Kian Joe Lie

The ultrastructure of the amebocyte-producing organ in Biomphalariaglabrata

The amebocyte-producing organ (APO) in normal and echinostome-sensitized Biomphalaria glabrata was studied at the ultrastructural level. The APO in unexposed snails consists of small clusters of primary ameboblasts resting on the epithelial cells lining the pericardium. The ameboblasts are held in a loose reticulum formed by extensions from smooth muscle and few fibroblastic cells. Secondary ameboblasts and amebocytes constitute further stages of this cell line. Amebocytes, resembling cells in the snails circulation, appear in the blood sinus coursing through the interior of the APO. Exposure of snails to echinostome miracidia results in significant morphological changes in the organ. Large clusters of primary and and secondary ameboblasts appear, many of these cells undergoing mitosis. Fully activated APOs consist of masses of cells loosely arranged in zones of progressive maturation. Blood cells in activated APOs were significantly larger than those seen in normal APOs.

The Life History of Echinostoma paraensei sp. n. (Trematoda: Echinostomatidae)

The life history of Echinostoma paraensei, a new Brazilian species with 37 collar spines, has been completed experimentally. The planorbid snail Biomphalaria glabrata serves as first intermediate host, and intramolluscan stages also develop in Physa rivalis. Sporocysts usually develop in the heart. Rediae are of two types, distinguishable by large or small pharynges. Cercariae released 25 or more days postexposure develop into metacercariae in the pericardial sac and kidney of snails, sometimes in various tissues of snails infected with rediae. Adults develop in hamsters, mice, or rats after they are fed metacercariae, but not in pigeons, chicks, or ducklings. The natural final host is not known. In recent papers (Basch and Lie, 1966; Lie, 1966a, 1967; Lie et al., 1965) we described the destructive antagonism of larval echinostomes to trematode sporocysts developing in the same snail. Various echinostome species naturally transmitted by Biomphalaria glabrata (Say) in Brazil are being sought and evaluated to determine their efficiency in destroying Schistosoma mansoni Sambon. This paper describes the life history of a new species of echinostome encountered during our studies. MATERIALS AND METHODS Cercariae of this species appeared in collections of B. glabrata snails made in Belo Horizonte (Bairro Sao Domingos), Caratinga, and Bambui, all in the State of Minas Gerais, Brazil. Snails were shipped by air to San Francisco, California, where we studied the life histories of their trematodes. Cercariae were permitted to encyst in laboratory-bred B. glabrata, and metacercariae were fed to hamsters and albino rats and mice. Eggs from washed hamster stools produced miracidia, which were used principally to infect laboratory-raised albino B. glabrata of a strain obtained from the National Institutes of Health, Bethesda, Maryland. Infected snails were kept in clear plastic, 1-gal aquaria at 24 to 27 C and fed on red leaf lettuce (Lactuca sativa). Techniques for the study of the parasite were the same as in previous studies (Lie, 1963a; 1965; 1966b, c). All measurements are in microns. Received for publication 12 June 1967. * Supported by the University of California International Center for Medical Research and Training (UC ICMRT, Hooper Foundation, San Francisco School of Medicine) with Research Grants TW 00144 from the Office of International Research, and AI-07054-01 from the NIAID, NIH, U. S. Public Health Service; and by Rockefeller Foundation Grant GA-MNS6654. RESULTS Egg and miracidium (Figs. 1, 2) Eggs appear in stools of hamsters and mice 11 to 13 days (in rats 14 to 16 days) after infection, in uncleaved condition, yellow-brown, 104 to 122 by 74 to 86, with thickening at nonoperculated end of shell. Eggs kept in distilled water in a petri dish at 28 C begin to hatch in 11 days. Exposure to light stimulates hatching, which usually occurs in the morning. Newly hatched miracidia swim rapidly in the water for several hours and are positively phototactic. They enter the snail host through its exposed parts while shedding their epidermal plates. Complete penetration takes about half an hour. Miracidia penetrate into B. glabrata and also into Physa rivalis (Maton and Rackett) (sometimes referred to as Aplexa marmorata (Guilding), but we prefer the more commonly used name P. rivalis in the absence of an overall revision of neotropical Physidae). In both snails they develop into sporocysts which produce rediae and these in turn pro-

Studies on resistance in snails: Specific resistance induced by irradiated miracidia of Echinostoma lindoense in Biomphalaria glabrata snails

Abstract In juvenile Biomphalaria glabrata snails exposed to irradiated Echinostoma lindoense miracidia, the sporocysts migrated to the heart at the same speed as did nonirradiated sporocysts in control snails. However, in each snail so exposed to irradiated miracidia, amebocyte clumps in the snails heart destroyed the sporocysts within 2–9 days post-exposure. This process induced a strong, highly specific resistance to homologous reinfection in these previously susceptible snails. The snails remained susceptible to Schistosoma mansoni and Paryphostomum segregatum (Echinostomatidae), but were partially resistant to Echinostoma paraensei and E. liei, two echinostome species closely related to E. lindoense.

Studies on resistance in snails: A specific tissue reaction to Echinostoma lindoense in Biomphalaria glabrata snails

Abstract In juvenile albino Biomphalaria glabrata snails exposed for the first time to Echinostoma lindoense miracidia, and observed to be resistant, the sporocysts migrated to the heart at the same speed as they did in susceptible snails. However, in resistant snails the sporocysts were soon destroyed in the heart by amebocyte clumps. When these snails were then re-exposed to miracidia of the same species of trematode, the sporocysts were quickly destroyed soon after miracidial penetration, chiefly in the head-foot region. This strongly accelerated tissue reaction appears to have been induced by the previous contact with the same parasite. The sensitization of the snail tissues was highly specific: the hosts remained susceptible to Schistosoma mansoni and Paryphostomum segregation (Echinostomatidae), although partial resistance was observed against Echinostoma paraensei and E. liei , which are closely related to E. lindoense .

Tissue reactions induced by Schistosoma mansoni in Biomphalaria glabrata.

In Biomphalaria glabrata with a strong natural resistance, Schistosoma mansoni sporocysts are rapidly encapsulated by granulocytes and killed, mainly by the strong phagocytic activity of the cells. Irradiated Echinostoma paraensei sporocysts seem able to suppress the function of the granulocytes. Tissue reactions in snails with self-cure demonstrate: involvement of two types of cells, granulocytes and hyalinocyte-like cells; formation of amoeba-fibrous capsules; limited tendency of granulocytes to become attracted to the parasites; a slow process of parasite destruction; and a possible involvement of humoral factors. It seems that there is partial suppression of the granulocyte function in smails with self-cure.

Schistosoma mansoni: temporary reduction of natural resistance in Biomphalaria glabrata induced by irradiated miracidia of Echinostoma paraensei.

Abstract Sporocysts developing in the heart of the snail host from irradiated Echinostoma paraensei miracidia are unable to form rediae and can survive for only a brief time. However, they still were able to reduce temporarily the strong natural resistance to Schistosoma mansoni in juveniles of a strain of Biomphalaria glabrata selected for genetic resistance to this parasite. S. mansoni primary sporocysts, unable to survive in single (control) infections in this host strain, developed successfully in most snails in which irradiated E. paraensei sporocysts were present. After the echinostome sporocysts were destroyed by host amebocytes, the snails regained their natural resistance against a new infection by miracadia of S. mansoni. Successful initiation of an infection with S. mansoni was achieved only in the presence of living irradiated echinostomes. Once the schistosome infection had become well established, however, developing primary and secondary sporocysts could survive and produce cercariae, although the protecting echinostomes had by then been destroyed. Early growth stages of the primary sporocysts apparently are more vulnerable to the snails defensive reaction and generally do not survive unless protected by irradiated E. paraensei sporocysts. After the S. mansoni sporocysts grow older, they develop their own capacity to interfere with the snails natural resistance and continue to survive and produce progeny without further protection by the echinostomes. Irradiated sporocysts have a lower capacity to interfere with the snails natural resistance than do nonirradiated sporocysts. Suitability, as distinguished in this paper from susceptibility, can be separated from resistance of the snail to trematodes by employing double infections.

Studies on resistance in snails. 7. Evidence of interference with the defense reaction in Biomphalaria glabrata by trematode larvae.

Echinostoma lindoense sporocysts that develop from irradiated miracidia normally are destroyed by amebocyte capsules in the ventricle of Biomphalaria glabrata within 10 days postexposure. The survival period of these ventricular sporocysts was considerably longer in snails that also harbored normal sporocysts of E. lindoense, Paryphostomum segregatum, or Schistosoma mansoni. Protection of irradiated E. lindoense sporocysts by the same of different trematode species is presumed to be the result of an active process by which normal sporocysts interfere with capsule formation and protect themselves and other trematode larvae from encapsulation. Homologous protection was stronger than heterologous.

Elevation of aminopeptidase activity in Biomphalaria glabrata (Mollusca) parasitized by Echinostoma lindoense (Trematoda)

Abstract Comparisons of the levels of aminopeptidase activity in the hemocytes and serum of Biomphalaria glabrata at 20 and 30 days postexposure to irradiated Echinostoma lindoense miracidia with enzyme levels in control snails have revealed that there are significant elevations in the serum of snails at both time periods postexposure. Furthermore, there is a significantly higher level of aminopeptidase activity in the serum of snails at 30 days than at 20 days postexposure. Although the biologic function of the elevated levels of serum aminopeptidase in sensitized snails remains uncertain, it is possible that this lysosomal enzyme may degrade the surface proteins of secondarily introduced parasites and thus act as a form of acquired humoral immunity.

Failure of Echinostoma lindoense to reinfect snails already harboring that species

In Biomphalaria glabrata snails already harboring Echinostoma lindoense, attempts at reinfection with the same trematode species failed when the interval between exposures was 6 days or more. With 2 days between exposures, the experimental snails were as susceptible to reinfection as the control snails. Between these limits, the reinfection failure rate increased with the exposure interval. Failure to reinfect the snails was probably due to cannibalism, young sporocysts from the second infection being swallowed by predatory rediae from the first infection. Development of sporocysts in successfully reinfected snails was normal.

Leucocytosis in Biomphalaria glabrata sensitized and resensitized to Echinostoma lindoense

Leucocytosis was shown to occur in the pulmonate gastropod Biomphalaria glabrata exposed to the trematode Echinostoma lindoense. In these sensitized snails, the leukocyte count in the hemolymph was elevated 3 to 5 days postexposure to miracidia, and prior to complete encapsulation of sporocysts. This increase continued 1 to 5 days after destruction of sensitizing, irradiated E. lindoense sporocysts. Counts returned to normal levels after this period. A significant and more rapid increase in numbers of circulating leukocytes occurred 1 to 6 hr after reexposure of snails to a sensitizing dose of nonirradiated E. lindoense sporocysts. The leukocyte counts usually returned to normal levels after this period, except in snails in which some resensitizing sporocysts remained alive.