Persistence of pierisin-1 activities in the adult cabbage white butterfly, Pieris rapae, during storage after killing

Abstract

Crude extracts from larvae, pupae and adults of cabbage white butterflies, Pieris rapae and Pieris brassicae, and green-veined butterfly, Pieris napi, have an ability to induce apoptosis in the human cancer cell lines. As apoptosis inducing protein, pierisin-1 and -2 have been isolated from pupae of P. rapae and P. brassicae, respectively, and shown to exhibit DNA ADP-ribosylating activity. Although the highest activity was detected in the late phase of larvae and early phase of pupae, certain activity was found in adult butterflies. In order to investigate distribution of substances having pierisin-like activities in butterflies, many species need to be analyzed. However, fresh samples of larvae and pupae are hard to obtain, especially if samples are of scarce species or from overseas. The usage of adult butterflies is practical to examine the distribution of pierisin-like activity in many species. In this study, we examined the cytotoxicity of crude extracts from adults of P. rapae against HeLa cells and DNA ADP-ribosylation ability during storage for 1, 2 and 8 weeks at room temperature after killing adult butterflies after eclosion. Body weights decreased to 18% for 8 weeks through dehydration. Cytotoxicity of samples from butterfly kept for 1, 2 and 8 weeks decreased to 47, 39 and 22%, respectively, of the control value. DNA ADP-ribosylating activity of the samples also decreased to 30, 27 and 23%. Similar reduction was observed on western blot analysis with anti-pierisin-1 antibody. Fortunately, these results suggest that cytotoxic and DNA ADP-ribosylating activity persists to some extent in the body after killing, at least for 8 weeks. Thus, butterfly adult samples kept for two months at room temperature can still be useful for examination of the presence of substance having pierisin-like activity.

Distribution of cytotoxic and DNA ADP-ribosylating activity in crude extracts from butterflies among the family Pieridae

“…Both activities were decreased by Ϸ20% compared with fresh samples (18). In the present study, we found that crude extracts from dried adult samples of Talbotia naganum, Aporia gigantea, Aporia hippia, Delias pasithoe, and Appias nero exhibited cytotoxicity and DNA ADP-ribosylating activity.…”

“…Recently, we reported that cytotoxic and DNA ADP-ribosylating activities of pierisin-1 in adults of Pieris rapae persisted to some extent in the body after killing. Both activities decreased to Ϸ20% for 8 weeks (18). This is valuable information in that adult specimens after death can still be used for examination of the presence of substance having pierisin-like activity, at least for a few months, thus increasing the availability of species in this study.…”

“…They were fed with 10% honey solution and oviposited on fresh natural host plants: Brassica oleracea for Pieris rapae, Arabis glabra for Pieris napi and Anthocharis scolymus, Rorippa indica for Pieris melete, Albizia julibrissin for Eurema hecabe, and Trifolium pratense for Colias erate in our laboratory. After hatching, larvae were reared on fresh natural diet at room temperature until pupation, except for the fourth and fifth larvae of Pieris rapae that reared on artificial diet, as reported (18). Final instar larvae of Pieris canidia, Aporia crataegi, and Appias lyncida were purchased from Eikoh Science Corp., and were reared on natural host plants: Brassica oleracea for Pieris canidia, Prunus yedoensis for Aporia crataegi, and Crataeva religiosa for Appias lyncida at room temperature until pupation in our laboratory.…”

“…Cytotoxicity of crude extract samples against HeLa cells was examined with WST-1 [2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium; Dojindo Laboratory] as described (2,3,18).…”

“…The DNA was recovered, and digested to deoxyribonucleoside 3Ј-phosphates by treatment with micrococcal nuclease and phosphodiesterase II, and were spotted onto TLC sheets. After development, these were exposed to Fuji Imaging Plates (Fujifilm) and DNA adducts were detected with a Bio-Image Analyzer (Fujifilm) (18). Levels of adduct formation were calculated by densitometric analysis of DNA adducts by using Scion Image software, values being normalized to that for 1 pg of pierisin-1 in each test.…”