Azadirachtin induced apoptosis in the prothoracic gland in Bombyx mori and a pronounced Ca²⁺ release effect in Sf9 cells

Abstract

Azadirachtin is a bio-rational insecticide used as an antifeedant and growth disruption agent against many insect species. However, recent studies have shown that there is a potential risk of this compound harming some beneficial insects. In such cases its application does not normally lead to death, but it may result in altered developmental regulation. Therefore, it is essential to obtain toxicological data to understand the mechanism of such sub-lethal effects, especially where they relate to important beneficial insects. Here, we found that azadirachtin could regulate growth and cocooning in silkworms, which may be associated with induced apoptotic effect on the prothoracic gland. However, azadirachtin treatment could not induce apoptosis in the prothoracic gland in vitro, in contrast to the effect of 20-hydroxyecdysone in vitro, which suggesting that the apoptosis might not be direct effect of azadirachtin. Then we examined the activity of Ca2+-Mg2+-ATPase and found that azadirachtin could trigger a significant increase in intracellular Ca2+ release in the Sf9 cell line, which suggested that the calcium signaling pathway might be involved in the process of apoptosis in prothoracic gland and growth regulation in vivo silkworms. Although more evidence is needed to fully understand the mechanism of azadirachtin in perturbing the growth of silkworms, this study provides some toxicological information and highlights the potential risks of azadirachtin in relation to silkworms.

Proteomic Profiling Analysis of Male Infertility inSpodoptera LituraLarvae Challenged with Azadirachtin and its Potential‐Regulated Pathways in the Following Stages

“…For TUNEL, after the testis were dissected from treated and control moths, they were collected and fixed with 4% paraformaldehyde overnight at 4 °C, washed four times with PBS for 5 min at room temperature and stored in 70% ethanol at –20 °C until use. After sectioned, the sample was detected by using a commercial kit (Apo‐Direct TUNEL assay kit, Millipore) as described previously . Then the treated samples were observed by transmission electron microscope (TEM).…”

Prodigiosin isolated from Serratia�marcescens in the Periplaneta�americana gut and its apoptosis‑inducing activity in HeLa cells

“…The HeLa cells were then harvested, washed with PBS and fixed with 2.5% glutaraldehyde at 4˚C for 12 h. Following washing with PBS, HeLa cells were dehydrated with ethanol and acetone, sequentially fixed with 1% osmium tetroxide (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 2 h at 37˚C, followed by embedding in epoxy resin for 12 h at 37˚C. Ultra-thin slices (80 nm) were then produced, stained with 1.0% uranylacetate for 5 min at room temperature and viewed on a transmission electron microscope (23,24).…”

Stability of selected reference genes in Sf9 cells treated with extrinsic apoptotic agents

“…Apoptosis has been reported in different insect cell lines treated with azadirachtin, including SL-1 ( S. litura ), BTI-Tn-5B1–4 ( Trichoplusia ni ) and S2 ( Drosophila melanogaster ) 26,29,30 . In addition, it has also been observed in prothoracic glands of Bombyx mori and in midgut of S. litura after treated with azadirachtin 31,32 . Our previous studies indicated that azadirachtin could activate mitochondrial and lysosomal pathways that lead to apoptosis in Sf9 cells 1,8,33 .…”