Europ. J. Protistol, 35, 417-425 (1999) December 30, 1999 http://www.urbanfischer.de/journalslejp
European Journal of
Ultrastructural Aspects of the Sporogony of Aggregata octopiana (Apicomplexa, Aggregatidae), a Coccidian Parasite of Octopus vulgaris (Mollusca, Cephalopoda) from NE Atlantic Coast Camino Gestal'. Santiago Pascual':", Laura Corral- and Carlos Azevedo1 Area
de Parasitologia, Grupo FEPMAR-PB2, Facultad de Ciencias, Universidad de Vigo, Apdo. 874, 36200 Vigo, Spain, e-mail: [email protected]
uvigo.es; Fax: 986-812556; Phone: 986-812394 2 Department of Cell Biology, Institute of Biomedical Sciences, and CIMAR, University of Oporto, P-4050 Porto, Portugal
Summary Scanning and Transmission Electron Microscopy, and Atomic Force Microscopy techniques were used to study different topographic and cytological aspects of the architecture of the sporogonial stages of the eimeriorin coccidian Aggregata octopiana. Ultrastructural studies concerning the formation of sporoblast, sporocysts and number of sporozoites were presented for the first time for this coccidian species. Sporocysts are 11 to 15 pm in diameter, the wall is 0.25-0.35 pm thickness in tangential section. The sporocyst wall surfaces present projections or spiny which are 0.32 urn long. Each sporocyst contains probably 8 sporozoites. The microscopic analysis of the spiny sporocyst cover in A octopiana, a very important diagnostic character for the taxon, makes it possible to clear up the diagnoses of the species, allowing the taxonomic reappraisal of A spinosa Moroff, 1908 as a synonymy of A octopiana Schneider, 1875. Key words: Aggregata octopiana; Coccidian; Topography; Cytology; Ultrastructure.
Introduction Coccidia within the genus Aggregata are intracellular parasites with two-host life cycle, which are transmitted through the food-web. Sexual stages, gametogony and sporogony, occurs in the digestive tract of cephalopods, as definitive host. Asexual stages, merogony, are present in the digestive tract of crustaceans, as intermediate host . "corresponding author © 1999 by Urban & FischerVerlag
After fertilisation, a wall around the zygote is formed which will be the oocyst cover, and a reductional division or meiosis occurs. After gametic reduction, an important mitotic activity is observed in the now early sporont characterised by cell membrane folding and alignment of numerous nuclei on the surface of the partitioned cytoplasm. Individual nuclei with accompanying cytoplasm later budded off, forming uninucleate spherical sporoblasts. The development of sporoblasts into sporocysts is characterised by an increase in the number of nuclei, and the further partitioning of nuclei and cytoplasm forming sporozoites . Lieberkiihn (1854) described for the first time the genus Aggregata as a gregarine. Lately, it was correctly defined as a coccidium by Schneider (1883) and brought into the family Aggregatidae by Labbe (1899). In European waters, the type-species of the genus Aggregata (Aggregata eberthi Labbe, 1895), infects the cuttlefish Sepia officinalis and the crab Portunus depurator in the Mediterranean Sea,"the English Channel and the North Sea . In Octopus vulgaris two additional Aggregata species have been described, Aggregata octopiana (Schneider, 1875), in the Mediterranean Sea, the English Channel and the North East (NE) Atlantic Ocean; and A. spinosa (Moroff 1908), in the Mediterranean Sea and the English Channel. Moroff (1908) described nine additional species which lately were considered to be synonymies of the three species described above. Old literature from Dobell (1925) provided comprehensive LM information on the life cycle and structure of A. octopiana. Otherwise, although A. eberthi has been extensively studied by TEM 0932-4739/99/35/04-417 $ 12.00/0
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[11, 12, 13, 14], no effort has been carried out to study the ultrastructure of A. octopiana, which it does not allow to compare the ultrastructure of the two accepted European species within the genus. Problems during fixation of the later species due to the nature of the protecting wall cyst was suggested as an important technical handicap. With the aid of three different microscopic techniques, Scanning (SEM), Transmission Electron (TEM), and Atomic Force Microscopes (AFM), new diagnostic characters which had not been seen till now due to a limited instrument performance by light microscopy, are examined. This allowed us to clarify the taxonomic confusion existing between A. octopiana and A. spinosa infecting the common octopus from the NE Atlantic waters.
Materials and Methods A total of 100 living common octopuses Octopus vulgaris Cuvier, 1798, parasitized with A. octopiana (Protozoa, Apicomplexa) were collected by artisanal gears in the"Ria of Vigo", Galicia, Spain (NE Atlantic Ocean: 42°15'N, 8°48'W). Numerous sporulated sporocysts of this coccidian were observed under differential interference contrast (Nomarski) optics from small fragments of parasitized intestinal and caecum tissues. For SEM study, host tissue extracts were prepared by homogenisation for posterior isolation and purification of the sporocysts (Gestal et al., in press). The isolated sporocyst suspension as well as fragments of parasitized intestinal and caecum tissues were fixed for 4 hr in 2.5% glutaraldehyde in 0.1 M Na-cacodylate buffer (pH 7.3 at 4 "C) and washed 30 min in the same buffer during. Samples were then dehydrated in an ethanol series, critical point-dried in
CO 2 using a Polaron E300(1~nclsputter-coatedin a Polaron SCSOO using 60% gold-palladium. They were examined with a Philips XC30 SEM operated atl0-20 kV. The AFM study was done by putting a drop of isolated sporocyst suspension (in distilled water) on a silicon stub. Subsequently, the sporocysts were air-dried at room temperature for a few minutes. Then, they were scanned in a Digital Instrument Topometrix Discoverer microscope AFM in air at room temperature and pressure. The study was carried out by the non-contact resonant method. With this method, the sensor or cantilever beam with a tip of 1-2 nm of radius, swing to the resonance frequency which allows to maximize the image resolution and minimize the damage to the sample surface. Measurements were performed with the device software utilities (TopoMetrix SPMLab Ver. 4.0) directly on the graphic display. For TEM study, small fragments of the infected tissues were collected and fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, for 4 h at 4°C, washed for 12 h at 4°C in the same buffer and post-fixed in buffered 2% OS04 for 3 h at the same temperature. After dehydration in a graded ethanol series, the infected tissue was embedded in Spurr. Semithin sections obtained by diamond knife were stained with methylene blue. Ultrathin sections were double stained with uranyl acetate and lead citrate, and observed using aJEOL 100CXII TEM operated at 60 kYo
Results Numerous sporocysts in parasitized tissue samples were observed in semithin sections under light microscopy with the interference contrast (Nomarski) (Fig. 1). The SEM analysis of heavily parasitized tissue showed the surface topography of the host-parasite in-
Fig. 1. Aggregata octopiana. Oocyst containing several sporocysts observed under differential interference contrast microscopy (DIC-Nomarski). 1,525x.
Ultrastructure of the sporogony of Aggregata octopiana from Octopus vulgaris
teraction. Oocysts with different sizes ranging from 100 to 1,000 pm (depending of the maturation degree), emerge from the inner of the host tissue cells causing surface tissue disruption (Fig. 2, 3, 4). Oocystscontain between 1,000 and 60,000 sporocysts which get off together; they are covered by a residuum tissue-like membrane (Fig. 5, 6). The sporocysts look irregularly spherical in shape, with a diameter 11 to 15 pm in size. They are formed by two hemispheric valves closely attached together with a prominent, longitudinal suture or dehiscenc mechanism (Fig. 7). Characteristically, sporocysts present a double-layered wall: the outer layer represents a regular, smooth cover; and the inner one consists of a regular, spiny cover (Fig. 8, 9, 10,11). The mature sporocysts probably contain 8 sporozoites. When they become mature and the dehiscence mechanism of the sporocyst is opened, they go out to the lumen of the host intestine and then go to the sea within the faeces (Fig. 12, 13). Observed by high-resolution section analysis in the AFM, unfixed sporocysts bear small tiny ornamentations externally projected spiny-like (Fig. 14, 15, 16), fully covering the sporocyst surface. The projections or spiny were more distinct when the stage was tilted about 45° (Fig. 17). The spiny showed an irregular distribution pattern, 397.02 ± 194.82 nm (n = 25) in height, and 1.64 ± 0.86 pm (n = 25) in distance berween each spiny. Individual spines are 0.05 ± 0.05 prrr' (n = 15) in volume, representing an average 0047 ± 0.22 unr' (n = 15) surface area, an average 3048 ± 1.02 pm (n = 15) perimeter, and an aierage 99.79 ± 32.60 nm (n = 15) height. All the above data allow us to calculate a single roughness surface measurement of A. octopiana sporocysts as being 295.84 nm. The analysis of the oocyst wall by TEM showed a coccidian typical cyst wall, with an internal electrondense cover and an external one with numerous cyst projections covered by an outer membrane (Fig. 18). Surrounded by the oocyst wall, an important mitotic activity, cell membrane folding and alignment of numerous nuclei on the surface of the partitioned cytoplasm occurs (Fig. 19). The early sporoblast contains in the cytoplasm several reserve of proteins, lipids and few mitochondria. The spherical nucleus contains scattered chromatin (Fig. 20), and in favorable sections, a prominent nucleolus. During the sporoblastogenesis, a simple wavy membrane with numerous micropores appeared. This simpie membrane will grow towards outside becoming an electron-dense layer which will be later the sporocyst cover with a great stiffness. It is externally skirted by two united membranes. The nucleus, still unique, is 5 pm in size. The cytoplasm is completely filled with lipid vacuoles which were pushed towards the surface of the sporocyst. In addition to these dominating vac-
uoles numerous vesicles appeared at the sporogony: large, dark bodies, probably proteins, with or without electron-lucent spaces, bodies of medium or irregular densities, small vacuoles and scattered granules of paraglycogen which surround other different granules. It is possible to see too few mitochondria and RER (Fig. 21). The development of sporoblasts into sporocysts was characterised by an increase in the number of nuclei (probably 8 at this species), and the further partitioning of nuclei and cytoplasm forming elongate sporozoites. At this time, the sporocyst wall is completely formed (Fig. 22). Through a favorable tangential section it is possible to see an electron-dense wall ranging from 0.25 to 0.35 pm in size, composed by regular transversal grooves within a period of 0.13 pm. Regularly intercalated between the above electron-dense grooves, this cover also presents thin grooves. Between two followed electron-dense grooves, a distance of 15-17 nm was measured (Fig. 23). The sporocyst wall exhibited a particular differentiation located in two diametrically opposite points on the surface wall, the dehiscence mechanism or suture line, passing transversally through the striated wall (Fig. 22). Externally at the sporocyst wall surface we observed some small conical projections (ornamentation-like protrusions). Those .p rojections are 0.32 pm long and 0.30 pm wide, presenting an inner granular .an d electron-dense structure (Fig. 24,25). By serial ultrathin sections, it also was observed that each sporocyst contained probably 8 sporozoites (Fig. 26).
Discussion UItrastructure Though the fixation procedure was revealed as a critical factor during technique performance due to the impermeability of the cyst wall, the present study provided us a correct vision on the ultrgstructure of the different sporogonic stages of A . opopiana. Although many ultrastructural features of 4 /octopiana herein recorded are similar to the fine structure previously recorded in other Aggregata species [11, 13] or even fish coccidians , some structures and their measurements requires some care. Porcher-Hennere and Richard (1969) noted an immature sporocyst grooved wall with a thickness of 700 A in A. eberthi. Mature sporocysts of A. octopiana are 0040-0.50 pm. The distance between two electrondense grooves is 19 nm for A. eberthi and 15-17 nm for A. octopiana. In the sporoblasts of A. octopiana it is not possible to see a fully -developed crystaloide, as it occurs in A. eberthi. To date, it is unclear whether these morphometric and morphological differences are due
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Figs. 2-13. A. octopiana observed by SEM. 2, 3, 4. Appearance of the parasitized intestinal surface. Eme rge ooc yst (OC) deforming and causing surface tissue disruption. 5, 6. Oocyst with sporocysts covered by a residual tissue-like membrane. 7. Sporocyst with the longirudinal surure line or dehiscence mechanism (SL). 8,9,10. Visualization of the sporocyst with a doublelayered wall, the outer smooth, and the inner with a spiny cover. 11. Detail of a single spiny (SP) of the inner cover. 12, 13. Way of opening of the sporocyst (SC) valves for sending the sp orozo ites (S2) off.
Ultrastrudure of the sporogony of Aggregata oaopiana from Octopus vulgaris
160 kV 26
to different developmental stages for each species, or simply they are specific taxonomic structures. The surface of the sporoc yst, completely smooth in A. eberthi, and ro ugh (spiny cover) in A. octopiana, seems the most dist inctive ultrastru cture character sufficiently developed , represented, and important enough to be used to distinguish both European Aggregata species.
? IJIIl CACTI
The applicab ility of AFM, as a new technique added to traditional ones, to the stud y on the sporocyst fine topological structure provides a double-aid: first, it allows to get the real 3-D topography on the sporocyst cover by using a non-invasive method (i. e. cysts are simpl y not passed through fixation, dehydration, point-drying or gold cover with a subsequent distor-
22 .0 4
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Figs. 18-25. A. octopiana. Ultrastructural aspects observed by TEM. Fig. 18. Oocyst wall with numerous small projections (arrows). Nearly one sporocyst (Sc). 10,600x. 19. Beginning of the sporoblast formation with partitioned cytoplasm. Nucleus (N). 3,100x. 20. Early sporoblast. Nucleus (N), scattered chromatin (arrow heads). Lipid droplets (L). 7,lS0x. 21. Late sporoblast. Aspect of the wall (Wa) during the development of the sporocyst starting to sporoblast. Lipid droplets (L). 6,100x. 22. Ultrastructural aspect of a sporocyst showing part of the internal sporozoites (",-) and the wall (Wa) with the suture line (arrow). 8,OOOx. 23. Sporocyst wall (Wa) completely formed showing a regular transverse groovetion, striation (arrow). 71,SOOx. 24. Protrusions or spiny (arrows) in the sporocyst wall. 32,OOOx. 25. Detail of the inner granular structure of a spiny (arrow). 48,OOOx. 26. A single sporocyst containing 8 sporozoites C') as seen in serial ultrathin sections. The surrounding host cell (He) shows evident signs of lysis. 10,600x.
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Height. Max.: 1238.8032 nm
O \.llll-f-_ _.-........................:..ai oIi..,.
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Figs. 14-17. A. octopiana. Sporocysts surface observed by AFM. 14, 15, 16. Sporocyst fully covered by ornamentations like spiny. Detail of the disposition and shape of the spiny. 17. Sporocyst shape and measurement of the roughness surface of the sporocyst wall.
Ultrastructure of the sporogony of Aggregata oetopiana from Octopus vulgaris
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tion of surface morphology) ; secondly, in coccidian taxonomy shape traditionally has been described in qualitative terms for taxonomic purposes. Again , classical shape description vocabularies are rife with ambiguity, and AFM profile sections provide a set of fine morphometric measurements on the surface topography on the sporocyst cover. Therefore, AFM can be considered as a valuable diagnostic tool during the taxonomic study of Aggregatidae, uncovering fundamental processes in cellular function.
Host-parasite interaction Oocyst low-magnification micrographs by SEM, showing its structure, site, progressive growth and development into the cells of the host tissue, provided interesting data on parasitic-caused pathology via host tissue destruction. In massive infections (which are common in nature), the mechanical compression effects give rise to a deformed parasitized tissue which hinder the blood circulation and the muscular activity on the infected organs [5, 3]. Furthermore, in those cases of heaviest infection, almost all the host tissue is replaced by oocysts, which is probably due to a great increase in size to the end of the developmental cycle of the parasite within the octopus. Then, the parasite distends the host cell until the outbreak occurs . Therefore, infected hosts are subjected to a great limitation during intestinal absorption of the nutrients. Though the coccidian infections are not believed to be a primary cause of death in wild common octopus populations , it is likely that parasites cause loss of condition. Then, under stress situations, cephalopod populations are susceptible of epizootic outbreaks by other more virulent microparasites and/or xenobiotics .
Taxonomic reappraisal of A.
The classification and nomenclature within the coccidian genusAggregata has historically been controversial . This has also been true for th e two pre viou sly described, accepted Aggregata species in European wa ters (Table 1). MoroH (1908), beside a great number of Aggregata species which are now recognised as synonymies of the already described A. octopiana, was the first author to deal with the other species A. spinosa. Studies by Moroff distinguished the above two species by the existence of spin y on the cover of the sporocyst of A. spinosa, whereas A. octopian a was described as having a smooth sporocyst cover. The present study shows that all the sporocyst cover of eimeriorins belonging to the genus Aggregata from o. v ulgaris in European waters, regardless of their specific status, bear spines recovered by a membrane or tis-
sue layer whi ch makes its visualiz ation impossible before the cover is washed during the isolation and purification procedures (Gestal et al., in press). Nevertheless, in some cases when we opened the sporocyst for bringing out the sporozoites, we have observed this cover deta ching from the sporocyst. In some other cases during persisting the membranous cover, and when the sporocyst starts to clean it, those protrusions can be guessed like bulkiness. This could be the spin y structure that Moroff (1908) described by light microscopy for the species A. spinosa. The SEM images of both sporocyst samples, from a single octopus and other samples obtained from a "tissue-pool" of octopuses, showed different sporocysts surface architectures. Some sporocysts appear with a fully-developed cover; in other sporocyst samples the cover only appears in certain regions on the sporocyst surface (probably due to processing of removal), or even without the external cover. This fact does prove the existence of a single Aggregata species for wh ich the topological phenotypic diagnostic character, the sporocysts spiny cover, is not always visible depending on cell preparation procedure evolved. The use of various combined ultrastructural techniques allowed us a better understanding on the morphology, morphometry and surface topology of Aggre:gata species, which had not been reported till now. More over, it brings light out to clarify the taxonomic status of A. spinosa, and thus its emendation as a synon ym y of A. octopiana, the only valid Aggregata species in European common octopuses till now. Those species formerly attributed to A. spinosa, should also be synonymized with A. octopiana . Furthermore, since the phenotypic characters originally used to delimit species within the Aggregatidae have been proven unstabl e (i.e. with 90% of names for Aggregata proposed during the last two centuries regarded as synonym s, see Table 1), the use of molecular data to supplement traditional methods as a reliable method to' discriminate cephalopod coccidians based on the species concept is much needed. Acknowledgements: We are grateful to Carmen Serra and Jesus Mendez (CACTI, University of Vigo) for technical assistance at the AFM and SEM studies. This work was partially supported by the Spanish Governm ent under Proj ect CICYTMAR 95-1919-C05-03, and the A. Almeida Foundation (Port o, Portugal ).
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