Identification of Naegleria fowleri and other Naegleria spp. (free-living amoebae) using cellulose acetate membrane electrophoresis of glucose phosphate isomerase

Abstract A simple isoenzyme cellulose acetate membrane electrophoresis method with respect to glucose phosphate isomerase (GPI) was developed for the differentiation of the human pathogenic free-living amoeba Naegleria fowleri from other Naegleria spp. A single GPI band was detected in all the species tested, the relative mobility of which could be used to identify N. fowleri . Of the other Naegleria spp., only N. italica and N. jadini shared a common GPI mobility. No intraspecies variation in GPI profile was detected, regardless of whether the strains were cultured in monoxenic or axenic media. The technique is proposed as a useful means of identifying N. fowleri soon after isolation from the environment.     

Morphological and phylogenetic analysis of Raillietina spp. in indigenous chickens (Gallus gallus domesticus) in Bangladesh

Raillietina spp. (Cestoda: Davaineidae), the most common cestodes in indigenous chickens, cause a substantial production loss in poultry industry in Bangladesh. Here, we estimated the prevalence, confirmed the species and determined the genetic pattern of species of Raillietina using molecular tools. We collected and examined 375 chickens randomly from household of different villages of Mymensingh sadar and Gouripur upazila, Mymensingh district and adult parasites were isolated and identified. Genomic DNA was extracted from collected parasites, amplified ITS-2 and ND-1 genes, sequenced and analyzed. Out of 375 samples, 270 (72.0%) were found positive with Raillietina species and mean worm burden was 10.46 ± 0.56. Microscopically, three species of Raillietina, such as R. cesticillus (37.9%), R. echinobothrida (41.1%) and R. tetragona (52.8%) were detected on the basis of their morphological features. The total length, length and width of scolex, sucker and rostellum were also measured. Among different factors, age, farming nature and flock size of chickens were significantly (p < 0.05) influenced Raillietina infections. For further validation, the sequences of ITS-2 gene generated in this study were matched with reference sequences of R. cesticillus, R. echinobothrida and R. tetragona and found 99.63% − 100% similarity. The phylogenetic analyses of ITS-2 and ND-1 sequences were clustered together with the reference sequences of R. cesticillus, R. echinobothrida and R. tetragona confirming microscopic identification. This is the first confirmation of species of Raillietina along with the prevalence of the species, which will be helpful for the formulation of a control strategy and provide basic information for further molecular study.     

Distribution and phylogenetic relationships of Australian glow-worms Arachnocampa (Diptera, Keroplatidae)

Glow-worms are bioluminescent fly larvae (Order Diptera, genus Arachnocampa) found only in Australia and New Zealand. Their core habitat is rainforest gullies and wet caves. Eight species are present in Australia; five of them have been recently described. The geographic distribution of species in Australia encompasses the montane regions of the eastern Australian coastline from the Wet Tropics region of northern Queensland to the cool temperate and montane rainforests of southern Australia and Tasmania. Phylogenetic trees based upon partial sequences of the mitochondrial genes cytochrome oxidase II and 16S mtDNA show that populations tend to be clustered into allopatric geographic groups showing overall concordance with the known species distributions. The deepest division is between the cool-adapted southern subgenus, Lucifera, and the more widespread subgenus, Campara. Lucifera comprises the sister groups, A. tasmaniensis, from Tasmania and the newly described species, A. buffaloensis, found in a high-altitude cave at Mt Buffalo in the Australian Alps in Victoria. The remaining Australian glow-worms in subgenus Campara are distributed in a swathe of geographic clusters that extend from the Wet Tropics in northern Queensland to the temperate forests of southern Victoria. Samples from caves and rainforests within any one geographic location tended to cluster together within a clade. We suggest that the morphological differences between hypogean (cave) and epigean (surface) glow-worm larvae are facultative adaptations to local microclimatic conditions rather than due to the presence of cryptic species in caves.     

The maintenance of free-living amoebae by cryopreservation.

We have successfully cryopreserved free-living amoebae in order to maintain them feasibly under the conditions in our laboratory. The viability of trophozoites was higher when frozen by slow cooling (overall 0.7 degree C/min) than by fast cooling (overall 1.3 degrees C/min). Glycerol and dimethylsulfoxide at the final concentration of 7.5% each was used for cryopreservation of free-living amoebae trophozoites. The survival rate was 2-39% after storage in the liquid nitrogen for 60 days. Gross cultural or morphological changes were not noted in trophozoites thawed from frozen suspensions.     

Entamoeba histolytica: rapid identification and differentiation of Indian isolates by riboprinting

Entamoeba histolytica infection still remains one of the major public health problem for developing countries like India. A rapid and accurate detection of this parasite is essential for prevention and control of amoebiasis. In this study, using the method of 'riboprinting' (PCR-RFLP of rRNA genes from amoeba) we have analysed 15 stool samples from symptomatic patients of amoebiasis. All 15 patients of clinical amoebiasis had E. histolytica in their stool and two of the samples also showed mixed infection of E. dispar. Apart from the known restriction enzyme sites within the amoeba SSU-rRNA genes, a new Sau3A site having a discriminatory value is identified in these E. histolytica isolates from India. Hence, it is possible to rapidly identify E. histolytica DNA and differentiating it from E. dispar using minute amounts of clinical stool samples, thus eliminating the laborious parasite culturing process. Thus, riboprinting is advantageous for clearcut identification of E. histolytica in order to decide an effective antiamoebic therapy.     

The phylogenetic relationships and generic limits of finches (Fringillidae)

Phylogenetic relationships among the true finches (Fringillidae) have been confounded by the recurrence of similar plumage patterns and use of similar feeding niches. Using a dense taxon sampling and a combination of nuclear and mitochondrial sequences we reconstructed a well resolved and strongly supported phylogenetic hypothesis for this family. We identified three well supported, subfamily level clades: the Holoarctic genus Fringilla (subfamly Fringillinae), the Neotropical Euphonia and Chlorophonia (subfamily Euphoniinae), and the more widespread subfamily Carduelinae for the remaining taxa. Although usually separated in a different family-group taxon (Drepanidinae), the Hawaiian honeycreepers are deeply nested within the Carduelinae and sister to a group of Asian Carpodacus. Other new relationships recovered by this analysis include the placement of the extinct Chaunoproctus ferreorostris as sister to some Asian Carpodacus, a clade combining greenfinches (Carduelis chloris and allies), Rhodospiza and Rhynchostruthus, and a well-supported clade with the aberrant Callacanthis and Pyrrhoplectes together with Carpodacus rubescens. Although part of the large Carduelis-Serinus complex, the poorly known Serinus estherae forms a distinct lineage without close relatives. The traditionally delimited genera Carduelis, Serinus, Carpodacus, Pinicola and Euphonia are polyphyletic or paraphyletic. Based on our results we propose a revised generic classification of finches and describe a new monotypic genus for Carpodacus rubescens.     

MALDI-TOF质谱技术对克罗诺杆菌的鉴定与分型

以基质辅助激光解析电离飞行时间质谱(MALDI-TOFMS)技术用于克罗诺杆菌的鉴定与分型。通过对获得的克罗诺杆菌属典型菌株、阴沟肠杆菌和产气肠杆菌近似菌株以及克罗诺杆菌分离株的蛋白质质量图谱进行对比分析,找出克罗诺杆菌特征性离子峰,将其作为鉴定克罗诺杆菌的生物标识物;对全细菌蛋白质质量图谱进行聚类分析,将克罗诺杆菌属进一步划分为不同类型,结果显示,4株克罗诺杆菌参考菌株质量图谱约在5740(m/z)离子质荷比处出现1个相近离子峰,28株克罗诺杆菌分离株中27株(占96.4%)表现出相同结果;32株克罗诺杆菌被分为6种类型(以50%距离水平为分类界限)。MALDI-TOFMS作为一种新的技术,不仅能够用于克罗诺杆菌的鉴定,而且根据获得的细菌蛋白质质量图谱可将克罗诺杆菌划分为不同类型。     

Mitochondrial DNA variation and phylogenetic relationships of Jasus spp. (Decapoda: Palinuridae)

The seven species of rock lobster in the genus Jasus have a fragmented circumpolar distribution, inhabiting continental or island waters of the Southern Ocean. Gene flow between nominal species is possible as the planktonic larval stages of Jasus are widely dispersed in major oceanic gyres. Restriction endonuclease analysis of mitochondrial DNA (mtDNA) from five species ( J. verreauxi, J. novaehollandiae, J. edwardsii, J. lalandii and J. tristani ) was used to assess taxa previously defined only by morphological criteria. Intraspecific mtDNA nucleotide sequence diversity was generally high (0˙33–0˙99%). An absence of episodic population bottlenecks and extinctions, attributable to a teleplanic (far wandering) and prolonged pelagic stage, may be a significant factor contributing to this variation. New Zealand ( J. edwardsii ) and Australian ( J. novaehollandiae ) populations appear to be conspecific and should be referred to as J. edwardsii ; however, a significant difference in the magnitude of mean sequence diversities between these populations may indicate restrictions to gene flow across the Tasman Sea. The genome of J. verreauxi is highly distinct from the genomes of the other species (nucleotide sequence diversity: 14˙92–16˙67%), supporting the existence of 'verreauxi' and 'lalandii' groups within Jasus . High sequence diversities separating J. edwardsii, J. lalandii and J. tristani (4˙41–7˙36%) indicates longterm reproductive isolation. Hypotheses for the evolution of 'lalandii' group Jasus , which suggest a relatively recent divergence of J. lalandii and J. tristani , are not supported by phylogenetic reconstruction. Instead, it gives systematic validity to the grouping of J. lalandii with J. edwardsii as proposed by the existing taxonomy.     

Redefinition and phylogenetic relationships of the family Pinguipedidae (Teleostei: Perciformes)

The phylogenetic relationships of the family Pinguipedidae plus the genus Cheimarrichthys von Haast, 1874, were studied to redefine the family. Based on a phylogenetic analysis using derived characters belonging to 21 transformation series, accepting the monophyly of Pinguipedidae plus Cheimarrichthys provisionally for the operational procedure of the analysis, it was hypothesized that Cheimarrichthys is not closely related to Parapercis Bleeker, 1863, although these genera had been considered as having a close affinity. Although the Pinguipedidae and Cheimarrichthys share nine derived characters, it was determined that these characters are also found in other trachinoid families. In addition, several families, such as the Leptoscopidae, Uranoscopidae, and Trichodontidae, have many more derived characters in common with Cheimarrichthys than the pinguipedids have with that genus. The conclusion drawn here is that it is not parsimonious to recognize the monophyly of the Pinguipedidae and Cheimarrichthys based on these nine derived characters, and that these characters are not compelling enough to link these groups. The redefined Pinguipedidae is proposed to include the following five genera: Parapercis, Prolatilus Gill, 1865, Pinguipes Cuvier, 1829, Pseudopercis de Miranda-Ribeiro, 1903, and Kochichthys Kamohara, 1960. Cheimarrichthys, excluded from the Pinguipedidae, is put into its own family, Cheimarrichthyidae.     

Identification of polyhydroxyalkanoate (PHA)-producing Bacillus spp. using the polymerase chain reaction (PCR)

AIMS: The aim of the work was to develop efficient method to identify polyhydroxyalkanoate (PHA)-producing species of Bacillus from numerous soil isolates of bacteria. Identification of the isolates and characterization of the PHA produced by strains positive on the polymerase chain reaction (PCR) was envisaged. METHODS AND RESULTS: Different bacteria isolated from soil were screened by PCR using two sets of primers designed for Bacillus megaterium. Amongst 23 isolates examined, the DNA of 12 isolates reacted positively with the primers giving amplicons identical in size to that obtained from B. megaterium. The isolates which were identified as strains of B. sphaericus, B. circulans, B. brevis and B. licheniformis, produced 11- 41% of PHA in biomass, in sucrose-containing medium, over a growth period of 24-72 h. The nature of the PHA thus produced was analyzed by Fourier transform infrared spectroscopy, gas chromatography and by nuclear magnetic resonance (NMR) and found to contain polyhydroxy butyrate and polyhydroxyvalerate. CONCLUSIONS: The results indicate that most of our isolates from different species contained the B. megaterium type of PHA synthase. Bacillus licheniformis appeared to belong to another group as it did not react with both sets of primers. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows the universality of the B. megaterium type of PHA synthase in soil isolates of Bacillus. Some variations were also found.     

Cocultivation of Legionella pneumophila and free-living amoebae.

Studies of the interaction of Legionella pneumophila with free-living amoebae showed that Naegleria lovaniensis and Acanthamoeba royreba could use L. pneumophila as a sole food source. However, growth of the amoebae on nonnutrient agar plates seeded with L. pneumophila was slower than growth on nonnutrient agar plates seeded with Escherichia coli. On inoculation of L. pneumophila into axenic cultures of N. lovaniensis and A. royreba, 99.9% of the L. pneumophila was destroyed within 24 h. After several weeks, however, some amoeba cultures became chronically infected and supported the growth of L. pneumophila. Amoebae exposed to L. pneumophila and containing adhered L. pneumophila, L. pneumophila antigens, or both, showed no increased pathogenic potential on intranasal inoculation of weanling mice. Similarly, L. pneumophila propagated in chronically infected amoeba cultures showed no increase in virulence on intraperitoneal inoculation of guinea pigs relative to L. pneumophila grown in yeast extract broth.     

Carboniferous aulate corals from Azrou-Khenifra Basin (Morocco): Distribution and phylogenetic relationships

The Carboniferous outcrops from the Azrou-Khenifra Basin (central Morocco) have yielded solitary and fasciculate aulate corals. Although mostly rare, their presence has significance for extending the known stratigraphic range of two genera. Both Solenodendron and Semenoffia which were previously restricted to the Tournaisian-Viséan interval are now recognised to range up into the Serpukhovian. Moreover, Solenodendron is now last recorded in lower Bashkirian strata, having survived the end-Serpukhovian (mid-Carboniferous) faunal crisis. New phylogenetic relationships are proposed within this aulate group of corals for the western Palaeotethys, based on detailed examination of their morphology, microstructure and stratigraphic range. The distribution of solitary and fasciculate aulate corals in the Azrou-Khenifra Basin (Morocco) is comparable with that of the Béchar Basin (Algeria), and some species are commonly recorded in Europe. A new species, Aulokoninckophyllum potyi is described.     

鸻形目15种鸟类线粒体ND6基因序列差异及其系统进化关系

采用PCR和质粒克隆测序方法,首次获得鸻形目l5种鸟类线粒体基因组的ND6基因全长522bp的序列。经对位排列,序列间末见有插入和缺失,共有216个变异位点,种间序列差异为5．17％—19．92％。以白鹤为外群,用NJ法构建15种鸟类的进化关系树。研究结果表明：构建的系统树将鸻形目15种鸟类分为2个支系：第1支系包括蒙古沙鸻、环颈鸻、灰斑鸻和反嘴鹬。第2支系包括红脚鹬、林鹬、青脚鹬、翘嘴鹬、翻石鹬、大滨鹬、尖尾滨鹬、斑尾塍鹬、中杓鹬、大杓鹬和白腰杓鹬,其中鹬属的3个种和杓鹬属的3个种分别组成一个单系;翘嘴鹬和翻石鹬、大滨鹬和尖尾滨鹬分别聚为姊妹群,表现出较近的亲缘关系;斑尾塍鹬独立分支出来。分子证据提示：鹬科中的塍鹬属、鸻科中的斑鸻属应提升为亚科分类阶元;反嘴鹬与鸻科鸟类亲缘关系较近,组成一个单系,将其归入鸻科下属的一个类群更为合理,与核型研究结果及Sibley新分类体系的观点相一致。     

The phylogenetic diversity of thermophilic members of the genus Bacillus as revealed by 16S rDNA analysis

Abstract Sixteen thermophilic strains of the genus Bacillus , representing eight validly described and six invalidly described species, as well as one unassigned strain, were investigated by comparative 16S rDNA analyses and the sequences compared to the existing database for the genera Bacillus and Alicyclobacillus . The majority of strains were found to cluster in two groups represented by B. stearothermophilus and B. pallidus. Bacillus smithii, B. thermocloacae , and B. thermoruber are phylogenetically well separated and cluster within the radiation of mesophilic bacilli. The as yet undescribed taxon 'B. flavothermus' warrants species status. B. schlegelii and B. tusciae group peripherally with members of Alicyclobacillus and may be reclassified when more phenotypic data support their phylogenetic position.     

Comparative mitochondrial genomics and phylogenetic relationships of the Crossoptilon species (Phasianidae,Galliformes)

Background

Phasianidae is a family of Galliformes containing 38 genera and approximately 138 species, which is grouped into two tribes based on their morphological features, the Pheasants and Partridges. Several studies have attempted to reconstruct the phylogenetic relationships of the Phasianidae, but many questions still remain unaddressed, such as the taxonomic status and phylogenetic relationships among Crossoptilon species. The mitochondrial genome (mitogenome) has been extensively used to infer avian genetic diversification with reasonable resolution. Here, we sequenced the entire mitogenomes of three Crossoptilon species (C. harmani, C. mantchuricum and C. crossoptilon) to investigate their evolutionary relationship among Crossoptilon species.

Results

The complete mitogenomes of C. harmani, C. mantchuricum and C. crossoptilon are 16682 bp, 16690 bp and 16680 bp in length, respectively, encoding a standard set of 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes, and a putative control region. C. auritum and C. mantchuricum are more closely related genetically, whereas C. harmani is more closely related to C. crossoptilon. Crossoptilon has a closer relationship with Lophura, and the following phylogenetic relationship was reconstructed: ((Crossoptilon + Lophura) + (Phasianus + Chrysolophus)). The divergence time between the clades C. harmani-C. crossoptilon and C. mantchuricum-C. auritum is consistent with the uplift of the Tibetan Plateau during the Tertiary Pliocene. The Ka/Ks analysis showed that atp8 gene in the Crossoptilon likely experienced a strong selective pressure in adaptation to the plateau environment.

Conclusions

C. auritum with C. mantchuricum and C. harmani with C. crossoptilon form two pairs of sister groups. The genetic distance between C. harmani and C. crossoptilon is far less than the interspecific distance and is close to the intraspecific distance of Crossoptilon, indicating that C. harmani is much more closely related to C. crossoptilon. Our mito-phylogenomic analysis supports the monophyly of Crossoptilon and its closer relationship with Lophura. The uplift of Tibetan Plateau is suggested to impact the divergence between C. harmani-C. crossoptilon clade and C. mantchuricum-C. auritum clade during the Tertiary Pliocene. Atp8 gene in the Crossoptilon species might have experienced a strong selective pressure for adaptation to the plateau environment.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1234-9) contains supplementary material, which is available to authorized users.     

Differentiation of vocalizations in bushbabies (Galaginae, Prosimiae, Primates) and the significance for assessing phylogenetic relationships

A comparative bioacustic analysis of vocalizations of the prosimian subfamily Galaginae revealed that morphologically similar sibling taxa within the main groups of the lesser galagos and the greater galagos can be reliably identified phenotypically on the basis of the acoustic structure of their loud call or advertisement call. Results confirm the separation of two distinct species of greater galagos, Galago crassicaudatus and Galago garnettii, and strongly suggest the discrimination of three distinct species from the senegalensis lesser bushbaby group, Galago senegalensis, Galago moholi and Galao zanzibaricus. An investigation of the ontogenetic development of the loud call indicated that it is derived from the infant's isolation call, displaying in all studied bushbaby taxa a fairly similar acoustic pattern. Shared acoustic characters of the loud call among the different taxa as well as the infant's isolation call were used to propose a hypothesis about the phylogenetic affinities in bushbabies. The results seem to be supported by recent fossil records.     

Grossuana codreanui (Grossu, 1946) and the phylogenetic relationships of the East Balkan genus Grossuana (Radoman, 1973) (Gastropoda: Rissooidea)

The rich rissooidean fauna of Balkan Peninsula, the history of which began at the time of Paratethys, is not sufficiently studied yet. It includes, among others, many taxa of minute gastropods with a moderately high-spired shell, a penis with a double lobe on its left edge and female reproductive organs with a loop of coiled oviduct and two receptacles, the morphology of which does not allow for a certain determination. In the article we deal with five such populations. We describe the shell, radula, soft part morphology and anatomy of the reproductive organs of Grossuana codreanui (Grossu, 1946) from its type locality at the Techirghiol Lake, Dobrogea, Romania. The species is compared molecularly (fragments of two genes: mitochondrial CO1 and ribosomal 18 S) with G. codreanui from Bulgaria, G. serbica (Radoman, 1973) from its type locality, Belgrandiella haesitans (Westerlund, 1881) from the spring of the Louros River, and Orientalina delphica (Radoman, 1973) from the spring Kastalia at Delphi. All the five populations were found to belong to the genus Grossuana: G. codreanui, G. serbica, G. vurliana (Radoman, 1966), and G. dephica (Radoman, 1973), respectively. We applied 12 morphological characters of Grossuana and 17 other rissooidean genera (mostly of the Hydrobiidae s. stricto) to infer phylogeny, which has shown the position of the genus within the Hydrobiidae s. stricto, most close to Orientalina. All the molecular data confirm that Grossuana is a distinct genus belonging to the Hydrobiidae, Horatiinae, whose closest relatives are Belgrandia, Orientalina and Horatia. Handling editor: C. Sturmbauer     

形目15种鸟类线粒体ND6基因序列差异及其系统进化关系

采用PCR和质粒克隆测序方法 ,首次获得形目 15种鸟类线粒体基因组的ND6基因全长 5 2 2bp的序列。经对位排列 ,序列间未见有插入和缺失 ,共有 2 16个变异位点 ,种间序列差异为 5 17%～ 19 92 %。以白鹳为外群 ,用NJ法构建 15种鸟类的进化关系树。研究结果表明 :构建的系统树将形目 15种鸟类分为 2个支系。第 1支系包括蒙古沙、环颈、灰斑和反嘴鹬。第 2支系包括红脚鹬、林鹬、青脚鹬、翘嘴鹬、翻石鹬、大滨鹬、尖尾滨鹬、斑尾塍鹬、中杓鹬、大杓鹬和白腰杓鹬 ,其中鹬属的 3个种和杓鹬属的 3个种分别组成一个单系 ;翘嘴鹬和翻石鹬、大滨鹬和尖尾滨鹬分别聚为姊妹群 ,表现出较近的亲缘关系 ;斑尾塍鹬独立分支出来。分子证据提示 :鹬科中的塍鹬属、科中的斑属应提升为亚科分类阶元 ;反嘴鹬与科鸟类亲缘关系较近 ,组成一个单系 ,将其归入科下属的一个类群更为合理 ,与核型研究结果及Sibley新分类体系的观点相一致 [动物学报 49(1) :6 1～ 6 7,2 0 0 3]。     

Toxoplasma gondii: uptake and survival of oocysts in free-living amoebae

Waterborne transmission of the oocyst stage of Toxoplasma gondii can cause outbreaks of clinical toxoplasmosis in humans and infection of marine mammals. In water-related environments and soil, free-living amoebae are considered potential carriers of various pathogens, but knowledge on interactions with parasitic protozoa remains elusive. In the present study, we assessed whether the free-living Acanthamoebacastellanii, due to its phagocytic activity, can interact with T. gondii oocysts. We report that amoebae can internalize T. gondii oocysts by active uptake. Intracellular oocysts in amoebae rarely underwent phagocytic lysis, retained viability and established infection in mice. Interaction of T. gondii with amoebae did not reduce the infectivity and pathogenicity of oocysts even after prolonged co-cultivation. Our results show that uptake of oocysts by A. castellanii does not restrain the transmission of T. gondii in a murine infection model.