Helicoverpa armigera HÜbner is one of the most important agricultural crop pests in the world causing heavy crop yield losses. The continued and indiscriminate use of synthetic insecticides in agriculture for their control has received wide public apprehension because of multifarious problems, including insecticide resistance, resurgence of pest species, environmental pollution, and toxic hazards to humans and nontarget organisms. These problems have necessitated the need to explore and develop alternative strategies using eco-friendly and biodegradable plant products. In view of this, the efficacy of Thevetia neriifolia methanol stem extract was evaluated against the early fourth instars of H. armigera as an antifeedant and stomach poison agent. Feeding of larvae with the diet containing 0.005%–5.0% extract resulted in 2.06%–37.35% antifeedant index; the diet with 5.0% extract caused 54.3% reduced consumption. The negative impact of extract on larval feeding resulted in 37.5%–77.7% starvation, causing adverse effects on the larval weight. Choice between control and experimental diet resulted in feeding preference of larvae for the control diet, leading to 7.3%–42.9% reduced consumption of extract-containing diet. The only exception was the diet with 0.005% extract, which could not cause any deterrence. The midgut histological architecture of H. armigera larvae fed with 0.005%–0.05% extract-containing diet with negligible antifeedant potential showed significant damage, shrinkage, and distortion and vacuolization of gut tissues and peritrophic membrane, causing the disintegration of epithelial, goblet, and regenerative cells; the damage increased with the increase in concentration. These changes in the gut caused negative impact on the digestion and absorption of food and thus nutritional deficiency in the larvae, which could probably affect their growth and development. This study reveal the appreciable stomach poison potential of T. neriifolia stem methanol extract against H. armigera larvae, which can be explored as an eco-friendly pest control strategy.
Introduction
Helicoverpa armigera HÜbner (Lepidoptera: Noctuidae), cotton bollworm, is one of the most important agricultural crop pests in the world and inhabits diverse ecological habitats, leading to heavy yield losses in a diverse range of crops. It is also widely distributed in India and attacks varied plant species, leading to substantial losses.1 In the last few decades, the repeated and indiscriminate use of synthetic chemicals in the crop field of India has led to the resurgence and outbreak of bollworm with resistance to insecticides, elimination of existing natural enemies and environmental pollution. Reports are available that reveal the development of resistance in the pest to almost all the insecticides used for its control.2 Environmental hazards posed by synthetic pesticides provide an impetus for investigations into more plant-based alternatives.3
Plants have been widely known to be gifted with the potential to produce an extensive range of phytochemicals, which protect them from phytophagous pests. Plant-derived phytochemicals have been used in the management of agricultural pests, though their production varies from plant to plant.4 The selective though variable toxic potential of these chemicals against target pests, rapid degradability in the field, and low toxicity to beneficial organisms have made them appealing options for agricultural use. Certain parameters, such as age, location within, and response to the pest attack, can influence the production of phytochemicals and thus affect the insect feeding and oviposition on the plants.5 Various plants, such as Tephrosia vogelii (family: Fabaceae) and Solanum pseudocapsicum (family: Solanaceae), have been reported to be efficient in the management of H. armigera.6,7 The efficacy of certain plant extracts, such as larvicides, ovicides, antifeedant, and growth regulators against H. armigera, has supported their use in the field.89–10 The antifeedant activity of leaf aqueous extracts of 25 medicinal plants has been revealed against VI instars of H. armigera (HÜbner).11 The aqueous extracts of Gnidia glauca and Toddalia asiatica have also been evaluated for their antifeedant activity against H. armigera.12 Abbaszadeh et al13 reported that the extracts of Indian bhant tree, Clerodendron infortunatum L., could be used by the local farmers to control H. armigera populations due to the powerful antifeedant activity of clerodane diterpenoids present in the extracts. In addition to the impact of extracts on the feeding potential of H. armigera, it has been reported that feeding H. armigera larvae with plant extract-treated diet can cause alteration in the midgut tissues, suggesting their actions as stomach poisons.14 Similar effects have been reported in other insects, such as Spodoptera frugiperda, fall armyworm, and Anticarsia gemmatalis, velvetbean caterpillar, when their immature stages were fed with diets treated with plant extracts or isolated phytochemicals.15,16 The results of such feeding studies suggest that as the midgut region of insects is the principal site for the secretion of digestive enzymes, digestion of food, and absorption of nutrients, alteration in its morphology and histology can lead to considerable physiological changes affecting the growth and development of the insect pest.1718–19
Thevetia neriifolia (family: Apocynaceae), commonly called as yellow oleander plant, is an evergreen tropical shrub or small tree that grows well in warm subtropical regions. The plant has been reported to contain cardiac glycosides and cardenolides, while its seed oil has been used to make paints with antifungal and antibacterial properties.20 A number of phytochemicals, specifically cardiac glycosides, have been isolated from T. neriifolia with digitoxin and found to be the most potent.21 Though the plant has been reported as toxic against rats,22 the efficacy of the plant has not been explored much against insect pests. As T. neriifolia is commonly found in India, the objective of this study is to evaluate the effects of methanol extract, prepared from the stems of T. neriifolia, on the feeding behavior and the gut histology of H. armigera.
Materials and Methods
Rearing and Maintenance of H. Armigera under Controlled Conditions
The colony of H. armigera was maintained in the laboratory under the controlled conditions of 27 ± 1°C and 80 ± 5% relative humidity (RH) with a 12-hour:12-hour light/dark photoregime. Larvae of H. armigera were reared on artificial diet prepared using chickpea flour, casein, and yeast as the main components and certain necessary nutrients in traces and agar as the solidifying agents.23
The neonates of H. armigera were reared in groups for about seven days till they develop into third instars. Thereafter, to prevent cannibalism, individual larva was transferred to separate clean and sterilized Petri dishes (7 cm diameter) for further rearing. Pupae were sterilized with 0.5% sodium hypochlorite solution, washed with distilled water, and kept inside the plastic cages (15 X 15 cm) for adult emergence. Adults were fed on 10% sucrose solution that was kept in small vials in the cages. After a period of two days, 10 pairs of moths were selected, and each pair was released in a separate oviposition jar (2000 mL) covered with clean muslin cloth on the top for oviposition. The egg-laden cloth was replaced each day with the fresh cloth till the oviposition continued. The eggs collected were then allowed to hatch in a tight-lid box having tiny holes on the lid's surface.
Plant Collection and Preparation of Extracts
The stems of the T. neriifolia plant were collected from the premises of Acharya Narendra Dev College, New Delhi, India. Healthy and disease-free stems were separated for assays and carefully washed with water. After drying under the shade at 27 ± 2°C for ∼20 days, the stems were crushed, finely powdered, and sieved (150 µm) thoroughly. The fine powder of the stems thus obtained was weighed, and 200 g was extracted with 1000 mL of methanol for 24 hours at a temperature not exceeding the boiling point of the solvent (64.7°C) using Soxhlet apparatus. The crude extract (7.21 g) produced was concentrated using a Buchi-type rotator vacuum evaporator at 45°C under low pressure and stored in a refrigerator at 4°C as the stock solution for further use.
Preparation of Extract-Containing Artificial Diet
The artificial diet containing T. neriifolia stem methanol extract was prepared by replacing 1 mL of the 200 mL dry diet with that of the desired concentration of the extract. The diet was left undisturbed for the evaporation of solvent after which it was solidified using agar. The diet prepared by replacing 1 mL of the 200 mL with only solvent instead of the stem extract served as the negative control.
Antifeedant (no-choice) bioassay against early fourth instars of H. armigera.
The antifeedant potential of T. neriifolia stem methanol extract was evaluated against the early IV instars of H. armigera, the early fourth instar of H. armigera being the most actively feeding and thus the most damaging stage. The larvae were provided with 1 g of the diet containing extracts at 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1.0%, and 5.0% concentrations separately. The diet containing only solvent, instead of extract, served as the control diet. The diet, weighed using Shimadzu-BL220H electronic balance, was kept in the Petri plates lined with moist filter paper to avoid drying of the diet. For each concentration, 10 Petri plates were prepared.
A total of 10 IV instar larvae were selected, starved for four hours, and weighed. Individual larva was released at the center of separate Petri plates and kept in biochemical oxygen demand incubator under the controlled conditions of rearing. After a feeding period of 24 hours, the weight of the diet left in the Petri plates was measured. A total of five replicates were carried out for each assay, making a total of 50 larvae investigated at each concentration. The antifeedant index was calculated using the following formula24:
where C represents the weight of control diet (0.0% extract) consumed and T value indicates the weight of the extract-containing diet consumed by the early fourth instars. The results of % consumption of control diet and extract-containing diet were analyzed by one-way analysis of variance (ANOVA), and means were separated using Tukey's test for statistical significance considered for P ≤ 0.05.
Food Preference (Choice) Bioassay against Early Fourth Instars of H. Armigera.
The food preference assay was also carried out against early fourth instars of H. armigera by providing them a feeding choice between control diet and a diet containing stem methanol extract of T. neriifolia. Each Petri plate was divided into two halves: one half with 1 g of control diet and the other half with 1 g of experimental diet.
Individual early fourth instars of H. armigera was released in the center of each Petri plate and allowed to feed for 24 hours. The side of diet was changed after every six hours to negate positional error. After 24 hours, weight of each larva and the diets remaining in the Petri dish were recorded for all the dishes. For each assay, five replicates, each with 10 larvae, were carried out. Percent consumption of each diet was calculated as follows25:
The preference or choice index was estimated by dividing % consumption of experimental diet by % consumption of control food. The values >1 indicate the preference for extract-containing diet, whereas the values <1 indicate the preference for control diet. The antifeedant index was estimated as described in the choice assay.
The % diet consumption in control at each concentration was analyzed by one-way ANOVA, and means were separated using Tukey's test for statistical significance considered for P ≤ 0.05.
Starvation Index Assay
A total of 50 larvae of IV instars of H. armigera were selected and weighed. The larvae were starved for 24 hours and reweighed. The weights of these larvae were compared with the initial and final weights of each larva during no-choice bioassay carried out at 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1.0%, and 5.0%. Percent starvation for each assay was calculated according to the following formula:
where E is the mean weight gain of larvae fed on experimental diet at each assayed concentration within 24 hours, C represents the mean weight gain of larvae fed on control diet within 24 hours, and S is the mean weight gain of starved control larvae within 24 hours.
Impact of the Extract-Treated Diet on Midgut Epithelium
The impact of the T. neriifolia stem methanol extract as stomach poison against IV instars of H. armigera was investigated by evaluating the changes in their gut epithelium. The extract concentrations with antifeedant potential, 0.1%, 0.5%, 1.0%, and 5.0%, were rejected for the assay. The evaluation was carried out at 0.005%, 0.008%, 0.01%, 0.02%, and 0.05%. A total of 10 larvae fed with diet containing selected concentration of extract and 10 larvae fed with control diet were selected randomly and sacrificed. The alimentary canal was exposed, and the midgut was separated. After fixation in the Bouin's solution, standard histological techniques were used to cut midgut sections using a microtome. The thickness of the sections was preset between 4 and 6 µm. The sections were carefully transferred on a clean slide and stained with hematoxylin and counterstained with 70% eosin. The sections were observed under light microscope and photographed using a Canon PowerShot SX50 HS digital camera.
Results
The results of the antifeedant potential of the methanol stem extract of T. neriifolia investigated against H. armigera larvae presented in Tables 1 and 2 reveal the moderate antifeedant efficacy of the extract. The antifeedant assay carried out with diet containing 0.005%–5.0% stem methanol extract of T. neriifolia revealed the antifeedant index ranging from 2.06 to 37.35 (Table 1). The diet added with 0.005%, 0.01%, and 0.05% extract did not show significant antifeedant potential (P > 0.05), causing only 4.1%–20.1% reduced diet consumption. On the other hand, the larvae exhibited significant reduced consumption of 54.3% (P < 0.0001), as compared to the consumption of the control diet, when diet was added with 5% stem methanol extract of T. neriifolia.
Table 1.
Antifeedant no-choice assay against early IV instars of Helicoverpa armigera when fed on 1 g artificial diet containing stem methanol extract of Thevetia neriifolia.
When the early fourth instars of H. armigera were given a feeding choice between control diet and the diet containing T. neriifolia stem methanol extract, the larvae exhibited appreciable feeding preference over control diet. The only exception observed was with the diet containing 0.005% extract that could not deter feeding in the larvae (P > 0.05). Furthermore, the addition of diet with 0.05% extract caused only 7.3% reduction in diet consumption (P > 0.05) as compared to the control diet indicating insignificant antifeedant potential (Table 2). In contrast, the diet containing 0.5%–5.0% T. neriifolia stem methanol extract reduced feeding considerably from 10.9% (P < 0.05) to 42.9% (P < 0.0001). Our results show that at higher concentrations, T. neriifolia methanol stem extract showed higher antifeedant potential during choice bioassay in comparison to the no-choice bioassay, whereas at lower concentrations, the extract exhibited more antifeedancy during no-choice assays than that in choice assays.
Table 2.
Food preference (choice) assay against early IV instars of Helicoverpa armigera when fed on 1 g artificial diet containing stem methanol extract of Thevetia neriifolia.
Present studies revealed that the diet containing 0.005% and 0.05% T. neriifolia stem methanol extract did not show significant antifeedant potential (P < 0.05) in the larvae. Thus, the extracts in the range of 0.005%–0.05% were selected to evaluate their effects on the growth and development of larvae.
When the early fourth instars of H. armigera were starved for 24 hours, a weight decrease of 13% was observed, while feeding the larvae with the control diet resulted in 354% increase in their weight. However, when the larval weight was recorded after they were fed with extract-containing diet for 24 hours, a significant reduction in the larval weight, resulting in 37.5%–77.7% starvation, was observed. The larval weight further decreased with increase in the concentration of stem methanol extract in the diet (P < 0.05) (Table 3).
Table 3.
Starvation Index of early IV instars of Helicoverpa armigera when fed on 1 g artificial diet containing stem methanol extract of Thevetia neriifolia.
The study of the midgut tissues of H. armigera larvae fed on control diet and a diet with extracts showed remarkable changes in the epithelium amplifying with the increase in the concentration of the extract. The larvae fed on the control diet exhibited large, elongated, and columnar epithelial cells (ECs) of uniform sizes surrounding the wide gut lumen (GL) that was lined by peritrophic membrane (PM). Each EC was distinctly nucleated and had smooth and complete base (Fig. 1A and B). The epithelial layer was in close contact with the basement membrane (BM) with negligible space in between (Fig. 1B). Groups of large spongy digestive mucous secreting goblet cells (GCs) were observed at the bases of columnar cells with their inner ends projecting into the GL. Smaller basal cells, regenerative cells (RCs), were found in the form of patches at the bases of ECs (Fig. 1A).
Figure 1.
(A) T.S. of the midgut of IV instars of Helicoverpa armigera fed on control diet (40X); (B) Enlarged view of the midgut section.
Abbreviations: BM, Basement membrane; EC, Epithelial cell; GC, Goblet cell; GL, Gut lumen; MV, Microvilli; NU, Nucleus; PM, Peritrophic membrane; RC, Regenerative cell.
The midgut of the larvae fed on the diet with 0.005% and 0.008% stem methanol extracts of T. neriifolia showed slight but distinct shrinkage and distortion of the absorptive ECs coupled with altered shape and structure (Fig. 2A and B). The GCs and RCs were not distinctly visible, but the coherent sheet of epithelial layer was found to be in close contact with the BM. The increased concentration of the extract in the diet resulted in further decreased absorptive surface area and increased GL (Fig. 2C). More severe results were obtained when larvae were fed on the diet with 0.02% extract, ECs showing increased disruption with augmented vacuolization (Fig. 2D). Detachment of the epithelial layer from the BM resulted in the appearance of the large space detached area (DA).
Figure 2.
T.S. of the midgut of IV instars of Helicoverpa armigera fed on the diet treated with Thevetia neriifolia stem methanol extract (40X) at (A) 0.005%; (B) 0.008%; (C) 0.01%; (D) 0.02% and (E) 0.05%; (F) Enlarged view of the midgut section of the larva fed on 0.05% Thevetia neriifolia stem methanol extract.
Abbreviations: BM, Basement membrane; DA, Detached area; DBL, Disappeared boundary lines; DEC, Disintegrated epithelial cell; EC, Epithelial cell; GC, Goblet cell; GL, Gut lumen; PM, Peritrophic membrane.
When the larvae of H. armigera were fed on the diet added with 0.05% extract, the ECs were nonuniform, shrunken, and strongly vacuolated with complete disappearance of intercellular lines (Fig. 2E). The most conspicuous observation was the complete loss of PM and wide separation and exfoliation of the midgut epithelium from its BM, resulting in its complete destruction (Fig. 2F).
Discussion
Natural products in insect pest management programs are gaining recognition in recent years due to environmental pollution, pest resistance and resurgence, and undesirable effects to the nontarget organisms caused by unsystematic use of synthetic pesticides. Several plant extracts or isolated active compounds have been shown to possess antifeedant,11 growth-inhibitory,26 and toxic effects27 against a number of economically important insects, such as H. armigera and Spodoptera littoralis.28,29
T. neriifolia, commonly called as yellow oleander, has been reported to contain a number of toxic principles in all parts, the kernels being the most toxic in comparison to the other parts.22 Antifeedant assay carried out with the diet containing 0.005%–5.0% stem methanol extract of T. neriifolia revealed the antifeedant index ranging from 2.06 to 37.35. On the other hand, feeding deterrence in H. armigera was found to be 100% when fed on 1.0% Crataegus crenulata and Xylosma longifolium.30 Nevertheless, aqueous extracts of Calotropis procera and Datura stramonium have displayed about 90% feeding protection against H. armigera,31 while varied antifeedant potentials of leaf aqueous extracts of 25 medicinal plants have been reported against VI instars of H. armigera.11 The antifeedant activity of acetone, chloroform, ethyl acetate, hexane, and methanol extracts prepared from the peel, leaf, and flowers of Citrus sinensis, Ocimum canum, Ocimum sanctum, and Rhinacanthus nasutus has also been confirmed against the fourth instars of H. armigera.32 Likewise, 0.2% and 0.5% petroleum ether extracts of Parthenium hysterophorus have been found to exhibit 100% feeding deterrence in H. armigera.33 Our studies on choice bioassays also revealed feeding preference of H. armigera larvae for control diet than the diet containing T. neriifolia extracts. Nevertheless, the addition of 0.005% T. neriifolia extract could not deter larval feeding and the addition of 0.05% extract caused only 7.3% reduced consumption. However, feeding on 5.0% ethyl acetate crude extracts of Syzygium lineare leaves resulted in significant antifeedant activity of 79.4% against the fourth instars of Spodoptera litura.34
Feeding on experimental diet resulted in significant reduction in the weight of H. armigera larvae, which decreased drastically with the increase in the concentration of extract. The larvae exhibited 37.5%–77.7% starvation after feeding with 0.005%–0.05% methanol stem extract-containing diet. These results are in agreement with that of Barakat35 who showed increased percent starvation in the larvae of S. littoralis when provided with castor leaf discs treated with increased concentration of acetone extract of Casimiroa edulis (Rutaceae) leaves. They observed 3.2% starvation on treatment with 10.4 mg/cm2 extract, while the highest tested concentration of 332.8 mg/cm2 extract resulted in 75.2% starvation. Similarly, 354.50–396.66 mg reduced weight in III instars of H. armigera was reported when fed with seed extracts of Pongamia pinnata.36
It is well known that the insects' digestive system is one of the main physiochemical barriers against many toxins and pathogenic agents. Hence, the toxins taken by insects during feeding that overcome these barriers should damage the cellular architecture.14 The gut is the main organ responsible for digestion, assimilation, and absorption of food; any anomaly in the gut region could affect the growth and development of the insect pest and its survival. Our results showed damaged absorptive ECs, atrophied nuclei, and distorted epithelium of the larvae when fed with extract-containing diet. Similar results of completely dystrophied midgut tissues with shrunken columnar ECs and atrophied nuclei have been reported in H. armigera when fed with diet containing ethanolic leaf extracts of Lantana camara.14 The histopathological effects of Artemisia monosperma extract have also been revealed on the midgut of S. littoralis, resulting in the destruction of midgut epithelium.37 Comparable effects were reported on the midgut cells of Schistocerca gregaria and Locusta migratoria when fed with azadirachtin, causing various pathological changes, including slow necrosis, destruction of midgut cells, and damaged circular and longitudinal muscles with separation from BM.38 The histopathological disturbances in the midgut cells of S. littoralis with vacuolization and destruction of nuclear content, degenerated columnar ECs, and their detachment from the BM have been elucidated when fed on castor leaves dipped in crude extracts of Azadirachta indica and Citrullus colocynthis.39
Our studies suggest that the toxic effects of T. neriifolia methanol stem extracts on the midgut tissues of H. armigera are concentration dependent with the most damage observed at a diet with 0.05% extract and the highest concentration tested. The atrophied RCs, fading of cell boundaries, and degradation of PM indicate the increased entry of plant toxins into the midgut, leading to the damage of ECs. It further suggests that toxins present in the T. neriifolia stem methanol extracts had destructed the RCs, which thus could not replace the damaged epithelium. PONNEEM has been reported to cause disintegration of peritoneal membrane in the midgut region of the IV instars of H. armigera with the dilution of circular and longitudinal muscles, suggesting the inability of midgut to push the food into the hindgut.40
Our studies showed that though the stem methanol extract of T. neriifolia exhibited moderate antifeedant potential against the early fourth instars of H. armigera, they led to remarkable damage to the larval midgut, thus causing appreciable nutritional deficiency in the larvae that could probably affect their growth and development. This suggests the possible use of T. neriifolia stem methanol extracts in the field against H. armigera larvae as an eco-friendly product. Further investigations are, however, needed to explore the potential of extract as growth-inhibitory and growth-arresting agent against H. armigera in the fields for formulating preparations with enhanced potency and stability.
Author Contributions
Conceived and designed the experiments: MM, KKG, and SK. Analyzed the data: MM. Wrote the first draft of the manuscript: MM. Contributed to the writing of the manuscript: KKG and SK. Agreed with manuscript results and conclusions: KKG and SK. Jointly developed the structure and arguments for the paper: SK and MM. Made critical revisions and approved the final version: SK. All authors reviewed and approved the final manuscript.
Acknowledgments
The authors are highly grateful to the Council of Scientific and Industrial Research, New Delhi, for providing research fellowship to carry out the present investigations. Thanks are extended to Dr. Savithri Singh, Principal, Acharya Narendra Dev College, for providing infrastructure and research facilities.
