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Studies on Hydatigera taeniaeformis. III. The water content of larval and adult worms
EXPERIMENTAL
PARASITOLOGY
Studies
on
9,
Hydatigera taeniaeformis. of Larval and Adult C. A.
Department
257-263 (1960)
Hopkins
of Zoology, Uniuersity Science and
(Submitted
and
W.
III. The Worms M.
Content
Hutchison
of Glasgow, Scotland, and The Technology, Glasgow, Scotland
for publication,
Water
30 November
Royal
College
of
1959)
1. The water content of H. taeniaejormi larvae developing in mice, and aduIts from the intestine of cats, has been determined at various ages. 2. In larvae up to 8 months old the water content is 72 f 3%, but in some older strobilocerci it rises to 80% 3. A plot of the water content of different regions down the larvae shows that there is little change until the pseudostrobila is reached. 4. In adult worms the water content gradually increases with age during the first 18 days to about 78-80%. Variation after this period appears to be due to inter- and intra-host factors which are discussed, and not age of the worm. 5. It is concluded that within certain specified age limits a ‘normal’ water content exists and that. variation from this may be used by an experimenter as a criterion of abnormality. 6. Comnarison with the water content of other cestodes is made and the validity of an apparent dichotomy discussed.
The water content as a percentage of the total weight has been recorded for several species of cestode (reviewed by v. Brand, 1952). However, the reports only record the water content of a cestode at one, often unspecified, stage of its life-cycle, or give a figure calculated from a group of worms of different and unknown ages. Such information is of limited use. Analysis figures only become of value when they are associated with a particular stage of development or physiological change, and can be shown to be reproducible. In an earlier paper (Hopkins and Hutchison, 1958) it was shown that the percentage nitrogen content of H. taeniueformis followed a specific pattern during development of the larval and adult worms, and that characteristic “breaks” in the rate of change were associated with major physiological changes. In the present paper we report the results of a similar investigation to determine the mean, and normal variation in, water content of H. taeniaeformis at different’ phases of its life-cycle. An attempt is made to correlate the observed changeswith
other physiological and morphological changes, and to assessthe importance of various factors which influence the variation shown by worms of the same age. MATERIAL
AND
METHODS
Larval H. taeniaeformis were removed from strain A albino mice killed at intervals after infecting. The worms were immediately washed in balanced saline (A = 0.56%) and then pressed lightly between filter paper to remove surface moisture and weighed (fresh weight). In this process the bladder burst and often a small part was lost, but by the 8th week, the earliest stage estimated, the bladder is a negligible fraction of the total. Immediately after weighing, the larvae were placed on porcelain slabs at 105°C and dried for 18 hours, then re-weighed (dry weight). In order to investigate the water content along the length of the strobilocercus, worms were removed from a mouse liver and divided into anterior and posterior portions which were estimated separately for water content. The point’ of division in the 46
257
258
HOPKINS
AND
strobilocerci was varied to form a series in which the dry weight of the posterior portion varied from 5-95s of that of the whole worm. Adult worms were obtained from the small intestine of laboratory infected cats immediately after killing. The worms were treated in the same way as the larvae. RESULTS
1. Water Content During Development of
the Larva in the Liver of a Mouse a. Effect of age of larva. In Fig. 1, the mean dry fresh weight ratio of strobilocerci recovered from mice between 8 to 61 weeks after infecting is plotted, together with the standard deviations where the sample was of five or more worms. The number of worms in the mice varied from 1 to 164; sixty-six of the 75 mice examined had relatively light infections (<20 worms), the other nine had medium or heavy infections. No significant difference was found between the water content of larvae from heavy and light infections. b. The water gradient clown the strobilocercus. Before a comparison could be made between water content of larval and adult worms, it was necessary to determine the water content of not only the whole larva
HUTCHISON
but that part of the larva which survives after ingestion by the cat and gives rise to the adult worm. The amount of the larva that constitutes the true larval strobila as distinct from pseudostrobila depends on the age of the larva, varying from 80 to 30% by weight of the larva (Hutchison, 1959). It was decided, therefore, to combine this investigation with a more general one to dctermine the amount of water in each region, from which could be calculated the amount in whatever fraction constituted the true larval strobila. Figure 2 shows the data as measured and needs some explanation. Forty-six larvae, aged 32 weeks, were each cut into two pieces as described in “Methods.” This procedure of cutting the worms only once instead of dividing the worms by several transverse cuts into small fragments kept weighing errors, and errors due to loss on cutting, to a minimum. However, the results obtained are not in a very useful form until they are transformed mathematically. Looking at Fig. 2 the first impression is that the water content gradually increases as one proceeds posteriorly from the scolex, but this is not reallv so, as can be seen in Fig. 3. In this figure the dry/fresh weight ratio expressed as a percentage has been plotted for every 10% portion of the strobi-
40
0 ;
32 -
2 l-
28-
3z 3
a 0
24 -
12
I 0
4
8
12 AGE
16 20 24 28 32 OF STROEILOCERCUS
FIG. 1. The dry/fresh weight at different ages. l = estimation of a worm ; 0 of 5 or more worms and standard
ratio,
plotted
= means deviation.
36
40
*
I
I
a
I
,
44 48 [WEEKg
.
52
56
60
64
68
H.
taeniaeformis,
as a percentage,
of 24
worms;
l
of larval cut by
vertical
line
= mean
STUDIES
ON
HYDATIGERA
TAENIAEFORMIS.
259
III
32
1 100
RATIO STFi%LO%RC”:~~ D.;:] FIG. 2. The dry/fresh weight ratio of portions of a strobilocercus. The ratio is plotted against the dry weight of the fraction expressed as a percentage of the dry weight of the whole worm. 0 = the mean dry/fresh weight ratio of the 46 complete strobilocerci.
locercus. This was calculated from Fig. 2. Reading from the graph the posterior 10% of the worm had a dry weight content of 13.3% (i.e. the dry weight of the posterior 10% of a worm whose fresh weight is 100 mg would be 1.33 mgi. Similarly the posterior 20% has a dry weight content of 17.3% (i.e. dry matter constitutes 3.46 mg in a worm of 100 mg fresh weight). Subtraction of these two figures, 1.33 from 3.46, shows that the region between 10 and 20% up from the posterior end of the larval strobila has 2.13 mg of dry matter in it, and hence that the dry weight is 21.3% of the fresh weight. This calculation was repeated by taking readings from the graph in Fig. 2 at 10% steps. The transformed data 1)lotted in Fig. 3 show in a much clearer manner the percentage dry matter, and hence water content, of different parts of the strobila. R. Water Content During Development of the Adult Worm in the Intestine of a Cut In view of the variation in the dry/fresh weight ratio in larvae over 30 weeks old
(Fig. 1)) only strobilocerci between 10 and 26 weeks old were used to infect cats. All infections in the cats were light ones of 1 to 6 worms; the average burden was 4. a. Changes associated with increasing age of the worm. The percentage dry matter in adult worms is shown in Fig. 4. The total number of worms estimated at a particular age is shown by the index; where t,he worms were obtained from more than one cat, the individual means, calculated from worms recovered from each cat, are shown as closed points ( l ) , and an overall mean as an open circle (0). 6. Water content of worms of the same age. (i) Variation within a single cut. Variation in water content of worms in a cat were usually small, e.g. the 6 worms in cat 126 (39 day infection, Fig. 4) had a mean of 78.9% water with a range from 78 to 80.1%. The average range in water content of worms was 2.7% in the 26 cats infected with 3 or more worms, aged between 3 to 42 days. Only rarely did the range exceed 4%. (ii) Variation between.worms in different
260
HOPKINS
AND
HUTCHISON
PSEUDOSTROBILA
TRUE
LARVAL
STROBILA
>
. \
TAIL
IO
20
30
IO% FIG. 3. The of strobilocerci.
dry For
weight, further
40
50
60
70
OF
THE
FRACTIONS
expressed explanation
as a percentage see text.
IMMATURE RAPID GROWTH.
of the
80
90
SCOLEX
STROBILA fresh
weight,
of 10%
portions
MATURE SLOW GROWTH.
I
I”
0
4
6
I2
I6
20
24
AGE OF WORM
28
32
36
40
44
IN CAT DAYg
FIG. 4. The dry/fresh weight ratio, plotted as a percentage, oping in the intestine of a cat. l = mean based on worms from a cat. 0 = mean of worms recovered from two or more cats. Total number of worms at each age is shown by the indices.
of H.
taeniaeformis
devel-
STUDIES
ON
HYDATIGERA
cats. The mean water content of worm groups recovered from different cats sometimes varied considerably (Fig. 4). For instance the means of the five 12-day-old infections ranged from 71.7 to 75.2%, and in the three 27-day-old infections a difference of 7.8% occurred between individual means. This was exceptionally large, but considering all the age groups at which infections from three or more cats were examined, it is apparent that the means at any particular age may vary over a range of about 5%). DISCUSSION
Although larval H. taeniaeformis can be seen and removed from the liver of mice after the second week, the difficulties involved in weighing such smaI1 worms coupled with the difficulty in getting rid of surface moisture made it impossible to obtain an accurate dry/fresh weight ratio before the end of the eighth week. Between the 8th to 30th week, only minor variations in the dry/fresh weight ratio occur, nearly all worms lie in the range 28 * 3% (Fig. 1). If there is any trend during this period it is of a minor order which is concealed by experimental errors and variation that exists between larvae of the same age. After the 30th week there was a greater scatter in means. At first, it was thought that a drift downwards in the dry/fresh weight ratio of the larvae could be detected but there are several exceptions. The increase in water content sometimes occurred in all the larvae, but in other cases only some of the larvae showed a low dry/fresh weight ratio, which resulted in a large standard deviation, e.g. 46 week infection, Fig. 1. It is probable that this increase in water is due to degenerative changes in these older larvae. The fact that it does not appear to bc correlated with a loss of infectivity is not surprising as degenerative changes are most likely to be occurring in the pseudostrobila. This structure forms 50 to 70% of the larva at this age. It is, however, a useless appendage and is lost during the first day in the cat’s intestine (Hutchison, 1959). One of the most interesting stages in the physiological development of a cestode is the switchover from larval to adult metabolisni, which occurs on transfer t,o the defini-
TAENIAEFORMIS.
III
261
tive host. When comparing the chemical composition of adult and larval H. taeniaeformis it is, of course, necessary to make the comparison between adult and true larval strobila, not the whole larva. Thus, the mean dry matter content of worms after 1 day in a cat is approximaely 31%, compared with 28% in the larva. Both these figures are based on a large number of observations and although the difference is not very great, a comparison of the data in Figs. 1 and 4 leaves no doubt that it is real. However, the conclusion that there is an increase in percentage dry matter is wrong. Reference to Fig. 3 shows that in a larva having an overall dry weight ratio of 28.40/o, the true strobila has a dry weight ratio of over 31%. The initia1 “rise” in percentage dry weight is, therefore, merely a result of the loss of the pseudostrobila. The extent to which the change from true strobila to pseudostrobila is marked by increased water content cannot be precisely determined, as there is no way, morphologically, of distinguishing the line of demarcation, but Hutchison (1959) records that the posterior 40% approximately is pseudostrobila in larvae of 130 to 150 mg fresh weight (the size of the worms on which the data in Figs. 2 and 3 are based). Figure 3 shows the change which occurs in the water content of the tissue at a point which almost coincides with the expected point of change from true larval strobila to pseudostrobila. In the anterior 60% of the worm the results indicate a slight increase in percentage dry matter in the tissues, proceeding posteriorly from the scolex. Such a rise agrees with the general concepts of biochemical heterauxesis (Needham, 1950), in that8 animal t’issue drys as it ages. In this case, following accepted principles that cestode growth occurs in the neck region, the furt’her the tissue is from the neck the older it is, and hence the drier it, may be expected to be. But, at a point 60% down the worm new controlling factors come into operation which bring about a rapid increase in the water content. It seems reasonable to conclude, therefore, that tissues of the pseudostrobila are physiologically different, as is shown by their inability to withstand the intest,inal environment, and
262
HOPKINS
AND
in having a higher water content. What these factors are which cause this change is not known, but one might speculate that they are degenerative changes leading concomitantly to swelling of the tissues and loss of viability in the definitive host. The problem we have attempted to solve with regard to the water content of adult H. taeniaeformis is whether there is a specific composition at a particular age, and if so, does it change in relationship to age of worm. The principle difficulty in resolving this problem is that variation due to factors other than age may be so large as to mask, or at least make doubtful, changes due to age. If the water content of all H. taeniaeformis recovered is considered, an estimation of the total variance can be made. This can then be broken down into variance due to different factors. Firstly, there is the variation between worms in a single cat, i.e. intra-host variation. This was small, relative to total variation. The average range was 2.7% and only rarely did it exceed 4%. It is due to a number of factors, such as, genetical variation, experimental errors, position of the worms relative to one another in the intestine. We have not attempted to assess the importance of these and other possible factors, but merely refer to the variation between worms within one infection as the “basic variation.” Secondly, there is variation between worms taken from different cats, i.e. interhost variation. This can be assessed by comparing the mean water content of worms recovered from two or more cats, with the same age of infection (Fig. 4). The difference between means is often considerable, and it is apparent that one or more new factors are operating which superimpose their effect on the basic variation. These “inter-host” factors affect all the worms in an infection to a similar extent, as far as can be told from our data. Their effect is not to increase the basic variation but to alter the absolute value of the mean. There are several possible causes for this difference in the dry/fresh weight ratio between urorms from different cats. The most probable cause, in these experiments, was the time the cats were killed in relation to their last
HUTCHISON
meal. It is well established that even a few hours of starvation effects the glycogen content of worms considerably, which causes, to some extent at least, a fall in the dry,/ fresh weight ratio (Reid, 1942; Archer and Hopkins, 1958). It is also possible that variations in the osmotic pressure of the intestine may be partly responsible for changes in the dry/fresh weight ratio, but this is open to more doubt, as mammals maintain very close isotonicity between intestinal contents and plasma. Other factors which may influence the worm population as a whole and lead to inter-host variation are differences such as age and sex of the host, and hence gut diameter, mucus production, hormone secretions etc,. There are also the differences between the strobilocerci fed to each cat, though this was kept small by using larvae of a narrow age range. Thirdly, there is variation in water content due to ageing of the worms. To determine this it is necessary to eliminate as far as possible intra- and inter-host variation at a particular age. Ideally, a large number of cats should be examined at each age of infection, a mean and degree of scatter calculated, and a regression coefficient determined. We have not done this as the object of the investigation was not to determine a statistic but to discover whether an experimenter working with H. taeniaeformis either in vivo or in vitro, could talk about a “normal” water-level and if so, what absolute values might be interpreted as norma or abnormal, and, what degree of variation to expect in a small sample. Examination of the data in Fig. 4 shows that age does have a marked effect, a considerable fall in the dry/fresh weight ratio occurs during the first 3 weeks in the cat. However, whether this is a gradual changeover as implied by the plot is questionable. The figures show that by the end of the first day the dry/fresh ratio is between 30 to 35%, which does not appear to alter appreciably for 2 to 3 days. Between the 5th and 17th day the dry/fresh weight ratio lies between 24 to 29%; after the 18th day and up to the end of the period investigated the ratio lies between 19 to 25%. The water content, therefore, rises during the pre-patent period from an initial level of 68 to 70% to
STUDIES
ON HYDATIGERA
rather less than 80%. It will also be observed that this change from a low larval level to a high adult level takes place during the rapid growth phase (Hutchison, 1959) of the first 18 days. By the time the worms are mature and egg production has commenced the new adult level has been reached. Thereafter variation results only from intra- and inter-host factors discussed above. When these result,s for the dry matter content of adult H. taeniaeformis are compared with those of other adult cyclophyllidean tapeworms it is found that they agree closely with one set of figures (Raillietina cesticillus, 20.5% (Reid, 1942) ; Dipylidium caninum, 20.4$%, Taenia hydatigena (marginata) , 23.5%, Anoplocephala magna (T. plicata), 27.5% (v. Brand, 1933))) but differ very greatly from the other figures (Taenia saginata, 12.2% (Smorodinzew et al., 1933) ; T. solium, 8.7% (Smorodinzew and Bebeschin, 1936) ; Moniexia expansa, about lo%, various authors, see v. Brand (1952) ) . The question arises, is there a genuine dichotomy in the Cyclophyllidea? Examination of the “low group” fails to support such a dichotomy. The figures of Smorodinzew and co-workers for T. saginata and T. solium have no biological significance. The procedures used by these workers have been discussed by Archer and Hopkins (1958) and in retrospect at least it is apparent that they were measuring water content of moribund worms. With regard to the other figures insufficient detail is given about the method used for a critical appraisal, but it seems a noteworthy coincidence that high water figures, characteristic of degenerating tissue, are found in just those h&ninths which come from hosts unlikely to be killed in a laboratory to supply fresh cestodes! It seems fair to conclude, therefore, that there is no reliable evidence that adult cyclophyllidean cestodes in healthy condition ever have such low dry matter content; all recent work suggests a percentage dry matter content of between 20 to 25%. The position with regard to water content, which has long been regarded as showing wide variation in cestodes, is beginning to be reversed. Differences do occur, but when allowance is made for the factors discussed
TAENIAEFORMIS.
III
263
in this paper the striking point is the similarity, not the difference. This similarity, however, exists only between cyclophyllidean worms. The data available on pseudophyllideans which mature in warm blooded hosts (summarized by Archer and Hopkins, 1958) show that Diphyllobothrium and allied genera have a dry/fresh weight ratio about 30%, i.e. nearly 50% greater than that of cyclophyllideans. k!KNOWLEDGMENTS
Our thanks arc due to Professor C. M. Ponge in whose Department this work was carried out and to t,he Royal Society (London) for a grant-in-aid. We would also like to thank Professor J. P. Todd of the School of Pharmacy for kindly providing laboratory facilities for one of US (W.M.H.) at the R.C.S.T., Glasgow. REFERENCES
AHCHER, D. M., AND HOPKISS, C. A. 1958. Studies on Cestode Metabolism. V. The chemical composition of Diphyllobothrium sp. in the pleroccrcoid and adult stages. Exptl. Parasitol. 7, 542-554.
vos BRAND, T. 1933. Untersuchungen iiber den Stoffbestand einiger Cestoden und den Stoffwechse1 von Moniezia expansn. Z. ve~gleich. Physid.
18,562~596.
VON BR~XD, T. 1952. Chemical Physiology of Endoparasitic Animals. $cademic Press Inc., Kew York. HOPKINS, C. A., ASD HUTCHISON, W. M. 1958. Studies on Cestode Metabolism. IV. The nit,rogen fraction in the large cat tapeworm, Hyrlatigera (Taenin) taeniaeformis. Exptl. Pnrasitol.
7, 349-365.
HUTCHISOS, W. M. 1959. Studies on Hydatigera (Tnenin) tacniaeformis. II. Growth of the adult phase. Exptl. Parasitol. 8, 557-567. NEEDHA~W,J. 1950. Biochemistry and Morphogenesis. Cambridge University Press, England. REID, W. M. 1942. Certain nutritional requirements of the fowl cestode Raillietiua cesticillus (Molin) as demonstrated by short periods of starvation of t,he host. J. Parasitol. 28, 319-340. SMORODINZEW,I. A., BEBESCHIN, K. W., ASD PAWLOWA, P. I. 1933. BeitrLge zur Chemie der Helminthen. I. Mitt&lung: Die Chemische Zusammensetzung von Taenia saginata. Biothem.
Z. 261,
176-178.
S.~ORODIXZEW,I. A., AND BEBESCHIN, K. W. 1936. Beitrage zur Chemie der Helminthen. Mitt. III. Die Chemische Zusammensetzung des Taenia
soliztm.
J. Biochem.
23,
No.
1, 19-20.
PARASITOLOGY
Studies
on
9,
Hydatigera taeniaeformis. of Larval and Adult C. A.
Department
257-263 (1960)
Hopkins
of Zoology, Uniuersity Science and
(Submitted
and
W.
III. The Worms M.
Content
Hutchison
of Glasgow, Scotland, and The Technology, Glasgow, Scotland
for publication,
Water
30 November
Royal
College
of
1959)
1. The water content of H. taeniaejormi larvae developing in mice, and aduIts from the intestine of cats, has been determined at various ages. 2. In larvae up to 8 months old the water content is 72 f 3%, but in some older strobilocerci it rises to 80% 3. A plot of the water content of different regions down the larvae shows that there is little change until the pseudostrobila is reached. 4. In adult worms the water content gradually increases with age during the first 18 days to about 78-80%. Variation after this period appears to be due to inter- and intra-host factors which are discussed, and not age of the worm. 5. It is concluded that within certain specified age limits a ‘normal’ water content exists and that. variation from this may be used by an experimenter as a criterion of abnormality. 6. Comnarison with the water content of other cestodes is made and the validity of an apparent dichotomy discussed.
The water content as a percentage of the total weight has been recorded for several species of cestode (reviewed by v. Brand, 1952). However, the reports only record the water content of a cestode at one, often unspecified, stage of its life-cycle, or give a figure calculated from a group of worms of different and unknown ages. Such information is of limited use. Analysis figures only become of value when they are associated with a particular stage of development or physiological change, and can be shown to be reproducible. In an earlier paper (Hopkins and Hutchison, 1958) it was shown that the percentage nitrogen content of H. taeniueformis followed a specific pattern during development of the larval and adult worms, and that characteristic “breaks” in the rate of change were associated with major physiological changes. In the present paper we report the results of a similar investigation to determine the mean, and normal variation in, water content of H. taeniaeformis at different’ phases of its life-cycle. An attempt is made to correlate the observed changeswith
other physiological and morphological changes, and to assessthe importance of various factors which influence the variation shown by worms of the same age. MATERIAL
AND
METHODS
Larval H. taeniaeformis were removed from strain A albino mice killed at intervals after infecting. The worms were immediately washed in balanced saline (A = 0.56%) and then pressed lightly between filter paper to remove surface moisture and weighed (fresh weight). In this process the bladder burst and often a small part was lost, but by the 8th week, the earliest stage estimated, the bladder is a negligible fraction of the total. Immediately after weighing, the larvae were placed on porcelain slabs at 105°C and dried for 18 hours, then re-weighed (dry weight). In order to investigate the water content along the length of the strobilocercus, worms were removed from a mouse liver and divided into anterior and posterior portions which were estimated separately for water content. The point’ of division in the 46
257
258
HOPKINS
AND
strobilocerci was varied to form a series in which the dry weight of the posterior portion varied from 5-95s of that of the whole worm. Adult worms were obtained from the small intestine of laboratory infected cats immediately after killing. The worms were treated in the same way as the larvae. RESULTS
1. Water Content During Development of
the Larva in the Liver of a Mouse a. Effect of age of larva. In Fig. 1, the mean dry fresh weight ratio of strobilocerci recovered from mice between 8 to 61 weeks after infecting is plotted, together with the standard deviations where the sample was of five or more worms. The number of worms in the mice varied from 1 to 164; sixty-six of the 75 mice examined had relatively light infections (<20 worms), the other nine had medium or heavy infections. No significant difference was found between the water content of larvae from heavy and light infections. b. The water gradient clown the strobilocercus. Before a comparison could be made between water content of larval and adult worms, it was necessary to determine the water content of not only the whole larva
HUTCHISON
but that part of the larva which survives after ingestion by the cat and gives rise to the adult worm. The amount of the larva that constitutes the true larval strobila as distinct from pseudostrobila depends on the age of the larva, varying from 80 to 30% by weight of the larva (Hutchison, 1959). It was decided, therefore, to combine this investigation with a more general one to dctermine the amount of water in each region, from which could be calculated the amount in whatever fraction constituted the true larval strobila. Figure 2 shows the data as measured and needs some explanation. Forty-six larvae, aged 32 weeks, were each cut into two pieces as described in “Methods.” This procedure of cutting the worms only once instead of dividing the worms by several transverse cuts into small fragments kept weighing errors, and errors due to loss on cutting, to a minimum. However, the results obtained are not in a very useful form until they are transformed mathematically. Looking at Fig. 2 the first impression is that the water content gradually increases as one proceeds posteriorly from the scolex, but this is not reallv so, as can be seen in Fig. 3. In this figure the dry/fresh weight ratio expressed as a percentage has been plotted for every 10% portion of the strobi-
40
0 ;
32 -
2 l-
28-
3z 3
a 0
24 -
12
I 0
4
8
12 AGE
16 20 24 28 32 OF STROEILOCERCUS
FIG. 1. The dry/fresh weight at different ages. l = estimation of a worm ; 0 of 5 or more worms and standard
ratio,
plotted
= means deviation.
36
40
*
I
I
a
I
,
44 48 [WEEKg
.
52
56
60
64
68
H.
taeniaeformis,
as a percentage,
of 24
worms;
l
of larval cut by
vertical
line
= mean
STUDIES
ON
HYDATIGERA
TAENIAEFORMIS.
259
III
32
1 100
RATIO STFi%LO%RC”:~~ D.;:] FIG. 2. The dry/fresh weight ratio of portions of a strobilocercus. The ratio is plotted against the dry weight of the fraction expressed as a percentage of the dry weight of the whole worm. 0 = the mean dry/fresh weight ratio of the 46 complete strobilocerci.
locercus. This was calculated from Fig. 2. Reading from the graph the posterior 10% of the worm had a dry weight content of 13.3% (i.e. the dry weight of the posterior 10% of a worm whose fresh weight is 100 mg would be 1.33 mgi. Similarly the posterior 20% has a dry weight content of 17.3% (i.e. dry matter constitutes 3.46 mg in a worm of 100 mg fresh weight). Subtraction of these two figures, 1.33 from 3.46, shows that the region between 10 and 20% up from the posterior end of the larval strobila has 2.13 mg of dry matter in it, and hence that the dry weight is 21.3% of the fresh weight. This calculation was repeated by taking readings from the graph in Fig. 2 at 10% steps. The transformed data 1)lotted in Fig. 3 show in a much clearer manner the percentage dry matter, and hence water content, of different parts of the strobila. R. Water Content During Development of the Adult Worm in the Intestine of a Cut In view of the variation in the dry/fresh weight ratio in larvae over 30 weeks old
(Fig. 1)) only strobilocerci between 10 and 26 weeks old were used to infect cats. All infections in the cats were light ones of 1 to 6 worms; the average burden was 4. a. Changes associated with increasing age of the worm. The percentage dry matter in adult worms is shown in Fig. 4. The total number of worms estimated at a particular age is shown by the index; where t,he worms were obtained from more than one cat, the individual means, calculated from worms recovered from each cat, are shown as closed points ( l ) , and an overall mean as an open circle (0). 6. Water content of worms of the same age. (i) Variation within a single cut. Variation in water content of worms in a cat were usually small, e.g. the 6 worms in cat 126 (39 day infection, Fig. 4) had a mean of 78.9% water with a range from 78 to 80.1%. The average range in water content of worms was 2.7% in the 26 cats infected with 3 or more worms, aged between 3 to 42 days. Only rarely did the range exceed 4%. (ii) Variation between.worms in different
260
HOPKINS
AND
HUTCHISON
PSEUDOSTROBILA
TRUE
LARVAL
STROBILA
>
. \
TAIL
IO
20
30
IO% FIG. 3. The of strobilocerci.
dry For
weight, further
40
50
60
70
OF
THE
FRACTIONS
expressed explanation
as a percentage see text.
IMMATURE RAPID GROWTH.
of the
80
90
SCOLEX
STROBILA fresh
weight,
of 10%
portions
MATURE SLOW GROWTH.
I
I”
0
4
6
I2
I6
20
24
AGE OF WORM
28
32
36
40
44
IN CAT DAYg
FIG. 4. The dry/fresh weight ratio, plotted as a percentage, oping in the intestine of a cat. l = mean based on worms from a cat. 0 = mean of worms recovered from two or more cats. Total number of worms at each age is shown by the indices.
of H.
taeniaeformis
devel-
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HYDATIGERA
cats. The mean water content of worm groups recovered from different cats sometimes varied considerably (Fig. 4). For instance the means of the five 12-day-old infections ranged from 71.7 to 75.2%, and in the three 27-day-old infections a difference of 7.8% occurred between individual means. This was exceptionally large, but considering all the age groups at which infections from three or more cats were examined, it is apparent that the means at any particular age may vary over a range of about 5%). DISCUSSION
Although larval H. taeniaeformis can be seen and removed from the liver of mice after the second week, the difficulties involved in weighing such smaI1 worms coupled with the difficulty in getting rid of surface moisture made it impossible to obtain an accurate dry/fresh weight ratio before the end of the eighth week. Between the 8th to 30th week, only minor variations in the dry/fresh weight ratio occur, nearly all worms lie in the range 28 * 3% (Fig. 1). If there is any trend during this period it is of a minor order which is concealed by experimental errors and variation that exists between larvae of the same age. After the 30th week there was a greater scatter in means. At first, it was thought that a drift downwards in the dry/fresh weight ratio of the larvae could be detected but there are several exceptions. The increase in water content sometimes occurred in all the larvae, but in other cases only some of the larvae showed a low dry/fresh weight ratio, which resulted in a large standard deviation, e.g. 46 week infection, Fig. 1. It is probable that this increase in water is due to degenerative changes in these older larvae. The fact that it does not appear to bc correlated with a loss of infectivity is not surprising as degenerative changes are most likely to be occurring in the pseudostrobila. This structure forms 50 to 70% of the larva at this age. It is, however, a useless appendage and is lost during the first day in the cat’s intestine (Hutchison, 1959). One of the most interesting stages in the physiological development of a cestode is the switchover from larval to adult metabolisni, which occurs on transfer t,o the defini-
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tive host. When comparing the chemical composition of adult and larval H. taeniaeformis it is, of course, necessary to make the comparison between adult and true larval strobila, not the whole larva. Thus, the mean dry matter content of worms after 1 day in a cat is approximaely 31%, compared with 28% in the larva. Both these figures are based on a large number of observations and although the difference is not very great, a comparison of the data in Figs. 1 and 4 leaves no doubt that it is real. However, the conclusion that there is an increase in percentage dry matter is wrong. Reference to Fig. 3 shows that in a larva having an overall dry weight ratio of 28.40/o, the true strobila has a dry weight ratio of over 31%. The initia1 “rise” in percentage dry weight is, therefore, merely a result of the loss of the pseudostrobila. The extent to which the change from true strobila to pseudostrobila is marked by increased water content cannot be precisely determined, as there is no way, morphologically, of distinguishing the line of demarcation, but Hutchison (1959) records that the posterior 40% approximately is pseudostrobila in larvae of 130 to 150 mg fresh weight (the size of the worms on which the data in Figs. 2 and 3 are based). Figure 3 shows the change which occurs in the water content of the tissue at a point which almost coincides with the expected point of change from true larval strobila to pseudostrobila. In the anterior 60% of the worm the results indicate a slight increase in percentage dry matter in the tissues, proceeding posteriorly from the scolex. Such a rise agrees with the general concepts of biochemical heterauxesis (Needham, 1950), in that8 animal t’issue drys as it ages. In this case, following accepted principles that cestode growth occurs in the neck region, the furt’her the tissue is from the neck the older it is, and hence the drier it, may be expected to be. But, at a point 60% down the worm new controlling factors come into operation which bring about a rapid increase in the water content. It seems reasonable to conclude, therefore, that tissues of the pseudostrobila are physiologically different, as is shown by their inability to withstand the intest,inal environment, and
262
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AND
in having a higher water content. What these factors are which cause this change is not known, but one might speculate that they are degenerative changes leading concomitantly to swelling of the tissues and loss of viability in the definitive host. The problem we have attempted to solve with regard to the water content of adult H. taeniaeformis is whether there is a specific composition at a particular age, and if so, does it change in relationship to age of worm. The principle difficulty in resolving this problem is that variation due to factors other than age may be so large as to mask, or at least make doubtful, changes due to age. If the water content of all H. taeniaeformis recovered is considered, an estimation of the total variance can be made. This can then be broken down into variance due to different factors. Firstly, there is the variation between worms in a single cat, i.e. intra-host variation. This was small, relative to total variation. The average range was 2.7% and only rarely did it exceed 4%. It is due to a number of factors, such as, genetical variation, experimental errors, position of the worms relative to one another in the intestine. We have not attempted to assess the importance of these and other possible factors, but merely refer to the variation between worms within one infection as the “basic variation.” Secondly, there is variation between worms taken from different cats, i.e. interhost variation. This can be assessed by comparing the mean water content of worms recovered from two or more cats, with the same age of infection (Fig. 4). The difference between means is often considerable, and it is apparent that one or more new factors are operating which superimpose their effect on the basic variation. These “inter-host” factors affect all the worms in an infection to a similar extent, as far as can be told from our data. Their effect is not to increase the basic variation but to alter the absolute value of the mean. There are several possible causes for this difference in the dry/fresh weight ratio between urorms from different cats. The most probable cause, in these experiments, was the time the cats were killed in relation to their last
HUTCHISON
meal. It is well established that even a few hours of starvation effects the glycogen content of worms considerably, which causes, to some extent at least, a fall in the dry,/ fresh weight ratio (Reid, 1942; Archer and Hopkins, 1958). It is also possible that variations in the osmotic pressure of the intestine may be partly responsible for changes in the dry/fresh weight ratio, but this is open to more doubt, as mammals maintain very close isotonicity between intestinal contents and plasma. Other factors which may influence the worm population as a whole and lead to inter-host variation are differences such as age and sex of the host, and hence gut diameter, mucus production, hormone secretions etc,. There are also the differences between the strobilocerci fed to each cat, though this was kept small by using larvae of a narrow age range. Thirdly, there is variation in water content due to ageing of the worms. To determine this it is necessary to eliminate as far as possible intra- and inter-host variation at a particular age. Ideally, a large number of cats should be examined at each age of infection, a mean and degree of scatter calculated, and a regression coefficient determined. We have not done this as the object of the investigation was not to determine a statistic but to discover whether an experimenter working with H. taeniaeformis either in vivo or in vitro, could talk about a “normal” water-level and if so, what absolute values might be interpreted as norma or abnormal, and, what degree of variation to expect in a small sample. Examination of the data in Fig. 4 shows that age does have a marked effect, a considerable fall in the dry/fresh weight ratio occurs during the first 3 weeks in the cat. However, whether this is a gradual changeover as implied by the plot is questionable. The figures show that by the end of the first day the dry/fresh ratio is between 30 to 35%, which does not appear to alter appreciably for 2 to 3 days. Between the 5th and 17th day the dry/fresh weight ratio lies between 24 to 29%; after the 18th day and up to the end of the period investigated the ratio lies between 19 to 25%. The water content, therefore, rises during the pre-patent period from an initial level of 68 to 70% to
STUDIES
ON HYDATIGERA
rather less than 80%. It will also be observed that this change from a low larval level to a high adult level takes place during the rapid growth phase (Hutchison, 1959) of the first 18 days. By the time the worms are mature and egg production has commenced the new adult level has been reached. Thereafter variation results only from intra- and inter-host factors discussed above. When these result,s for the dry matter content of adult H. taeniaeformis are compared with those of other adult cyclophyllidean tapeworms it is found that they agree closely with one set of figures (Raillietina cesticillus, 20.5% (Reid, 1942) ; Dipylidium caninum, 20.4$%, Taenia hydatigena (marginata) , 23.5%, Anoplocephala magna (T. plicata), 27.5% (v. Brand, 1933))) but differ very greatly from the other figures (Taenia saginata, 12.2% (Smorodinzew et al., 1933) ; T. solium, 8.7% (Smorodinzew and Bebeschin, 1936) ; Moniexia expansa, about lo%, various authors, see v. Brand (1952) ) . The question arises, is there a genuine dichotomy in the Cyclophyllidea? Examination of the “low group” fails to support such a dichotomy. The figures of Smorodinzew and co-workers for T. saginata and T. solium have no biological significance. The procedures used by these workers have been discussed by Archer and Hopkins (1958) and in retrospect at least it is apparent that they were measuring water content of moribund worms. With regard to the other figures insufficient detail is given about the method used for a critical appraisal, but it seems a noteworthy coincidence that high water figures, characteristic of degenerating tissue, are found in just those h&ninths which come from hosts unlikely to be killed in a laboratory to supply fresh cestodes! It seems fair to conclude, therefore, that there is no reliable evidence that adult cyclophyllidean cestodes in healthy condition ever have such low dry matter content; all recent work suggests a percentage dry matter content of between 20 to 25%. The position with regard to water content, which has long been regarded as showing wide variation in cestodes, is beginning to be reversed. Differences do occur, but when allowance is made for the factors discussed
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in this paper the striking point is the similarity, not the difference. This similarity, however, exists only between cyclophyllidean worms. The data available on pseudophyllideans which mature in warm blooded hosts (summarized by Archer and Hopkins, 1958) show that Diphyllobothrium and allied genera have a dry/fresh weight ratio about 30%, i.e. nearly 50% greater than that of cyclophyllideans. k!KNOWLEDGMENTS
Our thanks arc due to Professor C. M. Ponge in whose Department this work was carried out and to t,he Royal Society (London) for a grant-in-aid. We would also like to thank Professor J. P. Todd of the School of Pharmacy for kindly providing laboratory facilities for one of US (W.M.H.) at the R.C.S.T., Glasgow. REFERENCES
AHCHER, D. M., AND HOPKISS, C. A. 1958. Studies on Cestode Metabolism. V. The chemical composition of Diphyllobothrium sp. in the pleroccrcoid and adult stages. Exptl. Parasitol. 7, 542-554.
vos BRAND, T. 1933. Untersuchungen iiber den Stoffbestand einiger Cestoden und den Stoffwechse1 von Moniezia expansn. Z. ve~gleich. Physid.
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VON BR~XD, T. 1952. Chemical Physiology of Endoparasitic Animals. $cademic Press Inc., Kew York. HOPKINS, C. A., ASD HUTCHISON, W. M. 1958. Studies on Cestode Metabolism. IV. The nit,rogen fraction in the large cat tapeworm, Hyrlatigera (Taenin) taeniaeformis. Exptl. Pnrasitol.
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HUTCHISOS, W. M. 1959. Studies on Hydatigera (Tnenin) tacniaeformis. II. Growth of the adult phase. Exptl. Parasitol. 8, 557-567. NEEDHA~W,J. 1950. Biochemistry and Morphogenesis. Cambridge University Press, England. REID, W. M. 1942. Certain nutritional requirements of the fowl cestode Raillietiua cesticillus (Molin) as demonstrated by short periods of starvation of t,he host. J. Parasitol. 28, 319-340. SMORODINZEW,I. A., BEBESCHIN, K. W., ASD PAWLOWA, P. I. 1933. BeitrLge zur Chemie der Helminthen. I. Mitt&lung: Die Chemische Zusammensetzung von Taenia saginata. Biothem.
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