Lessons from leeches: a call for DNA barcoding in the lab

Many evolution of development labs study organisms that must be periodically collected from the wild. Whenever this is the case, there is the risk that different field collections will recover genetically different strains or cryptic species. Ignoring this potential for genetic variation may introduce an uncontrolled source of experimental variability, leading to confusion or misinterpretation of the results. Leeches in the genus Helobdella have been a workhorse of annelid developmental biology for 30 years. Nearly all early Helobdella research was based on a single isolate, but in recent years isolates from multiple field collections and multiple sites across the country have been used. To assess the genetic distinctness of different isolates, we obtained specimens from most Helobdella laboratory cultures currently or recently in use and from some of their source field sites. From these samples, we sequenced part of the mitochondrial gene cytochrome oxidase I (COI). Sequence divergences and phylogenetic analyses reveal that, collectively, the Helobdella development community has worked on five distinct species from two major clades. Morphologically similar isolates that were thought to represent the same species (H. robusta) actually represent three species, two of which coexist at the same locality. Another isolate represents part of a species complex (the "H. triserialis" complex), and yet another is an invasive species (H. europaea). We caution researchers similarly working on multiple wild-collected isolates to preserve voucher specimens and to obtain from these a molecular "barcode," such as a COI gene sequence, to reveal genetic variation in animals used for research.     

Discrimination of Bacillus anthracis and closely related microorganisms by analysis of 16S and 23S rRNA with oligonucleotide microarray

Analysis of 16S rRNA sequences is a commonly used method for the identification and discrimination of microorganisms. However, the high similarity of 16S and 23S rRNA sequences of Bacillus cereus group organisms (up to 99-100%) and repeatedly failed attempts to develop molecular typing systems that would use DNA sequences to discriminate between species within this group have resulted in several suggestions to consider B. cereus and B. thuringiensis, or these two species together with B. anthracis, as one species. Recently, we divided the B. cereus group into seven subgroups, Anthracis, Cereus A and B, Thuringiensis A and B, and Mycoides A and B, based on 16S rRNA, 23S rRNA and gyrB gene sequences and identified subgroup-specific makers in each of these three genes. Here we for the first time demonstrated discrimination of these seven subgroups, including subgroup Anthracis, with a 3D gel element microarray of oligonucleotide probes targeting 16S and 23S rRNA markers. This is the first microarray enabled identification of B. anthracis and discrimination of these seven subgroups in pure cell cultures and in environmental samples using rRNA sequences. The microarray bearing perfect match/mismatch (p/mm) probe pairs was specific enough to discriminate single nucleotide polymorphisms (SNPs) and was able to identify targeted organisms in 5min. We also demonstrated the ability of the microarray to determine subgroup affiliations for B. cereus group isolates without rRNA sequencing. Correlation of these seven subgroups with groupings based on multilocus sequence typing (MLST), fluorescent amplified fragment length polymorphism analysis (AFLP) and multilocus enzyme electrophoresis (MME) analysis of a wide spectrum of different genes, and the demonstration of subgroup-specific differences in toxin profiles, psychrotolerance, and the ability to harbor some plasmids, suggest that these seven subgroups are not based solely on neutral genomic polymorphisms, but instead reflect differences in both the genotypes and phenotypes of the B. cereus group organisms.     

Dominance and diet are unrelated within a population of invasive crayfish

Laboratory behavioral experiments are an important tool in ecology and evolution, but whether these behaviors reflect the field function of organisms is not always clear. Directly connecting laboratory behaviors to field interactions would increase understanding of a variety of organisms. A recent study proposed using stable isotopes to link laboratory behaviors to field function of individuals, but failed to find any such links within a population of the invasive rusty crayfish Faxonius rusticus (Girard, 1852). Here, we assessed whether methodological decisions around tissue analyzed for stable isotopes, laboratory acclimation time, and timing of primary consumer collection may have affected the result, hypothesizing that more dominant crayfish would have higher trophic positions and tissue with faster turnover rates may exhibit a stronger association between laboratory behavior and recent field function. We tested this relationship using F. rusticus individuals from a single population, and related laboratory dominance to stable isotope-derived trophic position using linear regression. We failed to find a relationship between dominance and trophic position, regardless of our different methodologies. Future studies should consider alternative behaviors that may better relate to function in the field and also investigate whether laboratory behavior and field function are related between, rather than within, populations or species.

     

A DNA microarray for identification of selected Korean birds based on mitochondrial cytochrome c oxidase I gene sequences

DNA barcoding with the gene encoding cytochrome c oxidase I (COI) in the mitochondrial genome has been proposed as a standard marker to identify and discover animal species. Some migratory wild birds are suspected of transmitting avian influenza and pose a threat to aircraft safety because of bird strikes. We have previously reported the COI gene sequences of 92 Korean bird species. In the present study, we developed a DNA microarray to identify 17 selected bird species on the basis of nucleotide diversity. We designed and synthesized 19 specific oligonucleotide probes; these probes were arrayed on a silylated glass slide. The length of the probes was 19-24 bps. The COI sequences amplified from the tissues of the selected birds were labeled with a fluorescent probe for microarray hybridization, and unique hybridization patterns were detected for each selected species. These patterns may be considered diagnostic patterns for species identification. This microarray system will provide a sensitive and a high-throughput method for identification of Korean birds.     

Genetic connectivity between north and south Mid-Atlantic Ridge chemosynthetic bivalves and their symbionts

Transform faults are geological structures that interrupt the continuity of mid-ocean ridges and can act as dispersal barriers for hydrothermal vent organisms. In the equatorial Atlantic Ocean, it has been hypothesized that long transform faults impede gene flow between the northern and the southern Mid-Atlantic Ridge (MAR) and disconnect a northern from a southern biogeographic province. To test if there is a barrier effect in the equatorial Atlantic, we examined phylogenetic relationships of chemosynthetic bivalves and their bacterial symbionts from the recently discovered southern MAR hydrothermal vents at 5°S and 9°S. We examined Bathymodiolus spp. mussels and Abyssogena southwardae clams using the mitochondrial cytochrome c oxidase subunit I (COI) gene as a phylogenetic marker for the hosts and the bacterial 16S rRNA gene as a marker for the symbionts. Bathymodiolus spp. from the two southern sites were genetically divergent from the northern MAR species B. azoricus and B. puteoserpentis but all four host lineages form a monophyletic group indicating that they radiated after divergence from their northern Atlantic sister group, the B. boomerang species complex. This suggests dispersal of Bathymodiolus species from north to south across the equatorial belt. 16S rRNA genealogies of chemoautotrophic and methanotrophic symbionts of Bathymodiolus spp. were inconsistent and did not match the host COI genealogy indicating disconnected biogeography patterns. The vesicomyid clam Abyssogena southwardae from 5°S shared an identical COI haplotype with A. southwardae from the Logatchev vent field on the northern MAR and their symbionts shared identical 16S phylotypes, suggesting gene flow across the Equator. Our results indicate genetic connectivity between the northern and southern MAR and suggest that a strict dispersal barrier does not exist.     

Metagenomics: DNA sequencing of environmental samples

Although genomics has classically focused on pure, easy-to-obtain samples, such as microbes that grow readily in culture or large animals and plants, these organisms represent only a fraction of the living or once-living organisms of interest. Many species are difficult to study in isolation because they fail to grow in laboratory culture, depend on other organisms for critical processes, or have become extinct. Methods that are based on DNA sequencing circumvent these obstacles, as DNA can be isolated directly from living or dead cells in various contexts. Such methods have led to the emergence of a new field, which is referred to as metagenomics.     

Detection of Mycoplasma ovipneumoniae and M. arginini in bighorn sheep using enrichment culture coupled with genus- and species-specific polymerase chain reaction

Mycoplasma species are of interest as possible primary pathogens in the pneumonia complex of bighorn sheep (Ovis canadensis). Previous investigations have not commonly detected low frequencies of Mycoplasma spp. from free-ranging bighorn sheep, possibly due to the fastidious and slow growth of these organisms. We developed a culture protocol that employed an average initial 3-day enrichment culture in liquid Hayflick broth in a CO(2)-enhanced atmosphere. The broth was plated to solid Hayflick medium and the cultures observed for growth for up to 30 days. Polymerase chain reaction (PCR) was performed on DNA isolated from the enrichment broth and on isolates obtained from culture using Mycoplasma genus-specific PCR assays and species-specific PCR assays for M. arginini and M. ovipneumoniae. Some cultures that grew on Hayflick plates were picked as single colonies but were mixed because two organisms may grow together and appear as a single colony. Culture and PCR tests produced similar results for M. arginini, but for M. ovipneumoniae, culture alone was less accurate than PCR. Use of genus-specific primers also may allow detection of other species in samples negative for M. arginini and M. ovipneumoniae. Two methods of transport from field to laboratory (Port-a-Cul? tubes, cryoprotectant in liquid N(2) and Fisher Transport System) gave similar results under our study conditions.     

Laboratory and microcosm experiments testing the toxicity of chlorinated hydrocarbons on a cyanobacterium strain (Synechococcus PCC 6301) and on natural phytoplankton assemblages

In the last few years, halogenated hydrocarbons have been detected in the soil, in the aquatic environment, in organisms, and even in drinking water. The toxic effects of three chlorinated aliphatic hydrocarbons (trichloroethylene, tetrachloroethylene and tetrachloroethane) were studied in laboratory experiments (using the cyanobacterium Synecococcus elongatus PCC 6301 as test organism) and in field-like circumstances (natural phytoplankton assemblages enclosed in microcosms). The results of the laboratory experiments showed that all of the tested compounds significantly inhibited the growth of the cultures within the first 4 h. Enzymatic changes of the treated cultures suggested that oxidative stress occured—all of the three compounds caused an increase in the activity of peroxidases and superoxide dismutase, and also increased the levels of lipid peroxidation. Observed changes in microcosms were comparable with the results of the laboratory experiments: the number of individuals and chlorophyll contents decreased in the treated assemblages. The elevated levels of peroxidation on the second day in the assemblages treated with tetrachloroethane and tetrachloroetylene suggest that oxidative stress could occur in field conditions. One of the most important findings is the decrease in species number. Our results showed that cryptomonads, some green algae species and the cyanobacterium Limnothrix gradually disappeared from the treated beakers during the experiment.     

Identifying Fishes through DNA Barcodes and Microarrays

Background

International fish trade reached an import value of 62.8 billion Euro in 2006, of which 44.6% are covered by the European Union. Species identification is a key problem throughout the life cycle of fishes: from eggs and larvae to adults in fisheries research and control, as well as processed fish products in consumer protection.

Methodology/Principal Findings

This study aims to evaluate the applicability of the three mitochondrial genes 16S rRNA (16S), cytochrome b (cyt b), and cytochrome oxidase subunit I (COI) for the identification of 50 European marine fish species by combining techniques of “DNA barcoding” and microarrays. In a DNA barcoding approach, neighbour Joining (NJ) phylogenetic trees of 369 16S, 212 cyt b, and 447 COI sequences indicated that cyt b and COI are suitable for unambiguous identification, whereas 16S failed to discriminate closely related flatfish and gurnard species. In course of probe design for DNA microarray development, each of the markers yielded a high number of potentially species-specific probes in silico, although many of them were rejected based on microarray hybridisation experiments. None of the markers provided probes to discriminate the sibling flatfish and gurnard species. However, since 16S-probes were less negatively influenced by the “position of label” effect and showed the lowest rejection rate and the highest mean signal intensity, 16S is more suitable for DNA microarray probe design than cty b and COI. The large portion of rejected COI-probes after hybridisation experiments (>90%) renders the DNA barcoding marker as rather unsuitable for this high-throughput technology.

Conclusions/Significance

Based on these data, a DNA microarray containing 64 functional oligonucleotide probes for the identification of 30 out of the 50 fish species investigated was developed. It represents the next step towards an automated and easy-to-handle method to identify fish, ichthyoplankton, and fish products.     

COI1 links jasmonate signalling and fertility to the SCF ubiquitin-ligase complex in Arabidopsis

Jasmonates (JAs) regulate Arabidopsis thaliana wound and defence responses, pollen development, and stress-related growth inhibition. Significantly, each of these responses requires COI1, an F-box protein. Other F-box proteins interact with SKP1 and cullin proteins to form SCF complexes that selectively recruit regulatory proteins targeted for ubiquitination. To determine whether COI1 also functions in an SCF complex, we have characterized Arabidopsis proteins that bind to COI1. An Arabidopsis cDNA expression library was screened in yeast for clones that produce proteins which can bind to COI1. We recovered two SKP1 homologues and a histone deacetylase. The Arabidopsis F-box protein TIR1 interacted with SKP1 proteins, but not with the histone deacetylase. Mutant COI1 proteins revealed that the F-box is required for interaction with SKP1s, but that sequences in leucine-rich repeat domains are required for interaction with the histone deacetylase. Epitope-tagged COI1 was introduced into Arabidopsis plants and cell cultures. Co-immunoprecipitation experiments confirmed the interaction in planta of COI1 with SKP1-like proteins and histone deacetylase, and also indicated that COI1 interacted with cullin. These results suggest that COI1 forms an SCFCOI1 complex in vivo. COI1 is therefore expected to form a functional E3-type ubiquitin ligase in plants and to regulate expression of jasmonate responsive genes, possibly by targeted ubiquitination of a histone deacetylase.     

DNA条形码技术在植物中的研究现状

DNA条形码技术（DNA barcoding）是用短的DNA片段对物种进行识别和鉴定的分子生物学技术。在动物研究中该技术已经成功应用于利用线粒体细胞色素c氧化酶亚基I（COI）进行物种鉴定和发现隐种或新物种。相对于动物, COI基因在高等植物中进化速率较慢, 因此植物条形码研究以叶绿体基因组作为重点, 但目前还处于寻找合适的基因片段阶段。许多学者对此进行了积极的探索, 报道了多种植物条形码的候选片段或组合, 但还没有获得满足所有标准的特征位点片段。该文介绍了DNA条形码的标准、优点、工作流程及数据分析方法, 总结了DNA条形码在植物中的研究现状。     

DNA barcoding of stygofauna uncovers cryptic amphipod diversity in a calcrete aquifer in Western Australia's arid zone

The arid Yilgarn region of Western Australia contains numerous subterranean calcrete aquifers with unique assemblages of obligate groundwater invertebrates (stygofauna). We aimed to establish a DNA barcoding framework for the macro-invertebrates present in a single calcrete, as a basis for future assessment of biodiversity of the Yilgarn calcretes and for investigating food webs. Intense sampling of a bore field grid in the Sturt Meadows calcrete was undertaken to obtain representatives of the entire macro-invertebrate ecosystem. A 623-bp fragment of the mitochondrial cytochrome c oxidase 1 (COI) gene was used to provide DNA barcodes for stygobiont macro-invertebrates plus terrestrial organisms that are found in the calcrete. Phylogenetic analyses revealed the existence of 12 divergent monophyletic groups of haplotypes. Subterranean amphipods (Chiltoniidae) showed three groups of COI haplotypes with sequence divergences between them of >11%. Allozyme analyses found a large number of fixed allelic differences between these three amphipod groups, indicating that there are three morphologically cryptic species within the Sturt Meadows calcrete. Unlike the sister triplet of dytiscid beetles present, the amphipods are not sister clades and are more closely related to other Yilgarn and non-Yilgarn amphipods than to each other. Our results show that the aquifer contains at least 12 macro-invertebrate species and DNA barcoding provides a useful means for discriminating species in this system.     

Locomotor compensation creates a mismatch between laboratory and field estimates of escape speed in lizards: a cautionary tale for performance-to-fitness studies

A key assumption in evolutionary studies of locomotor adaptation is that standard laboratory measures of performance accurately reflect what animals do under natural circumstances. One widely examined measure of performance is maximum sprint speed, which is believed to be important for eluding predators, capturing prey, and defending territories. Previous studies linking maximum sprint speed to fitness have focused on laboratory measurements, and we suggest that such analyses may be appropriate for some species and intraspecific classes, but not others. We provide evidence for a general inverse relationship between maximum laboratory sprint speed and the percentage of maximum capacity that animals use when escaping from a threat in the field (the model of locomotor compensation). Further, absolute values of field escape speed and maximum laboratory speed are not significantly related when comparing across a diverse group of Anolis and lacertid lizards. We show that this pattern of locomotor compensation holds both within (i.e., among intraspecific classes) and among lizard species (with some exceptions). We propose a simple method of plotting field escape speed (y-axis) versus maximum laboratory speed (x-axis) among species and/or intraspecific classes that allows researchers to determine whether their study organisms are good candidates for relating laboratory performance to fitness. We suggest that species that reside directly on, or near the "best fitness line" (field escape speed = maximum laboratory speed) are most likely to bear fruit for such studies.     

Carbon metabolism of intracellular bacteria

Bacterial metabolism has been studied intensively since the first observations of these 'animalcules' by Leeuwenhoek and their isolation in pure cultures by Pasteur. Metabolic studies have traditionally focused on a small number of model organisms, primarily the Gram negative bacillus Escherichia coli, adapted to artificial culture conditions in the laboratory. Comparatively little is known about the physiology and metabolism of wild microorganisms living in their natural habitats. For approximately 500-1000 species of commensals and symbionts, and a smaller number of pathogenic bacteria, that habitat is the human body. Emerging evidence suggests that the metabolism of bacteria grown in vivo differs profoundly from their metabolism in axenic cultures.     

Phylogenetic Analysis of Brine Shrimp (Artemia) in China Using DNA Barcoding

DNA barcoding is a powerful approach for characterizing species of organisms,especially those with almost identical morphological features, thereby helping to to establish phylogenetic relationships and reveal evolutionary histories. In this study, we chose a 648-bp segment of the mitochondrial gene, cytochrome c oxidase subunit 1 (COI), as a standard barcode region to establish phylogenetic relationships among brine shrimp (Artemia) species from major habitats around the world and further focused on the biodiversity of Artemia species in China, especially in the Tibetan Plateau. Samples from five major salt lakes of the Tibetan Plateau located at altitudes over 4,000 m showed clear differences from other Artemia populations in China. We also observed two consistent amino acid changes, 153A/V and 183L/F, in the COI gene between the high and low altitude species in China.Moreover, indels in the COI sequence were identified in cyst and adult samples unique to the Co Qen population from the Tibetan Plateau, demonstrating the need for additional investigations of the mitochondrial genome among Tibetan Artemia populations.     

Adaptive laboratory evolution--harnessing the power of biology for metabolic engineering

Adaptive laboratory evolution (ALE) strategies allow for the metabolic engineering of microorganisms by combining genetic variation with the selection of beneficial mutations in an unbiased fashion. These ALE strategies have been proven highly effective in the optimization of production strains. In contrast to rational engineering strategies and directed modification of specific enzymes, ALE has the advantage of letting nonintuitive beneficial mutations occur in many different genes and regulatory regions in parallel. So far, the majority of applications of ALE in metabolic engineering have used well-characterized platform organisms such as Saccharomyces cerevisiae and Escherichia coli; however, applications for other microorganisms are on the rise. This review will focus on current applications of ALE as a tool for metabolic engineering and discuss advancements and achievements that have been made in this field.     

Discrimination of two Japanese water pennies,Eubrianax granicollis Lewis and E. ramicornis Kiesenwetter (Coleoptera: Psephenidae), based on laboratory rearing and molecular taxonomy

Two psephenid beetles, Eubrianax granicollis Lewis and E. ramicornis Kiesenwetter, are common species on the main islands of Japan (i.e. Honshu, Shikoku and Kyushu), but diagnostic characters for larval identification are unknown. Two types of field‐collected Eubrianax larvae from Honshu and Kyushu were discriminated based on the distributions of granules on the dorsal surface. These larvae were assigned to E. granicollis and E. ramicornis by comparing them with larvae of the two species obtained via laboratory rearing. The two species were also identified unambiguously on the basis of their mitochondrial cytochrome oxidase subunit I (COI) gene sequences. Larval and pupal morphology are described based on laboratory‐reared specimens.     

Trapped in freshwater: the internal anatomy of the entoproct Loxosomatoides sirindhornae

Background

The identification of vast numbers of unknown organisms using DNA sequences becomes more and more important in ecological and biodiversity studies. In this context, a fragment of the mitochondrial cytochrome c oxidase I (COI) gene has been proposed as standard DNA barcoding marker for the identification of organisms. Limitations of the COI barcoding approach can arise from its single-locus identification system, the effect of introgression events, incomplete lineage sorting, numts, heteroplasmy and maternal inheritance of intracellular endosymbionts. Consequently, the analysis of a supplementary nuclear marker system could be advantageous.

Results

We tested the effectiveness of the COI barcoding region and of three nuclear ribosomal expansion segments in discriminating ground beetles of Central Europe, a diverse and well-studied invertebrate taxon. As nuclear markers we determined the 18S rDNA: V4, 18S rDNA: V7 and 28S rDNA: D3 expansion segments for 344 specimens of 75 species. Seventy-three species (97%) of the analysed species could be accurately identified using COI, while the combined approach of all three nuclear markers provided resolution among 71 (95%) of the studied Carabidae.

Conclusion

Our results confirm that the analysed nuclear ribosomal expansion segments in combination constitute a valuable and efficient supplement for classical DNA barcoding to avoid potential pitfalls when only mitochondrial data are being used. We also demonstrate the high potential of COI barcodes for the identification of even closely related carabid species.     

Barcoding quarantine nematodes and their close relatives: an update on the QBOL-project

Identification of plant pests, in particular quarantine species, needs to be fast and accurate to enable timely plant protection measures. In addition, a false diagnosis can cause serious financial losses for trade and producers. It is now well established that genetically based diagnosis is a reliable alternative to the classical identification procedures generally based on morphological features, which usually require expert taxonomic skills. On the other hand, genetic diagnosis through the use of DNA-barcodes, i.e. stretches of DNA that contain taxon-specific information, can be performed by any skilled laboratory worker. The European Union 7th framework project QBOL aims to establish DNA-barcodes for all European quarantine organisms as well as their close relatives. The results and protocols will be disseminated in the publicly available and curated database Q-BANK. To enable genetically based identification requires knowledge of the genetic variation both within and between the species of interest as well as their close relatives. For the nematodes, several gene regions (the COI, COII, SSU, LSU and RNA polymerase subunit II) are being evaluated for their barcoding potential.     

Comparative performance of the 16S rRNA gene in DNA barcoding of amphibians

BACKGROUND: Identifying species of organisms by short sequences of DNA has been in the center of ongoing discussions under the terms DNA barcoding or DNA taxonomy. A C-terminal fragment of the mitochondrial gene for cytochrome oxidase subunit I (COI) has been proposed as universal marker for this purpose among animals. RESULTS: Herein we present experimental evidence that the mitochondrial 16S rRNA gene fulfills the requirements for a universal DNA barcoding marker in amphibians. In terms of universality of priming sites and identification of major vertebrate clades the studied 16S fragment is superior to COI. Amplification success was 100% for 16S in a subset of fresh and well-preserved samples of Madagascan frogs, while various combination of COI primers had lower success rates.COI priming sites showed high variability among amphibians both at the level of groups and closely related species, whereas 16S priming sites were highly conserved among vertebrates. Interspecific pairwise 16S divergences in a test group of Madagascan frogs were at a level suitable for assignment of larval stages to species (1-17%), with low degrees of pairwise haplotype divergence within populations (0-1%). CONCLUSION: We strongly advocate the use of 16S rRNA as standard DNA barcoding marker for vertebrates to complement COI, especially if samples a priori could belong to various phylogenetically distant taxa and false negatives would constitute a major problem.