Effect of gallic acid on the larvae of Spodoptera litura and its parasitoid Bracon hebetor

The antibiosis effect of gallic acid on Spodoptera litura F. (Lepidoptera: Noctuidae) and its parasitoid evaluated by feeding six days old larvae on artificial diet incorporated with different concentrations (5 ppm, 25 ppm, 125 ppm, 625 ppm, 3125 ppm) of the phenolic compound revealed higher concentration (LC50) of gallic acid had a negative impact on the survival and physiology of S. litura and its parasitoid Bracon hebetor (Say) (Hymenoptera:Braconidae). The mortality of S. litura larvae was increased whereas adult emergence declined with increasing concentration of gallic acid. The developmental period was delayed significantly and all the nutritional indices were reduced significantly with increase in concentration. Higher concentration (LC50) of gallic acid adversely affected egg hatching, larval mortality, adult emergence and total development period of B. hebetor. At lower concentration (LC30) the effect on B. hebetor adults and larvae was non-significant with respect to control. Gene expression for the enzymes viz., Superoxide dismutase, Glutathione peroxidase, Peroxidase, Esterases and Glutathione S transferases increased while the total hemocyte count of S. litura larvae decreased with treatment. Our findings suggest that gallic acid even at lower concentration (LC30) can impair the growth of S. litura larvae without causing any significant harm to its parasitoid B. hebetor and has immense potential to be used as biopesticides.


Results
Effect on growth and development of S. litura larvae. Gallic acid incorporated diet when fed to the six days old larvae of S. litura significantly affected the growth, development and survival of S. litura larvae ( Table 1). In gallic acid treated S. litura larvae, the larval mortality significantly increased in a dose dependent manner from 3.33% in control to 70% at 3125 ppm. The percentage adult emergence decreased with increase in concentration. No adult emerged at the highest concentration of 3125 ppm. At 625 ppm the adult emergence was inhibited by 73.33% when compared with control. The larval period and total developmental period were delayed significantly after feeding larvae with diet having gallic acid when compared with control. The larval period was delayed by 13.52 days at 3125 ppm concentration while delay of 10.37 days in total developmental period was observed at 625 ppm as compared to control. The pupal weight was also significantly lower at all the concentrations when compared with control. The LC 30 and LC 50 calculated on the basis of larval mortality were 52.44 ppm and 402.80 ppm concentrations, respectively. Growth and survival of azadirachtin treated positive control S. litura larvae was more adversely affected when compared to gallic acid fed larvae. At higher concentration of 3125 ppm, 93.33% larval mortality was observed in azadirachtin fed larvae ( Table 2). The larval period and total developmental period were also more prolonged in azadirachtin treated larvae. The LC 30 and LC 50 of azadirachtin fed larvae was 5.981 ppm and 26.551 ppm, respectively.
Effect on the nutritional physiology of S. litura. The nutritional physiology of the larvae was also negatively affected in gallic acid treated larvae as evident from observations recorded for various nutritional indices ( Table 3). The AD (approximate digestibility) of the larvae declined with treatment in comparison to control. The ECD (efficiency of conversion of digested food) and ECI (efficiency of conversion of ingested food) showed a significant concentration dependent decline. At the highest concentration of 3125 ppm the ECD decreased by 56.69% and ECI decreased by 64.16% respectively when compared with control. The RGR (relative growth rate) and RCR (relative consumption rate) also decreased significantly in the larvae of S. litura with increasing concentration of gallic acid incorporated in diet. At 3125 ppm, a 87.88% reduction in RGR and 65.37% reduction in RCR was noticed as compared to control. The nutritional physiology of the larvae was more adversely affected when fed on azadirachtin treated diet when compared to control and gallic acid treated larvae (Table 4). Table 1. Larval mortality, adult emergence, larval period, pupal period, total developmental period, pupal weight of S. litura when 6 days old instar larvae were fed on different concentrations of gallic acid. **Significant at 1%, ns Non-Significant. Values are Mean ± SE. Means followed by the same letter within the columns are not significantly different according to Tukey's test at p ≤ 0.05.

Concentrations (ppm)
Larval mortality (%) Adult emergence (%) Larval period (days) Pupal period (days)  Effect on B. hebetor adults. Gallic acid showed no significant effect on female fecundity, percent parasitization and the eggs laid per host at both LC 50 and LC 30 concentrations when compared to control (   www.nature.com/scientificreports/      30, it was reduced by only 13.52% as compared to control. The inhibition in female emergence was also greater at LC 50 than at LC 30 but when compared to positive control an increase in emergence was observed. The larval period and the pupal period were not significantly affected at both the concentrations when compared with control but significant increase in total development period was observed with increase in concentration from LC 30 to LC 50 concentration of gallic acid provided to the host with respect to control. Azadirachtin treated S. ltura larvae had more significant adverse affect on growth and survival of B. hebetor at both LC 30 and LC 50 when compared with gallic acid fed larvae. Azadirachtin was found to have more toxic effect at third trophic level (Table 8).

Discussion
The artificial diet containing gallic acid when fed to 6 days old S. litura larvae had negative effects on the larvae as well as on its parasitoid, B. hebetor. The larval mortality increased and adult emergence decreased under the influence of gallic acid in a concentration dependent manner. Selim-Rani et al. 21 had also reported that quercetin isolated from Euphorbia hirta L. caused mortality and cellular deformities in second, fourth and fifth instar larvae of S. litura. Ghumare and Mukherjee 22 had reported that S. litura larvae develop better on plants having lower phenolic content. Ananthakrishnan et al. 23 had also reported the excessive defecation along with formation of muscular lesions in the posterior half of the S. litura larvae after consumption of gallic acid supplemented diet. Prolonged larval and total developmental period of S. litura larvae was also noticed. Inability of S. litura larvae to develop on artificial diet containing phenolic rich extracts of red gram (Cajanus cajan L.) and Acacia nilotica (L.) has been reported by Bhattacharya and Chenchiah 24 and by Gautam et al. 25 , respectively. Gallic acid, hydrolysable tannin can interact with metal ions and macromolecules such as polysaccharides. It can also form soluble complexes with proteins and can inhibit digestive enzymes and is thus toxic to insects 26 . The nutritional indices were reduced significantly with increase in concentration of gallic acid. Ingestion of gallic acid incorporated diet decreased with a consistent decrease in RGR of S. litura larvae. Gallic acid seems to have inhibited growth by either exerting an antifeedant or toxic effect on the S. litura larvae. Both ECI and ECD declined with increase in concentration of gallic acid. The decrease in ECD and ECI of S. litura larvae reflect increased metabolic cost which might have occurred due to the energy required for detoxification of the compound. These findings indicate toxicity of gallic acid to S. litura larvae was due to the inhibitory effect on digestion and reduced efficiency of conversion of assimilated food into biomass. Toxic effects of gallic acid have also been reported against other insects such as melon fruit fly, Bactrocera cucurbitae (Coquillett) larvae 27 . Our findings indicated that azadirachtin incorporated diet was more toxic to S. litura as compared to gallic acid incorporated diet. The toxic effects of azadirachtin on feeding and mortality of S. litura was also reported by Nathan and Kalaivani 28 and Deota and Upadhyay 29 . Azadirachtin induced structural changes in the S. litura larval midgut by activation of apoptosis 30 .
Studies on tritrophic interactions involving plants, insects and their parasitoids have made significant contribution towards understanding the role of plant traits such as induced volatiles on host location and acceptance behavior by natural insect enemies 31 . However, reports linking plant allelochemicals and parasitoids are very few. Most of these reports have highlighted the adverse effects of plant allelochemicals on parasitoid development. Camphell and Duffey 32,33 had reported higher mortality rates, morphological deformities, increased development time, decreased adult weight and longevity of Hyposoter exiguae (Viereck) developing in caterpillars of tomato fruitworm, Heliothis zea (Boddie) fed on artificial diet containing α tomatine, a glycol alkaloid as compared to those fed on control diet. Rutin, a phenolic compound also exerted indirect negative effects on H. exigua fed on treated H. armigera 34 . Linear furanocoumarin too was reported to increase larval mortality of the host S. exigua and its parasitoid Archytas marmoratus (Townsend) 8 . Corroboratory results were obtained for the parasitoid, B. hebetor when it was allowed to parasitize S. litura larvae which had been fed gallic acid incorporated diet. However, it was noticed that these effects were significantly greater at higher concentration of gallic acid (LC 50 ) than at lower concentration (LC 30 ) when compared to control. Larval mortality of B. hebetor larvae increased and adult emergence decreased as the concentration of gallic acid supplemented in diet of S. litura larvae increased from LC 30 to LC 50 . While the larval period and pupal period of B. hebetor larvae was not significantly affected, the time taken by the larvae to develop into adults also showed a concentration dependent increase. These findings Table 8. Development and survival of B. hebetor larvae emerged from S. litura larvae fed on gallic acid and azadirachtin supplemented diet. **Significant at 1%, *Significant at 5%, ns Non significant. Values are Mean ± SE. Means followed by the same letter within the columns are not significantly different according to Tukey's test at p ≤ 0.05.GA (Gallic acid), AZA (Azadirachtin).

Concentrations (ppm)
Hatching (%) Larval mortality (%) Larval period (days) www.nature.com/scientificreports/ indicated that toxicity of gallic acid to S. litura as well as its parasitoid increased with increase in concentration. Similar to our findings, El-Heneidy et al. 35 had reported that high levels of nicotine in artificial diet of S. frugiperda (JE smith) lowered the survival rate of larvae of its parasitoid, Hyposter annulipas (Coresson), prolonged the developmental time and resulted in small sized adults. Reitz and Trumble 36 had also observed that fewer adults of parasitoid, Copidosonma floridarum (Ashmead) emerged from broods developing on cabbage looper, Trichoplusia ni (Hubner) fed on artificial diet containing higher concentration of three furanocoumarins viz., psoralen, xanthotoxin and bergapten when compared to diet having lower furanocoumarin concentrations. Gallic acid also affected the hatching of B. hebetor larvae which was considerably less in S. litura larvae fed LC 50 concentration than at LC 30 concentration when compared to control. Mondy et al. 37 had observed that nutrients obtained from host affect egg viability and hatching in parasitoids. S. litura larvae feeding on diet having higher levels of gallic acid may have been a poor quality host which could have been due to diversion of energy and resources to detoxify the phenolic acid or could have resulted from gallic acid toxicity. Punia et al. 38 also reported deterrence of host quality under the influence of ellagic acid which indirectly impacted the survival of its parasitoid, B. hebetor. Barbosa and Saunders 39 had reported that toxic substances in plants retard growth, reduce vigour or kill susceptible herbivores and can cause physiological or metabolic changes in parasitoids.
Increasing concentration of gallic acid provided to S. litura larvae decreased parasitization by B. hebetor adults. The eggs laid per host as well as fecundity of B. hebetor adults decreased in the treated larvae. However the decrease was not significant. Furanocoumarins viz., isopimpinellin and xanthotoxin were also found to decrease the parasitization rate and clutch size of both male and female of polyembryonic parasitoid, Copidosoma sosares (Walker) when reared on hosts fed on higher concentrations of the compounds 40 .
Growth inhibitory effects of gallic acid were less severe on parasitoid when compared to positive control (azadirachtin). The toxic effect of azadirachtin on larval stages and adult emergence of Trichogramma chilonis Ishii was also reported by Narendra et al. 41 . Commercial formulations of azadirachtin had also been reported to negatively affect the life table parameters of Habrobracon hebetor (Say) by Abedi et al. 42 . Our findings showed 93.33% mortality of S. litura larvae at higher concentration of azadirachtin. In field conditions this may indirectly affect the survival of natural enemies as the mortality of prey population is high. This would result in reduction of food source for natural enemies and thus lead to decline in natural enemy population through starvation 43,44 .
Hemocytes form an important component of immune system in insects. Plant metabolites can impact parasitoid survival by altering the immune response of insects 45 . In the present study a significant decline in total hemocyte count and increase in hemocyte deformity was observed in S. litura larvae when fed on gallic acid treated diet when compared with control. Similarly, Ayyangar and Rao 46 had also reported significant decline in total haemocyte count in the haemolymph of final instar larvae of S. litura when treated with azadirachtin. Zibaee and Bandani 47 had also reported a significant dose dependent decline in total hemocyte count of adults of sunn pest, Eurygaster integriceps (Puton) when fed on diet containing Artemisia annua extract. Crude aqueous leaf extract of C. inerme had also resulted in lower haemocyte count in the 6th instar larvae of the cotton bollworm, Helicoverpa armigera (Hubner) 48 . Plumbagin, a phytochemical, was also found to drastically reduce the haemocytes of red cotton bug, Dysdercus koenigii (F.) 49 . The findings indicate that feeding on gallic acid incorporated diet compromised the cellular immune response of S. litura larvae and thereby increased its susceptibility to attack by the parasitoid.
Oxidation of phenolic compounds generate free radicals which can affect the growth and survival of insects 50,51 . Upregulated expression of genes for the antioxidant enzymes, SOD, Glutathione peroxidae and POX indicate their involvement in mitigating oxidative stress in the larvae generated by the ingestion of gallic acid incorporated diet. Also the expression of the genes encoding esterases and GSTs increased in S. litura larvae with treatment. Insects show resistance to allelochemicals via short term induction of detoxification enzymes 52 . The involvement of the GST super family in the detoxification of various plant xenobiotics has been reported by Ref. 53 . Various xenobiotics and endogenous compounds, including insecticides, drugs, insect hormones, organic solvents, allelochemicals and host plants lead to the induction of detoxification enzymes viz., glutathione transferases and esterases 54 . Li et al. 55 also reported the involvement of esterase in allelochemical metabolism.
It can be concluded from the present findings that gallic acid at lower concentrations impaired the growth of S. litura but only slightly affected the development of B. hebetor. However higher concentrations of gallic acid were toxic to both the host as well as its parasitoid. This study could provide baseline data to plant breeders for enhancing resistance traits in plants against insect pests without causing any significant harm to their parasitoids.
Spodoptera litura rearing. The S. litura were reared on fresh castor leaves, Ricinus communis (L.) under standard conditions in the Insect Physiology laboratory of Guru Nanak Dev University, Amritsar. The glass jars (15 cm × 10 cm) having fresh castor leaves, R. communis were used for culturing until pupation. On pupation, the pupae were transferred to pupation jars (15 cm × 10 cm) which had 4-5 cm of moist sand covered with filter paper. On emergence, the adults were shifted to oviposition jars (15 cm × 10 cm) lined with filter paper to facilitate egg laying, secured with muslin cloth to check their escape and provided with cotton swab soaked with water and honey solution (4:1) to serve as food.
Bracon hebetor rearing. The parasitoid, B. hebetor was reared on 5th instar larvae of C. cephalonica. The culture of C. cephalonica was reared on partially crushed sorghum grains at standard conditions of temperature (25 ± 2 °C) and humidity (65 ± 5% RH). The freshly emerged adult parasitoids in ratio 1:2 (male:female) were Scientific Reports | (2021) 11:531 | https://doi.org/10.1038/s41598-020-80232-1 www.nature.com/scientificreports/ transferred to glass chimneys having a cotton swab soaked in water and honey (4:1) solution to serve as food. The parasitoid, B. hebetor was provided with healthy 5th instar larvae of C. cephalonica for parasitization and parasitized larvae were observed for egg laying under microscope and were then kept inside tissue papers in plastic petri plates (90 mm × 15 mm) until cocoon formation. The cocoons formed were shifted into sterile solo cups (4 cm × 6 cm) and the adults emerged were allowed to mate for 24 h. These newly emerged B. hebetor larvae were then used for bioassay studies.
Chemicals used. Gallic acid and azadirachtin with 95% purity were obtained from Sigma Aldrich Pvt. Ltd., India. Nutritional assays with S. litura. A 3 days experiment was conducted with 6 days old larvae of S. litura according to Koul et al. 56 using different concentrations of gallic acid and azadirachtin (5 ppm, 25 ppm, 125 ppm, 625 ppm and 3125 ppm) along with control. The larvae were weighed and released into sterilized plastic container already containing weighed treated and control diet. The larvae were allowed to feed for 72 h and then annotations were made for final larval weight, diet left and fecal matter (in mg) and the dry weights for the same were taken after incubating for 72 h at 60 °C inside an incubator. The dry weight readings served the purpose of water loss under controlled conditions. From this data, the following nutritional indices were calculated on dry weight basis after 3 days of feeding as proposed by Waldbauer 57 . Each concentration including control was replicated 6 times with 5 larvae in each replicate and the experiment was repeated twice.

Conduction of experiment.
where RGR = Relative growth rate, RCR = Relative consumption rate, ECI = Efficiency of conversion of ingested food, ECD = Efficiency of conversion of digested food, AD = Approximate digestibility.
Bioassays with B. hebetor. The effect of gallic acid and azadirachtin on parasitization and fecundity of B.
hebetor was studied by allowing the newly emerged adults to mate for 24 h and the mated adults were then transferred to glass chimney in the ratio female: male (2:1). The rearing of S. litura larvae upto 3rd instar stage was done on diet amended with LC 30 and LC 50 concentrations of gallic acid as well as on control (unamended diet). A single treated 12 days old S. litura larvae was then exposed to parasitoid wasp for 24 h in the chimney and then the parasitized larvae were transferred on tissue paper in the petriplates (90 mm × 14 mm). The paralyzed larvae were observed daily under stereo microscope (Magnus) at 40X magnification to observe parasitoid eggs for various parameters. In this way, S. litura larvae fed on different concentrations of gallic acid in artificial diet were exposed daily till the female died in each treatment. www.nature.com/scientificreports/ fresh hosts were provided to the wasps daily. The larvae removed from the chimney were placed individually in petri plates (90 mm × 14 mm) and were allowed to complete development. The petri plates were checked daily for development of parasitoid larvae. Cocoons of the parasitoid were also checked daily and the time of emergence of wasps was recorded. On emergence, the number and sex ratio of the progeny were noted. The experiment was replicated six times with two females per replicate and a total of 60 larvae were exposed to parasitoid wasps for each treatment and control.

Cellular immune response of S. litura.
To evaluate the effect of gallic acid on immune response of S. litura, 3rd instar larvae (12 days old) were fed on artificial diet supplemented with LC 30 and LC 50 concentrations for different time intervals i.e. 24, 48, 72, 96 and 120 h. The treated larvae along with the larvae fed on control diet were kept at standard conditions of temperature and humidity i.e. 25 ± 2 °C and 65 ± 5%, respectively. The hemolymph was collected by piercing the prothoracic legs with a sterile needle. For each time interval, ten larvae were randomly selected from each treatment group and the hemolymph was collected. The pooled hemolymph was used to study the total haemocyte count (THC) and deformities in haemocytes according to the protocol of Tauber and Yeager 58 and Arnold and Hinks 59 , respectively. All experiments were replicated twice.
Gene expression of antioxidant enzymes. The relative expression of genes related with different antioxidant and detoxifying enzymes was measured using quantitative RT-PCR in the larvae of S. litura treated with the LC 50 concentration of gallic acid at different intervals (48 and 72hours). The total RNA was extracted using Trizol method (Invitrogen). The quality and concentration of total RNA was checked using agarose gel electrophoresis (1%) and nanodrop spectrophotometer. The iScript cDNA synthesis kit of Biorad was used to synthesize cDNA from 1.0 μg of total RNA. The cDNA obtained was stored at − 20 °C for further use after a dilution of tenfold. The mRNA sequences of various genes used to design primers of genes of interest were obtained from NCBI and actin was used as an internal reference gene ( Ethical approval. This article does not contain any studies with human participants or animals performed by any of the authors. Table 9. Primer sequences used for gene expression analysis using qRT-PCR.