Experimental studies on morphological variation in the cestode genus Hymenolepis

EXPERIMENTAL

PARASITOLOGY

Experimental in III.

the

8,

427-470 (1959)

Studies Cestode

X-Irradiation Facilitating

on Morphological Genus Hymenolepis as a Mechanism Analyses in H. nana*

Everett Department The

of Pathobiology, Johns Hopkins (Submitted

for

L. Schiller

School University, for

Variation

publication,

of Hygiene Baltimore, 8 May

and Public Maryland

Health

1958)

Individual variation has been the source of much confusion to taxonomists. Mayr, Linsley and Usinger (1953) estimated that more than half of the thousands of synonyms in zoological nomenclature owe their origin to an underestimation of this phenomenon. The importance of this problem in the field of parasitology has been well emphasized by Manwell et al. (1957) in a recent symposium on intraspecific variation in parasitic animals. A series of investigations have been undertaken in an attempt to elucidate something of the nature, extent and directional trends of morphological variations in the cestode genus Hymenolepis Weinland, 1858 through experimental procedures involving H. nana (von Siebold, 1852) as the subject. The first two papers in this series (Schiller, 1959a, b) were concerned with the morphology and development of the cysticercoid in the insect intermediate host, and the influence of different definitive host species on the growth rate, egg productivity and life span of the strobilate phase of this cestode. A major limitation in the analysis of variation by comparative morphological studies is encountered in the laborious process of examining the great number of specimens required to ascertain whether regular intergradations between extreme morphological differences occur, as well as to detect those variant characters which are infrequently expressed. A knowledge of the latter is of conthe out

* This investigation National Institutes during the tenure

was supported in part by a research grant E-1508, from of Health, U. S. Public Health Service and was carried of a U. S. Public Health Service Research Fellowship.

428

SCHILLER

siderable importance to the taxonomist since it is in the lower range of variation that overlapping in the characteristics of morphologically similar species may, and often does occur. According to the literature pertaining to radiation-biology (Lea, 1947; Dobzhansky, 1955; Hollaender, 1955), ionizing radiations are known to increase greatly the rate at which the usual variant characters are expressed in a number of different organisms. Preliminary studies (Schiller, 1957) with H. nana indicated that cestodes probably do not differ from other organisms in this respect. The present paper deals with some qualitative and quantitative effects of exposure to different high doses of X-irradiation on the morphology of the cysticercoid and strobila of this worm. MATERIALS

AND

METHODS

Details of methodology relative to the establishment and maintenance of infections in the intermediate and definitive host species and procedures for the preparation and study of resultant specimens have been presented in the preceding papers (Schiller, 1959a, b). The general methods for the preparation and exposure of eggs of H. nuna to X-irradiation for the purpose of determining the effects on the morphology of both the cysticercoid and the adult cestode were as follows. Gravid proglottids obtained at autopsy of experimentally infected albino mice were kept in tap-water at a temperature of 4°C for a period of 24 hours. They were then segregated into groups and each group, with the exception of one which served as a control, was exposed to a different roentgen (r) dosage. The dosage range employed was from 5 to 40 kiloroentgens (kr), with the exposures usually given in increments of 5 or 10 kr. During exposure the gravid proglottids were in water of just sufficient volume to cover their surfaces. Throughout all of the X-irradiation experiments the exposure constant was 100 kilovolts at 5 milliamperes without a filter. The time of exposure was varied to provide the desired roentgen dosage. Princeton Swiss mice were used as the definitive hosts for both the indirect and direct experimental infections and T. confusum served as the intermediate host in the indirect infections. The initial experimental infections were carried out immediately after irradiation of the gravid proglottids. The morphological determinations were made from the microscopic study of whole-mount specimens of adult cestodes recovered at autopsy of the mice and/or the cysticercoids dissected from the beetles.

MORPHOLOGICAL

VARIATION

IN

HYMENOLEPIS.

III

429

TABLEI Experiment Record

of Injections

R-dose,

Xl08

5

10 15 20 Control*

with First Generation Proglottids Exposed

N;g$p examined

75 60 55 45 703

N;~;feyf infected

20 30 21 26 400

Per cent infected

26.6 50.0 38.2 57.7 57.0

I. Part I Cysticercoids in to X-irradiation Total number of cysts obtained

365 609 560 726 7,630

T. confusum Fed Gravid

Range in number of cysts

M%Ul number ‘;,em&-

fi:t’ad beetle

beetle

l-83 l-66 1-143 1-137 1-161

18.2 20.3 26.6 28.0 19.0

SE. of mean

f4.94 h3.0 f8.38 ~~6.87 f1.34

* Record of infections with cysticercoids of H. nana in T. confusum fed gravid proglottids unexposed to X-irradiition-combined results from infections established in beetles over a period of 16 months. (Fed in same manner a8 irradiited proglottids.)

Experimental

The following studies of the effect of X-irradiation on this specieswere based on specimens resulting from two experiments, each having three parts. In Experiment I the dosage employed was from 5 to 20 kr, increased in 5 kr increments. In Part I of this experiment four groups of beetles were infected with X-irradiated gravid proglottids. Each of these groups received proglottids exposed to a different r-dosage and a fifth group serving as a control was given non-irradiated proglottids. The beetles were dissected 15 days after infection and the cysticercoids were recovered for subsequent indirect infections as well as for morphological study. The data concerning infectivity for the beetles are presented in Table I. Part II of this experiment involved the direct infections of mice with X-irradiated and non-irradiated gravid proglottids from the same lots of material used to infect the beetles in Part I. Thirty mice were segregated into groups of six animals. About 200 of the non-irradiated gravid proglottids were fed to each animal in one group which served as a control. Each of the remaining four groups were infected with proglottids which had been exposed to the various dosages of X-irradiation. The number given each animal was approximately 200. All animals were autopsied 15 days after infection and the worms were recovered. To make possiblea study of the effects of this irradiation on the second

430

SCHILLER

generation, some of the terminal gravid proglottids were removed from each of these worms for subsequent infections and the remainders of the strobilae were fixed for morphological examination. After being kept in tap-water for 24 hours at a temperature of 4°C these gravid proglottids were fed to appropriate groups of T. con&sum. The cysticercoids which developed in these beetles were recovered by dissection 15 days later and fed to corresponding groups of Princeton Swiss mice. The resultant second generation adult worms were recovered from these animals at autopsy 15 days after infection and prepared for morphological study. The data concerning infectivity for the beetles are summarized in Table II and those concerning infectivity of the second generation cysticercoids for the mice are given in Table III. In Part III of this experiment indirect infections of mice were carried TABLE

II

Experiment Record

-

I. Part

II

of Cysticercoid Infections in T. confusum Given Eggs from First Generation Adults Obtained in Direct Infections with X-irradiated Material

Original r-dose, Xl08

Number ‘$$,$$

5 10 15 20

25 25 20 20

* This number study.

N”3berPerinfectedcent ,beetles Infected

19 15 14 12 constitutes

Total numbe~b~fi$~ts

Range in number of cysts per infected beetle

infected beetle

863 740 961 134

1-143 l-72 15-164* 145

45.4 49.3 61.5 11.1

76.0 60.0 70.0 60.0 the heaviest

oyaticercoid

infection

TABLE Experiment Results

of Infections, Originated from

Group

I

Original r-dose, X10”

recorded

SE. of mean

f8.82 f5.24 f11.36 f2.97

for this host during

the present

III I. Part

II

in Albino Mice, with Second Eggs Exposed to X-irradiation First Generation

Generation Cysticercoids which at the Beginning of the

Number of mice exposed

Number of cysts given

Number of mice infected

Per cent infected

Number of cestodes recovered

Range in mm,,4e;&

MealI number in&\;d

S.E. of mean

II

5 10

10 10

25 25

7 0

70.0 0.0

32 0

2-7 -

4.5 -

f.78 -

III IV

15 20

10 10

25 25

9 9

90.0 90.0

35 13

l-11 l-3

3.9 1.4

fl.1 A.22

MORPHOLOGICAL

VARIATION

IN

HYMENOLEPIS.

431

III

TABLE IV Experiment I. Part III Record

of Infections

confusum Which

in Albino Had

Original r-dose, xl03

Been

Mice Given First Generation Cysticercoids from Fed Gravid Proglottids Exposed to X-irradiation in Experiment I, Part I

NUIIIbar of “FX: amined

Number i$e$~~

Per cent infected

Number Range of ces- in numtodes ob- ber of tained restodes

3 33.3 5 I 5 9* II 10 10 8 80.0 19 100.0 24 III 15 10 10 IV 20 IO 3 30.0 3 v Control 10 5 50.0 17 Results of experiments in which 5 and 10 cysticercoids, the life cycle were fed (data from Table I, p. 217 10 cysticercoids fed: 60 40 66.6 140 5 cysticercoids fed : 72 38 52.7 54 * One animal

l-3 l-5 l-5 1 l-6 with no Schiller, l-8 l-3

Meall number per in$$sf

T.

S.E. of mean

1.6 f.60 2.4 f.50 2.4 f.41 1.0 3.4 A.96 X-irradiation in 1959b). 3.5 A.12 1.4 f.19

died.

out with first generation cysticercoids obtained at the dissection of the beetles in Part I. Fifty mice were segregatedinto five groups corresponding to the four r-dosages and the non-irradiated control. Each animal was given 10 cysticercoids from the appropriate group of beetles. All animals were autopsied 15 days after infection and the worms were removed. The data obtained concerning infectivity for the mice are presented in Table IV. In experiment II the dosage range employed was from 15 to 40 kr. This experiment was also carried out in three parts according to the same procedures used in the preceding experiment, except that in this case no attempt was made to produce a second generation of worms. The findings on infectivity obtained at dissection of the beetles in Part I, Experiment II, as well as those resulting from an analysis of the frequency of occurrence of abnormal cysticercoids, are presented in Table V. The results concerned with the indirect infections in the animals in Part III, Experiment II, are presented in Table VI. Qualitative Effects on the Morphology of the Cysticercoid after X-irradiation of the Egg The more obvious effects of X-irradiation on the morphology of the first generation cysticercoids are malformations and arrest of development. The organisms in which arrest of development has occurred are

432

SCHILLER

TABLE

V

Experiment

II.

Part

Record of Infections with First Generation T. confusum Fed Gravid Proglottids

15 20 30 40 Control (combined expts. nonirradiated material)

50 50 50 18 702

48 45 33 9 400

96.0 90.0 66.0 50.0 57.0

1,598 639 207 24 7,630

TABLE Experiment Record

I

Cysticercoids of H. nana Exposed to X-irradiation

1-136 l-74 l-32 l-10 l-161

33.2 14.0 6.3 2.6 19.0

f4.54 f2.47 f1.31 fl.O ~1~1.34

Part

III

Number

I II III Iv* V

Original r-dose, X10”

15 20 30 40 Control

Per cent infected

10 10 10 2 10

7 4 2 0 4

353 335 162 22 38

VI II.

of Infections in Albino Mice Given First Generation Cysticercoids from T. confusum Which Had Been Fed Gravid Proglottids Exposed X-irradiation in Experiment II, Part I

Group

in

70.0 40.0 20.0 0 40.0

(See Table IV for comparison with results of experiments used.) * Only 12 cystioercoids were available for these infections; cysticercoids.

cesF:des obtai ned

MeaIl Range in number number c&odes Of wr EJectiin-

56 8 14 0 16

1-16 l-3 1-13 0 l-5

in which

non-irradiated

therefore

Obtained to

two animals

8.0 2.0 7.0 0 4.0

SE of me&

f2.1 f0.47 f4.88 f.95

cysticercoida

were

were each given

morphologically identical with organisms obtained at various stages ranging from 24 to 144 hours during the study of normal development. Microscopic examination of the other anomalous forms indicated that the normal growth and development of these organisms have been altered in three main ways. These include: (1) inhibition of cell division; (2)

6

MORPHOLOGICAL

VARIATION

IN

HYMENOLEPIS.

III

433

failure of the cells to differentiate; and (3) interference with structural organization. The appearance of some forms indicates that only one of these developmental functions has been interfered with and the effect may be restricted to a specific structure of the cysticercoid or the entire organism may be involved. Others show evidence of varying degrees of interference with all three of these functions. The capsule and cercomer, though frequently distorted in shape, are usually present, but the cestode larva within the capsule may show considerable structural disorganization or may be absent altogether. This suggests that the cells responsible for the production of the larva are comparatively more vulnerable to irradiation than those responsible for the production of its enclosing membranes. Cysticercoids in which the scolex has failed to develop usually contain several large kidney-shaped calcareous bodies. Examples of the various anomalies obtained in these experiments are illustrated in Plates I, II and III. The frequency of occurrence of abnormal cysticercoids was determined in accordance with the r-dose received and the resultant data are presented (see Table V or Fig. 1). The curve was fitted by inspection as it did not appear to be of value to analyze these data further. None of the abnormal forms, including those in which development had been arrested, developed to the adult stage when fed to mice. It appears that, in the case of the abnormal cysticercoid, the effect of irradiation may be considered lethal as far as perpetuation of the species is concerned, even though these organisms were viable when dissected from the beetles 15 days after infection. Degeneration is apparent in most of the abnormal forms recovered after 30 days in this intermediate host. There is some evidence that the products of degeneration may have a lethal effect on the beetle itself; however, this requires further investigation. Inasmuch as the abnormal cysticercoids fail to survive, the curve (Fig. l), may represent some measure of the lethal effect within this range of X-irradiation. Most of the anomalies are difficult to classify into meaningful categories because of the diversity and complexity of the morphological changes produced. However, some general grouping was attempted for the purpose of further analysis of the relationship between the frequency of occurrence of certain characteristics and the r-dose given. One form of abnormality, other than the arrest of development, which seemed to show some general pattern in the morphological effect, is that in which the scolex has developed external to the capsule of the cysticercoid (Figs.

PLATE

I

Photomicrographs of abnormal first generation cysticercoids of H. nanu from !Z’. conjusum experimentally infected with X-irradiated eggs. (X200). (Beetles dissected 15 days after infection.) FIG. 1. Cysticercoid with development arrested at about the 120-hour stage. FIGS. 2-4. Malformed cysticercoids. 434

MORPHOLOGICAL

VARIATION

IN

HYMENOLEPIS.

III

435

PLATE II Photomicrographs of abnormal first generation cysticercoids of H. nana from T. confusum experimentally infected with X-irradiated eggs. FIGS. 1, 2 and 4. Abnormal cysticercoids showing external development of structure normally destined to become the scolex. (X210) a. Malformed scolex. b. Capsule within which scolex normally develops. c. Cercomer. FIG. 3. Enlargement of malformed scolex. Note absence of suckers, rostellum and rostellar hooks. (X540) a. Calcareous bodies.

PLATE

III

Photomicrographs of abnormal first, generation cysticercoids of H. nana from T. conf~sum experimentally infected with X-irradiated eggs. FIG. 1. Abnormal cysticercoid showing distortion of capsule and failure of larva to develop within it. (x150) a. Capsule. b. Cercomer. FIG. 2. Abnormal cysticercoid showing capsular membrane formation without larval development. (x210) a. Capsule. b. Cercomer. FIG. 3. Enlargement of anterior portion of capsule from abnormal cysticercoid. (~337.5) a. Incompletely developed sucker. b. Incompletely developed rostellar hooks. c. Calcareous bodies. FIQ. 4. Enlargement, of anterior portion of capsule from abnormal cysticerooid. a. Accumulations of characteristically shaped calcareous bodies. Note absence of suckers and rostellar hooks. (X337.5) 436

MORPHOLOGICAL

VARIATION

R- DOSE (THOUSAND

IN

HYMENOLEPIS.

III

437

ROENTGENS)

between the frequency of abnormal cysticercoids of infected T. confusum and the radiation dose received by the eggs. Curve drawn by inspection from point on ordinate representing frequency of occurrence of abnormal cysticercoids as determined on the basis of cyst,icercoids which developed from non-irradiat,ed eggs. FIG.

1. Relationship

H. nana in experimentally

l-4, Plate II). The results obtained in the analysis of this characteristic as well as those for the analysis of arrested development, are summarized in Table VII. These findings indicate that the proportion of cysticercoids developing a non-functional external scolex is more or less directly related to the r-dose received, whereas the percentage of organisms showing arrest of development doesnot follow this relationship. In the latter case, the analysis included all forms showing arrest, regardless of the stage at which it had occurred. It seemsprobable that the elicitation of this condition may be relatively non-specific with a number of different causes involved, some operating at one stage in development and others at another. This could account for the lack of any apparent relationship between the number of cysticercoids affected and the r-dose received. Infectivity of the X-irradiated eggs does not seem to be significantly different from that of non-irradiated controls in this dosage range, except for the doses15 and 20 kr in Table V. The high rates of infection at these two dosagessuggest a possible stimulatory effect. It may be noted, however, that the mean numbers of cysticercoids per infected beetle decreaseas the dosageincreasesbeyond 15 kr.

438

SCHJLLER

TABLE Frequency

from

VII

of

Occurrence of Gross Morphological T. confusum Fed Eggs from X-irradiated Arrested

5 10 15 20 30 40

123 381 1,066 828 300 12

Changes Gravid

in Cysticercoids Proglottids of

development

42 105 132 39 54 6

External

34.1 27.5 12.3 4.7 18.0 50.0

3 3 69 168 93 0

Obtained nana

H.

SC&X

2.43 .79 6.47 20.28 31.0 -

63.4 71.7 81.1 75.0 51.0 50.0

Since cysticercoids exhibiting gross morphological abnormalities were found to be incapable of completing their development to adult worms in mice, only cysticercoids whose morphology appeared to be normal were used to establish the infections in Part III of both experiments. Although infectivity of these forms was determined, emphasis was placed on obtaining the strobilate phase of the worm for the purposes of studying the effects of irradiation on the morphology of the adult. The percentage of animals in which infections were established is highly variable (Tables IV and VI). Infectivity is significantly reduced at 30 kr and it appears that 40 kr is completely lethal. The high percentage of infections in the animals receiving cysticercoids which develop from eggs exposed to 15 kr again suggests a stimulatory effect consistent with that observed in the case of the egg infections in the beetles at this r-dose. Qualitative E$ects on the Morphology of the Adult Cestode after X-irradiation of the Egg The normal morphology of mature and gravid proglottids of H. nana is illustrated in Figs. 1 and 2 of Plate IV. A study of adult specimens from both direct and indirect infections in the foregoing irradiation experiments revealed a number of alterations in the characteristics of this worm. Some of the more conspicuous effects are shown in Plates IV, V and VI. These include duplication of cirri with and without distal confluence at a common genital atrium (Figs. 3,4, Plate IV), incomplete development of the genitalia resulting in sterility (Fig. 1, Plate V; 1-4,

MORPHOLOGICAL

VARIATION

PLATE

IN

HYMENOLEPIS.

439

III

IV

Photomicrographs of portions of H. nana strobilae illustrating effects of X-irradiating the eggs. FIG. 1. Mature region, normal morphology. (Non-irradiated.) a. Testes. b. Cirrus sac. c. Ovary. FIG. 2. Gravid region, normal morphology. (Non-irradiated.) FIGS. 3, 4. Mature region showing duplication of cirri with (X 128) a. Duplication of cirri in same segment.

morphological (X240)

(X128) distal confluence.

/

PLATE

V

morphological Photomicrographs of portions of H. nana strobilae illustrating effects of X-irradiating the eggs. FIG. 1. Proglottids in early gravid region. a. Imperfect segmentation and fusion of cirri from adjoining proglottids. (~37.5) b. Normal appearing early gravid proglottid. c. Degenerating testis in sterile proglottid. FIG. 2. Immature region of strobila. (x37.5) a. Proglottids showing irregular alternation of genital atria. FIG. 3. Mature region of strobila. (x150) a. Proglottids with three testes in normal arrangement. b. Proglottids with only two testes. Middle testis absent. c. Vitelline gland. FIQ. 4. Mature region of strobila. (x240) a. Proglottids with normal distribution of testes. b. Proglottids with abnormal distribution of testes. 440

MORPHOLOGICAL

VhRIATIOX

PLATE

IN

HYMENOLEPIS.

III

VI

Photomicrographs of portions of H. nana strobilae illustrating morphologica :ffects of X-irradiating the eggs. (X60) FIG. 1. Gravid region. a. Normal appearing gravid proglottid. b. Duplication of external seminal vesicle. c. Sterile proglottid. Note absence of reproductive organs, FIG. 2. Gravid region. a. Normal appearing early gravid proglottids. b. Degenerating testes. c. Sterile proglottid. Note absence of female genitalia. FIG. 3. Gravid region. a. Incompletely developed ovary. b. Incompletely developed vitelline gland. FIG. 4. Gravid region. a. Uterus containing malformed eggs. b. Cirri in abnormal positions.

441

442

SCHILLER

Plate VI), imperfect segmentation (Fig. 1, Plate V), irregular alternation of the genital atrium (Fig. 2, Plate V), reduction in testes numbers (Fig. 3, Plate V) and changes in relative position of the testes (Fig. 4, Plate V). Duplication of the cirri may occur on the same side of the proglottid or with one on either side. In all respects these latter proglottids resemble those in cestodes of the genus Diploposthe Jacobi, 1896, which Wardle and McLeod (1952) classify in another family, the Diploposthidae Poche, emended Southwell, 1929. Sterility is probably the consequence of incomplete development of some or all of the reproductive organs. As far as can be determined from microscopic examination of these strobilae, a change in the position of the testes, or reduction of the testes numbers from three to two, does not have a marked effect on egg production; however, these characters have considerable taxonomic value at both the specific and generic levels. This will be discussed in greater detail in the section following the analysis of the frequency of occurrence of these conditions. The position of the atrium also has been considered as an important specific cha.racter in differentiating cestodes of the genus Hymenolepis. Normally the position of the genital atrium is unilateral and dextral in H. nuna. It should be pointed out that any given observable effect, such as those listed above, seldom occurs in a series of adjoining proglottids, but usually is found at irregular intervals throughout the strobila. This seems to indicate that this cestode develops as an individual rather than as a colony as some biologists are inclined to believe. It also should be emphasized that, below the lethal level in X-irradiated material, the writer has observed no abnormalities which cannot be found in nonirradiated cestodes if enough specimens are examined. These observations indicate that sublethal irradiation does not produce new types of morphological alterations but only causes an increase in the frequency with which the usual alterations normally occur. Qualitative E$ects on the Morphology of the Adult Cestode after X-irradiation of the Cysticercoid

For purposes of comparison, an experiment was undertaken to determine the effect on the morphology of the adult when the cysticercoid was similarly X-irradiated. Fully developed cysticercoids which had been dissected from experimentally infected T. confusum were exposed to X-irradiation by the same procedure used in the case of gravid proglottids, except that the organisms were in 0.85 % NaCl solution instead

MORPHOLOGICAL

VARIATION

TABLE Infections

in Albino

Mice

IN

Group

I II III IV V

r-Dose,

5 10 15 20 Control

X10”

5 5 5 5 5

443

III

VIII with

X-irradiated

“gg’ infected

4 3 4 2 4

Cysticercoids

NUIIP

NUllINumber of mice examined

HYMENOLEPIS.

Per cent infected

80.0 60.0 80.0 40.0 80.0

berof cestodes obtained

R”y~b: of cestodes

,yzb& Per infected mouse

14 4 8 6 13

2-5 l-2 l-4 l-6 2-6

3.5 1.3 2.0 3.0 3.2

S.E. of mean

f.71 f.30 rt.71 f2.08 A.95

of water during the exposure period. The dosage range employed was from 5 to 20 kr and the exposure was in 5 kr increments. One lot which received no irradiation served as a control. Immediately after exposure the irradiated and non-irradiated cysticercoids were fed to appropriate groups of Princeton Swiss mice. Each group consisted of five animals and each animal received 20 cysticercoids. All animals were autopsied 15 days after infection and the cestodes prepared for study. Infectivity data, obtained at autopsy, are presented in Table VIII. Microscopic study of these specimens revealed that the morphological effects are more radical than those observed in worms which developed from X-irradiated eggs. Examples of some of the more extreme abnormalities observed are illustrated in Plate VII. In contrast to the effect produced by X-irradiating the eggs, the anomalous proglottids in this case often occur together in LLblocks” with the blocks occurring at irregular intervals in the strobila. Although the ratio of abnormal to normal proglottids in the strobila can be correlated with the r-dose received by the cysticercoid, the damage to the reproductive organs within those affected is usually so widespread that no particular kinds of effects can be classified. The testes and ovary appear to be especially vulnerable. Instead of forming the usual discrete structures, individual cells of these organs are often widely distributed in the proglottid, or occur as irregular deeply staining granular masses in the places where the testes and ovary normally are located. When completely dissociated, these cells are spherical to ovoid and range in size from 14 to 21 microns. It is inconceivable that normal function could be retained under these conditions. The fact that the numbers of sterile proglottids in the gravid region are proportionately greater than the numbers of proglottids in the mature region in which abnormalities

PLATE

VII

Photomicrographs of portions of H. nana strobilae illustrating morphological effects of X-irradiating mature cysticercoids. (X60) FIG-. 1. Early gravid region showing “blocks” of sterile proglottids. a. Development of external seminal vesicles without cirri. b. Aggregation of granular cells where testis normally is located. c. Dissociated group of cells where ovary normally is located. FIG. 2. Early gravid region showing “blocks” of sterile proglottids. a. Seminal receptacle appears normal despite absence of male genitalia. b. Uterus containing abnormal eggs. c. Incompletely developed cirrus sac with normal appearing external seminal vesicle. Note absence of other reproductive organs. FIG. 3. Gravid region. a. External seminal vesicle without cirrus sac. b. Partially developed uterus containing abnormal eggs. c. Confluence of cirri from adjoining segments. FIG. 4. Mature region showing fenestration of strobila and irregularities in testis-position. a. Discrete opening between proglottids.

MORPHOLOGICAL

VARIATION

IN

HYMENOLEPIS.

III

445

can be detected, indicates that reproduction is interfered with although observable morphological changes may not be found in these organs, The cells responsible for the formation of the cirrus and external seminal vesicle appear to be more resistant to the effects of X-irradiation, since these organs persist in proglottids which otherwise manifest the most extreme abnormalities. Duplication of genital organs was not seen in this material. The observations on these specimens again suggest that cells destined to divide and differentiate into specialized organs during the process of development are the most seriously affected by X-irradiation. Morphological structures which have already attained complete development in the cysticercoid stage before irradiation, such as those of the scolex, exhibit no observable morphological alterations. Lea (1947, p. 132) referred to radiation-induced phenomena of this nature in Drosophila as a “mosaic” effect. In explaining this effect he stated that “If a mutation occurs in a cell of the developing organism at some stage intermediate between the zygote and the adult, then in the adult a mosaic effect may be noticeable, as a result of the mutant character appearing in the group of cells which have developed from the cell in which the mutation occurred, the remaining cells being unaffected.” Although the diverse and heterogeneous morphological alterations observed in specimens of H. nana obtained from X-irradiated cysticercoids suggest a mosaic effect, there is no evidence, at present, to indicate whether the effect is due to radiation-induced somatic mutation or is the result of direct multiple damage to primordial cells in the embryo. Quantitative

Efects on the Morphology of the Adult Cestode after X-irradiation of the Egg

Preliminary analyses on specimens of H. nana from the experiments involving irradiation of the eggs indicated that the yield of visible variant characters was proportional to the amount of X-irradiation received. This observation suggested that X-irradiation might be useful for taxonomic purposes in evaluating the relative stability of any given morphological character as well as for obtaining an estimate of the rate at which certain variant characters might be expected to occur spontaneously in these cestodes, if any regularity in the pattern of variability existed. If the proportionality were direct, it was considered that graphical extrapolation to the ordinate, representing zero irradiation, of the line expressing the relationship between the frequencies of occurrence of

446

SCHILLER

the variant character and the different r-doses should indicate the frequency with which the variant character occurs in the non-irradiated population. Five of the variant characteristics observed in the adult worm were analyzed to test this hypothesis. To obtain supplementary material for these analyses, a third series of infections with X-irradiated eggs was undertaken. The procedures were the same as described for Experiments I and II but, in this case, the eggs were irradiated at 5 kr increments throughout the range encompassed in the two previous experiments (5 to 40 kr). Again no adult worms were obtained, either directly or indirectly, from eggs which had received more than 30 kr, indicating that the dosage which is lethal to 100% of these organisms lies between 30 and 35 kr. The following analyses were based upon adult specimens from all three experiments, the worms having been grouped according to the r-dose received by the eggs from which they developed. 1. Method of Analysis. The frequency of occurrence of the variant characteristics was determined by counting the total number of proglottids in the strobilae of the worms in each group and calculating the percentage of proglottids exhibiting the condition under study. The resultant data were plotted arithmetically and the frequency with which the variant characteristic could be expected to occur in the non-irradiated population was estimated by extrapolation. The estimate obtained was then checked by determining the frequency of occurrence of the same condition in a large series of non-irradiated specimens from the same host species. 2. Characteristics

Analyzed

a. Fusion of cirri. Because of the unusual and conspicuous nature of this abnormality, it was selected as one of the subjects for analysis. In this condition, the two cirri, usually one from each of two adjacent proglottids, are joined distally and terminate at a common genital atrium (Figs. 2, 3, Plate IV, Fig. 1, Plate V). The data resulting from the analysis of this condition in both irradiated and non-irradiated material are summarized in Table IX and presented graphically in Fig. 2. According to these data, the rise in the rate at which this condition occurs is directly proportional to the r-dose. When the line expressing this relationship is extrapolated to the ordinate, an estimate of about 0.25 % is obtained. The percentage (0.224) obtained in the analysis of this condition in non-irradiated specimens tends to verify the estimate.

MORPHOLOGICAL

VARIATION

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TABLE IX Frequency Obtained

of Occurrence of Proglottids Showing “Fusion” of by Direct Injections, in Albino Mice, with Eggs from Gravid Proglottids of H. nana

Total No. of proglottids examined

r-Dose, X103

Cirri

in Cestodes

X-irradiated

Number of proglottids showing fusion of cirri

3,088 3,718 2,372 1,182 1,377 735 8,023

5 10 15 20 25 30 Non-irradiated

30 62 56 38 51 31 18

The percentage (4.21) recorded at 30 kr suggests that this rate may be near the upper extreme at which the worm is capable of expressing this variant characteristic. The associated changes wrought by the drastic treatment necessary to induce an increase in the rate beyond this extent

I

0

I

I

5

IO R-DOSE

15 (THOUSAND

20

25

30

35

ROENTGENS)

FIG. 2. Relationship between the frequency of proglottids with “fused” cirri in strobilae of H. nana and the radiation dose received by the eggs. Note: When the linear relationship is extrapolated, the ordinate is intersected at a point representing a frequency of occurrence which coincides closely with that found by actual determinations based on non-irradiated specimens (see Table IX).

448

SCHILLER

TABLE

X

Frequency of Occurrence of Proglottids Showing Reversal of Genital Atrium Cestodes Obtained by Direct Infections, in Albino Mice with Eggs from X-irradiated Gravid Proglottids of H. nana

r-dose, X103 5 10 15 20 25 30 Non-irradiated

Total number of proglottids examined 2,772 2,206 1,469 920 913 764 2,880

in

Number with reversed atrium 8 31 34 32 34 35 3

probably cause the death of the organism. With the range in the frequency of occurrence of proglottids having fused cirri presumably limited to lessthan 5 % of the total number of proglottids in the strobila, this type of morphological variation in the character of the cirrus would be of little taxonomic significance. 6. Reversal of genital atrium. As indicated previously, the usual position of the genital atrium in this species is unilateral and dextral. Proglottids with the genital atrium in the alternative position were considered to be variant forms (Fig. 2, Plate V). The data resulting from the analysis of this condition in both irradiated and non-irradiated material are summarized in Table X and presented graphically in Fig. 3. The relationship between the frequency of occurrence of this variant and the amount of radiation involved again appears to be direct throughout the range employed. The figure obtained by determining the frequency of occurrence of this variant in non-irradiated specimens confirms the estimate arrived at by extrapolation, as in the previous case. c. Reversal in the position of the testes. In view of the various unsuccessful attempts to reclassify the genus Hymenolepis on the basis of the position of the three testes (Cohn, 1901, 1904; Clerc, 1902, 1903; Fuhrmann, 1906, 1932; Mayhew, 1925), it seemedof interest to analyze the variability in testicular arrangement according to the method under consideration. In H. nana the testes are usually arranged in a straight line across the proglottid, with one poral and two aporal to the ovary and vitelline gland (Fig. 1, Plate IV). Although the variations from this arrangement are characterized by a number of different configurations,

MORPHOLOGICAL

VARIATION

R-DOSE

FIG.

3. Relationship

between

(THOUSAND

IN

HYMENOLEPIS.

449

III

ROENTGEN3

the frequency

of proglottids

with

irregular

position of genital atrium in strobilaeof H. nana and the radiation dosereceived by the eggs. Note: When the linear relationship

is extrapolated,

the ordinate

is intersected

at, a point representinga frequency of occurrencewhich coincidesclosely with that found by actual determinationsbased on non-irradiated specimens(see Table X) .

the one selected for analysis was that in which the testes occur in a straight line, but with two poral and one aporal to the aforementioned organs (Fig. 4, Plate V). The data resulting from the determinations on

the frequency of occurrence of this variant arrangement

are summarized

in Table XI and presented graphically in Fig. 4. In this case it appears

that the rate of increase in the frequency of occurrence remains constant only for the first three dosage intervals. Although the abrupt change in the rate at 20 kr represents a significant deviation from the expected proportionality, extrapolation of the linear relationship obtained in the lower range, where the rate of increase is constant, still provides an estimate of the spontaneous rate which appears to be reliable when

compared with the determined

rate.

The precipitous decline in the rate after 20 kr seemsto be correlated

with the reduced number of specimens available for study at the higher dose levels. The increased frequency of the combined effects, induced in this range, may have contributed to the death of a large percentage of the grossly affected individuals so that the survivors represent only a

450

SCHILLER

TABLE Frequency of Occurrence of Proglottids to the Ovary in Cestodes Obtained Eggs from X-irradiated

r-Dose, X103 5 10 15 20 25 30 Non-irradiated

R-DOSE

XI

Showing Testes Arrangement with Two Poral by Direct Infections, in Albino Mice, with Gravid Proglottids of II. nana

Total number of proglottids examined

Number with two testes poral

7,832 9,808 7,352 4,368 1,792 1,114 10,290

792 1,116 1,048 848 272 77 765

(THOUSANDROENTGENS)

FIG. 4. Relationship between frequency of proglottids with testis-position reversed in strobilae of H. nana and the radiation dose received by the eggs. Note: When the linear relationship is extrapolated, the ordinate is intersected at a point representing a frequency of occurrence which coincides closely with that found by actual determinations based on non-irradiated specimens (see Table XI). Broken line indicates departure from linearity.

MORPHOLOGICAL

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451

biologically selected sample of the population. This would cause a bias in the data. As pointed out in the beginning of this section, the particular testicular arrangement chosen as the subject for analysis is only one of the several variants in testis-position found in this species. Therefore, the frequency of this variant represents only a fraction of the total variability in the arrangement of these organs. d. Reduction in the number of testes. Among the testicular variants observed in proglottids of H. nana was one characterized by the presence of only two of these organs instead of the usual three (Fig. 3, Plate V). Almost invariably it was the middle testis which was missing from these proglottids. Although a series of morphologically intergrading forms of this organ occurred in numerous other proglottids of a strobila, only those lacking this testis altogether were considered for the purpose of analysis. In view of the taxonomic importance of testis numbers in the family Hymenolepididae and the possible evolutionary significance the phenomenon of testis-deletion may have, if heritable, it was considered desirable to determine, insofar as possible, the consequences of the original irradiations with respect to this variant, not only on the first generation of adults but on their descendant’s as well. For the purpose of ascertaining whether the frequency of occurrence of this type of variant might be influenced by the different modes of infection (i.e., direct or indirect), the following determinations were based on specimens obtained from (1) direct infections with X-irradiated material; (2) indirect infections with X-irradiated material; (3) direct infections with nonirradiated material; and (4) indirect infections with non-irradiated material. (1) Frequencies with which proglottids containing only two testes occurred in the jirst generation. The data obtained in the analysis of specimens in the first generation following irradiation of the eggs are summarized in Table XII and the relationships between the frequencies of occurrence and the different r-doses are illustrated graphically in Fig, 5. In the case of each method of infection it is again noted that the estimated spontaneous rates obtained by extrapolation closely coincide with the respective rates determined from non-irradiated material. When the rate in specimens from the direct infections is compared with that in specimens from the indirect route, a difference is apparent. However, a chi-square test of this difference, based on the data with regard to non-irradiated material, shows that it is not statistically

452

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TABLE XII Analyses of Testes Numbers in H. nana r-Dose, X103 A. Cestodes resulting 5 10 15 20 25 30 Non-irradiated

No. of mature proglottids examined

from direct infections

in albino mice with X-irradiated 976 1,141 1,344 1,021 890 1,364 2,594

B. Cestodes resulting from indirect infections with X-irradiated eggs 5 10 15 20 25 30 Non-irradiated

No. with 2 testes

1,412 1,551 1,762 1,146 949 837 2,322

eggs

46 74 110 103 108 64 76 in albino mice 42 60 81 65 59 57 54

significant (P = 0.20). This does not rule out the possibility that variation in this characteristic may be under the influence of host-parasite relationships involved in the different modes of infection. Since it has been shown that there is a significant difference when the direct and indirect infections were established with irradiated material, the failure to detect it in non-irradiated specimensmay be due to the fact that this difference in the latter is extremely small. (2) Frequencies with which proglottids containing only two testes occurred in the secondgeneration. The specimenscomprising the material for the analysis of testis-deletion in the second generation were direct descendants of the worms discussed in the preceding paragraph (see Part II of Experiment I). For some undetermined reason, no cestodes were recovered from animals in Group II of this experiment (see Table III), therefore, the specimens studied are representative of only three of the roentgen-dose increments originally employed. Microscopic examination of this material has revealed that the testes are proportionately much more markedly affected. In the great majority of mature

MORPHOLOGICAL ANALYSIS

VARIATION

IN

OFTESTES

NUMBERS

HYMENOLEPIS.

III

453

IN H. NANA

1::

L 0

I 5 R-DOSE

I

I

IO

15

ITHOUSANDS

I 20

25

I

OF ROENTGENS)

FIG. 5. Relationship between frequency of proglottids having only two testes in strobilae of H. nana from direct and indirect infections and the radiation dose received by the eggs compared with the frequency in specimens from direct and indirect infections with no irradiation in the life cycle (see Table XII).

proglottids, one or more of these organs are unusually small, malformed and granular in appearance. Despite this condition, almost all of the specimenscontain gravid proglottids with eggs which appear to be fully developed. The frequency of occurrence of proglottids with only two testes is given according to the r-dose in Table XIII. According to these figures, the proportionate number of proglottids showing testis-deletion is considerably higher in worms of the second generation following irradiation than in worms receiving no irradiation. It also may be noted that the percentage of proglottids affected in this manner is approximately the same, regardless of the original amounts of irradiation. When compared with the results obtained from the analysis of this condition in the parent worms (Table XII) it is seen that these percentages are slightly higher than the maximum recorded in specimens from direct infections with eggs irradiated at 25 kr. The significance of this apparent relationship is not understood, although there can be little

454

SCHILLER

TABLE Frequency

XIII

of Occurrence of Proglottids with only Two Testes in Cestodes Originating from Eggs Exposed to X-irradiation Beginning of the First Generation

A. Without Original

r-dose, X103

5 10 20 Non-irradiated

respect to individual

r-dose, X103

5 10 20 Non-irradiated

at the

cestodes:

No. of mature proglottids examined

No. with 2 testes

Per cent of total

2,104 3,426 1,959 2,594

305 483 270 76

14.5 14.2 13.8 2.9

B. With respect to individual Original

Second Generation

No. of cestodes examined 10 10 10 30

cestodes: Mean per cent of proglottids with 2 testes

Standard error of the mean

13.3 11.2 11.5 1.2

2.7 3.1 3.1 0.2

doubt that the testis-deletion effect is, in some way, transferred to the second generation individuals. Contrary to what might be expected, no second generation individuals were found in which proglottids containing only two testes predominated (Table XIII B). This seemsto suggest that testis-deletion does not “breed true,” at least in the second generation, despite the relatively high frequency of occurrence induced by X-irradiation in the parent worms. It seemsnoteworthy that extensive infections by a microsporidian parasite, tentatively identified as Nosema helminthorum Moniez, 1887, occurred in practically all of these second generation cestodes. This organism was not found in the parent worms, nor in any of the other specimens obtained experimentally during this investigation. This observation suggeststhat X-irradiation of the eggs, from which the first generation adults originated, in some way altered these organisms so that their progeny were highly susceptible to infection with this sporozoan. In mammals, a lowered resistance to infection through direct damage to tissues responsible for the maintenance of disease-defense

MORPHOLOGICAL

VARIATION

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455

mechanisms is recognized as one of the principal effects of exposure to high doses of ionizing radiations; but the transfer of an increased susceptibility to the progeny, particularly when the effect has not been manifested in the parent, is of considerable interest. Investigation of this phenomenon would seem desirable. An attempt to produce a third generation of adult worms in this series proved unsuccessful due to the fact that, of 150 beetles (three groups of 50) exposed to gravid proglottids from the strobilae of second generation worms, 145 died during the 15-day interval prior to dissection. Of the five surviving beetles, two contained cysticercoids, all of which were The cause of this unusually high abnormal, and three were negative. rate of mortality in the beetles could not be determined with certainty. However, it is suspected that a large proportion of the cysticercoids developing in them were abnormal and that death and degeneration of these abnormal cysticercoids may have resulted in the liberation of toxic products which were lethal to the host. Further investigation of this question would be necessary in order to determine whether it would be feasible to carry out these studies for more than two generations with material from indirect infections. e. Sterility. This condition probably is much less specific in nature than either of those considered above, since it may result from a number of different functional disturbances in the reproductive system. For the purposes of analysis, a proglottid in the gravid region of the worm was recorded as sterile only if it was completely devoid of eggs; however, the extreme variation in the number of eggs per gravid proglot,tid observed in the affected individuals may also be attributed to the effects of irradiation. As in the previous case, this analysis was based upon specimensfrom direct as well as indirect infections established in mice both with X-irradiated and non-irradiated eggs. A summary of the results derived from these determinations is given in Table XIV and the relationships between the frequencies of occurrence of sterile proglottids and the corresponding r-doses are plotted graphically in Fig. 6. The apparent differences in the frequency of occurrence of the variant condition, depending upon whether the infection was direct or indirect, is not statistically significant according to the chi-square test (P = 0.50). When compared with the rate at which sterile proglottids occur in nonirradiated specimens, the estimate obtained by extrapolation of the results in the lower range of irradiation again appears to be reliable. There is a general similarity in the linear pattern obtained from the

456

SCHILLER

Analyses

of Frequency

r-Dose, X lo3 A. Cestodes resulting 5 10 15 20 25 30 Non-irradiated

TABLE of Occurrence

XIV of Sterile

No. of gravid proglottids examined from direct

infections 1,395 1,871 1,646 1,314 1,528 1,326 6,234

B. Cestodes resulting from indirect with X-irradiated 5 10 15 20 25 30 Non-irradiated

1,780 2,620 3,297 1,357 1,241 1,034 3,724

Proglottids

Number with eggs present

in

H. nana

Per cent of total

No. with no eggs present

in albino mice with X-irradiated 1,390 1,860 1,632 1,278 1,522 1,321 6,219

99.64 99.39 99.15 97.26 99.61 99.62 99.76

infections eggs

in albino mice

1,762 2,578 3,218 1,291 1,225 1,023 3,712

99.0 98.4 97.6 95.1 98.7 98.9 99.68

eggs 5 11 14 36 6 5 15

18 42 79 66 16 11 12

data with respect to irradiated material in this instance and that resulting from the analysis of testis-position (Fig. 4). Both of these variant characteristics show: (1) a consistent rate of increase through the first three r-doses; (2) an abrupt increase in the rate at 20 kr; and (3) a sharp decline at doses thereafter. The reason for the rate change at 20 kr is not clear. In these more complicated types of structural alteration, this change in the rate may be a manifestation of the cumulative effect due to multiple ionizations within the tissue when the radiation reaches a certain intensity, but there is inadequate evidence at present to permit interpretation of this rate change on the basis of theories (Lea, 1947) concerning the effect of irradiation on genetic mechanisms. f. Rostellar hooks. Workers generally have regarded

the number, size and shape of the rostellar hooks in cestodes as being fairly constant within a species but differing significantly between species. For this reason these characters have been given considerable weight in hymenolepidid taxonomy. In marked contrast to the prevalence of variant characteristics observed in other morphological structures of H. nana,

MORPHOLOGICdL

VARIATION OF NUMBERS

ANALYSIS

IN I

IN

HYMENOLEPIS.

OF STERILE H. NANA n

III

457

PROGLOTTIDS

6 :: ::

‘I4

X-IRRADIATED -

DIRECT

----

NON-IRRADIATED OlRECTlNFECTlONS DlRECTlNFECTlONS

---

NON-IRRADIATED INDIRECT INFECTIONS

/

INFECTIONS

:

:

X-IRRADIATED xIRRADIb.TEO ,NOlRECT INFECTIONS

t

’

:

;

;

: :

\

‘: 0

I 00

55

IO IO

R-DOSE

I5 I5

(THOUSANDS

I

I

20

25

OF ROENTGENS)

FIG. 6. Relationship between frequency of sterile proglottids in strobilae of H. nana from direct and indirect infections and the radiation dose received by the eggs compared with the frequency in specimens from direct and indirect infections with no irradiation in the life cycle (see Table XIV). Broken lines in x-irradiation curves indicate departure from linearity.

examinsltion of the rostellar hooks in adult specimensfrom the irradiation experiments revealed no distinctive alterations which could be correlated with the radiation dose. According to the hypothesis upon which the method of analysis used in this study is based, the fact that irradiation did not increase the variation rate to a detectable level indicates that significant deviations in these structures are probably of very rare occurrence in the normal population. The opinions with respect to the stability of rostellar hook characters, gained more or less empirically by the earlier taxonomists, tend to support this assumption. However, because of the taxonomic importance of these characters, it was microscopic

458

SCHILLER

necessary to examine the problem of morphological variation more closely. The differences, which once were sufficiently great to be considered highly significant in distinguishing the known species, are rapidly becoming less dependable because of the introduction of intermediates as systematists continue to describe new species, The illustrations presented by Hughes (1941) exemplify some of the similarities in hook morphology which prevail among different species in the genus Hymenolepis. Because of these close resemblances, even minor variations could be extremely confusing in attempting to formulate a sound system of classification. Where hook shape is concerned, lesser variations are often difficult to analyze critically by the usual procedures in microscopy, primarily because, to date, no satisfactory method has been devised for expressing, in precise terms, a configuration of this type (Plate VIII). In an effort to standardize procedures for measuring hymenolepidid hooks, Meggitt (1927) proposed a method of measurement based on a formula of five values, but this formula does not give an accurate repre-

PLATE

Photomicrograph

of rostellar

VIII hooks

of

H. nana.

(X 4000)

MORPHOLOGICAL

VARIATION

IN

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459

III

sentation of hook shape since the linear measurements do not, take into account the curvatures of these structures. Because of this discrepancy, a somewhat, different method of measurement. was used for the purpose of analyzing variation in hook shape in this study. The procedure was as follows. A random sample of nonirradiated specimens of H. nana was obtained at the autopsy of a large series of naturally infected albino mice. The scolices were removed from these cestodes and prepared as whole-mounts according to the conventional method, except that in the process of mounting, the specimens were flattened by applying sufficient pressure on the cover glass to cause the rostellar hooks to be spread out, in the same plane. Photomicrographs were then taken of these mounted specimens under oil immersion (Plate VIII). The photographs were enlarged when printed and the subsequent measurements were made on the printed subject. Total length was measured in a straight line from the point of the blade to the end of the handle and planar area was determined with the aid of a planimeter. Area was expressed in terms of the average of three planimeter measurements made on each hook. For the purpose of statistical analysis, hook shape was represented by the ratio between the total length of the hook and its planar area. No statistically significant differences were found in an analysis of variance of these data:

Sauce of variation

Total Between specimens Within specimens

Sum of squares 1.67 0.16 1.51

Analysis

of variance

Degrees of Mean freedom squares 148 28 120

0.0113 0.0057 0.0126

F

0.452

Inasmuch as this result indicates that, according to this criterion, the variation between specimens is no greater than the variation occurring within specimens, hook shape in H. nana appears to be a very reliable taxonomic character. This finding relative to rostellar hook stability in the non-irradiated population tends to confirm the prediction based on microscopic examination of the irradiated material. DISCUSSION

Variability in the morphological characters upon which the taxonomy of cestodes is based, has received very little study. Although the literature contains several reports in which character variants are described,

460

SCHILLER

few investigators have a.ttempted to determine the frequencies with which these variants occurred in the species exhibiting them. In the family Hymenolepididae, where some attempts have been made, the analyses have been undertaken with specimens collected from naturally infected host animals. Of the several variant characters known in hymenolepidid cestodes, those involving the testes have received the most attention. The number, size, shape and relative position of these organs constitute important taxonomic characters in this family and consequently they have been the most satisfactorily described of the internal structures. The importance of these organs as taxonomic characters is indicated by the fact that generic differentiation depends primarily upon the number of testes occurring within each proglottid of the strobila. The first attempt to determine the stability of testis characteristics in this family was made by Mayhew (1925). In a careful study of Hymenolepis sacciperium from the American scaup duck, Matila murila, he found that the testes were invariably located in the same relative position with reference to each other in all proglottids of a strobila as well as in other individuals of the same species. From these observations Mayhew concluded that testis-position was relatively stable and proposed that this characteristic be used as a criterion in further subdividing the complex genus Hymenolepis. In contrast, Voge (1952) found testis-position to be highly variable, both in Hymenolepis citelli from ground squirrels, Citellus beecheyi, and in H. diminuta from Norway rats, Rattus norvegicus, but the two species of cestodes differed significantly in the relative frequencies with which each of six classes of variants in testis-position occurred. Because the observed frequencies also differed significantly from the probability expected if they were due entirely to chance, Voge postulated that the differences in relative frequency of testicular variants are genetically determined. The writer (1952) made a somewhat similar analysis of morphological variation in Hymenolepis horrida obtained from a large series of different microtine rodent hosts collected over a relatively wide geographical range. Determinations of the frequency of occurrence of several types of organ variants revealed that the extreme differences in the range of variation of a given character were usually interconnected by a series of intergrading forms. Although variants in several characters occurred concomitantly in this species, variation in one character appeared to be completely independent of variation in any other character. The data obtained in the study of H. horrida indi-

MORPHOLOGICAL

VARIATION

IN

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III

461

cated that variation within the species population occurs with a certain pattern of regularity. These observations seemed to exclude the probability that variant characters are mere “accidents” in development of the organism and would tend to support Voge’s concept that the relative frequencies with which variant characters are expressed by the species may be intrinsically governed. However, the possible influence of the host on the frequency of occurrence of variant characters in the parasite cannot be properly evaluated in the analysis of specimens from naturally infected animals. Representatives of both extremes in the range of variation may be found in a large collection of material from such sources and unless an intergrading series of morphological differences intermediate between the extremes occurs within the sample, the investigator cannot always be certain that some of the extreme forms actually belong to the population with which he is concerned. For these reasons the studies of intraspecific variation reported herein were based upon specimens from experimentally produced infections in laboratory animals. With this approach, the specific identity of each individual specimen is assured, all of the stages in the life cycle of the organism are available for study, and information concerning the host and its diet can be taken into consideration relative to any observed effect on the morphology of the parasite. The foregoing studies have shown that the frequency with which certain variants occur in H. nana can be increased by exposing the eggs of this species to X-irradiation and that the rate of increase is proportional to the amount of radiation employed. In the case of the variant characteristics analyzed, the relationship between the frequency of occurrence and the radiation dose tends to be direct either through most or all of the range of sublethal exposure. It also has been shown that, when these direct relationships are extrapolated to zero irradiation, the percentages derived from determinations of the frequency of occurrence of these variants in non-irradiated specimens conform to linearity. Thus, by measuring the effects of several different large doses and extrapolating the results, one can obtain a reasonably accurate estimate of the frequency with which the variant may be expected to occur in the natural population. Obtaining reliable data concerning the kinds of variants and the frequency with which they occur is prerequisite to the proper evaluation of the relative stability of morphological characters used for taxonomic purposes. This information is of particular importance to the taxonomist when he is faced with the problem of deciding

462

SCHILLER

whether questionable forms belong to the same or different populations at the species level. The conventional method of acquiring these data involves laborious analyses of a very large number of “natural” specimens in order to insure that the sample is adequately representative of the population. The results reported herein indicate that, by increasing the rate of occurrence of the variants through the use of X-irradiation, the number of measurements needed to provide the same information can be reduced appreciably. Thus, it would seem that the use of radiation would be of considerable value in facilitating studies of normal variation. Insofar as the writer is aware, the literature contains only one report of work in which an attempt was made to determine the effects of X-rays on cestodes. Palais (1933), in a somewhat similar study with HymenoZepis diminuta, compared the frequencies of abnormalities in X-irradiated and non-irradiated worms and concluded that radiation had no effect in producing a greater frequency of abnormal segments. Unfortunately, it has not been possible to compare the absolute amounts of radiation used in the present study with those used by Palais because the information on dosage given in the description of her procedure is meaningless without a knowledge of the original source of energy: “Avec les Rayons X, l’irradiation, dans les diverses experiences, a varie de 10 a 17 minutes avec une intensite de 0 milliampere, 5 a 1 milliampere, une distance de 0 m. 20 a 1 metre de l’anticathode et des etincelles de 0 m. 04 a 0 m. 10.” Not even a relative dosage range can be compared since she reported no lethality as a result of her experiments. This suggests that, with organisms such as H. nana which are capable of withstanding extremely high amounts of radiation before the effect is completely lethal, the doses required to produce a detectable increase in the rate at which certain morphological variants occur, also must be very high. From the viewpoint of proportionality, lower doses would produce correspondingly smaller changes in the rate of occurrence. If the doses were too low, the rate change may not be detectable on the basis of morphological criteria. Therefore, insufficient radiation may account for the failure of Palais to observe any differences between the control and experimental worms. Variations are known to have different causes. Permanent and transmissible variations involving chromosome aberration (e.g. breaks, inversions, translocations, losses and polyploidy), gene mutation, or segregation have been referred to as genotypic. Variations which occur

MORPHOLOGICAL

VARIATION

IN

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in response to the nature of the external environment, the physiological state of the internal environment, or the interactions of both have been classified as developmental or temporary changes. Difficulties in methodology are involved in arriving at a decision regarding the origin of a variation as a genotypic change in cestode. Although some cytological studies have been reported for a few species (Child, 1904, 1911, in Moniezia; Harman, 1913, in Tuenia; Young, 1908, 1912, 1919, 1935, in Taeniidae; Motomura, 1929, in Archigetes; Jones, 1945, in Hymenolepididae and Dilepididae; Jones and Ciordia, 1956, in Tueniu; and Jones and Wyant, 1957, in Tueniu) comparatively little is known about heredity in these organisms. In the present state of knowledge with regard to these worms it is not possible to know with certainty if events such as chromosome or gene mutations occur and how important a contribution they may make to the observed cases of variation. The problem is not simple since one must depend upon a phenotypic expression to indicate the change in genotype. Although the results of these preliminary investigations with X-irradiation have not permitted the separation of variations in H. nunu according to their basic causes, certain aspects of the observed effects suggest that some of the changes may be analogous to the phenomena of radiation-induced gene and chromosome mutations as observed in other organisms. For example, since Muller (1927) discovered that X-rays cause mutations in the fruit-fly, Drosophila, at a rate greatly in excess of the spontaneous rate, numerous experiments reported in the literature (reviewed by Lea, 1947, Dobzhansky, 1955; Hollaender, 1955) have indicated that the radiation-induced rise in the rate of gene mutations is directly proportional to the amount of additional exposure. For such organisms as have been studied, this principle is known to hold good when the radiation dose is fairly high and, at present, there does not appear to be sufficient evidence to warrant the assumption that there is any real departure from this principle even at the lowest doses. Accordingly, the American National Academy of Sciences, National Research Council (1956) and the British Medical Research Council (1956) committees, in reviewing the existing scientific evidence on the medical aspects of nuclear and allied radiations, have accepted this proportionality as a basis for attempting to assess, in terms of the incidence of certain specific grossly harmful conditions, the consequences for the individual and society of increasing the rate of mutation and to define the levels of dose which might be expected to bring about such an increase. In view of the foregoing, the fact that direct propor-

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tionalities were similarly observed in the case of certain variant characteristics in H. nuna may be suggestive that they also are phenotypic expressions of genotypic changes. Important criteria that require fulfillment in judging whether these variants are genetically based are those concerning the permanency and transmissibility of the observed changes. Unfortunately the number of generations of H. nana available for examination in this connection have been too few to permit an evaluation of these factors. Although there is fairly good evidence, in the case of testis-deletion, to indicate that the irradiation effect has been transmitted to the second generation, it has not been possible to determine the permanency of this change. It would be necessary to follow the effect in a number of subsequent generations to clarify this. Some other observations concerning the family Hymenolepididae may be worthy of mention in considering the likelihood that changes in testisnumber may have a genetic basis. Mayhew (1925), in a study of this family, was of the opinion that the ancestral form, from which the various species assigned to the genus Hymenulepis have evolved, contained at least six and as many as twelve testes which eventually became united, resulting in three compound organs with the different patterns of arrangement found in the present species. His evidence for the compound nature of the testes, which formed the basis for his opinion, was (1) the irregularities in the number and branching of the vasa efferentia, which he found in five species; (2) the lobing of the testes; and (3) the irregularities in the number of testes, which he found in one species. Mayhew’s concept implies that evolution in the family Hymenolepididae has proceeded from species with multiple testes through a series of intermediates toward species with a single testis. Although there are irregularities in the numbers of testes in H. nuncz, nothing was observed during the present study which would indicate that the testes are compound. However, assuming that the ancestral form from which the family Hymenolepididae evolved did contain multiple testes, the unstable nature of the middle testis in H. nunu may be an indication that reduction in testes numbers during the process of evolution may have occurred through a series of mutations leading to complete deletion instead of through union of these organs as thought by Mayhew. On the other hand, the observations with regard to testis-variability in H. nunu could be interpreted to mean that the middle testis has been added relatively recently (in terms of the evolutionary history of this species) and has not yet

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become stabilized. Addition, rather than reduction of testes would imply that evolution in the family Hymenolepididae may have been in the opposite direction. Considered from the phylogenetic point of view that the most highly developed cestodes may be expected to occur in the most highly developed hosts, host-occurrence in the family Hymenolepididae would tend to support the “loss” concept since the genera containing one and two testes (Haploparaxis and Diorchis) have been found only in birds, whereas the genera which contain more than two testes (Hymenolepis and Oligorchis) occur not only in birds, but in mammals as well. Indications that the evolution of the family Hymenolepididae has proceeded by such transitions from one genus to another may be found by a further consideration of some specific examples. Jones (1944) described Diorchis reynoldsi from the short-tailed shrew, Blarina brevicauda. This species was unique in the sense that it was the only species of the genus Diorchis reported from other than an avian host. In consideration of this, in addition to differences in the rostellar hooks, the Russian workers Skrjabin and Matevosian (1948) erected the new hymenolepidid genus Pseudodiorchis with Jones’ species as the type and only species. The writer (1953), unaware of the report of Skrjabin and Matevosian, examined the type specimen of Diorchis reynoldsi and found that it had three testes rather than two as reported in the original diagnosis. On the basis of this finding and the fact that the rostellar armature of this species is more characteristic of the genus Hymenolepis than it is of Diorchis, it was recommended that D. reynoldsi be assigned to the genus Hymenolepis. More recently, Oswald (1957) redescribed Pseudodiorchis reynoldsi (Jones, 1944) from specimens occurring in Blarina brevicauda in Ohio and presented data on variability of the number of testes in this cestode. In view of this new information concerning the characteristics of this species, the writer is in agreement with the need for a new genus to contain it. The anterior (middle) testis in this worm appears to be highly unstable and is either missing or shows a retarded development. Oswald found that, in ten strobilae of which 465 proglottids were examined, 61.1 per cent contained only two testes while 37.4 per cent contained three testes. He considered the retarded development of this organ and the fact that it was missing in over half of the proglottids, to be indicative that P. reynoldsi is evolving in the direction of a Z-testicular form. Hymenolepis diorchis Fuhrmann, 1913, a parasite of anseriform birds,

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is somewhat similar to P. reynoldsi in that only two testes are well develloped, the third being rudimentary and non-functional. Fuhrmann believed that this species clearly illustrated the transition from Hymenolepis to the 2-testicular genus Diorchis. Mayhew (1925), in the same study mentioned earlier in this report, found that, although a relatively small number of proglottids in Hymenolepis sacciperium contained only two testes, groups of cells, which stained in a manner similar to those of reproductive cells, usually occurred in the position of the missing testis. It will be recalled that similar observations were made with regard to the effect of X-irradiation on these organs in H. nana. Oswald (lot. cit.) is also of the opinion that the evidence shown by Pseudodiorchis reynoldsi, Hymenolepis diorchis and H. sacciperium tends to support the hypothesis that the reduction in testicular number in the genera of Hymenolepididae was caused by a gradual loss of testicular primordia rather than by a fusion of two or more primordia as postulated by Mayhew. In attempting to understand the basis for the variations observed in H. nana, consideration also must be given to the fact that many types of anomalous development are characterized, in one phase or another, by degeneration of tissue. The destructive changes may be merely visible expressions of aberration in some physiological process. Mitchell (1943) has shown that at least one of the disturbances preceding cellular degeneration, after X-ray exposure, is the inability of cells to synthesize desoxyribonucleic acid, although ribonucleic acid formation is not interfered with. Degeneration may be extensive and result in the destruction of the entire individual or it may be sharply localized and involve only a given region or structure. Such degenerative processes occur rather frequently as a step in normal morphogenesis. Experimental embryologists have demonstrated that normal development depends on an harmonious sequence of closely interdependent events. Such development is the end product of the expression of intrinsic potentialities of cells or groups of cells as conditioned and modified by their relationship to each other and to the rest of the embryo. Zwilling (1955) points out that in a situation where such a vast array of orderly interactions must occur in order that a normal structure be formed it is not surprising that deviations from the normal are frequently encountered. Disturbances in either the spatial or the temporal synchronization of the many developmental interrelationships may lead to abnormal individuals and structures.

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In the absence of more specific information, rather general hypotheses have been advanced to explain the action of agencies which produce abnormalities. One of the oldest of these is the concept of arrested development. Initially this hypothesis was utilized to describe the fact that, in many anomalies, development seems to have stopped at an early stage in the formation of the embryo or one of its component structures and that primitive features are retained. Later authors have extended the implications of this concept to include situations in which one does not necessarily find persistent embryonic conditions. In such instances the anomaly is supposedly preceded by temporary arrest at some stage and this arrest is the prime contributing factor to the subsequent abnormal development. Wolff (1948) considers arrests of development of sufficient importance in the production of anomalies that he postulates this as the first of his laws of teratogenesis. It has been seen in the present study that arrest of development, as observed in cysticercoids of H. nunu, is one of the more conspicuous effects of X-irradiating the eggs. Whether the subsequent morphological alterations exhibited by the adult cestode are in any way related to irradiation-induced but undetected temporary arrests at some stage in the early development of the larva, is unknown. Holtfreter and Hamburger (1955)) in discussing the various factors of embryological organization, apply the term “organizer”, in a provisional sense, to the determinative interactions between the germ layers during development. It seems possible that certain of the abnormalities observed in H. nuna, particularly those which are characterized by the absence of one or more of the reproductive organs and consequent sterility, or those involving changes in the positions of these structures, might also be attributable to the disturbance of “organizer” systems and that radiation only serves to increase the frequency of these disturbances. It is difficult, however, to believe that damage to a so-called “organizer” could be responsible for such phenomena as duplication of those organs which normally occur singly in the proglottids of H. nana such as that seen in the case of doubling of the cirri. According to Willier, Weiss, and Hamburger (1955), many of the teratologists of the past have become strong proponents of either environmental or hereditary factors as the exclusive cause of abnormalities. These authors are of the opinion, however, that observations of the last few decades indicate strongly that a compromise between the two is more representative of the facts-that not only may anomalies be mediated by both hereditary and environmental factors, but similar

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kinds of anomalies may be produced by either. The writer is inclmed to believe that this is the case where H. nuna is concerned. &J&NARY

1. Observations have been presented on the morphological effects of exposing eggs of Hymolepis nana to different doses of X-irradiation ranging from 5 to 40 kiloroentgens. Conspicuous effects in cysticercoids were malformations and arrested development. Abnormal appearing cysticercoids failed to develop to adults when fed to albino mice. 2. No adult cestodes were obtained, either directly or indirectly, from eggs exposed to more than 30 kr. 3. Below the lethal level of radiation, no abnormalities were observed which could not be found in non-irradiated specimens. Analyses indicated that X-irradiation induced an increase in the frequency with which a given variant characteristic occurred and that the frequency tended to be proportional to the dose received. 4. Data on frequency of occurrence, according to the r-dose received, have been presented for each of five variant characteristics. Comparisons made with non-irradiated specimens indicated that reliable estimates of the frequency with which a given morphological variant characteristic occurs in the normal population can be obtained by extrapolation of the irradiation data. Experimental evidence has been given in support of the proposed hypothesis that X-irradiation may serve as a useful tool in facilitating determinations of the nature, extent and possible directional trends of morphological variations in a population of cestodes. ACKNOWLEDGMENTS I wish to express my sincere appreciation to Dr. Clark P. Read for his friendly counsel during the course of this investigation. My thanks also are extended to Dr. Robert Dickson and members of his staff for providing radiological facilities, advice and technical aid. I am grateful to Dr. Helen Abbey for checking the statistical applications and to Dr. Bentley Glass for valuable suggestions in the preparation of the manuscript.

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