Histopathological Effects of the Protein Toxin from Xenorhabdus nematophila on the Midgut of Helicoverpa armigera

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Agricultural Sciences in China 2006, 5(9): 685-690

September 2006

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Histopathological Effects of the Protein Toxin from Xenorhabdus nematophla on the Midgut of Helicoverpa armigera NANGONG Zi-yan, WANG Qin-ying, SONG Ping, YANG Jun and MA0 Wen-jie College of Plant Protection, Hebei Agricultural UniversityIBiocontrol Center of Plant Diseases and Plant Pests of Hebei Province, Baoding 071001, P.R.China

Abstract Xenorhabdus nernatophiZa HJ33 10, which is highly virulent for many insects, is symbiotic with Steinernerna carpocapsae HB310. Toxin I1 was obtained using methods such as salting out and native-PAGE from the cells of X . nematophila HB310. The histopathology of toxin I1 on H. armigera larvae was studied by dissecting an olefin slice of the midgut. The symptoms showed that the histopathology of the H. armigera midgut was similar to that of other novel midgut-active toxins such as the &endotoxins from Bacillus thuringiensis, as well as Tca from Photorhabdus luminescens W14. The midgut tissues of H. armigera fourth-instar larvae began to transform after the oral intake of the toxin 11 over 6 h. First, the anterior region of the peritrophic membrane (PM) began to degrade followed by the elongation of the columnar cells. The epithelium decomposed gradually, and the midgut tissues were either loose or disordered. The PM disappeared after 12 h but reappeared after 72 h following transient or sublethal exposure to the toxin 11. Toxin 11 also directly destroyed in vitro PMs of H . armigera.

Key words: Xenorhabdus nematophila, toxin, Helicoverpa armigera, midgut, histopathology, peritrophic membrane

INTRODUCTION Xenorhabdus spp. and Photorhabdus spp. are symbiotically associated with nematodes of the families, Steinernematidae and Heterorhabditidae, respectively. Being motile gram-negativebacteria, they belong to the family Enterobacteriaceae (Forst et al. 1997). Under natural conditions, the bacteria are carried and released into the insect hemocoel upon nematode invasion. The insect is killed by the combined action of the nematodes and the bacteria, presumably via a combination of the toxin action and direct infection (Forst et al. 1997). A lot of toxin proteins isolated from several strains of Xenorhabdus and Photorhabdus have direct injectable toxicity on the insect hosts. Unexpectedly,

several of the toxin complexes (Tc) also show oral activity against the insects (Bowen et al. 1998). Bowen et al. (1998) isolated several kinds of Tc from Photorhabdus Zuminescens W14. These Tcs were orally active against the Manduca sexta. They reported that the histopathology of the M. sexta midgut following oral Tca treatment was very similar to that described for the &endotoxins from Bacillus thuriqiensis (Bowen et al. 1998; Blackburn et al. 1998). These observations highlight the utility of Xenorhabdus as a biological control method, Xenorhabdw nematophila HB310 was isolated from Steinernema carpocapsae HB310. It had high oral and injectable toxicity on a lot of insects (Li et al. 2004). Wang et al. (2005) isolated a toxin complex with oral activity from X . nematophila HB310. Researches on toxin gene showed that toxin gene cluster

This paper is translated from its Chinese version in Scientia Agricultura Sinica. NAN GONG Zi-yan. Ph D, E-mail: [email protected]; Correspondence WANG Qin-ying, Professor, Ph D, Tel: +86-312-7528150, E-mail: wqinying8yahoo. com.cn

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and toxin gene products of Xenorhabdus were different from those of Photurhabdus (Cui et al. 2003; Sergeant et al. 2003). Although Tc toxins from P . luminescens strain W14 were studied in detail, no much study has been done on toxins from Xenorhubdus. The insecticidal mechanism on toxins of Xenorhabdus is still not clear. This article describes the influence of the toxin protein isolated from X. nematophila HB310 on the midgut tissues of Helicuverpa armigera larvae.

MATERIALSAND METHODS Stains of bacteria and insect H . armigera larvae and X . nernatophila HB310 were obtained from the Pest Biocontrol Insectary, Hebei Agricultural University.

Toxin I1 purification The purified toxin IT was obtained using the methods such as salting out and native-PAGE from the cells of X . nematophila HB310 (Wang et uE. 2005). The cells of X . nematophila HB3 10 were separated by centrifugation from culture broth. The total intracellular protein was isolated from the cells of X . nematophila HB310 by salting out. Toxin I1 was obtained by native-PAGE from the total intracellular protein. The purified toxin was stored at -70°C until use.

insect bioassays by oral delivery H . armigera neonates and fourth-instar larvae were placed in the individual wells of a 24-well cell-culture cluster plate similarly filled with diet and held in an incubator at (26 f l)OC. The diet was either treated with 100 @ of sterile water per 1 g of diet alone as an untreated control, or was treated with 100 pL, of toxin I1 (protein density 51.9 pg mL-') per 1 g of diet. Symptoms of toxicity were noted and survivor weight was gained over 120 h. Three replicates were used for each treatment, 72 insects per treatment.

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der starvation for more than 24 h were transferred to the artificial diet (0.15 g per larva) and held in an incubator at (26 & 1 ) T . The diet was either treated only with 20 pL of phosphate (PBS) buffer as an untreated control or with 20 pL of toxin 11(protein density 51.9 pg mL-I). After the treated diet was consumed by larvae, certain amount of fresh untoxin diet was added. The PMs were obtained by dissecting the treated larvae midguts followed by washing individually in cold PBS buffer at 6, 12, 24, 36, 48, 60, 72, and 96 h. The difference between the control PMs, and the toxintreated PMs was observed and compared over the same period of time. Ten insects were dissected per treatment.

In vitro bioassay of PMs with toxin I1 PMs from the healthy fourth-instar H. armigera larvae were individually placed in a centrifuge tube (0.5 mL) with 20 yL of toxin I1 (protein density 5 1.9 yg mL-') and held in an incubator at (26 f 1)OC. The control PM was treated with 20 pL of PBS buffer. After treating for 12, 24, 48, and 60 h, the transformation of PMs was observed. Ten insects were dissected per treatment.

Sectioning and staining Midguts from the larvae of oral bioassays were chilled on ice (20 min) before sectioning. The midguts were then dissected and immediately fixed in Bouin's fluid (24 h). Following fixation, the midguts were dehydrated in an ethanol-tetrahydrofuran-xyleneseries and embedded in paraffin. Embedded midguts were sectioned (5 pm). The sections were then deparaffinized, rehydrated, and stained in haematoxylin (12 min) followed by eosin Y ( 5 min). The sections were dehydrated, cleared in xylene, and then mounted in neutral balsam. The mounted sections were examined using a Nikon light microscopy (Wang 1998).

RESULTS Larvae toxicity symptoms

In vivo bioassayof PMswith toxin I1 by oral delivery Fourth-instar larvae of H. arrnigera that was kept un-

The data show that the inhibitory effect of toxin I1 on the growth of H . urmigera larvae was very obvious.

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HistopathologicalEffects of the Protein Toxin from Xenorhabdus nemafophila on the Midgut of Helicoverpa armigera

Following exposure of H . amzigera larvae to the toxintreated diet, larvae often fed for a short period of time and then ceased feeding. In addition, the frass of the toxin-treated larvae was red and few; however, the normal frass was usually brownish yellow. Although the larvae exhibited low mortality, the weight of the toxin-treated larvae was dramatically lower than that of the control larvae. Four-instar-treated larvae could progress to the pupal stage, but the pupae showed comparatively lesser weight than the normal pupae, and the adults resulting from the toxin-treated larvae could not expand their wings completely.

In vivo or in vifro transformationof PM after toxin exposure After 6 h of exposure to the toxin-treated diet, the colour of PMs turned from transparency to milk-white. There

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were certain fissures on the front of the milk-white PMs. However, PMs were still flexible and did not rupture into fragments when placed in water (Fig.1-2). After 12 h, PMs ruptured into several fragments in water; however, the larger pieces were still flexible (Fig.l-3). PMs ruptured into many debris after 24 h of treatment. However, recovery of the PMs back to the complete structure as well as transparent membrane clarity was observed after 72 h of treatment (Fig.1-4). The control PMs dissected at the same time as the treated ones were flexible, complete, and clear in water (Fig.1-I). Symptoms of PMs that were in vitro treated with toxin I1 were similar to those treated by oral delivery. PMs at 12 h after treatment with toxin I1 loosened but retained their whole shape (Fig.2-2). After 24 h, the treated PMs disintegrated into debris (Fig.2-3). The control PMs did not occur transformation under the same condition (Fig.2-I).

Fig. 1 The effects of toxin TI on the peritrophic membrane (PM) of H. urmigeru. 1, control; 2, PM after 6 h on treated diet; 3, PM after 12 h on treated diet; 4, PM after 72 h on treated diet.

Fig. 2 The transformation of the excised PM with toxin 11. 1, control; 2, PM after 12 h on toxin I1 ; 3, PM after 12 h on toxin 11.

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Table Oral toxicity of toxin 11 against H.armigeru larvae (120 h) Sample

.-

Neonate 9.3722.20 a’ 0.33i0.01 d

CK Toxin I1

Average weight Fouth-instar larvae

PuDae

2 8 5 . 6 ~ 4 . 6 a0 133.5k2.95 b

289.45259 a 154.4k5.41 h

~~

The data in the table indicate mean*SE, the means in the same column followed by different letters are significantly different at P
Fig. 3 The histopathological effects of toxin I1 on the midgut of H.armigera (400x). 1, control; 2, midgut after 6 h on toxin 11 treated diet; 3, midgut after 12 h on toxin TI treated diet; 4, midgut after 24 h on toxin I1 treated diet; 5, midgut after 48 h on toxin 11 treated diet; 6, midgut after 72 h on toxin I1 treated diet.

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Histopathological Effects of the Protein Toxin from Xenorhabdus nematophila on the Midgut of Helicoverpa annigera

Histopathology of the insect midgut after oral delivery Columnar cells were ellipsoidal and arranged closely, and PMs could be recognized clearly in the control midguts of larvae fed with untreated diet (Fig.3-1). Initial observations of toxin-treated midguts showed tissue levels similar to those of control midguts. After being exposed to toxin-treated diet for 6 h, PMs and the apical microvilli still existed but the columnar cells of the anterior midgut elongated from the basal membrane (Fig. 3-2). At 12 h, the columnar cells of toxin-treated midgut swelled apically and began to extrude large cytoplasmic vesicles into the gut lumen. PMs disappeared completely (Fig.3-3). However, the apically-formed blebs often contained nuclei as well as large vacuoles. The columnar cells extruded their contents into the gut lumen until the nucleus was ejected at 24-60 h (Fig.34,5). Although the gut epitheliumwas still disorganized, the PMs reappeared at 72 h (Fig.3-6).

DISCUSSION The study indicates that toxin IT existed both in cells and broth (Wang et al. 2005). Compared with other novel, orally active toxins such as the Gendotoxins from Bacillus thuringiensis,as well as Tca from Photorhabdus lurninescens W14 (Bowen et al. 1998; Blackburn et at. 1998), toxin I1 has similar effects on the midgut epithelium of H. armigera. After being fed with toxintreated diet, the effect on columnar cells of midgut epithelium was the most degenerating. Because columnar cells are located at the outer boundary, these cells are primarily affected when PMs are destroyed by oral activity. Goblet cells were also destroyed. The toxin had little effect on the revival cells because these cells lay in the basal membrane of the midgut epithelium. They were capable of restoring the destroyed columnar cells and goblet cells (Mu et al. 1996). The regenerated cells secreted PMs again. As a result of compensatory activity of the revival cells, toxin-treated insects could be fed with the diet and could grow up as pupae following the disappearance of toxicity after being exposed to fresh diet again. This could be due to the revival of PM and the epithelial cells. The effects of toxin I1 on the PMs had begun to

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occur transformation at 6 h after being fed with toxintreated diet and were broken into pieces after 12 h. Similar symptoms were found in toxin-treated PMs in vitro and histopathology staining of the midgut. PMs of H. armigera larvae belong to type 1 ,which is continuously secreted by all midgut epithelial cells. The PMs serve as the first line of defense in the midgut. When the PMs were destroyed, the toxin penetrated into the midgut epithelial cells and continued to destroy the cells. When the insects were subjected to transient or sublethal exposure to the toxin, the epithelial cells were gradually restored and excreted, and the PMs were renewed with the disappearance of the toxin activity (Mu et al. 1996). Although toxin I1 has low insecticidal activity on the older-instar larvae of H. armigera, it has high oral toxicity on H. annigera neonates, Pieris rapae, and Plutella xybstella larvae (Wang et al. 2004). Unlike other toxins, such as B. thuringiensis endotoxins, toxin I1 has high oral toxicity against a wide range of insects. These often exhibit specificity for a given insect group. Hence, it has the potential to be used as a kind of bacterial insecticide or as an alternative to Bt for transgenic deployment. Toxin I1 can also be used synergically with other insecticides.

Acknowledgements This work was supported by the National Natural Science Foundation of China (30400296), Natural Science Foundation of Hebei Province, China (C2006000443), and Fund from Hebei Agricultural University (9816).

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