Daniel Campo

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.

Molecular Organization and Evolution of 5S rDNA in the Genus Merluccius and Their Phylogenetic Implications

The molecular organization of the 5S rRNA gene family has been studied in a wide variety of animal taxa, including many bony fish species. It is arranged in tandemly repeated units consisting of a highly conserved 120 base pair–long region, which encodes for the 5S rRNA, and a nontranscribed spacer (NTS) of variable length, which contains regulatory elements for the transcription of the coding sequence. In this work, a comparative analysis of 5S ribosomal DNA (rDNA) organization and evolution in the 12 species of the genus Merluccius, which are distributed in the Atlantic and Pacific oceans, was carried out. Two main types of 5S rDNA (types A and M) were identified, as differentiated by the absence or presence of a simple sequence repeat within the NTS. Four species exhibited the 2 types of 5S rDNA, whereas the rest showed only 1 type. In addition, the species M. albidus and M. bilinearis showed 2 variants (S and L) of type-M 5S rDNA, which differentiated by length. The results obtained here support the hypothesis of a 5S rRNA dual system as an ancient condition of the Piscine genome. In contrast, some inconsistencies were found between the phylogeny of the genus Merluccius based on mitochondrial genes and that obtained from nuclear markers (5S rDNA, microsatellite loci, and allozyme data). Hybrid origin of the American species M. australis is suggested based on these results.

Strong genetic differentiation of the Austral hake (Merluccius australis) across the species range.

Population structuring of marine organisms is not always easily understood. In open marine areas without apparent barriers to migration, species with high dispersal capacity are expected to be organized in large populations that, ideally, could represent the best example of panmictic units more or less genetically homogeneous. However, there are a number of examples contradicting this panmixia theory. In some cases, physical barriers like straits (e.g. the Gibraltar Strait separating Mediterranean and Atlantic populations, see a review in Patarnello et al. (2007)) or currents (e.g. the East Australian Current for sea urchin (Banks et al., 2007)) can be identified as obstacles for population homogenization across a species range. As there is much variation in DNA markers which is easily detected, there has been a growing interest in their application to better understand population structuring. Microsatellite loci, for example, can detect weak differentiation in species with high gene flow (Waples, 1998). On the other hand, large-scale geographical differences are better detected employing mitochondrial DNA polymorphisms because, due to maternal inheritance, effective population size for mtDNA is a quarter of that of nuclear genes, and consequently genetic drift may produce higher population differentiation (Birky et al., 1989). Therefore combining nuclear and mitochondrial loci is a good strategy for detecting even weak regional differences in population structure.

Mislabeling of Two Commercial North American Hake Species Suggests Underreported Exploitation of Offshore Hake

Abstract Mislabeling of North American merlucciid hakes in stock surveys and commercial market samples was detected by employing nuclear 5S ribosomal DNA (rDNA) and mitochondrial cytochrome b variation as molecular markers. Results showed that offshore hake Merluccius albidus is sold in European markets but is labeled as the morphologically similar silver hake M. bilinearis, which is the target species of the fishery. This suggests that offshore hake may be inadvertently included within silver hake landings, as the two species overlap in the southern area of silver hake distribution (approximately 41°-35°N latitude near North American coasts). An inexpensive and technically easy technique based on polymerase chain reaction (PCR) amplification of a fragment of 5S rDNA and visualization of PCR products in agarose gels is recommended for routine species assignation in landings for purposes of exploitation estimates and for authentication of commercial hake species.

Inquiry-Based Learning of Molecular Phylogenetics.

Reconstructing phylogenies from nucleotide sequences is a challenge for students because it strongly depends on evolutionary models and computer tools that are frequently updated. We present here an inquiry-based course aimed at learning how to trace a phylogeny based on sequences existing in public databases. Computer tools are freely available on the Internet. Student feedback has been more than satisfactory in the six years (2002–2007) when this course was active at the University of Oviedo (Spain).

Inquiry-based learning of molecular phylogenetics II: the phylogeny of Camelidae

This article is the output of a doctorate course on Molecular Evolution held at the University of Oviedo in Spain in 2005. Students chose by consensus a taxonomic group of their interest (Camelidae), collected sequences from public databases, gathered information on morphological traits, built phylogenies employing different methodologies, compared the trees obtained and discussed the results.

A20 regulates canonical wnt-signaling through an interaction with RIPK4

A20 is a ubiquitin-editing enzyme that is known to regulate inflammatory signaling and cell death. However, A20 mutations are also frequently found in multiple malignancies suggesting a potential role as a tumor suppressor as well. We recently described a novel role for A20 in regulating the wnt-beta-catenin signaling pathway and suppressing colonic tumor development in mice. The underlying mechanisms for this phenomenon are unclear. To study this, we first generated A20 knockout cell lines by genome-editing techniques. Using these cells, we show that loss of A20 causes dysregulation of wnt-dependent gene expression by RNAseq. Mechanistically, A20 interacts with a proximal signaling component of the wnt-signaling pathway, receptor interacting protein kinase 4 (RIPK4), and regulation of wnt-signaling by A20 occurs through RIPK4. Finally, similar to the mechanism by which A20 regulates other members of the receptor interacting protein kinase family, A20 modifies ubiquitin chains on RIPK4 suggesting a possible molecular mechanism for A20’s control over the wnt-signaling pathway.