The Physiology of Infective Processes of Nematode Parasite; the Stimulus from the Animal Host Author(s): W. P.

Rogers Source: Proceedings of the Royal Society of London. Series B, Biological Sciences, Vol. 152, No. 948 (Jun. 14, 1960), pp. 367-386 Published by: The Royal Society Stable URL: http://www.jstor.org/stable/75342 . Accessed: 20/05/2011 09:10
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The physiology of infective processes of nematode parasites; the stimulus from the animal host
BY W. P. ROGERS The Department of Zoology, University of Adelaide, South Australia (Communicated by D. Keilin, F.R.S.-Received Revised 8 December 1959) 13 August 1959-

The processes by which larvae of Trichostrongylus axei, T. colubriformis and Haemonchus contortus, and eggs of Ascaris lumbricoides (pig strain), Ascaridia galli, and Toxocara mystax infect the host have been studied. A stimulus from the host, depending for its activity on the concentration of undissociated carbonic acid, and on the Eh and pH, was required to start development of the parasite. The stimulus caused larvae to produce 'exsheathing fluid' which completed the second moult, or it caused the production of a 'hatching fluid' so that the eggs hatched. In bicarbonate-carbon dioxide buffers containing reducing agents under mixtures of nitrogen and carbon dioxide the exsheathment of larvae of Trichostrongylus axei and Haemonchus contortus increased as the concentration of the undissociated carbonic acid was increased. A concentration of 05 x 10-3M caused 70 % of the larvae of Trichostrongylus axei to exsheath at pH 7-3 in 0-02M-sodium dithionite in 3 h at 37 ?C. More than 1.5 x 10-3M was necessary to get similar results with Haemonchus contortus. These species differed from all the others because their exsheathment was not inhibited by high concentrations of undissociated carbonic acid. At pH 6-0 the relative activity of reducing agents was sodium dithionite > cysteine > ascorbic acid. At pH 7-3 the activity of cysteine and ascorbic acid relative to sodium dithionite was increased. Larvae of Trichostrongylus colubriformis exsheathed readily in hydrochloric acid between pH 1-5 and 2.5 when the concentration of undissociated carbonic acid was 5 x 10-3M. Cysteine did not increase the exsheathment. The eggs of Ascaris lumbricoides, Ascaridia galli and Toxocara mystax hatched in bicarbonate-carbon dioxide buffers containing reducing agents under nitrogen-carbon dioxide if the concentration of undissociated carbonic acid was about 0.25 x 10-3M at pH 7*3. For the hatching of eggs of Ascaris lumbricoides at pH 7*3 the activity of the reducing agents was sodium dithionite > cysteine > ascorbic acid. In all the species which were examined except Trichostrongylus colubriformis the undissociated carbonic acid was more effective at a higher pH. And, as a rule, reducing agents were relatively less effective at higher concentrations of undissociated carbonic acid. The addition of sodium chloride up to 01 M and sodium taurocholate up to 0-05 usually increased activity. The stimulus for exsheathment was effective within 15 min for larvae of T. axei and 30 min for Haemonchus contortus. Though at these times only a few larvae had exsheathed, during subsequent incubation in water exsheathment often rose to 80 % in 3 h. Longer periods were necessary for the stimulus to act on eggs of Ascaris lumbricoides and hatching seldom rose by more than 30 % after the stimulus was removed. The process whereby the host provides a stimulus for the resumption of development of the parasite is related to specificity and the site of infection. It is suggested that suspension of development in the infective stage and the dependence upon the host for restarting development may be an adaptation to parasitism. INTRODUCTION

Processes in the life cycle of free-living nematodes such as the hatching of eggs and the moulting of larvae must be controlled and co-ordinated largely by some sort of mechanism within the nematodes. This might be achieved by a system of 'internal secretions' which regulates the time of production of substances which cause hatching or moulting. In parasitic species part of this mechanism might be
[ 367 ]

368

W. P. Rogers

lost so that the parasite would be dependent on the host to replace it (figure 1). For instance, the infective stage, lacking part of this system for restarting development, would remain in a 'resting' condition until, in the right host, the missing components were provided. The host might function by providing: (I) a stimulus which causes the infective stage to produce the internal secretions; or (II) by providing the substances which replace the missing internal secretions; or (III) by producing substances which cause exsheathment or hatching by their direct action on the sheath or egg shell. The strongest argument against (III) stems from the fact that the hosts do not normally produce the chitinases or the particular proteases needed to break down
infective eggorlarva

(I hlost .
.-

>

internal I secretion

(II)
(III)

hatching orexsheathing fluid (chitinase,esterase,protease) FIGURE 1. Host-parasite relationships in the process of infection. The host may start infection (I) which stimulates the infective agent to produce, by providing an environment directly or indirectly, hatching or exsheathing fluids which contain certain enzymes. There is no evidence, as yet, to show that the host may function as in (II) or (III).

the outer coverings of most eggs or infective larvae. It is possible that a host could produce substances which play a highly specific role in the physiology of a parasite as suggested in (II). Indeed the unsaturated lactone which serves as the 'hatching factor' for Heterodera rostochiensis (Calam, Todd & Waring I949; Ellenby & Gilbert I957) might act in this way. There is, however, no evidence to support this view. The suggestion (I) that the host stimulates the production of a fluid which completes the process of moulting (exsheathment) in infective larvae of trichostrongyle parasites of sheep was made by Rogers & Somerville (I957) who used the rumen fluid of the host to stimulate the larvae to produce exsheathing fluid. Rogers (I958) gave more details of the chemistry of the stimulus which induced infective eggs of Ascaris lumbricoides to produce a 'hatching fluid' which contained the enzymes which attacked the egg shell. In this paper the components of the stimulus which cause the exsheathment of infective larvae of Trichostrongylus axei, T. colubriformis, Haemonchus contortus, and the hatching of infective eggs of Ascaris lumbricoides (pig strain), Ascaridia galli and Toxocara mystax are given. Trichostrongylus axei and Haemonchus contortus commonly exsheath in the rumen of the host though the adults live in the abomasum. With Trichostrongylus colubriformis exsheathment takes place in the abomasum, and the adults live in the small intestine. The eggs of Ascaris lumbricoides, Ascaridia galli and Toxocara mystax hatch in the small intestine of the pig, fowl and cat, respectively, where the adults also live.

Infective processes of nematode parasites

369

Most of the experiments which are described here were carried out to examine the three chief components of the stimulus. To make the interpretation of results easier the less important factors which enhanced activity were seldom added. Thus the activity reported for many experiments was less than that which could have been obtained under ideal conditions in vitro. When infection occurs, the processes of stimulating the infective stages to produce exsheathing and hatching fluids, and the enzymic breakdown of the sheath or egg shell, take place in the one medium. Nevertheless, it is necessary that these two processes should be studied separately, otherwise it would not be known if a given condition was affecting the stimulus or the enzymes. In this work, it was convenient to carry out most of the experiments so that both processes took place in the one medium. From the results so obtained more critical experiments were carried out in which the second process, during which the enzymes in the exsheathing and hatching fluids attacked the sheaths and egg shells, took place in buffer or water. In this way it was found that the major components of the stimulus normally had little effect on the second process.
MATERIALS AND METHODS

Larvae were obtained from cultures of faeces from sheep infected with Trichostrongylus axei, T. colubriformis or Haemonchus contortus. Eggs were dissected from the posterior 2 cm of the uteri of Ascaris lumbricoides. Ascaridia galli and Toxocara mystax were incubated for 24 h in saline and the eggs laid during this period were collected. The eggs of Ascaris lumbricoides were washed for 2 h in 0-5 N-sodium hydroxide at room temperature (Fairbairn 1955); the eggs of the other species were washed for 10 min. Thereafter they were incubated at 28 ?C either in 01 N-sulphuric acid or in 1 % formalin. The eggs were either shaken continuously throughout the incubation period or else they were kept in layers less than 2 mm deep. Larvae were stored in water at 5 ?C. They were about 2 to 3 weeks old when used. To estimate the degree of exsheathment larvae were mixed with Lugol's iodine and examined with a microscope. A larva which had emerged even partly from the sheath was regarded as exsheathed. At least 100 larvae were counted for each experiment; those which obviously had been dead for some time were not counted. After incubation for 20 days at 28 ?C eggs were stored in 0-lN-sulphuric acid or in water at 5 ?Cuntil they were needed. It was best to use eggs of A. lumbricoides within 10 days otherwise the results were sometimes variable. The eggs of the other species could be stored for 30 days before use. The proportion of eggs which had hatched in an experiment was measured by counting at least 100 of the eggs or larvae with a microscope. If the larva was free from the egg, or even if the embryo had emerged sufficiently to break the inner membrane of the egg, it was considered to be hatched. Unembryonated eggs were not counted. It was necessary to mix the eggs and larvae to be counted with a thick solution of methyl cellulose before placing the coverslip in position. Otherwise they were distributed unevenly.

370

W. P. Rogers

Bicarbonate-carbon dioxide buffers (Umbreit, Burris & Stauffer 1957) were checked with a glass electrode. In early experiments gas mixtures were passed over copper turnings at 400 ?C before use. The removal of traces of oxygen in this way did not affect the results and in later experiments gas was used directly as it came from cylinders. Solutions of cysteine and ascorbic acid were brought to the appropriate pH with sodium hydroxide. Sodium dithionite (British Drug Houses) was dissolved in oxygen-free water under mixtures of nitrogen and carbon dioxide immediately before use. The solutions of sodium dithionite were added, under nitrogen or nitrogen and carbon dioxide, to the rest of the system after it had been gassed to remove oxygen. In this way the formation of acid in the solutions was kept low and the buffers were not disturbed appreciably. The sum of the concentrations of undissociated carbonic acid and dissolved gaseous carbon dioxide in the bicarbonate-carbon dioxide buffers was calculated with the formula

1 [H2C03][ +
where

1 ( + [1

)1

Pco255 5

[12CO31 = the molar concentration of undissociated carbonic acid plus

gaseous dissolved carbon dioxide; K- = Henry's law constant; Pco, = the partial pressure of carbon dioxide; in the gas phase in mm of mercury; and K, and K2 = the first and second dissociation constants, respectively. The effect of ionic strength on the dissociation constants was neglected. The value of pK, at 37 ?C was taken as 6-317 (Shedlovsky & Maclnnes I935) and the correction at maximum ionic strength (pH 7.3, Pco, 285 mm of mercury, 0-05 Msodium chloride, 004 M-sodium dithionite) would have been less than 0-03 (Umbreit et al. I957). The consequent change in K, would have corrected an error of less than 4%. The solubility of carbon dioxide in dilute hydrochloric acid was taken from graphs of Van Slyke, Sendroy, Hastings & Neill (1928). For calculating the total carbon dioxide in solution it was assumed that the carbonic acid was undissociated when the pH was below 3. The effects of varying the period the larvae were in the stimulating medium were examined. After exposure to the medium at 37? C, chilled water or 1 x 10-3irmagnesium chloride in 001 m-phosphate buffer at pH 7 2 was added and the larvae were concentrated by centrifuging. This process was repeated three times after which larvae were incubated for a further period in water or buffer. Exsheathment was measured after both periods of incubation. Similar experiments were carried out with eggs. After exposure to the stimulus, however, the eggs were washed in warm saline and then incubated. Unless otherwise stated experiments were carried out at 37 ?C and the period of incubation was 3 h.

Infective processes of nematode parasites

RESULTS

371

Some results from preliminary experiments are shown in table 1. From the results it appeared that carbon dioxide was a necessary part of the stimulus. Sodium bicarbonate alone was not sufficient but some activity could be obtained without sodium bicarbonate if carbon dioxide were present. It seems then, that the undissociated carbonic acid or the dissolved gaseous carbon dioxide rather than carbonate or bicarbonate ions was a component of the stimulus. It has not been possible to separate the actions of these un-ionized components nor have their separate concentrations been calculated. For convenience, therefore, the undissociated carbonic acid referred to in the rest of this paper will be regarded as including the dissolved gaseous carbon dioxide.
TABLE 1. THE GENERAL EFFECT OF GASES, PH, AND
REDUCING AGENTS ON LARVAE AND EGGS

exsheathment of larvae

(%)
composition of medium 'buffer' phosphate phosphate phosphate phosphate bicarbonate bicarbonate bicarbonate bicarbonate bicarbonate bicarbonate was used gas phase air N2 5% C2-N2 5% C02-N2 5% CO2--N2
5 % C02-N2 5% C02-N2

hatching of eggs (%) Toxocara mystax 0 0

1

pH 6-0 6-0 c. 60 c. 6-0 6-0

6-0 80

5% C02-N2 N2 N2

8-0 c. 8-3 c. 8-3

Trichoreducing Haemonchus strongylus contortus axei agent 0 0 0 0 + 4 1 6 0 + 3 5 54 14 + 3 2 21 5 + 0 0 3 0 +

Ascaris lumbricoides 0 0
1

3 5 16 40 62 0 0

1 6 38 7 64 0 0

The reducing agent was 0O02M-sodium dithionite

except for Toxocara mystax for which 0O02M-cysteine

Activity was low when the nitrogen in the gas phase was replaced by air or oxygen. Reducing agents under nitrogen without carbon dioxide had little activity. Their main action seemed to be the enhancement of activity due to the undissociated carbonic acid. The pH was important because it affected the concentration of undissociated carbonic acid; but it seemed to have other actions also. Sodium chloride, wetting agents and blood serum, which are not shown in table 1, affected activity slightly. (1) The action of undissociated carbonic acid (a) Experiments with larvae The exsheathment of larvae of Haemonchus contortus, Trichostrongylus axei and T. colubriformis was examined in sodium bicarbonate-carbon dioxide buffers with and without 0-02M-sodium dithionite. The gas mixtures consisted of nitrogen

372

W. P. Rogers

containing 1, 2-5, 5, 10, 20 and 40 % carbon dioxide (vol./vol.) and the concentrations of sodium bicarbonate were chosen to give pH 6*0, 6.9, 7-3, and 8*0 for all concentrations of carbon dioxide except 40 % for which pH 7 3 was the highest used.
80o? -

c 60~ /

40'6, 40

)20

0
FIGURE

rS

46

[H2CO3]x 10M 2. The effect of different concentrations of undissociated carbonic acid on the exsheathment of larvae of Trichostrongylus axei. Bicarbonate-carbon dioxide buffers under nitrogen containing carbon dioxide at different partial pressures were used. Sodium dithionite, 0.02M, was present in experiments which gave the results for the upper curves; 0.05M-sodium chloride was present in all experiments. El, I, pH 7.3; 0, 0, pH 6-0.
o

60-

1 40-

20-

0
FIGURE

l{

I ..... ,

[H2CO3]x 103M 3. The effect of different concentrations of undissociated carbonic acid on the exsheathment of larvae of Haemonchus contortus. Bicarbonate-carbon dioxide buffers under nitrogen containing carbon dioxide at different partial pressures were used. Sodium dithionite, 0.02M, was present in experiments which gave the results for the upper curves; 0-05N-sodium chloride was present in all experiments. l, S, pH 7.3; 0, e, pH 6.0.

Infective processes of nematode parasites

373

The results from one set of experiments with T. axei and Haemonchus contortus at pH 6*0 and 7-3 are shown in figures 2 and 3. Some variation was obtained with different lots of larvae; in particular, older larvae were more sensitive to undissociated carbonic acid. However, the general relationships shown in figures 2 and 3 were consistent. Never more than 18 % of Trichostrongylus colubriformis exsheathed under these conditions (table 2). Activity was greatest at the lowest pH. For this reason, and because T. colubriformis normally exsheaths in the abomasum, experiments were
TABLE 2. THE EXSHEATHMENT OF LARVAE OF TRICHOSTRONGYLU SCOLUBRIFORMIS

composition of the medium sodium chloride,

pH 1.6 gas phase air air N2 0-1M + +

cysteine,
0-02M -

exsheathed
(%) 0 4 1

(HC1)
1.6

(HC1)
1'6

(HC1)
1-6 N2
20%C02-N2 20%C02-N2

+
+ -

+
-

7
71 21

(HC1)
1-6

(HCI)
1*6

(HC1)
1.6 20%CO2-N2
20 % CO2-N

+
+

+
-

62
12

(HC1)
6'0

(NaHCO3) 6.0 (NaHCO,)

20%CO2-N2

18

conducted in 0-025N-hydrochloric as shown in table 2. Exsheathment was enhanced by carbon dioxide in the gas phase so the effect of varying the concentration of undissociated carbonic acid was examined by using mixtures of nitrogen and carbon dioxide (figure 4). This experiment was only repeated once; greatest activity, 91 %, was again obtained at 4-8 x 10-3vr. (b) Experiments with infective eggs The hatching of eggs was examined in sodium bicarbonate under nitrogen or mixtures of nitrogen and carbon dioxide within the range pH 6-0 to 8.3 with and without reducing agents. Some of the results are shown in figure 5 and tables 3 and 4. With Ascaris lumbricoides the optimum concentration of undissociated carbonic acid was about 0-25 to 0-5 x 10-3 at pH 7-3 and about 1 to 2 x 10-3M at pH 6-0. At high concentrations no eggs hatched. At a given concentration of undissociated carbonic acid and pH the addition of reducing agents always increased activity. The eggs of the other two species, which were examined in two

374

W. P. Rogers

experiments only, needed somewhat lower concentrations of undissociated carbonic acid. The results of these and other experiments with the eggs and larvae showed certain features about the action of the undissociated carbonic acid.

40-

? 20
I

I 1 0

I 2

l 4

l 6

l 8

10

[H2CO3] x 103M FIGURE 4. The effect of different concentrations of undissociated carbonic acid on exsheathment of larvae of Trichostrongylus colubriformis. The medium consisted of 0-025N hydrochloric acid, 0 05M-sodium chloride under mixtures of nitrogen and carbon dioxide.

I t i
40 -I

Xe IA
g
o I

I
/

\
\

, 20+-

I\

I.1

0 0

2
2 [Hl2CO3] x 103M 4 6

FIGURE 5. The hatching of eggs of Ascaris lumbricoides at different concentrations of undisdioxide sociated carbonic acid and hydrogen ions. The medium was bicarbonate-carbon buffer at pH 7-3 (broken line) and pH 6.0 (continuous line) containing 0.02M-sodium dithionite under different mixtures of nitrogen and carbon dioxide.

(i) For larvae of Haemonchus contortus and Trichostrongylus axei activity of the stimulus increased with increasing concentration of the undissociated carbonic acid at least up to 6-2 x 10-3M. And inhibition did not occur at high concentrations; high activity was even obtained under 100 % carbon dioxide at pH 6-0.

Infective processes of nematode parasites

375

(ii) With the other species high concentrations of undissociated carbonic acid decreased the activity of the stimulus so it would appear that only a small proportion of the larvae or eggs would infect a host if the concentration was above about 4 x 10-3M in solutions below pH 7-3. (iii) Under conditions that were otherwise similar, the exsheathment of Haemonchus contortus required much higher concentrations of undissociated carbonic acid than was necessary for the infective agents of the other species.
TABLE 3. THE EFFECT OF 0-02M-CYSTEINE MYSTAX AT DIFFERENT ON THE HATCHING OF EGGS OF TOXOCARA CARBONIC ACID

CONCENTRATIONS OF UNDISSOCIATED

carbonic acid
pH x 104M

--without
cysteine

% hatched in 3 h

with
cysteine

6.0
6.3 6.9 8.0 TABLE

1.6
1.1 0.5 0.05

5
8 8 40

16
19 28 62 ON THE HATCHING OF EGGS OF

4. THE EFFECT OF SODIUM DITHIONITE

ASCARIS LUMBRICOIDES AT DIFFERENT CARBONIC ACID

CONCENTRATIONS OF UNDISSOCIATED

% of eggs hatched concentration of undissociated carbonic acid, x 104M at pH 8-0 at pH 6-0

-7-9 2 15 38 47 48 159 16 31 45 58 45 159 < 2 < 2 024 5 24 43 54 54 0-47 24 37 40 50 53

sodium
dithionite x 102 M 0 1 2 4 8

(2) The action of reducing agents (a) Experiments with larvae When undissociated carbonic acid was present, reducing agents generally increased the proportion of larvae of Trichostrongylus axei and Haemonchus contortus

which exsheathed (figures 2, 3). The optimum concentration of sodium dithionite for exsheathment of larvae of Trichostrongylus axei in a medium containing 0 16 x 10-3 or 1-6 x 10-3M-undissociated carbonic acid and 0-05M-sodium chloride at pH 6-0 was 0-04M. Only one experiment was carried out with each of ascorbic acid and cysteine; the optimum concentrations were 0.02 and 0-04M, respectively. With Haemonchus contortus the optimum concentrations of the three reducing agents always fell between 0-02 and 0-06M. The relative increase in the activity of the stimulus due to sodium dithionite was always greater at the lower concentrations of undissociated carbonic acid (table 5, figures 2, 3). This was generally true of ascorbic acid and cysteine also (table 5).

376

W. P. Rogers

The activity of the reducing agents at pH 6-0 for stimulating exsheathment of larvae of Trichostrongylus axei was sodium dithionite > cysteine > ascorbic acid; i.e. activity increased as the oxidation-reduction potential fell. Similar results were obtained with larvae of Haemonchus contortus though sometimes the activity of cysteine and sodium dithionite was about the same. The change in the relative activity of the reducing agents when the pH was raised from 6-0 to 7-3 was in accordance with the fall in the oxidation-reduction potential which would occur under such circumstances (table 5). As exsheathment of Trichostrongylus colubriformis could not be obtained except at relatively high hydrogen ion concentrations, 0-02 M-cysteine was tested instead of sodium dithionite (see, for example, table 2). It had no effect.
TABLE FOR THE (0-04M) EXSHEATHMENT OF LARVAE OF TRICHOSTRONGYLUS AXEI AND HAIEMONCHUS 5. THE RELATIVE ACTIVITY OF REDUCING AGENTS CONTORTUS relative activity: sodium dithionite/ control 11 3.5 100 10 14 1.6 1.3 3.8 1.8 1.6

species T. axei

pH 6.0 6.0 7.3 7.3 6.0 6.0 6*0 7.3 7.3 7.3

carbonic acid, x 104M 7.9 15.9 1.1 2.2 7.9 15.9 31.8 1.1 2.2 4.4

control 9 28 1 10 7 64 74 26 55 63

ascorbic acid 9 62 25 12 60 77 88 100 124 122

cysteine 25 72 130 120 110 94 97 107 89 81

sodium dithionite 100 100 100 100 100 100 100 100 100 100

H. contortus

(b) The hatching of eggs Reducing agents increased the proportion of eggs of Ascaris lumbricoides which hatched in bicarbonate-carbon dioxide buffers. The optimum concentrations and the relative activity of the reducing agents were similar to those obtained with Trichostrongylus axei and Haemonchus contortus (figure 6) but the relative activity of cysteine and sodium dithionite was not affected by changes in pH (figure 8). The effect of adding sodium dithionite at concentrations between 0.01 and 0-08M was always relatively less at higher concentrations of undissociated carbonic acid both at pH 6-0 and 8*0 (table 4). The addition of reducing agents also increased the proportion of eggs of Toxocara mystax (table 3) and Ascaridia galli which hatched. These and other results showed that the reducing agents had similar actions on eggs and larvae. Except in experiments with Trichostrongylus colubriformis the addition of a reducing agent always increased activity at a given concentration of undissociated carbonic acid. But the effect of the reducing agent was always decreased at a given pH when the concentration of the carbonic acid was increased.

Infective processes of nematodeparasites

377

Increases in the concentration of sodium dithionite had little effect on exsheathment or hatching unless carbon dioxide was present in the gas phase. As the three reducing agents are different in structure their action on the eggs and larvae cannot be attributed to any of their individual properties. It seems likely, therefore, that it was the oxidation-reduction potential of the system which was important in the stimulus.

60-

40-

bD

.^/

._

X20

1 time (h)

FIGURE6. The effect of reducing agents on the hatching of eggs of Ascaris lumbricoides. The medium consisted of bicarbonate-carbon dioxide buffer at pH 7-3 with a gas phase of nitrogen containing 5% carbon dioxide. The reducing agents were OOl1M-sodiumdithionite (upper curve), OOl M-cysteine (middle curve) and 0Ol M-ascorbic acid (lower
curve).

(3) The effect of pH with larvae (a) Experiments Exsheathment of Trichostrongylus axei and Haemonchus contortusin bicarbonatecarbon dioxide buffers under a given concentration of carbon dioxide was greatest at the lower end of the range pH 6-0 to 8-0 (figure 9). This was because increases in the concentration of undissociated carbonic acid always lead to increased exsheathment with these species. But the pH also had an effect on exsheathment independent of its effect on the concentration of the carbonic acid; at a given concentration of undissociated carbonic acid increases in pH increased activity both with and without a reducing agent (figures 2, 3). The exsheathment of larvae of Trichostrongylus axei and Haemonchus contortus was also examined in solutions of 0-025N-hydrochloric acid at different concentrations of undissociated carbonic acid and with and without cysteine. Larvae of H. contortus did not exsheath. The highest proportion of Trichostrongylus axei which exsheathed, 11 %, was obtained under nitrogen-40 % carbon dioxide with cysteine present. Within the usual range of bicarbonate-carbon dioxide buffers, pH 6-0 to 8-0, exsheathing of T. colubriformis was low. Only when the hydrogen ion concentration

378

W. P. Rogers

exceeded 10-3M was appreciable activity obtained. When the concentration of undissociated carbonic acid was about 5 x 10-3M, optimum activity was obtained in the range pH 1-5 to 2-5 (figure 7).
40-

2020-/

pH
of Trichostrongylus colubriformis. The FIGURE 7. The effect of pH on the exsheathment medium consisted of dilute hydrochloric acid containing 0.lM-sodium chloride under of undissociated nitrogen containing 20% carbon dioxide so that the concentration carbonic acid was about 4-80 to 4-85 x 10-3M (upper curve). The results shown in the lower curve were obtained when there was no carbon dioxide in the gas phase. 80-

T?

/^

0 b\

0 6

. . .. 7 pH 8

FIGURE 8. The effect of pH on the hatching

of eggs of Ascaris lumbricoides with 0.02M-sodium dithionite (upper curve) and 0.02M-cysteine (lower curve). The medium consisted of bicarbonate under nitrogen (pH 8.3) or bicarbonate-carbon dioxide buffers under nitrogen containing 5 % carbon dioxide. Sodium chloride was added in amounts ranging from 0-025M at pH 8-3 and pH 8-0 to 0-1M at pH 6-0.

(b) The hatching of eggs The hatching of eggs of Ascaris lumbricoides increased as the pH was raised from 6-0 to 8-0 in bicarbonate-carbon dioxide buffers containing ascorbic acid, cysteine or sodium dithionite under nitrogen-5 % carbon dioxide. At pH 8*3,

Infective processes of nematode parasites

379

when nitrogen alone formed the gas phase, activity was greatly decreased (figure 8). This was probably due to the low concentration of undissociated carbonic acid rather than the low hydrogen ion concentration. The stimulus for the hatching

of eggs of A. lumbricoides, Toxocara mystax and Ascaridia galli at a given

concentration of undissociated carbon dioxide was always greatest at the higher pH (figure 5). The results obtained with eggs and larvae indicated that hydrogen ions formed an important part of the stimulus. The pH influenced the action of the stimulus by affecting: (a) the concentration of undissociated carbonic acid in the medium,
80

6 pH PH
FIGURE

9. The effect of pH on the exsheathment of Trichostrongylus axei. Experiments were carried out in bicarbonate-carbon dioxide buffers containing 0.02m-sodium dithionite and under nitrogen containing 5 % carbon dioxide. In one set of experiments (continuous line) a constant amount of sodium chloride, 0.05m, was added; in the other (broken line) the concentration of sodium chloride was decreased in steps from 0.1M at pH 6.0 to 0025M at p60-80. 0w025M at pE 8B0.

(b) the larvae and eggs so that a given concentration of the carbonic acid was more effective at a higher pH, and (c), the oxidation-reduction potential of the reducing agents. (9) Miscelloneous factors in the stimulus (4) Miscellaneousfctors in the stimulus Though bicarbonate or carbonate ions had little direct effect on larvae or eggs, it seemed possible that the different concentrations of sodium bicarbonate in buffers may have had an indirect effect by chaingig the osmotic pressure. This buIfers may have had an indirect effect by changing the osmotic pressure. This was examined by adding sodium chloride to the bicarbonate-carbon dioxide 0-1M buffers containing a reducing agent, to give a concentration of At pHi 6@0 bufers containing concentration of 0-IM at pH 6.0 reducing agent, give xei. erence s osmotic in concentration of 0o025M at pH 8 Diff T025M 8m0. Differences falling falling in steps to a bicarbonate were thus amounts pressure due to the varying amounts of sodium bicarbonate were thus largely removed. These experiments,experiments, with Haemonchus contortus and Trichoconducted Hcaemonc7hu,s n str ongylus a whenhe constant amount of similar strongylus axei, gave results similar to those obtained w resent sodium 05M, was sodium chloride,chloride, was present (figure 9). The relationship between pH and 0 0.05m, p
25
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W. P. Rogers

the hatching of eggs of Ascaris lumbricoides was also little affected when appropriate amounts of sodium chloride were added to avoid large changes in osmotic pressure of the buffers (figure 8). When the exsheathment of larvae was compared under similar conditions, but with and without 01 M-sodium chloride, it was found that activity was generally increased by 5 to 15 %. The effect of sodium chloride was greatest in experiments with Trichostrongylus colubriformis in 0-025 N-hydrochloric acid. At a concentration of 0-05M, the salt sometimes increased exsheathment by 20 %. With al three species of larvae 0-4M-sodium chloride inhibited exsheathment. Sodium chloride in concentrations up to 0-1 M increased the hatching of eggs of Ascaris lumbricoides by 15 to 30 % in bicarbonate-carbon dioxide buffer at pH 7.3 (0*013M-sodium bicarbonate) containing 0-02M-sodium dithionate. At 0-2M, sodium chloride was less effective. But even when the concentration was 0-3M the proportion of eggs which hatched was often greater than 30 %, In the two experiments which were carried out, 0-1 and 0.2M-potassium chloride increased the hatching of eggs between 5 and 15 %, as did 0-1 M-ammonium chloride. The hatching of eggs decreased slightly when 0*2M-ammonium chloride or 0-4Msucrose was added. Concentrations of sodium taurocholate up to 0.05M in bicarbonate-carbon dioxide buffers containing reducing agents, increased the exsheathing of Trichostrongylus axei and Haemonchus contortus by 5 to 15 %. Similarly, horse serum, 10 %, and 'Tween 80', 0-01 to 0-001 %, increased the hatching of eggs of Ascaris lumbricoides, but octyl alcohol and a commercial 'anti-foaming' agent were strong inhibitors. (5) The time relations of the stimulus If larvae of Trichostrongylus axei were incubated in bicarbonate-carbon dioxide buffer at pH 6-9 containing 0-04M-sodium dithionite under 100 % carbon dioxide for 15 min and then washed and incubated in water or phosphate buffer containing magnesium chloride the subsequent exsheathment of larvae reached 75 % after 2| h. About the same proportion of larvae exsheathed if they were incubated continuously in the stimulating medium (figure 10). The proportion of larvae of Trichostrongylus axei 'triggered' for exsheathment decreased as the time of exposure to the stimulus was reduced below 12 min. With larvae of Haemonchus contortus exposure to the stimulus for about 30 min was necessary to stimulate the proportion of larvae which would exsheath if they were exposed to the stimulus continuously for 3 h. Eggs of Ascaris lumbricoides were incubated in bicarbonate-carbon dioxide buffer at pH 8-0 containing 0-04M-sodium dithionite under nitrogen-5 % carbon dioxide. After 1, 2 and 3 h samples were taken and the proportions of eggs which had hatched were measured. The hatching of these eggs when they were subsequently incubated in saline was measured at intervals up to 19 h after the beginning of the experiment (figure 11). This experiment was varied by using different stimulating media and by using a 'balanced salt' medium (Fenwick 1939a), known to favour the survival of the hatched larvae, instead of saline. As before, the eggs continued to hatch, but at a reduced rate.

Infective processes of nematode parasites

(6) The stimulus in vitro and in vivo

381

For comparing the response of infective agents to the stimulus in vivo and in vitro the eggs of Ascaris lumbricoides were used (table 6). The capacity of different lots of eggs to hatch in the small intestine of the mouse closely paralleled the
80-

0v
__

q~

.
M I

4(

3h

h 45min

3h

3h

ca
20,
A

31mir

15min

I
FIGURE 10. The time relations of the stimulus for the exsheathment of larvae of Trichostrongylus axei. The proportion of larvae which exsheathed when they were incubated in bicarbonate-carbon dioxide buffer at pH 6*9 containing 0.02M-sodium dithionite and 0-05 M-sodium chloride under 100 % carbon dioxide is shown by the lengths of the black areas. The exsheathment which took place subsequently in 0001 M-magnesium chloride in 001M-phosphate buffer at pH 7 2 is shown by the lengths of the open areas.

0 61

oX 0-

I- !

ta) 41 0
C)

I~
0i i ._ -.-

I
Co

021

\I

I 4

I
1'

1, 16

jI

I 20

time (h)
FIGURE 11. The time relations for the hatching of eggs of Ascaris lumbricoides. The proportion

of eggs which hatched in bicarbonate-carbon dioxide buffer containing 0.04M-sodium dithionite under nitrogen-5 % carbon dioxide is indicated by the arrows. The subsequent hatching of the eggs occurred when they were incubated in saline.
25-2

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W. P. Rogers

hatching of eggs in vitro. Thus the eggs of lot B 10 which hatched only in small numbers in vitro also failed to hatch in vivo.
TABLE 6. THE HATCHING OF EGGS OF ASCARIS LUMBRICOIDES IN VIVO AND IN VIT?RO

% of eggs hatched incubation time (h) 2 3 4 in vitro Al 73 A2 76 in vivo -Al B10 9 8 48 6

r
B8 24 31 58 BiO 7 5 12

r
B8 29 26 67

A2 62 -

A 1 and A2 were tested about a week after Four different lots of eggs were examined. embryonation was complete; eggs in lots B 8 and B 10 were much older. DISCUSSION

(1) The nature of the stimulus in vivo Larvae have been exsheathed in vitro in a variety of media. Lapage (1935 a, b) removed the sheaths of third-stage larvae of trichostrongyle parasites with hypochlorites, sodium sulphide, or organic compounds containing sulphur. More physiological media were used by Crofton (1947) who exsheathed larvae of Trichostrongylus retortaeformis in acid solutions containing pepsin but incubation had to be continued for 60 h before the majority of the larvae exsheathed. Poynter (1954a, b) found that larvae of Trichostrongylus axei exsheathed slowly in vitro in the duodenal contents of the horse; when coliform organisms from horses were present exsheathment was rapid. Rogers & Sommerville (1957) showed that cultures of Escherichia coli, when they had reached low oxidation-reduction potentials, caused exsheathment of Trichostrongylus axei. The concentration of undissociated carbonic acid in these cultures was probably high; this would explain the activity which was obtained. Infective eggs have been caused to hatch by detergents, hypochlorite and the action of abrasives or pressure (Jaskoski I952; Fenwick I939b; Pitts 1948). More physiological media have been used (Pick 1948) but long periods of incubation were needed. It is probable that the processes of exsheathment and hatching described in this paper are similar to the processes in vivo. Thus the hatching of eggs in vitro paralleled the hatching of eggs in vivo (table 6). Moreover, the capacity to hatch in vitro and in vivo developed at the same time (Rogers 1958). And when eggs were incubated in the fungicide 'Shirlan' (salicyl anilide) the delay in development of the infective stage measured by tests in vitro gave the same results as tests in vivo. The eggs of Ascaris lumbricoides did not appear to hatch in vitro until after the first moult had occurred, as also was shown to be the case in vivo by Alicata (I934)Morphological changes during exsheathment of larvae in vitro followed the same stages as those which occur in vivo (Veglia 1916; Lapage I935 b; Rogers & Sommerville 1960). The changes in the egg shell of A. lumbricoides during hatching

Infective processes of nematode parasites

383

in vitro showed the same two forms that were seen in eggs which hatched in the small intestine of the mouse (Rogers I958). (2) Characteristic properties of gut fluids It seems reasonable that undissociated carbonic acid, low oxidation-reduction potentials and low oxygen pressures should be the important components of the environment which start infection in the alimentary canals of animals. These properties distinguish regions in the alimentary canal from most other habitats, and ensure that development from the infective stage would not start until the infective agent was eaten by the host. The factors which enhance the activity of the stimulus, such as sodium taurocholate and salts, also occur at appropriate concentrations in gut fluids. The instability of rumen fluid for inducing exsheathment of larvae of Trichostrongylus axei and Haemonchus contortus in vitro (Rogers & Sommerville i960) can now be explained because all three components of the stimulus would change when the fluid was exposed to air. Moreover, the need for high concentrations of undissociated carbonic acid for the exsheathment of H. contortus explains why reducing agents partly restored the activity of rumen fluid for the exsheathment of Trichostrongylus axei but failed for Haemonchus contortus. (3) The stimulus for infection in different parts of the alimentary canal The high concentration of undissociated carbonic acid and the low oxidationreduction potential needed for the exsheathment of Haemonchus contortus suggests that the rumen is one of the few organs where infection with this species could be started. Indeed the rumen of the sheep, and presumably of other ruminants also, is a habitat which must be almost unique. Thus the rumen contents of six of the sheep examined by Turner & Hodgetts (I955) had a pH range of 6-6 to 7.3 and the carbon dioxide in the gas phase varied from about 65 to 39 % so that the undissociated carbonic acid may have reached concentrations from 3 x 10-3 to 7 x 10-3M. Oxidation-reduction potentials in rumen fluid may range from -210 to -260 mV (Dewey, Lee & Marston 1958). Rumen fluid would thus be suitable for causing the exsheathment of larvae of Trichostrongylus axei and Hlaemonchus contortus. The high pH and concentration of undissociated carbonic acid in the rumen fluid would inhibit exsheathment of Trichostrongylus colubriformis and the high concentration of undissociated carbonic acid alone would prevent the hatching of the eggs of Ascaris lumbricoides, Ascaridia galli and Toxocara mystax. This conclusion has been verified experimentally with eggs of Ascaris lumbricoides. Similarly Sommerville (I957) showed that Trichostrongylus colubriformis did not exsheath in the rumen. The pH of fluid in the abomasum ranges from 1-05 to 3-6 in sheep (Spector I956). The concentration of undissociated carbonic acid may be high because the Pco2 in the mucous membrane of the cat and rabbit lies between 40 and 60 mm of mercury (Campbell 1933). In the bulk of the stomach contents where exsheathment would occur the po2 would be very low (Rogers I949) but the oxidationreduction potential, + 150 mV in the rat (Bergeim, Kleinberg & Kirch I945) is

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W. P. Rogers

probably somewhat higher than in many other parts of the gut. Conditions of this sort in vitro especially favoured the exsheathment of larvae of T. colubriformis; and though a small proportion of T. axei also exsheathed, very few larvae of Haemonchus contortus were affected. No eggs would be expected to hatch under these conditions. These results agree very largely with those obtained from studies on life cycles of the parasites. Thus the eggs of Ascaris lumbricoides, Ascaridia galli and Toxocara mystax hatch in the small intestine of the host-not in the stomach. Trichostrongylus colubriformis exsheathed very rapidly when placed in a Cellophane sac in a fistula in the abomasum of the sheep. Under the same conditions exsheathment of T. axei and Haemonchus contortus was much slower. And when tested in vivo by conventional methods H. contortus did not exsheath at all; Trichostrongylus axei was not tested in vivo (Sommerville I957). The pattern of the components which stimulates the hatching of eggs would be found in the fluids of the small intestine in vivo. Thus the Po2 would be low except close to the mucosa (von Brand I946; Rogers I949). The Eh would also be low; it is about -100 mV for the small intestine and -200 mV for the caecum and colon of the rat (Jahn i933; Bergeim et al. I945). In the intestinal juices of the dog the pH in the jejunum ranged from 6-1 to 7-3 (Robinson, Luckey & Mills 1943) and the corresponding concentrations of undissociated carbonic acid would have been about 1 to 2 x 10-3M at pH 6-8. Under conditions like these the eggs of Ascaris lumbricoides, Ascaridia galli and Toxocara mystax, which would be unaffected in the anterior parts of the gut, would be expected to hatch. (4) The stimulus of infection in relation to host specificity It is difficult to discuss the relationship between the nature of the stimulus for starting infection and specificity because little is known about the range of hosts of many parasites. Also specificity may often be determined partly by the behaviour of the infective stage and the host, and not wholly by the ability of the parasite to survive within a particular host. Thus behaviour may determine specificity between broad limits. The capacity of the host to provide a pattern of components necessary to start development of the infective stage within the host might narrow these limits. Finally, the development of the parasite to different stages within a host would depend upon a number of factors concerning the anatomy and physiology of the host. The lack of a high degree of specificity in the stimuli which start infection with different species of parasites is thus not surprising. Moreover, specificity of many nematode parasites is not high (Chandler 1932). Thus Trichostrongylus axei and T. colubriformis have a wide range of hosts (see, for example, Roth 194I; Lie Kian I946; Bearup & Bolliger 1949; Drudge, Leland, Wyant & Elam I955; Tromba & Douvres I957). On the other hand, Haemonchus contortus is seldom found except in ruminants. Is this because the stimulus for infection with H. contortus rarely occurs outside the rumen Eggs of Ascaris lumbricoides and Toxocara mystax hatch in the small intestine of a variety of hosts. This would be expected because the stimuli needed in vitro

Infective processes of nematode parasites

385

are not exacting. The range of hosts in which Ascaridia galli will hatch has not been examined extensively. (5) The process of infection as an adaptation to parasitism In parasitic nematodes the infective stage forms a 'bridge' by which the parasite passes from one environment to another. The suspension of development in this stage, and the requirement that a stimulus from the host is necessary to restart it, is an adaptation to parasitism. It ensures that protective structures such as the sheath or egg shell are not discarded until an environment which might support future development is reached. The hatching of eggs of Trichostrongylus retortaeformis, which occurs outside the host, is retarded by concentrations of salts as low as 0-05M (Wilson I958). But higher concentrations did not seriously inhibit the hatching of eggs of Ascaris lumbricoides. This may be regarded as an adaptation to parasitism because it allows the hatching of infective eggs in vivo under conditions where eggs like those of Trichostrongylus retortaeformis would be inhibited. (6) How does the stimulus act? The egg shell of Ascaris lumbricoides is impermeable to some ions; cyanide and azide ions do not affect development or respiration whereas hydrogen cyanide and hydrazoic acids inhibit (Resnitschenko I927, I928; Passey & Fairbairn I955). During respiration oxygen passes into the egg and carbon dioxide is produced. Labelled carbon dioxide (14CO2) passes in through the shell in some form or other Fairbairn 1957). The results which suggest that undissociated carbonic (Passey & acid or dissolved gaseous carbon dioxide rather than bicarbonate or carbonate ions is a component of the stimulus thus seem reasonable. It seems that undissociated carbonic acid or dissolved gaseous carbon dioxide is the principle component of the stimulus and that other factors, chiefly the Eh and pH, simply enhance or decrease its effect. The action of pH on the relative activity of the different reducing agents (table 5) was probably due to changes in the values of Eo which would fall as the pH: was raised from 6-0 to 7-3. Dissolved gaseous carbon dioxide affects the rate of cell division and differentiation in some organisms. It also controls sexual differentiation in Hydra (Loomis I957). It is not known how carbon dioxide causes these changes. The importance of the stimulus from the host for starting the development of the infective stage of nematode parasites generally is difficult to assess. But it seems possible that when infection takes place in the gut of the host a process similar to that described here may operate even for parasites like Trichinella spiralis which have no free-living stages. On the other hand the culture of Haemonchus contortus throughout its life cycle in media composed of embryo- and liverextract, casein hydrolysate and serum (Silverman 1959) suggests that under certain conditions a stimulus which is necessary for infection in vivo is not required. I wish to thank Mrs M. Ross for technical assistance. My thanks are also due to Dr M. Creeth and Dr P. Dunlop of the Department of Physical Chemistry, University of Adelaide for advice. Mr R. I. Sommerville of C.S.I.R.O., McMaster

386

W. P. Rogers

Laboratory, Sydney, with whom I had several helpful discussions, supplied the larvae. Part of this work was carried out during the tenure of a visiting professorship at McGill University and I wish to thank Professor T. W. M. Cameron and Dr D. Fairbairn of the Institute of Parasitology, McGill University, who arranged my visit and gave helpful advice. Grants in aid of this work from the George Aitken Pastoral Research Trust, Melbourne, and the Rockefeller Foundation are gratefully acknowledged.
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Alicata, J. E. 1934 Proc. Helm. Soc. Wash. 1, 12. Bearup, A. J. & Bolliger, A. I949 Aust. J. Sci. 12, 75. Bergeim, O., Kleinberg, J. & Kirch, E. R. I945 J. Bact. 49, 453.

Calam, C. T., Todd, A. R. & Waring, W. S. 1949 Biochem. J. 45, 520. Campbell, A. I933 Quart. J. Exp. Physiol. 22, 159. Chandler, A. C. 1932 J. Parasit. 18, 135. Crofton, H. D. I947 Parasitology, 38, 101. Dewey, D. W., Lee, H. J. & Marston, H. R. I958 Nature, Lond. 181, 1367. Drudge, J. H1.,Leland, S. E., Wyant, Z. N. & Elam, G. W. 1955 J. Parasit. 41, 505. Ellenby, C. & Gilbert, A. B. I957 Nature, Lond. 180, 1105. Fairbairn, D. I955 Canad. J. Biochem. Physiol. 33, 122. Fenwick, D. W. I939a J. Helminth. 17, 211. Fenwick, D. W. I939b J. Helminth. 17, 69.
Jahn, T. L. I933 J. Parasit. 20, 129.

Jaskoski, B. J. 1952 Exp. Parasit. 1, 291. Lapage, G. 1935a Parasitology, 27, 186. Lapage, G. I935 b Univ. Cambridge, Inst. Animal Path., Rept. Director, 4th Rep., p. 208. Lie Kian Joe I946 Naturk. Tijdschp. Ned-Ind. 102, 41.
Loomis, W. F. 1957 Science, 126, 735.

F. Passey, BR. & Fairbairn, D. 1955 Canad. J. Biochem. Physiol. 33, 1033. Passey, R. F. & Fairbairn, D. I957 Canad. J. Biochem. Physiol. 35, 511. Pick, F. 1948 Bull. Soc. Path. exot. 41, 208. Pitts, T. D. 1948 Proc. Soc. Exp. Biol., N.Y., 69, 348. Poynter, D. i954b Nature, Lond. 177, 481. Resnitschenko, M. S. 1927 Biochem. Z. 191, 345. Resnitschenko, M. S. I928 Biochem. Z. 201, 110. Robinson, C. S., Luckey, H. & Mills, H. 1943 J. Biol. Chem. 147, 175. Rogers, W. P. 1949 Aust. J. Sci. Res. B2, 157. Rogers, W. P. I958 Nature, Lond. 181, 1410. Rogers, W. P. & Sommerville, R. I. I957 Nature, Lond. 179, 619. Rogers, W. P. & Sommerville, R. I. I960 Parasitology. (In the Press.)
Roth, H. Silverman, 194I J. Parasit. 27, 363. 1959 Nature, Lond. 183, 197. Poynter, D. 1954a Nature, Lond. 173, 781.

Shedlovsky, T. & Maclnnes, D. A. I935 J. Amer. Chem. Soc. 57, 1705.

P. H.

Sommerville, R. I. 1957 Exp. Parasit. 6, 18. Spector, W. S. I956 Handbook of biological data. Springfield: Carpenter Lithio and Printing Company. Tromba, F. G. & Douvres, F. W. 1957 J. Parasit. 43, Suppl. 12. Turner, A. W. & Hodgetts, V. E. 1955 Aust. J. Agric. Res. 6, 115.
Umbreit, W. W., Burris, R. H. & Stauffer, J. F. 1957 Manometric techniques. Minneapolis:

Burgess Publishing Company. Van Slyke, D. D., Sendroy, J., Hastings, A. B. & Neill, J. M. I928 J. Biol. Chem. 78, 765. Veglia, F. 1916 Rept. Director Vet. Education Research, Onderstepoort, 3rd and 4th Rep., 1915-16, p. 349. Von Brand, T. I946 Anaerobiasis in invertebrates. Normandy, Missouri: Biodynamica. Wilson, P. A. G. I958 J. Exp. Biol. 35, 584.