Development of Eimeria tenella in MDBK cell culture with a note on enhancing effect of preincubation with chicken spleen cells

Eimeria tenella, an intracellular protozoan parasite infecting the epithelial cells of the ceca of chickens, causes severe diarrhea and bleeding that can lead its host to death. It is of interest that E. tenella first penetrate into the mucosal intraepithelial lymphocytes (IEL) before they parasitize crypt or villous epithelial cells. This in vitro study was undertaken to know whether the penetration of E. tenella into such a lymphoid cell is a beneficial step for the parasite survival and development. Three sequential experiments were performed. First, the in vitro established bovine kidney cell line, MDBK cells, were evaluated for use as host cells for E. tenella, through morphological observation. Second, the degree of parasite development and multiplication in MDBK cells was quantitatively assayed using radioisotope-labelled uracil (3H-uracil). Third, the E. tenella sporozoites viability was assayed after preincubation of them with chicken spleen cells. E. tenella o?cysts obtained from the ceca of the infected chickens were used for the source of the sporozoites. Spleen cells (E) obtained from normal chickens (FP strain) were preincubated with the sporozoites (T) at the E:T ratio of 100:1, 50:1 or 25:1 for 4 or 12 hours, and then the mixture was inoculated into the MDBK cell monolayer. Morphologically the infected MDBK cells revealed active schizogonic cycle of E. tenella in 3-4 days, which was characterized by the appearance of trophozoites, and immature and mature schizonts containing merozoites. The 3H-uracil uptake by E. tenella increased gradually in the MDBK cells, which made a plateau after 48-60 hours, and decreased thereafter. The uptake amount of 3H-uracil depended not only upon the inoculum size of the sporozoites but also on the degree of time delay (preincubation; sporozoites only) from excystation to inoculation into MDBK cells. The 3H-uracil uptake became lower as the preincubation time was prolonged. In comparison, after preincubation of sporozoites with spleen cells for 4 or 12 hours, the 3H-uracil uptake was significantly increased compared with that of control group. From the results, it was inferred that, although the penetration of E. tenella sporozoites into the lymphoid cells such as IEL is not an essential step, it should be at least a beneficial one for the survival and development of sporozoites in the chicken intestine.     

Cystoisospora belli: in vitro multiplication in mammalian cells

Intracellular development of Cystoisospora belli was demonstrated in 4 different mammalian cell lines. Human ileocecal adenocarcinoma (HCT-8), epithelial carcinoma of lung (A549), Madin-Darby bovine kidney (MDBK), and African green monkey kidney (VERO) were exposed in vitro to C. belli sporozoites, which had been isolated from the feces of HIV-AIDS patients. Parasites invaded all the cellular types between 4 and 12h after exposure and multiplication was demonstrated after 24 h. Grater number of merozoites formed in VERO cells, followed by HCT-8. In the MDBK and HCT-8 cells, the parasitophorous vacuole was less evident and immobile merozoites were observed in the cytoplasm. In VERO cells, one or several parasitophorous vacuoles contained up to 16 mobile sporozoites. No oocysts were found in any of the cell types used. VERO cells may be suitable for studies of the interaction between parasite and host cells.     

Microbial adhesion of Cryptosporidium parvum sporozoites: purification of an inhibitory lipid from bovine mucosa

Cryptosporidium parvum is a protozoan pathogen of humans and livestock worldwide. Its ability to infect a wide range of species raises questions as to the involvement of a specific host cell receptor for parasite-host recognition. To investigate the mechanism of parasite-host cell recognition, we have developed an in vitro cell suspension binding assay to investigate adhesion of C. parvum sporozoites to host cells. Morphologic features of binding events observed with this assay were identical to those described in natural infections. Glycoconjugates, Madin Darby bovine kidney (MDBK) cell fractions, and plasma membrane vesicles (PMVs) were screened for their ability to block binding of sporozoites to MDBK cells. Mucins, MDBK cell fractions, and PMVs exhibited dose-dependent inhibition of sporozoite binding. The major inhibitory fraction from MDBK cells was found to be insoluble in aqueous medium, nonsaponifiable, and lacking carbohydrate moieties, nitrogen, and phosphorus. Its inhibitory effect was resistant to heat, protease digestion, and glycosidase treatment, suggesting that the inhibitory activity is a lipid or a lipid-like component. The inhibitory activity was purified from MDBK cells, and in larger amounts from bovine small intestinal mucosa, by organic solvent extraction, semipreparative high-pressure liquid chromatography, and preparative high-performance thin-layer chromatography. Biochemical analyses, thin-layer chromatography staining techniques, mass spectrometry, and elemental analysis were used to partially characterize the purified lipid. These results indicate that a host intestinal lipid(s) or a lipid-like component(s) may play an important role in the early stages of host cell invasion by C. parvum.     

Changes in the Cytoplasmic Elements of Cultured Cells Infected with Eimeria vermiformis Sporozoites

Epithelial-type (PK-15) and fibroblast-type (MDBK) mammalian cell cultures were inoculated with purified Eimeria vermiformis sporozoites. Matched samples from 0 to 93 h after inoculation (HAI) were processed for electron microscopy; half of the sample preparations were extracted with non-ionic detergent prior to fixation. Specimens were examined by both transmission and scanning electron microscopy. Numerous sporozoites were attached to the cultured cells from 2 to 93 HAI, usually near the cell periphery. Some host cell microvilli extended up and appeared attached to the sporozoites. Sporozoites fixed during the penetration process were markedly constricted at the site of entry; however, no noticeable changes occurred in the host cell membrane or surface microvilli during sporozoite invasion or in sporozoite-infected cells. In cells extracted with 1% Triton X-100, the host cytoskeleton was progressively reorganized about the parasites but changes were limited to the immediate area of the sporozoite. Around resident sporozoites, the cytoskeleton became less dense but also more ordered, which contrasted with adjacent cell areas. Cytoskeletal elements passed both over and under the parasites. The appearance of the cytoskeleton suggested that the host cell formed a loose, basket-like net of cytoskeletal elements about the parasite.     

Changes in the cytoplasmic elements of cultured cells infected with Eimeria vermiformis sporozoites

Epithelial-type (PK-15) and fibroblast-type (MDBK) mammalian cell cultures were inoculated with purified Eimeria vermiformis sporozoites. Matched samples from 0 to 93 h after inoculation (HAI) were processed for electron microscopy; half of the sample preparations were extracted with non-ionic detergent prior to fixation. Specimens were examined by both transmission and scanning electron microscopy. Numerous sporozoites were attached to the cultured cells from 2 to 93 HAI, usually near the cell periphery. Some host cell microvilli extended up and appeared attached to the sporozoites. Sporozoites fixed during the penetration process were markedly constricted at the site of entry; however, no noticeable changes occurred in the host cell membrane or surface microvilli during sporozoite invasion or in sporozoite-infected cells. In cells extracted with 1% Triton X-100, the host cytoskeleton was progressively reorganized about the parasites but changes were limited to the immediate area of the sporozoite. Around resident sporozoites, the cytoskeleton became less dense but also more ordered, which contrasted with adjacent cell areas. Cytoskeletal elements passed both over and under the parasites. The appearance of the cytoskeleton suggested that the host cell formed a loose, basket-like net of cytoskeletal elements about the parasite.     

High-titer replication of nondefective Sendai virus in MDBK cells.

Egg-grown Sendai virus was adapted to growth in a bovine kidney cell line (MDBK cells) by serial passage under defined conditions. The adapted virus contained only 50S RNA and was highly infectious for MDBK cells. Infection of these cells with a high multiplicity of adapted virus resulted in a yield of 10(8) MDBK-infectious units/ml by 18 h, accompanied by severe cytopathic changes in the host. Cell fusion did not occur. Examination of the proteins of the adapted virus revealed that despite the high infectivity of this virus for MDBK cells the virions contained considerable quantities of Fo, the precursor to the F glycoprotein that is responsible for cell fusion and high infectivity in other systems.     

Host-Dependent Restriction of Mengovirus Replication

Mengovirus infection of a restrictive cell line, Maden's bovine kidney (MDBK), results in a virus yield 1,000-fold less than that obtained from productively infected cell lines such as L cells or Ehrlich ascites tumor cells (EAT). Cells of both types of host systems are infected with comparable efficiencies and are completely killed as a consequence of infection. Infective center assays, coupled with the observation of total cell killing, suggest that comparable numbers of cells synthesize viral antigen and release virus in both types of host system. Viral-specific ribonucleic acid (RNA) synthesis is initiated and proceeds in an identical fashion for approximately 4 hr after the infection of MDBK, EAT, or L-cells. At this time, viral RNA synthesis in MDBK ceases, whereas viral RNA synthesis in EAT and L-cells continues at a linear rate. These results indicate that none of the early viral events leading to the initiation of viral-specific RNA synthesis constitutes the primary site of mengovirus restriction in MDBK. Rather it appears that the cessation of viral RNA synthesis in restrictive cells constitutes the primary limiting event. Based on its delayed interaction with mengovirus RNA synthesis, it appears that the host-related restrictive agent is initially compartmentalized and then released as a consequence of infection subsequent to those early events in mengovirus infection leading to the initiation and continued synthesis of viral RNA.     

Development and characterization of bovine x human hybrid cell lines that efficiently support the replication of both wild-type bovine and human adenoviruses and those with E1 deleted

The 293 cell line that was generated by transforming human embryonic kidney cells with human adenovirus type 5 (HAV5) early region 1 (E1) sequences is an excellent host for generating and growing HAV5 recombinants with E1 deleted, but it does not support the replication of bovine adenovirus type 3 (BAV3). Madin-Darby bovine kidney (MDBK), an established bovine cell line, is an excellent host for growing and plaquing BAV3. For the purpose of combining the unique characteristics of these two cell lines (293 and MDBK), we generated a number of bovine x human hybrid (BHH) cell lines. Comparison of three BHH hybrid clones-BHH3, BHH8, and BHH2C-with 293-Puro (puromycin-resistant 293 cells) and MDBK-Neo (G418-resistant MDBK cells) cell lines for total cellular DNA content, species-specific surface markers, isoenzyme analysis, and karyotyping indicate that they are hybrid in nature. BHH clones constitutively expressed the E1 proteins (E1A, E1B-21kDa, and E1B-55kDa) of HAV5 and efficiently supported the replication of both wild-type and replication-incompetent bovine or human adenoviruses. Transient gene expression experiments with a plasmid encoding the bacterial beta-galactosidase gene demonstrated that BHH cell hybrids seem to have better transfection efficiencies than either of the parental cell lines. These cell lines will be useful for isolating and growing replication-competent human or bovine adenovirus recombinants with E1 deleted and for the study of cellular or viral factors important for viral replication. The development of somatic cell hybrids appears to be a simple way of combining some of the desirable characteristics present separately in two parental cell lines.     

COMPARATIVE DEVELOPMENT OF THE RED ALGAL PARASITES BOSTRYCHIOCOLAX AUSTRALIS GEN. ET SP. NOV. AND DAWSONIOCOLAX BOSTRYCHIAE (CHOREOCOLACACEAE,RHODOPHYTA)1

The development of two red algal parasites was examined in laboratory culture. The red algal parasite Bostrychiocolax australis gen. et sp. nov., from Australia, originally misidentified as Dawsoniocolax bostrychiae (Joly et Yamaguishi-Tomita) Joly et Yamaguishi-Tomita, completes its life history in 6 weeks on its host Bostrychia radicans (Montagne) Montagne. Initially the spores divide to form a small lenticular cell, and then a germ tube grows from the opposite pole. Upon contact with the host cuticle, the germ tube penetrates the host cell wall. The tip of the germ tube expands, and the spore cytoplasm moves into this expanded tip. The expanded germ tube tip becomes the first endophytic cell from which a parasite cell is cut off that fuses with a host tier cell. The nuclei of this infected host cell enlarge. As parasite development continues, other host-parasite cell fusions are formed, transferring more parasite nuclei into host cells. The erumpent colorless multicellular parasite develops externally on the host, and reproductive structures are visible within 2 weeks. Tetrasporangia are superficial and cruciately or tetra-hedrally divided. Spermatia are formed in clusters. The carpogonial branches are four-celled, and the carpogonium fuses directly with the auxiliary (support) cell. The mature carposporophyte has a large central fusion cell and sympodially branched gonimoblast filaments. Early stages of development differ markedly in Dawsoniocolax bostrychiae from Brazil. Upon contact with the host, the spore undergoes a nearly equal division, and a germ tube elongates from the more basal of the two spore cells, penetrates the host cell wall, and fuses with a host tier cell. Subsequent development involves enlargement of the original spore body and division to form a multicellular cushion, from which descending rhizoidal filaments form that fuse with underlying host cells. This radically different development is in marked contrast to the final reproductive morphology, which is similar to B. australis and has lead to taxonomic confusion between these two entities. The different spore germination patterns and early germ-ling development of B. australis and D. bostrychiae warrant the formation of a new genus for the Australian parasite.     

Development of Hammondia heydorni in cultured bovine and ovine cells

Sporozoites were excysted from oocysts of Hammondia heydorni obtained from a naturally-infected dog and inoculated into monolayer cultures of bovine pulmonary artery endothelial cells (CPA), Madin-Darby bovine kidney (MDBK) cells, bovine monocytes (M617), or ovine monocytes (WOMO). Sporozoites penetrated all four cell lines and underwent asexual reproduction by endodyogeny (as determined by electron microscopy) to form cyst-like structures at four to nine days after sporozoite inoculation (DAI). At 4-10 DAI, considerably more zoites were harvested from M617 cultures (80.1 x 10(6) zoites) than from CPA (17.4 x 10(6], MDBK (47.3 x 10(6], and WOMO (53.5 x 10(6]. Little or no parasite multiplication occurred at 10-16 DAI. Zoites harvested at 7 DAI and transferred to freshly prepared cultures did not penetrate cells nor develop further.     

Host-dependent restriction of mengovirus replication. II. Effect of host restriction on late viral RNA synthesis and viral maturation.

Restricted mengovirus replication in Mandin-Darby bovine kidney (MDBK) cells is characterized by a 400-fold reduction in infectious virus yield and a 40-fold increase in the production of noninfectious virus. Using conditions which insure that all MDBK cells are infected, virus-specific RNA and protein synthesis were measured in the restrictive host and in a permissive host for mengovirus, HeLa cells. Labeling kinetics and sucrose gradient analysis of mengovirus-specific RNA from MDBK cells show a reduction of 10-fold in virion RNA, 5-fold in double-stranded RNA, and 12.5-fold in single-stranded RNA. The viral RNA biosynthetic processes which occur late in the replicative cycle and result in the production of 90% of the single-stranded viral RNA that is packaged into capsid proteins in the permissive host are absent in restrictive MDBK cells. Viral protein synthesis as measured by labeled viral-specific polysome is decreased, and there is an accumulation of 80S subviral particles in the restricted host. It is suggested that restriction may act at a number of stages of viral replication and maturation.     

The SH integral membrane protein of the paramyxovirus simian virus 5 is required to block apoptosis in MDBK cells

In some cell types the paramyxovirus simian virus 5 (SV5) causes little cytopathic effect (CPE) and infection continues productively for long periods of time; e.g., SV5 can be produced from MDBK cells for up to 40 days with little CPE. SV5 differs from most paramyxoviruses in that it encodes a small (44-amino-acid) hydrophobic integral membrane protein (SH). When MDBK cells were infected with a recombinant SV5 containing a deletion of the SH gene (rSV5DeltaSH), the MDBK cells exhibited an increase in CPE compared to cells infected with wild-type SV5 (recovered from cDNA; rSV5). The increased CPE correlated with an increase in apoptosis in rSV5DeltaSH-infected cells over mock-infected and rSV5-infected cells when assayed for annexin V binding, DNA content (propidium iodide staining), and DNA fragmentation (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling assay). In rSV5DeltaSH-infected MDBK cells an increase in caspase-2 and caspase-3 activities was observed. By using peptide inhibitors of individual caspases it was found that caspase-2 and caspase-3 were activated separately in rSV5DeltaSH-infected cells. Expression of caspase-2 and -3 in rSV5DeltaSH-infected MDBK cells appeared not to require STAT1 protein, as STAT1 protein could not be detected in SV5-infected MDBK cells. When mutant mice homologous for a targeted disruption of STAT1 were used as a model animal system and infected with the viruses it was found that rSV5DeltaSH caused less mortality than wild-type rSV5, consistent with the notion of clearance of apoptotic cells in a host species.     

Development and Structure of the Haustorium of the Parasite Rhamphicarpa fistulosa (Scrophulariaceae)

Rhamphicarpa fistulosa (Hochst.) Benth. (Scrophulariaceae), a parasite of African cereals, develops secondary haustoria which penetrate the roots of the host plant. Light and electron microscopy have been used to study the structure and development of haustoria in this species, which, until now, have not been well characterized. Haustoria are initiated in the hypodermis of the parasite roots. A meristematic strand is developed between the parasite root stele and the host-parasite interface. From this strand, cells differentiate into xylem elements after penetration of the host root. Xylem differentiation follows an acropetal pattern. Mature haustoria are characterized by a continuous xylem bridge between water conducting elements of parasite and host. A detailed study of the hostparasite interface revealed the presence of collapsed and compressed host cells at the lateral interface (between parasite cells and host cortex), whereas the central interface between parasite cells and the host stele is almost devoid of host cell remnants. Implications of these observations for the penetration mechanisms are discussed.     

Host cell apoptosis impairs Cryptosporidium parvum development in vitro

The absence of a self-sustaining in vitro propagation method for Cryptosporidium parvum is a major obstacle for research on this parasite. Conventional cell monolayers are unsuitable for long-term parasite propagation because the level of infection decreases over time and few oocysts, if any, are produced. The interaction between parasite and host cell was studied to identify factors limiting parasite development in vitro. Loss of substrate adherence and death of parasitized host cells was observed in 2 epithelial cell lines. Nuclear morphology, DNA laddering, annexin V binding, and terminal deoxytransferase-mediated dUTP nick end labeling indicated that host cell death occurred by apoptosis. At 6 hr postinfection, only a minority of infected cells remained in the monolayer, and few survived the initial phase of parasite development without losing adherence. Treatment of infected monolayers with caspase inhibitors drastically reduced cell detachment but failed to increase the number of parasites in monolayers. In contrast, cell cultures grown on laminin-coated plates showed a higher proportion of infected cells. These observations indicate that cell detachment and apoptosis in C. parvum-infected cell culture negatively affect parasite survival in vitro.     

Development of Eimeria alabamensis from Cattle in Mammalian Cell Cultures*

SYNOPSIS. Monolayer primary cultures of cells from bovine embryonic intestine (BEInt), kidney (BEK), spleen (BES), and thyroid (BETy) and cell line cultures of embryonic bovine trachea (EBTr) and synovium (BESy) as well as established cell line cultures of bovine kidney (Madin-Darby, MDBK), human intestine (Int 407) and Syrian hamster kidney (BHK) were inoculated with freshly excysted sporozoites of Eimeria alabamensis and observed for 4–5 days. Sporozoites penetrated all cell types; during the 1st 24 hr, intracellular sporozoites, trophozoites and binucleate schizonts were seen in all cell cultures. Mature schizonts were more numerous in BES and MDBK cells than in the others. Large schizonts, 14.2 (11–18.5) by 10.2 μ (8.5–11), with 6–14 short, stubby merozoites (each with 2 refractile bodies) occurred at 2 and 3 days in all cells except BESy, Int 407, and BHK. Small schizonts, 9.7 (5.5–13) by 6 μ (5–8.5), with 6–10 long, slender merozoites (each with 2 refractile bodies) were found 3 days after inoculation in all cell types. At 4 days, some intracytoplasmic merozoites and a few intranuclear 2nd generation trophozoites were found. After 4 days post-inoculation, intracellular parasites were rarely seen and these were apparently degenerate. Development within the host cell nucleus, the normal site of development in the host animal, was observed infrequently in cell cultures. Intranuclear sporozoites, found no earlier than 2 days after inoculation, developed similarly to those in the cytoplasm, and small intranuclear schizonts with 6–10 merozoites (each with 2 refractile bodies) occurred after 3 days in culture.     

THE EVOLUTION OF PARASITISM IN RED ALGAE: CELLULAR INTERACTIONS OF ADELPHOPARASITES AND THEIR HOSTS1

In the initial stages of cell–cell interactions (spore germination and host penetration), the adelphoparasites Gardneriella tuberifera Kyl. and Gracilariophila oryzoides Setch. & Wilson form infection rhizoids that fuse directly with underlying host epidermal or cortical cells. In so doing, parasite nuclei and other organelles enter the cytoplasm of the host. The resulting heterokaryon may fuse with adjacent host cells either directly, via secondary pit connections, or by the dissolution or dislodgment of pit plugs from existing pit connections. The cell fusion events result in a heterokaryotic syncytium in which parasite nuclei replicate. In Gardneriella, formation of the syncytium induces surrounding host tissues to divide to form a photosynthetic callus. The internalized syncytium forms conjunctor and rhizoidal cells that fuse with host callus, eventually transforming the host callus into cells containing parasite nuclei. Gracilariophila does not induce surrounding host tissue to divide. Rather, division of the initial heterokaryotic tissue gives rise to the colorless mantle that protrudes from the host and forms reproductive structures. The heterokaryotic tissue also fuses with underlying host cells, thereby spreading parasite nuclei throughout adjacent host cells. In both these adelphoparasites, transformation of host cells by parasite nuclear invasion results in plastid dedifferentiation, an increase in mitochondria, autolysis of organelles, and accumulation of large amounts of floridean starch. The development and physiology of these parasites is similar to normal post-fertilization processes in the hosts that give rise to carposporophytes and suggests that these adelphoparasites may have originated from perturbations of developmental pathways involved in their host's post-fertilization development.     

口蹄疫病毒前导蛋白(Lpro)致牛肾细胞(MDBK)作用的形态学观察

为探讨口蹄疫病毒Lpro致MDBK细胞病变效应中的形态学变化,本实验在成功构建可稳定表达口蹄疫病毒Lpro目的基因的MDBK细胞系的基础上,人工诱导Lpro表达后,采用光学显微镜观察、Hoechst33258染色、AO-EB染色、DNALadder等进行检测,研究口蹄疫病毒Lpro致MDBK细胞的病变效应。结果显示,MDBK细胞系在诱导表达口蹄疫病毒Lpro24h后,光学显微镜下细胞形态表现为细胞体积缩小、核浓缩、细胞周围出现透明圈等现象;Hoechst33258染色检测呈现典型的细胞核浓缩和梅花状核碎裂;诱导表达Lpro36h后,AO-EB染色显示早期病变细胞核染亮绿色呈致密斑块或碎片状,晚期病变细胞核染橘黄色呈致密斑块;DNA凝胶电泳显示可见的DNALadder"梯状"条带。证明口蹄疫病毒Lpro在体外可诱导MDBK细胞发生凋亡。     

Bovine respiratory syncytial virus nonstructural proteins NS1 and NS2 cooperatively antagonize alpha/beta interferon-induced antiviral response

The functions of bovine respiratory syncytial virus (BRSV) nonstructural proteins NS1 and NS2 were studied by generation and analysis of recombinant BRSV carrying single and double gene deletions. Whereas in MDBK cells the lack of either or both NS genes resulted in a 5,000- to 10,000-fold reduction of virus titers, in Vero cells a moderate (10-fold) reduction was observed. Interestingly, cell culture supernatants from infected MDBK cells were able to restrain the growth of NS deletion mutants in Vero cells, suggesting the involvement of NS proteins in escape from cytokine-mediated host cell responses. The responsible factors in MDBK supernatants were identified as type I interferons by neutralization of the inhibitory effect with antibodies blocking the alpha interferon (IFN-alpha) receptor. Treatment of cells with recombinant universal IFN-alpha A/D or IFN-beta revealed severe inhibition of single and double deletion mutants, whereas growth of full-length BRSV was not greatly affected. Surprisingly, all NS deletion mutants were equally repressed, indicating an obligatory cooperation of NS1 and NS2 in antagonizing IFN-mediated antiviral mechanisms. To verify this finding, we generated recombinant rabies virus (rRV) expressing either NS1 or NS2 and determined their IFN sensitivity. In cells coinfected with NS1- and NS2-expressing rRVs, virus replication was resistant to doses of IFN which caused a 1,000-fold reduction of replication in cells infected with wild-type RV or with each of the NS-expressing rRVs alone. Thus, BRSV NS proteins have the potential to cooperatively protect an unrelated virus from IFN-alpha/beta mediated antiviral responses. Interestingly, BRSV NS proteins provided a more pronounced resistance to IFN in the bovine cell line MDBK than in cell lines of other origins, suggesting adaptation to host-specific antiviral responses. The findings described have a major impact on the design of live recombinant BRSV and HRSV vaccines.     

DEVELOPMENT OF JANCZEWSKIA MORIMOTOI (CERAMIALES) ON ITS HOST LAURENCIA NIPPONICA (CERAMIALES,RHODOPHYCEAE)1

Janczewskia morimotoi Tokida was successfully cultured from spore to reproductive maturity on its host Laurencia nipponica Yamada. The spore penetrates the host without requirement for wound or abrasion sites, growing between host cortical cells and developing a superficial and an endophytic system simultaneously. During the juvenile period, when the parasite is nonpigmented, it differentiates a cortex and the proliferating endophytic filaments enlarge causing a displacement of layers of host cells into the parasitic tissue. Host cells contacted by cells of the parasite exhibit increased wall thickness, cytoplasmic density and vesicle formation. Pit connections between host and parasite cells were rarely observed whereas penetration of host cell walls was seen commonly. As the parasite increases in size, its cells become pigmented evenly throughout the cortex and host cells show less obvious reactions to the parasite. At this same time, the parasite develops branches and reproductive structures. Host plant segments less than 3 cm long failed to grow when infected with spores of the parasite whereas longer segments were not significantly affected by the parasite. In the absence of the host, the parasite cannot complete its development. Although J. morimotoi is well pigmented at maturity, the absence of pigmentation in the juvenile stage, penetration of host cells, and effect on host growth in culture strongly suggest that it is parasitic during at least its early development.