Arthur W. Jones

Resistance of mice to reinfection with the bile-duct cestode, Hymenolepis microstoma.

Abstract Mice, immunized with lethally irradiated 7-day-old Hymenolepis microstoma , were challenged with cysticercoids 8, 50, 100, and 150 days after inoculation. The challenge worms were removed after 10 days, counted, and measured. Worms in immunized mice were significantly fewer and shorter than controls. This inhibitory response arose between 8 and 50 days after immunization, and began to decline between 100 and 150 days, a pattern of response which has not previously been reported as due to lumen-inhabiting cestodes.

Autoelimination by means of x-rays: distinguishing the crowding factor from others in premunition caused by the mouse bile duct cestode, Hymenolepis microstoma.

Abstract Seven-day-old specimens of H. microstoma have been irradiated heavily in vitro (40 kr and 20 kr, X-rays) and implanted into the duodenum of recipient hosts (5 or 10 worms per mouse, respectively). After different intervals of incubation (8 and 20 days) following implantation, the hosts were challenged with cysticercoids by means of a stomach tube (5 or 10 cysticercoids per mouse, respectively). Autopsy after a week then revealed that in those hosts receiving the 40 kr-5 worms treatment, with 8 days incubation, the challenge of secondary worms were unaffected, although 11% of the primary worms was still present. In those hosts receiving the 20 kr-10 worms treatment, followed by 20 days incubation, both infection rate and length of the challenge worms were greatly reduced, compared with normal controls. Preliminary experiments had demonstrated a crowding effect: inhibition of establishment and growth of individual worms by pre-existing and coexisting infections. By autoelimination of pre-existing infections we showed that host resistance to H. microstoma occurs in the absence of any worms. Therefore this study succeeded in distinguishing a simple crowding effect from protective host response to a cestode.

X-ray induced abnormalities and recovery in Hymenolepis microstoma.

Abstract Seven-day old specimens of Hymenolepis microstoma were subjected to single doses of 2500, 5000, and 7500 R, X-rays, respectively, in vitro . The worms were transplanted into mice. Unirradiated worms were similarly transplanted as controls. After intervals of 2, 4, 6, 8, and 32 days, respectively, the hosts were sacrificed and the worms collected for study. Data were obtained on the morphology, number, and location of worms in each host; the morphology data were obtained only for 8-day and 32-day posttransplantation groups. Data on morphology suggest that tissues of the neck region may be radioresistant near the scolex, but relatively radiosensitive in the region where proglottids are forming. Radiosensitivity extends throughout the regions where proglottids are maturing and is evidenced by abnormalities of organ and segment formation. Damage is proportional to dose. Apparently complete recovery occurs by the thirty-second day after irradiation, the damaged strobila being replaced by a normal one. Both damage and recovery support the general view that radiation damage to growing organisms is largely a mitotic death effect. Cells which survive, in either an embryonic tissue, a bacterial population, or a cestode neck region, can mitotically replace the cells lost by radiation injury.

The chromosomes of Hymenolepis microstoma (Dujardin 1845).

The chromosomes of Hymenolepis microstoma, studied in Feulgen-stained squash preparations of germ cells and early embryos, are 12 diploid. Their average size in first cleavage metaphase is 2 to 5 p. The inequality in size of some homologous chromosomes is explained in terms of origin from unequally contracted pronuclear chromosomes. Centromeres are terminal; heterochromatic regions exist near them. Meiosis in the oocytes and mitosis and cleavage in the early embryos are briefly described. Papers by Jones et al. (1945, 1951, 1956, 1957), Kisner (1961), and Douglas (1962) on the chromosomes of various cestodes have created interest in further cytological investigation of this group. Hymenolepis microstoma, the bile duct tapeworm of mice, became available in 1959 for laboratory use. The present study is part of a series of investigations into the biology of this worm (Dvorak et al., 1961). MATERIALS AND METHODS For the analysis of chromosomes, 20 normal worms belonging to a strain which has been maintained in our laboratory for the past 3 years were removed from two mice 14 days after infection and fixed in Carnoys fixative (6:3:1 absolute alcoholchloroform-glacial acetic) for at least 12 hr. The specimens were then hydrated, hydrolyzed in water bath at 60 C in 1 N HCI for 12 min, rinsed in cold 1 N HC1, and stained in Feulgens leukobasic fuchsin (prepared according to Gray, 1954) for at least 1 hr. The stained materials were preserved in distilled water in the refrigerator. Squash preparations were made of a small region of the anterior third of each worm using a drop of 45% acetic acid and sealing the cover glass with clear fingernail polish. The chromosomes of selected cells were drawn with the aid of an oil immersion objective (N.A. 1.40) and a camera lucida. Chromosomes from about 50 slides of normal oocytes and early embryos were studied. Since chromosomal details were observed best in oocytes and early embryos, the present study was largely confined to these stages. RESULTS Normal chromosome complements in somatic cells of the early embryo typically consist of 6 pairs, or 12 diploid. Of these, four pairs of homologues are of small size (2 to 3 4), one pair is of intermediate size (4 p), and one pair is definitely larger than the rest (5 4). The average size range, therefore, in first cleavage metaphase is 2 to 5 p. In some cells the two members of the largest pair are somewhat unequal in size (Fig. 10); in others, this difference in size cannot be clearly observed. The location of the centromere is not clearly visible in the metaphase chromosomes, but from the observations f early anaphase of first cleavage it is obvious t at all centromeres are terminal or very nearly so. Separation in early anaphase always begins at one end of the metaphase chromosomes. Also, in anaphase all chromosomes are rod-shaped (Fig. 8) as expected if centromeres are terminal. In some metaphase figures of first cleavage, there appear some lightly stained regions near one end of the chromosomes. These regions resemble subterminal centromeres, a suggestion which is not supported by studies of anaphase figures. The lightly stained portions should be considered heterochromatic regions extending from the terminal centromeres. Because the chromosomes of the testes are quite small, little was learned from testis material other than confirmation of the haploid number from meiotic metaphase in primary spermatocytes (Fig. 1). As the ovaries mature, they become lobed and packed with germ cells. The germ cells Received for publication 16 August 1962. * This study was supported in part by U. S. AEC contract AT(40-1) 1749. t On deputation from the Pakistan Atomic Energy Commission in a doctoral program sponsored by the U. S. Agency for International Development.

Hatchability, viability, and infectivity of Hydatigera taeniaeformis eggs.

Eggs of Hydatigera taeniaeformis were tested in vitro for hatchability in an effort to predict in vivo results in laboratory animals. Hatching was accomplished in solutions containing cholesterol, trypsin, pancreatin, bile and sodium tauroglycolate. Twelve hatching solutions were used. Each differed from the others in either bile concentration, pancreatin concentration or hydrogen ion concentration. The hatching medium containing five % bile concentration gave the largest percentage of hatched eggs. There were no appreciable effects of changes in pancreatin concentrations and hydrogen ion concentrations. Viability was determined by observing motility in the hatched oncospheres and in the ability of the oncospheres to take up vital stains. In vivo tests were made using the best hatching medium. The per cent eggs hatched was compared to the per cent infectivity in the rat. The correlation coefficient was 0.73 for the results obtained. Although this was not a high correlation, it is thought that this method of determining infectivities should be further investigated.

Prolonged selfing in Hymenolepis microstoma (Cestoda)

Abstract A single cysticercoid of Hymenolepis microstoma, the mouse bile duct tapeworm, was fed via stomach tube to an HA/ICR strain albino mouse. After 20 days, gravid proglottids of the resulting adult worm (parental, or P generation) were fed to flour beetles, Tribolium confusum; 30 days later, one F1 cysticercoid from the beetles was fed to each of 10 mice. Gravid F1proglottids were subsequently recovered and fed to beetles as above. This procedure was repeated for 14 selfed generations without an apparent change in viability of either eggs or cysticercoids. The infection rate for 14 generations averaged 89%, varying from 80 to 100%. This rate approximated the 70–80% infection rate obtained from multiplyinfected mice. Morphological analysis of worms of all generations showed change in one parameter only, a slight increase in number of gravid proglottids with repeated selfing. The 14th generation resembled random-bred controls in all characteristics measured. The viability of selfed H. microstoma contrasts with that of selfed H. nana, as reported by earlier workers AND this difference may have evolutionary and immunological significance.

Chromosome analysis of Hymenolepis microstoma

Abstract The chromosomes of Hymenolepis microstoma were prepared by both micre-spread-dispersion and squash methods. For the former, an individual worm was macerated in distilled water and the neck cells and young embryos after hypotonic treatment were fixed in Carnoys fixative and dropped as a cell-fixative suspension on precleaned water-wet slides which were then air-dried. For squashes, whole worms were hypotonically treated and fixed in 50% acetic acid. Bits of tissue were broken up on slides in a drop of fixative, large fragments were removed, and cells and embryos were squashed under considerable pressure. The coverslip was removed after immersion in a Dry Ice-methanol bath. Following anhydrous methanol treatment, the slides were airdried. All slides were stained with Giemsa and made permanent with balsam. Chromosome analysis confirmed diploid 12, all acrocentric. Observations suggested the possibility of anaphase “pairing.” In the spread-dispersion slides the six longest chromosomes could be arranged in descending order of length, while the six smaller formed three “pairs,” statistically. Toughness of the embryophore necessitated pressure to obtain flat squashes; this treatment may have caused the observed variation in measured chromosome length.

HOST-RELATIONSHIPS OF RADIATION-INDUCED MUTANT STRAINS OF HYMENOLEPIS DIMINUTA.

Studies on the structure and behavior of radioinduced mutant strains of Hymenolepis diminuta are reviewed. The effects of radiation upon cestodes is classified as morphological, physiological, and cytological. (P.C.H.)

In vivo incorporation of tritiated cytidine and tritiated thymidine by the cestode, Hymenolepis microstoma.

The mouse bile duct cestode, Hymenolepis microstoma, was exposed in vivo in separate experiments to tiitiated cytidine and tritiated thymidine, respectively. Nucleolar and nuclear labeling, by cytidine, and nuclear labeling, by thymidine, as revealed by autoradiographic methods, occurred in both host and cestode tissues. As expected, only mitotically or synthetically active nuclei were labeled, those of the neck, developing organs, and embryos of the cestode. Bile-duct epithelium and pancreatic acinar cells, autoradiographed as a control, showed the same degree and localization of cytidine labeling as the active cells of the cestode. The assimilation of cytidine amd thymidine by direct absorption from the fluid contents of the bile duct implies that cestodes can absorb such materials from their immediate environment. (auth)

Establishment of Tribolium confusum populations after stress by parasitism with Hymenolepis microstoma.

Abstract The hypothesis that parasitism of Tribolium confusum by the cysticercoids of Hymenolepis microstoma reduces the reproduction potential of T. confusum was tested. Before this measurement (an equilibrium experiment) could be made, it was necessary to estimate the level of infestation of T. confusum (an infestation experiment). Mixed size, large and small T. confusumlarvae were used for this purpose. Each sample consisted of 30 larvae. Three replicates were used. The “small larvae” investigation from this infestation experiment was run concurrently with the next (equilibrium) experiment. The equilibrium experiment, for which the “small larvae” group provided an estimate of level of infestation, used a randomized complete block design. There were three groups, each with an original sample size of 30, and three replicates. This design allowed measurement of the numbers of adults at a given time (when the non-parasitized group was in equilibrium). From the analysis of this measurement, the hypothesis could be accepted or rejected. Four things were done to carry out this experimental design. First, pure stock cultures of T. confusum and H. microstoma were obtained. Second, sexing of T. confusum larvae was made unnecessary by using a large enough sample size to insure that a normal distribution of sexes would result. Third, infestation was accomplished by removing gravid proglottids from adult worms, macerating them on moist filter paper, and then leaving the beetle larvae on the filter paper for 48 hours with no other source of food. Finally, the numbers of larvae, pupae and adults in each of the 24 groups were counted twice weekly for 97 days. The infestation experiments compared large larvae with small larvae and larvae of mixed sizes as to degree and uniformity of infestation. While there were no significant differences between different sizes of larvae in either respect, the small larvae were most uniformly infested and were used concurrently and subsequently in the equilibrium experiments. When the numbers of adults that survived the process of infestation were analyzed, no significant difference was found between parasitized and non-parasitized groups. Any later differences in adult number could not be due to differences in initial numbers of adults, for there were none. When the numbers of adult beetles in each group at the time when the non-parasitized groups were in equilibrium were analyzed, the non-parasitized and parasitized groups were significantly different.