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.

Keywords: Ca2+ release; apoptosis; azadirachtin; growth regulation; prothoracic gland; silkworm.

Fig 1

Morphological characteristics of silkworms after treated by 2.5 mg/L azadirachtin. a. Fifth instar larvae of silkworms treated by 2.5 mg/L azadirachtin produced smaller cocoon compared with control group. b. The development could be influenced by treatment with 2.5 mg/L azadirachtin on 5^th instar larvae. c. The cocooning rate after treated by different concentrations of azadirachtin on 5^th instar larvae of silkworms. Every group has 50 silkworms, and repeated for three times. Different letters indicated statistically significant difference when compared with control (DMRT). d. The eclosion rate after treated by different concentrations of azadirachtin on 5^th instar larvae. Every group has 50 silkworms, and repeated for three times. Different letters indicated statistically significant difference when compared with control (DMRT).

Fig 2

Morphological characteristics of prothoracic gland of 5^th instar larvae of silkworm after treated by 2.5 mg/L azadirachtin observed by optical microscopy. a. The morphological characteristics of prothoracic gland in 5^th instar larvae silkworm in control group. b. The morphological characteristics of prothoracic gland in 5^th instar larvae of silkworm after treated by 2.5 mg/L azadirachtin. c. The full length of prothoracic gland in control and treated group. d. The diameter of prothoracic gland in control and treated group. e. The length of widest part of prothoracic gland in control and treated group. f. The length of narrowest part of prothoracic gland in control and treated group. Every treated and control group has 50 silkworm, and every group was repeated for 3 times. * indicated statistically significant difference when compared with control (DMRT) (*, P<0.5, **, P<0.01).

Fig 3

Morphological changes of prothoracic gland of 5th instar larvae after treated by 2.5 mg/L azadirachtin by transmission electron microscopy. a, c, e, g indicated morphological characteristics in control group; b, d, f, h indicated morphological characteristics in treated group. C: chromatin, V: vacuole, NE: nuclear membrane, M: mitochondria, AT: authophagosome. The bar in a, b, c, d, e, f, g, h represented 5 μm, 5um, 0.5μm, 1μm, 200 nm, 100 nm, 5 μm and 1μm.

Fig 4

TUNEL assays. a, The prothoracic gland in control group was dissected and was observed under fluorescence microscopy (Olympus BX51, Japan) by stained with DAB solution and mounted in Fluoromount-G. b, The prothoracic gland in treated group.

Fig 5

Agarose gel electrophoretic pattern of prothoracic gland after treated by azadirachtin. Lane M: marker. Lane 1: DNA sample of the prothoracic gland in control group. Lane 2: DNA sample of the prothoracic gland in control group after 36 h. Lane 3: DNA sample of the prothoracic gland after treated by azadirachtin for 0 h. Lane 4: DNA sample of the prothoracic gland after treated by azadirachtin for 12 h. Lane 5: DNA sample of the prothoracic gland after treated by azadirachtin for 36 h.

Fig 6

TUNEL assays of prothoracic gland in vitro. The prothoracic gland after treated by 20-hydrocyecdysone in vitro with 100× magnification (a) and 200× magnification (b). The prothoracic gland after treated by azadirachtin in vitro with 100× magnification (c) and 200× magnification (d).

Fig 7

Measurement of enzyme activities. “*” indicated significant difference (P<0.05), “**” indicated significant difference (P<0.01).

Fig 8

Dynamic curves of Ca2+ in the Sf9 treated by azadirachtin. a. The dynamic curves of Ca2+ without any treatment in DHank's solution. b. The dynamic curves of Ca2+ after treated by azadirachtin in DHank's solution. 100 μg/ml of azadirachtin was added. c. The fluorescence intensity of cells after treated by azadirachtin for different time intervals.