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Studies on Schistocephalus solidus. I. The correlation of development in the plerocercoid with infectivity to the definitive host
ORIGINAL EXPERIMENTAL
PARASITOLOGY
13, 235-243
RESEARCH
PAPERS
(1963)
Studies on Schistocephalus solidus. I. The of Development in the Plerocercoid with to the Definitive Host C. Adrian
Hopkins
Department (Submitted
of Zoology,
and
Morag
Glasgow
for publication,
L.
University, 29 January
0.
Correlation Infectivity
McCaig
Scotland 1962)
Histological examination of plerocercoids showed traces of segmentally arranged genital primordia preceded external signs of segmentation. The various genital organs become apparent in plerocercoids between 3-19 mg in weight. Plerocercoids weighing as little as 6 mg are capable of becoming established in ducklings under 4 weeks old. Establishment was 3% in plerocercoids below lOmg, 40% in worms from IO-30 mg, and 80% in worms > 30 mg. A lower percentage at all sizes became established in pigeons. In ducklings (rectal temperature 42°C) egg production commenced within 48 hrs in worms down to 10 mg in weight; below 10 mg maturation was incomplete in 48 hrs. Similar development was found in pigeons, but in mice (rectal temperature 365°C) development was slower, eggs were found only in worms > 23 mg. Schistocephahs loses weight in a duckling, about 10% in the first 48 hrs and 20% in 96 hrs. The interrelationship of infectivity, size of plerocercoid and development of the genitalia is discussed.
The life-cycle of Schistocephalus solidus involves several phases, a procercoid stage in the hemoceleof a copepod, a plerocercoid stage in the perivisceral cavity of a fish (usually Gasterosteusaculeatus), and a short adult life. Maturation occurs in 48 hours, the adult rarely surviving more than two weeks in a warm blooded host. In recent years a considerable amount of information about the adult stage has been obtained by Smyth, who succeeded in inducing plerocercoids to mature in vitro and produce fertile eggs (Smyth, 1954, 1959). The next step to the ultimate goal of carrying the life-cycle through completely in vitro, under known chemical and physical conditions, is the cultivation of the larval stages.In the course of culturing the plerocercoid, certain problems have arisen. 235
Plerocercoids of Schistocephalusmay grow to at least 200-300mg, but it is not at all certain how much development is essential within the fish before the plerocercoid becomescapable of maturing at 40°C. It would appear, however, to be quite small, since Clarke (1952) reported an instance of a 12 mg plerocercoid maturing and producing eggs in the perivisceral cavity of a mouse. The experiments described in the first section of this paper are an amplification of Clarke’s work, aimed at determining the weight at which plerocercoids become infective. It was soon found that the infectivity of small plerocercoids was greatly reduced by removal from the fish and weighing, before feeding to the final host (McCaig and Hopkins). It was necessary, therefore, to feed whole fish. This introduced the problem
236
HOPKINS
AND
MC CAIG
of the relationship between the weight of cut at 8~ and stained in Heidenhain’s hemathe worm found in the intestine and the toxylin; and by staining the remaining weight of the plerocercoid from which it had anterior and posterior parts in catechol developed (section 2). (Johri and Smyth, 1956). Infectivity is not necessarily the same as Pigeons and ducklings were infected by ability to mature, and the third section force feeding infected fish or plerocercoids. reports a histological examination of worms, Intra-celomic infection of mice was carried carried out to determine the extent to which out by laparotomy using pento-barbitone infectivity and ability to produce eggs and (Nembutal) as anaesthetic. sperm are synchronized. RESULTS Lastly, having determined the approximate size at which plerocercoids are infective and 1. The Effect of Size of Plerocercoid able to mature, an investigation was made to on Infectivity see whether these stages could be correlated Table I shows the weight of worms rewith the development of any part of the covered 48 hrs after infecting ducks and genital primordia in the plerocercoid (secpigeons. The purpose of the experiment was tion 4). to discover what relationship existed between MATERIAL AND METHODS size, and ability to become established in Schistoceph’alus solidus plerocercoids were the intestine. The results indicate that worms between 10 and 30 mg have only about found in sticklebacks (Gasterosteus aculeatus) occurring in a reservoir 10 miles north half the chance of becoming established as those over 30 mg, and plerocercoids of less of Glasgow. The fresh weight of worms was measured than 10 mg rarely become established. by weighing in metal foil to prevent moisture In the case of birds fed intact fish, the loss. Weighing errors of up to I+ 1 mg percentage recovery was estimated. This was occurred, which were of little significance in done by dissecting l/3 of the fish caught, plerocercoids above 20 mg, but introduced and recording the size and number of pleromore substantial errors when working with cercoids. From this the approximate number very small plerocercoids. and weight of the plerocercoids fed in 72 Histological development was determined fish to pigeons, and in 120 fish to ducks, was by examining serial sections of proglottids calculated. from a region l/2-2/3 down the strobila, A minor correction was applied by exTABLE The
Numbers
of
Worms,
of
Inoculation,
Host
NO.
Different Shown
Infective dose
Weight
Groups,
as Q Proportion
of the
from
Definitive
Hosts
of Plerocevcoids
Number
No. of worms
recovered/No.
Two
Days
Fed
plerocercoids
fed
10 mp
IO-19 mg
20-29 mg
30 mg
2/16 1370
S/23 22%
23/53 44%
12/27 447%
16/38 42%
74/88 84%
6
12 fish
o/40
Ducka
10
12 fish
2/67 3%
Duckb
5
plerocercoids (see Table 2)
O/7
Pigeon”
I Recovered
0%
0%
0 The number of plerocercoids fed is an estimate, b Includes two ducks killed 96 hr after infecting.
see text.
l/5 rzo%l
2/6 33%
18/21 86%
after
STUDIES
ON
SCHISTOCEPHALUS
pressing the number of adult worms recovered in the four weight categories < 10, 10-19, 20-29 and > 30mg as a percentage of the estimated number of plerocercoids in the < 11, 11-21, 22-33 and > 33mg weight categories. This was done to allow for the loss of approximately 10% in weight which occurs during 48 hr development in the duck (see next section). 2. Weight Change in Schistocephalus solidus during Development in a Definitive Host Table II shows the weight of plerocercoids fed to five ducklings, and the weight (column 4) of the worms recovered. The percentage change shown in column 5 is only an indication of the order of weight change to be expected. Where the adult worm recovered could not be identified with a specific plerocercoid but only with a weight group, the percentage change was calculated by comparing with the mean weight of the plerocercoids fed in that group, e.g. the 24 mg worm in Duck 1 was calculated as having lost
The Bird no.
Change
in Weight
Hours in duck
1
of plerocercoids fed 7 25
45
3
-
75
-6 -1
21
21
22
63 100
63 103
64
12
13
44
45
77
81 85
73
79
6 8 63 64 65
46
50
5
-
26 55
~.-
lost.
68
53
18 53 55 60 96
58d
61
-16 -13 -5
40
16
of worm
Change in weight (%)
73
25
portion
Development
-6
96
-
96Hour
Wt. of adult worms recovered
78
65
53
-10 -5
56
-19
29
Will
Table III summarizes the development seen by examining serial transverse sections of proglottids. For details of the histology of Schistocephalus plerocercoid and adult, as seen in transverse section, see Smyth (1946)) and Hopkins and Smyth (1951). The weight of the worms recovered from the perivisceral cavity of mice, or intestine of birds, is shown
32 73 55d
62
d = damaged
to Which Small Worms Mature in 48 Hours
24
96
5
3. Degree
3
45
4
1.5 mg, and the 18 mg worm in duck 2 as having lost 3.3 mg. The average loss in weight of the 13 intact worms recovered after 45 hrs was 970, and of the 6 worms recovered after 96 hrs, 20%. The change in weight of Schistocephalus in the perivisceral cavity of mice also appears to be small and probably indicative of a slight loss. Of five plerocercoids, weighing 19, 12.4, 11, 6.6 and 4.9 mg respectively, inserted individually into the perivisceral cavity of mice, the weights on recovery 48 hrs later were 18.2, 12.5, 9.2, 4.8 and 4.0mg. The 4 mg worm was degenerating.
26
34 73 2
237
I.
TABLE II of Schistocephalus solidus after 4.5- and in a Dejinitive Host, the Duck Wt.
45
SOLIDUS.
21
-21
43
-22
238
HOPKINS
Level
of Genital
Development after
Weight mg
plug
Seminal vesicle Pr cirrus
Mouse 33 Mouse 11 Duck 57 Mouse 66 Duck 58 Duck 59 Mouse 29165 Pigeon 3 Mouse 68/32 Pigeon 7 Mouse 67
1.3 3.3 6 9 9 10.5 12/13 13 14/15 17 18
C C 0 0 0 0 0 0 0 0 0
0 0 C C + + + + + + +
Mouse
23/29
0
+
10/9
MC
column
2. Weights
of
the
48-hour
adults
are probably slightly less than the weights of the corresponding plerocercoids (see section 2). The letters YF in column 9 indicate that some of the cells in the vitelline follicles have matured, enlarging considerably in the process, with the deposition of yolk and shell material in their cytoplasm. Such cells, instead of showing only a darkly staining nucleus, have a large number of basophil granules. (For a full description of the maturation of the vitelline cells in Schistocephalus, see Smyth (1956).) In mice, worms of 23 mg and over produced sperm and eggs. The 18 mg worm showed a borderline condition; the testes contained a few sperm, the seminal vesicle and uterus were large hollow, but empty, ducts opening by the genital pores, and a few cells in the vitelline follicles had matured. The 12-15 mg worms were less well developed. Although the seminal vesicle, cirrus sac and uterus were identifiable, and in the 14 and 1.5mg worms lumina had formed in these ducts, no sperm or mature vitelline cells had been produced. As was to be expected the worms gave a negative reaction with
solidus Hosts
of Different
Weights
d&0 pore
Testes
Ovary
0 0 C C + + + + + + +
0 0 0 0 0 + 0 t + + +
0 l-2 6 14 16 S R S R S S
0 0 + + + + + + + + +
0 0 ? ? l-3 YF 1-5 YF 4-9 YF YF
0 0 0 0 0 + 0 + 0 + 0
+
+
S
+
YF
+
Uterus
C = concentration of nuclei; R = spermatocyte rosettes; vitelline cells (see text) ; 0 = absent; + = present; numerals testis and vitelline follicles; genital plug-see text, section 4. in
CAIG
TABLE III in “Adult” Schistocephalus 48 hr in Various Laboratory
Host and reference no.
Genital
AND
Vitellaria
Eggs
S = sperm; YF = follicles containing mature = the average number of cells in sections of
catechol, which showed the absence of polyphenol oxidase, an enzyme associated with shell formation, present in mature vitelline cells (Johri and Smyth, 1956). In the 9 mg worm, apart from some multiplication of the testicular cells, the only signs of “adult development” was the differentiation of the central mass of cells into two concentrations of nuclei, identifiable as the male and uterine duct, and the appearance of the ovary as a thin ribbon of cells. Below 9 mg, development was restricted to the appearance of testes consisting of one or two cells. The 1.3 mg worm was alive but enmeshed by host cells and probably degenerating. Development of small worms in pigeons could not be studied, as worms less than 13 mg were not found (Table I). The 13 mg worm was fully mature, except that it, like the 17 mg worm, had nearly 20 terminal proglottids without eggs, instead of the usual 1 or 2 proglottids without eggs which often occurs in larger worms. Smaller worms were found in ducks and the important point shown in Table III is that the transition from infective but incapable of maturing in 48 hours, to infective
STUDIES
Level wt. mg.
of Genital Genital plug
ON
TABLE in Schistocephalus
Development Seminal vesicle and cirrus
SCHISTOCEPHALUS
Uterus
SOLIDUS.
IV solidus
239
I.
of Different
Plerocercoids
Uterine and 8 Pores
Testes
Weights
Vitellaria
Ovary
Ref.
3.1
C
0
0
0
0
0
0
6.5
+
0
0
0
0
0
0
Feb. S 36 Feb.
9.0
+
0
0
0
l-3
0
0
s 34/5 Oct.
11
C
C
0
8
0
1
Nov.
12
+
+
M
10
+
1
Nov.
14
+
+
D
13
+
2
Nov.
19
+
+
+
18
+
4
S 26 Nov.
20
+
+
+
25
+
5-10
24
+
+
+
30
+
8
+
50
+
S 64 s 20 s 22
s 25 Nov. s 24
+
230
With
For
Column other
5 -ducts abbreviations
lumen
+
With
Aug. s 50 Sl
30
lumen
extending out to muscle see Table III.
layer
and capable of maturing, occurs between 6 and 10 mg. The 10.5 mg worm (Duck 59) was clearly on the borderline, the uterus containing only a few, 2-20 eggs. In some proglottids the pore of the male opening did not appear to have been formed, however, small massesof sperm were found in the seminal receptacula of these proglottids.
(M),
to sub-cuticular
layer
(D),
to surface
pore
(+).
The numbers have little precise significance but indicate trends. The smallest (3.1 mg) plerocercoid sectioned shows faint traces of densely nucleated cells forming a central mass. These cells are arranged in segmental blocks. This segmental distribution precedes any external sign of proglottid formation (cf. Proteocephah fiZicoZZis,Hopkins (1959)). Traces of 4. Development of Genitalia in Plerocercoids tubular organization and clusters of cells The levels of development of the genital forming testes can be seenas early as the 11 primordia in plerocercoids of different weight mg plerocercoid and by 19 mg the cells of the are shown in Table IV. The term genital plug central mass have differentiated to form the is used to denote the massof densely nucleat- uterus, seminal vesicle, cirrus etc. The numed cells in the mid line which later differen- ber of testes visible in a section of a 19 mg tiate to form various genital structures; it is plerocercoid is similar to that in larger plerothe most obvious structure in a transverse cercoids, e.g. a 230 mg plerocercoid. Although section of a small plerocercoid. In order to in- thl4 method of estimating the number of testroduce some quantitative criteria the ap- tes is open to several sourcesof error, it sugproximate size of the vitellaria and testeswas gests the number of follicles does not indetermined by counting the number of cells crease. If this is so it appears that after the forming a follicle, as seen in an 8 p section. plerocercoid has grown to 19 mg, develop-
240
HOPKINS
ment of the genitalia is restricted to an increase in size of the existing primordia, and the formation of lumina in the ducts. The testes are the first genital organs to be clearly identifiable; situated ventrally to the dorsal transverse muscle fibres they are easily distinguishable from the surrounding parenchymatous cells. By the 14 mg stage the vitelline cells are identifiable as small groups of 2-3 cells; earlier identification is difficult as many nuclei are found in the cortical region outside the longitudinal muscle bundles. It is equally difficult to determine precisely the stage at which an ovary may be said to exist. In a small plerocercoid the ovarian primordium is indistinguishable from the central mass of cells and it is only as this central mass differentiates that a thin row of cells becomes apparent on the ventral side. The time of year the plerocercoid was collected is shown (Column 9, Table IV.) This has been done because plerocercoids grow quicker in summer than winter and no evidence exists to indicate whether the time taken to reach a certain size is also important in determining the level of differentiation. DISCUSSION
The development of the genital primordia in plerocercoids of Schistocephalus solidus (Table IV) may be summarised as follows: (i) the process commences at a very early stage; (ii) by 19 mg all the genitalia are formed; (iii) after 19 mg only quantitative changes occur (i.e. testes, vitelline follicles, etc., increase in size) ; (iv) as in tapeworms in which genital development is confined to the final host the male system appears first. The three principal variables to which differentiation may be correlated are weight, age, and region of the strobila. This last point is critical in very small plerocercoids as a development gradient extends both anteriorly and posteriorly from a central region in the strobila. Thus, in an adult of about 10 mg, only the central proglottids produce eggs. The extent to which age, as distinct from
AND
MC
CAIG
size or weight, affects the level of differentiation has not been determined, as the plerocercoids were obtained from a natural population in which the age was not known. The precise relationship of development with size and age can only be determined by using plerocercoids reared in laboratory-infected fish under controlled conditions. Two principal difficulties were encountered in investigating the capability of plerocercoids to mature. Firstly, plerocercoids of the critical size, i.e. < 20 mg are unlikely to become established, especially if they are removed from the fish before feeding to a duck or pigeon (Table I). Secondly, while plerocercoids of < 1 mg were available in our supply near Glasgow, (this being the overwintering size range in most fish), worms of 5-20 mg on the contrary were scarce, as the majority of plerocercoids passed quickly through this size range in early summer. To overcome the first of these difficulties the plerocercoids were matured in the perivisceral cavity of mice (Clarke, 1952). Using this technique, eggs and sperm were formed in plerocercoids of 23 mg and over within 48 hours. Smaller plerocercoids showed progressively less development. The levels of genital differentiation in a few are shown in Table III. In contrast to Clarke (1952) who found that small worms (8 and 3 mg) disintegrated, active worms down to 3.3 mg were found. Nevertheless, since they rapidly become enmeshed by host tissue, 48 hours would appear to be about the maximum time small plerocercoids can survive in the perivisceral cavity. The minimum size of worm producing eggs agrees fairly closely with that found by Clarke (1952) except that he found eggs in a 1.5 mg worm, whereas we failed to obtain eggs in worms less than 23 mg (Table III). From these experiments it was inferred that worms of 23 mg and upwards, provided they become established, can be expected to mature in 48 hours in the definitive host. From Smyth’s (1952) observations 48 hrs at 36.5
STUDIES
0~
~CHISTOCEPHALUS
C [rectal temperature of a mouse (Spector, 1956)] would appear to be the minimum time necessary even for large plerocercoids to mature. This is an interesting point as it suggests the level of development of the genitalia is essentially the same in a 23 mg plerocercoid as in larger plerocercoids. It was found later that worms down to 10 mg could mature in ducklings. The reason why smaller plerocercoids should mature, in 48 hrs, in a duck than in a mouseis probably merely a temperature effect. The rectal temperature of a duck is 41.5 C (Spector, 1956), five degreeshigher than that of a mouse.The hnding of mature worms down to 10 mg indicates that the genital primordia, even in such small plerocercoids, are capable of maturing in responseto a rise in temperature. However, as was to be expected from the weak development of genitalia in plerocercoids of that size (Table IV), only the most developed proglottids in the centre of the strobila matured within 48 hours. Although worms of lessthan 10 mg did not produce eggsand sperm in 48 hours, it is still clear that the rise in temperature evoked a switch to adult development. Thus the testes in the 9 mg worms (M66 and D58, Table III) consisted of about 16 cells in section. Such a size would only be reached in plerocercoids of about 19 mg (Table IV). The effect of temperature on development is therefore far more profound than can be accounted for by a QIO effect. Even in small plerocercoids weighing as little as 6 mg, a rise in temperature causesa switch from a metabolic pathway in which size of genitalia increasesconcomitantly with somatic tissue, to a metabolic pathway in which somatic growth ceases and only genital development takes place (Table II). (It is not wished to anticipate here a more detailed consideration of growth changes in Schistocephalus which is in preparation, but the interpretation of the datum in Table 11 as indicating a cessationof somatic growth in all or nearly all of the worm is based on two
SOLIDUS.
I.
241
reasons. Firstly, the dry weight of plerocercoids of 40-70 mg increasesby at least lo20% in 48 hours at 22’ C; at 40” C an even greater increase in body weight would be espetted unless growth is inhibited. Secondly, if it is assumed that no growth has taken place at 40°C and allowance is made for a glycogen loss similar to that which occurs in Schistocephalus during 48 hours in the pigeon’s intestine (Hopkins, 1950), and for the small loss in water which occurs (unpublished)! the calculated weight of the worm after 48 hours is similar to that found experimentally (Table II) ) . Furthermore, since worms down to 6 mg have been found in the intestine, plerocercoids of this weight must in addition to switching their metabolism, be capable of responding to the stimulus of the gut environment and triggering off their 5etention mechanism” to counteract peristaltic pressure. The fact that few worms of lessthan 10 mg were found (Table I) indicates that factors, other than the intrinsic ability of a plerocercoid to trigger off a retention response, are involved in determining whether a plerocercoid will become established. These extrinsic factors will be discussedelsewhere (McCaig and Hopkins). In general, the decreasing percentage of plerocercoids to become establishedas the weight falls below 30 mg suggests a progressively less favorable host/parasite relationship. Below 6 mg, conditions are so unfavorable, apparently, as to make establishment highly improbable. However, worms below 6 mg while incapable of retaining their position in the intestine might still be intrinsically capable of responding to the external stimulus which evokes the switch from plerocercoid to adult development and behavior. The point at which a pIerocercoid becomescapable of responding and altering its metabolism is presumably a constant depending on its genetical constitution, but the point at which a plerocercoid becomes infective appears to be a variable depending on the host environment. Compare
242
HOPKINS
AND
MC
CAIG
for instance the “take” in pigeons with that in ducks (Table I). The host/parasite relations are less favorable to Schistocephalus in pigeons at all stages, and the threshold below which it becomes improbable that plerocercoids will become established is higher than in ducks. In summary, entry into the duck’s intestine initiates the following changes in a plerocercoid:
(i)
the cessation (Table II) ;
of
somatic
(ii)
the stimulation of a response mechanism by the worm to stay in the intestine, which is effective in worms over 30 mg, less effective between 6-30 mg, and ineffective below 6 mg (Table I) ;
DEVELOPMENT
IN
PLEROCERCOID
DEVELOPMENT IN
ADULT
growth
(iii)
in worms over 10 mg, the stimulation of maturation with egg and sperm production occurring within 48 hours (Table III) ; or
(iv)
in worms of 6-10 mg, the stimulation of maturation, but without development progressing to egg and sperm production.
The course of development and suggested control mechanisms are expressed diagrammatically in Fig. 1. The weights indicate the size of the plerocercoids in the two phases. 0.3 micrograms is an estimate of the weight of a plerocercoid immediately after its establishment in the fish host. Synthesis of both somatic and genital tissue occurs in the plerocercoid. In the case of the genitalia, as has been discussed, there are two phases; the qualitative, during which the various genital rudiments become differentiated, and the quantitative, during which the genital rudiments increase in size but show little further progress towards maturation. The final phases of oogenesis, vitellogenesis and spermatogenesis are inhibited at low temperatures and can only take place when stimulated by a high temperature (Smyth, 1952). It is gener-
FIG.
1.
cephalus
The
solidus
pattern during
of the
development in Schistoplerocercoid and adult
phases. ally accepted that with the onset of maturation somatic growth ceases (Wardle and McLeod, 1952, p. 563). This is the case in worms developing in ducks (Table II), and probably in worms maturing in the perivisceral cavity of mice (section 2, Results). Whether this cessation of growth occurs as a result of an internal hormonal mechanism acting as a feed-back from the maturing genitalia, or because the environment does not contain suitable metabolites, or for some other reason, can only be determined by further experiment. To return to the problems posed in the Introduction, plerocercoids become infective to ducklings and pigeons between 6-l 5 mg, but there is no reason to believe that in some other hosts even smaller worms could not become established. The minimum obligate amount of development which must occur in the fish is therefore small. Infectivity and the ability to produce eggs appears to be nearly synchronized. This synchronization, however, is not necessarily evidence of a direct physiological link. More probably it arises from genetical selection pressure, in that it is advantageous to the species that a plerocercoid should be capable of maturing as soon as it is infective. The “target” in the plerocercoid which on
STUDIES
ON
SCHISTOCEPHALUS
stimulation initiates the retention response mechanism and hence brings about infectivity, exists in the plerocercoid at a very early stage. It does not appear to be a genital structure, but, as will be discussed elsewhere (McCaig and Hopkins), is part of, or controlled by, the scolex. ACKNOWLEDGMENTS
The authors gratefully assistance given by Miss mond Stoddart.
acknowledge Beryl Tagg
the technical and Mr. Ray-
REFERENCES
A. S. 1952. Maturation of the plerocercoid of the pseudophyllidean cestode Schistocephalus solidus in alien hosts. Experimental Parasitology 2, 223-229. HOPKINS, C. A. 1950. Studies on cestode metabolism. 1. Glycogen metabolism in Schistocephalus solidus in vivo. Journal of Parasitology 36, 384-390. HOPKINS, C. A. 1959. Seasonal variations in the incidence and development of the cestode PYOteocephalus filicollis (Rud. 1810) in Gasterosteus aculeatus (L. 1766). Parasitology 49, 529-542. HOPKINS, C. A., AND SMYTH, J. D. 1951. Notes on the morphology and life history of Schistocephalus solidus. Parasitology 41, 283-291. JOIIRI, L. N., AND SMYTH, J. D. 1956. A histoCLARKE,
SOLIDUS.
I.
243
chemical approach to the study of helminth morphology. Parasitology 46, 107-116. MCCAIC, M. L. O., AND HOPKINS, C. A. Studies on Schistocephalus solidus. 2. Establishment and longevity in the definitive host. (In preparation). SMYTH, J. D. 1946. Studies on tapeworm physiology. I. The cultivation of Schistocephalus solidus in vitro. Journal of Experimental Bio!ogy 23, 47.70. SMUT-H, J. D. 1952. Studies on tapeworm physiology. VI. Effect of temperature on the maturation in vitro of Schistocepkalus solidus. Journal of Experimental Biology 29, 304-309. SMYTH, J. D. 1954. Studies on tapeworm physiology. VII. Fertilization of Schistocephalus solidus in vitro. Experimental Parasitology 3, 64-71. SMYTH, J. D. 1956. Studies on tapeworm physiology. IX. A histochemical study of egg-shell formation in Schistocephalus solidus (Pseudophyllidea) Experimental Parasitology 5, 519540. SMYTH, J. D. 1959. Maturation of larval pseudophyllidean cestodes and strigeid trematodes under axenic conditions; the significance of nutritional levels in platyhelminth development. Annals of the New York Academy of Science 77, 102-12s. SPECTOR, W. S. 1956. Handbook of Biological Data. W. B. Saunders, Philadelphia and London. WARDLE, R. A., AND MCLEOD, J. A. 1952. The Zoology of Tapeworms. The University of Minnesota Press, Minneapolis.
PARASITOLOGY
13, 235-243
RESEARCH
PAPERS
(1963)
Studies on Schistocephalus solidus. I. The of Development in the Plerocercoid with to the Definitive Host C. Adrian
Hopkins
Department (Submitted
of Zoology,
and
Morag
Glasgow
for publication,
L.
University, 29 January
0.
Correlation Infectivity
McCaig
Scotland 1962)
Histological examination of plerocercoids showed traces of segmentally arranged genital primordia preceded external signs of segmentation. The various genital organs become apparent in plerocercoids between 3-19 mg in weight. Plerocercoids weighing as little as 6 mg are capable of becoming established in ducklings under 4 weeks old. Establishment was 3% in plerocercoids below lOmg, 40% in worms from IO-30 mg, and 80% in worms > 30 mg. A lower percentage at all sizes became established in pigeons. In ducklings (rectal temperature 42°C) egg production commenced within 48 hrs in worms down to 10 mg in weight; below 10 mg maturation was incomplete in 48 hrs. Similar development was found in pigeons, but in mice (rectal temperature 365°C) development was slower, eggs were found only in worms > 23 mg. Schistocephahs loses weight in a duckling, about 10% in the first 48 hrs and 20% in 96 hrs. The interrelationship of infectivity, size of plerocercoid and development of the genitalia is discussed.
The life-cycle of Schistocephalus solidus involves several phases, a procercoid stage in the hemoceleof a copepod, a plerocercoid stage in the perivisceral cavity of a fish (usually Gasterosteusaculeatus), and a short adult life. Maturation occurs in 48 hours, the adult rarely surviving more than two weeks in a warm blooded host. In recent years a considerable amount of information about the adult stage has been obtained by Smyth, who succeeded in inducing plerocercoids to mature in vitro and produce fertile eggs (Smyth, 1954, 1959). The next step to the ultimate goal of carrying the life-cycle through completely in vitro, under known chemical and physical conditions, is the cultivation of the larval stages.In the course of culturing the plerocercoid, certain problems have arisen. 235
Plerocercoids of Schistocephalusmay grow to at least 200-300mg, but it is not at all certain how much development is essential within the fish before the plerocercoid becomescapable of maturing at 40°C. It would appear, however, to be quite small, since Clarke (1952) reported an instance of a 12 mg plerocercoid maturing and producing eggs in the perivisceral cavity of a mouse. The experiments described in the first section of this paper are an amplification of Clarke’s work, aimed at determining the weight at which plerocercoids become infective. It was soon found that the infectivity of small plerocercoids was greatly reduced by removal from the fish and weighing, before feeding to the final host (McCaig and Hopkins). It was necessary, therefore, to feed whole fish. This introduced the problem
236
HOPKINS
AND
MC CAIG
of the relationship between the weight of cut at 8~ and stained in Heidenhain’s hemathe worm found in the intestine and the toxylin; and by staining the remaining weight of the plerocercoid from which it had anterior and posterior parts in catechol developed (section 2). (Johri and Smyth, 1956). Infectivity is not necessarily the same as Pigeons and ducklings were infected by ability to mature, and the third section force feeding infected fish or plerocercoids. reports a histological examination of worms, Intra-celomic infection of mice was carried carried out to determine the extent to which out by laparotomy using pento-barbitone infectivity and ability to produce eggs and (Nembutal) as anaesthetic. sperm are synchronized. RESULTS Lastly, having determined the approximate size at which plerocercoids are infective and 1. The Effect of Size of Plerocercoid able to mature, an investigation was made to on Infectivity see whether these stages could be correlated Table I shows the weight of worms rewith the development of any part of the covered 48 hrs after infecting ducks and genital primordia in the plerocercoid (secpigeons. The purpose of the experiment was tion 4). to discover what relationship existed between MATERIAL AND METHODS size, and ability to become established in Schistoceph’alus solidus plerocercoids were the intestine. The results indicate that worms between 10 and 30 mg have only about found in sticklebacks (Gasterosteus aculeatus) occurring in a reservoir 10 miles north half the chance of becoming established as those over 30 mg, and plerocercoids of less of Glasgow. The fresh weight of worms was measured than 10 mg rarely become established. by weighing in metal foil to prevent moisture In the case of birds fed intact fish, the loss. Weighing errors of up to I+ 1 mg percentage recovery was estimated. This was occurred, which were of little significance in done by dissecting l/3 of the fish caught, plerocercoids above 20 mg, but introduced and recording the size and number of pleromore substantial errors when working with cercoids. From this the approximate number very small plerocercoids. and weight of the plerocercoids fed in 72 Histological development was determined fish to pigeons, and in 120 fish to ducks, was by examining serial sections of proglottids calculated. from a region l/2-2/3 down the strobila, A minor correction was applied by exTABLE The
Numbers
of
Worms,
of
Inoculation,
Host
NO.
Different Shown
Infective dose
Weight
Groups,
as Q Proportion
of the
from
Definitive
Hosts
of Plerocevcoids
Number
No. of worms
recovered/No.
Two
Days
Fed
plerocercoids
fed
10 mp
IO-19 mg
20-29 mg
30 mg
2/16 1370
S/23 22%
23/53 44%
12/27 447%
16/38 42%
74/88 84%
6
12 fish
o/40
Ducka
10
12 fish
2/67 3%
Duckb
5
plerocercoids (see Table 2)
O/7
Pigeon”
I Recovered
0%
0%
0 The number of plerocercoids fed is an estimate, b Includes two ducks killed 96 hr after infecting.
see text.
l/5 rzo%l
2/6 33%
18/21 86%
after
STUDIES
ON
SCHISTOCEPHALUS
pressing the number of adult worms recovered in the four weight categories < 10, 10-19, 20-29 and > 30mg as a percentage of the estimated number of plerocercoids in the < 11, 11-21, 22-33 and > 33mg weight categories. This was done to allow for the loss of approximately 10% in weight which occurs during 48 hr development in the duck (see next section). 2. Weight Change in Schistocephalus solidus during Development in a Definitive Host Table II shows the weight of plerocercoids fed to five ducklings, and the weight (column 4) of the worms recovered. The percentage change shown in column 5 is only an indication of the order of weight change to be expected. Where the adult worm recovered could not be identified with a specific plerocercoid but only with a weight group, the percentage change was calculated by comparing with the mean weight of the plerocercoids fed in that group, e.g. the 24 mg worm in Duck 1 was calculated as having lost
The Bird no.
Change
in Weight
Hours in duck
1
of plerocercoids fed 7 25
45
3
-
75
-6 -1
21
21
22
63 100
63 103
64
12
13
44
45
77
81 85
73
79
6 8 63 64 65
46
50
5
-
26 55
~.-
lost.
68
53
18 53 55 60 96
58d
61
-16 -13 -5
40
16
of worm
Change in weight (%)
73
25
portion
Development
-6
96
-
96Hour
Wt. of adult worms recovered
78
65
53
-10 -5
56
-19
29
Will
Table III summarizes the development seen by examining serial transverse sections of proglottids. For details of the histology of Schistocephalus plerocercoid and adult, as seen in transverse section, see Smyth (1946)) and Hopkins and Smyth (1951). The weight of the worms recovered from the perivisceral cavity of mice, or intestine of birds, is shown
32 73 55d
62
d = damaged
to Which Small Worms Mature in 48 Hours
24
96
5
3. Degree
3
45
4
1.5 mg, and the 18 mg worm in duck 2 as having lost 3.3 mg. The average loss in weight of the 13 intact worms recovered after 45 hrs was 970, and of the 6 worms recovered after 96 hrs, 20%. The change in weight of Schistocephalus in the perivisceral cavity of mice also appears to be small and probably indicative of a slight loss. Of five plerocercoids, weighing 19, 12.4, 11, 6.6 and 4.9 mg respectively, inserted individually into the perivisceral cavity of mice, the weights on recovery 48 hrs later were 18.2, 12.5, 9.2, 4.8 and 4.0mg. The 4 mg worm was degenerating.
26
34 73 2
237
I.
TABLE II of Schistocephalus solidus after 4.5- and in a Dejinitive Host, the Duck Wt.
45
SOLIDUS.
21
-21
43
-22
238
HOPKINS
Level
of Genital
Development after
Weight mg
plug
Seminal vesicle Pr cirrus
Mouse 33 Mouse 11 Duck 57 Mouse 66 Duck 58 Duck 59 Mouse 29165 Pigeon 3 Mouse 68/32 Pigeon 7 Mouse 67
1.3 3.3 6 9 9 10.5 12/13 13 14/15 17 18
C C 0 0 0 0 0 0 0 0 0
0 0 C C + + + + + + +
Mouse
23/29
0
+
10/9
MC
column
2. Weights
of
the
48-hour
adults
are probably slightly less than the weights of the corresponding plerocercoids (see section 2). The letters YF in column 9 indicate that some of the cells in the vitelline follicles have matured, enlarging considerably in the process, with the deposition of yolk and shell material in their cytoplasm. Such cells, instead of showing only a darkly staining nucleus, have a large number of basophil granules. (For a full description of the maturation of the vitelline cells in Schistocephalus, see Smyth (1956).) In mice, worms of 23 mg and over produced sperm and eggs. The 18 mg worm showed a borderline condition; the testes contained a few sperm, the seminal vesicle and uterus were large hollow, but empty, ducts opening by the genital pores, and a few cells in the vitelline follicles had matured. The 12-15 mg worms were less well developed. Although the seminal vesicle, cirrus sac and uterus were identifiable, and in the 14 and 1.5mg worms lumina had formed in these ducts, no sperm or mature vitelline cells had been produced. As was to be expected the worms gave a negative reaction with
solidus Hosts
of Different
Weights
d&0 pore
Testes
Ovary
0 0 C C + + + + + + +
0 0 0 0 0 + 0 t + + +
0 l-2 6 14 16 S R S R S S
0 0 + + + + + + + + +
0 0 ? ? l-3 YF 1-5 YF 4-9 YF YF
0 0 0 0 0 + 0 + 0 + 0
+
+
S
+
YF
+
Uterus
C = concentration of nuclei; R = spermatocyte rosettes; vitelline cells (see text) ; 0 = absent; + = present; numerals testis and vitelline follicles; genital plug-see text, section 4. in
CAIG
TABLE III in “Adult” Schistocephalus 48 hr in Various Laboratory
Host and reference no.
Genital
AND
Vitellaria
Eggs
S = sperm; YF = follicles containing mature = the average number of cells in sections of
catechol, which showed the absence of polyphenol oxidase, an enzyme associated with shell formation, present in mature vitelline cells (Johri and Smyth, 1956). In the 9 mg worm, apart from some multiplication of the testicular cells, the only signs of “adult development” was the differentiation of the central mass of cells into two concentrations of nuclei, identifiable as the male and uterine duct, and the appearance of the ovary as a thin ribbon of cells. Below 9 mg, development was restricted to the appearance of testes consisting of one or two cells. The 1.3 mg worm was alive but enmeshed by host cells and probably degenerating. Development of small worms in pigeons could not be studied, as worms less than 13 mg were not found (Table I). The 13 mg worm was fully mature, except that it, like the 17 mg worm, had nearly 20 terminal proglottids without eggs, instead of the usual 1 or 2 proglottids without eggs which often occurs in larger worms. Smaller worms were found in ducks and the important point shown in Table III is that the transition from infective but incapable of maturing in 48 hours, to infective
STUDIES
Level wt. mg.
of Genital Genital plug
ON
TABLE in Schistocephalus
Development Seminal vesicle and cirrus
SCHISTOCEPHALUS
Uterus
SOLIDUS.
IV solidus
239
I.
of Different
Plerocercoids
Uterine and 8 Pores
Testes
Weights
Vitellaria
Ovary
Ref.
3.1
C
0
0
0
0
0
0
6.5
+
0
0
0
0
0
0
Feb. S 36 Feb.
9.0
+
0
0
0
l-3
0
0
s 34/5 Oct.
11
C
C
0
8
0
1
Nov.
12
+
+
M
10
+
1
Nov.
14
+
+
D
13
+
2
Nov.
19
+
+
+
18
+
4
S 26 Nov.
20
+
+
+
25
+
5-10
24
+
+
+
30
+
8
+
50
+
S 64 s 20 s 22
s 25 Nov. s 24
+
230
With
For
Column other
5 -ducts abbreviations
lumen
+
With
Aug. s 50 Sl
30
lumen
extending out to muscle see Table III.
layer
and capable of maturing, occurs between 6 and 10 mg. The 10.5 mg worm (Duck 59) was clearly on the borderline, the uterus containing only a few, 2-20 eggs. In some proglottids the pore of the male opening did not appear to have been formed, however, small massesof sperm were found in the seminal receptacula of these proglottids.
(M),
to sub-cuticular
layer
(D),
to surface
pore
(+).
The numbers have little precise significance but indicate trends. The smallest (3.1 mg) plerocercoid sectioned shows faint traces of densely nucleated cells forming a central mass. These cells are arranged in segmental blocks. This segmental distribution precedes any external sign of proglottid formation (cf. Proteocephah fiZicoZZis,Hopkins (1959)). Traces of 4. Development of Genitalia in Plerocercoids tubular organization and clusters of cells The levels of development of the genital forming testes can be seenas early as the 11 primordia in plerocercoids of different weight mg plerocercoid and by 19 mg the cells of the are shown in Table IV. The term genital plug central mass have differentiated to form the is used to denote the massof densely nucleat- uterus, seminal vesicle, cirrus etc. The numed cells in the mid line which later differen- ber of testes visible in a section of a 19 mg tiate to form various genital structures; it is plerocercoid is similar to that in larger plerothe most obvious structure in a transverse cercoids, e.g. a 230 mg plerocercoid. Although section of a small plerocercoid. In order to in- thl4 method of estimating the number of testroduce some quantitative criteria the ap- tes is open to several sourcesof error, it sugproximate size of the vitellaria and testeswas gests the number of follicles does not indetermined by counting the number of cells crease. If this is so it appears that after the forming a follicle, as seen in an 8 p section. plerocercoid has grown to 19 mg, develop-
240
HOPKINS
ment of the genitalia is restricted to an increase in size of the existing primordia, and the formation of lumina in the ducts. The testes are the first genital organs to be clearly identifiable; situated ventrally to the dorsal transverse muscle fibres they are easily distinguishable from the surrounding parenchymatous cells. By the 14 mg stage the vitelline cells are identifiable as small groups of 2-3 cells; earlier identification is difficult as many nuclei are found in the cortical region outside the longitudinal muscle bundles. It is equally difficult to determine precisely the stage at which an ovary may be said to exist. In a small plerocercoid the ovarian primordium is indistinguishable from the central mass of cells and it is only as this central mass differentiates that a thin row of cells becomes apparent on the ventral side. The time of year the plerocercoid was collected is shown (Column 9, Table IV.) This has been done because plerocercoids grow quicker in summer than winter and no evidence exists to indicate whether the time taken to reach a certain size is also important in determining the level of differentiation. DISCUSSION
The development of the genital primordia in plerocercoids of Schistocephalus solidus (Table IV) may be summarised as follows: (i) the process commences at a very early stage; (ii) by 19 mg all the genitalia are formed; (iii) after 19 mg only quantitative changes occur (i.e. testes, vitelline follicles, etc., increase in size) ; (iv) as in tapeworms in which genital development is confined to the final host the male system appears first. The three principal variables to which differentiation may be correlated are weight, age, and region of the strobila. This last point is critical in very small plerocercoids as a development gradient extends both anteriorly and posteriorly from a central region in the strobila. Thus, in an adult of about 10 mg, only the central proglottids produce eggs. The extent to which age, as distinct from
AND
MC
CAIG
size or weight, affects the level of differentiation has not been determined, as the plerocercoids were obtained from a natural population in which the age was not known. The precise relationship of development with size and age can only be determined by using plerocercoids reared in laboratory-infected fish under controlled conditions. Two principal difficulties were encountered in investigating the capability of plerocercoids to mature. Firstly, plerocercoids of the critical size, i.e. < 20 mg are unlikely to become established, especially if they are removed from the fish before feeding to a duck or pigeon (Table I). Secondly, while plerocercoids of < 1 mg were available in our supply near Glasgow, (this being the overwintering size range in most fish), worms of 5-20 mg on the contrary were scarce, as the majority of plerocercoids passed quickly through this size range in early summer. To overcome the first of these difficulties the plerocercoids were matured in the perivisceral cavity of mice (Clarke, 1952). Using this technique, eggs and sperm were formed in plerocercoids of 23 mg and over within 48 hours. Smaller plerocercoids showed progressively less development. The levels of genital differentiation in a few are shown in Table III. In contrast to Clarke (1952) who found that small worms (8 and 3 mg) disintegrated, active worms down to 3.3 mg were found. Nevertheless, since they rapidly become enmeshed by host tissue, 48 hours would appear to be about the maximum time small plerocercoids can survive in the perivisceral cavity. The minimum size of worm producing eggs agrees fairly closely with that found by Clarke (1952) except that he found eggs in a 1.5 mg worm, whereas we failed to obtain eggs in worms less than 23 mg (Table III). From these experiments it was inferred that worms of 23 mg and upwards, provided they become established, can be expected to mature in 48 hours in the definitive host. From Smyth’s (1952) observations 48 hrs at 36.5
STUDIES
0~
~CHISTOCEPHALUS
C [rectal temperature of a mouse (Spector, 1956)] would appear to be the minimum time necessary even for large plerocercoids to mature. This is an interesting point as it suggests the level of development of the genitalia is essentially the same in a 23 mg plerocercoid as in larger plerocercoids. It was found later that worms down to 10 mg could mature in ducklings. The reason why smaller plerocercoids should mature, in 48 hrs, in a duck than in a mouseis probably merely a temperature effect. The rectal temperature of a duck is 41.5 C (Spector, 1956), five degreeshigher than that of a mouse.The hnding of mature worms down to 10 mg indicates that the genital primordia, even in such small plerocercoids, are capable of maturing in responseto a rise in temperature. However, as was to be expected from the weak development of genitalia in plerocercoids of that size (Table IV), only the most developed proglottids in the centre of the strobila matured within 48 hours. Although worms of lessthan 10 mg did not produce eggsand sperm in 48 hours, it is still clear that the rise in temperature evoked a switch to adult development. Thus the testes in the 9 mg worms (M66 and D58, Table III) consisted of about 16 cells in section. Such a size would only be reached in plerocercoids of about 19 mg (Table IV). The effect of temperature on development is therefore far more profound than can be accounted for by a QIO effect. Even in small plerocercoids weighing as little as 6 mg, a rise in temperature causesa switch from a metabolic pathway in which size of genitalia increasesconcomitantly with somatic tissue, to a metabolic pathway in which somatic growth ceases and only genital development takes place (Table II). (It is not wished to anticipate here a more detailed consideration of growth changes in Schistocephalus which is in preparation, but the interpretation of the datum in Table 11 as indicating a cessationof somatic growth in all or nearly all of the worm is based on two
SOLIDUS.
I.
241
reasons. Firstly, the dry weight of plerocercoids of 40-70 mg increasesby at least lo20% in 48 hours at 22’ C; at 40” C an even greater increase in body weight would be espetted unless growth is inhibited. Secondly, if it is assumed that no growth has taken place at 40°C and allowance is made for a glycogen loss similar to that which occurs in Schistocephalus during 48 hours in the pigeon’s intestine (Hopkins, 1950), and for the small loss in water which occurs (unpublished)! the calculated weight of the worm after 48 hours is similar to that found experimentally (Table II) ) . Furthermore, since worms down to 6 mg have been found in the intestine, plerocercoids of this weight must in addition to switching their metabolism, be capable of responding to the stimulus of the gut environment and triggering off their 5etention mechanism” to counteract peristaltic pressure. The fact that few worms of lessthan 10 mg were found (Table I) indicates that factors, other than the intrinsic ability of a plerocercoid to trigger off a retention response, are involved in determining whether a plerocercoid will become established. These extrinsic factors will be discussedelsewhere (McCaig and Hopkins). In general, the decreasing percentage of plerocercoids to become establishedas the weight falls below 30 mg suggests a progressively less favorable host/parasite relationship. Below 6 mg, conditions are so unfavorable, apparently, as to make establishment highly improbable. However, worms below 6 mg while incapable of retaining their position in the intestine might still be intrinsically capable of responding to the external stimulus which evokes the switch from plerocercoid to adult development and behavior. The point at which a pIerocercoid becomescapable of responding and altering its metabolism is presumably a constant depending on its genetical constitution, but the point at which a plerocercoid becomes infective appears to be a variable depending on the host environment. Compare
242
HOPKINS
AND
MC
CAIG
for instance the “take” in pigeons with that in ducks (Table I). The host/parasite relations are less favorable to Schistocephalus in pigeons at all stages, and the threshold below which it becomes improbable that plerocercoids will become established is higher than in ducks. In summary, entry into the duck’s intestine initiates the following changes in a plerocercoid:
(i)
the cessation (Table II) ;
of
somatic
(ii)
the stimulation of a response mechanism by the worm to stay in the intestine, which is effective in worms over 30 mg, less effective between 6-30 mg, and ineffective below 6 mg (Table I) ;
DEVELOPMENT
IN
PLEROCERCOID
DEVELOPMENT IN
ADULT
growth
(iii)
in worms over 10 mg, the stimulation of maturation with egg and sperm production occurring within 48 hours (Table III) ; or
(iv)
in worms of 6-10 mg, the stimulation of maturation, but without development progressing to egg and sperm production.
The course of development and suggested control mechanisms are expressed diagrammatically in Fig. 1. The weights indicate the size of the plerocercoids in the two phases. 0.3 micrograms is an estimate of the weight of a plerocercoid immediately after its establishment in the fish host. Synthesis of both somatic and genital tissue occurs in the plerocercoid. In the case of the genitalia, as has been discussed, there are two phases; the qualitative, during which the various genital rudiments become differentiated, and the quantitative, during which the genital rudiments increase in size but show little further progress towards maturation. The final phases of oogenesis, vitellogenesis and spermatogenesis are inhibited at low temperatures and can only take place when stimulated by a high temperature (Smyth, 1952). It is gener-
FIG.
1.
cephalus
The
solidus
pattern during
of the
development in Schistoplerocercoid and adult
phases. ally accepted that with the onset of maturation somatic growth ceases (Wardle and McLeod, 1952, p. 563). This is the case in worms developing in ducks (Table II), and probably in worms maturing in the perivisceral cavity of mice (section 2, Results). Whether this cessation of growth occurs as a result of an internal hormonal mechanism acting as a feed-back from the maturing genitalia, or because the environment does not contain suitable metabolites, or for some other reason, can only be determined by further experiment. To return to the problems posed in the Introduction, plerocercoids become infective to ducklings and pigeons between 6-l 5 mg, but there is no reason to believe that in some other hosts even smaller worms could not become established. The minimum obligate amount of development which must occur in the fish is therefore small. Infectivity and the ability to produce eggs appears to be nearly synchronized. This synchronization, however, is not necessarily evidence of a direct physiological link. More probably it arises from genetical selection pressure, in that it is advantageous to the species that a plerocercoid should be capable of maturing as soon as it is infective. The “target” in the plerocercoid which on
STUDIES
ON
SCHISTOCEPHALUS
stimulation initiates the retention response mechanism and hence brings about infectivity, exists in the plerocercoid at a very early stage. It does not appear to be a genital structure, but, as will be discussed elsewhere (McCaig and Hopkins), is part of, or controlled by, the scolex. ACKNOWLEDGMENTS
The authors gratefully assistance given by Miss mond Stoddart.
acknowledge Beryl Tagg
the technical and Mr. Ray-
REFERENCES
A. S. 1952. Maturation of the plerocercoid of the pseudophyllidean cestode Schistocephalus solidus in alien hosts. Experimental Parasitology 2, 223-229. HOPKINS, C. A. 1950. Studies on cestode metabolism. 1. Glycogen metabolism in Schistocephalus solidus in vivo. Journal of Parasitology 36, 384-390. HOPKINS, C. A. 1959. Seasonal variations in the incidence and development of the cestode PYOteocephalus filicollis (Rud. 1810) in Gasterosteus aculeatus (L. 1766). Parasitology 49, 529-542. HOPKINS, C. A., AND SMYTH, J. D. 1951. Notes on the morphology and life history of Schistocephalus solidus. Parasitology 41, 283-291. JOIIRI, L. N., AND SMYTH, J. D. 1956. A histoCLARKE,
SOLIDUS.
I.
243
chemical approach to the study of helminth morphology. Parasitology 46, 107-116. MCCAIC, M. L. O., AND HOPKINS, C. A. Studies on Schistocephalus solidus. 2. Establishment and longevity in the definitive host. (In preparation). SMYTH, J. D. 1946. Studies on tapeworm physiology. I. The cultivation of Schistocephalus solidus in vitro. Journal of Experimental Bio!ogy 23, 47.70. SMUT-H, J. D. 1952. Studies on tapeworm physiology. VI. Effect of temperature on the maturation in vitro of Schistocepkalus solidus. Journal of Experimental Biology 29, 304-309. SMYTH, J. D. 1954. Studies on tapeworm physiology. VII. Fertilization of Schistocephalus solidus in vitro. Experimental Parasitology 3, 64-71. SMYTH, J. D. 1956. Studies on tapeworm physiology. IX. A histochemical study of egg-shell formation in Schistocephalus solidus (Pseudophyllidea) Experimental Parasitology 5, 519540. SMYTH, J. D. 1959. Maturation of larval pseudophyllidean cestodes and strigeid trematodes under axenic conditions; the significance of nutritional levels in platyhelminth development. Annals of the New York Academy of Science 77, 102-12s. SPECTOR, W. S. 1956. Handbook of Biological Data. W. B. Saunders, Philadelphia and London. WARDLE, R. A., AND MCLEOD, J. A. 1952. The Zoology of Tapeworms. The University of Minnesota Press, Minneapolis.