Sea lice (Lepeophtherius salmonis) detection and quantification around aquaculture installations using environmental DNA.

The naturally occurring ectoparasite salmon lice (Lepeophtherirus salmonis) poses a great challenge for the salmon farming industry, as well as for wild salmonids in the Northern hemisphere. To better control the infestation pressure and protect the production, there is a need to provide fish farmers with sensitive and efficient tools for rapid early detection and monitoring of the parasitic load. This can be achieved by targeting L. salmonis DNA in environmental samples. Here, we developed and tested a new L. salmonis specific DNA-based assay (qPCR assay) for detection and quantification from seawater samples using an analytical pipeline compatible with the Environmental Sample Processor (ESP) for autonomous water sample analysis of gene targets. Specificity of the L. salmonis qPCR assay was demonstrated through in-silico DNA analyses covering sequences of different L. salmonis isolates. Seawater was spiked with known numbers of nauplii and copepodite free-swimming (planktonic) stages of L. salmonis to investigate the relationship with the number of marker gene copies (MGC). Finally, field samples collected at different times of the year in the vicinity of a salmon production farm in Western Norway were analyzed for L. salmonis detection and quantification. The assay specificity was high and a high correlation between MGC and planktonic stages of L. salmonis was established in the laboratory conditions. In the field, L. salmonis DNA was consequently detected, but with MGC number below that expected for one copepodite or nauplii. We concluded that only L. salmonis tissue or eDNA residues were detected. This novel study opens for a fully automatized L. salmonis DNA quantification using ESP robotic to monitor the parasitic load, but challenges remain to exactly transfer information about eDNA quantities to decisions by the farmers and possible interventions.

An evaluation of coral lophelia pertusa mucus as an analytical matrix for environmental monitoring: A preliminary proteomic study.

For the environmental monitoring of coral, mucus appears to be an appropriate biological matrix due to its array of functions in coral biology and the non-intrusive manner in which it can be collected. The aim of the present study was to evaluate the feasibility of using mucus of the stony coral Lophelia pertusa (L. pertusa) as an analytical matrix for discovery of biomarkers used for environmental monitoring. More specifically, to assess whether a mass-spectrometry-based proteomic approach can be applied to characterize the protein composition of coral mucus and changes related to petroleum discharges at the seafloor. Surface-enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF MS) screening analyses of orange and white L. pertusa showed that the mucosal protein composition varies significantly with color phenotype, a pattern not reported prior to this study. Hence, to reduce variability from phenotype difference, L. pertusa white individuals only were selected to characterize in more detail the basal protein composition in mucus using liquid chromatography, mass spectrometry, mass spectrometry (LC-MS/MS). In total, 297 proteins were identified in L. pertusa mucus of unexposed coral individuals. Individuals exposed to drill cuttings in the range 2 to 12 mg/L showed modifications in coral mucus protein composition compared to unexposed corals. Although the results were somewhat inconsistent between individuals and require further validation in both the lab and the field, this study demonstrated preliminary encouraging results for discovery of protein markers in coral mucus that might provide more comprehensive insight into potential consequences attributed to anthropogenic stressors and may be used in future monitoring of coral health.

Detection of small bioactive peptides from Atlantic herring (Clupea harengus L.).

Recent research has shown that fish residual materials contain a range of components with interesting biological activity. Therefore, there is a great potential in the marine bioprocess industry to utilize these by-products as starting material for generating more valuable products. The aim of the present study was to search for bioactive peptides (in particular small natural bioactive peptides with molecular weight lower than 10 kDa) in Atlantic herring (Clupea harengus L.) by-products such as skin and more general residual materials. By such means a range of peptides with claimed interesting biological activities was found. Herein the activity of the detected bioactive peptides and strategies for isolating peptide fragments containing the bioactive motif is discussed. Identification of bioactive peptides in crude peptide/protein sources (skin and residual materials) was performed directly using a combination of mass spectrometry (Orbitrap), bioinformatics and database search. This method was a good angle of approach in order to map the potential in new species and species that have been very little studied.

Mass spectrometric profiling - a diagnostic tool in fish?

The development of rapid and sensitive diagnostic tools to assess the effect of stressors on organisms is a principal objective of environmental proteomics. This study is focused on evaluating the potential of using surface-enhanced laser desorption/ionisation time-of-flight mass spectrometry (SELDI-TOF MS) to assess stress in Atlantic salmon (Salmo salar). Plasma and mucus samples were taken from fish that had previously been maintained in a range of high density conditions, together with control fish maintained under low density conditions. Samples were collected during the post-density stress period for protein profile analysis. The mass spectra were analysed to evaluate reproducibility and to search for condition specific changes in protein expression. Multivariate analysis of the peak relative intensity data indicated a segregation of the data into three entities in accordance with the density level fish had been subjected to during the density stress period. This segregation was seen in both plasma and mucus data.

Is nitrate reductase a major player in the plant NO (nitric oxide) game?

Nitric oxide (NO) is a diffusible, very reactive gas that is involved in the regulation of many processes in plants. Several enzymatic sources of NO production have been identified in recent years. Nitrate reductase (NR) is one of them and it has been shown that this well-known plant protein, apart from its role in nitrate reduction and assimilation, can also catalyse the reduction of nitrite to NO. This reaction can produce large amounts of NO, or at least more than is needed for signalling, as some escape of NO to the outside medium can be detected after NR activation. A role for NO and NR in stomata functioning in response to abscisic acid has also been proposed. The question that remains is whether this NR-derived NO is a signalling molecule or the mere product of an enzymatic side reaction like the products generated by the oxygenase activity of RuBisCO.

Mechanism and importance of post-translational regulation of nitrate reductase.

In higher plants, nitrate reductase (NR) is inactivated by the phosphorylation of a conserved Ser residue and binding of 14-3-3 proteins in the presence of divalent cations or polyamines. A transgenic Nicotiana plumbaginifolia line (S521) has been constructed where the regulatory, conserved Ser 521 of tobacco NR (corresponding to Ser 534 in Arabidopsis) was mutated into Asp. This mutation resulted in the complete abolition of activation/inactivation in response to light/dark transitions or other treatments known to regulate the activation state of NR. Analysis of the transgenic plants showed that, under certain conditions, when whole plants or cut tissues are exposed to high nitrate supply, post-translational regulation is necessary to avoid nitrite accumulation. Abolition of the post-translational regulation of NR also results in an increased flux of nitric oxide from the leaves and roots. In view of the results obtained from examining the different transgenic N. plumbaginifolia lines, compartmentation of nitrate into an active metabolic pool and a large storage pool appears to be an important factor for regulating nitrate reduction. The complex regulation of nitrate reduction is likely to have evolved not only to optimize nitrogen assimilation, but also to prevent and control the formation of toxic, and possibly regulatory, products of NR activities. Phos phorylation of NR has previously been found to influence the degradation of NR in spinach leaves and Arabidopsis cell cultures. However, experiments with whole plants of N. plumbaginifolia, Arabidopsis, or squash are in favour of NR degradation being the same in light and darkness and independent of phosphorylation at the regulatory Ser.

Mutation of the regulatory phosphorylation site of tobacco nitrate reductase results in high nitrite excretion and NO emission from leaf and root tissue.

In wild-type Nicotiana plumbaginifolia Viv. and other higher plants, nitrate reductase (NR) is regulated at the post-translational level and is rapidly inactivated in response to, for example, a light-to-dark transition. This inactivation is caused by phosphorylation of a conserved regulatory serine residue, Ser 521 in tobacco, and interaction with divalent cations or polyamines, and 14-3-3 proteins. The physiological importance of the post-translational NR modulation is presently under investigation using a transgenic N. plumbaginifolia line. This line expresses a mutated tobacco NR where Ser 521 has been changed into aspartic acid (Asp) by site-directed mutagenesis, resulting in a permanently active NR enzyme. When cut leaves or roots of this line (S(521)) were placed in darkness in a buffer containing 50 mM KNO(3), nitrite was excreted from the tissue at rates of 0.08-0.2 micromol (g FW)(-1) h(-1) for at least 5 h. For the control transgenic plant (C1), which had the regulatory serine of NR intact, nitrite excretion was low and halted completely after 1-3 h. Without nitrate in the buffer in which the tissue was immersed, nitrite excretion was also low for S(521), although 20-40 micromol (g FW)(-1) nitrate was present inside the tissue. Apparently, stored nitrate was not readily available for reduction in darkness. Leaf tissue and root segments of S(521) also emitted much more nitric oxide (NO) than the control. Importantly, NO emission from leaf tissue of S(521) was higher in the dark than in the light, opposite to what was usually observed when post-translational NR modulation was operating.