1. The osmotic pressure in the environment of Polymorphus minutus in the alimentary tract of ducks has been measured. Samples of intestinal contents were collected through cannulae inserted at known positions into the parasite’ s environment.

  2. Samples were recovered from two healthy living ducks, without inducing anaesthesia, from points at 60% and 80% of the distance along the intestine, and from ten freshly killed ducks at a point 70 % of the distance along the intestine.

  3. The mean osmotic pressures at 60%, 70% and 80% of the distance along the intestine were found to be 170·5, 171·6 and 178·2 mM-NaCl/1. respectively and the variance was found to be 154·84, 84·41 and 30·13 for the three positions respectively.

  4. It is postulated that the greater fluctuations in osmotic pressure at the anterior end of the environment may be responsible for the posterior migration of the parasite during the course of the infection.

  5. The method here described for investigating osmoregulation in P. minutus may be of general application for studying aspects of the biology of small intestinal helminths in vivo.

Many of the attempts to culture intestinal helminths in vitro appear to have been made with relatively little knowledge of the physico chemical conditions which occur in the alimentary tract of vertebrates. For example, only a few measurements have been made on the osmotic pressure of the contents of the intestinal lumen and even fewer have been made on the degree of variation of the osmotic pressure in a given region of the intestine over a period of time. The aims, therefore, of the work described in this paper were to measure the osmotic pressure at different positions in the environment of Polymorphus minutus in the intestine of domestic ducks and to obtain an estimate of the range of osmotic pressures to which the parasite is subjected. These data are required to facilitate the in vitro cultivation of P. minutus and to initiate a study of the osmoregulation of the parasite.

The location of the environment of P. minutus in the small intestine of domestic ducks has been described in detail by Crompton & Whitfield (1968). The majority of the worms are found to be attached at positions between 60% and 80% of the distance along the intestine with a mean attachment position at about 70% of the distance along the intestine During the course of the infection, the mean attachment position changes to about 74% of the distance along the intestine, a fact indicating that the population of worms migrates posteriorly.

The measurements of the osmotic pressure were made by the cryoscopic method of Ramsay & Brown (1955) on small samples of intestinal contents which had been withdrawn through cannulae inserted at known positions into the alimentary tract of ducks. In this way, data were obtained from different regions of the environment of P. minutus over a period of several weeks. A method for studying certain aspects of the physiology of P. minutus and other small intestinal helminths was developed during the course of the work and is also described in this paper.

Ducks

Thirteen Khaki Campbell ducks of ages varying from 3 to 107 days old were used. The ducks were reared in the laboratory and fed ad libitum on water and BOCM baby chick crumbs.

Surgical procedures

Three ducks were cannulated by the method described by Crompton, Shrimpton & Silver (1965). The type of Perspex cannula used for many of the measurements is shown in Text-fig. 1. Plate 1, which is a photograph of the alimentary tract of one of the ducks with the cannula in position, shows that the intestine remained free from adhesions and other defects during the course of the experiment. Further evidence of the success of the cannulation technique is provided by the fact that cannulated ducks are satisfactory hosts for P. minutus (Crompton & Whitfield, 1968). A cannula of the type shown in Plate 1 and Text-fig. 1 was positioned at 60% of the distance along the intestinal length in one bird (duck 1) and at 80% in another (duck 2) ; thus samples were obtained from the limits of the principal attachment zone of P. minutus. Another cannula, which is shown in Text-fig. 2, was positioned at 60% of the intestinal length of a third bird (duck 3). This preparation was used to obtain simultaneously (i) samples of the intestinal fluid in the immediate environment of the parasite without killing or anaesthetizing the host, and (ii) individual P. minutus, from which samples of pseudocoelomic fluid were withdrawn. Details of this procedure are given in the section on results. Samples of intestinal contents were also obtained from known positions in the environment of P. minutus within 1·5 min. of the deaths of ten other ducks.

Text-fig. 1 and 2.

Diagrama of the Perspex cannulae used to obtain samples of intestinal contents. Note the size of Polymorphut minutui drawn to the same scale. The arrow indicates intestinal flow, d., disk; g., groove; h., head; i.l., intestinal lumen; i. w., intestinal wall; n., nylon bag; pl., plunger; sr., shaft; w., worm.

Text-fig. 1 and 2.

Diagrama of the Perspex cannulae used to obtain samples of intestinal contents. Note the size of Polymorphut minutui drawn to the same scale. The arrow indicates intestinal flow, d., disk; g., groove; h., head; i.l., intestinal lumen; i. w., intestinal wall; n., nylon bag; pl., plunger; sr., shaft; w., worm.

Sampling procedure

Samples were recovered from ducks 1 and 2 with a clean dry Pasteur pipette after withdrawal of the solid glass plunger which occupied the shaft of the cannula (Text-fig. 1, pl.). The plunger, which extended as far as the end of the cannula shaft and not into the intestinal lumen, was held in position by a piece of thin rubber sheet tied round the head of the cannula. The sample of intestinal contents was either used immediately for determination of osmotic pressure or was rapidly frozen and stored under liquid paraffin at 20 °C. All samples were collected and frozen within 1 min. of the removal of the plunger from the cannula.

Determination of osmotic pressure

Osmotic pressure determinations were made by the cryoscopic method of Ramsay & Brown (1955). Throughout the paper, osmotic pressure is expressed in terms of the concentration of NaCl (in mM/1.) having the same freezing-point depression. Standard curves, from which the osmotic pressure of the intestinal samples was estimated, were constructed by plotting the freezing-point depressions against the concentrations of prepared solutions of NaCl. The reproducibility of the standard curve was checked regularly during the course of the work.

Two determinations were made for each sample and sometimes two samples were collected at a given time. Thus each value of the osmotic pressure given in Tables 1–3 is the mean of either two or four determinations and 288 determinations were made on samples from the thirteen ducks used.

Table 1.

The osmotic pressure of intestinal contents of a duck cannulated at a point 60% of the distance along the intestine

The osmotic pressure of intestinal contents of a duck cannulated at a point 60% of the distance along the intestine
The osmotic pressure of intestinal contents of a duck cannulated at a point 60% of the distance along the intestine
Duck 1

The results of the determinations of the osmotic pressure of samples from the intestine of this duck are given in Table 1. The duck was 50 days old when it was cannulated at a point 60 % of the distance along the intestinal length. Determinations were made over a period of 22 days. The mean osmotic pressure was found to be 170·5 mM –NaCl/1. and the data in Table 1 gives an indication of the range of osmotic pressures to which P. minutus, attached in this position, may be exposed. These results, however, do not give any indication of the length of the time for which the osmotic pressure remains constant. In an attempt to elucidate this problem, samples were collected every 12 hr. for approximately 36 hr. from duck 2, which had been cannulated at a point 80 % of the distance along its intestinal length.

Duck 2

The results of the determinations of the osmotic pressure of samples obtained at 30 min. intervals from the posterior part of the environment of P. minutus are given in Table 2. The results are also shown graphically in Text-fig. 3. The mean osmotic pressure was found to be 178·2 mM-NaCl/1. No satisfactory explanation can be provided for the failure to obtain a value for the osmotic pressure of samples 15, 32 and 70 (Table 2, Text-fig. 3).The samples froze rapidly, but it was impossible to determine accurately the point at which the ice-crystals melted and thus the depression of freezing-point could not be measured.

Table 2.

The osmotic pressure of intestinal contents of a duck cannulated at a point 80 % of the distance along the intestine

The osmotic pressure of intestinal contents of a duck cannulated at a point 80 % of the distance along the intestine
The osmotic pressure of intestinal contents of a duck cannulated at a point 80 % of the distance along the intestine
Text-fig. 3.

Graphical representation of the results given in Table 2 ◖, Sunset; ◠ sunrise.

Text-fig. 3.

Graphical representation of the results given in Table 2 ◖, Sunset; ◠ sunrise.

A comparison was made between the results for ducks 1 and 2 to determine the range of the osmotic pressures prevailing throughout the environment. It was assumed that differences between the ducks would be negligible since both were male ducks of the same age. The variance for the results from duck I was determined and found to be 15·84. In this case, the need to remove the first-order autocorrelation was ignored because the readings were taken at never less than 2 hr. intervals with one exception. The residual variance, after removal of the first-order autocorrelation, which was found to be significant, was then determined for duck 2 and found to be 30·13 Comparison of these variances by the F-test showed them to be significantly different at the 0·1% level. This result may be interpreted as signifying that the fluctuations in the osmotic pressure are much greater at 60% of the distance along the duck’ s intestine than at 80%. Consequently, P. minutus attached in the anterior portion of their environment will be subjected to greater changes in osmotic pressure than those living in the posterior portion, which appears to be a more stable osmotic environment. On the other hand, the osmotic pressure in the posterior portion of the environment is slightly higher than in the anterior portion as can be seen from the mean values of 178·2 and 170·5 mM-NaCl/1. respectively.

Other ducks

Table 3 gives the results of measurements of the osmotic pressure at a point 70 % of the distance along the intestinal length for eight ducks of varying ages. The ducks were all killed before samples were obtained, but each sample was collected and frozen within 1·5 min. of death. These results are within the same range of osmotic pressures obtained from ducks 1 and 2. A comparison of these results was also made with those from ducks 1 and 2 after assuming that differences between the ducks were negligible. The mean osmotic pressure was 171·6 mM-NaCl/1. and the variance was found to be 84·41. Thus, if it be tentatively accepted that the comparisons between these ducks and ducks 1 and 2 are valid, it can be seen that the mean osmotic pressure is similar to that existing at 60% of the distance along the intestinal length (duck 1) and that the degree of fluctuation in the osmotic pressure is intermediate between those found at 60% and 80%. Values of 180 and 175 mM-NaCl/1. were obtained from the 70% position for two other ducks aged 123 and 128 days respectively.

Table 3.

The osmotic pressure of the intestinal contents of 8 ducks

The osmotic pressure of the intestinal contents of 8 ducks
The osmotic pressure of the intestinal contents of 8 ducks

An important conclusion from the measurements made on ducks which had been killed before samples were taken is that postmortem changes appear to be insignificant when samples are obtained within a few minutes of death. Hobson, Stephenson & Beadle (1952) pointed out that the osmotic pressure of the intestinal fluid of pigs rises if the fluid is left to stand at room temperature. The osmotic pressure of the intestinal fluid may be expected to rise even more rapidly if it is left for many minutes in a freshly killed host.

A method for studying Polymorphus minutus in vivo

During the work described in this paper it was found that as many as six parasites could be suspended, in a small bag of Nylon net in the cannula shaft, so that they were in a position very similar to that which they occupy in a natural infection. A special cannula was required to accommodate the Nylon bag and the details are shown in Text-fig. 2. The following procedure was adopted. First, the parasites were recovered from the intestinal wall of a freshly killed duck and washed thoroughly in Hanks’ s saline kept at 42 °C. The parasites were next placed in the Nylon net bag (n) which was attached by means of cotton to the groove (g) near the base of the glass plunger. The plunger was pushed into position and the bag of worms came to rest in the position shown in Text-fig. 2. It was found that the bag could be removed from the duck, a worm recovered from the bag and the unit reassembled and replaced in the duck in approximately 2 min. This procedure was used in a preliminary experiment on the osmoregulatory characteristics of P. minutus in the following manner. Five, day-old worms were obtained from a duck and a sample of pseudocoelomic fluid was taken from one of the worms immediately and found to have an osmotic pressure of 185 mM-NaCl/1. The other worms were kept in Hanks’ s saline, having an osmotic pressure of about 160 mM-NaCl/1., for 12 hr. before three of them were placed in the intestine in a Nylon bag at a point 60 % of the distance along the intestinal length. The osmotic pressure of the pseudocoelomic fluid of the fourth worm was determined when the other worms entered the intestine and found to be 179 mM-NaCl/1. Samples of intestinal contents were taken through the cannula after 1, 2 and 4 hr. and individual worms were recovered from the bag at the same time. The osmotic pressures of the intestinal contents were found to 175 (1 hr.), 169 (2 hr.) and 172 mM-NaCl/1. (4 hr.) and the osmotic pressures of the pseudocoelomic fluid of the worms were found to be 178, 182 and 175 mM-NaCl/1. for the same times respectively. There are as yet too few data to indicate whether P. minutus is an osmoregulator or an osmoconformer, but this method should enable the problem to be investigated since it permits almost simultaneous sampling of the environment and the parasite’ s pseudocoelomic fluid. Crompton & Lockwood (unpublished results) found that the osmotic pressure of the pseudocoelomic fluid of P. minutus was about 185 ± 5 mM-NaCl/1. This result was also obtained using the cryoscopic method of Ramsay & Brown, but the worms were kept in vitro, under conditions differing from those in the duck’ s small intestine, before samples of pseudocoelomic fluid were obtained.

The results of the determinations of the osmotic pressure, in the environment of P. minutus in healthy, living ducks have shown that there is a gradient of increasing osmotic pressure from the middle to the posterior region of the environment. The gradient represents an increase from about 170 to 178 mM-NaCl/1., and since the osmotic pressure at the position 70% is very similar to that at 60%, it will be experienced over a distance of about 15 cm. in an adult duck. There also exists a gradient of fluctuations in the osmotic pressure in the environment, where changes in osmotic pressure about the mean value at 60% are greater than the changes at 80% of the distance along the intestine.

These results suggest an explanation to account for the posterior migration of P. minutus during the course of its infection in ducks. Crompton & Whitfield (1968) found that, initially, most worms in an infection become attached between 60 % and 80% of the distance along the intestine and that the mean attachment point was at about 70%. Analysis of the data during the course of the infection revealed that the populations moved posteriorly for a short distance, resulting in the mean attachment point being at 74 % of the distance along the intestine. There also appeared to be a tendency for worms attached anteriorly to 60% to move before other worms. The osmotic conditions in the worm’ s environment may be responsible for the migration of the worms. It may be postulated that the fluctuations in osmotic pressure which exist at 60% produce too great an osmotic stress for the worms with the result that they move a short distance to a region where the fluctuations are less but the mean osmotic pressure is relatively unchanged. The mean value for the mean osmotic pressures at the points 60%, 70% is 173·4 mM-NaCl/L, and since the principal attachment point changes to 74% of the distance along the intestinal length where the mean osmotic pressure can be predicted to be about 174 mM-NaCl/1., it would appear that this value represents the optimum environmental osmotic pressure for the worms.

We wish to thank Dr R. C. Campbell, School of Agriculture, University of Cambridge, for his help and advice on statistical procedures, and Mr David Barnard and Miss Maria Yates for their excellent technical help. Thanks are also due to Dr A. P. M. Lockwood and Dr P. Tate for their criticism of the manuscript.

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Plate 1

Photograph of the cannulated small intestine of duck 2, 22 days after the insertion of the cannula. C., caecum; D., duodenum; P., pancreas; R., rectum; Y.S., yolk stalk (Meckel’ s diverticulum).

Plate 1

Photograph of the cannulated small intestine of duck 2, 22 days after the insertion of the cannula. C., caecum; D., duodenum; P., pancreas; R., rectum; Y.S., yolk stalk (Meckel’ s diverticulum).

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