Toxicity and cytopathology mediated by Bacillus thuringiensis in the midgut of Anticarsia gemmatalis (Lepidoptera: Noctuidae)

Bioinsecticides and transgenic plants, based on Bacillus thuringiensis (Bt) toxins are important when managing Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae), a soybean defoliator pest. The interaction of these toxins with the caterpillar’s midgut cells determines their efficacy as an insecticide. The objective was to evaluate the toxicity of B. thuringiensis, subsp. kurstaki strain HD-1 and cytopathological changes mediated by these bacterial toxins in the midgut of A. gemmatalis caterpillars. Insecticidal efficacy was determined by calculating lethal concentration values (LC25, LC50, LC75, LC90 and LC99) in the laboratory. Midgut fragments from A. gemmatalis were extracted after bacterial ingestion and evaluated by light, transmission electron and confocal microscopy. The Bt median lethal concentrations showed toxicity [LC50 = 0.46 (0.43–0.49) mg mL−1] to fourth instar A. gemmatalis caterpillars after 108 hours. Bt induces severe cytotoxicity to A. gemmatalis midgut epithelial cells with increasing exposure over time, causing cellular disorganization, microvillus degeneration, cell fragmentation and protrusion, peritrophic membrane rupture, and cell vacuolization. The cell nuclei presented condensed chromatin and an increase in lysosome numbers. Apoptosis occurred in the midgut cells of caterpillars exposed to Bt. A regenerative response in A. gemmatalis caterpillars was observed 8 hours after exposure to Bt, however this response was not continuous. Toxins produced by Bt are harmful to A. gemmatalis at median concentration with structural damage and death of the midgut epithelial cells of this insect.

The velvetbean caterpillar, Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae) is the main defoliator on soybean plants (Glycine max L. Merrill, Fabaceae) 1 . In Brazil, this pest occurs throughout the year, especially in the vegetative phase of plants and its control is realized mainly with synthetic insecticides [2][3][4] . Integrated Pest Management (IPM) programs aim at reducing the use of chemicals in pest control 1 due to the negative effects of these products on non-target organisms 5,6 and on the environment 7,8 . Biological insecticides, such as Bacillus thuringiensis (Bt) Berliner (Bacillaceae) strains, specific to target pests, with no toxic effects on other animals or the environment 9 are an alternative to chemical control 10,11 . The wide Bt strain and toxin variety allow the production of bioinsecticides and the development of transgenic plants 12 . Histopathology. The A. gemmatalis caterpillar midgut not exposed to Bt presented epithelium composed of high columnar cells, goblet cells and evident peritrophic matrix. The cytoplasm of columnar and goblet cells had few vacuoles, vesicles and small granules. The nucleus was elongated, occupying the medial-basal cell portion, predominantly with decondensed chromatin (Fig. 2A).
Histological changes were observed in the midgut of A. gemmatalis caterpillars two hours after exposure to Bt (Figs 2B-3F). The epithelium presented irregular shapes, cellular degeneration and cellular fragments started in the lumen. The vacuolization of the cytoplasm was high and the peritrophic membrane was ruptured (Fig. 2B). At 4 h of exposure to the entomopathogen, the amount and size of the vacuoles increased, occupying much of the Ultrastructure. The midgut cell ultrastructure of A. gemmatalis caterpillars fed on non-Bt diet was well organized with dense cytoplasm and intact plasma membrane (Fig. 3A).  www.nature.com/scientificreports www.nature.com/scientificreports/ Immunofluorescence. Cleaved caspase-3 was randomly distributed in the A. gemmatalis caterpillar midgut exposed or not to Bt (Fig. 4), but an increase of this protease was observed in the midgut of caterpillars at 8, 16, and 32 h after exposure to Bt ( Fig. 4B-D).
An increase in the number of proliferating cells in the midgut of A. gemmatalis caterpillars was observed at 8 h after Bt exposure. However, this regenerative response was not observed at 16 and 32 h following bioinsecticide ingestion (Fig. 5).

Discussion
Anticarsia gemmatalis susceptibility to Bt confirms bacterium efficacy when controlling this pest, however, this can vary according to the insect species 31,32 . Spodoptera frugiperda J. E. Smith 33 , Helicoverpa armigera Hübner 34 and Spodoptera litura Fabricius 35 (Lepidoptera: Noctuidae) are susceptible to different Bt concentrations. The mortality of A. gemmatalis caterpillars, due to Bt toxins, depends on the bioinsecticide concentration demonstrating the toxicity of this bacterium through ingestion.
Irregularly shaped epithelium, increased cytoplasmic vacuolization, nuclear chromatin condensation and cellular fragments with cytoplasmic and nuclear contents being released into the midgut lumen were typical characteristics of cell degeneration observed in midgut of A. gemmatalis fed on Bt toxin contaminated diet. Cellular degeneration in the midgut due to exposure to toxic compounds has been reported for Alabama argillacea Hübner (Lepidoptera: Noctuidae) 19 and Plutella xylostella L. (Lepidoptera: Plutellidae) 36 . The release of cellular fragments, including nuclei, from epithelium into the midgut lumen observed in A. gemmatalis midgut after 2 h of Bt exposure can reduce the digestive capacity of insects as observed for H. armigera 37 and suggests a detoxification response to the toxic effect of Bt and the cell death process 38 . The higher vacuolization in the A. gemmatalis digestive cells, exposed to the entomopathogen, suggests cell death 39 . The vacuole presence in the midgut cells is common in insects 40,41 , but its greater numbers in the cytoplasm has been characterized as autophagy 42,43 . The histological effects observed in A. gemmatalis midgut suggest an attempt to detoxify the entomopathogen infected cells. Bt induced the A. gemmatalis midgut peritrophic membrane rupture. This membrane was also destroyed in some midgut parts in Alabama argillacea (Lepidoptera: Noctuidae) fed on Bt cotton leaves 19 . Nutrient absorption is reduced due to the damage to the peritrophic membrane that plays a fundamental role in digestion 45 and protects the epithelial cells from mechanical damage caused by the food bolus [45][46][47] , hindering pathogen entry and partitioning the digestion process 45,48 . The peritrophic membrane acts as a barrier against Bt toxins 49,50 delaying contact with digestive cells 51 . However, these toxins can penetrate the peritrophic membrane 52 , bind to the receptors of the columnar cell microvilli and infect A. gemmatalis midgut epithelial cells.
Microvilli degeneration in A. gemmatalis columnar cells can be explained by the toxin effect on the cytoskeleton actin, therefore Bt can interact with membrane proteins during initial action stages 53 inducing cytoplasm leakage into the midgut lumen 54 . Cellular protrusions released into the midgut lumen of A. gemmatalis caterpillars fed on Bt contaminated diet suggest a cytotoxic effect of this bacterium causing apoptosis, a morphological pattern of programmed cell death 55 . Elimination of cells by death 38 would be a response to damage to midgut epithelial cells after Bt ingestion. Donut-shaped mitochondria were observed in the insect intestine exposed to Bt. This change in shape is caused by respiratory chain inhibition and is an early marker of cellular stress 56 caused by entomopathogen.
The higher number of caspase-3 positive cells cleaved in the caterpillar midgut that ingested the bioinsecticide indicates apoptosis occurrence 43,57 . Cells showing a positive result for cleaved caspase-3 in the midgut of caterpillars fed on uncontaminated diet indicate normal cell renewal 43,58 .
The increase of proliferating cell numbers in the A. gemmatalis midgut after 8 hours of bacterial ingestion was indicated by anti-PH3 antibody, a mitosis cell-specific marker 59,60 . Damage to the insect's digestive system by Bt toxins activating defensive responses were reported for Heliothis virescens Fabricius (Lepidoptera: Noctuidae) 61,62 . www.nature.com/scientificreports www.nature.com/scientificreports/ Epithelium regeneration with dead cells replaced by newly differentiated ones depends on the proliferation and differentiation of the regenerative cells and allows resistant insects to recover and survive after exposure to the biotic agent 63 . Cell replacement is important for the homeostatic maintenance of midgut integrity [64][65][66] . Bombyx mori Linnaeus (Lepidoptera: Bombycidae) responds to Bt infection with a regenerative mechanism 67,68 by the asymmetric division of regenerative cells 44 . Anticarsia gemmatalis caterpillars do not have a continuous regenerative response as observed by the absence of cellular proliferation process in the midgut epithelium after 16 and 32 hours of Bt ingestion, possibly due to cell lysis and epithelial rupture providing a favorable medium for spore germination leading to severe septicemia and insect death 16,69 .
Toxins produced by Bacillus thuringiensis subsp. kurstaki strain HD-1 are harmful to A. gemmatalis at median lethal concentration and cause severe histological and ultrastructural changes degenerating the epithelium and causing the death of midgut epithelial cells in this insect.

Material and Methods
Insects. Anticarsia gemmatalis caterpillars were obtained from the insect biological control laboratory (LCBI) of the Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil and maintained at 25 ± 2 °C, 75 ± 5% relative humidity and 12-hour photophase. These caterpillars were fed on an artificial diet consisting of 10 g of agar, 15.6 g of brewer's yeast, 25 g of wheat germ, 25 g of soy protein, 31.2 g of beans, 12.5 g of casein, and 2.5 mL of vitamin solution (1.2% ascorbic acid, 0.03% calcium pantothenate, 0.015% niacin, 0.008%, riboflavin, 0.004% thiamine and 0.004% HCl) 70 . Twenty A. gemmatalis caterpillar groups were placed per polystyrene pot (15 × 9 cm) until pupa stage. Cleaning the pots and food replacement were performed every 48 hours. Anticarsia gemmatalis fourth instar larvae without amputations or apparent malformations were used in the bioassays. Ultrastructure. Twenty fourth-instar A. gemmatalis larvae were fed on Bt contaminated diet with the median lethal concentration (LC 50 ) for 32 h and cryoanesthesiated at −4 °C. The midgut of these caterpillars was dissected and transferred to 2.5% glutaraldehyd in 0.2 M sodium cacodylate buffer, pH 7.2 containing 0.2 M sucrose for 4 h at room temperature. Samples were post-fixed in 1% osmium tetroxide in the same buffer for 2 h, washed in buffer, dehydrated in an increasing ethanol series (70, 80, 90 and 99%) and soaked in LR White resin (London Resin Company Ltd.). Ultra-fine sections (80-90 nm thick) were obtained with a diamond power razor in Power Tome-X ultramicrotome (Boeckeler Instruments, Tucson, AZ, USA), contrasted with 1% aqueous uranyl acetate and lead citrate 72 and examined under transmission electron microscope Zeiss Libra 120 (Carl Zeiss, Jena, Germany).  73 (Finney, 1971) with the PROC PROBIT procedure of the SAS User v. Program. 9.0 for Windows 74 .