Leiocanthus quinquenudus sp. nov. and L. satanicus sp. nov., two new species of pycnophyid Kinorhyncha (Allomalorhagida: Pycnophyidae) from the Gulf of Mexico.

Two new species of pycnophyid Kinorhyncha, Leiocanthus quinquenudus sp. nov. and L. satanicus sp. nov., are described from soft seafloor sediment samples in the Gulf of Mexico. Leiocanthus quinquenudus sp. nov. is easily distinguished from the other congeners by the absence of ventromedial setae on segment 5, a structure otherwise present in all the other known Leiocanthus. Leiocanthus satanicus sp. nov. lacks lateral terminal spines, a feature only shared by L. langi and L. mainensis among the congeners; otherwise the new species is easily discernible by the arrangement of the paradorsal, laterodorsal and ventromedial setae, and the number of ventral sensory spots per segment. These findings are a significant contribution to the knowledge of the Kinorhyncha biodiversity from the Gulf of Mexico, which has recently been explored in several taxonomical and ecological studies mainly focused on cyclorhagid Kinorhyncha, not pycnophyid kinorhynchs.

Hyalophysa lynni n. sp. (Ciliophora, Apostomatida), a new pathogenic ciliate and causative agent of shrimp black gill in penaeid shrimp.

The parasitic ciliate causing shrimp black gill (sBG) infections in penaeid shrimp has been identified. The sBG ciliate has a unique life cycle that includes an encysted divisional stage on the host's gills. The ciliature of the encysted trophont stage has been determined and is quite similar to that of the closely related apostomes Hyalophysa bradburyae and H. chattoni. Hyalophysa bradburyae is a commensal ciliate associated with freshwater caridean shrimp and crayfish, while H. chattoni is a common commensal found on North American marine decapods. Based on 18S rRNA gene sequence comparisons, the sBG ciliate is more closely related to the marine species H. chattoni than to the freshwater species H. bradburyae. The unique life cycle, morphology, 18S rRNA gene sequence, hosts, location, and pathology of the sBG ciliate distinguish this organism as a new species, Hyalophysa lynni n. sp.

The complete life cycle of the unusual apostome Hyalophysa clampi (Ciliophora, Apostomatida), a symbiont of crayfish in Alabama (USA).

Hyalophysa clampi Browning and Landers, 2012 was reexamined to determine all stages in the life cycle of this symbiotic ciliate. The cell feeds as a normal exuviotroph within the exoskeleton of its molted crayfish host but does not encyst following the trophont stage. Trophonts transform into swimming tomont stages, which divide by palintomy over successive divisions, splitting to two cells, separating, and repeating. The divisions cease when the daughter cells attain the size of the infestive tomite stage, which attaches to a new crayfish. This unique life cycle is most similar to the European hermit crab symbiont Polyspira delagei, which forms chains of daughter cells during division. Scanning electron microscopy confirmed the unusual presence of two contractile vacuoles in H. clampi, unique among the Apostomatida, and provided ultrastructural details to better understand light microscopy silver staining. The genus diagnosis for Hyalophysa is modified herein to accommodate this new life cycle.

Black spot gill syndrome in the northern shrimp, Pandalus borealis, caused by the parasitic ciliate Synophrya sp.

Black spot gill syndrome in the northern shrimp, Pandalus borealis, is caused by an apostome ciliate, Synophrya sp., found within the gill lamellae. Whole mount staining, thin section histology, electron microscopy, and molecular studies were carried out on infected gills. The Synophrya 18S rRNA from Pandalus borealis (Genbank accession no. KX906568) and from two portunid crab species, Achelous spinimanus (Genbank accession no. MH395150) and Achelous gibbesii (Genbank accession no. MH395151) was sequenced. Phylogenetic analyses confirmed the identity of these ciliates as apostomes. The 18S rRNA sequence recovered from P. borealis shared 95% nucleotide similarity with the sequences recovered from the portunid crab species suggesting that it is a different species of Synophrya. The invasive hypertrophont stages, with a distinctive macronuclear reticulum, ranged in size from 300 to 400 µm with as many as 5 large forms/mm2 of gill tissue. Histotrophic hypertrophont stages and hypertomont stages were observed in these studies. The presence of the parasite was linked to the formation of melanized nodules (up to 9 nodules/mm2 of gill tissue) by the host and in some cases to extensive necrosis. Other studies have reported Synophrya sp. infections in P. borealis from Greenland, Labrador and Newfoundland, but further studies are necessary to determine the prevalence of this parasite in the dense schools of northern shrimp in the North Atlantic. Questions remain as to the possibility of epizootics of this pathogen and its impact on northern shrimp populations.

Description of a new kinorhynch species, Paracentrophyes sanchezae n. sp. (Kinorhyncha: Allomalorhagida) from the Gulf of Mexico, with differential notes on one additional, yet undescribed species of the genus.

A new kinorhynch species, Paracentrophyes sanchezae n. sp., is described from the Gulf of Mexico. The new species is distinguished from its congeners by its relatively low number of perispinal setae. In addition to the new species, the existence of one undescribed species of Paracentrophyes in the Gulf of Mexico is also reported. This undescribed species has very short lateral terminal spines, and the number of perispinal setae is lower than in any other known species of Paracentrophyes. Diagnostic notes are provided for this unnamed species, but the available material is too limited to provide a formal description.

Two new species of Echinoderes (Kinorhyncha, Cyclorhagida), E. romanoi sp. n. and E. joyceae sp. n., from the Gulf of Mexico.

Meiofauna sampling on the continental shelf of the northern Gulf of Mexico has been ongoing since 2007, on annual cruises in collaboration with the National Marine Fisheries Service laboratory in Pascagoula, Mississippi. This sampling has resulted in numerous new species of kinorhynchs from the shelf sediment, two of which are described in detail in this paper. Other species descriptions from this research effort include Echinoderes augustae, Echinoderes skipperae, and Echinoderes charlotteae. We now describe Echinoderes romanoi sp. n. and Echinoderes joyceae sp. n., which are unique in their spine, tube, and glandular cell outlet patterns.

Histochemical Study of the Progenetic Trematode Alloglossidium renale.

A histochemical study of the progenetic trematode Alloglossidium renale has demonstrated the absence of any secreted material between the adult worm and the host (freshwater shrimp) antennal gland tubules. Host tissue is affected only by the compression, abrasion, and ingestion by the parasite, and host tubule cells near the worm have the same staining patterns as those distant from the parasite. The trematode sometimes dies within the host, leaving a necrotic mass whose histochemical staining differs significantly from the living organism. In the necrotic mass, the only recognizable features were the ova and the vitellarium, which atrophied and resulted in tyrosine-positive staining within the mass. A melanin reaction was not observed in the host using a specialized ferro-ferricyanide stain. The only apparent host response to infection was a layer of damaged squamous host cells adhering to the necrotic worm. The results confirm benign host-parasite effects and a highly evolved relationship between the host and parasite, perhaps bordering on commensalism.

Exuviotrophic apostome ciliates from freshwater decapods in southern Alabama (USA) and a description of Hyalophysa clampi n. sp. (Ciliophora, Apostomatida).

Apostome ciliates from crayfish and freshwater shrimp in southern and central Alabama were surveyed in this study. Hyalophysa bradburyae was found on both crayfish and shrimp from 16 sites in eight drainages. A new species, Hyalophysa clampi n. sp., was found infesting crayfish at one site and simultaneously infesting crayfish with H. bradburyae at two sites. Characteristics of the trophont ciliature of H. clampi n. sp. separate it from other species in the genus. Particularly, the contractile vacuole pore is oriented posterior to the ventral kineties xyz, kineties 1 and 2 are undivided, an apparent second contractile vacuole pore is present between the ventral portions of kineties 1 and 2, the anterior ventral field is tightly arranged, and there is an apical field of kineties between kineties 4 through 6. This report expands the known range and diversity of the genus Hyalophysa in the state of Alabama.

The fine structure of the hypertrophont of the parasitic apostome Synophrya (Ciliophora, Apostomatida).

The hypertrophont stage of the parasitic apostome ciliate Synophrya was studied by light microscopy and transmission electron microscopy. This invasive stage of the ciliate was found within the gill lamellae and gill raphes of the longspine swimming crab Portunus spinicarpus. The ciliates elicited a melanized host reaction that walled off the parasite from the host. Additionally the ciliate produced a cyst wall of approximately 0.16-2.0 microm in thickness that further isolated the parasite. A mouth was not observed, as the internal stage of this ciliate takes in material via endocytosis across the entire surface. The outer surface was irregular, with folds, membrane pillows, and vesicles connected to the outer membrane. The hypertrophont had a sparce ciliature, with well developed kinetodesmal fibers connecting the kinetosomes. Within the cytoplasm the cell had numerous vacuoles, lipid droplets, and large plaquettes of material. The massive reticulate macronucleus had globular and elongated chromatin bodies, and was the most distinctive organelle within the cell.

The fine structure of the developing oocyst of Pterospora floridiensis (Apicomplexa, Urosporidae).

Developing oocysts of the gregarine Pterospora floridiensis Landers 2001 were examined by transmission electron microscopy. Each oocyst had an outer capsule and an inner capsule that contained 8 sporozoites. In early stages of development the inner capsular wall was separated from the developing sporozoites and residual mass, and was not appressed to the sporozoites. Early stage sporozoites were connected to a residual mass and were filled with endoplasmic reticulum, golgi and numerous developing secretory vesicles. In late stages of oocyst and sporozoite development, the inner capsular wall was closely appressed to the sporozoite surface. The inner capsular wall was approximately 60-100 nm thick and the outer capsular wall was approximately 160-320 nm thick. There were no extensions on the outer wall for which the genus was named. Late stage sporozoites had no residual mass connection, were more electron dense, and contained three distinct types of dense secretory structures: 1) small oval/spherical dense vesicles, 2) large (350-400 nm) vesicles near the anterior end, and 3) elongated dense tubular bodies that converged at the apex. Few ultrastructural reports exist of developing gregarine oocysts and sporozoites, and as more studies are completed these morphological characteristics may be important in interpreting molecular phylogenetic analyses.

Salinity tolerance in Hyalophysa chattoni (Ciliophora, Apostomatida), a symbiont of the estuarine grass shrimp Palaemonetes pugio.

The apostome ciliate Hyalophysa chattoni, a symbiont of the estuarine grass shrimp Palaemonetes pugio, was tested for its growth and reproductive ability in a wide range of salinities from 0.1 to 55 ppt. Shrimp, with their attached ciliates, were slowly acclimated to different salinities in order to assess protozoan cell size and division. The trophont and tomont stages of the ciliate life cycle were analyzed. In both stages, cell size increased with salinity from 0.1 to 20 ppt. Cell size leveled in the 20-35 ppt range, and decreased at higher salinities. The number of daughter cells produced per tomont cyst correlated with increased cell size, and also correlated with increased salinity. Additionally, increased salinity correlated with an increase in the percentage of cells able to divide and excyst as tomite stages. These results indicate that H. chattoni is able to grow and divide more effectively at salinities closer to seawater than in the estuarine environment from which they were collected. Though able to survive salinities from 0.1 to 55 ppt, the species is better adapted for an existence in the higher salt concentrations.

The fine structure of the phoront of Gymnodinioides pacifica, a ciliated protozoan (Ciliophora, Apostomatida) from euphausiids of the Northeastern Pacific.

Phoretic stages of the exuviotrophic apostome Gymnodinioides pacifica were examined using transmission and scanning electron microscopy (TEM and SEM). TEM revealed that the mature cyst wall possesses 2 or 3 layers differing by the presence or absence of the third inner layer. This inner layer may represent a different form of the middle wall material. The inner cyst layer is approximately 0.15 microm thick and has striations with a periodicity of approximately 19 nm. The middle cyst layer has a variable thickness and the outer dense layer is approximately 0.1 microm thick. The 3 layered cyst wall had a thickness of 0.3-0.7 microm and averaged 0.5 microm. Advanced phoront stages were enclosed by fully formed cyst walls or by cyst walls thinned to approximately 0.1 microm, as the phoronts prepared to excyst prior to host ecdysis. Additionally, we report the fine structure of the rosette, trichocysts, nuclei, food plaquettes, oral fiber, and other cytoplasmic inclusions. SEM revealed an outer cyst wall layer connected to the secreted peduncle material, which was observed to extend over a wide (15 microm) area on the host setae. Cysts were usually attached at their posterior ends or, less frequently, along their side.

Gymnodinioides pacifica, n. sp., an exuviotrophic ciliated protozoan (Ciliophora, Apostomatida) from euphausiids of the Northeastern Pacific.

A new species of apostome ciliate, Gymnodinioides pacifica n. sp. from the euphausiid Euphausia pacifica is described. The ciliated protozoan encysts on the setae of the appendages, telson and antennae. It excysts and enters the exoskeleton of the host after moulting, where it feeds on exuvial fluid. The phoront and trophont stages of this ciliate are described. The ciliature of the trophont has the following characteristics that distinguish it from the other species of Gymnodinioides: (1) a short kinety 9a (ciliary row 9a or K9a), approximately half of the length of falciform field 8, that extends from the anterior end of the cell posteriorly to the level of K1, (2) a straight K5a, perpendicular to K5b, K6, and K7, and (3) two distinct bands of kineties, K9b-K5b and K4-K1. In addition to those on the host E. pacifica, phoront cysts of similar shape were observed on Thysanoessa spinifera, T. gregaria, T. inspinata, T. longipes, and Nematoscelis difficilis. High prevalence rates were recorded (83%) for these apostome cysts on E. pacifica and T. spinifera during summer collections from the Oregon and Washington coasts. Additionally, we report other apostome ciliate symbionts of euphausiids, including Phtorophrya sp., which preys on apostomes in the genus Gymnodinioides.

Phylogeny of marine Gregarines (Apicomplexa)--Pterospora, Lithocystis and Lankesteria--and the origin(s) of coelomic parasitism.

Gregarines constitute a large group of apicomplexans with diverse modes of nutrition and locomotion that are associated with different host compartments (e.g. intestinal lumena and coelomic cavities). A broad molecular phylogenetic framework for gregarines is needed to infer the early evolutionary history of apicomplexans as a whole and the evolutionary relationships between the diverse ultrastructural and behavioral characteristics found in intestinal and coelomic gregarines. To this end, we sequenced the SSU rRNA gene from (1) Lankesteria abbotti from the intestines of two Pacific appendicularians, (2) Pterospora schizosoma from the coelom of a Pacific maldanid polychaete, (3) Pterospora floridiensis from the coelom of a Gulf Atlantic maldanid polychaete and (4) Lithocystis sp. from the coelom of a Pacific heart urchin. Molecular phylogenetic analyses including the new sequences demonstrated that several environmental and misattributed sequences are derived from gregarines. The analyses also demonstrated a clade of environmental sequences that was affiliated with gregarines, but as yet none of the constituent organisms have been described at the ultrastructural level (apicomplexan clade I). Lankesteria spp. (intestinal parasites of appendicularians) grouped closely with other marine intestinal eugregarines, particularly Lecudina tuzetae, from polychaetes. The sequences from all three coelomic gregarines branched within a larger clade of intestinal eugregarines and were similarly highly divergent. A close relationship between Pterospora schizosoma (Pacific) and Pterospora floridiensis (Gulf Atlantic) was strongly supported by the data. Lithocystis sp. was more closely related to a clade of marine intestinal gregarines consisting of Lankesteria spp. and Lecudina spp. than it was to the Pterospora clade. These data suggested that coelomic parasitism evolved more than once from different marine intestinal eugregarines, although a larger taxon sample is needed to further explore this inference.

Comparative surface morphology of marine coelomic gregarines (Apicomplexa, Urosporidae): Pterospora floridiensis and Pterospora schizosoma.

Two species in the aseptate gregarine genus Pterospora from the Pacific and Gulf coasts were analyzed by scanning electron microscopy, which revealed characteristics not reported in other gregarines. The gamonts of these species had branching trunks that ended in terminal digits, and both species moved by cytoplasmic streaming and peristalsis. Pterospora floridiensis had surface pits and tracts of parallel ridges that bended and connected with one another. Pterospora schizosoma had irregular-shaped surface swellings that were usually arranged in rosette patterns. These unique surface features have not been reported for other gregarines, and are strikingly different from the surface features of many septate and aseptate gregarines that inhabit the intestinal lumena of their hosts and move by gliding. The correlation of Pterospora's unique pellicular features to the habitat and cytoplasmic streaming characteristic of the genus may be significant, and may reflect an adaptation for development in coelomic environments.

Exuviotrophic apostome ciliates from crustaceans of St. Andrew Bay, Florida, and a description of Gymnodinioides kozloffi n. sp.

Gymnodinioides kozloffi n. sp. is described from the eelgrass broken-back shrimp Hippolyte zostericola. The species is distinct from others in the apostome genus Gymnodinioides in that the trophont ciliature has a small group of kinetosomes located to the right of Kinety 9a, and Kinety 1 and 2 are divided. Other apostome morphologies are described from many decapod crustaceans from St. Andrew Bay, Florida, including Gymnodinioides inkystans, Hyalophysa chattoni, and variants of both H. chattoni and G. kozloffi. All of these apostome ciliates are exuviotrophic, found feeding on exuvial fluid within the exoskeleton of the host after ecdysis. The hosts surveyed for this study are the following: Callinectes sapidus, Eurypanopeus depressus, Hippolyte zostericola, Farfantepenaeus spp., Palaemonetes intermedius, Palaemon floridanus, Portunus spp., Tozeuma carolinense, and Sicyonia laevigata, which revealed a number of new host-apostome records.

The fine structure of the gamont of Pterospora floridiensis (Apicomplexa: Eugregarinida).

Transmission electron microscopy of the gamont stage of Pterospora floridiensis has revealed a number of features. The gamont's surface varies from smooth to crenulate, with numerous pockets and folds. The pellicle is composed of an outer membrane, a middle lucent region, and an inner dense layer comprised of two tightly appressed membranes. Short ridges on the pellicle are 200-300+ nm long, 75-100 nm wide, and have a height of approximately 50 nm. The thickness of the pellicle is 100 nm when measured from the inner membrane to the top of a ridge. The ridges are formed by the plasma membrane and an underlying structure that is circular in cross-section. The surface folds and the pellicular ridges are distributed over the soma and the cell's unusual branching arms, though both are reduced near the junction between two gamonts in syzygy, and are absent at the central area of the junctional site. The cell has numerous active Golgi complexes associated with vesicles, as well as scattered dense mitochondria, lipid droplets, and paraglycogen granules. The nucleus has a large (13 microm) endosome, eccentrically located, and peripheral chromatin along the inner nuclear membrane.