EXPERIMENTAL PARASITOLOGY 37, 380-387

Trichinella

(1975)

spirdis: Morphological Characteristics and Female Intestine-Infecting Larvae

of Male

J. KOZEK~

WIESLAW

of Immunology and Medical Microbiology, University of Florida, Gainesville, Florida; Department of Microbiology, University of Chicago,

Department

Chicago,

Illinois; and Department of Parasitology, Tulane University Medical School, New Orleans, Louisiana Accepted

September

19, 1974

KOZEK, W. J. 1975. Trichinella spiralis: Morphological characteristics of male and female intestine-infecting larvae. Experimental Parasitology 37, 380-387. The sex of encysted and excysted intestine-infecting T. spiralis larvae can be distinguished by the following morphological characteristics: the male larva has a long (approx 50 pm) rectum, and the anterior part of the testis is curved posteriorly. The female larva has a shorter rectum (approx 25 pm), a telogonic ovary, coiled uterine and seminal receptacle primordia, and a vaginal primordium. In paraffin sections males can be recognized by the spermatocytes which are of the same size. The oocytes vary in size: the smallest are located in the ventral portion, the largest on the dorsal portion of the ovary. Sex of the larvae can be differentiated by the length of the rectum as early as the tenth day, by the curvature of the anterior part of the testis and by the uterine primordium by ,the eleventh day, and by the presence of the vaginal primordium by the thirteenth day of intramuscular development. Farm’s Organ is believed to be the primordium of the seminal receptacle. INDEX DESCRIPTORS: Trichinella spiralis; Intestine-infecting larvae; Morphological characteristics; Rectum; Testis; Spermatocytes; Ovary; Oocytes; Uterine primordium; Farre’s Vaginal primordium; Seminal vesicle primordium; Intramuscular development; Organ; Swiss female mice.

One unique characteristic of intestineinfecting larvae (IIL) of Trichin&z spiral& is that their sexual maturity is comparable to that observed in the fourth- or early fifthstage larvae of other nematodes. This advanced development of the primary sex characteristics could be used to rapidly and accurately distinguish encapsulated, or excysted, male from the female larvae. Since the ability to administer inocula of determined sex ratio could be exploited to elucidate many questions of the basic biology of this parasite, these studies were 1 Present address: California Center, University of California, 95616.

Primate Research Davis, California

380 Copyright Q 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.

conducted (1) to review the morphological features by which the sex of the larvae can bc distinguished, (2) to determine when some of these characteristics can be recognized in the larvae during the intramuscular development, and (3) to identify the Farre’s Organ. MATERIALS

AND METHODS

Materials used in these investigations consisted of ( 1) living encapsulated IILs, (2) hematoxylin and eosin (H and E) stained sections of rat muscle containing encysted IILs, (3) developing larvae in muscles of rats, and (4) developing enteric stages in the mouse.

T. Sphdk:

MALE

AND FEMALE

LARVAE

MORPHOLOGY

381

FIGS. l-4. Encapsulated IILs of Trichinella spiralis. X 880. S-stichosome, I-intestine, T-testis. Fig. 1. Sharp tapering, sinusoid uterine primordium (UP) and granular, refractile seminal receptable primordium (SRP) = Farre’s Organ. Fig. 2. Blunt anterior pole of the testis (T). Posterior end of female (Fig. 3) and male larva (Fig. 4), arrows indicate intestine rectal junction. R-rectum.

Capsules ,containing 1-yr-old IILs were teased from rat muscles, placed in a drop of saline on a micros,cope slide, covered with a cover glass, and examined alive with a compound microscope.

H and E stained sections of infected rat muscle were part of the collection of Dr. R. M. Lewert, Department of Microbiology, University of Chicago, Chicago, Illinois. Larvae developing intramuscularly were

382

WIESLAW

J. KOZEK

FIGS. 5 and 6. Posterior ends of intramuscular Trichindlu spiralis larvae 15 days after X 940. Fig. 5. Male. inoculation. Arrows indicate intestine-rectal junction, R-rectum. Fig. 6. Female.

T. spiralis:

MALE AND FEMALE LARVAE MORPHOLOGY

obtained daily from rat diaphragms, from 6 to 29 days inclusive after oral inoculation of IILs, by the technique which has been described in another report (Kozek, 1971). Enteric stages were collected at hourly intervals up to 30 hr after inoculation from the intestine of Swiss female mice which received approximately 306 IILs ,per OS. The method of recovery was the same as that used to recover enteric stages from rats (Kozek, 1971), except that the worms were examined and photographed alive. RESULTS

The most striking differences between the male and female larvae can be observed in the region of the junction of the stichosome with the intestine, and in the region of the rectum (Figs. 14). At the intestinestichosome junction the anterior pole of the gonads differ in shape. The testis (Fig. 2) has a blunt anterior pole since the tip of the testis curves posteriorly. Due to the plane of its orientation the anterior hook of the testis is difficult to see in the mature IIL but it is relatively easy to see in developing larvae (Fig. 9). In the corresponding region of the female one can recognize three parts of the female genital system: the ovary, Farre’s Organ, and the uterine primordium (Fig. 1). The ovary is the largest organ. The Farre’s Organ, a group of smaller cells which contain refractile nuclei, is located between the anterior pole of the ovary and the base of the uterine primordium. The latter extends as a tapering, sinously bent, compressed cellular rod, anteriad from the Farre’s Organ. The rectum of the female is approximately 25 pm long; its length is approximately equal to the diameter of the larva

383

at the junction of the rectum with the intestine (Fig. 3). The rectum of the male is approximately 50 pm long, being about twice the diameter of the larva at the junction of the intestine with the rectum (Fig. 4). The vaginal primordium, another characteristic by which females can be distinguished from the males, is a collection of cells within the hypodermis on the ventral aspect of the larva, approximately 350 pm from the anterior end. When viewed en face, the primordium appears as an ovoid cellular plane, but from the lateral aspect, it appears as a bulge of the body wall protruding into the perienteric cavity against the stichosome (Fig. 8). The sex of the larvae within the muscles can be determined as early as 15 days after inoculation (= approx after 10 days of intramuscular development ) by the length of the rectum, which is longer in the male (Figs. 7, 8). In these and older larvae the ratio of the body diameter at the intestinerecta1 junction to the length of the rectum closely approximates that observed in mature IILs. At 16 days after inoculation the males can be differentiated from the females by the Aexure of the anterior pole of the testis (Fig. 9), which contrasts with the straight, anteriorly growing uterine primordium of the females (Fig. 12). Vaginal primordium is discernible at 18 days after inoculation (Fig. IO). The larvae can be sexed in paraffin sections by the morphology of the oocytes and the spermatocytes. In the telogonic ovary the oocytes are of uneven size, the smallest oocytes, with correspondingly small nuclei and nucleoli, are located in the dorsal portion, while the largest oocytes are present in the ventral

FIG. 7. Developing vaginal primordium (delineated by arrows) in intramuscular Trichinek spiral larva 18 days after inoculation. X 940. FIG. 8. Vaginal primordium (arrows) of mature TTichine&x spimlk IIL. X 940. FIG. 9. Flexure (arrows) of the anterior pole of testis in intramuscular T~ichinelk spir& larva 16 days after inoculation. X 940. FIG. 10. Straight uterine primordium in intramuscular Ttichinella spiralis larva 16 days after inoculation. X 940.

384

WIESLAW

FIGS. 11-14. tine, O-ovary, muscular larva, after inoculation postinocuIation. at the base of

J. KOZEK

Seminal receptacIe primordium (SRP) in Triclzinella spif .ali.s larvae. I- -inl FeesS-stichosome, U-uterus, UP-uterine primordium. X Fig. 1 1 . Inl ira25 days after inoculation. Fig. 12. Enteric stage before the first mIOlf:, 9 hr n. Fig. 13. Enteric stage, 19 hr postinoculation. Fig. 14. Ent eric stal 9, 25 llr Seminal receptacle primordium (Iarge arrow) appears as aL po1mh-like St1 -uct ure i:he uterus (U). Oviduct (small arrow) joins the ovary to the uterus.

T. spiralis: MALE AND FEMALE LARVAE MORPHOLOGY portion of the ovary (Fig. 15). In the hologonic testis the spermatocytes, their nuclei and nucleoli, are of uniform size in ventral, dorsal and lateral regions (Fig. 16). DISCUSSION

In contrast to other parasitic nematodes, the infective stage of which is either a first-, second-, or #third-stage larva, the intestineinfecting larva of T. spiralis is almost an adult. This high level of maturity attained by the larvae in the muscles, especially the development of the gonads, provides sufficient number of morphological characteristics to distinguish the male from the females, regardless whether the larvae examined are encapsulated, free, or in tissue sections. Since the recognition of the sex of larvae in tissue sections is primarily of academic interest, emphasis will be placed on the features and methods by which the living larvae can be sexed. Examination of an encapsulated or a free larva through a compound microscope at low magnification (100 X ) is usually sufficient to sex the larvae, although examination at higher magnifications may be necessary to ascertain the identification in some cases. In the experience of this writer, the characteristic which is most striking, easiest to recognize, and most useful, is the length of ‘the rectum. If one bears in mind that in the female the length of the rectum is approximately equal to the diameter of the larva at the intestine-rectal junction, whereas the rectum of the male is 1.5-2 times the diameter of the larvae at the intestine-rectal junction, it is not necessary to measure the length of the rectum with the aid of an ocular micrometer. The second most useful characteristic is the shape of the anterior pole of the gonad: blunt in the male, tapering to a fine ‘point in the female.

385

Sirme the vaginal primordium is best seen at higher magnifications, it is not as useful as the other two features. After considerable experience, it is possible to sex the larvae accurately with the aid of a dissecting microscope ( at approx 50 X ). The first published attempt to sex the larvae of T. spiralis can be attributed to Farre (1835) who, within months after Owen ( 1835) published the description of T. spimh larva, distinguished the female from the male larvae by the presensce, in the females, of a structure which he believed to be the ovary. This structure has been referred to as the “Farre’s Organ” by subsequent investigators. Pagenstecher ( 1865) has not only differentiated the sexes by the shape of the gonads, but described the coils of the uterine primordium which he interpreted to represent eggs in the uterus. This opinion was shared by Bugge ( 1934), Hemmert-Halswick and Bugge ( 1934)) and Thomas ( 1965). Leuckart (1876) mentioned the flexure of the anterior ‘pole of the testis. Bugge (1934) and HemmertHalswick and Bugge (1934) described the vagina1 primordium, and summarized the characteristics, known up to that time, of the male and female larvae. Their summary has been published in English by Gould (1945), and Table I represents an updated modification of Gould’s list, including Villella’s (1966) criterion based on the number of stichocytes, but omitting others, e.g., distance of the posterior tip of the gonad to the anus, since this distance is partly determined by the length of the rectum. The features by which the larvae could be sexed during the intramuscular development were discerned in the current study earlier than the observations reported by other investigators, but the differences are

FIGS. 15 and 16. Cross sections through the gonad of mature encapsulated T&&E& spiralis IILs. X 1250. Fig. 15. Female, note small oocytes (small arrows) in the dorsal portion of the ovary, large oocytes (large arrow) at the ventral portion. Fig. 16. Male, note uniform size of spermatocytes (arrows), nuclei and nucleoli in all portins of the testis.

386

WIESLAW

J. KOZEK

TABLE Morphological

Characteristics

Feature Vaginal

primordium

No. of stichocytes stichosome Shape of anterior Location

of gonad

Uterine

primordium

Seminal receptacle

Length

I

qf Male and Female Intestine-Infecting -

Larvae of Trichinella

spiralis

Males

Females

-

On ventral aspect of body wall, approximately 350 pm from the anterior tip of larva

49 (47-52)

43 (37745)

in the pole of gonad

Blrmt,

test,is curves posteriorly

Dorsal

to intestinal

Sharp, anteriorly primordium \‘entral

ampulla

-

Gonad (in cross section)

t.0 intestinal

Group of located uterine pole of Approximately

50 pm

Hologonic testis : spermatocytes, nrmlei and nucleoli of uniform

not considered to be significant. Ali Khan (1966) could sex the larvae by the length of the rectum at 16 days p.i.; Thomas (1965) could distinguish the males by the flexion of the anterior pole of the testis at 17.5 days p.i. Moreover, the present study revealed that the vaginal primordium was discernible at 18 days p-i. The structure observed and described by Farre (1835) as “. . . aggregation of small round granules, about dozen in number, situated at about one-fifth of the length of the animal from the larger extremity, and generally close to that side of the coil which is outermost,” has not been previously identified with any degree of certainty. Farre believed it to be the ovary, but Pagenstecher (1865) speculated it to be either the anterior end of the ovary or the base of the uterus. Results presented in this communkation indicate that the relationship of the Farre’s Organ to the uterus and the ovary is ‘constant during the enteric development of the larvae: this granular

nterine

ampulla

Tapering, sinuous cellular rod anterior to the seminal receptacle primordium

primordium

of rectum

tapering

size

highly refringent cells between the base of primordiiim and anterior t,he ovary

Approximately

2-i pm

Telogonic ovary oocytes, large size of nuclei parallels size

: small ventral dorsal oocytes ; and nncleoli of oocytes

structure always occupies the space corresponding to the base of the uterus. Since Fig. 14 indicates that the Organ is not the oviduct, and the only other component of the female reproductive system which has the same relationship is the seminal receptacle (cf. Figs. 7 and 8 of Wu, 1955), there seems to be no reason to doubt that the Farre’s Organ is the primordium of the seminal receptacle. ACKNOWLEDGMENTS

Presented at the Third International Conference on Trichinellosis. Miami Beach, Florida, November 2-4, 1972. Supported in part by USPHS Postdoctoral Fellowship 5F2-AI-44, 307 and by Public Health Service Grants Al-00884, Al-05345, AI-04919, and AI-00002 from the NIAID. The author is grateful to Drs. Robert M. Lewert, Richard B. Cranclall, and Catherine A. Crandall for their stimulus and encouragement during these investigations, and would like to thank Ms. Rina Girard Kaminsky for the translation of pertinent sections of the German references.

T. spiralis:

MALE

AND FEMALE

REFERI~N~I~S ALI KHAN, Z. 1966. The postembryonic development of TrzUzinelZu spiralis with special reference to ecdysis. Journal of Parasitology 52, 248-259. BUGGE, G. 1934. Trichinen im Darm. Archiv fiir Wissenschaftliche und Praktixhe Tierheilkunde 68,24-32. FAME, A. 1835. Observations on the Trichina spiralis. London Medical Gazette 17, 382-387. GOULD, S. E. 1945. “Trichinosis.” Charles C Thomas, Springfield, Illinois, 356 pp. HEMMERT-HALSWICK, A., AND BUGGE, G. 1934. Trichinen und Trichinose. Ergebnisse der Augemeinen Pathologie und Pathologischen Anatomie des Menschen und der Tiere 28, 313-392. KOZEK, W. J. 1971. The molting pattern in Trichinella spiralis. I. A light microscope study. 57, 1015-1028. .IozrrnuZ of Parasitology

LARVAE

MORPHOLOGY

387

LEUCKART, R. 1876. “Die Parasiten des Menschen und die von ihren Herruhrenden Krankheiten,” Vol. II. C. F. Winter’sche Verlagshandlung. Leipzig and Heidelberg, pp. 512-609. OWEN, R. 1835. Description of a microscopic entozoon infesting the muscles of the human body. Transactions of the Zoological Society (London) 1,315-324. PAGENSTECHER, H. A. 1865. “Die Trichinen.” Verlag von Wilhelm EngeImann. Leipzig, 116 pp. THOMAS, H. 1965. BeitrLge zur Biologie und mikroskopischen Anatomie von Trichinella spiral% Owen 1835. Zeitschrift fiir Tropemedicin und Parasitologie 16, 148-180. VILLELLA, J. B. 1966. Morphologic criteria for distinguishing the sex of Trichinella spiralis larvae from muscle. Journal of Parasitology 52,908-910. WV, L.-Y. 1955. Studies on TrichinelIa spiralis. I. Male and female reproductive systems. Journal of Parasitology 41, l-8.

Trichinella spiralis: morphological characteristics of male and female intestine-infecting larvae.

EXPERIMENTAL PARASITOLOGY 37, 380-387 Trichinella (1975) spirdis: Morphological Characteristics and Female Intestine-Infecting Larvae of Male J...
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