A Description of a New ‘‘Amoebozoan’’ Isolated from the American Lobster,Homarus americanus

JEFFREY COLE,a,1 O. ROGER ANDERSON,b YONAS I. TEKLE,c JESSICA GRANT,c LAURA A. KATZc and THOMAS NERADd

aAmerican Type Culture Collection, Protistology Collection, 10801 University Blvd., Manassas, Virginia 20110, andbBiology, Lamont-Doherty Earth Observatory of Columbia University, Palisades, New York 10964, and

cDepartment of Biological Sciences, Smith College, Northampton, Massachusetts 01063, anddGeorge Mason University, PWII, Manassas, Virginia 10801

ABSTRACT. Our knowledge of the diversity of amoeboid protists is rapidly expanding as new and old habitats are more fully explored.In 2003, while investigating the cause of an amoeboid disease afflicting lobsters on the East Coast, samples were examined for thepresence of amoebae from the carapace washings of the American lobster, Homarus americanus. During this survey a unique communityof gymnamoebae was discovered. Among the new taxa discovered was a small Thecamoeba-like organism with a single posteriorlydirected pseudopodium. Although resembling Parvamoeba rugata, this amoeba displayed distinctive morphology from that isolate or anyother amoebozoan. Phylogenetic analysis shows this amoeba is distantly related to the Thecamoebidae. In this paper we describe theunique morphology of a second species of Parvamoeba and discuss its phylogenetic position with respect to the ‘‘Amoebozoa.’’

Key Words. Amoebozoa, epibionts, fine structure, isolate ATCCs PRA-35TM, Parvamoeba monoura, phylogenetic analysis, SSU rDNA,SSU rRNA secondary structure.

THE vast marine environment provides numerous novel nichesfor microbial eukaryotes. Therefore, it is no surprise that

many new amoebozoans have been isolated from various diversemarine habitats (Amaral-Zettler et al. 2006; Anderson, Rogerson,and Hannah 1997; Kudryavtsev et al. 2005; Moran et al. 2007;Nikolaev et al. 2006; O’Kelly et al. 2003; Page 1983, 1987; Peglaret al. 2003; Rogerson and Hauer 2002; Smirnov 1999; Smirnovand Goodkov 1993; Smirnov et al. 2007). In addition, there is anincreasing awareness of the ecological importance and the possi-ble role of many diverse amoebae as opportunistic pathogens ofanimals. For example, many amoebae may be primary or second-ary invading pathogens of crustacea (Johnson 1977; Jones 1985;Sawyer 1976; Sawyer and MacLean 1978; Sprague, Beckett, andSawyer 1969) or vertebrates, including fish (for a review, seeDykova and Lom 2004; Dykova et al. 2005). Moreover, cysts ofmany species of the genus Acanthamoeba, the causative agent ofgranulomatous amoebic encephalitis and amoebic keratitis in hu-mans, have been isolated from marine habitats, although theyhave often been found in soils and freshwaters (Booton et al.2004). In an effort to characterize the amoebae associated withdisease in the American lobster Homarus americanus Milne-Edwards, 1837 (Mullen et al. 2004) off the coast of Connecticut,the surface of a lobster carapace was sampled. A unique commu-nity of protists was isolated from the carapace, including theamoeba described here. While preparing to describe this am-oebozoan and establish a new genus, the authors were made awareof a significant small subunit ribosomal DNA (SSU rDNA) se-quence identity (499% identity) with Parvamoeba rugata Rog-erson, 1993 (Berney, pers. commun.). Comparison of the datafrom the two isolates showed them to be similar in general ap-pearance, but identifiable by distinctive morphological characters.We propose that this new isolate, ATCCs PRA-35TM is a newmember of the until now monotypic genus Parvamoeba describedby Rogerson (1993) and that a new species is established for thisamoeba based on light and electron microscopical morphologicalstudies and a phylogenetic analysis of its SSU rDNA genesequence.

MATERIALS AND METHODS

Isolation, culture, and cryopreservation. The amoeba wasrecovered from the carapace of the American lobster, H. ameri-canus, collected in a survey trawl in 2002 in Long Island Soundnear Oyster Bay, New York (center point of trawl: 41.02501N,73.36541W). The carapaces of six male lobsters that were eithersuspected or confirmed of being infected with Paramoeba weresampled. The carapace of each lobster was swabbed with a sterilecotton swab that was then immersed in 10 ml of Seawater 802medium (American Type Culture Collection, Manassas, VA[ATCCs medium 1525TM] bacterized with Klebsiella pneumo-niae subsp. pneumoniae (ATCCs 700831TM) that was containedin a 25-cm2-tissue culture flask. The swab was vigorously agitatedto dislodge any amoebae. The amoeba of interest was establishedin presumptive clonal culture in the above medium. Although theculture was fed only a single species of bacterium, other uniden-tified environmental bacteria also were present. The amoeba strainwas cultivated in 25-cm2-tissue culture flasks containing 10 ml ofbacterized medium and incubated horizontally at 20–25 1C withthe caps screwed on tightly. In addition, cultures of the amoebawere incubated at 4 1C, 10 1C, 15 1C, 20–25 1C, 30 1C, and 37 1Cfor 1–3 days at each temperature before successively transferringcultured amoebae to the next higher or lower temperature in theseries. Amoebae were also transferred through a successive seriesof culture media of decreasing salinity in 10% increments (90–0%of full-strength sea salts) using the incubation regimen as above.

The amoebae were cryopreserved using 7.5% (v/v) dimethylsulfoxide prepared in ATCCs medium 1525TM and distributed in0.5 ml aliquots to 1.2 ml conical bottom Nalge Nuncs vials(VWR International, Bridgeport, NJ). The vials were then placedin a 1 1C/min freezing apparatus (Nalgene Catalog #5100-0001)for 3 h and then plunged into liquid nitrogen. After 24 h, a frozenampoule was thawed for 3 min in a 35 1C water bath and the con-tents added to 10 ml of ATCCs medium 1525TM bacterized withK. pneumoniae subsp. pneumoniae (ATCCs 700831TM) in a25-cm2-tissue culture flask. The flask was incubated at 20–25 1C.

Light microscopy. Light microscopical observations were madeon live cells using a Zeiss Axioskop compound microscope (CarlZeiss Jena GmbH, Zeiss Gruppe, Unternehmensbereich MicroskopieD-7740, Jena) equipped with an Axiocam HR digital camera (CarlZeiss Microlmaging Inc., Thornwood, NY) and using phase contrastand differential interference contrast (DIC) optics at 400–1,000�magnification. Images of locomotive and floating cells were captured

1Present Address: 1015 Madison Lane, Blackburg, VA 24060Corresponding Author: J. Cole, American Type Culture Collection,

Protistology Collection, 10801 University Blvd., Manassas, Virginia20110—Telephone number: 1701 365 2700; FAX number: 1703 3652730; e-mail: jcole@atcc.org

40

J. Eukaryot. Microbiol., 57(1), 2010 pp. 40–47r 2009 The Author(s)Journal compilation r 2009 by the International Society of ProtistologistsDOI: 10.1111/j.1550-7408.2009.00445.x

and measurements were made from these images using the interac-tive measurement module of the Axiovision version 2.0 software.

Transmission electron microscopy. Samples were preparedaccording to the methods of Anderson et al. (1997). Equal vol-umes (� 5 ml) of suspended cells in culture medium and TEMgrade glutaraldehyde (6% (v/v) in 0.02 M cacodylate bufferedmedium, pH 7.2) were mixed to produce a final 3% (v/v) glut-araldehyde fixative at room temperature and plunged in an icebath for 20 min. The fixed cells were sedimented by centrifugationand the pellet fixed at 5 1C for 1 h in 2% (v/v) osmium tetroxide inthe same buffer as the primary fixative. The osmium-fixed cellswere sedimented by centrifugation, and the pellet enrobed in 0.8%(w/v) agar. Small segments (3 mm cubes) were cut from the en-robed mass of fixed cells, washed in deionized water, dehydratedin a graded acetone/aqueous series, infiltrated, and embedded withlow viscosity TAAB epoxy resin (Energy Beam Sciences, Gran-by, CT.), and polymerized at 70 1C overnight. Ultrathin sections,obtained with a Porter-Blum MT-2 (Sorvall Scientific, Norwalk,CT) ultramicrotome fitted with a diamond knife, were collected onuncoated 200-mesh copper grids, post-stained with Reynold’s al-kaline lead citrate, and observed with a Philips TEM 201 (PhillipsElectronics, Einthoven, the Netherlands) electron microscope op-erated at 60 kV accelerating voltage.

Molecular genetics. Total cellular DNA was extracted usingFastDNA Kit (Bio 101 Inc., Carlsbad, CA, Catalogue 6540-40) perthe manufacturer’s instructions. Primers for SSU rDNA genes arefrom Medlin et al. (1988) with three additional primers used togenerate overlapping sequences from each clone as described inSnoeyenbos-West et al. (2002). Polymerase chain reaction (PCR)products were cleaned using the Qiaquick PCR Purification System(Qiagen, Valencia, CA), and cloned using the TOPO TA CloningKit (Invitrogen, Carlsbad, CA). Plasmid DNA was purified usingthe Qiagen MiniPrep Kit (Qiagen). Direct sequencing of PCR prod-ucts or cloned plasmid DNA was accomplished in both directionsusing gene-specific primers and the BigDye terminator kit (AppliedBiosytems [ABI], Foster City, CA). Sequences were run on an ABI310 automated sequencer. Sequences were submitted to GenBankwith the following accession numbers: EF455775 (SSU), EF455789(b-tubulin), EF455756 (a-tubulin), and EF455773 (actin).

To align SSU-rDNA sequences, we used the Hmmer package(Eddy 1998), version 2.1.4 with default settings. Hmmer used aset of previously aligned sequences to determine (model) the sec-ondary structure of the unaligned sequences. The training align-ment for building the model, consisting of all availableamoebozoa SSU-rDNA sequences (as of October 2006) alignedaccording to their secondary structure were downloaded from TheEuropean Ribosomal Database (Wuyts, Perriere, and Van de Peer2004). The resulting alignment was further edited manually inMacClade v. 4.05 (Maddison and Maddison 2002).

Gene trees were constructed using MrBayes (Huelsenbeck et al.2001) and RA �ML (Stamatakis, Ludwig, and Meier 2005a;Stamatakis, Ott, and Ludwig 2005b). To assess the position ofour amoeba within the ‘‘Amoebozoa,’’ a total of 100 taxa wereused: 95 ingroup taxa (‘‘Amoebozoa’’ sequences including ourisolate) and five outgroup taxa (two animals, two fungi, andBreviata anathema). MrModeltest (Nylander 2004) was used toselect the appropriate model of sequence evolution for our data,which was the GTR1I1G model of evolution. Bayesian analyseswere then performed with the parallel version of MrBayes 3.1.2using the GTR1I1G model of evolution (Ronquist and Huelsen-beck 2003). Four simultaneous MCMC chains were run for10 � 106 generations sampling every 100 generations. Based onthe convergence of likelihood scores the first 2,500,000 genera-tions (25,000 trees) were discarded as ‘‘burnin.’’ The 50% ma-jority-rule consensus tree was determined to calculate theposterior probabilities for each node from the remaining trees.

Concordant gene trees were also inferred in RAxML v.2.0, amaximum likelihood method, using the rapid hill climbing algo-rithm under the GTRGAMMA model.

After 100 bootstrap replications, using the same model, a ma-jority rule consensus tree displaying the bootstrap frequencies wascalculated in PAUP� 4.0b10 (Swofford 2002).

Secondary structure prediction of the small subunit ribo-somal RNA (SSU rRNA). The predicted secondary structures forthe SSU rRNA of ATCCs PRA-35TM (Manassas, VA 20110),Thecamoeba, and the related Stenamoeba stenopodia were basedupon the model proposed by Wuyts et al. (2004). The programMfold was used to determine optimal foldings of stems and loops(Zuker 2003). The final structures were drawn using the programAdobe Illustrators.

RESULTS

Description of isolate ATCCs PRA-35TM. The major mor-phological and fine structural features, culture characteristics, andmolecular genetics are presented to augment the concise descrip-tions presented in the diagnoses.

Light microscopic morphology. The distinguishing featureof this amoeba at the light microscopical level is the temporarypresence of a short (range 5 0.9–2.7 mm, mean 5 1.7 � 0.43mm,N 5 7) solitary, posteriorly directed, fine pseudopod (Fig. 1, 2).Rarely, a second fine pseudopod appeared near the origin of thefirst pseudopod, but was soon resorbed (Fig. 3). Amoebae were 3–5.5 mm long (average 5 3.9 mm, SD 5 0.49, N 5 30) and 1.9–4.3 mm (average 5 3.2 mm, SD 5 0.04, N 5 30) in breadth. Theaverage length to breadth ratio was 1.2 (range 5 1–2.8,SD 5 0.53, N 5 30) and average area of the footprint of theamoeba was 9.25 mm2 (range 5 6.54–13.75 mm2, SD 5 1.54).

The amoebae firmly attach to the substrate and must be scrapedwith a cell scraper to be dislodged. The floating form is globular.One to three fine pseudopodia that can flex may be seen in thefloating form as it prepares to settle (Fig. 3). Amoebae glide alongthe substrate with very little change in shape. A hyaline zone maydevelop but is usually not pronounced. They present a slightlywrinkled or sculptured appearance and do not move in a linearfashion but tend to follow a curved or circular path. Actively mo-tile amoebae glided at a mean rate of 7.8 � 2.2mm/min (range4.7–10.0, N 5 5). These amoebae, however, are not always mo-tile. Many cells in a culture remain stationary for long periods oftime. This species forms cysts (Fig. 4), but excystment was neverobserved and could not be induced in old or neglected cultures.

Fine structure. The somewhat lobate nucleus (Fig. 5) occu-pies a substantial part of the cytoplasm and occasionally presents asomewhat granular nucleolus with an irregular margin (Fig. 8).The prominent Golgi body (Fig. 5, inset) is typically juxtanuclear,but is displaced toward the periphery in the encysted stage (Fig.10, 11). Mitochondria (� 0.5 mm) are tubulocristate (Fig. 6). Thecytoplasm contains dense granules of glycogen and scattered,more dense, larger granules, � 0.1–0.2 mm, (Fig. 8) typicallydistributed near the cell margin that is coated with a finely fibrousglycocalyx (� 30-nm thick). The outer fuzzy margin of the glyco-calyx is somewhat more electron dense (Fig. 7). The solitarypseudopodium emerges through a rupture in the glycocalyx (Fig.8, 9) and may extend rather rigidly from the surface (Fig. 8). Thespheroidal cyst (� 3 mm) is enclosed by a thin wall (� 40–50-nmthick) of fairly uniform, low electron density (Fig. 10, 11). Thenucleus of the cyst is located peripherally in the cytoplasm andsometimes appears more lobate (Fig. 10) than in the trophontstage (Fig. 5). A relatively large (� 1 mm) very electron-dense‘‘storage body’’ is centrally located in the cyst cytoplasm(Fig. 10) and the Golgi apparatus remains prominently organized(Fig. 11).

41COLE ET AL.—PARVAMOEBA MONOURA, N. SP.

Culture. At the temperatures tested, ATCCs PRA-35TM grewbest at 20 1C, but some growth occurred at temperatures as low as10 1C. At 37 1C, growth was not detected nor could active amoe-bae be recovered from these cultures. Amoebae remained motilein full strength seawater media and in all dilutions to 30% (v/v) offull-strength. Amoebae were not motile or recoverable after beingcultured in 20%, 10%, and 0% marine media.

Molecular data. Most of the ‘‘Amoebozoa’’ SSU rDNA genetree is relatively well resolved, although there is weak or no sup-port at deeper nodes. The phylogenetic placement of the newlydescribed amoeba within the ingroup taxa ‘‘Amoebozoa’’ is wellsupported. ATCCs PRA-35TM is one of the basal taxa in theTubulinean clade in most of our analyses, although this relation-ship is only weakly supported in our MrBayes analyses(PP 5 0.78) (Fig. 12). In all of our analyses the new taxondid not group with the two species of Thecamoeba, which areconsistently recovered as forming a sister group to Acanthopodidatogether with S. stenopodia (PP 5 1.00). Instead, the genus Parv-amoeba as represented by ATCCs PRA-35TM, is an ‘‘orphan’’in that there are no closely related SSU rDNA sequences pub-lished to date. It was only after this analysis was completed that itwas found ATCCs PRA-35TM shared an almost identicalSSU rDNA sequence with P. rugata CCAP 1556/1 (Berney, pers.commun.).

Ribosomal RNA secondary structure. A secondary struc-ture comparison of SSU rRNA with other Tubulineans indicatesthat the ATCCs PRA-35TM secondary structure is different fromthat of Thecamoeba and the related S. stenopodia: it has the E23-1and E23-4 stems typical of all tubulineans (Fig. 13) but the overallstructure of the E-23 region is not closely similar to any of the taxawithin the tubulinean clade. However, this region is quite variablewithin this clade.

DISCUSSION

The discovery of the new species of Parvamoeba from thecarapace of the American lobster suggests that there is a wealthof amoeboid diversity yet to be discovered. This Parvamoebashares a very similar SSU rDNA with its congener yet exhibits aunique suite of morphological characters. Under current taxo-nomic sampling, Parvamoeba monoura also presents an ‘‘or-phan’’ phylogenetic position in molecular phylogenetics. Theanalysis of the molecular data here (Fig. 12) and in two otherpublications where the lineage is referred to as Thecamoeba sp.ATCCs PRA-35TM (Tekle et al. 2008; Yoon et al. 2008) placesthis taxon as one of the most basal Tubulinea, but no clear mor-phological synapomorphies can be established. Alternatively,the new taxon described here may represent a novel clade within

Fig. 1–4. Light micrographs of Parvamoeba monoura n. sp. 1. Differential interference contrast (DIC) image of motile amoeba showing sculptedsurface appearance, central nucleus, and pseudopod (arrowhead). 2. Phase contrast image (� 400) of several motile amoebae showing extended pseu-dopods (arrowheads). 3. Phase contrast image of floating form preparing to settle on the substrate. A second pseudopodium is present (arrowhead). 4. DICimage of cyst. Scale bars 5 5.0 mm. All images at �1,000 except where otherwise noted.

42 J. EUKARYOT. MICROBIOL., 57, NO. 1, JANUARY– FEBRUARY 2010

Fig. 5–11. Transmission electron micrographs of Parvamoeba monoura n. sp. 5. A section through a cell showing the somewhat lobate and elongatednucleus (N) and mitochondria (M) with tubular cristae. The cytoplasm has abundant deposits of glycogen granules. Inset: Golgi body consisting of fivecisternae, typically juxtanuclear. Scale bars 5 0.5 mm. 6. A view of the peripheral cytoplasm containing tubulocristate mitochondria. Scale bar 5 0.5 mm.7. A high magnification view of the cell surface showing the fibrous glycocalyx (arrow), approximately 30-nm thick, with a somewhat more osmiophilic,thin outer layer. Scale bar 5 0.1 mm. 8, 9. The electron-dense, uncoated pseudopodium (Ps) emerges from the cell periphery through a ruptured region ofthe glycocalyx (arrows). Scale bars 5 0.5 mm. 10. A section through a cyst, enclosed by a thin, somewhat electron-translucent wall (arrow), showing thenucleus (N), tubulocristate mitochondria (M), and an electron-dense ‘‘reserve’’ body (B) characteristically observed near the center of the cyst cytoplasm.Scale bar 5 0.4 mm. 11. An enlarged view of the periphery of a cyst with details of the 40–50-nm-thick wall (arrow) and nearby cytoplasmic featuresincluding the Golgi body. Scale bar 5 0.1 mm.

43COLE ET AL.—PARVAMOEBA MONOURA, N. SP.

Fig. 12. Bayesian gene tree (ln L 5 � 39,639.825) based on the small subunit (SSU) rDNA of 1,426 characters showing the placement of Parv-amoeba monoura n. sp. within the ‘‘Amoebozoa’’ (arrow). Posterior probabilities (PP) and bootstrap values (100 replicates, with GTRGAMMA inRAxML v2.0) are shown above/below nodes, respectively. Black solid circles represent nodes with full support in both analyses and nodes below 0.5% or50% support values are represented by a dash. Tree branches are drawn to the scale bar (0.1 substitution) as shown above, except the five branches markedby asterisks, which are reduced by half for convenience.

44 J. EUKARYOT. MICROBIOL., 57, NO. 1, JANUARY– FEBRUARY 2010

the ‘‘Amoebozoa’’ and its placement near the Tubulinea may bedriven by limited taxonomic sampling. In previous studies, theSSU, actin, a-tubulin, and b-tubulin genes were analyzed in thecontext of a broad representative sampling of eukaryotes (Tekle

et al. 2008; Yoon et al. 2008). In concatenated analyses ofthese four genes, this taxon fell within the ‘‘Amoebozoa’’ withfull support under both Bayesian and maximum likelihoodalgorithms.

Fig. 13. The predicted secondary structure for the small subunit ribosomal RNA of Parvamoeba monura n. sp. Although the secondary structure isnot particularly distinctive, it has the typical E23-1 and E23-4 helices (see arrows) found in most amoebozoans. The secondary structure of the E23 regionis not closely similar to any of the tubulinean taxa in the tree in Fig. 12 but this region is quite variable within this clade.

45COLE ET AL.—PARVAMOEBA MONOURA, N. SP.

Differential diagnosis. Parvamoeba monoura n. sp. closelyresembles P. rugata Rogerson, 2003 in size and shape, but may bedistinguished from P. rugata by the more sculptured surface andpresence of a trailing pseudopod in the locomotive form. P. mon-oura exhibits a relatively fast motility of 7.8 � 2.2mm/min com-pared with 0.7 mm/min in P. rugata. The nucleolus of P. monourahas an irregular shape in contrast to the spherical, centrally locatednucleolus of P. rugata. The fine structure of the nucleus indicatesno internal fibrous lamina as reported in Thecamoeba spp. (e.g.Page 1983). The glycocalyx in P. monoura consists of a denserouter layer in contrast to the uniform electron density of the cellcoat of P. rugata. Another marked difference in the glycocalyx ofP. monoura is the presence of a rupture zone where the solitarypseudopodium emerges. There is no break in the glycocalyx forthe pseudopodia of P. rugata. Although the differences in finestructure of the glycocalyx in our isolate compared with that of P.rugata are subtle, we believe that they are sufficiently defined tobe diagnostically significant. We do not believe that the differ-ences are due to effects of different fixation protocols. The hyalinelayer of the glycocalyx of P. monoura is consistently present inour preparation, exhibits varying widths at different locations, andis more pronounced at the point of emergence of the pseudopo-dium. Hence, we conclude that it is structurally less substantialthan the more distal densely staining layer and becomes morefully separated from the cell surface during emergence of thepseudopodium. The distal more electron-dense layer of the glyco-calyx in our isolate is thinner and more fibrous in appearance thanthat of P. rugata. Hence, we conclude that the fine structural fea-tures of the glycocalyx are diagnostically discriminative for thetwo species, and name this isolate P. monoura n. sp. The capa-bility of cyst formation has not been observed in P. rugata. Withrespect to other possibly related genera, the fine structure of thenucleus indicates no internal fibrous lamina as reported in Thec-amoeba spp. (e.g. Page 1983), which in addition to the moleculargenetic data further separates this isolate from the Thecamoeb-idae.

The variety of growth conditions, including temperature andsalinity, in which P. monoura n. sp. can be cultivated suggests thatthis species should be able to exploit a variety of habitats and maybe quite widespread. Its isolation from the carapace of the Amer-ican lobster is probably coincidental and this species is most likelynot restricted to this habitat because the number of habitats thathave been adequately sampled is still very limited.

Phylogenetic position of P. monoura n. sp. Putative super-group: ‘‘Amoebozoa’’ (Luhe, 1913, emend. Cavalier-Smith,1998). Secondary Rank, possibly Tubulinea (Smirnov et al.2005), although there is only moderate support for this hypothe-sis and the genus Parvamoeba has no close relatives based uponthe SSU rDNA sequence data.

‘‘Amoebozoa’’ Luhe, 1913, emend. Cavalier-Smith, 1998Tubulinea Smirnov et al. 2005

P. monoura n. sp.

Diagnosis. Light microscopic morphology reveals a small dis-coidal amoebozoan, 3.0–5.5 mm long (mean 5 3.9 mm; length:-breath ratio 1.0:2.8 [mean 5 1.2] distinguished by the presenceof a solitary, short, fine pseudopodium (range 0.9–2.7 mm;mean 5 1.7 mm). The pseudopodium is posteriad (opposite to thedirection of movement) during locomotion and can be resorbed atany time. Locomotive amoebae show very little change in shapeas they move and may remain stationary for long periods of time.The floating form is globular and settles very rapidly on glass orplastic surfaces.

Fine structure includes the single lobate nucleus containing anoccasionally observed nucleolus with an irregular margin, tub-ulocristate mitochondria, a Golgi with five somewhat dilated sac-

cules, and a fibrous glycocalyx with a slightly more electron-denser outer layer. Cysts are spheroidal with a central electrondense ‘‘reserve’’ body, and peripherally located Golgi. The cystwall is finely fibrous and only moderately electron dense.

Etymology. The species name, monoura, is derived from theLatin meaning ‘‘one tail’’ and refers to the unique, thin posteriorlydirected pseudopodium.

Type locality and habitat. The species has been isolated onlyonce from washings from the carapace of the American lobster, H.americanus, taken in a trawl from Connecticut waters (centerpoint of trawl: 41.02501N, 73.36541W).

Reference material. The culture is maintained in the cryopre-served state in liquid nitrogen at the American Type Culture Col-lection (ATCCs) and has been accessioned as P. monouraATCCs PRA-35TM.

ACKNOWLEDGMENTS

This is Lamont-Doherty Earth Observatory Contribution Num-ber 7293. This work is supported by the National Science Foun-dation Assembling the Tree of Life grant (043115) to L.A.K. andto ATCC (0429848). The authors would like to thank Penny Ho-well and Colleen Giannini of the Marine Fisheries Office of Con-necticut CT Department of Environmental Protection for allowingus to sample the lobsters.

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Received: 03/26/09, 07/24/09; accepted: 07/29/09

47COLE ET AL.—PARVAMOEBA MONOURA, N. SP.