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A novel insecticidal toxin from photorhabdus luminescens, toxin complex a (Tca), and its histopathological effects on the midgut of manduca sexta - PubMed

A novel insecticidal toxin from photorhabdus luminescens, toxin complex a (Tca), and its histopathological effects on the midgut of manduca sexta

M Blackburn et al. Appl Environ Microbiol. 1998 Aug.

Abstract

Photorhabdus luminescens is a bacterium which is mutualistic with entomophagous nematodes and which secretes high-molecular-weight toxin complexes following its release into the insect hemocoel upon nematode invasion. Thus, unlike other protein toxins from Bacillus thuringiensis (delta-endotoxins and Vip's), P. luminescens toxin (Pht) normally acts from within the insect hemocoel. Unexpectedly, therefore, the toxin complex has both oral and injectable activities against a wide range of insects. We have recently fractionated the protein toxin and shown it to consist of several native complexes, the most abundant of which we have termed Toxin complex a (Tca). This complex is highly active against the lepidopteran Manduca sexta. In view of the difference in the normal mode of delivery of P. luminescens toxin and the apparent communality in the histopathological effects of other gut-active toxins from B. thuringiensis, as well as cholesterol oxidase, we were interested in investigating the effects of purified Tca protein on larvae of M. sexta. Here we report that the histopathology of the M. sexta midgut is similar to that for other novel midgut-active toxins. Following oral ingestion of Tca by M. sexta, we observed an acceleration in the blebbing of the midgut epithelium into the gut lumen and eventual lysis of the epithelium. The midgut shows a similar histopathology following injection of Tca into the insect hemocoel. These results not only show that Tca is a highly active oral insecticide but also confirm the similar histopathologies of a range of very different gut-active toxins, despite presumed differences in modes of action and/or delivery. The implications for the mode of action of Tca are discussed.

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Figures

FIG. 1
FIG. 1

The Toxin complex a (tca) locus and the encoded Tca complex. (A) The tca locus consists of three open reading frames (tcaA, tcaB, and tcaC) transcribed in one direction and a shorter terminal open reading frame (tcaZ) transcribed in the opposite direction (3). Note that TcaA and TcaB are proteolytically cleaved (to TcaAi, TcaAii, and TcaAiii and to TcaBi and TcaBii, respectively) while TcaC is uncleaved. (B) On a native agarose gel, all of the components of Tca migrate as a single native complex (termed complex A or Band 1 [by Bowen and Ensign {}]). (C) On a sodium dodecyl sulfate-polyacrylamide gel, the different polypeptides encoded by the tca locus can be resolved (except for the predicted TcaZ, which is not detectable).

FIG. 2
FIG. 2

Symptoms displayed by first-instar M. sexta larvae after 72 h of feeding on diet treated with a single component (Tca) of P. luminescens toxin. (A) Untreated control. Note normal frass production. (B) Larva exposed to treated diet. Note that the animal has not increased in size since hatching and that frass production is greatly reduced. Bar, 5 mm.

FIG. 3
FIG. 3

Time course of the histopathological effects of Tca on the anterior midgut of M. sexta larvae. (A) Control. Shown is the anterior midgut epithelium of a 24-h-old first-instar larva fed untreated diet. Note the columnar cells (CC) and goblet cells (GC) of the midgut epithelium and the apical microvilli (AMV) of the columnar cells which border the lumen. (B) Tca-treated larva sectioned after 3 h on diet. Arrows indicate the formation of apical vesicles (V) which are shed into the gut lumen. Note that at this early stage in poisoning, these vesicles appear to be shed through the apical microvilli (unlabelled arrows), which are still attached to the columnar cells. (C) Section after 6 h on treated diet. Vesicles continue to be shed into the gut lumen. Note the basal-apical elongation of the epithelial cells, the appearance of nuclei within the shedding vesicles (NV), and the absence of apical microvilli. (D) Section after 12 h on treated diet. By this time, the columnar cells are destroyed and all that remains of the anterior midgut epithelium is a disorganized matrix of cellular debris and a few isolated goblet cells. Note the clear view of the basal membrane (BM), which appears to thicken at this stage and is now largely devoid of intact attached cells. (E) At 24 h, cellular remains continue to be shed from the basal membrane (BM), often leaving it completely exposed to the gut lumen. Note that a few distorted goblet cells persist. (F) Detail of 6-h section. Note that the columnar cells extrude their contents into the gut lumen until the nucleus itself (N) is ejected, presumably resulting in cell death. Also note the large vacuoles (Va) seen in the apical regions of the budding cells. Bars, 50 μm in plate A (applies to plates A to E); 10 μm in plate F.

FIG. 4
FIG. 4

Color plate illustrating the fates of different cells in the midgut epithelium after Tca poisoning. Goblet cells are stained red, and columnar cells are stained blue. (A) Longitudinal section at 6 h after exposure to treated diet. Note the junction of the foregut (FG) and midgut (MG) and that the midgut lumen is occluded with extruded gut epithelial cells. The presence of red and blue vesicles in the lumen suggests blebbing of both the goblet cells (red) and the columnar cells (blue). The absence of vesicles from the anterior midgut is due to the presence of the esophageal diverticula, which can clearly be seen as nonstaining membranes in this region of the gut. (B) Detail of panel A. Note goblet cells (red) clearly forming apical cytoplasmic vesicles. (C) Control. Shown is a normal anterior midgut epithelium from a neonate fed untreated diet for 6 h. Bars, 100 μm in plate A; 50 μm in plate C (also applies to plate B).

FIG. 5
FIG. 5

Histopathology due to injected Tca in midgut of third-instar M. sexta larvae. (A) Control. Normal morphology of columnar cells (CC) and goblet cells (GC) is shown. (B) Injected larva. Anterior midgut at 72 h after injection of 550 ng of purified Tca is shown. Note the rounded appearance of epithelial cells (arrows) despite the fact that toxin delivery is from the hemocoel rather than from the gut lumen (see text). Bar, 50 μm.

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