Nitric oxide synthase in filariae: demonstration of nitric oxide production by embryos in Brugia malayi and Acanthocheilonema viteae.

The radical gas nitric oxide (NO) is synthesized by nitric oxide synthase (NOS) from l-arginine and molecular oxygen. Nitric oxide is an important signaling molecule in invertebrate and vertebrate systems. Previously we have shown that NOS is localized to more tissues in Brugia malayi than has been reported in Ascaris suum. In this paper, we analyze the distribution of NOS in Acanthocheilonema viteae, a filarial nematode that differs from B. malayi in that A. viteae females release microfilariae without a sheath. A. viteae is also one of a few filarial parasites without the Wolbachia intracellular endosymbiont. By use of a specific antibody, NOS was demonstrated in extracts of A. viteae and Dirofilaria immitis. The localization pattern of NOS in A. viteae was similar to that seen in B. malayi, with the enzyme localized to the body wall muscles of both sexes, developing spermatozoa, intrauterine sperm, and early embryos. By use of DAF-2, a fluorescent indicator specific for nitric oxide, the embryos of B. malayi and A. viteae were demonstrated to produce NO ex utero. The near identical staining patterns seen in A. viteae and B. malayi argue that NO is not produced by Wolbachia, nor is it produced by the nematodes in response to the infection. Localization of NOS to the sperm of filarial nematodes suggests a role for NO during fertilization as has been described for sea urchin and ascidian fertilization. Demonstration of the activity of embryonic NOS supports our earlier hypothesis that NO is a signaling molecule during embryogenesis in filarial nematodes.

Immunocytochemical studies on several developmental stages of Dipetalonema viteae (Filarioidea).

The binding of antibodies to infective larvae (L3), eggs, uterine contents and blood microfilariae was demonstrated by light microscopy employing the peroxidase-antiperoxidase (PAP) method. Antigen-antibody reactions were located on the shell of filarial eggs as well as on the cuticle and the interior of L3 and microfilariae. Using sera from Meriones unguiculatus, Mastomys natalensis and golden hamsters infected with D. viteae obtained at 7, 14, 21 and 28 weeks p.i. it was observed that the intensity of the immunostaining on several developmental stages of the filariae decreased with increasing duration of infection. This effect was more pronounced in the case of M. unguiculatus and M. natalensis than in the case of golden hamsters. For detection of surface antigenicity the simple procedure of drying the test material on microscopic slides proved to be time saving and equally specific as compared to the embedding in Epon of the filarial stages and the successive preparation of semithin sections. However, embedded filarial stages presented more intensive immunostaining than nonembedded stages. Moreover, sections allowed to demonstrate antigen-antibody reactions on the cuticle as well as inside the body cavity of the stages.

[R1 cell and musculature of Filariae; ultrastructural analysis (author's transl)]. / Cellule R1 et musculature des Filaires; analyse ultrastructurale.

Ultrastructural study of ontogenesis of a filaria, Dipetalonema (M.) dessetea, confirms Bain's (1970) hypothesis, following which the R1 cell plays a role in the elaboration of the musculature of the adult filaria. Anatomical observations related to the hypodermis and musculature of the larva (distribution of the cells, nature of their junctions, presence of intermediate cells between embryonic cells derived from R1 cell and muscle cells) show that the cells derived from R1, which form four longitudinal files, produced successively, from 5th to 13th day, about ten files of muscle cells in each submedian field. The primary musculature of the microfilaria, which corresponds to a single file of muscular cells in each field, is not ectodermic, as supposed before, but mesenchimatic. At the vicinity of R1 daughter cells, this musculature is slightly dedifferenciated (about 5 files of myofilaments instead of 10-20 in the microfilaria and the L3); then its evolution becomes identical to this of new formed muscle cells. The presence as well as the evolution of the R1 cell seem to be identical in every totally heteroxenous Phasmidian Nematodes; both suggest analogy with the imaginal discs of the holometabolic Insects.