Protection against Atlantic halibut nodavirus in turbot is induced by recombinant capsid protein vaccination but not following DNA vaccination

Fish nodaviruses (betanodaviruses) are small, non-enveloped icosahedral single-stranded positive-sense RNA viruses that can cause viral encephalopathy and retinopathy (VER) in a number of cultured marine teleost species, including Atlantic halibut (Hippoglossus hippoglossus). A recombinant protein vaccine and a DNA vaccine were produced, based on the same capsid-encoding region of the Atlantic halibut nodavirus (AHNV) genome, and tested for protection in juvenile turbot (Scophthalmus maximus). Vaccine efficacy was demonstrated in the fish vaccinated with recombinant capsid protein but not in the DNA-vaccinated fish, despite the fact that in vivo expression of the DNA vaccine-encoded antigen was confirmed by RNA in situ hybridisation and immunohistochemistry. Combined DNA and recombinant vaccine administration did not improve the effect of the latter. Surprisingly, fish vaccinated with 50 microg recombinant protein demonstrated a threefold lower survival rate than the two groups that received 10 microg recombinant protein. Neither the recombinant protein vaccine nor the DNA vaccine induced anti-viral antibodies 9 weeks after immunisation, while antibodies reactive with the recombinant protein were detectable mainly in fish vaccinated with 50 microg recombinant protein. The study also demonstrates evidence of viral replication inside the myocytes of intramuscularly challenged fish.     

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.     

Barriers to hybridisation and their conservation implications for a highly threatened Australian fish species

Hybridisation and introgression are natural phenomena that may lead to the transfer of adaptive alleles from one species to another and increased species diversity. At the same time, hybridisation and subsequent introgression threaten many species world‐wide through the loss of genetic and species diversity. In Australia, introgressive hybridisation between native and alien species has not typically been considered a significant threat to native biodiversity because of the taxonomic distance between native and alien biota. However, many native fish have been introduced outside their natural range. Recently, four taxa in the genus Melanotaenia have been nationally listed as threatened due to introgressive hybridisation with introduced Melanotaenia splendida. We examined pre‐ and post‐zygotic barriers to hybridisation between M. splendida and one of these threatened taxa—Running River rainbowfish (RRR)—to assess the potential for hybridisation to occur. We used dichotomous mate choice experiments to examine pre‐zygotic barriers and mating experiments to examine post‐zygotic barriers. Size was not a significant predictor of the proportion of time subjects spent with a potential mate, nor was there any significant difference in the amount of time subjects spent with potential mates of their own or the opposite species. Eggs from hybrid pairings with female RRR had a slightly higher hatching rate than those from hybrid pairings with female M. splendida, but neither were significantly different from intraspecies crosses. We could not identify any definite barriers to hybridisation, demonstrating that the introduction of “native” fish species outside their natural range poses a higher risk of hybridisation than previously thought. We call for better education around the consequences of moving “native” fish and the development of rapid response plans to deal with recently established alien populations of Australian fish species in order to prevent future extinctions due to introgressive hybridisation.     

Species relative abundance and direction of introgression in oaks

Successful hybridisation and subsequent introgression lead to the transfer of genetic material across species boundaries. In this process, species relative abundance can play a significant role. If one species is less abundant than the other, its females will receive many heterospecific gametes, increasing mate-recognition errors and thus hybridisation rate. Moreover, first-generation hybrids will also more likely mate with the more abundant species, leading to asymmetric introgression. These predictions have important fundamental consequences, especially during biological invasions or when a rare species threatened by extinction is surrounded by individuals from a related species. However, experimental tests in nature of the importance of the relative abundance of each species on hybridisation dynamics remain scarce. We assess here the impact of species relative abundance on hybridisation dynamics among four species from the European white oak species complex. A total of 2107 oak trees were genotyped at 10 microsatellite markers and Bayesian clustering methods were used to identify reference trees of each species. We then used these reference trees to simulate purebred and hybrid genotypes to determine optimal threshold for genetic assignment. With this approach, we found widespread evidence of hybridisation between all studied oak species, with high occurrence of hybrids, varying from 11% to 31% according to stand and sampling strategies. This finding suggests that hybridisation is a common phenomenon that plays a significant role in evolution of this oak species complex. In addition, we demonstrate a strong impact of species abundance on both hybridisation rate and introgression directionality.     

Transmission of lungworms of harbour porpoises and harbour seals: Molecular tools determine potential vertebrate intermediate hosts

Harbour porpoises (Phocoena phocoena) and harbour seals (Phoca vitulina) from German waters are infected by six species of lungworms (Metastrongyloidea). These nematodes parasitise the respiratory tract, are pathogenic and often cause secondary bacterial infections. In spite of their clinical and epidemiological significance, the life cycle and biology of lungworms in the marine environment is still largely unknown. Regions of ribosomal DNA (ITS-2) of all lungworms parasitising harbour porpoises and harbour seals in German waters were sequenced to characterise and compare the different species. The phylogenetic relationship among the lungworm species was analysed by means of their ITS-2 nucleotide sequences and the species-specific traits of the ITS-2 were used to screen wild fish as possible intermediate hosts for larval lungworms. Molecular markers were developed to identify larval nematodes via in-situ hybridisation of tissues of harbour porpoise and harbour seal prey fish. Potential wild intermediate fish hosts from the North Sea were dissected and found to harbour larval nematodes. Histological examination and in-situ hybridisation of tissue samples from these fish showed lungworm larvae within the intestinal wall. Based on larval ITS-2 nucleotide sequences, larval nematodes were identified as Pseudalius inflexus and Parafilaroides gymnurus. Turbot (Psetta maxima) bred and raised in captivity were experimentally infected with live L1s of Otostrongylus circumlitus and ensheathed larvae were recovered from the gastrointestinal tract of turbot and identified using molecular tools. Our results show that fish intermediate hosts play a role in the transmission of metastrongyloid nematodes of harbour porpoises and harbour seals.     

Evaluation of the relative effectiveness of hybridization probes prepared by action of hydrazides of biotin derivatives on DNA

Efficiencies of biotinylated DNAs as hybridisation probes in a model system of non-radioactive detection were compared. Probes were obtained by interaction of single-stranded DNA and each of four different hydrazides of biotin derivatives. The most sensitivity in detection of complementary target was obtained using (biocytin hydrazide)-treated DNA. Relations between hydrazide structures and sensitivity of biotinylated probes are discussed.     

Evidence for clustering of RNA polymerase and ribosomal protein genes in six species of Enterobacteria

Summary ³²P-labelled DNA fragments from the E. coli beta operon were used as hybridisation probes for homologous DNA sequences in chromosomal digests of six species of Enterobacteria. In all species the hybridisation pattern suggested clustering of the genes comprising the beta operon.     

Measurement of cytochrome P4501A induction in dab (Limanda limanda) and other teleosts with species-specific cDNA probes: isolation and characterisation of dab cDNA and its use in expression studies with β-naphthoflavone-treated fish

Induction of cytochrome P4501A (CYP1A) in fish is an important biomarker in marine monitoring programmes but a number of factors complicate interpretation of data based on catalytic activity. To provide additional analytical tools, we have cloned and sequenced entire (dab) and partial cDNAs (flounder, turbot, sand eel) from several fish species. A detailed analysis comparing the new sequences to those on the database (13 sequences) is presented and identifies an invariant, teleost-specific sequence (195-IVVSVANVICGMCFGRRYDH-214) which might be the basis for production of a species cross-reactive antibody. Northern and slot blots of fish RNA (sand eel, plaice, turbot, flounder and dab) showed extensive cross-species hybridisation with each of the cDNAs (sand eel, plaice, turbot, flounder and dab). The exception was turbot RNA, which only gave adequate hybridisation when the turbot probe was used. Attempts to normalise the hybridisation data to GAPDH mRNA were not satisfactory since there were significant species differences in expression of this gene and expression was suppressed (20–40%) by β-naphthoflavone treatment. The CYP1A probes indicated induction levels relative to untreated dab of: plaice (five-fold); turbot (12-fold); flounder (12-fold); and dab (10-fold). The study demonstrates the relative ease with which species-specific molecular probes can be generated and used.     

An oligonucleotide microarray for the identification and differentiation of trichothecene producing and non-producing Fusarium species occurring on cereal grain

Cereal grain may be infected with a number of Fusarium species some of which are producers of highly toxic compounds such as the trichothecenes. Correct identification of these species is essential for risk assessment of cereal grain for human or animal consumption. Most of the available methods for identification are either time consuming or aimed at only one or a few target species. Microarray technology offers parallel analysis of a high number of DNA targets. In this study 57 capture oligonucleotides (CO) were designed based upon Fusarium ITS2 rDNA sequences, and used for microarray production. From this array COs could be selected that were able to hybridise specifically to labelled PCR products from the ITS region of Fusarium graminearum/Fusarium culmorum, Fusarium pseudograminearum, Fusarium poae, Fusarium sporotrichioides, Fusarium equiseti, Fusarium langsethiae and Fusarium tricinctum/Fusarium avenaceum. A few COs showed some cross hybridisation to non-target species. In a preliminary experiment it was shown that this cross hybridisation could be eliminated by increasing hybridisation stringency. The array could be used to detect individual Fusarium species in mixed samples and in environmental samples. This study demonstrates the feasibility of oligonucleotide microarrays for parallel detection of a number of Fusarium species.     

Rapid and efficient screening of a Representational Difference Analysis library using reverse Southern hybridisation: identification of genetic differences between Haemophilus parasuis isolates

Representational Difference Analysis (RDA) is an established technique used for isolation of specific genetic differences between or within bacterial species. This method was used to investigate the genetic basis of serovar-specificity and the relationship between serovar and virulence in Haemophilus parasuis. An RDA clone library of 96 isolates was constructed using H. parasuis strains H425(P) (serovar 12) and HS1967 (serovar 4). To screen such a large clone library to determine which clones are strain-specific would typically involved separately labelling each clone for use in Southern hybridisation against genomic DNA from each of the strains. In this study, a novel application of reverse Southern hybridisation was used to screen the RDA library: genomic DNA from each strain was labelled and used to probe the library to identify strain-specific clones. This novel approach represents a significant improvement in methodology that is rapid and efficient.     

Demonstration of mRNAs for oxytocin and prolactin in porcine granulosa and luteal cells. Effects of these hormones on progesterone secretion in vitro

The relative levels of mRNAs for relaxin, prolactin, inhibin and oxytocin have been measured in porcine granulosa as well as luteal cells by hybridisation to single-stranded synthetic DNA. The likelihood of a paracrine function of oxytocin and prolactin in the porcine ovary was inferred from the in vitro effects of both hormones on progesterone secretion of ovarian cells. Both hormones were found to inhibit progesterone secretion of luteal cells. In contrast, only prolactin but not oxytocin stimulated progesterone secretion in granulosa cells.     

Hybridisation of surface-immobilised single-stranded oligonucleotides and polymer monitored by surface plasmon resonance

We have investigated the hybridisation of thiol-modified single-stranded DNA embedded in a polyacrylamide layer through the technique of surface plasmon resonance (SPR). Kinetic studies were carried out by two different immobilisation methods: (a) SH-ssDNA was firstly attached on gold and the remaining free space was filled with polymer and (b) SH-ssDNA and the polymer was attached onto the surface from the same solution. The immobilisation methods were compared for various concentrations of SH-ssDNA. Hybridisation was dependent on both the immobilisation method and the concentration of the components. The highest hybridisation was obtained when SH-ssDNA and the polymer was immobilised from the same solution at low SH-ssDNA concentration or when high concentrations of oligos were spread onto the surface and the surface was post-treated with polymer. The target response corresponded to a surface coverage of 100+/-15 ng/cm2. The same surface coverage on hybridisation was also obtained when low concentration of SH-ssDNA and polymer was attached onto the surface from the same solution. The non-specific binding of sample DNA was very low at optimal concentrations due to the polymer and the hybridisation was linearly dependent on target concentration.     

A Retrospective Approach to Testing the DNA Barcoding Method

A decade ago, DNA barcoding was proposed as a standardised method for identifying existing species and speeding the discovery of new species. Yet, despite its numerous successes across a range of taxa, its frequent failures have brought into question its accuracy as a short-cut taxonomic method. We use a retrospective approach, applying the method to the classification of New Zealand skinks as it stood in 1977 (primarily based upon morphological characters), and compare it to the current taxonomy reached using both morphological and molecular approaches. For the 1977 dataset, DNA barcoding had moderate-high success in identifying specimens (78-98%), and correctly flagging specimens that have since been confirmed as distinct taxa (77-100%). But most matching methods failed to detect the species complexes that were present in 1977. For the current dataset, there was moderate-high success in identifying specimens (53-99%). For both datasets, the capacity to discover new species was dependent on the methodological approach used. Species delimitation in New Zealand skinks was hindered by the absence of either a local or global barcoding gap, a result of recent speciation events and hybridisation. Whilst DNA barcoding is potentially useful for specimen identification and species discovery in New Zealand skinks, its error rate could hinder the progress of documenting biodiversity in this group. We suggest that integrated taxonomic approaches are more effective at discovering and describing biodiversity.     

Magnetic capture hybridisation for improved PCR detection ofNectria galligena from lignified apple extracts

In order to reduce the effects of inhibitors present in DNA extracts from lignified apple tissues, a magnetic capture-hybridisation PCR (MCH-PCR) technique was developed forNectria galligena using the ITS 1 region of the rRNA gene repeats as target. The trapping reagent used to coat the magnetic beads was an 81 bp single-stranded DNA oligonucleotide biotin-labelled on the 5é-terminal and designed to be complementary to part of the rRNA gene ITS 1 region ofN. galligena. For specificity, the probe was located from 14 bp downstream from the 3é-terminal nucleotide of theN. galligena forward primer Ch1 to the last ITS 1 nucleotide immediately upstream of the 5.8S rRNA gene. Following hybridisation in a total DNA extract of woody tissue, magnetic recovery of the bead-oligomer-template conjugate separated target template from other DNA species and inhibitory compounds. Magnetic capture-hybridisation was followed by PCR amplification with the previously designed species-specific primers, Ch1 and Ch2. Application of the MCH-PCR technique resulted in increased levels of sensitivity and reliability when compared to PCR without MCH when used on total DNA extracts from lignified tissues.     

Detection of invasive freshwater fish species using environmental DNA survey

Invasive species are one of the most significant problem in freshwater ecosystems. Most common non-native freshwater species in Turkish freshwater fish fauna are Prussian Carp (Carassius gibelio), North African Catfish (Clarias gariepinus), Nile Tilapia (Oreochromis niloticus) and Topmouth Gudgeon (Pseudorasbora parva).Recent studies showed that environmental DNA could be used to detect target species inhabiting the ecosystem with higher precision and less effort compared to traditional field surveys. In this study, eDNA approach was used to investigate non-native freshwater fish species from fifteen different locations of Upper Sakarya Basin. eDNA was successfully extracted from the water samples of locations where the species were visually observed. Mean amplification rate of eDNA was calculated as 77.03%.This study is the first environmental DNA study used in detection of four of the most common invasive freshwater fish species. Results clearly indicating that eDNA surveys could be used as an important molecular tool to monitor invasive fish species in freshwater ecosystems.     

Mini- and micro-satellites in the genome of rodent malaria parasites.

Higher eukaryotes contain within their DNA numerous arrays of repetitive DNA, many of which are known as satellite DNAs and display extensive variability. The presence of these repeats has been demonstrated for various species and they have been used for genetic identification and classification. Here, it is demonstrated that Southern hybridisation of DNA from rodent malaria parasites allows detection of micro- and minisatellite sequences in the genome of Plasmodium species. Closely related lines of malaria parasites exhibit a monomorphic hybridisation pattern, which is in contrast to the allelic variation observed in higher eukaryotes. Among different species, however, restriction-fragment length polymorphism was observed. Pulsed-field gel electrophoretic chromosome separation showed that the probes used in this study [33.15, 33.6, (CAC)n and (GT)n] detect several loci spread over different chromosomes.     

Labelfree fully electronic nucleic acid detection system based on a field-effect transistor device

The labelfree detection of nucleic acid sequences is one of the modern attempts to develop quick, cheap and miniaturised hand-held devices for the future genetic testing in biotechnology and medical diagnostics. We present an approach to detect the hybridisation of DNA sequences using electrolyte-oxide-semiconductor field-effect transistors (EOSFETs) with micrometer dimensions. These semiconductor devices are sensitive to electrical charge variations that occur at the surface/electrolyte interface, i.e. upon hybridisation of oligonucleotides with complementary single-stranded (ss) oligonucleotides, which are immobilised on the oxide surface of the transistor gate. This method allows direct, time-resolved and in situ detection of specific nucleic acid binding events without any labelling. We focus on the detection mechanism of our sensors by using oppositely charged polyelectrolytes (PAH and PSS) subsequently attached to the transistor structures. Our results indicate that the sensor output is charge sensitive and distance dependent from the gate surface, which pinpoints the need for very defined surface chemistry at the device surface. The hybridisation of natural 19 base-pair sequences has been successfully detected with the sensors. In combination with nano-transistors a PCR free detection system might be feasible in future.     

Comparative analysis of fish environmental DNA reveals higher sensitivity achieved through targeted sequence-based metabarcoding

Environmental DNA (eDNA)-based methods of species detection are enabling various applications in ecology and conservation including large-scale biomonitoring efforts. qPCR is widely used as the standard approach for species-specific detection, often targeting a fish species of interest from aquatic eDNA. However, DNA metabarcoding has the potential to displace qPCR in certain eDNA applications. In this study, we compare the sensitivity of the latest Illumina NovaSeq 6000 NGS platform to qPCR TaqMan assays by measuring limits of detection and by analysing eDNA from water samples collected from Churchill River and Lake Melville, NL, Canada. Species-specific, targeted next generation sequencing (NGS) assays had significantly higher sensitivity than qPCR, with limits of detection 14- to 29-fold lower. For example, when analysing eDNA, qPCR detected Gadus ogac (Greenland cod) in 21% of samples, but targeted NGS detected this species in 29% of samples. General NGS assays were as sensitive as qPCR, while simultaneously detecting 15 fish species from eDNA samples. With over 34,000 fish species on the planet, parallel and sensitive methods such as NGS will be required to support effective biomonitoring at both regional and global scales.     

Hybridisation,paternal leakage and mitochondrial DNA linearization in three anomalous fish (Scombridae)

Using mitochondrial DNA for species identification and population studies assumes that the genome is maternally inherited, circular, located in the cytoplasm and lacks recombination. This study explores the mitochondrial genomes of three anomalous mackerel. Complete mitochondrial genome sequencing plus nuclear microsatellite genotyping of these fish identified them as Scomberomorus munroi (spotted mackerel). Unlike normal S. munroi, these three fish also contained different linear, mitochondrial genomes of Scomberomorus semifasciatus (grey mackerel). The results are best explained by hybridisation, paternal leakage and mitochondrial DNA linearization. This unusual observation may provide an explanation for mtDNA outliers in animal population studies.