Morphology and Molecular Phylogeny of Two New Ciliates,Holostichides heterotypicus n. sp. and Holosticha muuiensis n. sp. (Ciliophora: Urostylida)

Two new urostylid species, Holostichides heterotypicus n. sp. and Holosticha muuiensis n. sp., were discovered in South Korea. Morphological and phylogenetic analyses were carried out to confirm that these species are new to science. Holostichides heterotypicus is mainly characterized by the following combination of features: 110–205 μm long in vivo; 5–10 frontoterminal cirri; 6–8 midventral pairs with 2–3 midventral cirral rows; cortical granules present; four bipolar dorsal kineties; and 6–9 caudal cirri. Ontogenetic features of H. heterotypicus are similar to those of H. typicus. Phylogenetic analyses revealed that H. heterotypicus was distantly separated from bakuellid genera Apobakuella, Bakuella, Metaurostylopsis, and Neobakuella. This result is supported by the following features: transverse cirri (present in the other four bakuellids vs. absent in Holostichides) and caudal cirri (absent in the other four bakuellids vs. present in Holostichides). Holosticha muuiensis n. sp. is mainly distinguished from its congeners by the following combination of features: 100–185 long in vivo; shortened undulating membrane; cortical granules lacking; contractile vacuole absent; 51–66 adoral zone of membranelles; 42–60 macronuclear nodules; and five bipolar dorsal kineties. In the phylogenetic tree, Holosticha muuiensis n. sp. clustered with a Holosticha group (containing Holosticha diademata, Holosticha foissneri, and Holosticha heterofoissneri).     

Longidorus carniolensis sp. n. (Nematoda, Longidoridae) from vineyard soil in Slovenia

A new needle nematode, Longidorus carniolensissp. n., recovered from the soil around the roots of grapevine Vitis vinifera L. from Slovenia, is described and illustrated. Longidorus carniolensisis an amphimictic species, characterised by females with a moderately long (L=5.6-8.2 mm) and plump (a=51-72.4, ave. 66.3) body, assuming a spiral to C-shape when heat relaxed. Head region continuous, anteriorly almost flat, lip region 23-25 μm wide; guiding ring situated posteriorly (42-47 μm, 43-50 μm in males), odontostyle long (ave. 146.6 (136-157) μm); pharyngeal glands with normal location, their nuclei of approximately equal size; tail bluntly conoidal to almost hemispherical. Males abundant, spicules slender and long (122-145 μm), ventromedian supplements 13-17, irregularly spaced, preceded by an adanal pair. Four juvenile stages present, the first stage juvenile with bluntly conoidal tail. Codes for identifying the new species when using the key by Chen et al. (1997) are: A 56, B 4, C 4, D 1, E 4, F 35, G 1, H 1, I 2. The new species is morphologically the most similar to Longidorus poessneckensis Altherr, 1974, Longidorus macrosoma Hooper, 1961, Longidorus caespiticola Hooper, 1961, Longidorus helveticus Lamberti et al., 2001, Longidorus macroteromucronatus Altherr, 1974, Longidorus pius Barsi & Lamberti, 2001, Longidorus raskii Lamberti & Agostinelli, 1993, Longidorus kheirii Pedram et al. 2008, Longidorus silvae Roca, 1993, Longidorus iuglandis Roca et al., 1985, Longidorus vinearum Bravo & Roca, 1995 and Longidorus major Roca & d'Erico, 1987, but differs from these species either by the body and odontostyle length, position of guide ring, head region and tail shape or the shape of the first stage juvenile tail. Sequence data from the D2-D3 region of the 28S rDNA distinguishes this new species from other speciesof the genus Longidorus with known sequences. Relationships of Longidorus carniolensissp. n. with other Longidorus species based on analysis of this DNA fragment and morphology are discussed.     

Isolation of a new vahlkampfiid amoeba from soil: Paravahlkampfia lenta n. sp.

When the genus Paravahlkampfia was distinguished from the genus Vahlkampfia on the basis of significant differences in small subunit ribosomal DNA sequences, Paravahlkampfia ustiana was the only described species of this new genus. More recently, a vahlkampfiid strain has been isolated (from soil from an upland farm in Scotland) which has trophozoite and cyst morphology more similar to P. ustiana than to other non-flagellating vahlkampfiid species. Also, it clusters with P. ustiana in phylogenetic trees derived from 5.8S ribosomal DNA sequences. However, it is significantly different from P. ustiana in both phenotype and internal transcribed spacer sequence. Consequently, a new species, Paravahlkampfia lenta, is proposed.     

Taxonomic characterization of two marine peritrichous ciliates, Pseudovorticella clampi n. sp. and Zoothamnium pararbuscula n. sp. (Ciliophora: Peritrichia), from North China

The morphology, infraciliature, and silverline system of two new peritrichous ciliates, Pseudovorticella clampi n. sp. and Zoothamnium pararbuscula n. sp., have been investigated based on both living and silver-impregnated specimens. Partial sequence of 18S-ITS1-5.8S rDNA of Z. pararbuscula is also determined in order to compare it with the closely related congener, Zoothamnium arbuscula. Zoothamnium pararbuscula can be distinguished from its close form Z. arbuscula by the different habitats, the appearance of the main stalk, the position of the contractile vacuole, and the information derived from 18S-ITS1-5.8S rDNA sequence analysis. Pseudovorticella clampi n. sp. is distinguished from its congeners by its body shape and size, pellicle granules, habitat, and number of transverse silverlines.     

Steinernema akhursti sp. n. (Nematoda: Steinernematidae) from Yunnan, China

A new species of entomopathogenic nematode, herein described as Steinernema akhursti sp. n., was recovered from soil samples collected from Yunnan Province, the People's Republic of China. Both morphological and molecular data show congruently that S. akhursti sp. n. belongs to the Steinernema feltiae group. It can be separated from all described Steinernema species by the combined morphological and morphometrical characters of various stages of the nematodes. For the first generation male, the new species can be recognized by spicule length 90 +/- 4.6 microm, spicule tip blunt with an aperture on the ventral side, gubernaculum with a long and needle-shaped cuneus, and tail conoid with a prominent mucron on the tip and a concave on ventral side. For the infective juvenile, the combination of the following characters: body length 812 +/- 19 microm, distance from anterior end to excretory pore 59 +/- 1.5 microm, tail length 73 +/- 2.9 microm, E% 77 +/- 4.5, lateral field with six evenly distributed and identical ridges at the middle body portion, and tail with long and slightly constrict hyaline portion can be used to separate the new species from other nematodes. For the female, the new species is characterized by: tail conoid with a short mucron and slightly swelling anal portion and a symmetrical, slightly protruding vulva with conspicuous double-flapped epiptygma. The nematode can be separated from other described species of Steinernema by DNA sequences of either a partial 28S rDNA or the internal transcribed spacer regions of rDNA and from the closely related species S. feltiae and Steinernema oregonense by cross-breeding tests.     

Alona stochi n.sp. – the third cave-dwelling cladoceran (Crustacea: Cladocera) from the Dinaric region

Nemerteans (phylum Nemertea) have been viewed by mostzoologists as descended from, or closely related to,the flatworms. This view is based mainly on theirsupposedly acoelomate body. Their ancestry, however,is a point of controversy and there is evidence for acoelomate, protostomous origin. Notwithstanding thesedifferent views, most zoologists consider nemerteansto be phylogenetically distant from the chordates.Four authors (Hubrecht, Macfarlane, Jensen, Willmer),however, have postulated that nemerteans instead areclosely related to the chordates and that they sharea most recent common ancestor with the vertebrates. We argue that this view is based on a flawed view ofhomology and of seeing evolution as a series ofprogressions, which has no support in modernevolutionary thinking. Since there are nomorphological synapomorphies supporting aChordata-Nemertea clade, these authors instead guesswhat characteristics in extant nemerteans gave rise tocharacters observed in recent chordates. For example,they propose that the nemertean proboscis sheath hasevolved into the notochord. This is mere speculation,lacking testable propositions and is hence void ofinformation, and thus becomes futile in our view. However, the idea of a nemertean-vertebrate sisterrelationship as such is a testable hypotheses, and wetest it by applying the parsimony criterion to a setof morphological characters, and a set of molecular(the 18S rRNA gene) characters. Both tests reject thehypothesis.     

A New Freshwater Naked Lobose Amoeba Korotnevella venosa n. sp. (Amoebozoa,Discosea)

A new freshwater species of naked lobose amoebae Korotnevella venosa n. sp. isolated from freshwater pond in St. Petersburg, Russia was studied with light and transmission electron microscopy. Basket scales of this species have six vertical columns supporting perforated rim. The latter has tongue‐like broadening with membranous region. Vertical columns bifurcate at both ends so that neighboring columns are connected by their bifurcations forming combined structure. Basket scales of K. venosa are similar to those of Korotnevella hemistylolepis in having six full‐length vertical columns and perforated rim. At the same time, they are different in having tongue‐like broadening of perforated rim with membranous region and absence of six half‐length columns and an intermediate crosspiece. Phylogenetic trees based on 18S rDNA gene placed K. venosa either at the base of the whole Korotnevella clade, next to K. hemistylolepis, or as a sister to the clade comprising Korotnevella species with latticework basket in large scales.     

甲烷氧化菌Methylomonas sp.GYJ3中甲烷单加氧酶基因与16S rRNA序列分析

甲烷氧化细菌中的关键酶系甲烷单加氧酶是一个含双核铁的多组份氧化酶,常温、常压下能够催化甲烷转化为甲醇。对甲烷氧化细菌Methylomonas sp.GYJ3中溶解性甲烷单加氧酶基因和16SrDNA进行了测序与分析。利用已知相关基因数据库信息,设计了PCR引物和测序引物,获得了满意的测序结果。全长的溶解性甲烷单加氧酶基因为5690bp,部分16S rDNA的序列长度为1280bp。与已发表的甲烷氧化细菌中甲烷单加氧酶进行了比较,结果表明MMOX组份中氨基酸序列的同一性为78%到99%,基因序列的同一性为71%到97%,6个组份中orfY片段的同一性相对较低。MMOX氨基酸序列的多序列联配表明,MMOX序列具有高度保守性,特别是在双核铁中心区域。16S rDNA进化分析显示Methylomonas sp.GYJ3与γ蛋白细菌是相关联的,基于MMOX氨基酸序列的进化分析证明,与Methylomonas sp.GYJ3最近似的菌株是Ⅰ型甲烷氧化细菌Methylomonas sp.KSWⅢ。综合分析表明,菌株GYJ3属于Ⅰ型甲烷氧化细菌Methylomonas sp.属。这个结果为Ⅰ型甲烷氧化细菌也能表达溶解性甲烷单加氧酶提供了新的证据。羟基化酶的理论等电点是6.28,理论分子量为248874.41Da。     

Morphology,Morphogenesis, and Molecular Phylogeny of Neobakuella aenigmatica n. sp. (Ciliophora,Spirotrichea, Bakuellidae)

The morphology and morphogenesis of a new ciliate species, Neobakuella aenigmatica n. sp., which was discovered in an estuary in Korea, were investigated, using live observation, protargol impregnation, and scanning electron microscopy. This new species is characterized by a large (185–300 × 55–105 μm in vivo), elongate‐ellipsoidal, flexible but not contractile body. It has ellipsoidal, yellowish cortical granules, 1.3 × 1.0 μm in size. The species has invariably 3 frontal and 2 frontoterminal cirri, about 5–10 buccal and 1–6 parabuccal cirri, 7 midventral rows, and 1 right and 2–4 left marginal rows. The outer left marginal row(s) consists of 1–7 short rows of cirri. The nuclear apparatus comprises 130 macronuclear nodules and 2 spherical micronuclei on average. The dorsal ciliature consists of 3 dorsal kineties. The leftmost left marginal row(s) likely develops from anlagen originating from both the rightmost and leftmost left marginal row(s). The molecular phylogenetic tree based on SSU rDNA suggests the nonmonophyly of the genus Neobakuella.     

Molecular Phylogeny and Surface Morphology of Thiriotia hyperdolphinae n. sp. and Cephaloidophora oradareae n. sp. (Gregarinasina,Apicomplexa) Isolated from a Deep Sea Oradarea sp. (Amphipoda) in the West Pacific

In an effort to broaden our understanding of the biodiversity and distribution of gregarines infecting crustaceans, this study describes two new species of gregarines, Thiriotia hyperdolphinae n. sp. and Cephaloidophora oradareae n. sp., parasitizing a deep sea amphipod (Oradarea sp.). Amphipods were collected using the ROV Hyper‐Dolphin at a depth of 855 m while on a cruise in Sagami Bay, Japan. Gregarine trophozoites and gamonts were isolated from the gut of the amphipod and studied with light and scanning electron microscopy, and phylogenetic analysis of 18S rDNA. Thiriotia hyperdolphinae n. sp. was distinguished from existing species based on morphology, phylogenetic position, as well as host niche and geographic locality. Cephaloidophora oradareae n. sp. distinguished itself from existing Cephaloidophora, based on a difference in host (Oradarea sp.), geographic location, and to a certain extent morphology. We established this latter new species with the understanding that a more comprehensive examination of diversity at the molecular level is necessary within Cephaloidophora. Results from the 18S rDNA molecular phylogeny showed that T. hyperdolphinae n. sp. was positioned within a clade consisting of Thiriotia spp., while C. oradareae n. sp. grouped within the Cephaloidophoridae. Still, supplemental genetic information from gregarines infecting crustaceans will be needed to better understand relationships within this group of apicomplexans.     

Longidorus ferrisi n. sp. from California Citrus

In October 1999, the authors received fixed specimens of a species of Longidorus from Howard Ferris found about the roots of a citrus tree in Oakville, Napa County, CA. After determining it to be new a species, we requested additional specimens. The samples contained roughly equal numbers of males and females. Longidorus ferrisi n. sp. is most similar to L. elongatus, but can be distinguished by a greater c-ratio (111-187 vs 73-141), a lesser c′ (0.7-1.1 vs 1.0-1.3), a more offset head, a more posterior guide ring (35-40 vs 30-33 μm), the presence of sperm in the uterus in mature females, and the approximate 1:1 ratio of females to males. Other similar species include L. artemisiae, L. crassus, L. glycines, and L. milanis. Longidorus ferrisi n. sp. differs from L. artemisiae by a lesser a-ratio (74-102 vs 109-155), a lesser c′ value (0.7-1.1 vs 1.0-1.6), a more posterior guide ring (35-40 vs 27-34 μm), a longer odontostyle (91-108 vs 84-98 μm), a wider lip region (16-19 vs 14-17 μm), wider mid-body (53-69 vs 41-52 μm), and longer spicules (57-65 vs 39-49 μm). The new species differs substantially from L. crassus by its lip shape and the presence of males, and differs from L. glycines by a shorter body (4.33-5.97 vs 6.14-8.31 mm), a lesser c′ value (0.7-1.1 vs 0.9-1.4), a narrower lip region (16-19 vs 20-23 μm), wider mid-body (53-69 vs 39-57 μm), longer spicules (53-69 vs 45-53 μm), and fewer supplements (7-11 vs 11-17). Longidorus ferrisi n. sp. differs from L. milanis by a longer body (4.33-5.97vs 3.00-4.90 mm), a greater c value (111-187 vs 86-130), a wider mid-body (53-69 vs 43-56 μm), a different head shape, and longer spicules (53-69 vs 41-54 μm). The nuclear 18S ribosomal DNA sequence of this species revealed that this species is unique with respect to all sequenced Longidorus species.     

Oxnerella micra sp. n. (Oxnerellidae fam. n.), a tiny naked centrohelid, and the diversity and evolution of heliozoa

We describe a new tiny naked centrohelid heliozoan, Oxnerella micra, and sequenced its 18S and 28S rRNA genes. Its extremely slender axopodia have prominent extrusomes and are normally stretched across the substratum like those of many tiny granofilosean Cercozoa. Phylogenetic analysis of 18S rDNA shows that Oxnerella does not branch within any of the six known centrohelid families but very deeply in the order Pterocystida, between Choanocystidae and Pterocystidae; therefore we place it in a new family, Oxnerellidae. Oxnerella arose from ancestors with siliceous scales by losing them; as independently did Heterophryidae and Marophryidae, which replaced them by organic spicules, and Chlamydaster that is not truly naked but retains a mucilage coat and nests extremely shallowly within Pterocystidae. 28S rDNA has a group I intron. Concatenated Bayesian 18S/28S rRNA phylogeny shows centrohelids weakly as sisters to the naked non-centrohelid heliozoan Microheliella maris (Microhelida: Heliozoa). The centrohelid Marophrys marina possesses an elongation factor α-like (EFL) protein related to that of Polyplacocystis; Microheliella also has EFL. We also analysed Hsp90 and 18S rDNA sequences from 'Pinaciophora sp.' ATCC50355; they must be from a centrohelid, probably misidentified as Pinaciophora, the rDNA sequence branching deeply within Pterocystida. We reclassify two Polyplacocystis, Luffisphaera, Phaeodaria and Rotosphaerida.     

Brasilonema lichenoides sp. nov. and Chroococcidiopsis lichenoides sp. nov. (Cyanobacteria): two novel cyanobacterial constituents isolated from a tripartite lichen of headstones

Cyanolichens are an assemblage of fungi and cyanobacteria from diverse, cosmopolitan habitats. Typically composed of a single species of cyanobacterium, with or without another eukaryotic alga, here we present two novel cyanobionts isolated from an undescribed tripartite lichen. This endolithic lichen was isolated from a granite cemetery tombstone from Jacksonville, FL, and contains two potentially nitrogen‐fixing cyanobionts. Employing a total evidence approach, we characterized the cyanobionts using molecular (the 16S rDNA and ITS gene region), morphological, and ecological data. Phylogenetic analyses revealed two novel taxa: Brasilonema lichenoides and Chroococcidiopsis lichenoides, both of which fell within well‐supported clades. To our knowledge, this represents the first instance of a tripartite lichen with two cyanobacterial and no eukaryotic members. These types of lichens may well represent an unexplored reservoir of cyanobacterial diversity. The specific epithets are proposed under the provisions of the International Code of Nomenclature for algae, fungi, and plants.     

Oscheius basothovii n. sp. (Nematoda: Rhabditidae), a new entomopathogenic nematode isolated from an uncultivated grassland in South Africa

During a recent soil sample survey in Heidelberg at the Suikerbosrand Nature Reserve near Johannesburg in South Africa, a new entomopathogenic nematode species, collected from the grassland was discovered. Oscheius basothovii was isolated from soil samples using the Galleria mellonella bait method. Amplification and Sanger sequencing of the 18S ribosomal DNA placed the nematode in genus Oscheius. Morphological studies with light and scanning electron microscopy confirmed its anatomical affinities with the genus. The new species is characterised by various traits including its original 18S rDNA sequence, six lips, and amphidelphic reproduction. Males were characterised by the presence of spicules towards their posterior end and short blunt tails. Females had a body with irregular ridges, a vulva opening, and long pointed tails. The infective juveniles of this new species are able to invade Galleria mellonela and Tenebrio molitor larvae and cause mortality within 24–72?h after inoculation under laboratory conditions.     

Steinernema aciari sp. n. (Nematoda: Steinernematidae), a new entomopathogenic nematode from Guangdong, China

A new species of entomopathogenic nematode, Steinernema aciari sp. n. was described. It was recovered from a soil sample collected from Haimen town, Shantou district in the eastern coast of Guangdong province, the People's Republic of China during a survey for entomopathogenic nematodes. S. aciari sp. n. belongs to the Steinernema glaseri group. It can be separated from all described Steinernema species by the combined morphological and morphometrical characters of various stages of the nematodes. For male, the new species can be recognized by spicule length (86+/-6.3 microm); spicule tip blunt with a hook-like structure; gubernaculum with a short and Y-shaped cuneus and corpus well-separated posteriorly. For infective juvenile, the combination of the following characters: body length (1113+/-68 microm), distance from anterior end to excretory pore (95+/-3.7 microm), tail length (78+/-5.2 microm), and E % (123+/-7) can be used to differentiate the new species from other nematodes. For female, the tail (conoid with a long mamillate terminus and a distinct postanal swelling) and vulva (slightly protruding from body surface with conspicuous double flapped epiptygma) shapes can be used as diagnostic characters for the new species. The new species can also be distinguished from other Steinernema species by DNA sequences of either a partial 28S rDNA or the internal transcribed spacer regions of rDNA, and from the close related species S. glaseri, Steinernema longicaudum CWL05, and Steinernema guangdongense by cross-breeding test.     

高抑菌活性乳酸杆菌的筛选及性质

从自酿酸奶中分离得到1株高抑菌活性菌株,经16S rDNA测序后鉴定为Lactobacillus sp.FSZ。以大肠杆菌、金黄色葡萄球菌为指示菌,取得良好抑菌效果。经组分分析及蛋白酶降解,抑菌活性物质确定为蛋白物质,推测其由一些高分子的蛋白类物质和低分子的多肽类物质组成。抑菌活性物质在酸性条件下显示出良好的抑菌活性,发酵液经60℃处理30 min后,活性基本没有下降,经100℃处理30 min仍保留83.9%的活性,表现出良好的热稳定性。     

Aggregata polibraxiona n. sp. (Apicomplexa: Aggregatidae) from Octopus bimaculatus Verrill, 1883 (Mollusca: Cephalopoda) from the Gulf of California,Mexico

The Apicomplexa Aggregata spp. are intracellular parasites of cephalopods that infect the intestinal tract of commercially important species such as Octopus bimaculatus, which sustains the octopus fishery in Baja California (B.C.), Mexico. In this study, Aggregata polibraxiona n. sp. was described from the cecum of O. bimaculatus collected from Bahia de Los Angeles, B. C. Light and electron microscopy revealed that oocysts and sporocysts were spherical to ovoid in shape. Sporulated oocysts (293–835 × 177–688 μm) contained 135–674 sporocysts (12–24 × 11–22 μm). The sporocyst wall was covered by tubular projections (0.55–2.19 μm in length) bifurcated in the top, unevenly distributed, covered by a thin membrane. Each sporocyst contains 11–13 sporozoites (16–26 × 1.20–3 μm). Three partial sequences of the 18S rDNA gene were obtained, and two phylogenetic approaches were performed according to Bayesian inference and Maximum Likelihood. In both phylogenetic reconstructions, the sequences of A. polibraxiona n. sp. were recovered as a monophyletic group within the genus Aggregata and placed as a sister group to Aggregata octopiana Lineage II. Aggregata polibraxiona n. sp. is the first Apicomplexa described from a cephalopod host from Mexico and extends the geographical range of Apicomplexa infecting cephalopods.     

Trichonosema algonquinensis n. sp. (Phylum microsporidia) in Pectinatella magnifica (Bryozoa: phylactolaemata) from Algonquin Park, Ontario, Canada

A new species of microsporidian, Trichonosema algonquinensis, is described from a freshwater bryozoan, Pectinatella magnifica from Ontario, Canada. The parasite develops in epithelial cells and appears as white, spherical masses throughout the tissues. Trichonosema algonquinensis is diplokaryotic, diploblastic and undergoes development in direct contact with the cytoplasm of the host cell. Mature spores are ovoid, tapered at one end, and measure 8.5 +/- 0.3 x 4.4 +/- 0.1 microm. The polar filament is wound in 20 to 23 helical coils. Although the parasite resembles T. pectinatellae described from the same host in Michigan and Ohio, it differs in the length of the spore and number of coils of the polar filament. Analysis of 16S rDNA by maximum likelihood, parsimony and Baysian inference, complements the morphological data in supporting the placement of T. algonquinensis as a sister species of T. pectinatellae.     

Labyrinthula diatomea n. sp.—A Labyrinthulid Associated with Marine Diatoms

Labyrinthulomycetes are mostly fungus‐like heterotrophic protists that absorb nutrients in an osmotrophic or phagotrophic manner. Members of order Labyrinthulida produce unique membrane‐bound ectoplasmic networks for movement and feeding. Among the various types of labyrinthulids’ food substrates, diatoms play an important role due to their ubiquitous distribution and abundant biomass. We isolated and cultivated new diatom consuming Labyrinthulida strains from shallow coastal marine sediments. We described Labyrinthula diatomea n. sp. that differs from all known labyrinthulids in both molecular and morphological features. We provided strain delimitation within the genus Labyrinthula based on ITS sequences via haplotype network construction and compared it with previous phylogenetic surveys.