Species-specific oligonucleotide probes for macroalgae: molecular discrimination of two marine fouling species of Enteromorpha (Ulvophyceae)

The green seaweeds Enteromorpha intestinalis and E. compressa are important fouling organisms commonly found in polluted and nutrient-enriched marine and brackish water habitats, where they are used in environmental monitoring. Discrimination of the two species is extremely difficult because of overlapping morphological characters. In this study a quick molecular method for species identification was developed based on the nuclear rDNA ITS2 sequence data of 54 E. intestinalis samples and 20 E. compressa samples from a wide geographical range. Oligonucleotide probes were designed for species-specific hybridization to dot-blots of the PCR-amplified ITS1, 5.8S gene and ITS2 fragment of both E. intestinalis and E. compressa. Specificity of the oligonucleotide probes was confirmed by tests with taxonomically diverse species that could morphologically be confused with E. intestinalis or E. compressa. This is the first use of species-specific probes for macroalgae. The restriction endonuclease NruI digested specifically the amplified PCR product from E. compressa into two fragments detectable on agarose gels, but no suitable restriction sites were identifiable in the PCR product of E. intestinalis.     

Identification of indicator microorganisms using a standardized PNA FISH method.

A standardized fluorescent in situ hybridization (FISH) method using Peptide Nucleic Acid (PNA) probes for analysis of gram-negative and gram-positive bacteria, as well as yeast, has been developed. Fluorescently labeled PNA probes targeting specific rRNA sequences of Escherichia coli, Pseudomonas aeruginosa, Staphyloccocus aureus, Salmonella were designed, as well as PNA probes targeting eubacteria and eucarya. These PNA probes were evaluated by PNA FISH using 27 bacterial and 1 yeast species, representing both phylogenetically closely related species, as well as species important to both clinical and industrial settings. The S. aureus and P. aeruginosa PNA probes did not cross react with any of the organisms tested, whereas the E. coli PNA probe, as expected from sequence data, also detected Shigella species. The Salmonella PNA probe reacted with all of the 13 Salmonella strains, representing the 7 subspecies of Salmonella, however, it is also complementary to a few other bacterial species. The eubacteria- and eucarya-specific PNA probes detected all bacterial species and one yeast species, respectively. The general applicability of the PNA FISH method made simultaneous identification of multiple species, both gram-negative and gram-positive, in a mixed population an attractive possibility never accomplished using DNA probes. Four color images using differently labeled PNA probes showed simultaneous identification of E. coli, P. aeruginosa, S. aureus and Salmonella, thereby demonstrating the potential of multiplex FISH for various diagnostic applications within both clinical and industrial microbiology.     

DNA Microarrays for Identifying Fishes

In many cases marine organisms and especially their diverse developmental stages are difficult to identify by morphological characters. DNA-based identification methods offer an analytically powerful addition or even an alternative. In this study, a DNA microarray has been developed to be able to investigate its potential as a tool for the identification of fish species from European seas based on mitochondrial 16S rDNA sequences. Eleven commercially important fish species were selected for a first prototype. Oligonucleotide probes were designed based on the 16S rDNA sequences obtained from 230 individuals of 27 fish species. In addition, more than 1200 sequences of 380 species served as sequence background against which the specificity of the probes was tested in silico. Single target hybridisations with Cy5-labelled, PCR-amplified 16S rDNA fragments from each of the 11 species on microarrays containing the complete set of probes confirmed their suitability. True-positive, fluorescence signals obtained were at least one order of magnitude stronger than false-positive cross-hybridisations. Single nontarget hybridisations resulted in cross-hybridisation signals at approximately 27% of the cases tested, but all of them were at least one order of magnitude lower than true-positive signals. This study demonstrates that the 16S rDNA gene is suitable for designing oligonucleotide probes, which can be used to differentiate 11 fish species. These data are a solid basis for the second step to create a "Fish Chip" for approximately 50 fish species relevant in marine environmental and fisheries research, as well as control of fisheries products.     

Comparative studies on species identification of Noctuoidea moths in two nature reserve conservation zones (Beijing,China) using DNA barcodes and thin‐film biosensor chips

Rapid and accurate identification of species is required for the biological control of pest Noctuoidea moths. DNA barcodes and thin‐film biosensor chips are two molecular approaches that have gained wide attention. Here, we compare these two methods for the identification of a limited number of Noctuoidea moth species. Based on the commonly used mitochondrial gene cytochrome c oxidase I (the standard DNA barcode for animal species), 14 probes were designed and synthesized for 14 species shared by two national nature reserves in Beijing and Hebei, China. Probes ranged in length from 18 to 27 bp and were designed as mismatch probes to guarantee that there were at least three base differences between the probe and nontarget sequences. The results on the chip could be detected by the naked eye without needing special equipment. No cross‐hybridizations were detected although we tested all probes on the 14 target and 24 nontarget Noctuoidea species. The neighbour‐joining tree of the 38 species based on COI sequences gave 38 highly supported independent groups. Both DNA barcoding and thin‐film biosensor chips, based on the COI gene, are able to accurately identify and discriminate the 14 targeted moth species in this study. Because of its speed, high accuracy and low cost, the thin‐film biosensor chip is a very practical means of species identification. Now, a more comprehensive chip will be developed for the identification of additional Noctuoidea moths for pest control and ecological protection.     

Genetic divergence and ecological specialisation of seed weevils (Exapion spp.) on gorses (Ulex spp.)

Abstract.  1. Reproductive isolation of sympatric populations may result from divergent selection of populations in different environments, and lead to ecological specialisation. In Brittany (France), the gorse Ulex europaeus (Fabaceae, Genisteae), may be encountered in sympatry with one of the two other gorse species present: U. gallii and U. minor . A recent study based on morphological identification of seed predators of gorse has shown that two weevil species (Curculionoidea, Apionidae) infest gorse pods at different seasons and have different host ranges: Exapion ulicis infests U. europaeus in spring, whereas E. lemovicinum infests U. gallii and U. minor in autumn. Weevil populations may thus have diverged in sympatry.
2. As morphological identification of weevils is often difficult and some of the characters used may exhibit individual or environmental variation, mitochondrial and nuclear sequences of weevils collected within pods of the three gorse species in 10 populations of Brittany were used to reconstruct their phylogeny.
3. The results reveal that species differentiation based on morphological characters is confirmed by the two molecular data sets, showing that E. ulicis and E. lemovicinum are distinct species, and suggesting the absence of host races. Finally, E. ulicis was able to use U. gallii and U. minor pods in spring in some years in some populations, which appeared to depend on the availability of pods present during its reproductive period.
4. Divergence between E. ulicis and E. lemovicinum may have resulted from temporal isolation of reproductive periods of weevil populations followed by specialisation of insects to host phenology.     

中国秋海棠属 (秋海棠科) 植物的DNA条形码评价

秋海棠属植物种类繁多,形态变异多样,导致种类的系统放置混乱,近缘种类鉴定困难。利用DNA条形码实现物种快速准确的鉴定技术具有不受形态特征约束的优势,为秋海棠属植物的分类鉴定提供了新的方法。本研究选择4个DNA条形码候选片段（rbcL,matK,trnH psbA,ITS）对中国秋海棠属26种136个个体进行了分析。结果显示：叶绿体基因rbcL,matK和trnH psbA种内和种间变异小,对秋海棠属植物的鉴别能力有限;ITS/ITS2种内和种间变异大,在本研究中物种正确鉴定率达到100%/96%,可考虑作为秋海棠属DNA条形码鉴定的候选片段。研究结果支持中国植物条形码研究组建议将核基因ITS/ITS2纳入种子植物DNA条形码核心片段中的观点。     

Noninvasive molecular methods to identify live scarab larvae: an example of sympatric pest and nonpest species in New Zealand

Despite the negative impact that many scarab larvae have on agro-ecosystems, very little attention has been paid to their taxonomy. Their often extremely similar morphological characteristics have probably contributed to this impediment, which has also meant that they are very difficult to identify in the field. Molecular methods can overcome this challenge and are particularly useful for the identification of larvae to enable management of pest species occurring sympatrically with nonpest species. However, the invasive collection of DNA samples for such molecular methods is not compatible with subsequent behavioural, developmental or fitness studies. Two noninvasive DNA sampling and DNA analysis methods suitable for the identification of larvae from closely related scarab species were developed here. Using the frass and larval exuviae as sources of DNA, field-collected larvae of Costelytra zealandica (White) and Costelytra brunneum (Broun) (Scarabaeidae: Melolonthinae) were identified by multiplex PCR based on the difference in size of the resulting PCR products. This study also showed that small quantities of frass can be used reliably even 7 days after excretion. This stability of the DNA is of major importance in ecological studies where timeframes rarely allow daily monitoring. The approach developed here is readily transferable to the study of any holometabolous insect species for which morphological identification of larval stages is difficult.     

Development of a DNA microarray for enterococcal species, virulence, and antibiotic resistance gene determinations among isolates from poultry

A DNA microarray (Enteroarray) was designed with probes targeting four species-specific taxonomic identifiers to discriminate among 18 different enterococcal species, while other probes were designed to identify 18 virulence factors and 174 antibiotic resistance genes. In total, 262 genes were utilized for rapid species identification of enterococcal isolates, while characterizing their virulence potential through the simultaneous identification of endogenous antibiotic resistance and virulence genes. Enterococcal isolates from broiler chicken farms were initially identified by using the API 20 Strep system, and the results were compared to those obtained with the taxonomic genes atpA, recA, pheS, and ddl represented on our microarray. Among the 171 isolates studied, five different enterococcal species were identified by using the API 20 Strep system: Enterococcus faecium, E. faecalis, E. durans, E. gallinarum, and E. avium. The Enteroarray detected the same species as API 20 Strep, as well as two more: E. casseliflavus and E. hirae. Species comparisons resulted in 15% (27 isolates) disagreement between the two methods among the five API 20 Strep identifiable species and 24% (42 isolates) disagreement when considering the seven Enteroarray identified species. The species specificity of key antibiotic and virulence genes identified by the Enteroarray were consistent with the literature adding further robustness to the redundant taxonomic probe data. Sequencing of the cpn60 gene further confirmed the complete accuracy of the microarray results. The new Enteroarray should prove to be a useful tool to accurately genotype strains of enterococci and assess their virulence potential.     

Thermal attributes of Chrysomya species

The correct identification of forensically important arthropods for post‐mortem interval estimation is crucial, as the rate of larval development can vary substantially between species. The identification of forensically important blowflies of the genus Chrysomya (Diptera: Calliphoridae) may be hampered by their close morphological similarities, especially as immatures. The aim of this study was to establish whether genetically closely related blowfly species would share similar developmental profiles. This could permit the application of developmental data to a number of closely related species, including those for which thermodevelopmental studies are lacking. If Australian Chrysomya were found to share developmental profiles, identification of the blowfly specimen to a level beyond genus may not be necessary, or at least it may not be necessary to distinguish morphologically similar sister species. The three Chrysomya species studied were collected from the same geographical location (Cairns, Australia), reducing the effects of acclimation and population‐level genetic variation. The experimental conditions in this study were virtually identical, which enabled direct comparisons to be made among the species. Blowfly larval lengths were obtained for 24‐hourly intervals at constant temperatures of 25, 30, and 35 °C. The thermal preferences of newly‐hatched feeding larvae were determined by their positions on a temperature gradient apparatus. This study established that all three species investigated differed significantly in their developmental profiles, despite the genetic closeness of the sister species Chrysomya megacephala (Fabricius) and Chrysomya saffranea (Bigot). Because of this, genetic distance was not considered to be a useful factor for predicting thermodevelopment profiles of closely related species within a genus, and highlights the necessity for correct species identification.     

贵州部分洞穴裸灶螽的鉴定及其遗传多样性分析

贵州是中国具有丰富喀斯特洞穴生态系统的省份之一,裸灶螽作为洞穴中的优势物种又为生态环境指示种,其遗传多样性能够反应洞穴生物多样性.为评估洞穴开发对洞穴生物多样性的影响,本研究通过形态鉴定和分子辅助鉴定的方式对来自贵州省毕节、黔西南、铜仁和黔南4个地区8个洞穴97号裸灶螽标本进行鉴定,并对其遗传多样性进行分析.结果共鉴定...     

Quenched autoligation probes allow discrimination of live bacterial species by single nucleotide differences in rRNA

Quenched autoligation (QUAL) probes are a class of self-reacting nucleic acid probes that give strong fluorescence signal in the presence of fully complementary RNAs and selectivity against single nucleotide differences in solution. Here, we describe experiments designed to test whether QUAL probes can discriminate between bacterial species by the detection of small differences in their 16S rRNA sequences. Probes were introduced into live cells using small amounts of detergent, thus eliminating the need for fixation, and fluorescence signal was monitored both by microscopy and by flow cytometry without any washing steps. The effects of probe length, modified backbone, probe concentration and growth state of the bacteria were investigated. The data demonstrate specific fluorescence discrimination between three closely related bacteria, Escherichia coli, Salmonella enterica and Pseudomonas putida, based on single nucleotide differences in their 16S rRNA. Discrimination was possible with cells in mid-log phase or in lag phase. These results suggest that QUAL probes may be useful for rapid identification of microorganisms in laboratory and clinical settings.     

Differentiation of two very similar glaucomid ciliate morphospecies (Ciliophora, Tetrahymenida) by fluorescence in situ hybridization with 18S rRNA targeted oligonucleotide probes

Conventional, morphological identification of ciliates and other protozoa needs considerable experience and often is difficult as various staining methods must be applied. New molecular techniques, such as fluorescence in situ hybridization (FISH) with gene probes, are powerful means to overcome this problem. As a test case, the morphology of two very similar, and thus difficult to differentiate ciliate morphospecies, Glaucoma scintillans and Glaucomides bromelicola, were compared. They were then distinguished by applying the Ciliate-FISH technique with a set of eight 18S rRNA targeted oligonucleotide probes, four of which have been developed for specific detection of G. scintillans. The remaining four probes were designed to detect G. bromelicola in order to prove probe specificities by binding to the homologous target region of the probes mentioned before. The tests resulted in a clear and easy differentiation of the two species by fluorescence signals of three of the four tested probe pairs. Thus, FISH techniques are very useful for the identification and detection of protozoa and might be of great help studying geographical distributions of known taxa.     

Fluorescence in situ hybridization with specific oligonucleotide rRNA probes distinguishes the sibling species Stylonychia lemnae and Stylonychia mytilus (Ciliophora, Spirotrichea)

Based on morphological and morphogenetic characters alone, the sibling species Stylonychia lemnae and Stylonychia mytilus, members of the Stylonychia mytilus complex, can hardly be distinguished. However, biochemical investigations of the isoenzyme pattern of different enzymes showed a distinct differentiation between these two species. In the last few years, fluorescence in situ hybridization (FISH) techniques have become a suitable and reliable tool for identification and differentiation of closely related species of protozoa, such as ciliates. To distinguish the sibling species, a set of specific oligonucleotide probes were developed. In the present study, the SSU rDNA of 7 clones of Stylonychia lemnae and 13 clones of Stylonychia mytilus, isolated from different geographic regions, were sequenced. Comparing all SSU rDNA sequences of both species, only one single difference within the whole gene was detected. Based on this difference, a set of two oligonucleotide probes, targeting the SSU rRNA of each species (Stylonychia mytilus and Stylonychia lemnae) was designed. These probes were successfully tested by applying the FISH techniques on preserved cells of different clones of both species.     

Identification of the pathogenic ciliate Pseudocohnilembus persalinus (Oligohymenophorea: Scuticociliatia) by fluorescence in situ hybridization

Many scuticociliatid ciliates are regarded as devastating pathogens in aquaculture. Among these, Pseudocohnilembus persalinus is a facultative pathogen that often results in refractory diseases of mariculture fish. Although traditional silver staining methods have been successfully used to identify these ciliates, their identification is hampered by their small size and their morphological similarity to closely related species. We designed an alternative method of identification of P. persalinus using an SSU-rDNA targeted oligonucleotide probe labeled with a fluorochrome, and optimized in a fluorescence in situ hybridization (FISH) protocol. The assay results in a clear identification by strong fluorescence signals from the oligonucleotide probe. The method can be used for quick and early detection of P. persalinus infections on host fish, as well as other susceptible organisms in aquiculture water. It may also be used in studies of the geographical distribution of this scuticociliate.     

A multiplex PCR assay for the simultaneous identification of three mealybug species (Hemiptera: Pseudococcidae)

Molecular species identification is becoming more wide-spread in diagnostics and ecological studies, particularly with regard to insects for which morphological identification is difficult or time-consuming. In this study, we describe the development and application of a single-step multiplex PCR for the identification of three mealybug species (Hemiptera: Pseudococcidae) associated with grapevine in South Africa: Planococcus ficus (vine mealybug), Planococcus citri (citrus mealybug) and Pseudococcus longispinus (longtailed mealybug). Mealybugs are pests on many commercial crops, including grapevine, in which they transmit viral diseases. Morphological identification of mealybug species is usually time-consuming, requires a high level of taxonomic expertise and usually only adult females can be identified. The single-step multiplex PCR developed here, based on the mitochondrial cytochrome c oxidase subunit 1 (CO I) gene, is rapid, reliable, sensitive, accurate and simple. The entire identification protocol (including DNA extraction, PCR and electrophoresis) can be completed in approximately four hours. Successful DNA extraction from laboratory and unparasitized field-collected individuals stored in absolute ethanol was 97%. Specimens from which DNA could be extracted were always correctly identified (100% accuracy). The technique developed is simple enough to be implemented in any molecular laboratory. The principles described here can be extended to any organism for which rapid, reliable identification is needed.     

Fluorescent in situ hybridization with rRNA-targeted oligonucleotide probes to identify small phytoplankton by flow cytometry.

Because of their tiny size (0.2 to 2 microns), oceanic picophytoplanktonic cells (either cultured strains or natural communities) are difficult to identify, and some basic questions concerning their taxonomy, physiology, and ecology are still largely unanswered. The present study was designed to test the suitability of in situ hybridization with rRNA fluorescent probes detected by flow cytometry for the identification of small photosynthetic eukaryotes. Oligonucleotide probes targeted against regions of the 18S rRNAs of Chlorophyta lineage (CHLO probe) and of non-Chlorophyta (NCHLO probe) algal species were designed. The CHLO and NCHLO probes, which differed by a single nucleotide, allowed discrimination of chlorophyte from nonchlorophyte cultured strains. The sensitivity of each probe was dependent upon the size of the cells and upon their growth stage. The mean fluorescence was 8 to 80 times higher for specifically labeled than for nonspecifically labeled cells in exponential growth phase, but it decreased sharply in stationary phase. Such taxon-specific probes should increase the applicability of flow cytometry for the rapid identification of cultured pico- and nanoplanktonic strains, especially those that lack taxonomically useful morphological features.     

Strain/Species-Specific Probe Design for Microbial Identification Microarrays

Specific identification of microorganisms in the environment is important but challenging, especially at the species/strain level. Here, we have developed a novel k-mer-based approach to select strain/species-specific probes for microbial identification with diagnostic microarrays. Application of this approach to human microbiome genomes showed that multiple (≥10 probes per strain) strain-specific 50-mer oligonucleotide probes could be designed for 2,012 of 3,421 bacterial strains of the human microbiome, and species-specific probes could be designed for most of the other strains. The method can also be used to select strain/species-specific probes for sequenced genomes in any environments, such as soil and water.     

PROBLEMS AND PROMISE IN CODING MORPHO-LOGICAL CHARACTERS FOR PHYLOGENETIC ANALYSIS: AN EXAMPLE FROM AULACOSEIRA (BACILLARIOPHYCEAE)

Edgar, S. M. & Theriot, E. C. Section of Integrative Biology, University of Texas at Austin, Austin TX 78713 USA Understanding the phylogenetic relationships among species of Aulacoseira Thwaites (formerly Melosira, in part) and their relationships to other genera is important for several reasons. Aulacoseira is globally distributed and is often a large component of planktonic communities in lakes, ponds and large rivers. Consequently, it plays a significant ecological role as a primary producer in grazer-based aquatic food webs. Although species of Aulacoseira (along with other diatom species) are used profitably as tools for reconstruction of paleo-environments, ecological monitoring, and other ecological studies which have generated a large amount of data, much of this data is not really informative for a systematic understanding of the group. There are currently around 60 extant and 20 extinct described species of Aulacoseira. However, there is no estimate of phylogeny within this genus that includes more than five species. Here we present the first comprehensive estimate of phylogeny for 30 species of Aulacoseira based on a cladistic analysis of morphological features gathered from the literature. Complications of gathering morphological data from the literature that was largely focused on refining species identifications for practical purposes of ecological identification will be addressed. Morphological characters chosen for the analysis and challenges associated with coding of characters will be discussed along with the resulting Aulacoseira phylogeny and our methods of its estimation.     

COI-based DNA barcoding of Arcoida species (Bivalvia: Pteriomorphia) along the coast of China

DNA barcoding is a promising tool for the rapid and unambiguous identification of species. Some arcoid species are particularly difficult to distinguish with traditional morphological identification owing to phenotypic variation and the existence of closely related taxa. Here, we apply DNA barcoding based on mitochondrial cytochrome c oxidase I gene (COI) to arcoid species collected from the coast along China. Combining morphology with molecular data indicates the 133 specimens of Arcoida could be assigned to 24 species. Because of the deep genetic divergence within Tegillarca granosa, there was an overlap between genetic variation within species and variation between species. Nevertheless, NJ and Bayesian trees showed that all species fell into reciprocally monophyletic clades with high bootstrap values. Our results evidence that the COI marker can efficiently identify species, correct mistakes caused by morphological identification and reveal genetic differentiation among populations within species. This study provides a clear example of the usefulness of barcoding for arcoid identification. Furthermore, it also lays a foundation for other biological and ecological studies of Arcoida.     

Toward a DNA taxonomy of Alpine Rhithrogena (Ephemeroptera: Heptageniidae) using a mixed Yule-coalescent analysis of mitochondrial and nuclear DNA

Aquatic larvae of many Rhithrogena mayflies (Ephemeroptera) inhabit sensitive Alpine environments. A number of species are on the IUCN Red List and many recognized species have restricted distributions and are of conservation interest. Despite their ecological and conservation importance, ambiguous morphological differences among closely related species suggest that the current taxonomy may not accurately reflect the evolutionary diversity of the group. Here we examined the species status of nearly 50% of European Rhithrogena diversity using a widespread sampling scheme of Alpine species that included 22 type localities, general mixed Yule-coalescent (GMYC) model analysis of one standard mtDNA marker and one newly developed nDNA marker, and morphological identification where possible. Using sequences from 533 individuals from 144 sampling localities, we observed significant clustering of the mitochondrial (cox1) marker into 31 GMYC species. Twenty-one of these could be identified based on the presence of topotypes (expertly identified specimens from the species' type locality) or unambiguous morphology. These results strongly suggest the presence of both cryptic diversity and taxonomic oversplitting in Rhithrogena. Significant clustering was not detected with protein-coding nuclear PEPCK, although nine GMYC species were congruent with well supported terminal clusters of nDNA. Lack of greater congruence in the two data sets may be the result of incomplete sorting of ancestral polymorphism. Bayesian phylogenetic analyses of both gene regions recovered four of the six recognized Rhithrogena species groups in our samples as monophyletic. Future development of more nuclear markers would facilitate multi-locus analysis of unresolved, closely related species pairs. The DNA taxonomy developed here lays the groundwork for a future revision of the important but cryptic Rhithrogena genus in Europe.