Histopathology of the larval midgut of Helicoverpa armigera (Hübner) fed on Bacillus thuringiensis crystals and Bt-tomato plants

Abstract

The histopathological effects of the spore-crystal complex of indigenous Bacillus thuringiensis (Bt) isolate, as well as Cry 2Ab gene expressed in transgenic tomato plants on the midgut of 4th instar larva of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidea) has been investigated using the transmission electron microscope (TEM). Remarkable ultrastructural changes were observed in the columnar and goblet cells of the larval midgut after feeding on either transgenic tomato leaves, or spore-crystal complex of Bt. The effects observed included breakdown of microvilli of epithelial cells, increase in the electron density of the cytoplasm and vacuolation associated with different sizes of lysosomes; interruption of the goblet cells and distorted goblet cavities which lost their cytoplasmic projections; destruction of the mitochondria which lost their cristae; degeneration of the endoplasmic reticulum; collapse of the nucleus associated with rupture of nuclear envelope and clumped chromatin. Feeding the larvae on transgenic Bt-tomato plants caused in addition to the aforementioned changes severe vacuolation and degeneration of the nucleus in both columnar and goblet cells and the nuclear membrane was broken into electron dense ring spheres.

1. Introduction

Studies on the ultrastructural changes of lepidopterous larvae fed Bt endotoxin received the attention of many workers [9], [19], [20], [5], [23], [13], [21], [8], [14]. In their investigations, the interaction between Bt toxins and the midgut of lepidopterous larvae using purified crystal toxins, was evaluated. These studies showed that crystal endotoxin caused the destruction of both goblet and columnar cells of the mid-gut together with notable changes in the microvilli, mitochondria and membranes associated with the Golgi complex [4], [7]. Therefore, a study has been done to investigate the effects of toxins expressed in transgenic cotton plants for comparison. In this concern, the histopathological effect of Cry 1AC toxin expressed by Bt – cotton plant fed to the lepidopterous insect Alabama argillacea (Hübner) was reported by Sousa et al. [22].

2. Materials and methods

2.1. Bacterial toxins

An indigenous Bt isolate containing Cry 2Ab; isolated from the soil of Qena governorate, Egypt was used [1]. Fourth instar larvae were isolated from the laboratory colonies of Helicoverpa armigera established on a semi-artificial diet [6]. The larvae were starved for several hours and then fed on a diet containing Bt at 500 μg/ml for 48 h. Similarly, another group of larvae of H. armigera were fed on transgenic Bt tomato plants for 1 day. These plants were developed via Agrobacterium-mediated transformation to express the insecticidal protein Cry 2Ab [18]. Agrobacterium tumefaciens LBA4404 strain harboring binary vector (Cry 2Ab gene) was used for tomato transformation. The plasmid contains insecticidal crystal gene (Cry 2Ab) under the transcriptional control of cauliflower mosaic virus (CaMV35S) promoter and nopaline synthase (NOS) terminator, and the selectable marker of hygromycin phosphotransferase gene (hpt) and β-glucuronidase (gus) as a reporter gene. Cotyledon and hypocotyl explants of 7–10 day-old seedlings were used. The transformation procedure was carried out as described by McCormick et al. [11], McCormick [10]. Putative transformed tomato plantlets were confirmed by histochemical analysis (GUS expression) and genomic analysis. Untreated larvae were used as control and reared on a semi-artificial diet. All experimental larvae were held at 28 ± 2 °C and 75% RH.

2.2. Electron microscopy

For electron microscopic investigations, the midgut of the control and treated larvae was dissected and fixed immediately in 2.5% glutaraldehyde sodium cocodylate buffer at pH 7.3 for 4–6 h, washed three times for 10 min each and kept in buffer for one day at 4 °C. Postfixation was done in 2% osmium tetraoxide buffer at pH 7.3 for 2 h at 4 °C (until the samples became dark in color), followed by 3 washes with glutaraldehyde buffer and kept for another day in this buffer at 4 °C. The tissues were subjected to dehydration in graded ethanol series (30–100%). Propylene oxide was substituted for the alcohol with two changes, of 2 min each. Embedding using 1:1 araldite–propylene oxide for 3 h, followed by the same mixture (2:1 ratio) for 6 h was carried out. Finally, the samples were transferred to a bath of fresh araldite and left overnight, and then the tissues were embedded in araldite-filled capsules. The capsules were allowed to polymerize at 4 °C for 2 h and transferred to an oven for 24 h at 45 °C and the temperature was then increased to 60 °C for another 24 h or until the blocks were adequately hardened [17], [12]. Semi-thin sections 1–3 μ thick, were stained with alkaline toluidine blue for preliminary examination by light microscope and for proper orientation. Ultra sections prepared using (Reichert-Jung) microtome were mounted on copper grids of 100–150 mesh size and double-stained with uranyl acetate (30 min) and lead citrate (3–5 min) before examination. The sections were examined and photographed using TEM-ten Zeiss 60 kV, resolution about 5 A°.

3. Results

It appears that the wall of the midgut in the control H. armigera larvae consists of a single layer of epithelial cells including columnar and goblet cells lying on a basement membrane, which is attached to the muscle connective tissue. Each epithelial cell has numerous microvilli (MV) which appear as striated borders on the apical surface of the columnar cells and apical intermicrovillar crypts (AC) (Fig. 1A). The cytoplasm is packed with numerous organelles such as mitochondria (M) with clear matrix, membranes and cristae, small Golgi bodies (GB) with few saccules, free ribosomes (R), and rough endoplasmic reticulum (rER). A clear rounded nucleus (N), regular nuclear envelope (NE) and central nucleolus (Nu) with normal chromatin appear in Fig. 1B.

Figure 1.

Midgut of the control 4th instar larvae of H. armigera. (A) (8000×): microvilli (MV), tracheoles (Tr), mitochondria (M), rough endoplasmic reticulum (rER), apical intermicrovillar crypts (Ac), golgi bodies (GB), free ribosomes (R). (B) (8000×): goblet cell (GC) and columnar cell (CC), round nucleus (N) with regular nuclear envelope (NE), normal chromatin and nucleolus (Nu). (C) (2500×): goblet cavity (GC1, 2, 3) lined by numerous cytoplasmic extensions inside, mitochondria (M) and nucleus (N) located at the lower portion of the cell.

The goblet cells are calyx shaped, and they lie between the columnar cells (Fig. 1C). The goblet cell is characterized by a large cavity called goblet cavity (GC1, 2, 3), with an apical opening into the lumen of the midgut. This cavity is lined by numerous cytoplasmic projections extending inside; each containing a mitochondrion. The nucleus (N) of the goblet cell is flat in shape lying in the basal portion of the cell beneath the goblet cavity.

3.1. Histopathological changes in the midgut of larvae fed on a diet containing Bt endotoxin

The midgut of the larvae fed on Bt endotoxin showed degenerative appearance of columnar cells (1, 2, 3) associated with destroyed microvilli (MV) and a remnant of peritrophic membrane (Tm) (Fig. 2A). Also, all cytoplasmic organelles appear with numerous vacuoles (V). Mitochondria are clearly vacuolated, destroyed, and associated with different sizes of lysosomes (L). Moreover, shrinkage of the nucleus (N), destroyed nuclear sheath, vacuoles formation and clumped chromatin appear with accumulation of ribosomes.

Figure 2.

Midgut of 4th instar larvae of H. armigera fed on Bt endotoxin (A–D), compared with other fed on transgenic Bt-tomato leaves (E–H). A (6300×): destroyed epithelial cells (Ep), degenerated microvilli (MV) and peritrophic membrane (Tm). Destroyed mitochondria (M), lysosomes (L) associated with different sized vacuoles (V), Nucleus (N), and nuclear envelope (NE). (B) (3150×): two goblet cells (GC1 and GC2), epithelial cells associated with lysosomes (L) suffering from vacuolation (V), detachment of microvilli (MV), collapsed nucleus (N), irregular nuclear envelope (NE), nuclei (Nu), destroyed mitochondria (M). (C) (12,500×): goblet cell with destroyed mitochondria (M), goblet cavity (GC) with vacuolated organelles (vo), and scattered lysosomes (L) inside. (D) (10,000×): degenerated nucleus (N) with irregular nuclear envelope (NE). (E) (6300×): destroyed gut epithelium; nucleus (N), mitochondria (M), goblet cavity (GC), vacuoles (V). (F) (3150×): destroyed apical borders of gut epithelium, destroyed peritrophic membrane (Pm) and microvilli (MV) as sloughed part (Slo) of degenerated epithelial cell towards the lumen (Lm), degenerated nucleus (N), lysosomes (L) and dense ring spheres (Rs). (G) (5000×): destroyed peritrophic membrane (Pm) and destruction of goblet cells (GC), vacuolar degeneration (Vd) of nuclei (N). (H) (8000×): distorted cytoplasm of epithelial cell associated with clear vacuolation of nucleus (N) and destroyed chromatin appeared inside.

The goblet cells suffered from degeneration in their cytoplasm (Fig. 2B). The microvilli (MV) were disrupted showing degenerative appearance at the apical region. The goblet cavity (GC1, GC2) appears losing its constituent organization. The cytoplasm is characterized by increased vacuolation (V) associated with different sizes of lysosomes (L) and destroyed mitochondria (M). An interrupted goblet cavity (GC) appears with cytoplasmic extensions scattered as mucigenous substances inside (Fig. 2C). Many vacuolated organelles on the periphery of the goblet cavity were observed. The nucleus, nuclear envelope, and nuclei lost their organization and clumped chromatin appeared inside (Fig. 2D).

3.2. Histopathological changes in the midgut of H. armigera larvae fed on transgenic Bt-tomato plants

When the larvae were fed on Cry2Ab, ultrastructural changes in the columnar and goblet cells occurred. Different significant changes were recorded in the microvillus, mitochondria, cytoplasmic vacuolation, and other parts (Fig. 2E). A sloughed part of degenerated epithelial cell appears (Slo) toward the lumen (Lm) (Fig. 2F). Distorted cytoplasmic organization was observed. The apical borders of epithelial cells showed destroyed microvilli (MV) and peritrophic membrane (Pm). In Fig. 2F, the cytoplasm showed increased vacuolation (V), destroyed peritrophic membrane and packed with scattered lysosomes (L) and distended mitochondria (M). The nucleus (N) of some columnar cells appeared containing degenerated nuclei replaced by dense ring spheres (Rs) in addition to destroyed nuclear sheath. The obvious vacuolation of the nucleus (N) showed to be a characteristic effect after feeding on transgenic plants.

Goblet cells with enlarged and distorted cavities (GC) were as observed in Fig. 2G. The cytoplasm of goblet cells became electron dense and granular. Damage of the cytoplasmic organelles and vacuolar degeneration of the nuclei were observed. Well distinct vacuolar degeneration (Vd) in the nucleus of the epithelial cells of the midgut occurred although the nucleolus is still intact (Fig. 2H). Moreover, the nuclear membrane (Nm) was broken down into electron dense ring spheres (Rs).

4. Discussion

The histopathological changes caused after feeding 4th instar larvae of H. armigera on transgenic tomato plants have been explored and compared with those changes that occurred after feeding on Bt endotoxin incorporated into an artificial diet. The effects after both treatments were mainly localized in the midgut. When the larvae were fed on Bt endotoxin, columnar and goblet cells were degenerated and microvilli were disrupted. Cytoplasmic organelles showed numerous distinguished vacuoles. The cytoplasm showed increased vacuolation associated with different sizes of lysosomes. Mitochondria were destroyed. Shrinkage of the nucleus and nuclear envelope destruction of nuclear sheath and chromatin clumping with accumulation of ribosomes were observed. These findings coincide with those of Salama and Sharaby [19], Ingle et al. [5], Salama et al. [21], Pandey et al. [14], Griego et al. [4], Endo and Nishiitsutsuji-Uwo [3], Percy and Fast [15], Abdel-Razek et al. [2], Rashmi and Singh [16] working on other lepidopterous insects. In their investigations, the mitochondria and microvilli showed rapid swelling and the connections between the epithelial cells were loosened by the separation of cell membranes. The electron density of the cytoplasm of goblet cells, breakdown of cytoplasmic projections lining the goblet cavity; destruction of the endoplasmic reticulum; increased vacuolation in cytoplasm occurred associated with lysosomes; deep infoldings of the basal membrane; and enlargement of nucleus associated with rupture of nuclear envelope and/or clumped chromatin.

On the other hand, when the larvae fed on transgenic Bt-tomato plants cytoplasmic organelles with increased vacuolation were observed with destroyed microvilli and peritrophic membrane. Nucleus of columnar cells appeared containing degenerated nuclei replaced by dense ring spheres in addition to destroyed nuclear sheath. Severe vacuolation of the nucleus showed to be a characteristic effect after feeding on transgenic plants. Enlarged and distorted cavities in the goblet cells were also observed. Well distinct vacuolar degeneration in the nucleus of the goblet cells occurred and the nuclear membrane was broken. Numerous ribosomal spheres were aggregated near the nucleus. The effects of ingesting Cry1Ac toxin expressed by Bt-cotton plants on A. argillacea midgut morpholophysiology were investigated by Sousa et al. [22]. They found that the larvae of this insect exhibited ultrastructural and morphological modification in the columnar cells with significant changes in the mitochondrial polymorphism, cytoplasmic vacuolation, microvillus and basal labyrinth. Expressive morphological alterations were also observed in the goblet cells indicating that the columnar cells’ ones are not the only target of the Cry 1Ac toxin but a significant reduction in the number of regeneration cells occurred.

In conclusion, it appears that the histopathological effects caused by feeding the larvae of H. armigera on Bt endotoxin or on transgenic Bt-tomato plants harboring Cry2Ab gene are mainly localized in the midgut and the effects were almost similar with some exceptions. The larvae fed on transgenic tomato plants, showed an obvious vacuolation of the nucleus and a loss of a part of its nuclear membrane in both the columnar and goblet cells.