Solanum nigrum Extract and Solasonine Affected Hemolymph Metabolites and Ultrastructure of the Fat Body and the Midgut in Galleria mellonella.

Glycoalkaloids, secondary metabolites abundant in plants belonging to the Solanaceae family, may affect the physiology of insect pests. This paper presents original results dealing with the influence of a crude extract obtained from Solanum nigrum unripe berries and its main constituent, solasonine, on the physiology of Galleria mellonella (Lepidoptera) that can be used as an alternative bioinsecticide. G. mellonella IV instar larvae were treated with S. nigrum extract and solasonine at different concentrations. The effects of extract and solasonine were evaluated analyzing changes in carbohydrate and amino acid composition in hemolymph by RP-HPLC and in the ultrastructure of the fat body cells by TEM. Both extract and solasonine changed the level of hemolymph metabolites and the ultrastructure of the fat body and the midgut cells. In particular, the extract increased the erythritol level in the hemolymph compared to control, enlarged the intracellular space in fat body cells, and decreased cytoplasm and lipid droplets electron density. The solasonine, tested with three concentrations, caused the decrease of cytoplasm electron density in both fat body and midgut cells. Obtained results highlighted the disturbance of the midgut and the fat body due to glycoalkaloids and the potential role of hemolymph ingredients in its detoxification. These findings suggest a possible application of glycoalkaloids as a natural insecticide in the pest control of G. mellonella larvae.

Histopathological effects and immunolocalization of periplocoside NW from Periploca sepium Bunge on the midgut epithelium of Mythimna separata Walker larvae.

Periplocoside NW (PSNW) with pregnane glycoside skeleton is a novel insecticidal compound isolated from the root bark of Periploca sepium Bunge. This compound has a potent stomach poisoning activity against several insect pests. In this study, we observed the intoxication symptoms, investigated the histopathological effects and carried out immuno-electron microscopic localization of PSNW on the midgut epithelium of oriental armyworm Mythimna separata Walker larvae for better understanding its action mechanism against insects. Ultrastructural observations showed that cell damages caused by PSNW in the midgut of M. separata larvae are related to the degeneration of brush border microvilli. The dissolution of cytoskeletal structures in the interior and on the surface of microvilli was responsible for the decrease in size and eventual disappearance of microvilli when bubbles of cytoplasmic substances protrude into the midgut lumen of M. separata, thus resulting in cell death. The immuno-electron microscopic localization research showed that gold particle appeared on the microvilli layer of the midgut of M. separate larvae firstly. The density of gold particle gradually added with the time, and finally microvilli layer was destructed severely. Meantime, the gold particles were also presented to the intracellular organelle membrane and the organelles also were destructed. Therefore, we proposed that this membrane system on insect midgut epithelium cells is the initial acting site of PSNW against insects.

Histopathology and effect on development of the PhopGV on larvae of the potato tubermoth, Phthorimaea operculella (Lepidoptera: Gelechiidae).

Larvae of the potato tubermoth (PTM), Phthorimaea operculella, feed on potato plants and tubers and are a major pest in the tropics and subtropics worldwide, causing up to 100% damage. The PTM granulovirus (PhopGV) provides significant potato protection, but little is known about its effect on larval development or its histopathology. Here we show that only 10% of larvae exited from PhopGV-treated tubers (1.4×10(8) granule/ml), lagging significantly behind controls, and most of these died by 72 h after emergence. Histopathology studies showed the fat body and epidermis were the principal tissues infected. PhopGV morphogenesis was similar to other GVs, the exception being small vesicles between mature granules.

Ultrastructural effects of Celangulin V on midgut cells of the oriental armyworm, Mythimna separata walker (Lepidoptera: Noctuidae).

The ultrastructural effects of ingested Celangulin V (CA-V), an insecticidal component extracted from the root bark of Chinese bittersweet (Celastrus angulatus Maxim), on the midgut epithelial cells of the oriental armyworm larva (Mythimna separata Walker) were studied. Transmission electron microscopy showed that CA-V could induce a severe, time-dependent cytotoxicity of the midgut epithelial cells, which had large cytoplasmic spaces, disrupted microvilli and swollen mitochondria. The cisternae of the rough endoplasmic reticulum were excessively dilated, vesiculated and then fragmented. The nuclei were pycnotic and contained one or two divided nucleoli and agglomerated chromatin. Numerous lysosome-like vacuoles and secretion granules were observed. Finally, the CA-V resulted in cell death by necrosis with plasma membrane lysis and the cytoplasm content leakage into the migdut lumen.

Synthesis of the complete 200K polyprotein encoded by cowpea mosaic virus B-RNA in insect cells.

The coding sequence for the entire 200K polyprotein of cowpea mosaic virus (CPMV) B-RNA was expressed in insect cells by using baculovirus expression vectors. The 200K polyprotein, which harbours all virus functions required for RNA replication, is completely cleaved into 170K and 32K products by the 24K protease activity contained within the polyprotein. Further processing of the 170K protein into CPMV-specific products of 60K, 84K, 87K, 110K and 112K occurred to a limited extent, similar to that observed in cowpea cells. Electron microscopy of insect cells in which the 200K protein was produced revealed the presence of membranous vesicles and electron-dense structures which were not seen in cells infected with wild-type baculovirus. Similar cytopathic structures develop in the cytoplasm of CPMV-infected cowpea cells and are thought to be the site of membrane-bound viral RNA replication. The electron-dense structures in insect cells could be preferentially labelled with several CPMV-specific antisera and Protein A-gold. Since electron-dense structures were not observed in cells in which the 170K protein only was produced, it seems that the 32K protein has a role in keeping the B-RNA-encoded proteins in these structures together. Membranous vesicles were also observed in insect cells in which the 60K protein only was produced. Use of specific antibodies and Protein A-gold showed that the 60K protein is associated with these vesicles, indicating that the 60K protein may induce the formation of vesicles. Although proteolytic processing of the 200K polyprotein and the induction of cytopathic structures indicate that the CPMV proteins produced in insect cells are functional, it has not been possible to demonstrate RNA polymerase activity in extracts of these cells using an oligo(U)-primed assay. The results indicate that in the assay an additional component is lacking and/or that the CPMV polymerase is not able to start RNA synthesis on an exogenous template.