Genetic characterization of three species of Onchocerca at 23 enzyme loci

The technique of allozyme electrophoresis was applied to species of Onchocerca from cattle to increase the number of enzyme loci established and therefore provide a genetic basis for a rational species-level taxonomy. Twenty-three enzyme loci were established and provided unequivocal genetic evidence for the taxonomic validity of Onchocerca gibsoni, O. gutturosa and O. lienalis. Furthermore, the diagnostic enzyme markers detected form the basis for identification of life-cycle stages, individuals and species and population structure analyses.     

Strain variation in Hymenolepis diminuta: enzyme profiles

In comparative study of respiratory metabolism, it was established that the relative proportions of respiratory end-products (succinic, acetic and lactic acids) differed consistently in two strains of Hymenolepis diminuta (Toronto and ANU). The ANU strain produced more lactic acid and less succinic acid under aerobic and anaerobic conditions. In the shift from aerobic to anaerobic conditions both strains compensated by increasing their outputs of succinic acid. The ANU strain possessed significantly higher activities of hexokinase, pyruvate kinase, lactate dehydrogenase, cytosolic and mitochondrial malic enzyme and cytosolic α-glycerophosphate dehy drogenase. The Toronto strain had significantly higher activities of fumarase, succinate dehydrogenase, and fumarate reductase. There were no significant differences in the activities of phosphoenolpyruvate carboxykinase and malic dehydrogenase between strains. The fumarase activity in the Toronto strain was 16 times that of the ANU strain, its K_m (malate) was 0.8mM, as opposed to 2.5 mM, and it was less sensitive to inhibition by NAD or ATP. These observations are consistent with the patterns of end-product formation in the two strains. Ratios of end-products and calculations of approximate redox balance suggest that the Toronto strain may have a greater capacity for aerobic metabolism.     

Molecular cloning and characterization of Hymenolepis diminuta alpha-tubulin gene.

To isolate a full-length alpha-tubulin cDNA from an eucestode, Hymenolepis diminuta, a lambda phage cDNA library was constructed. The alpha-tubulin gene was cloned, sequenced and characterized. The H. diminuta alpha-tubulin consisted of 450 amino acids. This protein contained putative sites for all posttranslational modifications as detyrosination/tyrosination at the carboxyl-terminal of protien, phosphorylation at residues R79 and K336, glycylation/glutamylation at residue G445 and acetylation at residue K40. Comparisons of H. diminuta alpha-tubulin with all full-length alpha-tubulin proteins revealed that H. diminuta alpha-tubulin possesses 10 distinctive residues, which are not found in any other alpha-tubulins. Phylogenetic analysis showed that H. diminuta alpha-tubulin has grouped in a separated branch adjacent eucestode and trematodes branch with 92% bootstrap value (1000 replicates). In conclusion, this is the first report of H. diminuta cDNA library construction, cloning and characterization of H. diminuta alpha-tubulin gene.     

Mucosal mast cell response to Hymenolepis diminuta infection in different rat strains.

Five-week-old DA male rats infected with 10 Hymenolepis diminuta cysticercoids showed significant mastocytosis 6 weeks post-infection and low persistence of worms. In F344/N rats, however, no mastocytosis and no worm loss occurred during a 6 week infection. Mucosal mast cells appear to be associated with the expulsion of H. diminuta from DA rats.     

Differential establishment and survival of Hymenolepis diminuta in syngeneic and outbred rat strains.

Experimental Hymenolepis diminuta infection was carried out in inbred strains of rats (F344/N, JAR-2, LOU/M, TM, DA and DA-bg/bg) and outbred Wistar rats. All strains became infected with this cestode, but clear strain-dependent variation in the susceptibility to H. diminuta infection was observed. Marked differences in worm persistence and worm weight were found at 6 weeks post-infection in TM and DA rats. These strains would be useful to clarify the interactions between H. diminuta and its rat host.     

Developmental physiology of cestodes: characterization of putative crowding factors in Hymenolepis diminuta

It was shown previously that worm-conditioned saline (WCS) prepared from crowded 10-day-old H. diminuta inhibited the incorporation of 3H-thymidine into DNA in the anterior regions of uncrowded worms and that the inhibition was partially accounted for by succinate and acetate excreted by the worms. The present study describes further characterization of the active components of WCS. An ultrafiltrate was fully as potent as untreated WCS, indicating that all detectable inhibitory components were less than about 500 daltons in molecular mass. Inhibitory factors in WCS were stable to heat (80 C for 30 min), cold (4 C for 48 hr), drying and reconstitution, alkaline pH (11 to 12 for 3 hr), and ethanolic extraction. Active compounds were probably not lipoidal in nature. Although the acidic ethanol extract of WCS was inhibitory, no activity was observed in fractions of WCS that contained basic, acidic and neutral amino acids. Amino compounds in the WCS were further investigated. Twenty-four amino acids were identified, 3 of which (phosphoserine, 1-methylhistidine, and 3-methylhistidine) have not been reported previously for H. diminuta. On a molar basis, alanine accounted for 40-50% of the amino acids released. The amino sugar, D-glucosaminic acid, was found in the WCS and also has not been heretofore reported from H. diminuta or any other cestode. In concentrations comparable to those in the WCS, D-glucosaminic acid inhibited incorporation of 3H-thymidine into the DNA of the tapeworms by 25-35%, suggesting that D-glucosaminic acid may be one of the crowding factors.     

Extensive heterozygosity at three enzyme loci in hybrid sunfish populations

Hybrid populations of sunfishes were produced in two different ponds, and the frequencies of allelic isozyme phenotypes were determined for three enzyme systems—malate dehydrogenase (NAD), esterases, and tetrazolium oxidase—in order to estimate the extent of heterozygosity at four different genetic loci. Interspecific F₁ hybrid fry (red-ear male × bluegill female) were produced in vitro. These fry were stocked in ponds at the free-swimming stage. When 1 year old, the F₁ hybrids produced a large F₂ hybrid population. Successful hybrid reproduction occurred each year thereafter. In one pond, a 1-year-old F₂ population exhibited all three isozyme phenotypes (red-ear, F₁, bluegill) at most loci in the approximate ratio of the 1:2:1 expected. In a second pond, 5-year-old individuals of the F₂ generation were morphologically like the F₁ and were all heterozygous for the enzyme loci studied. This unusual degree of heterozygosity in the older F₂ population appeared to be the result of differential survival of mature heterozygous individuals and not the result of early embryonic lethality. The increased heterozygosity at these unlinked loci was assumed to reflect the condition at other genetic loci in the F₂ hybrids. Several possible mechanisms are advanced to explain this apparent heterosis.This research was supported by NSF grant GB-16425 (G.S.W.) and by funds from the Illinois Natural History Survey (W.F.C.).     

Hymenolepis diminuta: immunogenicity of the strobilate worm

Mice were immunized against challenge with Hymenolepis diminuta by feeding cysticercoids or by surgically implanting into the duodenum strobilate worms of different ages. Young worms stimulate stronger immunity than older ones, although the latter presents the host with a greater amount of strobilar tissue per unit time. An increase in the number of immunizing worms is associated with an increase in the level of protection. It is concluded that the development of functional immunity against H. diminuta in mice has both quantitative and qualitative antigenic requirements; it is influenced by worm age and is independent of worm mass.