Immunocytochemical techniques were used to detect FMRFamide-like immunoreactivity in adults of the filarial nematodesDirofilaria immitis andBrugia pahangi. An FMRFamide-like peptide was also located in third-and fourth-stage larvae ofd. immitis. Positive immunoreactivity was observed in all parasites examined, irrespective of developmental stage. The major areas of positive immunoreactivity were located in the anterior nerve ring, lateral/dorso-ventral nerves, cephalic papillary ganglia and lateral ganglia. No staining was seen in the intestine or gonads of any parasite. These results indicate that filarial worms possess a peptidergic component in their nervous system. The possible role of an FMRFamide-like peptide in the control of certain physiological events is discussed.

When injected with [3H]ecdysone and maintained in vitro, the parasitic nematodes, Ascaris suum and Parascaris equorum each produced a series of polar and relatively apolar metabolites. A. suum metabolised the compound into ecdysonoic acid ([3H]EOIC), ecdysone 25-glucoside ([3H]E25gluc), putative ecdysone 22-phosphate([3H]E22P) and a series of at least six relatively apolar metabolites. All of these, except ecdysonoic acid, were hydrolysed by a crude enzyme preparation from Helix pomatia, releasing ecdysone. In a similar study, P. equorum produced ecdysone 25-glucoside, putative ecdysone 22-phosphate and a series of relatively apolar compounds all of which were hydrolysed by H. pomatia enzymes, releasing ecdysone. [3H]Ecdysone 25-glucoside was the most abundant single metabolite in both species, and in P. equorum, at least, was released into the culture medium in relatively large amounts. Apolar metabolites were present in worm samples and were the major, if not the only radiolabelled compounds detected in eggs of both species. Data indicated a metabolic relationship between some of the apolar conjugates found in both nematode species and ecdysone 25-glucoside.

The effects of the ecdysteroids ecdysone and 20-hydroxyecdysone on microfilarial release inBrugia pahangi and on meiotic reinitiation in the oocytes ofDirofilaria immitis were studied. Ecdysone was found to stimulate microfilarial release at 2×10−6M, but this effect was reduced at higher and lower concentrations. 20-Hydroxyecdysone was found to have no such effect. InD. immitis, ecdysone at 10−5M was also found to overcome the period of meiotic arrest that occurs during the pachytene stage of prophase 1. This effect was reduced when 10−6M ecdysone was used. These results help support the theory that the ecdysteroids play a hormonal role in filarial worms similar to that found in insects.

The excretion of ecdysteroids by the filarial nematode species, Dirofilaria immitis and Brugia pahangi, was examined both in vitro, by the analysis of culture medium, and in vivo, through analysis of serum samples from experimentally infected hosts. There was no evidence of ecdysteroid excretion by intact parasites of either species in vitro. Free ecdysteroids were detected in the serum of ferrets and dogs infected with D. immitis, but concentrations would be at or below the limit of detection in sub-millilitre serum samples. The detection of ecdysteroids in the serum of potential hosts is unlikely to be of value in the diagnosis of filarial infections due to a combination of low titre in the presence of current infection and measurable titre in its absence. Ecdysteroids of dietary origin may contribute to the latter.

Summary A series of compounds that apparently disrupt hormonally regulated processes in insects have been examined for effects on the viability and microfilarial production of adult Brugia pahangi cultured in vitro. The azasteroids, 25-azacoprostane and 25-azacholestane, inhibited the production of microfilariae at 5 ppm, the former also exhibiting macrofilaricidal activity at this concentration. The brassinosteroids examined inhibited microfilarial production at 5 ppm but did not affect worm viability. Azadirachtin also proved to be a significant inhibitor of microfilarial release without effect on worm motility or viability. Of all the compounds tested, the non-steroidal amines appeared to be the most promising as potential filaricides, several of them proving to be macrofilaricidal at 1 ppm and affecting microfilarial production at even lower concentrations.

Free ecdysteroids were detected in Onchocerca gibsoni, in tissues constituting O. volvulus and O. gibsoni nodules and in unrelated bovine tissues. Ecdysone and 20-hydroxyecdysone were identified by HPLC-RIA and GC/MS(SIM). The concentration of free ecdysteroids in the nodule tissue immediately surrounding the parasites was at least an order of magnitude higher than that detected in the worms themselves, or in adjacent nodular tissues or other bovine tissues.

Experiments were performed to investigate whether adult Dirofilaria immitis and Brugia pahangi were capable of synthesising ecdysteroids from cholesterol or various intermediates from the biosynthetic pathway functioning in insects. Metabolites of radioactively-labelled cholesterol and 5 beta-ketodiol (2,22,25-trideoxy-ecdysone) were detected in the filarial nematodes, but there was no radioactivity corresponding to ecdysteroid. Uptake of tritiated 2-deoxyecdysone was poor and metabolism was not observed. [3H]Ecdysone was absorbed sparingly by adult D. immitis, but was metabolised efficiently to several less polar products. There was no evidence of C-20 hydroxylation capability.

Adult males and females of the dog heartworm, Dirofilaria immitis, and of the swine parasite, Ascaris suum, were extracted, the free and polar conjugated ecdysteroid fractions separated and the latter hydrolysed enzymically. The ecdysteroids released by hydrolysis of the conjugates and the free hormones were analysed by radioimmunoassay, high-performance liquid chromatography on reversed phase and adsorption columns monitoring fractions by radioimmunoassay, and by gas-liquid chromatography/mass spectrometry (selected ion monitoring). In both species, males and females contained free and polar conjugated ecdysteroids, with evidence for the presence primarily of ecdysone and 20-hydroxyecdysone together with smaller amounts of 20,26-dihydroxyecdysone. Males and females of both species were then dissected into body fluid, reproductive system, gut and remaining body wall compartments, the ecdysteroids extracted, fractionated and analysed by radioimmunoassay and high-performance liquid chromatography monitoring fractions by radioimmunoassay. The reuslts for both sexes in the two species were similar and indicated that ecdysteroids were not detectable in body fluids and that free ecdysteroids occurred in the reproductive system and the body wall, whereas polar conjugated ecdysteroids were detected in the reproductive system and the gut; a minor portion of the free ecdysteroids in A. suum was also apparently present in the gut. Further localization of the ecdysteroids in the body wall of A. suum females suggested that negligible immunoreactivity was associated with the circumpharyngeal nerve ring. The possible significance of the results is discussed.

Protein synthesis in intact Plasmodium falciparum was 333 times more sensitive to cycloheximide than to chloramphenicol. The 50% inhibitory concentration (IC50) of cycloheximide in a 27-h assay in vitro was 6 × 10−7 M but no constant cycloheximide-insensitive fraction of total protein synthesis was observed at concentrations of this inhibitor between 10−2 and 10−2 M. 0.24% of total protein synthesis occurred in the presence of 10−3 M cycloheximide but the chloramphenicol sensitivity of this fraction was similar to that of overall protein synthesis (IC50 2 × 10−4 M). The major fraction of protein synthesis by P. falciparum, therefore, is assumed to be cytoplasmic and to occur on 80S ribosomes. Cycloheximide-insensitive, chloramphenicol-sensitive (70S ribosomal) protein synthesis being undetectable by the methods employed, mitochondrial protein synthesis in P. falciparum is presumed to constitute a considerably smaller fraction of the total protein synthetic capacity than observed in other lower eukaryotes.

The turnover of surface proteins in adults, fourth-stage and third-stage larvae of Brugia pahangi was measured using [125I]iodosulfanilic acid. Groups of worms (n = 10 adult, 20 L4, 50 L3) were labelled and surgically implanted into the peritoneal cavity of naive jirds. The amount of radioactivity remaining on worms recovered over a 7–8 day period was determined. Adult females showed no significant loss of label during a 7 day period. The recovery of fourth-stage larvae was low but the counts per minute remaining on each group of larvae recovered over an 8 day period, encompassing the major part of the instar, did not fall below the limits of the standard deviation of the time 0 groups, indicating that no significant loss of surface label had occurred. Third-stage larvae showed a significant loss of 125I-labelled proteins prior to the third moult, although it was not confirmed that these proteins occur on the worm surface. Electrophoresis and autoradiography of labelled homogenates of adult, fourth and third stage larvae suggested that [125I]iodosulfanilic acid labels polypeptides of different molecular weights on each life cycle stage of B. pahangi.

The specificity of a range of 125I labelling techniques (Chloramine T, Iodogen, Bolton and Hunter reagent, lactoperoxidase and iodosulfanilic acid) to the surface of the filarial nematode Brugia pahangi was evaluated by autoradiography of sections of labelled worms and of dried SDS-polyacrylamide gels following electrophoresis of homogenised worm extracts. It was concluded that Bolton and Hunter reagent was not surface specific but labelled proteins throughout the body of the worm. At the light microscope level autoradiography of worms labelled using Chloramine T, Iodogen, lactoperoxidase and iodosulfanilic acid demonstrated that the 125I labelling was restricted to the worm surface. Electrophoresis and autoradiography showed that each method produced a different pattern of labelled polypeptide. A polypeptide of molecular weight 30 kDa was labelled using each method except Bolton and Hunter reagent, and appears to be a major surface component.

Incubation in vitro of adult Brugia pahangi in an apparatus which permitted the separate exposure of the anterior, middle, or posterior region of the worms to medium-containing radioactively labeled d-glucose, l-leucine, and adenosine has provided evidence that these materials are taken up in physiologically significant amounts by a transcuticular route. No evidence for an oral ingestion of materials has been obtained from worms in vitro, but in vivo an oral uptake of Trypan blue has been demonstrated. The ultrastructure and cytochemical staining reactions for nonspecific esterase, acid phosphatase (EC-3.1.3.2), and leucine naphthylamidase of the gut and body wall are described.

The uptake and incorporation in vitro of various nucleic acid precursors by microfilariae, third-stage infective larvae, 10-day-old juveniles and adult worms of Brugia pahangi were investigated using scintillation counting and autoradiographic techniques. A significant uptake of uracil and of purines, including adenine, hypoxanthine, and guanine was demonstrated in this study. No evidence was obtained for the uptake and incorporation of thymine, cytosine, orotate, formate, folate or p-aminobenzoic acid by either micro- or macrofilariae of B. pahangi.