Molecular characterization of microalgae used in aquaculture with biotechnology potential

Microalgae are the main component of first tropic level in aquatic food chain; it is for this reason that they are used as food in aquaculture. Also due to its biotechnological potential properties, they are used in the production of diverse components, dyes, antioxidants, enzymes, emulsifiers, etc. The extended ways of microalgae applications require physiologically and genetically stable cultures as well as correctly identified organisms to guarantee reproducibility and reliability. But the variety of species and the morphological similarity between some of them make difficult the identification of some microalgae. The use of molecular markers has supplied a very useful tool for identification of microalgae in fast mode, such as in classification. The present study has worked on the molecular characterization of main species of microalgae used in aquaculture in base of the molecular markers 18S rRNA and 16S rRNA. Microalgae DNA has been amplified and sequenced, and the resultant sequences were analyzed and reflected in phylogenetic trees. The phylogenetic analyses obtained reflect as both molecular markers allow to differentiate the main genus used in aquaculture.     

Detection of land animal remains in fish meals by the polymerase chain reaction-restriction fragment length polymorphism technique

In the present study a technique was developed with the aim of guaranteeing the composition and security of fish meals, since it allows verification of whether these meals contain land animal remains. The method is based on polymerase chain reaction (PCR) and length polymorphism, followed by a restriction fragment length polymorphism (RFLP). Specific primers for every species were designed and calibrated, generating exclusively a PCR product with a specific size when DNA for each species was present in the sample. This technique allows the detection of land animal remains in fish meals, specifically cow, chicken, pig, horse, sheep, and goat. The identity of the PCR products can be confirmed by RFLP analysis using only one restriction enzyme. The selected restrictase generated one characteristic restriction profile for every species included in this study. The detection limit of this method was calculated by using mixtures of fish meals in different proportions and meal that exclusively contained remains of one of these land species studied. The analytical strategy herein proposed was applied to fish and meat meals, giving good results, both in the analyzed standards and in commercial samples.     

Development of a method for the genetic identification of mussel species belonging to Mytilus, Perna, Aulacomya, and other genera

Legislation regarding the labeling of processed products is an important issue in the protection of consumer rights. This labeling is especially important in products that cannot be identified on the basis of their morphological characters, because these are removed from the animal in the transformation process. The goal of this study was the identification of mussel species using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) and Forensically Informative Nucleotide Sequencing (FINS) methodologies. The molecular marker selected was 18S rDNA (nuclear small-subunit rDNA gene), which allows identification at the genus level and at the species level in some cases. The genera included in this study were Mytilus, Perna, Aulacomya, Semimytilus, Brachidontes, Choromytilus, and Perumytilus. Different markers were used for genetic identification at the species level. To identify the species included in the genus Perna and Choromytilus, a fragment of ITS 1 (Internal Transcribed Spacer 1) was amplified by multiplex PCR and digested with restrictases. The species of Mytilus were identified by length polymorphism and RFLP of the polyphenolic adhesive protein gene. This methodology was validated with products manufactured in the authors' pilot plant and applied to commercial samples. Therefore, this sequential method can be completely or partially used to determine the mussel genus or species present in any food product.     

Non‐invasive fast real‐time PCR assay for detection of the enteric parasite Enteromyxum scophthalmi in cultured turbot (Scophthalmus maximus L.)

Enteromyxum scophthalmi is a myxozoan parasite that causes severe parasitic diseases in cultured turbot affecting mainly the intestine of the host. It is characterized by producing acute enteritis, starvation and eventually death. Current diagnosis of E. scopthalmi use traditional techniques, based on the identification of the morphology of the parasite. These techniques take extended time to be carried out and do not favour the adoption of control measure at turbot farms and require the sacrifice of fish. This study develops a fast real‐time PCR molecular tool for the detection of E. scophthalmi in infected farmed turbot. This methodology is applicable for routine controls on the farm at every stage of the parasite infection. Results of the study demonstrate the robustness, specificity, efficiency and reliability of the technique. In addition, this study also provides a non‐invasive procedure of sampling through swaps. This allows control, prevention and diagnosis of the parasite infection at turbot farms while maintaining the welfare of the cultivated fish and avoiding sacrifice of the fish sampled.     

Genetic identification of squids (families Ommastrephidae and Loliginidae) by PCR-RFLP and FINS methodologies

Cephalopods are a taxonomic group that contains a great number of families, genera and species, with many of them very important at the commercial level. The existence of very similar species in this class added up to the transformation process applied to them makes it difficult or even impossible for species identification based on morphological characterization. Moreover, the global commerce makes it possible that one determined species can be marketed in its antipodes. These questions suggest the necessity of molecular techniques to solve this situation. In the present work, a genetic method was developed on the basis of the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and forensically informative nucleotide sequencing (FINS) technique and makes possible the identification of more than 20 species belonging to the families Ommastrephidae and Loliginidae, as well as some octopus and sepia species. The PCR was employed to amplify 651 and 208 bp fragments of the mitochondrial cytochrome b gene. These molecular systems were applied to fresh, frozen, precooked, even canned cephalopods, allowing for the identification of the species included in these products. Therefore, these molecular tools could be applied in questions related to correct labeling, traceability, and importation controls of squids, sepias, and octopuses.     

Rapid identification of seaweeds in food products by PCR combined with ALF-RFLP and FINS methodologies

In the present study, two methods for the genetic identification of the most important seaweed species used for human consumption were developed. Both are carried out through PCR amplification of an 18S rRNA gene fragment. The first one is based on the phylogenetic analysis of DNA sequences (FINS), while the second is based on length polymorphism and RFLP visualized by means of an ALF system. The main novelty of this work lies in the fact that it allows genetic identification of the main commercial species of seaweed. Moreover, the developed systems can be applied to all kinds of processed products, including those that have undergone intensive transformation, as for instance canned foods. These methodologies also permit the detection of species in complex matrixes where more than one algal species is present. The methods were validated using products manufactured in a pilot plant showing correct functioning. Finally, the methods were applied to 23 commercial samples including some that had been subjected to intensive thermal treatment, allowing the detection of those that were incorrectly labeled (30%). Therefore, these molecular tools can be used for clarifying questions related to the correct labeling and traceability of commercial products that include some seaweeds in their composition.     

Thrombosis of the portal vein in a miniature schnauzer

A miniature schnauzer with a history of apathy, anorexia and jaundice was presented to the Utrecht University Clinic for Companion Animals. Abnormal laboratory findings included highly increased levels of total bile acids and alkaline phosphatase, and hyponatraemia. Abdominal ultrasonography revealed that the right side of the liver was enlarged and the left side was small, together with a thrombus in the portal vein. Biopsies from the right side of the liver demonstrated subacute to chronic active hepatitis, for which the dog was treated with prednisolone (1 mg/kg/day for four weeks). No improvement was observed and the owner requested euthanasia. At necropsy the left lobes of the liver were found to be small and firm, while the right lobes were large and soft. There were two thrombi in the portal vein. Microscopic examination revealed chronic active hepatitis and cirrhosis.     

Authentication of anglerfish species (Lophius spp) by means of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and forensically informative nucleotide sequencing (FINS) methodologies

Lophius represents the most important genus of the family Lophiidae from a commercial point of view. The main marketing formats of the species included in this genus are tails and cheeks, making impossible the species identification on the basis of their morphological characters. In the present study, two methods based on the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and phylogenetic analysis of DNA sequences [forensically informative nucleotide sequencing (FINS)] were developed to differentiate the seven species contained in the genus Lophius. In both cases, the molecular marker studied was the cytochrome oxidase subunit I gene (COI). The RFLP analysis of the PCR products digested with the endonuclease Mbo I generated species-specific restriction profiles, and the phylogenetic analysis showing a neighbor-joining tree with independent nodes was strongly supported for all of the studied species. These methods were applied to 40 commercial samples, allowing us to detect the samples incorrectly labeled. The fraudulent labeling ratio was higher in processed products (68.75%) than whole fish (31.25%). The species subjected to mislabeling were L. budegassa (68.75%), L. vomerinus (18.75%), and L. piscatorius (12.5%). Therefore, both methodologies can be independently used to authenticate the species belonging to the genus Lophius, being useful to check the fulfillment of labeling regulations of seafood products and to verify the correct traceability of commercial trade and the control of fisheries.     

Fast real-time PCR for the detection of crustacean allergen in foods

Crustaceans are one of the most common allergens causing severe food reaction. These food allergens are a health problem, and they have become very important; there are various regulations that establish that labeling must be present regarding these allergens to warn consumers. In the present work a fast real-time PCR, by a LNA probe, was developed. This allows the detection of crustaceans in all kinds of products, including processed products in which very aggressive treatments of temperature and pressure during the manufacturing process are used. This methodology provides greater sensitivity and specificity and reduces the analysis time of real-time PCR to 40 min. This methodology was further validated by means of simulating products likely to contain this allergen. For this, products present on the market were spiked with crustacean cooking water. The assay is a potential tool in issues related to the labeling of products and food security to protect the allergic consumer.     

Immunohistochemical localisation of rabbit haemorrhagic disease virus VP-60 antigen in early infection of young and adult rabbits

This study evaluated the time course distribution of rabbit haemorrhagic disease virus (RHDV) structural protein VP60 in tissues from experimentally infected rabbits from three different age groups. Viral VP60 antigen could not be detected in tissue samples from animals under four weeks, and only a few hepatocytes (0.01 to 0.2 per cent) were stained in the 6-week-old animals. A 6-week-old rabbit euthanised at 72 hpi showed VP60-labelling in hepatocytes and macrophages close to areas of inflammation. Viral VP60 antigen was detected as early as 12 hpi in a few hepatocytes (0.03 per cent) from adult animals. Within this age group, the extent of hepatocyte labelling considerably increased at 18 (3.0 per cent), 24 (25.5 per cent), 36 (50 per cent) and 48 (60 per cent) hpi. Extrahepatic viral VP60 antigen was also detected at 36 and 48 hpi in spleen macrophages and lymphocytes from adult rabbits. These findings support the hypothesis that the hepatocyte is the only cell type in the liver able to support RHDV replication almost immediately after viral infection.