Photodynamic treatment of Chaoborus crystallinus larvae with chlorophyllin induces necrosis and apoptosis.

Chlorophyllin kills mosquito larvae (Culex, Aedes) in the aquatic habitat at low concentrations via photodynamic reactions under irradiation. The effects of chlorophyllin were investigated at the cellular level using the transparent larvae of Chaoborus crystallinus as a model system. Their transparency enabled in situ fluorescence investigation, showing that chlorophyllin accumulates in the intestine of the larvae. Uptake of chlorophyllin at room temperature took about 2 h. The fluorescence signal peaked after 5 h of incubation. Chlorophyllin accumulates up to about 15 ng per larvae. The intestine of treated larvae was dissected and stained with several dyes (acridine orange, Hoechst 33342 and propidium iodide). Apoptosis and necrosis increased with higher concentrations of chlorophyllin (to a smaller extent in dark controls) and were elevated in irradiated samples. Single cells from treated larvae were isolated and subjected to Annexin V flow cytometry. The fraction of apoptotic and necrotic cells increased significantly at a high chlorophyllin concentration (21.4 mg L(-1)) and under intensive irradiation. The activity of caspases-3, -8 and -9 as well as Bcl-2 and cytochrome c was investigated by means of western blot analysis. The data suggest a possible chlorophyllin concentration-dependent shift of the apoptotic pathway.

Optimizing conditions for the use of chlorophyll derivatives for photodynamic control of parasites in aquatic ecosystems.

Recently, it was demonstrated that mosquito larvae can be killed by means of photodynamic processes after the larvae have incorporated the photosensitizer chlorophyllin or pheophorbid, and were treated with light. The water-soluble substances were applied to and incorporated by the larvae in darkness. With Chaoborus sp. a dark incubation of about 3 h is sufficient to yield mortality of about 90% and ≥6 h resulted in almost 100% mortality during subsequent illumination. Temperature did not influence mortality of the larvae significantly in a treatment of 6 h dark incubation and subsequent 3 h illumination. At 10°C, 20°C, or 30°C, between 80% and 100% of the treated larvae died when the light intensity from a solar simulator was above 30 W/m(2). Lower irradiances were less effective. The LD(50) value of magnesium chlorophyllin was about 22.25 mg/l and for Zn chlorophyll 17.53 mg/l, while Cu chlorophyll (LD(50) 0.1 mg/l) was shown to be toxic also without light. Chlorophyllin, which was lyophilized immediately after extraction, was far more lethal to the larvae (LD(50) 14.88 mg/l) than air-dried Mg chlorophyllin.