Zinc as a co-factor for an enzyme involved in exsheathment of Haemonchus contortus

The activity of concentrated exsheathing fluid of Haemonchus contortus against isolated sheaths was not inhibited by ethylenediamine tetra-acetic acid (EDTA), 10^?2 M, even when the concentrations of Mg and Mn were < 4 × 10^?4 M and < 0·9 × 10^?6 M respectively. Purified or diluted solutions of exsheathing fluid, even in the presence of Mg²⁺, 10^?3 M, were inhibited. Leucine aminopeptidase (LAP) in exsheathing fluid was active even at concentrations of Mg < 1·3 × 10^?5M. Concentrated solutions were partially inhibited by EDTA, 10^?2 M, at low concentrations of Mg; inhibition was increased in diluted and purified preparations.1,10-phenanthroline (Ophen) strongly inhibited exsheathing activity (Zn < 1 × 10^?6 M). When Zn²⁺, 10^?3 M was added, the inhibition was abolished. The hydrolysis of l-leucinamide was greatly increased in the presence of Ophen, 10^?4 M; this effect was abolished by adding Zn²⁺, 10^?3 M.It is suggested that exsheathing fluid from at least some ‘strains’ of H. contortus contains a Zn metallo-enzyme, probably LAP, which is involved in the process of exsheathment.     

Enzymes in the exsheathing fluid of nematodes and their biological significance

Rogers W. P. 1982. Enzymes in the exsheathing fluid of nematodes and their biological significance. International Journal for Parasitology12: 495–502. The characteristics of an enzyme which hydrolysed denatured collagen and a lipase in exsheathing (ecdysal) fluid are described. A highly purified collagenase from Clostridium histolytica attacked isolated sheaths and reacted to additives in the same way as exsheathing fluid. However, relative to their activities with Azocoll or $\text{p-}\text{phenylazobenzyloxycarbonyl}\text{-}\text{L}\text{-}\text{Pro}\text{-}\text{L}\text{-}\text{Leu}\text{-}\text{Gly}\text{-}\text{L}\text{-}\text{Pro}\text{-}\text{D}\text{-}\text{Arg}$ as substrates, the enzyme of exsheathing fluid was >400 times as active as the bacterial collagenase in its action on isolated sheaths.It is suggested that the lipase in exsheathing and hatching fluids may, in association with the pseudocollagenase (and sometimes with chitinase also) have a function in the hatching of eggs. The pseudocollagenase alone may serve as the exsheathing enzyme. The leucine aminopeptidase in hatching and exsheathing fluids may be concerned in the breakdown of the excretory cell and the release of the fluids.     

Leucine aminopeptidase in exsheathing fluid of North American and Australian Haemonchus contortus

Rogers W. P. and Brooks F. 1978. Leucine aminopeptidase in exsheathing fluid of north American and Australian Haemonchus contortus. International Journal for Parasitology8: 55–58. Juveniles of Haemonchus contortus from north America and Australia produced exsheathing fluid containing leucine aminopeptidase when stimulated in tetraborate-carbon dioxide medium. Exsheathment in this medium was inhibited by 1, 10-phenanthroline, 10^?3M, and this inhibition was largely reversed by Zn²⁺, 10^?3M. This supports the view that the enzyme is produced by the juveniles and that it is concerned in exsheathment.     

The mechanism of hatching of eggs of Haemonchus contortus

Fluid collected from hatching eggs of Haemonchus contortus contained a lipase which hydrolysed 2-naphthyl laurate (about 0·7 μmol naphthol freed /h/10⁶ eggs). The fluid also hydrolysed l-leucinamide (about 2·3 μmol leucine freed/h/10⁶ eggs). The fluid when added to normal or heated eggs caused ‘hatching’. ‘Hatching’ also occurred in exsheathing fluid from infective juveniles and in a preparation of pancreatic lipase containing leucine aminopeptidase. A purified mammalian leucine aminopeptidase in combination with several different lipases did not attack egg shells.The ‘spontaneous’ hatching of eggs of H. contortus was strongly inhibited by 1,10-phenanthroline, 10^?3M, and this inhibition was reversed by Zn²⁺. However, the inhibition of ‘hatching’ of eggs in externally applied hatching fluid, or the hydrolysis of leucinamide in hatching fluid was generally less marked.     

Survival of Nippostrongylus brasiliensis larvae after freezing over liquid nitrogen

Survival of Nippostrongylus brasiliensis larvae after freezing over liquid nitrogen. International Journal for Parasitology4: 173–176. Third stage larvae of N. brasiliensis were frozen over liquid nitrogen and after storage for 7 days were thawed rapidly and inoculated subcutaneously into rats. When ensheathed larvae were used, none survived freezing as judged by motility and infectivity trials. Separate vials of exsheathed larvae survived freezing in proportions ranging from 10 to 64 per cent. Female worms, derived from frozen exsheathed larvae, had a normal complement of eggs in the uterus and both male and female worms had a normal histological appearance. Exsheathed larvae frozen in the presence of 10 per cent dimethylsulphoxide had the same survival rate as those frozen without the addition of cryoprotectant. The addition of 10 per cent glycerol adversely effected the survival of frozen exsheathed larvae.     

Secretion of anti-parasite substances and leukotrienes from ovine gastrointestinal tissues and isolated mucosal mast cells

The presence of larval migration inhibitory (LMI) compounds in the gastrointestinal mucus of nematode resistant sheep has been shown previously to be associated with increased numbers of gastrointestinal mucosal mast cells (MMC) and globule leukocytes (GL). This experiment was designed to determine if LMI compounds were secreted by MMC/GL in response to nematode antigenic challenge and if so, could secretion account for levels observed in mucus. Rommey sheep were immunized by repeated cycles of infection with Trichostrongylus colubriformis or Haemonchus contortus larvae and anthelmintic treatment. After slaughter, gastrointestinal tissue was taken for examination of histology and mucus anti-parasite activity. Segments of small intestine were ligatured to form sacs which were incubated with exsheathed nematode larvae or larval excretory/secretory antigens. Tissue slices from small intestine or abomasum were also incubated with nematode larvae or antigens. After homologous challenge, levels of leukotrienes secreted into small intestinal tissue sacs were significantly higher than levels in heterologously challenged sacs or unimmunized sheep intestinal sacs challenged with larvae of any nematode species (279.4±33.7, 141.0±27.8 and 39.5±15.2 ng h⁻¹ respectively). Tissue slices gave a similar pattern of leukotriene secretion. LMI activity was also significantly elevated in intestinal sacs from immunized sheep challenged homologously with nematode larvae or antigen (64±10 and 68±14% respectively cf. heterologous challenge 32±10% and unimmunized sheep sacs 15±6%). Histological examination of abomasal and small intestinal sections showed that immunized sheep had significantly greater numbers of MMC/GL than unimmunized sheep. MMC/GL isolated and purified from immunized sheep secreted leukotrienes and compounds having LMI activity when cultured with homologous nematode larvae or antigens. Secretion of leukotrienes and molecules having LMI activity from MMC/GL could account for the levels of these substances observed in small intestinal mucus.     

Leucine aminopeptidase and exsheathing activity in preparations from Haemonchus contortus

Rogers W.P. and Brooks F. 1978. Leucine aminopeptidase and exsheathing activity in preparations from Haemonchus contortus. International Journal for Parasitology 8: 449–452. Exsheathing activity relative to leucine aminopeptidase activity (LAP) was greater in exsheathing fluid of infective juveniles of Haemonchus contortus than extracts of homogenates of the same organism. In both preparations the biological and enzyme activities were precipitated with acetone 20 v/v and ammonium sulphate, 40% saturation. Broad peaks of exsheathing and LAP activities obtained by sucrose density-gradient centrifugation and on Sephadex G150 overlapped but the peak of biological activity was always found on the low mol. wt. side of the LAP peak. LAP in exsheathing fluid was separated into two sharp peaks in polyacrylamide gradient-pore electrophoresis. In four experiments the major peak gave a mol. wt. within the limits 345,000–354,500. A minor peak was obtained at 1,800,000. Exsheathing activity remained broadly distributed but fell mostly on the low mol. wt. side of the major LAP peak.It is concluded that LAP cannot be the sole agent involved in exsheathment a lipase may be necessary to expose the substrate attacked by LAP.     

Composition of High Molecular Weight Excretions-Secretions from Infective Larvae of Meloidogyne javanica

Infective larvae (J2) of Meloidogyne javanica were incubated in distilled water for up to 14 days, and their high molecular weight (> 1,000 daltons) excretions-secretions (ES) were isolated and partially characterized. The ES consisted of a mixture of proteins, glycoproteins, and proteoglycans or polysaccharides as revealed by differential staining on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and compositional analysis. Carbohydrate, with approximately equal amounts of neutral monosaccharides and hexosamines, was the major constituent of the ES, with only low levels of protein detected. Acidic sugar residues, including sialic acids, were not detected.     

Gain and loss of fluid in metamorphosing larvae of Manduca sexta

Larvae of Manduca sexta, (Sphingidae) increase their weight approx. 50% just before pupation and then secrete this fluid during the formation of their burrows. Time-lapse cinematography indicates that the fluid is ejected into the walls of the final burrow. It may offer some mechanical support; it is not particularly repellent to ants or mice, and it contains only small amounts of the alkaloids ingested from its preferred food plants. Comparison to other species indicates that the gain and loss of water is associated with burrowing behaviour; the fluid appears to be used in providing hydraulic pressure for burrowing, in forming and cementing the pupal chamber, and in acting as a CO₂ trap underground. The secretion is a hypertonic, highly alkaline solution containing KHCO₃ and small amounts of Na⁺, Ca²⁺, Mg²⁺, PO₄^?3 and some proteins. Haemolymph levels of K⁺ decrease, and those of Ca²⁺ increase, during the secretory phase. When radioactive calcium is injected into mature larvae, it appears promptly in the secretion. If however, the injection is given more than 24 hr before the animal begins secreting, then the calcium is bound to haemolymph protein and does not appear in the secretion.     

Changes in water content and volume accompanying exsheathment of Haemonchus contortus

Davey K. G. and Rogers W. P. 1982. Changes in water content and volume accompanying exsheathment of Haemonchus contortus, International Journal for Parasitology12: 93–96. The exchange water volume was determined for ensheathed larvae, for larvae exsheathed by exposure to CO₂ and for larvae desheathed by exposure to a solution of NaOCl. Larvae exsheathed by exposure to CO₂ lose approx. 66 pl. of water while those desheathed by exposure to NaOCl lose approx. 50 pl. Larvae first desheathed by exposure to NaOCl lose a further 20 pl of water when subsequently exposed to CO₂. Volumes calculated from linear measurements of larvae demonstrate that larvae exsheathed by exposure to CO₂ lose about 69 pl of their total volume, while those desheathed by NaOCl lose about 54 pl. It is concluded that exsheathment may involve at least two parallel processes. Both processes are initiated by CO₂, while one is mimicked by exposure to NaOCl.     

Exsheathment of the infective larva of Labiostrongylus eugenii, a nematode parasite of the Kangaroo Island wallaby Macropus eugenii.

The infective larva of L. eugenii is enveloped in two cuticles which are discarded when the larva exsheaths in the sacculated portion of the wallaby's stomach. In vitro larvae exsheathed in a 0·85% solution of sodium chloride at 37°C, buffered to pH 7 with bicarbonate ion and 40% carbon dioxide. Survival was enhanced if the liquid phase contained medium 199 and serum, and exsheathment was quicker if exposure to carbon dioxide was 1 h rather than 1 day or 7 days. As larvae exsheathed, contractions of the pharynx commenced, and medium was ingested, even when larvae were enveloped in both cuticles. The stimuli for exsheathment and the subsequent pattern of events are like those already recognised in some trichostrongyles.     

Nutrient absorption by the anal vesicle of the braconid wasp,Microplitis croceipes

The function of the anal vesicle of Microplitis croceipes in nutrient absorption was investigated. When larvae were incubated in low-concentrations of several ¹⁴C-labeled nutrient solutions, ¹⁴C-trehalose, -glucose and -amino acids accumulated in the body of control and head-ligated larvae but failed to accumulate in larvae with the anal vesicle ligated. When larvae were incubated in a more concentrated solution of trehalose, ¹⁴C-trehalose also accumulated in the body of control and head-ligated larvae and accumulation of trehalose was reduced in the body of anal-vesicle-ligated larvae. The results indicate that the anal vesicle functions in the absorption of trehalose, glucose and amino acids. Trehalose, when present in high concentrations, was also absorbed cutaneously to some extent. The lipid, triolein, appeared to be absorbed cutaneously and absorption was unaffected by vesicle ligation. The present study also indicates that disaccharides may be absorbed as disaccharides and rapidly converted to insoluble products in the parasitoid larvae.     

Skin penetration by ensheathed third-stage infective larvae of Necator americanus, and the host's immune response to larval antigens.

In vitro experiments were conducted to assess skin penetration by ensheathed third-stage infective larvae (L3) of Necator americanus. The fact that only a small proportion of larval sheaths was recoverable from the outer skin surface suggested that some larvae penetrate mouse skin without undergoing exsheathment. Penetration by ensheathed larvae was confirmed visually using a novel fluorescein isothiocyanate (FITC)-labelling technique in which viable ensheathed larvae were fluoresceinated, applied onto intact mouse skin, and their progress monitored in frozen skin sections. This direct observation that the L2-derived sheath can present antigens to the host's immune system was also monitored by immunoassay to provide confirmatory information regarding skin penetration by ensheathed larvae. Sera from humans infected with Necator americanus were shown to react in ELISA against antigens stripped by detergent (cetyltrimethylammonium bromide) from the sheath surface, and with antigens contained in L3-exsheathing fluid. These data suggest that the host's immune response, as a result of antigenic stimulation by the cast sheath and exsheathing fluid, could in fact be diverted away from the potentially vulnerable L3 stage.     

A novel method for the isolation of nematode larvae using pluronic F-68-treated cellulose strips.

A new method is described for the isolation of cultured nematode larvae. This allows effective separation of larvae from fecal contamination, exsheathed larvae from cast sheaths, and viable larvae from nonviable larvae. The method involves the use of cellulose strips and has been assessed using larvae from 2 hookworm species, Necator americanus and Ancylostoma ceylanicum. Pretreatment of the cellulose strips with 1.0% (w/v) of the nonionic surfactant, Pluronic F-68, significantly increased larval recovery of both species.     

Angiostrongylus cantonensis: in vitro cultivation from the first-stage to infective third-stage larvae

The first-stage larvae of Angiostrongylus cantonensis were cultured in various media at 27 degrees C. The most suitable medium for the development was Chernin's balanced salt solution supplemented with 10% L-15, 10% tryptose phosphate broth, 20% fetal calf serum, and 26 mM sodium bicarbonate. Addition of sodium bicarbonate to the medium facilitated early development of the first-stage larvae. When the first-stage larvae were cultured in the medium under 5% CO2 in air, the worms developed gradually to become quiescent and showed the C shape. Thereafter, the larvae developed to the second stage, retaining their first sheath. About 23 days later, the larvae began to develop to the third stage, being enclosed within the sheaths of the first and second molts. Under these conditions, the larvae developed uniformly and 82% of the larvae reached the third stage 50 days later. About 70% of the third-stage larvae discarded their two sheaths, showing almost the same size as those obtained in vivo. When these exsheathed larvae were inoculated into rats, they developed into adult worms and deposited numerous first-stage larvae.     

Purification and Some Biological Properties of Asparaginase from Azotobacter vinelandii

Asparaginase was found in the soluble fraction of cells of Azotobacter vinelandii, and its activity remained the same during growth of the organism in a nitrogen-free medium. The specific activity and the yield of A. vinelandii increased twofold in the presence of ammonium sulfate. Within limits, the temperature (30 to 37°C) and pH (6.5 to 8.0) of the medium showed little effect on the levels of enzyme activity. The enzyme was purified to near homogeneity by standard methods of enzyme purification, including affinity chromatography, and had optimum activity at pH 8.6 and 48°C. The approximate molecular weight was 84,000. The apparent K_m value for the substrate was 1.1 × 10^-4 M. Metal ions or sulfhydryl reagents were not required for enzyme activity. Cu²⁺, Zn²⁺, and Hg²⁺ showed concentration-dependent inhibition, whereas amino and keto acids had no effect on the enzyme activity. Asparaginase was stable when incubated with rat serum and ascites fluid. The enzyme had no effect on the membrane of sheep erythrocytes and did not inhibit the incorporation of radioactive precursors into deoxyribonucleic acid, ribonucleic acid, and protein in Yoshida ascites sarcoma cells. Asparaginase activity was not detected in the tumor cells.     

Observations on the coiled posture of trichostrongyle infective larvae using a freeze-substitution method and scanning electron microscopy

Wharton D. A. 1982. Observations on the coiled posture of trichostrongyle infective larvae using a freeze-substitution method and scanning electron microscopy. International Journal for Parasitology12: 335–343. A method for the preparation of the infective larvae of Trichostrongylus colubriformis for scanning electron microscopy is described. This involves freeze-substitution with methanol followed by critical-point drying. The method resulted in good preservation and the retention of the coiled posture. The morphology of ensheathed and exsheathed larvae is described and the mechanisms of coil formation discussed. Coiling was also observed in the infective larvae of nine other trichostrongyle species.     

Defense reaction of mosquitoes to filarial worms: role of the microfilarial sheath in the response of mosquitoes to inoculated Brugia pahangi microfilariae

The melanization response of Aedes trivittatus and A. aegypti (black-eyed Liverpool strain) against intrathoracically inoculated sheathed and chemically exsheathed Brugia pahangi microfilariae (mff) was assessed daily through 5 days postinoculation (PI). Response of A. aegypti against exsheathed mff was significantly reduced on all days compared with the response against sheathed mff, and a significantly greater percentage of exsheathed mff were alive through 4 days PI than were sheathed mff. The melanization response of A. trivittatus was nearly 100% effective against either sheathed or exsheathed mff by Day 2 PI. When mff were allowed to migrate through A. aegypti midguts in vitro before inoculation into intact A. aegypti, nearly 94% ($\text{120}\text{128}$) of the parasites recovered had avoided the response and were developing. Penetration of A. trivittatus midguts in vitro by mff before inoculation into intact A. trivittatus did not prevent a melanization response. Inoculation of mff into A. trivittatus following A. aegypti midgut penetration, however, resulted in almost 60% ($\text{98}\text{171}$) of the mff avoiding the response and developing as normal L₁ larvae after 5 days PI. The possibility of mff acquiring host antigens during midgut penetration and therefore avoiding recognition as nonself by mosquitoes, and (or) the possibility of the midgut environment modifying or stimulating mff to inhibit the response of mosquitoes are discussed.     

Phosphatidyl inositol-specific phospholipase C from Clostridium novyi type A

From the culture broth of Clostridium novyi type A, phosphatidyl inositol-specific phospholipase C was separated from the major part of phospholipase C (γ-toxin) which hydrolyzes phosphatidyl choline, phosphatidyl ethanolamine, and sphingomyelin. Sodium deoxycholate stimulated the activity of phosphatidyl inositol phospholipase C. The concentration of sodium deoxycholate for maximal stimulation was 0.2% with 2 mm phosphatidyl inositol. Divalent cations (Mg²⁺, Ca²⁺, and Zn²⁺) were rather inhibitory above 10^?3m. Phosphatidyl inositol phospholipase C was not inhibited by EDTA or o-phenanthroline. When phosphatidyl inositol phospholipase C was incubated with rat liver slices, not only alkaline phosphatase but also 5′-nucleotidase was liberated into the soluble fraction.     

Analysis of malpighian tubule membrane proteins from Drosophila melanogaster

A method had been developed for radioactively labelling and analyzing the membrane proteins of Malpighian tubules and other tissues obtained by dissections from Drosophila melanogaster larvae. A fraction was identified on sucrose gradients which binds concanavalin A, and is labelled by galactose oxidase reduction. This fraction was examined in the electron microscope and found to contain membraneous structures.The membrane proteins were analyzed following fractionation of dissected tissues using two dimensional gel electrophoresis and fluorography. Animals were made radioactive by feeding larvae on yeast which was grown in a medium containing [³²S]sulphate. The membrane fraction of Malpighian tubules contains approx. 125 major spots. Of these, about 50% seem to be common to the membranes of several cell types. The remainder of the membrane proteins appear to be tissue type specific.