Introduction

The effect of insecticides and other toxicants on insects have been traditionally assessed using measures of the acute mortality as a single endpoint and have relied on the determination of the acute lethal dose/concentration1. However, in addition to the direct effect on lethality these compounds may also impair various key biological traits of the individuals that survive exposure through physiological and behavioral effects1,2. Among physiological effects, developmental success, morphological deformities, adult longevity, sex ratio, fertility and fecundity are commonly estimated3,4. Behavioral effects may be manifested as impairment in insect mobility, learning ability, host finding, sexual communication as well as feeding and oviposition behavior5,6,7,8,9,10. An accurate assessment of these effects is crucial to acquire knowledge on the overall insecticide efficacy for long-term management of pest insect populations, as well as on their selectivity toward non-target species11. Indeed, when studying susceptibility of organisms towards insecticides, and beside the short term influences on the directly exposed individuals, it is important to take into account the entire life-history as a comprehensive method for evaluating the total effect on insect population, including the impacts on the next generation which have important implications for the success of an Integrated Pest Management (IPM) program2,4.

Among nowadays the insecticides used in sublethal effect studies, the botanical insecticides have been the subject of an increasing number of academic research as a potential option for an environment friendly pest management tools12,13 due to their rapid degradation in the environment, low mammalian toxicity, low risk of resistance development in target pest populations and good selectivity to non-target arthropods14,15,16,17,18. Azadirachtin (AZA), a natural tetranortriterpenoid compound extracted from the neem tree, Azadirachta indica19, is considered as one of the most promising plant compounds for pest control in organic agriculture14,20. AZA shows variable effects on insects including the model insect Drosophila melanogaster21,22. This triterpenoid acts as sterilant, insect growth regulators by disruption of the endocrine system, repellent, oviposition and feeding deterrent by activating bitter sensitive gustatory cells23,24. Larval exposure of D. melanogaster to sublethal doses of azadirachtin was found to affects various aspects of their physiology including digestive enzymes25 and this effect is also further observed in the adults10. This pre-imaginal exposure affects not only the physiology and the fitness of flies but also adults oviposition and feeding preference7,10.

Most studies on sublethal effects of insecticides are related to continuously or repeated exposure. This exposure provokes a generalized stress and activating a detoxification response such as up-regulated of cytochrome P450 genes which might lead to the detoxification of insecticide and even the development of resistance26. Moreover, the up-regulation is thought to provide versatility in environmental adaptation27. In botanical insecticide the potential fast desensitization to a feeding deterrent was reported28,29. Individual insects initially deterred by feeding inhibitor become increasingly tolerant due to repeated or continuous exposure29. Bomford and Isman15 reported an habitation to pure azadirachtin in the tobacco cutworms which become less sensitive to the antifeedant properties of azadirachtin, but not to a neem containing a same absolute amount of azadirachtin. This might have an important implication to avoid desensitization to commercial neem-based insecticides which contains additional non AZA-compounds15. Larval exposure to Neem Azal, a commercial Azadirachtin-rich based formulation, was found to enhance avoidances of this compound in adults of D. melanogaster surviving from previously treated larvae10,25. This long-lasting avoidance is related to conditioned aversion and may be related to another mechanism such as sensitization30,31 which also generally occurs after long term or repeated exposure and may increase avoidance to noxious stimulus32. Moreover, increasing evidence has highlighted the critical role of early life experience in adult physiology and behavior in insect33. Recent studies have revealed that insect can modulate their behavior on the basis of previous experiences early life and that various insecticide-mediated changes in the directly exposed generation can persist into the subsequent non-exposed generations34,35. Previously, we have focused on the impact of larval exposure to azadirachtin on adult’s fitness (fecundity, survival) and oviposition site preference of the parental generation of D. melanogaster as a model organism for testing insecticide activity7. Current study aimed to evaluate, the possible adverse effects of this prior single exposure to azadirachtin experienced by the preceding generations on life table and oviposition site preference of the filial generations. We monitored the oviposition site preference, fecundity, development, sex ratio, survival and morphological abnormalities of exposed and non-exposed generations. All these parameters were investigated over generations until their restoration to predict the outcome of azadirachtin use on pest management practices.

Results

Fecundity and oviposition site preference

Azadirachtin, topically applied on the 3rd instar larvae (LD25 and LD50 of immature stages) affect fecundity of females by a significant reduction of the number of eggs laid as compared to controls (KW = 24.73; p < 0.001). This reduction was observed over two successive generations (parental and F1), however, the total eggs laid was higher in the unexposed generation (F1) than in parental (P) ones (KW = 50.89; p < 0.001) (Fig. 1). Full restoration of affected fecundity was noted in the second generations (F2).

Figure 1
figure 1

Effect of azadirachtin (LD25 and LD50), topically applied on early third instars larvae of D. melanogaster on fecundity of females (number of eggs laid) subjected to non-choice experiments (m ± SE; n = 12 replicates of 3 flies). Different small letters indicate a significant difference between control and treated individuals of the same medium (P < 0.05). Different capital letters indicate a significant difference between generations of the same medium (P < 0.05).

Full size image

Results of oviposition preference in the no choice experiments (Fig. 1) revealed a clear preference for oviposition on untreated medium than in azadirachtin-treated ones.

For parental generation, Kruskal-Wallis test revealed significant effects in medium 0.1 μg/ml (KW = 29.42; p < 0.001) and medium 0.25 μg/ml (KW = 24.73; p < 0.001). In the first generation, a significant effect was also noted for medium 0.1 μg/ml (KW = 22.95; p < 0.001) and medium 0.25 μg/ml (KW = 27, 93; p < 0.001).

The results concerning the dual choice experiments (Fig. 2) revealed an oviposition preference in control medium than in treated medium for all tested generations (P, F1 and F2). Furthermore, flies previously exposed to azadirachtin (early 3rd instar larvae) showed a highest aversion to this substance compared to naïve flies and led fewer eggs for the two first generations (P and F1) with a more marked effects for parental generation (P < 0.001).

Figure 2
figure 2

Egg-laying preference (m ± SE; n = 12 replicates) of female adults of D. melanogaster subjected to a free-choice test on food treated with azadirachtin at two doses (0.1 µg/ml and 0, 25 µg/ml). Different small letters indicate a significant difference between control and treated individuals of medium untreated and treated (P < 0.05). Different capital letters indicate a significant difference between individuals of the same dose in the different medium (P < 0.05).

Full size image

The oviposition preference index (OPI) of adult females of D. melanogaster exposed, or not, to azadirachtin at larval stage of parental generation were always negative in all generations (Fig. 3).

Figure 3
figure 3

Oviposition preference index (m ± SE; n = 12 replicates) of female adults of D. melanogaster subjected to a free-choice test on food treated with azadirachtin at two doses (A: 0, 1 µg/ml; B: 0, 25 µg/ml). Different small letters indicate a significant difference between the same dose of different generations (P < 0.05). Different capital letters indicate a significant difference between difference tested doses of the same generation (P < 0.05).

Full size image

In the generation P, statistical analysis showed significant differences between OPI of previously treated flies and controls flies with a dose-dependent response (Fig. 3). In addition, for medium 0.1 μg/ml, Mann-Whitney revealed significant effects between LD25 of the parental generation and the first generation (Mann-Whitney test U = 8; P < 0.001), LD25 of parental and second generation (Mann-Whitney test U = 20; P = 0.0018) but there was no difference between the first and the second generations (Mann-Whitney test U = 42; P = 0.0887). Similar results were observed for the LD50, with significant effects observed between the parental generation and the F1 (Mann-Whitney test U = 19; P = 0.0014), also between P and F2 (Mann-Whitney test U = 34; P = 0.0284) but no difference between F1 and F2 (Mann-Whitney test U = 58; P = 0.4428). For control, there was no difference between all tested generations.

Similar results were obtained for medium 0.25 μg/ml, Mann-Whitney test revealed significant effects between LD25 of the parental generation and the first generation (Mann-Whitney test U = 25; P = 0.0045), LD25 of parental and second generation (Mann-Whitney test U = 24; P = 0.0045) but there was no difference between the first and the second generations (Mann-Whitney test U = 66; P = 0.5512). For the LD50, significant effects were observed between the parental generation and the F2 (Mann-Whitney test U = 25.50; P = 0.0025), also between F1 and F2 (Mann-Whitney test U = 34; P = 0.0028) but no difference was observed between F1 and P (Mann-Whitney test U = 49; P = 0.1974). There was no difference between controls for all generations.

Analyses of development

Results from development analysis of D. melanogaster are given in Tables 1 and 2, respectively for parental (exposed) and F1 (non-exposed) generation. Treatment of early third instar larvae at two tested doses (LD25 and LD50) decreased the number of larvae, pupae and the final number of organisms of parental generation with a dose-dependent relationship as expressed by the FNO which is always negative for the treated series. The development of F1 D. melanogaster doesn’t seem to be affecting by the early treatment of the parental generation. However, the FNO of tested flies (LD25, LD50 and control) in treated medium was significantly lower than in the control medium for both generations. There is no difference between the number of organisms reached the pupae stage and the final number of organism in both generations. In addition, treatment of early third instar larvae increased significantly (p < 0.001) the duration of larval and pupal development as expressed by T50, with dose-dependent manner only for the Parental generation (exposed) as compared to controls. There is no difference between the T50 of the tested flies in both treated and untreated medium.

Table 1 Effect of larval exposure to azadirachtin on development of parental generation (exposed) of D. melanogaster.
Full size table
Table 2 Effect of larval exposure to azadirachtin on development of first generation (non-exposed) of D. melanogaster.
Full size table

Larvae, pupae and imagoes of the parental generation showed several types of malformations and anomalies followed by death at each stage of development of D. melanogaster. The most prominent malformations detected are incomplete and malformed imagoes (malformed abdomen and wings), curved and smaller body shape, burned larvae, dead adults inside pupae (Fig. 4).

Figure 4
figure 4

Examples of the most frequent malformations of D. melanogaster (n = 50). (A) Malformed abdomen and wings curved and smaller body shape; (B) dead adults inside pupae; (C) malformed adult; (D) burned larvae.

Full size image

Pre-imaginal exposure of azadirachtin induced a male-biased sex ratio only for the parental generation with a dose-dependent relationship (Fig. 5). Kruskal-Wallis test revealed significant effects between the different tested insect (Control, LD25 and LD50) in untreated medium (KW = 9.30; p = 0.0095), medium 0.1 μg/ml (KW = 8.02; p < 0.0181) and medium 0.25 μg/ml (KW = 18.85; p < 0.0001) for the parental generation.

Figure 5
figure 5

Effect of azadirachtin (LD25 and LD50), topically applied on early third instars larvae of D. melanogaster on sex ratio of adults emerged. Different small letters indicate a significant difference between generations of the same medium (P < 0.05). Capital letters indicate a significant difference between control and treated individuals of the same medium (P < 0.05). (m ± SE; n = 15 replicates).

Full size image

Survival analysis of adults

A survival analyses during the 15 first days of adults previously treated with azadirachtin as 3rd instars larvae (Fig. 6) revealed a rapid reduction of adult surviving of the generation P (Male: Kaplan-Meier test, χ2 = 184, df = 2, P < 0.001; Female: Kaplan-Meier test, χ2 = 214, df = 2, P < 0.001). Lower mortality was noted for the generation F1 compared to parental. (Male: Kaplan-Meier test, χ2 = 39.1, df = 2, P < 0.001; Female: Kaplan-Meier test, χ2 = 63.1, df = 2, P < 0,001). Flies mortality was dose-dependent and the females were more affected by the treatment.

Figure 6
figure 6

Effect of azadirachtin (LD25 and LD50), topically applied on early third-instar larvae of D. melanogaster on the adult’s survival (male and female) of two generations tested (p < 0.05).

Full size image

For the control series no mortality was recorded for both tested generations. For the treated series, the lowest dose (LD25) decline the adult’s survival to 49% for males and 36% for females of the P generation versus 94% for males and 84% for females of the F1 generation. The highest dose (LD50) induced more marked effects on adult’s survival with 27% for males and 16% for females of the P generation and 81% for males and 64% in females for the F1 generation. Survival of 100% was noted for males and females of the F2 generation.

Discussion

Azadirachtin’s impact on reproduction have been reported on different insect species21,41,42,43,44,45. Our study has demonstrated that a single azadirachtin treatment (LD25/LD50) of D. melanogaster larvae reduced eggs number affecting negatively the fecundity of surviving females, not only through direct sublethal effects in exposed individuals, but also through transgenerational effects on F1individuals that were never directly exposed to the insecticide.

Oviposition is a complex and critical activity in the life cycle of an insect with a variety of factors that influence both physiology and subsequent behavior, that lead to egg deposition by an insect which tries to ensure safety to their progeny. Reduced fecundity and fertility after azadirachtin treatment has been reported in many insects including Spodoptera littoralis, D. melanogaster, Galleria mellonella, Dysdercus cingulatus, Tuta absoluta and Helicoverpa armigera17,41,43,44,45,46 and could be correlated to the negative action of azadirachtin on yolk protein synthesis and/or its uptake into oocytes21.

Ecdysteroids, JH and insulin/insulin-like growth factor signalling (IIS) regulation are crucial for reproduction of D. melanogaster47. Vitellogenesis in females was stimulated under JH action and has led to oocytes development, JH synergic action with 20E and IIS controls the nutrient-sensitive checkpoint necessary for oocytes formation47. Consequently, reduced fecundity may be related to the antagonist action of azadirachtin on major hormones controlling the reproductive process (JH/ecdysteroids)7.

In Anopheles stephensi, azadirachtin treatment has led to abnormal ovaries structure with a complete arrest of oogenesis, vitellogenesis and vitelline envelope formation impairment, as well as follicle cells degeneration48. Ovaries of azadirachtin-treated females of Heteracris littoralis also showed complete shrinkage with suppressed oocyte growth49, in addition to mitochondria disintegration and follicular cells destruction49. Moreover, Azadirachtin reduced mating success in D. melanogaster flies and negatively affected cyst and oocyte number and size45. Its treatment also affected food intake and digestive enzyme activity in the midgut, in these species10. This may disturb oogenesis and vitellogenesis since ecdysone and JH rates are affected by nutrient availability, which acts as positive regulator on insulin pathway conferring ovaries the necessary signalling for a normal oogenesis50,51.

In addition, tested flies of all generations preferred the control medium for oviposition avoiding the azadirachtin ones for the two tested doses and conditions (no-choice and free choice). A low oviposition rate of non-exposed (naïve) flies in azadirachtin-treated areas could be due to the known repellent, deterrent, and locomotor stimulation effects of azadirachtin and other neem-based insecticides, which were reported by Silva et al.52 for medflies Ceratitis capitata. Valencia-Botín et al.53 also suggest that the repellent property of neem extracts is the major factor responsible for the reduction of eggs number in Anastrepha ludens (Loew)53. The ovipository behavior inhibition may have a valuable impact in pest control.

In addition, flies who have already been treated (third instar larvae of P generation) showed an increased aversion to azadirachtin in comparison to the naïf flies. This continued for two successive generations (P and F1). When oviposition sites were treated with azadirachtin or other neem-based compounds, oviposition repellency, deterrency, or inhibition occurred in several insect species that are able to detect the bioinsecticide on the treated surface7,14,43,54,55. The capacity of insects to retain memory from early life exposure affecting the adult response was reported38,56,57,58. In D. melanogaster, females avoided oviposition on sites containing azadirachtin after larval exposure to the bio-insecticide7.

Here, we have reported for the first time that the negative effects of a single larval exposure to azadirachtin can also be passed on to the F1 generation (transgenerational effects). Environmental toxicants such as insecticide are able to provoke epigenetic alterations, which can be inherited in the next generations59. This may explain the reduced fecundity and oviposition avoidance in the non-exposed generation (F1).

Our study has also demonstrated that azadirachtin applied during the third larval instar of parental generation (LD25 and LD50) negatively affected various life traits of D. melanogaster, in a dose-dependent manner, as it significantly reduced larval, pupation, and emergence rate of the exposed generation. The biopesticide also significantly prolonged the larval and pupation period of development inducing important delays in immature stages development and affect sex ratios (with fewer females in the offspring) of the same generation. Additionally, the treatment induced morphological alterations of larvae, pupae and adults only in the exposed generation (P generation). The most prominent abnormalities were burned larvae, larva-pupa intermediate, pupa-adult intermediate, deformed wings, smaller body size and deformed abdomen. The recorded malformations finally resulted in insect dead. Similar results were noted in D. melanogaster37, Hyalomma anatolicum excavatum60 and Spodoptera litura22. Finally, a decline in adult’s survival was noted for the two successive generations with more marked effects among the P generation.

Azadirachtin is known to reduce pupation and eclosion rates of many insects like Aphis glycines61, Plodia interpunctella62, Aedes aegypti63 and D. melanogaster21. A negative impact of azadirachtin on the immature stages was expected due to its insect’s growth disruptor (IGD) action by suppressing haemolymph ecdysteroid and JH peaks25,64. Furthermore, azadirachtin is known to cause nucleus degeneration in the different endocrine glands (prothoracic gland, corpus allatum and corpus cardiacum) controlling insects moulting and ecdysis, which could act as generalised disruptor of neuroendocrine system24. Azadirachtin alters the growth and molting process of several insects by compromising their survival7,20,43,65,66. Lai et al.67 reported that azadirachtin down regulated the expression of different genes that are linked to hormonal regulation. This could explain the developmental aberrations observed in our results. Azadirachtin is also known to affect Drosophila nutrient intake and metabolism compromising the nutritional signals, which result in a decrease in insect weights and growth rates, and thus resulting in smaller body size impacting survival10,25,37,66. The male biased sex ratio under azadirachtin treatment was reported in literature67,68.

In conclusion, the present study indicated that pre-imaginal exposure to sublethal doses of azadirachtin affects the fecundity, oviposition preference, and the survival of D. melanogaster of parent generation as well as the non exposed F1 generation. The treatment triggered life history traits variation in the P generation.

Results demonstrated that a single azadirachtin application significantly reduced the survival of flies over two successive generations (P: exposed and F1: unexposed) while insects showed clear recovery in the survival rates in the second generation (F2). These findings reflect a long term effects through developmental stage and generations. This effect is consistent in the two first generations could be considered as advantage for pest control by compensating the well-known fast degradation by sunlight and low persistence of azadirachtin in environment (half-life DT50: 1.7–25d)23,69 and suggest a possible reduction both in application frequency and total amount of pesticide used.

Furthermore, the decreased fecundity and survival in P and F1 generations indicated an absence of resurgence induction in offspring, even after full restoration in F2, when parental flies were treated. This translated an absence of hermetic effect, which is considered as a serious problem of exposure to sublethal doses in agriculture.

In addition, the treatment extended the aversive effect induced by azadirachtin to over two successive generations. This could contribute as push-pull strategies that increase its insecticidal effects in integrated pest management programs.

Material and Methods

Flies

Wild-type Canton-S strain of D. melanogaster flies were reared on artificial fly food (cornmeal/agar/yeast) at 25 °C, 70% humidity and 12D-12 L cycle10.

Treatment

Neem Azal-TS (1% azadirachtin A, Trifolio-M GmbH, Lahnau, Germany) was solubilised in acetone for topical application (1 µl/larvae according to Bensebaa et al.36). The bioinsecticide was applied on D. melanogaster early third-instar larvae using two lethal doses of immature stages, 0.28 µg (LD25) and 0.67 µg (LD50)37. Controls received 1 µl acetone (solvent) and all flies were kept under the same conditions as cited above. All experiments were performed over two consecutive generations, the exposed (parental generation: P) and non-exposed (first generation: F1) generation.

Fecundity and oviposition site preference

We assessed the egg-laying performances of the females of D. melanogaster using a no-choice test. Three mated females (3 days old) that were pre-exposed to azadirachtin at the larval stage (LD25 and LD50) were tested for 24 h in a petri dish (Ø = 65 mm) filled with 3 ml medium containing azadirachtin at two concentrations 0.1 and 0.25 μg/ml according to Bezzar-Bendjazia et al.37 in addition to acetone as control medium. These concentrations were not lethal with the short exposure time (24 h) used. At the end of the test, flies were removed, and the number of eggs laid on each medium was counted. The control medium was used to test the possible effect of azadirachtin on female fecundity. The experiment was repeated 12 times for each medium and each generation. Oviposition site preference was measured by means of dual choice experiments. Three fertilized females (3 days old) from controls and treated series (LD25 and LD50) were allowed to oviposit for 24 h in a free choice egg-laying device. This device consisted of a two petri dishes either filled with control medium (acetone) or treated medium (azadirachtin: 0.1, 0.25 μg/ml). After 24 h, the egg-laying preference was assessed by counting the number of eggs laid in each medium. The test was performed for two successive generations with 12 replicates for each medium and generation.

Oviposition preference index (OPI) defined as (number of eggs on azadirachtin medium – number of eggs on control medium)/total number of eggs was calculated38.

Development assays

Ten controls or pre-exposed (LD25 and LD50) mated females (3 days old), named parental generation, were released into an oviposition box containing petri dishes filled with control (acetone) or treated medium (azadirachtin: 0.1 or 0.25 μg/ml) and left to lay eggs for 8 hours. At the end of the test, the flies were removed and a pool of 100 eggs for each experiment was transferred to a new petri dish containing the same medium. For all groups, we monitored the time course of larval development from egg to adult emergence by counting the number of third instar larvae, pupae, imagoes and their sex ratio, expressed as the number of males divided by the total number of emerged insects.

Next, ten parental flies from each condition (controls or treated) were crossed and the experiments were repeated for the non-exposed first generation (F1) as cited above with the same parameters recorded.

Furthermore, the developmental duration of each stage was recorded for the two tested generations expressed by T50 (time in hours, when 50% of population reached larval, pupal and imaginal developmental stage in vials). All insects were observed under stereo zoom microscope to find any morphological distortions and photographs were taken with Leica Z16 APO.

A factor describing the final number of organisms in comparison to control (FNO) according to Ventrella et al.39 was determined to compare the results:

$$FNO=\frac{{\rm{T}}-{\rm{C}}}{{\rm{C}}}\,\times 100$$

T = final number of organisms counted in treated medium.

C = final number of organisms counted in control medium.

Positive values of FNO show that number of organisms was higher in tested groups than within control, negative values mean that the number of individuals was higher in control than in exposed groups.

Survival analysis of adults

Survival analysis was performed according to Linford et al.40. For each generation (P: exposed (LD25 and LD50), F1: non-exposed) newly emerged adults were sexed and housed separately into a plastic vials (15 flies per vial) containing fresh food. Insects were transferred to new vial every 2 days. The flies were kept under observation for 15 days during which mortality was assessed every 24 h. Ten replicates were done for each dose and generation.

Statistical analyses

Data analysis was performed by R studio version 3.5.0 for Mac OS. The results were expressed as the means ± SE for each series of experiments. The homogeneity of variances was checked using Bartlett’s test. The Shapiro-Wilk statistic test was used for testing the normality.

Data from egg-laying preference and oviposition index preference was subjected to Kruskal–Wallis test and pairwise multiple comparisons using Dunn’s method. Development test were analysed with ANOVA followed by a post-hoc HSD Tukey test. Sex ratio was analysed using Kruskal–Wallis test and the FNO was calculated and shown. The results of the survival analysis were subjected to Kaplan–Meier survival test.