Allogeneic inhibition in a compound ascidian, Botryllus primigenus Oka. II. cellular and humoral responses in "nonfusion"reaction

Involvement of cellular and humoral responses in the “nonfusion” reaction (NFR) in a compound ascidian, Botryllus primigenus, was studied.NFR was irreversible and progressed at the same rate to completion even if one of the incompatible colonies was experimentally removed after NFR had been initiated between them. When two incompatible colonies were placed in a row with another compatible but genetically nonidentical colony between them, a rejection reaction was observed in blood vessels on the central colonies; or between the central colony and either one of the side colonies, which had fused with the central colony later than the other side colonies.It was tentatively supposed that NFR was mediated by an involvement of two types of factors. It was assumed that one was colony-specific, and the other was not necessarily colony-specific. The former was found contained in the test matrix and blood, which upon being introduced into a genetically different and incompatible colony, affected its test cells or granular amoebocytes which were then disintegrated. The latter was released from test cells upon their disintegration causing constriction of the ampullae or of the blood vessels forming an area of necrosis between reciprocally rejecting colonies.     

Colony specificity and chemotaxis in the compound ascidian Botryllus schlosseri

We re-investigated the behavior of hemocytes during the non-fusion (rejection) reaction between genetically incompatible colonies of the ascidian Botryllus schlosseri. In the course of the reaction, hemocytes - mainly morula cells - crowd inside the blind ends of marginal vascular vessels (known as ampullae) of the colonial leading edge (LE) facing the foreign colony which suggests the occurrence of chemotactic attraction of circulating hemocytes towards the ampullar lumen. Then, cells migrate, through the ampullar tips, into the partially fused tunics and contribute to the formation of the necrotic spots along the contact borders which characterize the reaction. Studies on histological sections clearly indicate that, although morula cell concentration is always higher in ampullae of the LE than in those of the lateral (L) part of the colony, their frequency significantly increases in LE ampullae of rejecting colonies with respect to LE ampullae of both fusing and isolated colonies. In addition, in vitro chemotaxis experiments demonstrated that blood plasma from incompatible colonies can stimulate morula cell migration through polycarbonate filters and this passage is inhibited by antibodies raised against mammalian pro-inflammatory cytokines. The possible nature and role of molecules recognized by anti-cytokine antibodies in hemocyte migration are discussed.     

Fusion between incompatible colonies of a viviparous ascidian, Botrylloides lentus

Abstract. The mode of allo-recognition among colonies was investigated in a viviparous ascidian, Botrylloides lentus . Each embryo is enveloped by a pouch of epithelial cells (brood pouch), and is brooded in the vascular lumen of the parental colony. That is, the parental colony tolerates the presence of semi-allogeneic conspecifics (embryos) in the vascular system. A rejection reaction occurs when incompatible colonies are brought into contact at their growing edges. The inflammatory rejection reaction is limited to a small area where the tunic of two colonies has partially fused. On the contrary, incompatible colonies fuse and their blood vessels become interconnected with one another, when they are brought into contact at artificially cut surfaces. This mode is the same as those of other viviparous species of Botrylloides, B. fuscus and B. violaceus . A relationship between viviparity and the loss of allo-recognition in the vascular system is suggested.     

Colony specificity in the xenogeneic combinations among four Botrylloides species (urochordata,ascidiacea)

Xenogeneic rejection reactions were histologically examined among four compound ascidians of the genus Botrylloides; B. simodensis, B. lentus, B. fuscus and B. violaceus, to compare with the allogeneic rejections of these species. When the incompatible conspecifics were brought into contact, hemolytic rejections occurred at the point where the tunic of the two colonies was partially fused. Xenogeneic contact at their growing edges induced hemolytic rejection in some combinations (B. simodensis-B. lentus, B. lentus-B. fuscus, and B. fuscus-B. violaceus), while conspicuous reaction was not found in the other combinations. Since the hemolytic rejection requires the partial fusion of tunic, the occurrence of hemolytic rejection suggests that the tunic cuticle of the colonies does not discriminate the facing colony from conspecifics. On the other hand, whereas cut surface contact between incompatible conspecifics induced intense rejection in B. simodensis, it resulted in fusion (formation of vascular connection) even in the combination in which the growing edge contact resulted in rejection. In xenogeneic combination, the cut surface contact of colonies always resulted in an intense rejection reaction except for B. fuscus-B. violaceus in which hemolytic reactions did not occur. The absence of hemolytic rejection suggests that the effector system for rejection reaction is not activated in this combination. Activity of phenoloxidase, a key enzyme of the rejection reaction, indicates lower reactivity in B. lentus, B. fuscus and B. violaceus than that in B. simodensis.     

Colonial allorecognition,hemolytic rejection,and viviparity in botryllid ascidians

Allorecognition is a fundamental system that animals use to maintain individuality. Although embryos are usually semiallogeneic with their mother, viviparous animals are required to allow these embryos to develop inside the maternal body, but must also eliminate an "invasion" by nonself. In colonial ascidians of the family Botryllidae, when two colonies are brought into contact at their growing edges, a hemolytic rejection reaction occurs between allogeneic colonies. Morula cells, a type of hemocyte, are the major effector cells in the hemolytic rejection. Morula cells infiltrate and aggregate where the two colonies make contact, and then discharge their vacuolar contents, which contain phenoloxidase and quinones. In viviparous botryllids, colonial contact at artificially cut surfaces always results in colonial fusion and establishment of a common vascular network even between allogeneic colonies in which the growing-edge contact results in rejection. This colonial fusion between incompatible colonies (surgical fusion) suggests that the allorecognition sites are not distributed in the vascular system in which the embryos are brooded. It is supposed that a common ancestor of the viviparous species lost the capacity for allorecognition in their vascular system to protect its embryos from alloreactivity, when it changed from ovoviviparous to viviparous in the course of evolution. The limited distribution of allorecognition sites would be a solution to the embryo-parent histoincompatibility in viviparity.     

Tunic phagocytes are involved in allorejection reaction in the colonial tunicate Aplidium yamazii (Polyclinidae, Ascidiacea)

The colonial ascidian Aplidium yamazii exhibited an allorejection reaction when two allogeneic colonies were brought into contact at their growing edges or at artificial cut surfaces. This species has no vascular network in the tunic, unlike the botryllid ascidians, which have a vascular network throughout the colony's common tunic. In the allorejection reaction induced by contact at the growing edges, some small, hard-packed tunic masses were formed at the contact points. Histological and electron microscopic investigation of these tunic masses revealed that they contained aggregates of tunic cells, with tunic phagocytes being the major cell type present. Some of the tunic phagocytes in these tunic masses appeared to be disintegrating. When allogeneic colonies were placed in contact at their artificial cut surfaces, the colonies partially fused, then separated. In this allorejection reaction, some loosely packed tunic masses remained in the gap between the two withdrawn colonies. These results strongly suggest that the tunic phagocytes are likely to be the major effector cells in the allorejection reaction. We also propose that the tunic phagocytes are not only the effector cells in the allorejection reaction but also bear the sites of allorecognition.     

Xenogeneic rejection among three botryllids (compound Ascidians)

Xenogeneic rejection was observed among colonies of three botryllids, Botryllus scalaris, Botryllus primigenus, and Botrylloides simodensis. Allogeneic recognition occurs in each of these species, but the manner of allogeneic rejection differs among them. We studied xenogeneic rejection reactions among these species under the following conditions: colony contact at natural growing edges, colony contact at artificially cut surfaces, and injection of xenogeneic blood plasma into a vascular vessel. In the first two cases, xenogeneic rejection occurred only in Botryllus primigenus and Botrylloides simodensis. The features of that xenogeneic rejection were similar to those of allogeneic rejection in each of these two botryllids. Injection of xenogeneic blood plasma induced responses similar to those of allogeneic rejection in all three botryllids. It is interesting to note that colonies of Botryllus scalaris never showed any response against injected blood plasma from allogeneic incompatible colonies, unlike the responses seen in colonies of the other two botryllids under the same conditions. On the basis of these results, the relationship between allogeneic and xenogeneic rejection in botryllids is discussed.     

Allorecognition responses in the soft coral Parerythropodium fulvum fulvum from the Red Sea

Allogeneic encounters were experimentally arranged in Eilat, Red Sea for the first time in the Alcyonacea with the soft coral Parerythropodium fulvum fulvum. All possible pairwise combination assays within two groups of five colonies each and one group of three colonies were setup in situ in 2–4 replicates each (a total of 76 assays). Control isogeneic encounters always resulted in complete tissue fusion. Two types of allogeneic responses were documented following tissue-totissue contacts. The first was retreat growth, in which contacting allografts started to retreat from each other a few days following direct contact until a bare area of a few mm separated them. In several assays the colonies repeatedly grew into contact and retreated again. The second allogeneic response was unilateral or reciprocal tissue overgrowth. In this type of response one colony overgrew the conspecific partner by several mm and then stopped. The underlying tissue of the overgrown partner died. No cytotoxicity was observed in allogeneic contacts either in growing parts or when assays were established between cut surface areas. Repeated assays of the same pair-combination were not consistent in terms of type and directionality of responses. We propose that effector mechanisms elicited following allogeneic encounters in P.f. fulvum may be affected by biological as well as non-biological parameters and are not specific to the type of allogeneic challenge. However, colony specificity in this species is restricted only to the level of self- and non-self discrimination.     

Studies on Japanese botryllid ascidians. IV. A new species of the genus Botryllus with a unique colony shape, from the vicinity of Shimoda

The morphology and life history of a strange and unidentified botryllid ascidian were investigated. This ascidian was first collected from the stony shore of Ebisu Island in Shimoda, a city on Izu peninsula in central Japan. Unlike other botryllid ascidians, whose colonies are flat and smooth, this ascidian's colonies are rugged. In each colony, zooids are arranged into several oval systems, each of which has a thick part containing zooids and very thin parts that do not. The arrangement of ovary and testis in this species is the same as in other species of the genus Botryllus; the ovary is situated anterior to the testis. The embryo of this ascidian develops in the peribranchial cavity of its mother zooid without any brooding organs, as is the case with Botryllus scalaris and Botryllus puniceus. Meanwhile, the results of cut colony assay experiments did not show the existence of colony specificity in this ascidian. Even when two syngeneic colonies were brought into contact at their growing edges, none fused together. On the other hand, when two colonies were brought into contact with each other at their cut surfaces, they always fused into a single colony, regardless of their origin. Therefore, this species may be the only species that lacks colony specificity among the botryllids studied so far.     

Mating type of isolates derived from the spermogonial state ofPuccinia coronata var.coronata

Hyphal confrontation between two haploid cultures originating from single basidiospores was used to determine the mating type ofPuccinia coronata var.coronata. Pairs of 15 single-basidiospore cultures were placed approximately 1 mm apart on the medium in all possible combinations. Hyphae of the pairs of colonies came into contact with each other in all combinations approximately two weeks after confrontations. When the nuclear number of hyphal cells in a contact zone was investigated one month after confrontation, monokaryotic hyphae were observed in selfing combination. On the other hand, dikaryotic hyphae were observed in 90.5% of crossing combinations between different cultures. Two isolates are considered compatible if dikaryotic hyphae are present in the contact zone but incompatible if they are absent. The mating type of the fungus was found to be characterized by multiple-allelomorphic tetrapolar incompatibility controlled by the “A” and “B” incompatible factors. Contribution No. 116, Laboratories of Plant Pathology and Mycology, Institute of Agriculture and Forestry, University of Tsukuba     

Studies on Japanese Botryllid Ascidians. III. A new species of the genus Botryllus with a vivid colony color from the vicinity of Shimoda

The morphology and life history of a new species of the genus Botryllus belonging to the family Botryllidae are described in detail. This ascidian was collected from the stony shore in the cove near Shimoda Marine Research Center, University of Tsukuba (Shimoda, Shizuoka Prefecture, Japan). The ascidian colony was easily distinguished from colonies of other botryllids because it was very thin and bright pink in color. The arrangement of ovary and testis in this ascidian was the same as that in other species of the genus Botryllus. This ascidian was prolific, with 1-5 embryos on each side of a zooid, and the embryos of this ascidian developed in the peribranchial cavity without any brooding organs as in Botryllus scalaris. We observed the processes and features of the allorecognition reaction in colony specificity and found that allorejection occurred after fusion of the vascular system between two incompatible colonies. This manner of allorejection is also shown in B. scalaris and Botryllus delicatus; however, the reaction speed of allorejection is faster than that of B. delicatus and similar to that of B. scalaris. These results indicate that this ascidian might be closely related to B. scalaris.     

A study into the sexual dimorphisms of the Ampullae of Lorenzini in the lesser-spotted catshark, Scyliorhinus canicula (Linnaeus, 1758)

The Ampullae of Lorenzini can vary in their size, shape and distribution patterns among elasmobranch species. However, no study has compared the ampullary characteristics between the sexes within a species. The present study found a sexual dimorphism in the Ampullae of Lorenzini of the lesser-spotted catshark, Scyliorhinus canicula. Male S. canicula were found to possess longer ampullae and alveoli, greater numbers of alveolar bulbs, larger sensory epithelial surface areas and greater numbers of sensory receptor cells in the ampullae than female S. canicula. Greater lengths of both ampullae and alveoli, numbers of alveoli, larger sensory epithelial surface areas and greater numbers of sensory receptor cells in male S. canicula could increase the capability of adult male S. canicula in detecting females. The presence of the sexual dimorphism in the alveoli of the Ampullae of Lorenzini could be directly related to reproductive behaviour and/or reflect the sexual segregation patterns of adult S. canicula.     

Functional micromorphology of petals of <Emphasis Type="Italic">Chaenomeles japonica</Emphasis> exposed to humid and cold season

In this study, micro- and nano-traits of petal epidermises of flowers of Chaenomeles japonica extended under environmental conditions, during the humid and cold period of the year, are presented. The outer (abaxial) and the inner (adaxial) epidermises of petals of C. japonica consist of convex and papillae cells, respectively, that are covered by epicuticular wrinkled relief further ornamented by submicron motifs, forming interfaces between floral tissues and environment. Structural epidermal features of the petal relief at the nanoscale level reveal different functionality on the two sides of the corolla. The cuticular folds of convex epidermal cells display declining water retention on the outer petal surface and the exposed side of the corolla to the environmental conditions. The cuticular folds of papillae epidermal cells increase in size the inner petal surface, in comparison with the outer surface; such traits facilitate light absorption and enhanced the contact area among folds and curvatures at the inner side of the corolla. It appears that nanometric surface structures of petals may be important adaptive features of C. japonica flowers, contributing to their performance in the field.     

Comamonas testosteroni colony phenotype influences exopolysaccharide production and coaggregation with yeast cells.

A Comamonas testosteroni strain was isolated from activated sludge on the basis of its ability to coaggregate with yeast cells. On agar plates the following two types of colonies were formed: colonies with a mucoid appearance and colonies with a nonmucoid appearance. On plates this strain alternated between the two forms, making sectored colonies. In liquid medium with constant agitation no such change was observed. In the absence of agitation and in contact with a glass surface a culture with predominantly nonmucoid-colony-forming cells very rapidly shifted to a culture dominated by mucoid-colony-forming cells. In liquid medium the reverse was observed under stress conditions imposed by hydrogen peroxide, sodium dodecyl sulfate, or starvation. Nonmucoid cells formed very rapidly settling flocs with yeast cells, while coaggregation of mucoid cells with yeast cells did not occur. These findings may be relevant to the behavior of activated sludge microbial communities.     

THE MECHANISM OF THE NEPHRIDIAL APPARATUS OF PARAMECIUM MULTIMICRONUCLEATUM : II. The Filling of the Vesicle by Action of the Ampullae

Our recent analysis of the nephridial apparatus of Paramecium multimicronucleatum by high-speed cinematography (300 fps at X 250) indicates that before the water expulsion vesicle ("contractile vacuole") is completely voided of fluid during expulsion, the ampullae surrounding and confluent with the vesicle swell with fluid entering from their respective nephridial tubules. Once the membranes of the excretory pore at the base of the excretory canal (leading from the vesicle proper to the outside) have constricted and resealed the excretory pore, the up till then constricted injection tubules of the ampullae which conduct fluid to the vesicle open as waves of contraction along the coacervate gel around the ampulla and proceed along each ampulla from distal to proximal end. The coacervate gel around any one ampulla does not necessarily contract in phase with that of any other ampulla. Each ampulla acts independently. The fluid from the ampullae is thus pumped sequentially, but not in predetermined order, into the water expulsion vesicle, refilling and distending it. Our previous studies (Organ et al., 1968a) suggest that an actomyosinoid ATP-using mechanism may be functional in the ampullary contractions.     

Oxidative stress induces cytotoxicity during rejection reaction in the compound ascidian Botryllus schlosseri

When genetically incompatible colonies of the compound ascidian Botryllus schlosseri contact each other, a rejection reaction occurs, characterised by the appearance of cytotoxic foci along the touching borders. In the course of this reaction, morula cells, a common haemocyte-type in ascidians, release their vacuolar content, mainly phenoloxidase and its polyphenol substrata, upon the recognition of soluble factors diffusing from the alien colony through the partially fused tunic. In a previous paper, we demonstrated the relationship between phenoloxidase and cytotoxicity. Here, we investigated the effects of superoxide dismutase, catalase and sorbitol (scavengers of superoxide anions, peroxides and hydroxyl radicals, respectively) on the cytotoxicity observed in haemocyte cultures incubated with heterologous blood plasma. Although the above compounds have no effects on morula cell degranulation and phenoloxidase activity, they suppress cell death, suggesting that oxidative stress plays a key role in in vitro cytotoxicity. In addition, sorbitol reduces the extent of the cytotoxicity occurring in the rejection reaction between incompatible colonies, which stresses the important role of hydroxyl radicals in this process. The observation of a decrease in total and non-protein thiols in haemocytes previously incubated with heterologous blood plasma fits the hypothesis of oxidative stress as the main cause of phenoloxidase-related cytotoxicity.     

Ultrastructural alteration of cytolytic T lymphocytes following their interaction with target cells. III. Plasmalemma, "membranosomas".

Ultrastructure of the plasma membranes of cytolytical T lymphocytes (CTL) in their interaction with target cells (TC) was studied. Thirty to sixty minutes after the beginning of interaction shedding of the CTL plasma membrane was observed: its fragments shedded from a local (50–100 nm in diam.) area on the lymphocyte surface at the site opposite to the CTL contact region with TC. Oval structures of high electron density 10 to 40 nm in diam. were detected on the CTL surface. We designated them as “membranosomas” (MS). MS were in close apposition to the inner surface of the plasma membrane and showed projections of 2 to 3 nm in diam. and 5 to 6 nm long towards the outer surface of the plasma membrane. MS were separated from the CTL surface during clasmatosis or as component parts of “shedding” plasma membranes.     

Effect of wounding or infection by Phytophthora infestans on the contents of terpenoids in potato tubers

Inoculation of the potato cut surfaces with an incompatiblerace of Phytophthora infestans induced an accumulation of rishitin,but only a trace occurred when infected by a compatible race.When the tuber was cut, a large amount of steroid-glycoalkaloids(solanine) accumulated in the cut surface tissue, although onlya trace was found in intact tissue. Only a small amount of solanine,if any, was contained in the wound periderm tissue. Most ofthe solanine seemed to be distributed in tissue neighbouringthe newly formed meristematic tissue zone. Distribution of solanineas a function of distance from the cut surface was exponential.Infection of the cut surface by an incompatible race of Phytophthorainfestans reduced the accumulation of solanine. The higher theconcentration of zoospore used, the less the solanine content.It has been reported that the higher the concentration of zoosporesused for inoculation of the cut surface, the less the numberof renewed meristematic cells in the wound tissue. In experimentsusing fresh and aged tubers, a good correlation between thenumber of renewed meristematic cells and solanine content wasfound. The accumulation of solanine in the wound tissue andits reduction due to infection by an incompatible race may berelated to renewed meristematic cells formation and its reductioncaused by the infection. No drastic change in carotenoids or sterol contents was found2 days after cutting or inoculation, when the tubers were cut,or cut and then infected by the incompatible race. ¹ Studies on the phytoalexin (No. 10). (6) in References constitutes"Studies on the phytoalexin No. 9". ² Present adress: Plant Pathology Laboratory, Faculty of Agriculture,Nagoya University, Chikusa-ku, Nagoya, 464, Japan. (Received April 1, 1972; )     

Ampulla Morphogenesis in Anural and Urodele Molgulid Ascidians

Vascular ampulla morphogenesis was studied in two anural-developing molgulids and one urodeledeveloping molgulid. In all three species, each juvenile developed one longer ampulla (termed the primary ampulla) and several shorter ampullae (termed secondary ampullae). Ampulla growth was accompanied by the formation of contraction rings that moved in a proximal to distal direction. Contraction ring formation was initiated by the epidermal cells situated in the proximal region of each ampulla. The formation of an expanding bubble-like region within each lumen preceded ring formation. These rings moved at approximately 120 μm/min. Rings were produced for several days until the ampullae retracted towards the body wall.
In all three species, hemoblast cells were evident at day 4 within the developing mantle sinus (hemocoel). Ampulla growth was studied by positioning chalk particles on the tunic surface near the tips of ampullae. During the growth phase, chalk particles moved towards the body of the zooid. This report is the first to describe contraction ring formation and the polarized transport of tunic components during ampulla morphogenesis in two anural ascidian species.