Synchronized Expression of Two Caspase Family Genes,ice-2andice-5, in Hydrogen Peroxide-Induced Cells of the Silkworm,Bombyx mori

Abstract

Caspase family proteins play important roles in different stages of the apoptotic pathway. To date, however, functions of Bombyx mori L. (Lepidoptera: Bombycidae) caspase family genes are poorly known. This paper focuses on the morphology, mitochondrial membrane potential, and expression profiles of two novel B. mori caspase family genes (ice-2 and ice-5) in 3 µM hydrogen peroxide (H2O2) damaged B. mori cells, which were separated from the ovary of B. mori. In addition, comparisons were made between damage caused by H2O2 and by ultraviolet (UV) irradiation. The results showed that the potential change of the mitochondrial membrane occurred at 0.5 h after H2O2 stimulation, which was sooner than occurred in the UV treated model where the obvious decrease appeared at 6 h after stimulation. In addition, the total change in the potential of the mitochondrial membrane in H2O2 treated B. mori cells was larger than with UV treated cells during the whole process. Analysis of fluorescent quantitative real-time PCR demonstrated that ice-2 and ice-5 might be involved in both H2O2 and UV-induced apoptosis in B. mori cells. Notably, after exposure to H2O2, the expression patterns of ice-5 were remarkably higher than those of ice-2, while the result was the opposite after exposure to UV irradiation. The data indicate that apoptosis induced by H2O2 was directly related to the mitochondrial pathway. The two isoforms of B. mori ice may play different roles in the mitochondrion associated apoptotic pathway in B. mori cells, and the apoptotic pathway in H2O2 induced B. mori cells is different from the UV induced apoptotic pathway.

Induction ofManduca sextaLarvae Caspases Expression in Midgut Cells byBacillus thuringiensisCry1Ab Toxin

“…The aim of our work was to explore if pyroptosis and apoptosis cell response processes, are activated in vivo in the midgut cells of Manduca sexta larvae in response to Cry1Ab intoxication. Here, we show that M. sexta larvae intoxicated with Cry1Ab induce a transient expression of cas-1, at low doses (LC 25 ) and short times. In contrast, the cas-3 is induced after longer time of toxin exposure depending also in the toxin dose.…”

“…In Panel B of Figure 4, we show the analysis of the changes in fold expression of cas-1 and cas-3 mRNA after Cry1Ab ingestion relative to control larvae fed with a noncontaminated diet. In this figure, it is clear that the highest expression of cas-1 in response to Cry1Ab intoxication was observed after 30 min intoxication with a LC 25 showing 1.5 fold induction, and then, the expression of this cas-1 gene was repressed after 24 h of toxin ingestion as well as with higher LC 50 dose of toxin administration, where it was repressed at all tested times, when compared with the control larvae fed with a normal diet (Figure 4(b), upper Panel). In contrast, the expression of cas-3 mRNA showed an induction of 2.3 fold over the control larvae at 2 h after intoxication with LC 25 toxin dose (Figure 4(b), lower Panel) and one fold induction after 2 h intoxication with LC 50 toxin dose.…”

“…Cry1Ab was applied onto the diet surface in 24-well plates as described [20]. For clarity, we added thirty-five microliters of Cry1Ab crystal suspensions, containing two different toxin concentrations (1 and 2 ng cm −2 ), corresponding to the lethal concentrations of LC 25 , and LC 50 , respectively, as previously described [20]. Once the surface was completely dry, a neonate first-instar M. sexta larvae was added per well, using 24 larvae per dose and time (from 0 min to 48 h) in order to analyze the initial response of the larvae to the toxin action.…”

“…Total RNA isolated from midgut tissue from control M. sexta or with intoxicated larvae with LC 25 and LC 50 concentrations of Cry1Ab toxin at different times from 0 to 48 h was used for cDNA synthesis as previously reported [22]. Control larvae were feed with same diet without toxin addition at the same times.…”

“…The Cas-1 group of Lepidopteran proteins contains B. mori (Q8I9V7), Spodoptera frugiperda (P89116), Plutella xylostella (D9IVD4), Trichoplusia ni (B6EEC1), and Helicoverpa armigera (A7L9Z3), and the Dipteran group includes Drosophila melanogaster (O01382), Culex quinquefasciatus (B0W0K2), and Musca domestica (B5AK94) Cas-1 proteins related to activation of cytokines during inflammation. The Cas-3 group of Dipteran sequences includes sequences from Aedes aegypti (Q178B6), C. quinquefasciatus (B0WZJ4), and D. melanogaster (Q7KHK9), while the Lepidopteran group contains two Cas-3 sequences from B. mori (Q2HZ04 and Q2HZ05) that are related to apoptosis [25]. We also add some other caspase proteins to this analysis, such as the Cas-6 from D. melanogaster (Q9NHF9), Cas-7 from Anopheles stephensi (Q86FL0), and two initiator Cas-8 sequences, one from An.…”