For the present study, larvae of A. janata Linn. (Noctuidae: Lepidoptera) were collected from the fields of Indian Institute of Oil Seeds Research, Hyderabad with no prior exposure to any synthetic or Bt pesticides, reared in the laboratory conditions (Budatha et al., 2007). They were maintained for three generations as to obtain homogenous population (in a growth chamber). Larvae were fed with sterile castor leaves and maintained at 27 ± 2°, 14:10 h (light: dark) photoperiod and relative humidity of 60–70% till pupation. Pupae were transferred to moist sand container for adult emergence. Adults were transferred to egg laying chamber and were supplemented with 10% honey containing Vitamin E. Adults laid their eggs on the leaves and the neonate larvae hatched were used for continuous culture as well as experiments.

DOR Bt-1 formulation was obtained from Indian Institute of Oil Seeds Research which exhibited high toxicity against A. janata larvae and various other lepidopteran pests. The PCR analysis of DOR Bt-1 isolate revealed presence of cry1 (cry1Aa, cry1Ab, and cry1Ac) and cry2 (cry2Aa and cry2Ab) genes, which provide broad-spectrum potential against lepidopteran, and dipteran insects (Reddy et al., 2012). The reported LC₅₀ value for the DOR Bt-1 formulation was 247.52 μg/ml for seven-day-old or 3rd instar larvae (Vimala Devi and Sudhakar, 2006). One tenth of LC₅₀ value was used as a sub-lethal dose (i.e., 24.75 μg/ml of water) for the present study. Toxin coated castor leaves were fed to the larvae. Triplicates were maintained, for each treatment 150 larvae were used. They were divided into two sets of experiments, wherein one batch was continuously exposed to sub-lethal dosage of toxin (0.170 μg/cm²) coated castor leaf discs (145 cm²) till 72 h, and a batch of 30 larvae were intermittently transferred to uncoated leaf discs after every 24 h period of toxin exposure for the recovery. Control larvae were maintained on water coated leaf discs. Furthermore, morphological changes occurring during the development and mortality were also recorded in toxin-exposed as well as recovered insects.

Third instar larvae (a batch of 100) exposed to toxin coated leaves were collected at every 12 h interval till 72 h. These were briefly narcotized on ice and dissected in insect Ringer solution (130 mM NaCl, 0.5 mM KCl, and 0.1 mM CaCl₂). An incision was made in the abdominal region and extended through the length of the larvae. The gut content was removed and the mid-gut region was specifically isolated. After removal of the gut, the yellowish-white perivisceral fat body was collected. The isolated tissues were used for histology, in-situ hybridization, genomic DNA and total RNA isolation.

Cell death/viability of mid-gut cells was primarily assessed using AnnexinV and Propidium Iodide (PI) staining (Vermes et al., 2000). For this experiment mid-gut was dissected out from both control and toxin exposed (24 h) larvae and maintained in Grace insect cell culture media (Invitrogen Corporation, USA) and incubated with AnnexinV and Propidium Iodide staining solutions (BD Biosciences, UK) and visualized under confocal microscope (LSM 880, Carl Zeiss, Jena). Furthermore, gut cells were strained through 70 μm mesh size MACS® smart strainer (Miltenyi Biotec, Singapore) using phosphate buffer saline (PBS) solution (pH 7.4). The cell reservoir was collected in the funnel and centrifuged at 1,500 rpm for 5 min at 4°C. The cell suspension was collected and immediately processed for FACS (fluorescence-activated cell sorting) analysis. An analysis was carried out using Annexin V-FITC Apoptosis Detection Kit according to the manufacturer's instructions (BD Biosciences, UK). In brief, AnnexinV and Propidium Iodide staining solutions were added to the sieved cell suspension and incubated at room temperature in the dark for 15 min. This was followed by addition of binding buffer and then analyzed using a FACS LSR Fortessa^TM flow cytometer (BD Biosciences, UK) and BD FACS Diva Software 6.0. For each sample, 10,000 events were acquired. The percentage of live cells vs. cells undergoing death was plotted.

To substantiate that cell death is due to necrosis but not because of apoptosis in larval mid-gut, DNA fragmentation assay was carried out following the protocol of Kasibhatla et al. (2006). Genomic DNA from the mid-gut was isolated using a standard protocol (Lagisz et al., 2010), with minor modifications. The tissue (100 mg) was homogenized using 500 μl of lysis buffer (100 mM Tris-HCl (pH8.0), 50 mM EDTA, 1.5 mM NaCl, 2% CTAB, and 0.1% 2-mercaptoethanol). DNA pellet thus obtained was solubilized in Tris-EDTA buffer (pH 8.0) and analyzed by 0.8% agarose gel electrophoresis.

The isolated mid-guts were fixed overnight in Bouin's fixative (saturated picric acid: formaldehyde: glacial acetic acid; 15:5:1), processed through a series of increasing ethanol concentrations, cleared in xylene and embedded in molten paraplast (Sigma-Aldrich, USA). The tissues were sectioned (~5 μm) using a rotatory microtome (Leica Microsystems, Germany) and stained with haematoxylin-eosin or toluidine blue (Sousa et al., 2010), visualized using Olympus IX-81 microscope (Olympus Corporation, Japan), and photographed.

Streptavidin-biotin based BrdU staining (Invitrogen Corporation, USA) was used for monitoring the cell proliferation in the mid-gut during toxin-induced damage following manufacturer's protocol. Larvae were injected with BrdU labeling reagent (10 μl/g body weight) 4 h prior to isolation of toxin exposed larval mid-gut using a Hamilton syringe. Dissected mid-guts were fixed and processed as mentioned in the previous Section Histological Analysis of Mid-Gut, tissue sections were spread on poly-L-lysine coated slides (Sigma-Aldrich, USA) and incubated overnight at 37°C following which deparaffinization was carried out using xylene and passed through a series of decreasing ethanol concentrations. Peroxidase quenching was performed using 30% H₂O₂ and absolute methanol (1:9). Denaturation and blocking steps were carried out using the kit based reagent prior to antibody incubation. Biotinylated mouse anti-BrdU was overlaid and the slides were incubated at room temperature (RT). After incubation, streptavidin peroxidase (Invitrogen Corporation, USA) was added to detect the incorporation. Color development was monitored after the addition of secondary antibody mixture provided by the manufacturer. Slides were counterstained and mounted using histomount™ (Invitrogen Corporation, USA) and visualized under Olympus IX-81 microscope.

For the preparation of arylphorin cRNA probe, a 160 bp fragment of A. janata arylphorin gene was positioned as forward locked in pGEM-T Easy Vector (Promega Corporation, USA). The vector was linearized with EcoRI and NdeI restriction enzymes (New England Biolabs®, UK). Sp6 promoter was used for the synthesis of antisense probe (positive), while T7 promoter was used for the synthesis of sense probe (negative). Further, RNA probe synthesis was carried out using DIG RNA labeling kit (Roche Diagnostics, Germany). Larval mid-gut and fat body were dissected out and fixed in 4% paraformaldehyde (PFA) (Sigma-Aldrich, USA) in 0.1 M PBS at 4°C overnight. Post-fixation, tissues were embedded in OCT compound medium (Leica Microsystems, Germany). Tissue cross sections were obtained using cryostat microtome (Leica CM1850, Leica Microsystems, Germany) and mounted on poly-L-lysine coated slides. The OCT compound was washed using PBST buffer and the sections were permeabilized using proteinase K (1 μg/ml). Once again 4% PFA was used for further tissue fixation and sections were incubated with 200 μl hybridization buffer (50% formamide, 5X SSC, 5 mM EDTA, 0.1% CHAPS, 1% SDS, 50 μg/ml yeast tRNA, 50 μg/ml heparin) for 1 h at 50°C. The digoxigenin labeled cRNA probe was mixed with 200 μl of hybridization buffer, heat denatured at 80°C for 5 min, draped and covered with parafilm and incubated at 50°C in a sterile incubator overnight. Post-hybridization, the slides were washed four times at 50°C (5 min each) with a wash buffer (SSC, 50% formamide, 0.1% Tween 20) in a series of decreasing SSC concentration. The slides were then processed with solution A (0.5 M NaCl, 5 mM EDTA, 10 mM Tris-HCl, 0.1% Tween 20, pH 8.0) and incubated at RT for 5 min followed by incubation with 20 μg/ml of RNase A for 20 min at RT. Later, the slides were again washed with solution A twice (5 min each) at RT.

For the antibody reaction, the slides were initially washed with maleic acid buffer (100 mM maleic acid, 150 mM NaCl, 0.1% Tween 20, pH 7.5) for 5 min at RT followed by blocking with 10% BSA for 1.5 h at RT. Anti-digoxigenin-ALP (Roche Diagnostics, Germany) was used for the detection of digoxigenin probe (1:2,000) and incubated overnight at 4°C. These slides were then washed with DIG wash buffer (Roche Diagnostics, Germany) for 4 times (15 min each). Incubation with 100 μl BCIP-NBT solution (G-Bioscience, USA) for 10 h at RT was carried out to develop the color. After color development, the slides were overlaid with detection buffer (Roche Diagnostics, Germany), counterstained with nuclear red (Vector Laboratories Inc., USA), and incubated in TE buffer (Ambion, Life Technologies, USA) for 5 min. After washing with PBST buffer, the slides were processed with increasing series of ethanol, mounted using DPX mountant, and visualized under Olympus IX-81 microscope.

Total RNA was isolated from the tissue (100 mg) using TRIzol method® (Ambion, Life Technologies, USA). The quantification of the RNA was carried out using NanoDrop spectrophotometer (ND-2000; NanoDrop Technologies, USA). Quality and integrity of RNA were assessed by formaldehyde denaturing gel electrophoresis. cDNA was synthesized using Revert Aid First Stand cDNA synthesis kit (Thermo Scientific, USA). For quantitative PCR analysis, a custom-made TaqMan gene expression assay was performed (Applied Biosystems, USA). Arylphorin primers of amplicon size 160 bp (Fwd: 5′-GTT AGA GAA GGG CCA GGT GTC TCA TG-3′; Rev: 5′- CTC TGG CTT GGT AGA CTC CTT GGT-3′) were designed from the arylphorin nucleotide sequence (KX462992). Insect rS7 gene (KF984201) was used as an internal control using primers (Fwd: 5′-ACG TGG ACG GTT CAC AAC TCA TCA-3′; Rev: 5′-TTC GCG GCC TGT TAG CTT CTT GTA-3′) which gave rise amplicon of 115 bp, to normalize the transcript expression levels. The real-time expression analysis was performed in triplicates. From the exponential phase of PCR amplification Cycle threshold (Ct) values were obtained. Arylphorin expression was normalized against the expression of rS7 rRNA to generate a ΔCt value (Ct of Arylphorin—Ct of rS7 rRNA). Comparative Ct method was used to calculate the change in the gene expression (Livak and Schmittgen, 2001).

Presented data was calculated using standard error of the mean (SEM) (n = 3) which passed homogeneity and normality tests. Statistical significance was verified using the SigmaPlot v12.3 software (SystatSoftware Inc., USA). Significance between groups was tested using One-Way ANOVA followed by Student-Newman-Keuls (SNK) test for pairwise multiple comparison analysis and the p-value was calculated for each set of experiments.

The larvae that fed on toxin-coated leaves exhibited reduced growth in a time-dependent manner (24–72 h; Figure 1A). The 24 h toxin-exposed larvae could recover and grow when transferred to normal leaves (Figure 1B), whereas the 48 and 72 h toxin-fed groups often showed slow and poor recovery. Further, the body weight of the toxin exposed larvae was fairly low and were only 50% of that of the age matched control groups after 72 h of recovery, when the larvae were exposed to toxin for 48 and 72 h (Figure 1B).

Confocal analysis revealed the presence of PI positive cells in toxin exposed larvae but not in controls (Figure 2A). Mid-gut cell suspension prepared from toxin exposed insects showed a distinct shift into the upper left quadrant (Q1) representing necrotic type of cell death (Figure 2B). Further, the cells undergoing necrosis have increased gradually from 12 to 36 h upon toxin-exposure. However, it was noteworthy that the number of necrotic cells reduced drastically in 48 and 72 h toxin-exposed samples (Figure 2B).

The result from DNA fragmentation assay revealed that the DNA integrity was preserved in both control and toxin-exposed mid-gut samples and the absence of DNA fragmentation in all the samples rules out the possibility of apoptotic form of cell death (Figure 2C). Microscopic, FACS and DNA fragmentation analyses indicated that necrosis was the most likely reason for cell death upon Cry intoxication.

The results presented in Figure 3 show few pockets of BrdU-positive cells (brown stained nuclei) were identified in the mid-gut collected from toxin exposed larvae (Figure 3A). The dividing cells which were mostly observed in the basal region of the gut epithelium could be ascribed as mid-gut stem cells (Figure 3B, inset).

Figure 4 shows the histological changes seen in toxin exposed larval mid-gut from 12 to 72 h. The mid-gut cells showed degeneration with a condensed nucleus and fairly low cytoplasmic content. In addition, a large number of vacuoles were seen in them. Maximum cell death was observed between 24 and 36 h (Figures 4C,D). However, it was interesting to note that active cell proliferation as depicted by toluidine blue was seen extensively in 36 h Cry toxin exposed larvae (Figures 4C,D). BrdU labeling, histological and toluidine blue staining studies in parallel confirmed that not only cell death but also regeneration occurred in larval mid-gut upon Cry intoxication in A. janata.

Detectable but low expression of arylphorin in the mid-gut as compared to the fat body was observed using qRT-PCR analysis (Figure 5A). In accordance to this, in situ hybridization results revealed the localization of arylphorin transcript in the gut epithelium primarily in the basal cells (Figure 5B).

In control insects, arylphorin transcript level was fairly low throughout the time points monitored, but a steep incline was observed at 72 h (Figure 6). On the other hand, arylphorin expression gradually increased from 6 h and reached maximum at 36 h in Cry intoxicated larvae, thereafter the mRNA levels declined significantly in 48 h and remained low till 72 h.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.