Reversible inhibition of hatching of infective eggs of Ascaris suum (Nematoda)

lnrernorronol Journolfor Printed in Greur Brimin.

Porosr~ology

Vol. 12, No. 5, PP. 463-465,

0020-7519/82/050463-03$03.00/O Pergomon Press Ltd. Sonety /or Parasrlology

1982

1982 Ausrralion

REVERSIBLE INHIBITION OF HATCHING OF INFECTIVE EGGS OF ASCARIS SUUA4 (NEMATODA) L. Department

C. HURLEY

of Zoology,

and R. I. SOMMERVILLE

University

of Adelaide,

(Received 20 November

Adelaide,

Australia

5000

1981)

Abstract-HuuEu L. C. and SOMMERVILLE R. I. 1982. Reversible inhibition of hatching of infective eggs of Ascuris sum (Nematoda). Infernntionaf Journal for Parasitology 12: 463-465. Dilute solutions of an oxidising agent, iodine, reversibly inhibit hatching of infective eggs of Ascuris suum. The capacity to hatch is restored by exposure to reducing agent, hydrogen sulphide. These observations add to known similarities between hatching of infective eggs and exsheathment of infective larvae. It is proposed that the regulatory mechanisms for both processes are similar. INDEX KEY WORDS: Parasitic nematodes; Ascuris suum; infective iodine; hydrogen sulphide; reversible inhibition.

INTRODUCTION

eggs; hatching;

exsheathment;

advocated to sterilize vegetables contaminated with eggs (Zaman & Visuvalingam, 1967) suggesting that under some circumstances at least, iodine may enter eggs at concentrations great enough to kill the larva. The iodide ion apparently forms lipid-soluble polyiodide complexes in which form it can permeate lipid membranes (Eisenman, Szabo, Cianai, McLaughlin & Krasne, 1973).

appropriate conditions, carbon dioxide induces infective larvae of Haemonchus contortus and Trichostrongylus colubriformis to exsheath. Experimentally this effect can be reversibly inhibited (Rogers, 1966a, b) by treating infective larvae with dilute solutions of iodine, while subsequent exposure of larvae to a reducing agent reverses the inhibition. Thus, larvae exposed to iodine, followed by a reducing agent will exsheath in response to carbon dioxide. The inhibition of exsheathment by iodine and therefore, the stimulation of exsheathment by carbon dioxide, appears to involve central mechanisms which control development generally. For example, iodine-treated sheaths are readily attacked by exsheathing enzymes. Moreover, infective larvae of Nematospiroides dubius, which have been artificially exsheathed, are less infective for mice after exposure to iodine. This reduction in infectivity is restored by a reducing agent (Rogers, 1966b). In addition to inducing exsheathment of infective larvae, carbon dioxide is also a major component of the hatching stimulus of some infective eggs, such as those of Ascaris suum (Rogers, 1958). If carbon dioxide induces hatching of eggs in the same way as it induces exsheathment of infective larvae, the hatching might be expected to be reversibly inhibited by oxidising agents. The range of these agents which might be used with eggs of A. suum is limited because the eggs are permeable only to lipid-soluble and respiratory gases, organic solvents and water (Fairbairn, 1957; Clarke & Perry, 1980). Although 0.04 M iodine seems not to penetrate either decoated or deshelled eggs (Barrett, 1976) the use of iodine has been UNDER

MATERIALS

AND METHODS

Preparation and hatching of eggs. Eggs were isolated and embryonated as described by Hinck and Ivey (1976). They were incubated for 20 days before use and experiments were conducted within 3 weeks of embryonation to avoid the effects of any deterioration with age. Viability was tested by exposing eggs to a hatching stimulus as described by Fairbairn (1961) except that 10% carbon dioxide-nitrogen was used. Between 75 and 95% of embryonated eggs hatched. Routinely however, reducing agent was not included in the medium and in its absence between 30 and 40% of eggs hatched. Exposure to iodine. Eggs were washed three times in distilled water and exposed to a freshly prepared solution of iodine, 0.04 M, for time intervals rang& from 15 min to 24 h. They were then washed three times in distilled water. Some eggs became stained with iodine. To check that the larva and not just the egg membranes were stained, the coverslip was pressed down quickly and firmly, cracking the chitinous layer and forcing the larva from the shell. Exposure to hydrogen sulphide. Hydrogen sulphide was prepared from 50% hydrochloric acid and powdered ferrous sulphide and passed through water before use. Eggs were exposed to a saturated solution of the gas in water for a total of 12 min, then washed six times in distilled water before exposure to the hatching stimulus. Determination of ‘70 hutch. A total of 500 unhatched, embryonated eggs and hatched larvae in a suitably diluted 463

I.J.P. VOL. 12. 1982

L. C. HURLEY and R. 1. SOMMERVILLE

464

drop were counted (Fairbairn, 1961). Two counts were made on each sample. Each experiment was carried out twice.

TABLE ~-REVERSAL

0~ THE INHIBITION OF HATCHING IN

Ascaris mum Treatment Control

RESULTS

Penetration hatching When proportion,

of iodine eggs

were which

into eggs and its effect

exposed to iodine a never exceeded 15%,

on

TABLE I-INFLUENCE OF TIME OF EXPOSURE TO IODINE ON HATCHING 0F E00s 0F Ascaris mum

% hatch, % hatch,

Expt. 1 Expt. 2

to 0.04 M 1, (min) 15 60 120

180

39.7

19.1

27.6

19.4

13.0

32.9

9.1

11.2

11.5

7.9

Hatching stimulus: 10% CO,-N,, 38”C, 3 h. Difference between % hatch of control group and those exposed to iodine is significant (P < 0.05), but there is no significant difference in hatching of those exposed to iodine for varying lengths of time (P > 0.05). Each value represents the mean of two counts. Differences between the two were < 5%.

Reversal of inhibition by hydrogen sulphide Inhibition of hatching by iodine was reversed by subsequent exposure to hydrogen sulphide (Table 2). Indeed hatching was greatly enhanced whether eggs had been exposed to iodine and hydrogen sulphide in succession, or to hydrogen sulphide alone. After in water saturated with hydrogen immersion sulphide, eggs appeared to have a black tinge, suggesting that some part of the egg membrane had reacted with the reducing agent. DISCUSSION

The reaction iodine followed

of infective by reducing

I,-H,S

H,S

% hatch,

Expt.

1

44.3

10.9

87.0

94.1

% hatch,

Expt.

2

36.7

13.3

94.0

95.3

variable

became stained. Sometimes the iodine penetrated slowly, so that at least 180 min was needed before stained eggs appeared, but in other batches of eggs, staining was apparent after only 30 min. Some eggs stained a pale yellow, while others evidently took up much larger amounts of iodine, becoming a dark reddish purple. Examination of larvae from fractured eggs showed that it was not just the membranes but also the enclosed larva which stained. Stained larvae were always dead. Although a majority of eggs showed no signs of staining even after exposure to iodine for 24 h, it was assumed that traces of iodine would be present in many and it might be expected that hatching would be reduced. This assumption was confirmed (Table 1). Although no stained eggs could be detected after exposure to iodine for 15 min, hatching was significantly reduced. Longer exposures, up to 180 min, did not significantly alter the proportion which hatched (P > 0.05).

Time of exposure 0

1,

eggs to iodine and to agent is similar to the

Hatching stimulus: 10% CO,-N,, 38°C. 3 h. Eggs were exposed to I,, 0.04 M, for 15 min, or to I, followed by H,S, 12. min, or fo H,S alone. The inhibition of hatching by iodine is significant (P < 0.05), but there is no significant differences-between hatching of eggs exposed to I, followed by H,S, or to H,S alone (P > 0.05). Each value represents the mean of two counts. Differences between the two were < 5%.

reaction of infective larvae to these reagents. The response of eggs and larvae to the stimulus for hatching or exsheathment involves a step which is reversibly inhibited by an oxidising agent, iodine: reversal is accomplished by a reducing agent, here hydrogen sulphide. These results imply an underlying similarity in the manner in which these infective stages restart development. Other similarities between infective eggs and infective larvae have already been established. For example, the stimulus which leads to the release of hatching fluid in infective eggs and exsheathing fluid in infective larvae is similar (Rogers & Sommerville, 1968), the major components being elevated temperatures and dissolved carbon dioxide. Hatching and exsheathing fluid, released in response to the stimulus, contain similar enzymes which attack each others natural substrates (Rogers & Brooks, 1977). A model describing changes which occur after the stimulus is received by the infective larva has been proposed (Davey, 1976; Rogers, 1978). The similarities between hatching of infective eggs and exsheathment of infective larvae justify the assumption that the model applies with equal validity to infective eggs. The way or ways in which iodine acts to reversibly inhibit hatching and exsheathing are unknown. We suggest that the iodine affects the larva inside the egg, rather than the egg membranes, in keeping with the observation that sheaths isolated from infective larvae which have been treated with iodine, are attacked by exsheathing fluid in the normal way (Rogers, 1966a). Several possible mechanisms for the way in which iodine acts exist. Rogers (1966a, b) has proposed that reactive thiol groups in a hypothetical receptor fol carbon dioxide are oxidised by iodine to block exsheathment. Treatment with reducing agent would restore the reactive groups. A second way in which iodine might act to inhibit hatching or exsheathing could involve the enzyme carbonic anhydrase. The hydration of carbon dioxide, which might be an important step in the stimulus for hatching or exsheathment, is catalysed

I.J.P. VOL. 12. 1982

Reversible inhibil tion of hatching

by carbonic anhydrase. Ethoxyzolamide, which inhibits this enzyme also inhibits exsheathment (Davey, Rogers & Sommerville, unpublished). Iodine inhibits carbonic anhydrase (Riese & Hastings, 1940), and this inhibition is reversed by the presence of a reducing agent. Alternatively, iodine might interfere with hatching and exsheathment by acting on noradrenaline, which is involved in exsheathing (Rogers & Head, 1972) and could be involved in hatching. The evidence linking iodine and noradrenaline comes from a histochemical technique used to distinguish between noradrenaline and adrenaline (Hillarp & Hbkfelt, 1953). Noradrenaline is oxidised by an iodate solution but it is not known whether this reaction is reversible. Iodide ion apparently forms lipid-soluble polyiodide complexes which can permeate lipid membranes (Eisenman et al., 1973). There is no doubt that iodine was able to pass through the membranes of many eggs of A. suum, sometimes in sufficient amounts to stain the enclosed embryo. All these eggs were fully embryonated and at least 20 days old. On the other hand O-day eggs are not penetrated by iodine, even after exposure for 24 h (Barrett, 1976). Barrett used O-day eggs because fully embryonated eggs might produce small amounts of hatching fluid, which would alter their permeability. It seems unlikely that even small amounts of hatching fluid would have been produced in our experiments, but the possibility that this does happen in embryonated eggs, so leading to gradual change in permeability, cannot be dismissed. Not only did exposure of iodine-treated eggs to hydrogen sulphide restore the capacity of these eggs to hatch, but hatching was greatly enhanced compared with the control groups. More than 85% of eggs hatched when exposed to hydrogen sulphide, with or without pre-treatment with iodine. This was comparable to hatching obtained in experiments using reducing agent, together with carbon dioxide and elevated temperatures as part of the stimulus (Fairbairn, 1961; Rogers, 1960). The observation that eggs treated with hydrogen sulphide developed a blackish tinge suggested that the gas combined with some component of the egg membranes. The hydrogen sulphide was not fully removed by repeated washing in distilled water. If washed eggs were exposed subsequently to osmium tetroxide, a black precipitate formed, indicative of the presence of a reducing agent (Porter & Kallman, 1953). If as seems likely, hydrogen sulphide was not fully removed from the eggs by repeated washing, then with carbon dioxide present at the appropriate pH and temperature, hatchings up to 95% might be expected. AcKNowleugrrnents-We arc grateful to Rogers, University of Adelaide and Prof.

Prof. W. p. K. G. Davcy,

465

York University, Toronto, Canada, for many helpful discussions. Support from the Australian University Grants Committee is gratefully acknowledged.

REFERENCES BARRETT J. 1976. Studies on the induction of permeability in Ascaris lumbricoides eggs. Parasitology 73: 109- 12 1. CLARKE A. J. & PERRY R. N. 1980. Egg-shell permeability and hatching of Ascaris suum. Parasitology 80: 447-456. DAVEY K. G. 1976. Hormones in nematodes. In: Organization of Nematodes (Edited by CROLL N. A.), pp. 273-291. Academic Press, New York. EISENMAN G., SZABO G., CIANI S., MCLAUGHLIN S. & KRASNE S. 1973. Ion binding and ion transport produced by neutral lipid-soluble molecules. Progress in Surface and Membrane Science 6: 139-241. FAIRBAIRN D. 1957. The biochemistry of Ascaris. Experimental Parasitology 6: 491-554. FAIRBAIRN D. 1961. The in vitro hatching of Ascaris lumbricoides eggs. Canadian Journal of Zoology 39: 153-162. HILLARP N. A & H~KFELT B. 1953. Evidence of adrenaline and noradrenaline in separate adrenal medullary cells. Acta Physiologica Scandinavica 30: 55-68. HINCK L. W. & IVEY M. H. 1976. Proteinase activity in Ascaris swum eggs, hatching fluid and excretionssecretions. Journal of Parasitology 62: 771-774. PORTER K. R. & KALLMAN F. 1953. The properties and effects of osmium tetroxide as a tissue fixative with special reference to its use for electron microscopy. Experimental Cell Research 4: 127-141. RIESE M. & HASTINGS A. B. 1940. Factors affecting the activity of carbonic anhydrase. Journal of Biological Chemistry 132: 281-292. ROGERS W. P. 1958. Physiology of the hatching of eggs of Ascaris lumbricoides. Nature (Land.) 181: 1410-1411. ROGERS W. P. 1960. The physiology of infective processes of nematode parasites: the stimulus from the animal host. Proceedings of the Royal Society, London 152B: 367-386. ROGERS W. P. 1966a. The reversible inhibition of exsheathment in some parasitic nematodes. Comparative Biochemistry and Physiology 17: 1103-l 110. ROGERS W. P. 1966b. Reversible inhibition of a receptor governing infection with some nematodes. Experimental Parasitology 19: 15-20. ROGERS W. P. 1978. The inhibitory action of insect juvenile hormone on the hatching of nematode eggs. Comparative Biochemistry and Physiology 61A: 187-190. ROGERS W. P. & BROOKS F. 1977. The mechanism of hatching of eggs of Haemonchus contortus. International Journal for Parasitology 7: 61-65. ROGERS W. P. & HEAD R. 1972. The effect of the stimulus for infection on hormones in Haemonchus contortus. Comparative and General Pharmacology 3: 6-10. ROGERS W. P. & SOMMERVILLER. I. 1968. The infectious process and its relation to the development of early stages of nematodes. Advances in Parasitology 6: 327-348. ZAMAN V. & VISUVALINGAMN. 1967. Action of aqueous iodine on ova of Ascaris lumbricoides and Ascaris suum. Transactions of the Royal Society of Tropical Medicine and Hygiene 61: 443-444.