Tailoring carotenoids by plant carotenoid cleavage dioxygenases (CCDs) generates various bioactive apocarotenoids. Recombinant CCD1 has been shown to catalyze symmetrical cleavage of C 40 carotenoid substrates at 9,10 and 9#,10# positions. The actual substrate(s) of the enzyme in planta, however, is still unknown. In this study, we have carried out RNA interference (RNAi)-mediated repression of a Medicago truncatula CCD1 gene in hairy roots colonized by the arbuscular mycorrhizal (AM) fungus Glomus intraradices. As a consequence, the normal AM-mediated accumulation of apocarotenoids (C 13 cyclohexenone and C 14 mycorradicin derivatives) was differentially modified. Mycorradicin derivatives were strongly reduced to 3% to 6% of the controls, while the cyclohexenone derivatives were only reduced to 30% to 47%. Concomitantly, a yellow-orange color appeared in RNAi roots. Based on ultraviolet light spectra and mass spectrometry analyses, the new compounds are C 27 apocarotenoic acid derivatives. These metabolic alterations did not lead to major changes in molecular markers of the AM symbiosis, although a moderate shift to more degenerating arbuscules was observed in RNAi roots. The unexpected outcome of the RNAi approach suggests C 27 apocarotenoids as the major substrates of CCD1 in mycorrhizal root cells. Moreover, literature data implicate C 27 apocarotenoid cleavage as the general functional role of CCD1 in planta. A revised scheme of plant carotenoid cleavage in two consecutive steps is proposed, in which CCD1 catalyzes only the second step in the cytosol (C 27 / C 14 + C 13 ), while the first step (C 40 / C 27 + C 13 ) may be catalyzed by CCD7 and/or CCD4 inside plastids.

Paramyosin, although a widely distributed muscle component among invertebrates, has hitherto not clearly been shown to occur in the muscles of schistosomes. Instead, it has been reported to occur in the tegument. In the present study, a specific antibody reacting with each of 10 isoforms of paramyosin was used for light microscopical immunolocalization in sections of Schistosoma mansoni. Specimens were fixed by a new method to immobilize antigens with uranyl acetate-trehalose-methanol. In cercariae, schistosomula, and adults, the circular and longitudinal muscles of the body wall, the dorsoventral muscles and those surrounding the gut and the pharynx as well as the fast moving cross-striated muscles of the tail of cercariae intensely reacted with the antibody. However, neither immunohistologically nor on Western blots of isolated tegument, were indications found for the presence of paramyosin in the tegument. In vivo phosphorylation and binding of anti-phospho-tyrosine and anti-phospho-serine antibodies show phosphorylation of paramyosin which probably is responsible for the generation of the isoforms.

The tegument of Ligula intestinalis plerocercoids is delimited by a membrane complex that in electron microscopy appears heptalaminate. We suggest that the plerocercoids are covered by two closely apposed lipid bilayers. Double membranes, which are well known in schistosomes, are thus not a unique feature of blood parasitic Digenea but could be documented for the first time in a cestode species that lives in the peritoneal cavity of its host. The surface-membrane complex of plerocercoids was lost for the most part after conventional preparation for electron microscopy but could be completely retained by improved fixation using osmium tetroxide plus potassium ferrocyanide. Furthermore, one type of vesicle in the tegumental syncytium of plerocercoids has been found to be enclosed by at least two membranes, which might indicate that these vesicles contribute to the renewal of the surface-membrane complex. Adult Ligula intestinalis removed from the gut of the final host Anas platyrhnychos or obtained by in vitro transformation exhibited a single surface membrane and lacked double membrane vesicles. The elongate electron-dense caps of the microtriches of plerocercoids were replaced by short caps in the course of worm maturation. Thus, the tegumental surface of this parasite is fundamentally altered following the change in its environment from the peritoneal cavity of the intermediate host to the intestine of the final host.

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