IDENTIFYING PHYTOPLANKTON IN SEAWATER BASED ON DISCRETE EXCITATION‐EMISSION FLUORESCENCE SPECTRA1

The feasibility of utilizing discrete excitation‐emission spectra (DEEMs) to identify dominant groups of phytoplankton at both the genus and division levels was investigated. First, the characteristics of in vivo DEEMs were extracted using Coif2 wavelet. Second, optimal characteristic spectra of scale vectors (SOCS) and time‐series vectors (TOCS) were selected by Fisher linear discriminant analysis (FLDA). Third, the SOCS and TOCS were sorted using hierarchical cluster analysis (HCA), and a two‐rank database was established according to their discrimination ability. Fourth, the discrimination of phytoplankton was established by nonnegative least squares (NNLS). For single‐species samples, the correct identification ratios (CIRs) were 62.9%–100% at the genus level and 95.1%–100% at the division level. The dominant species in the mixtures had corresponding CIRs of 87.5% and 97.9%, and 23 dominant species were correctly identified. Prorocentrum donghaiense D. Lu, Thalassiosira nordenskioeldi Cleve, Chaetoceros socialis Lauder (bloom‐forming species with a density of about 10⁷ cell·L^?1), and Skeletonema costatum (Grev.) Cleve (a dominant species with a density of 10⁴–10⁶ cell·L^?1 in seawater) were identified at the genus level. Other dominant species in seawater were identified at the division level if their density was 10⁵–10⁶ cell·L^?1.     

Tapping the potential of intact cell mass spectrometry with a combined data analytical approach applied to Yersinia spp.: detection, differentiation and identification of Y. pestis

In the everyday routine of an analytic lab, one is often confronted with the challenge to identify an unknown microbial sample lacking prior information to set the search limits.In the present work, we propose a workflow, which uses the spectral diversity of a commercial database (SARAMIS) to narrow down the search field at a certain taxonomic level, followed by a refined classification by supervised modelling. As supervised learning algorithm, we have chosen a shrinkage discriminant analysis approach, which takes collinearity of the data into account and provides a scoring system for biomarker ranking. This ranking can be used to tailor specific biomarker subsets, which optimize discrimination between subgroups, allowing a weighting of misclassification.The suitability of the approach was verified based on a dataset containing the mass spectra of three Yersinia species Yersinia enterocolitica, Y. pseudotuberculosis and Yersinia pestis. Thereby, we laid the emphasis on the discrimination between the highly related species Yersinia pseudotuberculosis and Y. pestis.All three species were correctly identified at the genus level by the commercial database. Whereas Y. enterocolitica was correctly identified at the species level, discrimination between the highly related Y. pseudotuberculosis and Y. pestis strains was ambiguous. With the use of the supervised modelling approach, we were able to accurately discriminate all the species even when grown under different culture conditions.     

Ameiurus melas (black bullhead): morphological characteristics of new introduced species and its comparison with Ameiurus nebulosus (brown bullhead)

In the present study we examined and compared external morphology as well as osteological characters of two species of the genus Ameiurus (Teleostei, Ictaluridae): the black bullhead (Ameiurus melas) and brown bullhead (Ameiurus nebulosus), two catfish species that are invasive to Europe. In total 83 (53 and 30) individuals were captured in the Czech Republic and the Slovak Republic and further analysed. For genetic screening of both Ameiurus species were chosen three DNA regions: part of nuclear Recombination activating gene 1 (RAG-1), mitochondrial gene for cytochrome b (cyt b) and mitochondrial sequence which includes 12S and 16S ribosomal RNA genes. The results presented in this paper concern specific osteological characteristics (original scheme of pectoral spine and pectoral girdle) used for the determination purposes and also verification that mathematical multivariate methods for external morphology analysis can be used for the determination of Ameiurus species. The summary of 80 out of 83 fish (96.4 %) was correctly classified using the discriminant analysis.     

Rapid MALDI biotyper-based identification and cluster analysis of <Emphasis Type="Italic">Streptococcus iniae</Emphasis>

Streptococcus iniae causes severe mortalities among cultured marine species, especially in the olive flounder (Paralichthys olivaceus), which is economically important in Korea and Japan. Recently, there has been growing concern regarding the emergence of S. iniae as a zoonotic pathogen. Here, 89 S. iniae isolates obtained from diseased olive flounders collected from 2003 to 2008 in Jeju Island, South Korea, were characterized using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The results were aligned both with the available Bruker Daltonics data-base and with a new set of S. iniae data entries developed in our laboratory, and the results were compared. When we used the Bruker Daltonics database, the 89 isolates yielded either “no reliable identification” or were incorrectly identified as Streptococcus pyogenes at the genus level. When we used the new data entries from our laboratory, in contrast, all of the isolates were correctly identified as S. iniae at the genus (100%) and species (96.6%) levels. We performed proteomic analysis, divided the 89 isolates into cluster I (51.7%), cluster II (20.2%), and cluster III (28.1%), and then used the MALDI Biotyper software to identify specific mass peaks that enabled discrimination between clusters and between Streptococcus species. Our results suggest that the use of MALDI TOF MS could outperform the conventional methods, proving easier, faster, cheaper and more efficient in properly identifying S. iniae. This strategy could facilitate the epidemiological and taxonomical study of this important fish pathogen.     

Morphological and molecular tools in identifying the Mediterranean limpets Patella caerulea, Patella aspera and Patella rustica

Allozyme electrophoresis, a partial nucleotide sequence of a mitochondrial gene for cytochrome oxidase I (COI) and discriminant analysis of shell morphometric characters were used to study the relationships among the Sicilian marine gastropods of the Patella genus. Allozyme and mtDNA markers unequivocally distinguished the species and were very useful markers in correctly classifying the different species when morphological characters overlapped each other. Several allozyme loci and many nucleotide positions were diagnostic of species. In contrast, the discriminant analysis of simple morphometric shell characters failed to adequately discriminate the species, suggesting that environmental factors influence colouration and morphological patterns in the Patella species. Our results underline the importance of a genetic approach, as compared to a morphological approach, in discriminating the Mediterranean Patella species.     

Phylogenetic relationships and DNA barcoding of nine endangered medicinal plant species endemic to Saint Katherine protectorate

A high degree of endemism has been recorded for several plant groups collectively in Saint Katherine Protectorate (SKP) in the Sinai Peninsula. Nine endangered endemic plant species in SKP were selected to test the variable abilities of three different DNA barcodes; Riboluse-1,5- Biphosphate Carboxylase/Oxygenase Large subunit (rbcL), Internal Transcribed Spacer (ITS), and the two regions of the plastid gene (ycf1) as well as Start Codon Targeted (SCoT) Polymorphism to find the phylogenetic relationships among them. The three barcodes were generally more capable of finding the genetic relationships among the plant species under study, new barcodes were introduced to the National Centre for Biotechnology Information (NCBI) for the first time through our work. The barcode sequences were efficient in finding the genetic relationships between the nine species. However, SCoT polymorphism could only cluster plant species belonging to the same genus together in one group, but it could not cluster plant species belonging to the same families except for some primers solely. RbcL was the most easily amplified and identified barcode in eight out of the nine species at the species level and the ninth barcode to the genus level. ITS identified all the species to the genus level. Finally, ycf1 identified six out of the eight species, but it could not identify two of the eight species to the genus level.     

Two New Potential Barcodes to Discriminate Dalbergia Species

DNA barcoding enables precise identification of species from analysis of unique DNA sequence of a target gene. The present study was undertaken to develop barcodes for different species of the genus Dalbergia, an economically important timber plant and is widely distributed in the tropics. Ten Dalbergia species selected from the Western Ghats of India were evaluated using three regions in the plastid genome (matK, rbcL, trnH-psbA), a nuclear transcribed spacer (nrITS) and their combinations, in order to discriminate them at species level. Five criteria: (i) inter and intraspecific distances, (ii) Neighbor Joining (NJ) trees, (iii) Best Match (BM) and Best Close Match (BCM), (iv) character based rank test and (v) Wilcoxon signed rank test were used for species discrimination. Among the evaluated loci, rbcL had the highest success rate for amplification and sequencing (97.6%), followed by matK (97.0%), trnH-psbA (94.7%) and nrITS (80.5%). The inter and intraspecific distances, along with Wilcoxon signed rank test, indicated a higher divergence for nrITS. The BM and BCM approaches revealed the highest rate of correct species identification (100%) with matK, matK+rbcL and matK+trnH-psb loci. These three loci, along with nrITS, were further supported by character based identification method. Considering the overall performance of these loci and their ranking with different approaches, we suggest matK and matK+rbcL as the most suitable barcodes to unambiguously differentiate Dalbergia species. These findings will potentially be helpful in delineating the various species of Dalbergia genus, as well as other related genera.     

Species-specific molecular signature of Commiphora species of Saudi Arabia inferred from internal transcribed spacer sequences of nuclear ribosomal DNA

The deciduous habit and tendency to produce flowers prior to developing leaves, and a predominantly dioecious system of breeding in the genus Commiphora leads to difficulties in its taxonomic identification at species level. The characteristics of easy amplification by universal primer, shorter length and higher discrimination power at the species level makes the internal transcribed spacer (ITS) sequence of nuclear ribosomal DNA (nrDNA) to a smart gene for generating species-specific phylogenetic inferences in most of the plants groups. The present study deals the ITS sequence of nrDNA based molecular genotyping of seven species of the genus Commiphora of Saudi Arabia. The molecular phylogenetic analysis of ITS sequences of nrDNA of Commiphora species distributed in Saudi Arabia reveals the the occurrence of C. madagascariens in Saudi Arabia.     

Candidatus Prosiliicoccus vernus,a spring phytoplankton bloom associated member of the Flavobacteriaceae

Microbial degradation of algal biomass following spring phytoplankton blooms has been characterised as a concerted effort among multiple clades of heterotrophic bacteria. Despite their significance to overall carbon turnover, many of these clades have resisted cultivation. One clade known from 16S rRNA gene sequencing surveys at Helgoland in the North Sea, was formerly identified as belonging to the genus Ulvibacter. This clade rapidly responds to algal blooms, transiently making up as much as 20% of the free-living bacterioplankton. Sequence similarity below 95% between the 16S rRNA genes of described Ulvibacter species and those from Helgoland suggest this is a novel genus. Analysis of 40 metagenome assembled genomes (MAGs) derived from samples collected during spring blooms at Helgoland support this conclusion. These MAGs represent three species, only one of which appears to bloom in response to phytoplankton. MAGs with estimated completeness greater than 90% could only be recovered for this abundant species. Additional, less complete, MAGs belonging to all three species were recovered from a mini-metagenome of cells sorted via flow cytometry using the genus specific ULV995 fluorescent rRNA probe. Metabolic reconstruction indicates this highly abundant species most likely degrades proteins and the polysaccharide laminarin. Fluorescence in situ hybridisation showed coccoid cells, with a mean diameter of 0.78 mm, with standard deviation of 0.12 μm. Based on the phylogenetic and genomic characteristics of this clade, we propose the novel candidate genus Candidatus Prosiliicoccus, and for the most abundant and well characterised of the three species the name Candidatus Prosiliicoccus vernus.     

Raman Spectroscopy and Chemometrics for Identification and Strain Discrimination of the Wine Spoilage Yeasts Saccharomyces cerevisiae,Zygosaccharomyces bailii,and Brettanomyces bruxellensis

The yeasts Zygosaccharomyces bailii, Dekkera bruxellensis (anamorph, Brettanomyces bruxellensis), and Saccharomyces cerevisiae are the major spoilage agents of finished wine. A novel method using Raman spectroscopy in combination with a chemometric classification tool has been developed for the identification of these yeast species and for strain discrimination of these yeasts. Raman spectra were collected for six strains of each of the yeasts Z. bailii, B. bruxellensis, and S. cerevisiae. The yeasts were classified with high sensitivity at the species level: 93.8% for Z. bailii, 92.3% for B. bruxellensis, and 98.6% for S. cerevisiae. Furthermore, we have demonstrated that it is possible to discriminate between strains of these species. These yeasts were classified at the strain level with an overall accuracy of 81.8%.     

Implications of Hybridization,NUMTs, and Overlooked Diversity for DNA Barcoding of Eurasian Ground Squirrels

The utility of DNA Barcoding for species identification and discovery has catalyzed a concerted effort to build the global reference library; however, many animal groups of economical or conservational importance remain poorly represented. This study aims to contribute DNA barcode records for all ground squirrel species (Xerinae, Sciuridae, Rodentia) inhabiting Eurasia and to test efficiency of this approach for species discrimination. Cytochrome c oxidase subunit 1 (COI) gene sequences were obtained for 97 individuals representing 16 ground squirrel species of which 12 were correctly identified. Taxonomic allocation of some specimens within four species was complicated by geographically restricted mtDNA introgression. Exclusion of individuals with introgressed mtDNA allowed reaching a 91.6% identification success rate. Significant COI divergence (3.5–4.4%) was observed within the most widespread ground squirrel species (Spermophilus erythrogenys, S. pygmaeus, S. suslicus, Urocitellus undulatus), suggesting the presence of cryptic species. A single putative NUMT (nuclear mitochondrial pseudogene) sequence was recovered during molecular analysis; mitochondrial COI from this sample was amplified following re-extraction of DNA. Our data show high discrimination ability of 100 bp COI fragments for Eurasian ground squirrels (84.3%) with no incorrect assessments, underscoring the potential utility of the existing reference librariy for the development of diagnostic ‘mini-barcodes’.     

Diversity of Environmental Mycobacterium Isolates from Hemodialysis Water as Shown by a Multigene Sequencing Approach

Here we used a multigene sequencing approach for the identification and molecular typing of environmental mycobacteria of the fast-growing subgroup. Strains were isolated from hemodialysis water and clinical samples. Eleven type strains of related species of the genus were also included in this study. To gain further insight into the diversity of the environmental mycobacteria, we analyzed several housekeeping genes (16S rRNA, ITS1, gyrB, hsp65, recA, rpoB, and sodA). No individual phylogenetic tree allowed good discrimination of all of the species studied. However, a concatenated and a consensus analysis, combining the genes, allowed better discrimination of each strain to the species level, and the increase in sequence size also led to greater tree robustness. This approach is useful not only for the discrimination and identification of environmental mycobacteria but also for their molecular typing and studies of population genetics. Our results demonstrate high genetic diversity among the isolates obtained, which are probably new species of the genus.     

The phytoplankton of Lake Atnsjøen, Norway – a long-term investigation

Quantitative samples were collected from Lake Atnsjøen five times per year in the growth seasons 1990–2000. The samples were analysed for variation in the phytoplankton composition, and the total volume and volume of the main groups of algae were calculated. Lake Atnsjøen is a large, deep and unregulated lake with a surface area of 4.8 km² and a maximum depth of 80.2 m. It is a nutrient-poor, oligotrophic lake with a maximum phytoplankton volume varying between 125–393 mm³/m³ in the years 1990–2000. The phytoplankton community is dominated by species of the groups Chrysophyceae and Cryptophyceae. The chrysophytes dominate the phytoplankton in the early part of the growth season (May–June) while the cryptophytes increase throughout the season and dominate in the autumn. Among the chrysophytes different species of chrysomonads were most frequent together with common species of the genus Dinobryon like D. borgei, D. cylindricum var. alpinum and D. crenulatum. A total of 22 species or taxa of chrysophytes were recorded in the samples. Common among the cryptomonads were several species of the genus Cryptomonas. Most important quantitatively, however, were Rhodomonas lacustris and Katablepharis ovalis. The succesion of the phytoplankton throughout the growth season was similar from year to year in quantitative as well as qualitative terms, but some changes were recorded after the great flood in 1995. Canonical Correspondence Analysis (CCA) shows a slight, but significant, phytoplankton community change over the succeeding years.     

Single strand conformation polymorphism analysis of PCR-tDNA fingerprinting to address the identification of Bacillus species

The suitability of tDNA-PCR fingerprinting to identify species of the genus Bacillus was tested on 75 strains. Strains belonging to the same species or the same phylogenetic cluster were correctly grouped. Among B. stearothermophilus strains, different pattern types were found. This could be due to the unclear taxonomic situation of these strains, rather than to a failure of the tDNA-PCR. Single strand conformation polymorphism (SSCP) of the PCR products allowed species discrimination within the `B. subtilis group', but not within the `B. cereus group'. The tDNA-PCR, alone or coupled with SSCP analysis, is useful to address Bacillus species identification, particularly for those species which are not phylogenetically tightly clustered.     

Start Codon Targeted (SCoT) markers provide new insights into the genetic diversity analysis and characterization of Tunisian Citrus species

Start Codon Targeted markers were used to establish phylogenetic relationship among seven species from Citrus L. genus. Twelve SCoT primers were used for their ability to reveal polymorphism of the targeted codon of initiation. A total of 132 amplicons were generated and 93.9% of them were polymorphic. The polymorphism information content of 0.884 and the resolving power of 75.22 illustrate the efficiency of the tested SCoT primers in highlighting polymorphism. The average Nei's (1973) gene diversity (0.376), the Schannon's index (0.548) and the Gst parameter (0.346) describe an important polymorphism at the interspecies level in Citrus genus. Analysis of molecular variance suggested significant genetic differences within species. In fact, 84% of variance occurs within the species, whereas 16% of the variation was recorded among the species of Citrus. The limited gene flow (Nm = 0.941) was recognized as a major factor to explain the partition of the observed diversity. The principal coordinates analyses, Neighbor Joining and the Bayesian clustering approach based on the SCoT markers also confirm the discrimination of the species of Citrus. Our results confirm the relevance and suggest the effectiveness of the SCoT markers for assessing genetic diversity, characterization and identification of the species of Citrus.     

Relationship between phytoplankton and environmental factors in landscape water supplemented with reclaimed water

The water quality of Feng-qing Lake, which is a landscape lake supplemented with reclaimed water, was surveyed to investigate the relationship between phytoplankton and environmental variables. A total of 29 water samples were collected to analyze temporal variations of phytoplankton and environmental factors from July 2013 to June 2014. Six phyla and 39 genera of phytoplankton were identified when the lake was supplied with reclaimed water. Among these, Cyanophyta and Chlorophyta account for 38.46% and 30.77% of phytoplankton, respectively. The dominant species in the lake are Pseudanabaena limnetica and Chlorella vulgaris, which are present the entire year. Other leading species include Cosmarium sp. and Raphidiopsis curvata. Principal component analysis (PCA) was conducted to analyze the relationship among environmental factors. Canonical correspondence analysis (CCA) was performed to investigate the relationship between environment factors and dominant species. The PCA result showed that temperature (T), total phosphorus (TP), total nitrogen (TN), transparency, and dissolved oxygen are the main factors that affect the eutrophication level of the lake. The CCA result revealed that TN, PO₄³⁻–P, chemical oxygen demand (COD), T, and chlorophyll a exhibit a close relation with dominant species. In particular, TN, salinity, and COD influence the growth of P. limnetica; T and COD influence the growth of R. curvata; and T, PO₄³⁻–P, NH₃–N, and pH influence the growth of C. vulgaris and Cosmarium sp.     

Using FAME Analysis to Compare, Differentiate, and Identify Multiple Nematode Species

We have adapted the Sherlock^® Microbial Identification system for identification of plant parasitic nematodes based on their fatty acid profiles. Fatty acid profiles of 12 separate plant parasitic nematode species have been determined using this system. Additionally, separate profiles have been developed for Rotylenchulus reniformis and Meloidogyne incognita based on their host plant, four species and three races within the Meloidogyne genus, and three life stages of Heterodera glycines. Statistically, 85% of these profiles can be delimited from one another; the specific comparisons between the cyst and vermiform stages of H. glycines, M. hapla and M. arenaria, and M. arenaria and M. javanica cannot be segregated using canonical analysis. By incorporating each of these fatty acid profiles into the Sherlock^® Analysis Software, 20 library entries were created. While there was some similarity among profiles, all entries correctly identified the proper organism to genus, species, race, life stage, and host at greater than 86% accuracy. The remaining 14% were correctly identified to genus, although species and race may not be correct due to the underlying variables of host or life stage. These results are promising and indicate that this library could be used for diagnostics labs to increase response time.