Noble hosts: effects of internal parasites on the physiology of an intertidal brooding Sea-star / Huéspedes nobles: los efectos de parásitos internos en la fisiología de una estrella de mar incubadora del intermareal

Abstract Introduction: The endoparasite Dendrogaster argentinensis infects the intertidal brooder sea star Anasterias antarctica. This sea-star species is in the highest trophic level in the Beagle Channel. Objective: To study the effects of parasitism by D. argentinensis on the fitness and reproduction of A. antarctica. Methods: Adults from the brooder sea-star were collected from the rocky intertidal of Ensenada Zaratiegui bay (54°51' S & 68°29' W), Argentina. Eight seasonal samplings were performed (four seasons in two years) in the upper and low intertidal. During dissection, parasites were removed, and all organs were extracted and weighed separately. Results: Dendrogaster argentinensis prevalence was the highest for the region (20.4 %). Parasitized individuals were more frequent in the low intertidal in all seasons, with a higher difference in summer, where it is likely that the higher temperatures and strong winds could make the upper intertidal more challenging for a parasitized individual. Five parasitized individuals were castrated. Generally, the gonadal (GI) and somatic (pyloric caeca, PCI; stomach, SI; body wall, WI) indexes were lower in parasitized than non-parasitized individuals. Conclusions: Parasitism by D. argentinensis negatively affects A. antarctica condition. It affects reproduction because it reduces the GI, and can also produce castration. The parasite competes for the sea-stars' energetic resources, also decreasing the individual's capacity for feeding (reduced stomach) and growth (reduced body wall).

Resumen Introducción: El endoparásito Dendrogaster argentinensis infecta a la estrella de mar Anasterias antarctica, especie que se encuentra en el nivel trófico más alto del Canal Beagle. Objetivo: Estudiar los efectos del parasitismo de D. argentinensis en la condición fisiológica y reproducción de A. antarctica. Métodos: Adultos de la estrella de mar incubadora fueron recogidos del intermareal rocoso de la bahía Ensenada Zaratiegui (54°51' S & 68°29' W). Se realizaron ocho muestreos estacionales (cuatro temporadas en dos años) en el intermareal superior y bajo. Durante la disección, se removieron los parásitos, y todos los órganos, los cuales fueron pesados por separado. Resultados: La prevalencia de D. argentinensis fue la más alta de la región (20.4 %). Los individuos parasitados fueron más frecuentes en el intermareal bajo en todas las estaciones, siendo la mayor diferencia en verano, donde es probable que las temperaturas más altas y los fuertes vientos puedan hacer que el intermareal superior sea más desafiante para un individuo parasitado. Se observaron cinco individuos parasitados que estaban castrados. Generalmente, los índices gonadales (GI) y somáticos (ciego pilórico, estómago, y pared del cuerpo) fueron menores en los individuos parasitados que no parasitados. Conclusiones: El parasitismo de D. argentinensis afecta negativamente la condición fisiológica de A. antarctica. Afecta a la reproducción en términos de bajo GI y puede causar castración. El parásito compite por los recursos energéticos de las estrellas de mar, disminuyendo también la capacidad del individuo para alimentarse (reducción del estómago) y crecer (reducción de la pared del cuerpo).

Prevalence of the scuticociliate Orchitophrya stellarum in seastars from the Pacific and Atlantic oceans.

As part of a study to investigate the use of the scuticociliate Orchitophrya stellarum as a biological control for the invasive seastar Asterias amurensis in Australia, we collected prevalence data for O. stellarum from 3 seastar species (A. amurensis, A. rubens, Pisaster ochraceus) between 1996 and 1999 from the Pacific (Australia, Japan, Korea, Canada) and Atlantic (France, Netherlands, Canada) oceans. In the Pacific Ocean, for the first time, we found O. stellarum in male A. amurensis in Korea and female A. amurensis in Japan. The parasite was not detected in the invasive A. amurensis from Australia. There was no significant difference between size of infected and uninfected male seastars, nor a correlation between biased sex ratio and parasite prevalence in populations in the Pacific or Atlantic oceans. Therefore, unlike other studies, we found size and sex ratio in seastar populations in the field are unreliable indicators of parasite impacts. Regular monitoring of infected seastar populations in the field would be useful to better understand how sex ratio varies with parasite prevalence. We recommend laboratory studies under controlled conditions to determine the effect of O. stellarum on seastar populations.

Surveillance of densoviruses and mesomycetozoans inhabiting grossly normal tissues of three Aotearoa New Zealand asteroid species.

Asteroid wasting events and mass mortality have occurred for over a century. We currently lack a fundamental understanding of the microbial ecology of asteroid disease, with disease investigations hindered by sparse information about the microorganisms associated with grossly normal specimens. We surveilled viruses and protists associated with grossly normal specimens of three asteroid species (Patiriella regularis, Stichaster australis, Coscinasterias muricata) on the North Island / Te Ika-a-Maui, Aotearoa New Zealand, using metagenomes prepared from virus and ribosome-sized material. We discovered several densovirus-like genome fragments in our RNA and DNA metagenomic libraries. Subsequent survey of their prevalence within populations by quantitative PCR (qPCR) demonstrated their occurrence in only a few (13%) specimens (n = 36). Survey of large and small subunit rRNAs in metagenomes revealed the presence of a mesomycete (most closely matching Ichthyosporea sp.). Survey of large subunit prevalence and load by qPCR revealed that it is widely detectable (80%) and present predominately in body wall tissues across all 3 species of asteroid. Our results raise interesting questions about the roles of these microbiome constituents in host ecology and pathogenesis under changing ocean conditions.

Isolation, characterization and virulence of Mesanophrys sp. (Ciliophora: Orchitophryidae) in farmed swimming crab (Portunus trituberculatus) in eastern China.

A disease outbreak occurred in swimming crab (Portunus trituberculatus) farmed in eastern China, with a mortality rate of more than 80%. To further investigate the characteristics and pathogenesis, we reported isolation, characterization and virulence of the causative agent of this disease from 10 sick crabs. Histopathological observation found that multiple tissues, especially haemolymph, contained lots of ciliates. The ciliate was isolated and cultured in vitro, and molecular and morphological studies were done. The results showed that SSU rDNA and LSU rDNA sequences of the ciliate were similar to Mesanophrys ciliates (>96.81%), while ITS1-5.8s-ITS2 sequence was similar to Mesanophrys pugettensis (95.37%) and identical to Orchitophrya stellarum (100%). Furthermore, the results of the morphological study confirmed that the ciliate was similar to Mesanophrys ciliates and O. stellarum cultured in supportive media, but different from O. stellarum cultured in living sperm cells of starfish (Leptasterias spp.). Also, the growth of the ciliate did not interfere with light, which was different from O. stellarum. Accordingly, the ciliate was classified as genus Mesanophrys and temporarily named as Mesanophrys sp. In addition, experimental infection confirmed that Mesanophrys sp. was the pathogen that infected farmed crabs. In summary, Mesanophrys sp. was first isolated and characterized in P. trituberculatus.

A newly discovered radiation of endoparasitic gastropods and their coevolution with asteroid hosts in Antarctica.

BACKGROUND: Marine invertebrates are abundant and diverse on the continental shelf in Antarctica, but little is known about their parasitic counterparts. Endoparasites are especially understudied because they often possess highly modified body plans that pose problems for their identification. Asterophila, a genus of endoparasitic gastropod in the family Eulimidae, forms cysts in the arms and central discs of asteroid sea stars. There are currently four known species in this genus, one of which has been described from the Antarctic Peninsula (A. perknasteri). This study employs molecular and morphological data to investigate the diversity of Asterophila in Antarctica and explore cophylogenetic patterns between host and parasite. RESULTS: A maximum-likelihood phylogeny of Asterophila and subsequent species-delimitation analysis uncovered nine well-supported putative species, eight of which are new to science. Most Asterophila species were found on a single host species, but four species were found on multiple hosts from one or two closely related genera, showing phylogenetic conservatism of host use. Both distance-based and event-based cophylogenetic analyses uncovered a strong signal of coevolution in this system, but most associations were explained by non-cospeciation events. DISCUSSION: The prevalence of duplication and host-switching events in Asterophila and its asteroid hosts suggests that synchronous evolution may be rare even in obligate endoparasitic systems. The apparent restricted distribution of Asterophila from around the Scotia Arc may be an artefact of concentrated sampling in the area and a low obvious prevalence of infection. Given the richness of parasites on a global scale, their role in promoting host diversification, and the threat of their loss through coextinction, future work should continue to investigate parasite diversity and coevolution in vulnerable ecosystems.

Rare endoparasitic Asteriomyzostomum (Annelida: Asteriomyzostomidae) from Japan, including three new species descriptions and their phylogenetic position within Myzostomida.

The rare myzostomid genus Asteriomyzostomum Jägersten, 1940 consists of two species, both parasitizing sea stars. The phylogenetic position of this genus among Myzostomida has not been previously shown using molecular data. In this study, three species of Asteriomyzostomum were collected from the Kumano Sea, Japan, and are described as A. hercules sp. nov., A. jinshou sp. nov., and A. monroeae sp. nov. Additional specimens of the genus Asteromyzostomum Wagin, 1954 were also collected from the Kumano Sea and briefly reported as Asteromyzostomum sp. A molecular phylogeny based on four gene markers (COI, 16S, 18S, H3) suggests that the three families Asteriomyzostomidae, Asteromyzostomidae, and Protomyzostomidae comprise a clade. The resulting topology of the tree indicates that a host change from Crinozoa (sea lilies and feather stars) to Asterozoa (asteroids and ophiuroids) occurred only once in the evolutionary history of Myzostomida.

A new commensal ostracod Asterositus ohtsukai n. g., n. sp. (Paradoxostomatidae) on the sea star Sclerasterias euplecta (Fisher) (Asteriidae) and its feeding habits.

A new commensal ostracod Asterositus ohtsukai n. g., n. sp. (Podocopida: Cytheroidea: Paradoxostomatidae) is described. This new taxon occurs on the ambulacral grooves and wreath of pedicellariae around superomarginal spines of the sea star Sclerasterias euplecta (Fisher) (Asteroidea: Forcipulatida: Asteriidae). Although the family Paradoxostomatidae Brady & Norman, 1889 contains four ectoparasitic or commensal genera, the association with asteroideans has never hitherto been reported. Morphological observations suggested that A. ohtsukai n. g., n. sp. is closely associated with the host sea star as it typically has distal hooks on the antenna and fifth to seventh limbs, and specialised mandibula and maxillula structures that are considered adaptations to a commensal life-style. In addition, this study discussed the feeding habits of A. ohtsukai n. g., n. sp. based on comparison with previous works. The extremely reduced palp and endites of maxillula of A. ohtsukai n. g., n. sp. imply that they have unique feeding habits that are not based on the feeding function of maxillula. From morphological comparisons with siphonostomatoid copepods, we suggest that A. ohtsukai n. g., n. sp. may feed on the body of host sea star by injuring them with styliform needle-like mandibular coxa and sucking the tissues, body fluid, or mucus with the suctorial disc on the oral cone.

Discovery of an opportunistic starfish pathogen, Orchitophrya stellarum, in captive blue crabs, Callinectes sapidus.

Histophagous scuticociliate infections were discovered in blue crabs, Callinectes sapidus, held in research facilities at the Virginia Institute of Marine Science. Ciliates were observed infecting every tissue examined including the gills, heart, muscle, hepatopancreas, and epidermis. Hemolymph smears and histological tissue sections indicated a morphological similarity to Mesanophrys chesapeakensis, the only recorded histophagous ciliate infecting blue crabs. However, subsequent analysis of the ribosomal ITS region (ITS1-5.8S-ITS2) of the ciliate indicated the parasite was Orchitophrya stellarum, a parasitic ciliate previously reported infecting sea stars from Europe, Australia, and North America. A simple Polymerase Chain Reaction (PCR-RFLP) assay was developed to detect and differentiate between O. stellarum and M. chesapeakensis. Its application confirmed the presence of O. stellarum infecting blue crabs held in an additional research facility in Maryland. For growth studies, cultures of O. stellarum grew optimally on 10% blue crab serum in crustacean saline held at 10-20°C. A field survey of blue crabs collected during the winters of 2011-2012 and sea stars (Asterias forbesi) during the winter of 2010 from the Chesapeake Bay and eastern shore of Virginia did not identify additional infected individuals.

Emerging infectious disease in sea stars: castrating ciliate parasites in Patiria miniata.

Orchitophrya stellarum is a holotrich ciliate that facultatively parasitizes and castrates male asteriid sea stars. We discovered a morphologically similar ciliate in testes of an asterinid sea star, the northeastern Pacific bat star Patiria miniata (Brandt, 1835). This parasite may represent a threat to Canadian populations of this iconic sea star. Confirmation that the parasite is O. stellarum would indicate a considerable host range expansion, and suggest that O. stellarum is a generalist sea star pathogen.

Association and distribution of the ciliate Orchitophrya stellarum with asteriid sea stars on the west coast of North America.

The association of the scuticociliate Orchitophrya stellarum with 3 species of asteriid sea stars from the west coast of North America was studied by flushing the gonopore region with seawater and spawning the sea stars, along a latitudinal gradient of 2549 km between Pigeon Point, California, and Kodiak, Alaska. Asterias forbesii and A. rubens from the Isles of Shoals, New Hampshire (east coast), were also sampled. The ciliate was found on the aboral surface of both sexes of reproductively ripe Evasterias troschelii, Leptasterias spp., and Pisaster ochraceus with a maximum intensity of association occurring at Cape Arago, Oregon, and Clallum Bay and Manchester Dock, Washington. A survey of gonad smears and hematoxylin and eosin-stained sections indicated that the ciliate was only present in males. Spring-spawning E. troschelii and P. ochraceus are more negatively impacted by the ciliate than are winter-spawning Leptasterias spp. as judged by a skewed sex ratio and sex size differences, which may be associated with seasonal differences in water temperature affecting the growth rate of O. stellarum. The external morphology of O. stellarum appears to be similar throughout the geographical range surveyed.

Anatomy and ecology of the shell-less endoparasitic gastropod Asterophila japonica Randall and Heath, 1912 (Mollusca: Eulimidae).

The shell-less, endoparasitic gastropod, Asterophila japonica , was collected from two species of sea star, Leptychaster anomalus Fisher, 1906 and Ctenodiscus crispatus (Retzius, 1805) in Toyama Bay, Japan. All observed individuals were located on the aboral side of the host's disk (except one specimen parasitizing the arm) between the epidermis and the coelomic epithelium. More than one large individual frequently co-occur on a single host. The body plan of A. japonica is surprisingly modified from that of general gastropods; organs unrelated to digestion and reproduction are greatly reduced, simplified or completely lost. Dimorphism of body size is striking between males and females: males are much smaller than females and are attached to the surface of the pseudopallium of females. Females deposit and brood an egg mass(es) in the pseudopallial cavity until the eggs develop to veliger larvae. At the late developmental stage, brooded larvae reduce the velum and develop the foot for crawling, suggesting lecithotrophic development with or without a short planktonic stage. It is uncertain as to how the larvae can find and parasitize the next generation of the host. The systematic placement of Asterophila in the family Eulimidae is supported by three characters, viz. parasitism on echinoderms, smooth hydrophobic protoconch, and the enclosure of the visceral mass with the pseudopallium.

Investigating the possible role of benthic macroinvertebrates and zooplankton in the life cycle of the haplosporidian Bonamia ostreae.

Bonamia ostreae is a protistan parasite of the European flat oyster, Ostrea edulis. Though direct transmission of the parasite can occur between oysters, it is unclear if this represents the complete life cycle of the parasite, and the role of a secondary or intermediate host or carrier species cannot be ruled out. In this preliminary study, benthic macroinvertebrates and zooplankton from a B. ostreae-endemic area were screened for the presence of parasite DNA, using polymerase chain reaction (PCR). Eight benthic macroinvertebrates and nineteen grouped zooplankton samples gave positive results. Certain species, found positive for the parasite DNA, were then used in laboratory transmission trials, to investigate if they could infect naïve oysters. Transmission of B. ostreae was effected to two naïve oysters cohabiting with the brittle star, Ophiothrix fragilis.

Patterns of asexual reproduction in the fissiparous seastar Coscinasterias acutispina (Asteroidea: Echinodermata) in Japan.

The morphological and anatomical characteristics of the fissiparous seastar Coscinasterias acutispina were investigated at 23 sites around Japan in order to clarify the environmental factors determining asexual reproduction. The frequency of recent fission among the sites differed, although no significant differences were evident among the three major sea areas around Japan (the Sea of Japan, Seto Inland Sea, and Pacific Ocean), indicating that the spatial variability in incidence of fission might be determined on a local scale. Morphological observations revealed that the seastars endoparasitized by Dendrogaster okadai had not undergone fission recently, suggesting that fission was suppressed by parasitism. Stepwise multiple regression analysis of the frequency of seastars that had recently undergone fission revealed that nutrient conditions affected the incidence of fission. The relationships between the pyloric caeca index and arm length ratio at the individual level suggested that good nutritional conditions were required before the occurrence of fission. Additionally, regression analysis showed that high temperature was one of the triggers of fission in C. acutispina. Thus, the process of asexual reproduction in C. acutispina appears to be governed by intricate interactions among several factors, including parasitism by D. okadai, nutrient conditions, and high temperature.

Environmental factors influencing the immune responses of the common European starfish (Asterias rubens).

The influence of handling, salinity, temperature, parasitism, and gender on the immune responses (reactive oxygen species (ROS) production and coelomic amoebocyte concentration (CAC) of the starfish Asterias rubens was investigated in experimental conditions. Additionally, a year-round monthly survey in two distant sites was conducted in order to understand which of these factors most influences the immunity of A. rubens in field conditions. All considered factors, except gender and handling stress, influenced the studied immune responses of A. rubens in experimental conditions. Amoebocyte ROS production was increased at low salinity and at the lowest temperature tested (6 degrees C). Amoebocyte concentration in the coelomic fluid was increased in starfish infested by the ciliate Orchitophrya stellarum. However, among all these factors, only temperature could be linked with the variability in ROS production measured in the field during the monthly survey. The variability in amoebocyte concentration in the field does not seem to be linked to any of the factors considered in this study; it appears to reflect mostly an inter-individual variation rather than seasonal fluctuations. Recommended periods and indicative values of immune responses are proposed for field studies using A. rubens.

Conservation of sequence in the internal transcribed spacers and 5.8S ribosomal RNA among geographically separated isolates of parasitic scuticociliates (Ciliophora, Orchitophryidae).

Nucleotide sequence from the internal transcribed spacers (ITS1 and ITS2) and the 5.8S gene from the ribosomal RNA gene cluster of isolates of the scuticociliate Orchitophrya stellarum from 4 asteroid hosts were compared. Surprisingly, these data (495 bp) were identical for O. stellarum isolated from the testes of Asterias amurensis from Japan; Pisaster ochraceus from British Columbia, Canada; Asterias rubens from The Netherlands; and Asterias vulgaris from Prince Edward Island, Canada. These sequence data were compared to those from 3 scuticociliates which parasitise crustaceans: Mesanophrys pugettensis, M. chesapeakensis and Anophryoides haemophila. No difference was found in this region between the nucleotide sequence of M. pugettensis and M. chesapeakensis. The sequence of Mesanophrys spp. differed by 9.2% in the ITS1 and 4.7% in the ITS2 from that of O. stellarum. The sequence from the ITS1 (135 bp) and ITS2 (233 bp) of A. haemophila differed by 42.6 and 20.5% respectively from those of O. stellarum. Therefore, nucleotide sequence of the ITS regions in these scuticociliates is highly conserved.

The ciliate Orchitophrya cf. stellarum and other parasites and commensals of the northern Pacific seastar Asterias amurensis from Japan.

To identify the pathogens and possible biological control agents for the introduced seastar Asterias amurensis, we examined seastars from source populations in central and northern Japan. In particular, we sought the scuticociliate Orchitophrya cf. stellarum. The ciliate was found in male A. amurensis from five sites. We also found the caprellid amphipod Caprella astericola on A. amurensis and Distolasterias nipon from Nemuro Bay. The copepod Scottomyzon gibberum was found on A. amurensis from Usujiri and polychaete scaleworms Arctonoe vittata were found on A. amurensis from Murohama and Nemuro Bay. Of these parasites and commensals, Orchitophrya cf. stellarum is the most likely agent for biological control of A. amurensis in Australian waters; however, its ability to regulate seastar populations is uncertain.

Visual behavior, eye and retina of the parasitic fish Carapus mourlani.

1. This is the first transmission electron microscope description of the retina of a parasitic fish. 2. The retina is well supplied with capillaries, some of which occur on the inner surface of the retina. An unusual and possibly primitive type of capillary junction is described. 3. The outer segments of the retina of Carapus mourlani, which measure approximately 2 micron in diameter, are of one type only. A certain degree of banking was observed. Outer segments are not isolated from each other by screening pigments; they are, however, surrounded by about 15 calycal processes. 4. Typical spheroids or conoids were not seen in the outer plexiform layer. The inner nuclear layer consists of only one or two layers of nuclei. The inner plexiform layer is unusually wide and some amacrine synapses can be identified. There are few ganglion cells and the ratio of nuclei in the outer nuclear layer to those of the inner nuclear layer to ganglion cells is approximately 100:10:1. 5. The eye of C. mourlani combines features of degenerated photoreceptors, characteristic of cave-organisms, with adaptations which are commonly found in nocturnal and deep-sea forms. 6. The visual behavior of the animal indicates that the eye is fully functional in spite of its corneal keratosis and small number of optic nerve fibers.

The embryonic development of the marine caddis fly. Philanisus plebeius Walker (Trichoptera: Chathamidae).

1. P. plebeius, a trichopteran with marine intertidal larvae, oviposits in the coelom of a starfish, Patiriella exigua. Oviposition occurs mainly in the spring and autumn months. 2. In spite of the intracoelomic location of the embryos, the development of P. plebeius follows an unmodified trichopteran mode, including the characteristic blastokinesis. Nutrients are not supplied to the caddis embryos by the host starfish. 3. Hatching takes place in the starfish coelum after 17-18 days. The newly hatched caddis larvae quickly escape to their rock pool habitat. 4. The form of the female ovipositor indicates that other species of Chathamidae utilize starfish species as oviposition hosts. 5. This mode of oviposition offers protection to the caddis embryos in the intertidal habitat.