Appraisal of growth inhibitory, biochemical and genotoxic effects of Allyl Isothiocyanate on different developmental stages of Zeugodacus cucurbitae (Coquillett) (Diptera: Tephritidae)

Allyl isothiocyanate (AITC), a glucosinolates’ hydrolytic product, was studied for its anti-insect potential against an economically important, destructive tephritid pest, Zeugodacus cucurbitae (Coquillett). The first, second and third instar maggots of the pest were fed on artificial diets amended with varied concentrations of AITC viz. 5 ppm, 25 ppm, 50 ppm, 100 ppm, 150 ppm and 200 ppm with DMSO (0.5%) as control. Results revealed high larval mortality, alteration of larval period, prolongation of pupal and total developmental periods in all instars of the maggots treated with AITC as compared to controls. Percent pupation and percent adult emergence decreased in all larval instars. Growth indices viz. Larval Growth Index (LGI) and Total Growth Index (TGI) were negatively affected. Anti-nutritional/post ingestive toxicity of AITC was also revealed by the decrease in Food Assimilation (FA) and Mean Relative Growth rate (MRGR) values with respect to control. Profiles of PO (Phenol oxidase) and other detoxifying enzymes including SOD (Superoxide dismutases), CAT (Catalases), GST (Glutathione-S-transferases), EST (Esterases), AKP (Alkaline phosphatases) and ACP (Acid phosphatases) were also significantly influenced. The genotoxic effect of AITC was also evaluated by conducting comet assays at LC30 and LC50. Significant DNA damage in hemocytes was reflected by increase in Tail length (μm), Percent Tail DNA, Tail Moment (TM) and Olive Tail Moment (OTM) as compared to controls. The results indicated high potential of AITC as biopesticide for pest management.

of herbivores such as birds, slugs and generalist insects are reportedly attributed to the actions of their derived hydrolysis products 7,8 . Among the various hydrolytic products, isothiocyanates (ITCs) have been documented to play a vital role in plant-pathogen interactions 9 . As these plant SMs or botanicals undergo biodegradation within a few hours of exposure 10 , a lot of research is underway to explore these botanical hydrolytic products especially ITCs against various insect pests so as to find compatible and ecologically safer alternatives to harmful synthetic pesticides. Vig et al. also reviewed such hydrolytic products as safer biofumigants in pest management studies as these are fully biodegradable and less toxic 4 .
ITCs can act as poisons or deterrents against bacteria 11 , fungi 12 and herbivores 13,14 . Allyl isothiocyanate (AITC) (Fig. 1) is used as an insecticide, bactericide, and nematicide possessing four active EPA registrations in the USA 15 . AITC has been reported to inhibit survival and growth of cabbage white fly, Pieris rapae (L.), a specialist lepidopteran insect pest 13 . It has also been reported that AITC generated suppressive effect on the reproductive cycles, particularly on larval and egg stages of two insect pests namely cigarette beetle, Lasioderma serricorne (Fabricius) and confused flour beetle, Tribolium confusum (Du Val) 16 . Insecticidal efficacy of AITC, extracted from Armoracia rusticana, has also been reported against four pests namely mason beetle Tribolium ferrugineum (F.), maize weevil Sitophilus zeamais (Motsch.), book louse Liposcelis entomophila (Enderlein) and lesser grain borer Rhizopertha dominica (F.) 17 . Bhushan et al. reported insecticidal efficacy of AITC against an insect pest, Spodoptera litura (Fabricius) 18 .
Zeugodacus cucurbitae (Coquillett) (earlier known by the name Bactrocera cucurbitae), commonly called as melon fruit fly, is one of the important agricultural pests that infests and damages a number of fruits and vegetables worldwide 19 . As fruitflies also have quarantine significance, trade is also adversely affected if fruitfly infestation occurs 20 . Native to the Indo-Malayan region, it is found in Burma, Kenya, Southern China, Malaya, Hawaii, India, Mauritius Island, Singapore, Philippines and Timor Island. Melon fruitflies have about 81 hosts including beans, cucumbers, melons, squashes, pumpkins, tomatoes, etc. and the common and usual hosts of melon fruit flies belong to Cucurbitacea family 19 . Voraciously feeding maggots happen to be the most damaging and cause serious damage to the infested crop wherever they occur causing serious economic loss 21 . Vegetation loss due to fruit fly infestation and damage ranges between 30 and100% and particularly in India, this pest has been reported to cause over 40-60% losses in vegetation 19,22 .
ITCs being GLTs hydrolytic products have already been reported to show biofumigant activity against a number of insect pests, especially stored grain pests, but there are no reports showing its post-ingestive insecticidal potency against the destructive tephritid pest, Z. cucurbitae. So, the present study aimed at ascertaining the effect of AITC on growth and developmental parameters of Z. cucurbitae. Moreover, the genotoxic effect of AITC on the insect pest was also evaluated to ascertain DNA damage induced by AITC in the hemocytes of the pest. Phenol Oxidase (PO), an important enzyme involved in insects' immune response along with some other detoxifying enzymes were also analyzed for their activity against AITC treatment.

Results
Insect bioassays. A significant inhibitory effect of AITC was observed on the various growth and developmental parameters of Z. cucurbitae. The percent pupation showed a significant concentration dependent decline (p ≤ 0.01) in all the three larval instars of Z. cucurbitae when fed with AITC amended artificial diet (Table 1). At 150 ppm, the decline in percent pupation was maximum in second instar maggots where it decreased by 75.91% as compared to controls. Complete larval mortality was observed at 200 ppm in all the three larval instars. The  The adult emergence too reduced significantly in all larval instars with increase in concentration of AITC. At highest concentration of 150 ppm, maximum reduction in adult emergence when compared to control was recorded in first larval instars (86.44%) (p ≤ 0.01) ( Table 2).
The observations for larval period also showed significant inhibitory effect of AITC treatment (Table 3). Compared to control, the larval period of first instar maggots shortened significantly with increase in concentration while that of second and third instar maggots prolonged significantly upto 50 ppm AITC concentration, and thereafter, was found to decrease. At highest AITC concentration, there was a shortening of larval period by 0.35 days and 0.19 days in the second and third instar Z. cucurbitae maggots, respectively when compared to control.
On the other hand, significant prolongation in pupal period and total development period was observed in all the three larval stages of Z. cucurbitae after the ingestion of AITC treated diet (p ≤ 0.01) (Tables 3 and 4).
After ingestion of AITC amended artificial diet, LGI and TGI was reduced in all the three larval instars of Z. cucurbitae (Table 5). Maximum reduction in the LGI was noticed in second instar maggots at highest  Nutritional assay. FA with respect to control decreased significantly from 20.26 mg at 5 ppm to 16.79 mg at 150 ppm (p ≤ 0.01) ( Table 6). Similar declining trend was observed in MRGR with maximum decrease at highest concentration (p ≤ 0.01). Larval weight gain (mg) per maggot decreased from 10.48 mg in control upto 6.62 mg at the highest concentration (p ≤ 0.01) ( Table 6).
Immune and detoxifying enzymes. The activity of PO, which plays a vital role in insect immunity, after 24 h of maggots feeding on AITC amended diet showed 38.83% inhibition as compared to control (p ≤ 0.01)   www.nature.com/scientificreports/ but thereafter, the activity was significantly induced in 48 h and 72 h treatment intervals as compared to control (p ≤ 0.01) (Fig. 4). The activity of antioxidant enzyme, SOD increased with increase in AITC exposure time with maximum increase (27.37%) observed after 72 h of AITC exposure as compared to control (p ≤ 0.01) (Fig. 4). CAT activity too increased after prolonged feeding of the maggots on AITC incorporated diet. At 48 h treatment interval, a 40.97% increase in CAT activity was recorded as compared to control (Fig. 4).
The activity of the detoxification enzyme, GST was significantly induced after consumption of AITC (p ≤ 0.01) (Fig. 4). With respect to control, its activity increased by 57.92% after 72 h treatment interval. On the other hand, the activity of EST was suppressed following AITC treatment of Z. cucurbitae maggots (Fig. 4). The suppression in enzyme activity was maximum after 24 h interval (43.79%) and was minimum at 72 h treatment interval (29.41%) with respect to controls (p ≤ 0.01).
The observations made for ACP revealed a decrease in ACP activity at 24 h and 48 h but at 72 h, a 9.45% increase in enzyme activity was observed as compared to control (p ≤ 0.01) (Fig. 4). AKP activity declined in a steady manner at all treatment intervals as compared to control groups (p ≤ 0.01) (Fig. 4).
Comet assay. AITC induced DNA damage was also investigated in hemocytes of Z. cucurbitae maggots using comet assay (Fig. 5).
The tail length (TL) was significantly increased in a concentration dependent manner in all the three treatment intervals (24 h, 48 h and 72 h) as compared to control but the increase in tail length was observed to be lesser in 48 h treatment groups at LC 50 concentration as compared to 24 h and 48 h exposure groups (Table 7). After 24 h treatment interval, it significantly increased from 10.45 μm in control to 25.25 μm and 41.50 μm in LC 30 and LC 50 concentrations of AITC, respectively (F = 2540.47, p ≤ 0.05). Similar trend was observed in 48 h (F = 405.07, p ≤ 0.05) and 72 h (F = 2270.06, p ≤ 0.05) exposure groups, however, the increase in tail length after 72 h treatment interval was maximum with a value of 42.47 μm in LC 50 treatment. Similar to tail length, the percent tail DNA values also increased significantly in both the treatment groups (LC 30 and LC 50 ) and at all the treatment intervals as compared to control (F 24h = 265.85, p ≤ 0.05; F 48h = 360.15, p ≤ 0.05; F 72h = 270.77, p ≤ 0.05) ( Table 7). In comparison to control, maximum value of 6.2% increase in percent tail DNA was observed in 72 h treatment interval.

Discussion
Bioassays. AITC, the hydrolysis product of aliphatic glucosinolates, has been reported to be toxic to a variety of organisms 4,23 . In our study, the exposure of AITC amended diet exhibited dose-dependent larval mortality resulting in a decline in percent pupation in all the larval stages of Z. cucurbitae. Although all the larval instars were lethally affected but the effect on early instars (first and second instar maggots) was greater than the third instar larval forms. Ingestive toxicity of GLTs containing extracts has previously been reported against larval forms of mosquito, Aedes aegypti (L.) (Diptera: Culicidae) and housefly, Musca Domestica (L.) (Diptera: Mus-  www.nature.com/scientificreports/ cidae) 24 . Bhushan et al. also reported insecticidal efficacy of AITC amended artificial diet against larval stages of lepidopteran insect pest, Spodoptera litura (Fab.) 18 . Li et al. 25 too reported insecticidal potential of AITC amended artificial diet against neonates of diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae) and southern armyworm, Spodoptera eridania (Cramer) (Lepidoptera: Noctuidae). Noble et al. exposed masked chafer beetle larvae, Cyclocephala spp., to soil amended with Brassica juncea L. tissue and reported that there existed a positive correlation between the AITC levels and larval mortality, with a mere 8% B. juncea demonstrating complete larval mortality containing mean AITC content of 11.4 mg/l of soil atmosphere 26 . AITC even at lower concentrations has been reported to be toxic to the immature larval stages of maize weevil, S. zeamais Motschulsky and cowpea weevil, Callosobruchus maculates (F.) (Coleoptera: Chrysomelidae) when applied as a fumigant 27,28 . Freitas et al. 29 and Jabeen et al. 30 also reported significant toxic and emergence inhibition effects of AITC on insects. The insecticidal toxicity of AITC has reportedly been attributed to its ability to disrupt the normal functioning of two mitochondrial complexes viz. Complex IV (primary target site) and Complex I (as secondary target site), thereby resulting in dysfunctioning of the tissue 31,32 . ITCs derived from GLTs have been reported to cause mortality, developmental retardation, and deformities in cotton bollworm, Helicoverpa armigera Hübner and in other lepidopteran insects 33,34 . In the present study, the larval period shortened with AITC treatment in first larval instar of Z. cucurbitae as compared to control but in the second and third larval instars, it showed prolongation at lower concentrations initially but at higher concentrations again got shortened with respect to controls. The shortening of larval period at higher AITC concentrations might be a defense strategy to avert its post-digestive toxicity. However, an overall delay in total developmental period of all the larval instars, attributed mainly to delayed pupal period, was observed when maggots were raised on AITC amended diet as compared to control groups. Agrawal and Kurashige 13 too reported that AITC decreased survival and growth, and prolonged development time in a dose-dependent manner in a specialist lepidopteran herbivore P. rapae (L.). Similar to our study, Jeschke et al. 35 also reported that the neonates of cotton bollworm, H. armigera, raised on GLTs demonstrated significantly prolonged developmental times for all stages.
The delay in developmental duration and late eclosion (i.e. delayed pupal period) with AITC exposure may also suggest quite a few detrimental inferences for the species fitness. A decline in growth rates (LGI and TGI) upon exposure of GLTs or their hydrolytic products, as in the present study for dipteran and in previous reports for different lepidopteran species 33 , may prove to be disadvantageous in a natural setting due to prolongation in exposure time to parasitoids and predators (Hypothesis: Slow growth-high mortality hypothesis) and also to prolonged time period in competing for the food resources. Moreover, late eclosion also comes with the overall fitness cost of producing lesser generations per season 33,36 . Nutritional assays. The decrease in MRGR, FA and Larval weight gain with respect to control in the present study also indicated either anti-feedant activity or post-ingestive toxicity induced by AITC amended diet. The delayed development observed in second and third instar maggots as compared to controls may also be correlated to decline in food assimilation efficiency in AITC treated maggots thereby reflecting the anti-nutritional efficacy of the test compound. The cotton bollworm larvae, H. armigera, while feeding on A. thaliana leaves demonstrated distressed feeding pattern avoiding the GLT-enriched parts particularly, midvein and the leaf edge confirming the deterrent activity of GLTs and their hydrolysis products 37 . Immune and detoxifying enzymes. AITC was observed to have a statistically significant influence on the activity of PO enzyme in Z. cucurbitae. The enzyme activity was inhibited during the initial 24 h treatment interval as compared to control. A study by Bai et al. 38 reported that the supression of PO activity resulted in delayed development in case of dipteran pest, Bactrocera dorsalis (Hendel). However, an upregulation in PO activity was observed on prolonged exposure (48 h and 72 h) of Z. cucurbitae maggots to AITC. Datta et al. 39 observed a similar induction in PO activity on prolonged exposure of S. litura (Fab.) to Alpinia galanga (L.) derived extracts and purified compounds eliciting an immune response. The ingestion of AITC in Z. cucurbitae larval gut stimulated the immune response via activation of PO system together with the activation of enzymes functioning upstream to prophenoloxidase (ProPO), for example ProPO-activating proteinase 40 and the consequent production of melanin may probably be linked to either mechanical or proteolytic damage of the epithelium of the gut rather endocytosis 41 . Activation of PO followed by melanisation process are related to production of toxic ROS, subsequent oxidative stress, and increased expenditure of energy, probably explaining the detrimental effects on developmental traits observed in Z. cucurbitae. Under natural conditions, these effects might compromise the survival fitness of the population by increasing the susceptibility of the pest to pathogens or other abiotic stressors 41 .
SODs play a key role in the antioxidant defense mechanism of insects and convert toxic O 2 − (superoxide anion) into O 2 (oxygen) and H 2 O 2 (hydrogen peroxide) 42,43 . In Z. cucurbitae maggots, the activity of SOD increased with increase in exposure time to AITC amended diet as compared to control. In accordance with our findings, Zhang et al. 44 also reported increased SOD expression following AITC exposure in S. zeamais.
In insects, in order to prevent H 2 O 2 generated via activity of SOD, from being transformed into a highly reactive -OH radical, CAT detoxifies this H 2 O 2 into H 2 O and O 2 45 thereby protecting an insect from detrimental effects of ROS that can attack and degrade various key biomolecules causing a functional decline 46 . In our study, CAT activity initially declined after 24 h AITC treatment compared to control but thereafter increased in 48 h treatment groups as compared to control. Wu et al. 31 reported similar variations in the CAT activity of AITC exposed adult maize weevil, S. zeamais. Zhang et al. 44  www.nature.com/scientificreports/ ITCs are usually detoxified via conjugation to the nucleophilic thiol (-SH) group of GSH (reduced glutathione) mediated by GSTs as reported in molluscs and generalist lepidopterans such as H. armigera, S. exigua, S. littoralis, cabbage moth, Mamestra brassicae (Linnaeus) and cabbage looper, Trichoplusia ni (Hübner) 47,48 . Interestingly, this pathway was also reported in dipteran Scaptomyza species larvae that are specialist leaf-miners on GLTs rich plants and do not utilize any extracellular biochemical mechanism to prevent formation of ITC, and instead intracellularly metabolize these compounds after being exposed 49 . In the present study, an induction in GST activity after consumption of AITC suggests its intracellular metabolization to some extent. Similar to our findings, GST activity was induced with AITC in adult S. zeamais 31 . AITC can irreversibly react with the protein and nucleic acid disulfide and thiol groups thereby causing inactivation of amine metabolic enzymes 31 .
The ESTs are vital detoxification enzymes protecting insects from toxic xenobiotics 50 . EST inhibiting activities, similar to our study, were reported for three ITCs including AITC in red imported fire ant, Solenopsis invicta Buren workers 23 . Similarly, ACP activity in the present study was significantly decreased with AITC at 24 h and 48 h treatment intervals and thereafter it increased in 72 h exposure group as compared to controls. AKP activity also declined in a steady manner at all treatment intervals as compared to control groups. Murfadunnisa et al. 51 reported that the sub-lethal dosage of phytochemicals from Sphaeranthus amaranthoides (Sa-EO) suppressed both ACP and AKP activities in S. litura larvae. ACP suppression or downregulation with AITC treatment suggests that the phytochemical had reduced the release of phosphorous for energy metabolism, probably resulting in reduced rate of metabolism 52 .
Comet assays. The comet assay has recently been adapted to assess the levels of genotoxicity or DNA damage in a number of insects' orders including Orthoptera, Diptera, Lepidoptera and Coleoptera 53 . In the present study, a statistically significant enhancement in all the comet parameters viz. TL (μm), Percent Tail DNA, TM and OTM was recorded in a dose dependent manner, thereby, advocating that the ingestion of AITC amended artificial diet caused significant DNA damage in the hemocytes of second instar maggots of the pest. Single strand breaks occur as a result of DNA damage thereby leading to cell apoptosis 54 and the formation or lengthening of tail is an indicator of the process of apoptosis as the cells undergoing apoptosis display nuclear fragmentation/ disintegration in the form of DNA tails 55 . Insect hemocytes are crucial in providing defensive functions during the growth period, and as such genotoxic damage to hemocytes might suppress the growth and developmental process 56 . Similar to our study, evaluation of genotoxicity of botanicals from A. galanga against S. litura also showed similar statistically significant increase in TL, percent tail DNA and OTM 57 . IGRs viz. pyriproxyfen and novaluron treated peach fruit fly, Bactrocera zonata (Saunders) (Diptera: Tephritidae) also exhibited a significant increase in DNA damage values of TL, % tail DNA and TM in the body cells as compared to control groups 58 .

Materials and methods
Flies. Zeugodacus cucurbitae culture was originally harvested from the fruit fly infested vegetables, pumpkins and bittergourds, taken from the vegetable markets and then maintained for many generations in the insect culture room of the Insect Physiology Division of the Department of Zoology, Guru Nanak Dev University, Amritsar, Punjab, India. A total of 7-8 insect rearing cages, each one carrying about 300-450 adult flies (males as well as females), were maintained. Every day, fresh pumpkin slices, 15-25% sugar/honey solution and proteinX were given as feed to the adult flies 59  www.nature.com/scientificreports/ Nutritional assay. The nutritional assays were performed using second larval instars only. Maggots were harvested, weighed and transferred to glass vials having artificial diet as in bioassays. For each concentration, there were 6 replications and a total of 15 maggots were released in each replica same as in bioassays. After 48 h of feeding, the maggots were taken out of the vials and weighed again to evaluate change in larval weight. The mean relative growth rate (MRGR) 62 and Food assimilated (FA) 63 were calculated as where, Ci = initial weight of control maggots, Cf = final weight of control maggots, Ti = initial weight of treated maggots, and Tf = final weight of treated maggots.
Immune and detoxifying enzymes. Enzymatic activities were estimated after 24 h, 48 h and 72 h feeding of second instar maggots on artificial diet amended with LC 50 concentration of AITC. There were 6 replications for each time interval and a total of 15 maggots were released in each replica same as in bioassays.
Phenol oxidase (PO). PO estimation was done using Zimmer's methodology 64 . Potassium sodium phosphate buffer (0.05 M, pH 6.2) was used to prepare the larval homogenate as enzyme extract. The assay mixture comprised catechol prepared in buffer (50 mM, pH 6.2) and the enzyme extract. At 340 nm and for 10 min, the increase in absorbance was recorded.
Catalases (CAT). For CAT estimation 66 , 5% (w/v) larval homogenate to be used as enzyme extract was prepared in potassium phosphate buffer (0.05 M, pH 7.0). The assay mixture consisted of enzyme extract and H 2 O 2 and at 240 nm and 25 °C, the decline in absorbance was recorded for a period of 3 min. www.nature.com/scientificreports/ Lysis buffer. Initially, 445 ml stock solution of the lysis buffer was prepared (73.01 g NaCl, 18.7 g EDTA, 0.6 g Tris and 4 g NaOH) in dH 2 O (pH 10) and then, DMSO (44.5 ml) and Triton X (4.95 ml) were added to the stock solution in order to prepare a working solution.

Glutathione-S-transferases (GST
Buffers: electrophoresis buffer and tris buffer. Again, Stock solutions of components of electrophoresis buffer viz. NaOH (40 g/100 ml ddH 2 O) and EDTA (7.44 g/100 ml ddH 2 O) were prepared separately. Then, 30 ml of NaOH and 5 ml of EDTA were taken and mixed with 965 ml of chilled ddH 2 O to form the working solution. Tris buffer (pH 7.4) comprised of 4.84 g of Tris buffer dissolved in 100 ml ddH 2 O.
Preparation of slide and electrophoresis. Base layer of 1% NMPA (Normal Melting Point Agarose) was applied over the glass slide and the slide was kept undisturbed for about 12-24 h. After 12-24 h, the second layer of 35 μl hemolymph sample mixed with 110 μl of 0.5% LMPA (Low Melting Point Agarose) was laid over the slide and the slide kept in refrigerator (4 °C) for about 15 min for gel casting. Finally, the top third layer of 0.5% LMPA was laid and again the slide was kept in refrigerator for the same period as in second layering. The slide was then completely immersed in the working lysis buffer for about 2-3 h at 4 °C in dark. After lysis, the slide was transferred to electrophoresis tank and then, electrophoresis buffer was poured upto a level that the slide was completely dipped in it. Following electrophoresis (300 mA, 20 V, 20 min), the slide was taken out and neutralized using Tris buffer. Finally, the slide was washed using chilled dH 2 O.
Staining and analysis. Staining was done using EtBr (ethidium bromide) and the slide was transferred under Fluorescent microscope (Nikon ECLIPSE E200), observed and photographed using camera (Nikon D5300). For each treatment, 150 cells were analysed using Casp Lab Software for Tail length (TL) (μm), Percent Tail DNA, Tail Moment (TM) and Olive Tail Moment (OTM) values to assess the degree of DNA damage.
Statistical analysis. The statistical analyses viz. ANOVA (One-way analysis of variance) and Tukey's test for comparison of means (p ≤ 0.05) for bioassays, nutritional assays and Comet assays were done using SPSS Statistical software Version no. 16. Data from biochemical work was also analysed using SPSS software by applying student 't' test.

Conclusion
The results of the present study, viz. larval mortality, growth inhibition, developmental delay, disruption of DNA integrity, alterations in profiles of immune and detoxifying enzymes and the reduced species fitness of the pest Z. cucurbitae after AITC treatment, indicated its high potential to be employed as a biopesticide for pest management studies especially for Z. cucurbitae. The study also provides baseline data that can be explored by plant breeders to make Z. cucurbitae resistant varieties by introducing AITC (or Allyl GLTs) encoding genes in those plants that are usually damaged by this destructive pest and do not form GLTs producing AITC.

Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.