The Second Ecdysis of Infective Nematode Larvae

Abstract

1. The second ecdyses experimentally produced in 1 in 20 and other watery dilutions of Milton hypochlorite are described. These occur without the detachment of a typical cap. The larva is passively propelled part of the way out through a hole at the anterior tip of the sheath, and completes its emergence by its own movements.The explanation of this is that the chlorine in the Milton solution alters the chemical composition of the sheath with two results: (a) the permeability of the sheath is altered so that increased internal pressure results, by which the larva is propelled out of the hole at the anterior tip, and (b) the sheath is rendered soluble in the NaOH in the Milton solution so that it is subsequently dissolved. The larva is expelled through a hole at the anterior tip before the whole sheath is dissolved because the sheath gives way here first to the combined effects of the increased internal pressure and the solvent action of the NaOH, this being its weakest point, where the remains of the mouth of the second larva are.The larva is only rarely propelled completely out by the raised internal pressure, namely, when the hole at the anterior tip is big enough to allow its widest part, at the base of its oesophageal region, to pass; or, alternatively, when the internal pressure is high enough to force this part of it through the hole. Usually it requires the help of its own movements to force this widest part of it through the hole.2. Neither chlorine, oxygen, nor caustic soda alone can cause ecdyses, although the latter may dissolve the sheaths of larvae kept long in cultures or exposed long upon pastures, so that a chemical change in their sheaths has occurred similar to that just described. Caustic soda may then also so weaken and thin the sheaths that larvae, if they are active enough, may break their way out.Neither carbonic nor hydrochloric acids, nor solutions of sodium chloride, could alone cause ecdyses; nor could solutions of sodium carbonate, although these acted upon “older” larvae in much the same way as solutions of caustic soda, which they must contain.3. Larvae, however, whether they were recently mature or had been long kept in cultures, showed, when they were treated with chlorine water or with hypochlorite solutions acidified with excess of either HCl or acetic acids, effects which indicated that the conclusion described in (a) above was correct. They never exsheathed, but their sheaths were blistered and thinned and frequently distended also, with the exception of the cap which is detached at the ecdysis; this was usually neither blistered, thinned nor distended. This distension was especially marked when these larvae were transferred, after this treatment with chlorine, to solutions hypotonic to them. In hypertonic solutions, on the other hand, the larvae were shrunken. This perhaps explains why larvae are often shrunken away from their sheaths in old cultures from which much evaporation has occurred or when they have been exposed upon pastures.Their sheaths were, moreover, rendered soluble in 0·4 or 0·8 per cent. NaOH by this treatment with chlorine, so that treatment with chlorine, followed by immersion in weak NaOH, will exsheath the larvae, whether they are active or not, by dissolving off their sheaths. This probably explains why larvae exposed on pastures or kept long in cultures, exsheath sometimes in alkaline media such as artificial pancreatic juice, or in the saliva of sheep.4. Observation of ecdyses that occurred at 38° C. in about 8 days in mixtures of 0·5 per cent. solutions of HCl and 0·5 per cent. NaOH or in 0·5 per cent. HCl followed by 0·5 or 0·8 per cent. NaOH, showed that these always occurred by the detachment of a typical cap, and that the process was essentially the same as that described above. An alteration of the permeability of the sheath occurred by which the pressure inside it was increased, so that the sheath was often distended. The sheath was at the same time rendered soluble in NaOH.The cap which was detached at the ecdysis resisted, however, this distension, perhaps because it had a slightly different chemical composition, so that tension existed at the base of this more rigid cap, between it and the more flexible and elastic part of the sheath behind it. The tension at this point was further observed to be increased by specific rotatory movements of the oral end of the larva, which incidentally also determined the length of the cap, and by movements of the more flexible and elastic part of the sheath behind the cap, by which it could be repeatedly rolled up on to the cap and back again, as rubber tubing may be rolled up on to a tap. All these movements, especially when they acted upon a distended sheath, ruptured first the inner layer of the sheath at the base of the cap, and then the much stronger outer layer.The larva then emerged by its own movements; or sometimes it was propelled out passively part of the way by the release of the high internal pressure.Both the rotatory movements of the oral end of the larva before the detachment of the cap, and the bending and swimming movements of the whole larva by which it extricates itself from the sheath after the cap has been detached, are essential to successful and normal ecdyses. The rotatory movements of the oral end are probably most effective when the oral tip of the sheath is fixed against some resistant object, as skin-penetrating larvae may, for example, fix it against the skin. This fixation of the oral end is not, however, necessary, because most of the ecdyses here recorded occurred in this way in drops quite free of any solid or semi-solid matter.5. Some experiments are briefly described which indicate that mechanical damage to the sheaths, or friction applied to them, cannot be more than secondary factors which influence only the ecdyses of so-called “older” larvae, whose sheaths are already chemically altered by climatic factors or long life in cultures, with results similar to those which follow treatment with chlorine.6. It is suggested that factors similar to those indicated above may be present in the stomach and duodenum of the host and may govern any second ecdyses that occur in these situations; and that changes in the sheaths, similar to those produced by chlorine in the experiments here described, may be produced outside the body of the host, by climatic factors, so that the sheaths become soluble in the saliva or other alkaline digestive juices.7. All attempts failed to induce the third ecdysis of larvae exsheathed and sterilised by 1 in 20 dilutions of Milton with water, and kept in sterile media until they were ready for this ecdysis. These larvae also, like the infective larvae, were susceptible to changes in the tonicity of the media in which they were kept, so that their sheaths could be alternately distended or collapsed upon them, and the larvae themselves could be shrunken or restored to normal by alterations in the concentration of the media. These third (or first parasitic) larvae showed a remarkable power of resistance to such treatment and seemed to be more resistant to it than infective larvae are.8. Further investigations of the second ecdyses by biophysical methods may bring all the ecdyses of parasitic nematodes under experimental control and so make possible the artificial cultivation of the adult parasites. They may also suggest methods of altering the permeability of the cuticle of adult nematodes, so that anthelmintics may be found which will penetrate this more readily and at the same time will not be toxic to the host.

Cloning of a Cysteine Protease Required for the Molting of Onchocerca volvulus Third Stage Larvae

“…A cathepsin L-like cysteine protease was recently shown to be also important for the molting of B. pahangi L3. 3 An endogenous cysteine protease that has a probable function in the excystment of Paragonimus westermani metacercariae was recently described by Chung et al (38). In this parasite, the authors have suggested that the dormant metacercariae begins to secrete the cysteine proteases on its arrival in duodenum, probably in response to the host signal of pH change.…”

The In Vitro Action of Immune Serum on the Juveniles of the Nematode, Oesophagostomum columbianum (Curtice, 1890) Stossich, 1899

“…The free-living, third stage juveniles were obtained from the cultures and washed repeatedly with distilled water to remove any faecal material adhering to them. They were exsheathed by treating them with 5% sodium hypochlorite solution (Lapage, 1935). The exsheathed juveniles were rinsed repeatedly in sterile saline to get rid of bacteria.…”

The protozoa occurring in the hind-gut of cockroaches

“…(a) Introduction The name Nydotherus velox was given by Leidy (1849) to a ciliate which he found in the hind-gut of the millipede Julus marginatus. In the following year, he added a second species to the new genus: N. ovalis, which he found in a similar site in the cockroach Blatta orientalis.…”

“…Neither Lucas nor Pai & Wang described the chemical constitution of the cyst wall. Lapage (1935) used 'Milton',* chlorine and NaOH to study the mechanism of the second ecdysis in Haemonchus contortus, Ostertagia circumcincta and Trichostrongylus spp. He found that 'Milton' caused ecdysis and subsequent dissolution of the sheath, but neither chlorine nor NaOH alone was effective.…”