DISEASES OF AQUATIC ORGANISMS Dis Aquat Org Vol. 91: 237–242, 2010 doi: 10.3354/dao02264 Published September 17 Aggregata bathytherma sp. nov. (Apicomplexa: Aggregatidae), a new coccidian parasite associated with a deep-sea hydrothermal vent octopus C. Gestal1,*, S. Pascual1, F. G. Hochberg2 1 Instituto de Investigaciones Marinas, Consejo Superior de Investigaciones Científicas, Eduardo Cabello 6, 36208 Vigo, Spain 2 Department of Invertebrate Zoology, Santa Barbara Museum of Natural History, 2559 Puesta del Sol, Santa Barbara, California 93105, USA ABSTRACT: Aggregata bathytherma sp. nov. is described from the digestive tract of Vulcanoctopus hydrothermalis, a deep-sea octopus recently discovered associated with hydrothermal vents in the northeast Pacific Ocean. Oocysts typically are spherical in shape, sometimes irregular, 163 to 356 µm in length, and 219 to 313 µm in width. Each oocyst contains from 50 to over 200 sporocysts. Sporocysts measure 27 to 32 µm in longest diameter. The cyst wall is smooth and 1 µm thick. Each sporocyst typically contains 14 to 17 sporozoites, 49 µm in length. Histological lesions associated with the presence of A. bathytherma include rupture of the basal membrane and detachment of the epithelial cells. In heavily infected areas, most of the tissue of the host digestive tract is replaced by parasites. A. bathytherma is the first Aggregata species described from a host that lives in association with hydrothermal vents, and the third species of Aggregata from eastern North Pacific waters. KEY WORDS: Aggregata bathytherma · Vulcanoctopus hydrothermalis · Coccidian parasite · Deep-sea hydrothermal vent Resale or republication not permitted without written consent of the publisher INTRODUCTION Species within the genus Aggregata are intracellular coccidian parasites with a 2-host life cycle. Sexual stages (gamogony and sporogony) occur in the digestive tracts of cephalopods, the definitive hosts, and asexual stages (merogony) in the digestive tracts of crustaceans, the intermediate hosts (Dobell 1925, Gestal et al. 2002a). The union of a microgamete and a macrogamete in the digestive tract of a cephalopod produces a zygote, which becomes an early sporont with numerous nuclei. Individual nuclei with accompanying cytoplasm later bud off, forming uninucleated spherical sporoblasts. The development of sporoblasts into sporocysts is characterized by an increase in the number of nuclei and further partitioning of nuclei- and cytoplasm-forming sporozoites. After formation and maturation, sporocysts containing infective sporozoites leave the host with the feces. In the sea sporocysts must be ingested by a suitable crustacean intermediate host to continue their life cycle. Sporocysts pass to the digestive tract of the crustacean, where they hatch as a result of the action of stomach acid, and release sporozoites. Sporozoites migrate through the midgut epithelium to the submucous connective tissue, where they grow and become meronts. Finally, meronts generate merozoites by schizogony in the same way as the sporozoites develop in the cephalopod host (Hochberg 1990, Gestal et al. 2002a). Experimental infections have concluded that coccidians of the genus Aggregata show a high degree of specificity in the definitive host; however, lower specificity was observed in the intermediate crustacean host (Gestal et al. 2002a). To date, coccidian infections have not been reported from deep-sea invertebrates. All previous records are known from shallow-water cephalopods and pelagic *Email: cgestal@iim.csic.es © Inter-Research 2010 · www.int-res.com 238 Dis Aquat Org 91: 237–242, 2010 and benthic crustaceans (Hochberg, 1990). In contrast, haematozoans have been described in deep-sea demersal fishes in the Atlantic Ocean (Khan et al. 1992), and haemogregarines infecting blood cells of Zeus capensis from deep waters of South Africa (Smit & Davies 2006). A diversity of myxosporidians has been reported from deep-water fishes, especially macrourids, in both the Atlantic and Pacific Oceans (Yoshino & Moser 1974, Threlfall & Khan 1990, Lom & Dyková 1992). With regard to crustaceans, 5 named and numerous unnamed species of Aggregata are known to occur in benthic and pelagic crustacean hosts (see Théodoridès & Desportes 1975, Hochberg 1990). However, no data exist on the presence of coccidian parasites in deep-sea crustaceans. Several years ago, González et al. (1998) described a new genus and species of a deep-sea octopus named Vulcanoctopus hydrothermalis. The species inhabits depths ranging from 2500 to 2700 m where it lives in close association with hydrothermal vents on the East Pacific Rise. Here we present morphological and morphometric characteristics on the sporogonial stages of a new species of coccidia parasite of the genus Aggregata found in this hydrothermal octopus. This is the third species of Aggregata to be described from the northeastern Pacific Ocean. calibrated ocular micrometer. All measurements are in micrometers (µm) unless otherwise indicated. Paraffinembedded tissue which contained Aggregata was sectioned at 4 µm and stained with hematoxylin and eosin following standard procedures (Culling et al. 1985). RESULTS Aggregata bathytherma sp. nov.: description Material examined: based on the examination of 5 infected octopuses (Table 1). Oocysts: shape typically spherical, sometimes irregular; sporocyst numbers ranged from 50 to over 200. Lengths ranged from 163 to 356 µm (mean = 245.6 µm); widths from 219 to 313 µm (mean = 255.0 µm) (n = 10, 3 hosts). Sporocysts: mature sporocysts large; shape subspherical to subovoid; surface smooth with thick wall (1 µm). Lengths ranged from 27 to 32 µm (mean = 28.7 µm), widths from 24 to 32 µm (mean = 27.9 µm) (n = 30, 3 hosts). Giant sporocysts not observed. Sporozoites: number of sporozoites typically 14 to 17 per sporocysts (n = 5, measured in 1 octopus); curled in spiral within sporocyst. Isolated sporozoites uniform in size, length 49 µm, width 5 µm (n = 5, measured in 1 octopus) (Fig. 1B,E,F,H,I). MATERIALS AND METHODS Samples of Vulcanoctopus hydrothermalis were Taxonomic summary caught during several dives of the deep-sea manned submersible ‘Alvin’ at the Genesis site on the East Pacific Type specimens (syntypes): histological sections of Rise at 12° 48.68’ N, 103° 56.39’ W. The octopod hosts host digestive tracts containing mature sporocysts were collected by a robotic arm grab at depths ranging were deposited in the Santa Barbara Museum of Natfrom 2595 to 2635 m. Host specimens were collected ural History: SBMNH 345335 (2 microslides) and near a high temperature hydrothermal vent. Octopus SBMNH 345347 (2 microslides). specimens were frozen immediately following capture. Type locality: northeastern Pacific Ocean, East PaciIn the laboratory they were fixed in 10% formalin and fic Rise, Genesis site, 12° 48.68’ N, 103° 56.39’ W, 2595 later transferred and preserved in 70% ethanol. Dorsal to 2635 m. mantle length (ML), total body weight (BW), sex and stage of maturation were Table 1. Vulcanoctopus hydrothermalis specimens examined for the presence of recorded for each octopus examined (see Aggregata bathytherma sp. nov. Host octopus ordered by mantle length (ML) Table 1). and body weight (BW). ++: A. bathytherma infection Aggregata oocysts were obtained from the digestive tracts, mainly caeSex Maturity ML BW Aggregata Host repository cum and intestine, of infected octopus (mm) (g) & catalog no. previously deposited at the Santa Barbara Museum of Natural History. Female Mature 38 16.6 ++ SBMNH 142880 Malea Mature 37 18.6 ++ SBMNH 142882 Squash preparations were examined Male Mature 52b 20.6 ++ FMNH 27864 by excising white cysts containing spoMale Mature 45 21.8 ++ SBMNH 142881 rocysts and crushing them between 2 Male Mature 53 30.6 ++ USNM 885672 microslides. Sporogonial stages were a Host symbiotype b measured under 100× magnification Mantle length distorted with an oil immersion objective using a Gestal et al.: Aggregata bathytherma in Vulcanoctopus hydrothermalis 239 Fig. 1. Aggregata bathytherma sp. nov. Histological sections of intestine and caecum of Vulcanoctopus hydrothermalis infected by parasite. (A) Oocyst containing a sporoblast with sporozoites in formation. Arrows show rupture of infected tissue from development of the coccidian detachment and loss of epithelial cells. (B) Detail of sporoblast development and division to form sporozoites. (C, D) Caecum villous infected with parasite showing oocysts containing sporocysts and destruction of the tissue organ architecture by replacement by parasites. Arrows show distension and rupture of the basal membrane and loss of digestive tract epithelium. (E) Oocyst containing sporocysts with mature sporozoites inside. (F) Detail of mature sporocysts containing 14 to 17 sporozoites. (G) Heavily infected intestinal area showing replacement of the infected host tissue by parasites, resulting in the loss of the digestive tract epithelium and destruction of the tissue organ architecture. (H) Detail of sporozoites inside the sporocyst. Transverse section. (I) Detail of sporozoites inside the sporocyst. Longitudinal section. Scale bars: (A, C, D, E, G) 30 µm; (B, F, H, I) 15 µm 9–22 8–10 8 4–8 Smooth Smooth Smooth – 21 (18–23) 24 (18–31) 18 17 8 Spiny 20 (16–24) Narasimhamurti (1979) 6–12 Smooth 16–18 Labbé (1895) 15–17 3 Smooth 12 4–8 Gestal et al. (2000) 16–20 3 Smooth, thick Smooth, thick 11–15 21 (15–27) 14 (11–17) 12 10 11–15 23 (18–31) 16 (12–20) 13 10 Enteroctopus dofleini O. bimaculoides E. megalocyatus O. tehuelchus A. dobelli A. millerorum A. patagonica A. valdesensis NE Atlantic, W Mediterranean NE Pacific NE Pacific SW Atlantic SW Atlantic A. octopiana – 12 (9–14) A. kudoi S. elliptica Octopodidae Octopus vulgaris – 8–9 Sepiidae Sepia officinalis A. eberthi NE Atlantic, W Mediterranean NW Indian 15 17 NE Atlantic Todarodes sagittatus A. andresi A. sagittata 9.7 SW Atlantic 8.2 28.7 (27–32) Vulcanoctopus hydrothermalis Ommastrephidae Martialia hyadesi A. bathytherma sp. nov. NE Pacific 27.9 (24–32) 49 14–17 Smooth, thick Gestal et al. (2005) Present study Source Sporozoites Length n Cyst wall Sporocysts Width Length Host Locality Other localities: additional material examined in the present study was collected in the region of the type locality and to the south at 9° 50.33’ N, 104° 17.48’ W. Cephalopod hosts were collected at depths ranging from 2512 to 2635 m. Symbiotype: Vulcanoctopus hydrothermalis González, Guerra, Pascual & Briand, 1998 (González et al. 1998) (Mollusca: Cephalopoda: Octopodidae). Symbiotype: mature male, 37 mm ML; SBMNH 142882. Additional host vouchers: see Table 1. Additional host species: none. Prevalence: the infection was confirmed in all 5 hosts examined (see Table 1). Site of infection: Sporogonial stages (sporoblasts and sporocysts containing sporozoites) were present in the intestine, spiral caecum and other non-cuticularized regions of the host digestive tract. Etymology: the specific name is derived from the Greek word bathytherma meaning ‘deep heat’ in reference to the host’s association with deep-sea hydrothermal vents. Histopathology Species Table 2. Aggregata spp. Comparative data on morphology and morphometry of Aggregata species based on sporogonial stages. Length and width measurements are given in µm as means (range), means or ranges. –: no data Schneider (1875) Gestal et al. (1999) Poynton et al. (1992) Poynton et al. (1992) Sardella et al. (2000) Sardella et al. (2000) Dis Aquat Org 91: 237–242, 2010 240 Marked distension of the infected tissue area (intestine and caecum) due to the development of the sporogonic stages was seen, causing rupture of the basal membrane and the detachment of the epithelial cells (Fig. 1A,C,D). In heavily infected areas, most of the infected host tissue was replaced by parasites, resulting in the loss of the digestive tract epithelium and destruction of the tissue organ architecture (Fig. 1G). DISCUSSION At present, 9 named and several more unnamed species of Aggregata have been reported in the literature to occur in cephalopod hosts (Sardella & Re 1988, Hochberg 1990, Poynton et al. 1992, Gestal et al. 1999, 2000, 2005). Additionally, 5 named and numerous unnamed species of Aggregata are known to occur in benthic and pelagic crustacean hosts (Théodoridès & Desportes 1975, Hochberg 1990). Of these, only 2 species have been previously described from octopuses in the northeastern Pacific Ocean (Poynton et al. 1992); namely, A. dobelli in Enteroctopus dofleini (Wülker, 1910) and A. millerorum in Octopus bimaculoides Pickford & McConnaughey, 1949. Furthermore, 2 other Aggregata species have been described in nerito-oceanic ommastrephid squids; namely, A. sagittata in Todar- Gestal et al.: Aggregata bathytherma in Vulcanoctopus hydrothermalis odes sagittatus (Lamarck, 1798) from the northeastern Atlantic Ocean (Gestal et al. 2000) and A. andresi in Martialia hyadesi Rochebrune & Mabille, 1889 from the southwestern Atlantic Ocean, at the Antarctic Polar Front Zone (Gestal et al. 2005). Traditionally, diagnostic characters among species of Aggregata include phenotypic aspects related to the sporocyst structure (shape, size and thickness of the outer surface wall), number and size of sporozoites contained within the sporocysts, as well as data on host specificity (Table 2). Aggregata bathytherma sp. nov. can be distinguished from all other known species in the genus by (1) a larger sporocyst size; (2) a larger size and larger number of sporozoites in each sporocyst; and (3) a thick, smooth sporocyst wall. The latter could be an adaptation to the greater depth (i.e. higher hydrostatic pressure) to which Vulcanoctopus hydrothermalis is exposed. This characteristic has been previously reported for A. sagitattus infecting neritooceanic ommastrephid squids from the NE Atlantic (Gestal et al. 2005). The histopathological analysis is coincident with the previously described for other Aggregata species. The damage depends upon the intensity of infection, as it is proportional to the degree of destruction of host cells (Gestal et al. 2002b). Similar to that observed in Octopus vulgaris infected by A. octopiana, the destructive effect of this parasite deduced by histopathological analysis may impair gastrointestinal functions, including the correct absorption of nutrients (Gestal et al. 2002c), and may have weakened the octopuses, making them more vulnerable to other biotic and abiotic effects. The description of a new Aggregata species for the first time in a deep-sea cephalopod host suggests a broader habitat and distributional range than previously expected, not only for the genus Aggregata, but also for any coccidian parasite. With regard to food habits, and determining potential intermediate hosts, very little information is available. Rocha et al. (2002) indicated that Vulcanoctopus hydroythermalis likely feed on hydrothermal vent crabs Bythograea thermydron Williams, 1980. Voight (2005) reported that remains of the bathypelagic amphipod Halice hesmonectes Martin, France & Van Dover, 1993 were found in the gut of the octopod. At present no deepsea crustaceans have been examined for the presence of Aggregata. Although traditional identification and characterization of Aggregata species has relied primarily on differences in well-standardized morphological features such as size and shape of life-cycle stages and host specificity (Hochberg 1990), molecular techniques provide alternative methods for taxonomic studies and are important tools in solving the problems of species 241 delimitation. The only up-to-date molecular reference refers to the nucleotide analysis of the small rDNA subunit of Aggregata species infecting the coastal cephalopods Octopus vulgaris and Sepia officinalis (Kopecná et al. 2006). 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