Purification and properties of the cyclic 3',5'-AMP-binding protein from the muscle of the Nematode Ascaris suum

The cyclic 3',5'-AMP-binding protein was isolated from the muscle of Ascaris suum and purified to apparent homogeneity. It migrated as a protein with a relative Mr 54,000 on electrophoresis under denaturing conditions. On gel filtration columns it was eluted at a volume corresponding to a protein of Mr greater than 200,000 under conditions which kept the cyclic 3',5'-AMP-binding property intact. The purified catalytic subunit of protein kinase from Ascaris and the C subunit of cyclic 3',5'-AMP-dependent protein kinase from bovine heart were inhibited by the cyclic 3',5'-AMP-binding protein. Gel filtration studies indicated the formation of a stable protein complex between the protein kinase and the cyclic 3',5'-AMP-binding protein from Ascaris.     

Optimization of the Agar-gel Method for Isolation of Migrating Ascaris suum Larvae From the Liver and Lungs of Pigs

Experiments on use of an agar-gel method for recovery of migrating Ascaris suum larvae from the liver and lungs of pigs were conducted to obtain fast standardized methods. Subsamples of blended tissues of pig liver and lungs were mixed with agar to a final concentration of 1% agar and the larvae allowed to migrate out of the agar-gel into 0.9% NaCl at 38°C. The results showed that within 3 h more than 88% of the recoverable larvae migrated out of the liver agar-gel and more than 83% of the obtained larvae migrated out of the lung agar-gel. The larvae were subsequently available in a very clean suspension which reduced the sample counting time. Blending the liver for 60 sec in a commercial blender showed significantly higher larvae recovery than blending for 30 sec. Addition of gentamycin to reduce bacterial growth during incubation, glucose to increase larval motility during migration or ice to increase sedimentation of migrated larvae did not influence larvae recovery significantly.     

Catalase activity during the development of the parasitic nematode, Ascaris suum

1. Catalase activity was partially purified from body wall muscle of the parasitic nematode, Ascaris suum, and was similar to catalases isolated from mammalian tissues. It exhibited a broad pH optimum and was unaffected by 2 mM ethylenediaminetetra-acetate. In contrast, it was inhibited reversibly by 1 mM cyanide and irreversibly by prior incubation in 40 mM 3-amino-1:2:4-triazole for 1 hr or heating at 80 degrees C for 15 min. 2. Catalase activity was highest in the unembryonated "egg" and decreased dramatically as development proceeded. 3. Catalase activity in adult body wall muscle was similar to that in rat skeletal muscle, but dramatically lower than that in rat liver. Catalase activity was barely detectable in A. suum testis. 4. Cytochrome-c peroxidase activity did not appear to be present in adult A. suum muscle mitochondria.     

Alternative migration routes of Ascaris suum in the pig

Experiments were conducted to investigate possible alternative routes of extraintestinal migration of Ascaris suum larvae in the pig. Pigs were infected with A. suum via injection of newly hatched larvae into cecal veins (i.v.), into cecal lymph nodes (LN), or intraperitoneally (i.p.), and control animals were inoculated orally with infective eggs (p.o.). Two pigs per inoculation route were necropsied on days 1, 4, and 13 postinoculation. The numbers of liver lesions and the percentage of larvae recovered was considerably greater in pigs inoculated i.v. or p.o. on each necropsy day. However, irrespective of inoculation route, at least a proportion of larvae passed through the livers and were able to complete migration to the small intestine by day 13. The results indicate that larval penetration of the intestinal wall is not necessary for liver-lung migration and that passage through the liver may be favorable for migrating A. suum larvae, although a delayed arrival in the small intestine cannot be ruled out for larvae following alternative routes.     

Intrapopulation variation in carbon and nitrogen stable isotope ratios in the earthworm Aporrectodea longa

The natural abundance variations in carbon and nitrogen stable isotope ratios in a population of the earthworm Aporrectodea longa, a species known to feed on both soil and plant litter, is reported in this paper. Worms were collected from a small land area of an old white clover field and body tissue and mucus were analyzed separately. The range of isotopic values was small, but patterns of variation were not random. Tissue carbon and nitrogen isotope ratios were significantly higher in adult than in juvenile A. longa and tissue nitrogen isotope ratios tended to increase with increasing biomass of individuals. Further, carbon and nitrogen isotope ratios were positively correlated in both tissue and mucus. Possible causes of the observed patterns, including physiological effects, body composition and assimilation of C and N from different plant, soil and microbial sources are discussed. It is concluded that the causes of natural variability in isotopic composition must be understood and validated experimentally before natural abundance stable isotope methods can be used for the analysis of trophic relations among detritivorous soil invertebrates.     

Extracellular recordings from the motor nervous system of the nematode,Ascaris suum

1. The close association of muscle and neurons in Ascaris suum makes it difficult to determine whether spikes recorded from nerve cords originate in muscle or neurons. We have developed criteria that distinguish muscle and neuronal activity. There are two categories of extracellular spikes. 2. The first category consists of spikes with a wide range of amplitudes, marked by large spikes. These spikes, which can be recorded over lateral muscle and over the dorsal and ventral nerve cords, are abolished when muscle is disrupted or removed, or when curare is applied. Large spikes are relatively infrequent, are correlated with intracellularly recorded muscle events, and respond to polarizations of motor neurons, implying that they originate in muscle. 3. The second spike category, small amplitude spikes, is exclusive to the ventral nerve cord, occurs more frequently than large spikes and displays patterned firing. Small spikes are not affected by muscle removal or by curare, and are correlated with motor neuronal post-synaptic potentials, but not with intracellularly recorded muscle events. We infer that they originate in neurons. 4. Low level activity recorded extracellularly over nerve cords may represent muscle activity due to tonic motor neuronal synaptic transmission. It responds to motor neuronal polarization and is suppressed by curare or muscle removal.     

Proteinase activity in Ascaris suum eggs, hatching fluid, and excretions-secretions.

Hatching fluid and the excretions and secretions (E.S.) of hatched larvae of Ascaris suum revealed proteinase activity when assayed by 2 different procedures employing collagen or casein as substrates. Both assays apparently detected similar levels of proteinase activity in hatching fluid and E.S. of hatched larvae. The Anson (casein substrate) assay worked best in 0.05 M phosphate buffer, pH 8.0. The Azocoll (collagen substrate) assay worked best in 0.05 M borate buffer at pH 8.8. Azocoll assays done at temperatures ranging from 25 to 65 C revealed maximal proteinase activity at 55 C. Analysis of hatching fluid from 18-, 21-, and 28-day-old embryos and of extracts from sonicated 0- to 28-day-old developmental stages showed that proteinase activity increased markedly 18 days after embryonation had begun. Prior to the 18th day of embryonation proteinase levels were relatively low.     

Genetic and Immunological Comparison of the Cladoceran Parasite Pasteuria ramosa with the Nematode Parasite Pasteuria penetrans

Pasteuria penetrans, an obligate endospore-forming parasite of Meloidogyne spp. (root knot nematodes), has been identified as a promising agent for biocontrol of these destructive agricultural crop pests. Pasteuria ramosa, an obligate parasite of water fleas (Daphnia spp.), has been shown to modulate cladoceran populations in natural ecosystems. Selected sporulation genes and an epitope associated with the spore envelope of these related species were compared. The sigE and spoIIAA/spoIIAB genes differentiate the two species to a greater extent than 16S rRNA and may serve as probes to differentiate the species. Single-nucleotide variations were observed in several conserved genes of five distinct populations of P. ramosa, and while most of these variations are silent single-nucleotide polymorphisms, a few result in conservative amino acid substitutions. A monoclonal antibody directed against an adhesin epitope present on P. penetrans P20 endospores, previously determined to be specific for Pasteuria spp. associated with several phytopathogenic nematodes, also detects an epitope associated with P. ramosa endospores. Immunoblotting provided patterns that differentiate P. ramosa from other Pasteuria spp. This monoclonal antibody thus provides a probe with which to detect and discriminate endospores of different Pasteuria spp. The presence of a shared adhesin epitope in two species with such ecologically distant hosts suggests that there is an ancient and ecologically significant recognition process in these endospore-forming bacilli that contributes to the virulence of both species in their respective hosts.     

Rhabditis pellio Schneider (Nematoda) from the Earthworm,Aporrectodea trapezoides Duges (Annelida)

Studies were conducted on the behak, ior of the nematode, Rhabditis pellio, in the earthworm, Aporrectodea trapezoides, from southern California. Juvenile and adult nematodes were found in the bladders and tubules of the metanephridia of the host. Similar nematodes that entered the coelom were encapsulated and incorporated into multiple capsules ("brown bodies"). It was demonstrated that this host response is an effective defense reaction since dead and dying nematodes, as well as living forms, were found in the capsules.