. However, taking into account the results obtained in the choice experiment regarding pupae production, additional effects on reproductive processes might be occurring too.

Azadirachtin is a botanical pesticide obtained from the seeds of the neem tree Azadirachta indica A. Juss. that is considered nontoxic to mammals, fish, birds and pollinators (Isman 2006; Morgan 2009). This product has shown efficacy on numerous insect pests, including Diptera (Singh 2003; Bezzar-Bendjazia et al. 2016; Zhou et al. 2020) and, although much is known about its effects on arthropod physiology (Morgan 2009), its specific mode of action is still considered to be unknown or uncertain (IRAC 2022). It produces a high variety of effects such as repellent, feeding and oviposition deterrent, growth regulator, inhibitor of reproduction and sterilizing agent (Schmutterer 1990; Morgan 2009). The effect of this compound on adult and immature mortality of D. suzukii has been tested, having shown moderate effects (Bruck et al. 2011; Andreazza et al. 2017b; Pavlova et al. 2017; Cahenzli et al. 2018). Here, offspring production by D. suzukii was reduced by more than a half compared to the control when adults fed continuously with diet treated with this compound. Nevertheless, the feeding for three days did not have any persistent effect on the number of offspring pupae and adults produced. These observed effects might be due to the feeding of the larvae on the treated diet or to effects on reproduction of adults. Pavlova et al. (2017) observed a reduction in the number of adults emerging from treated nutritive medium compared with the untreated control when mated females were allowed to lay eggs for 24 h before or after the treatment and pointed out that this effect would correspond to the activity of azadirachtin as insect growth regulator affecting metamorphosis. Similarly, Andreazza et al. (2017b) registered a significant increase in mortality of D. suzukii larvae when strawberry fruit previously infested were dipped in a solution of azadirachtin and Cuthbertson et al. (2014) did not report any increase in mortality during development of eggs laid on blueberries treated by immersion in a solution of neem oil, although a retarded development was observed. Therefore, the effects observed seem to be mainly related to the ingestion of treated food by the larvae.

Tebufenozide produced similar effects than azadirachtin, with a reduction in the number of descendants produced only in the continuous exposure experiment. This compound is a moulting hormone mimic that produces disruptive effects on cuticle formation of lepidopteran larvae, although it can also reduce the egg production in various lepidopteran, coleopteran, and dipteran insects (Dhadialla et al. 1998). However, lethal effects on larvae of different dipteran species have been reported too (Beckage et al. 2004; Gelbič and Roszypalová, 2012), as well as effects on the hatching of eggs (Tassou and Schulz 2013). With the results obtained here it is not possible to discern which effect is acting against D. suzukii, so more specific assays should be designed to establish it. Nevertheless, the reduced activity observed make that unprofitable.

In conclusion, the results presented here show that some of the IGRs tested could be useful for the control of D. suzukii. Thus, lufenuron and cyromazine could be included into chemosterilant bait stations, although the need for a continuous exposure is a limiting fact, because is not likely that flies feed in the field always from the same food sources. Also, as reported for lufenuron, cyromazine and pyriproxyfen might prevent larvae development when applied to fruits. However, this should be confirmed in additional laboratory experiments. In addition, field assays should be performed to check the feasibility of these substances to fight against D. suzukii under real crop conditions.

Author contributions

ISR and MGN conceived and designed the research. ISR, MGN and CEF conducted the experiments. ISR analysed the data. ISR and MGN wrote the manuscript. All authors read and approved the manuscript.