4.2. METHODS TO IDENTIFY (Gadus morhua L.)
Cite this article as: Uglem I, Black K, Berg M, Varne R, Nilsen R, Mork J, Bjørn PA (2012) Methods to identify Atlantic escaped cod (Gadus morhua L.). In: PREVENT ESCAPE Project Compendium. Chapter 4.2. Commission of the European Communities, 7th Research Framework Program. www.preventescape.eu
authors: Ingebrigt Uglem1, Kenny Black2, Marius Berg1, Rebecca Varne3, Rune Nilsen4, Jarle Mork3 & Pål Arne Bjørn4 Norwegian Institute of Nature Research, Tungasletta 2, NO-7485 Trondheim, Norway Scottish Association for Marine Science, Dunstaffnage Marine Laboratory, Oban PA34 1QA, UK 3 Trondheim Biological Station, Department of Biology, Norwegian University of Science and Technology, N-7491 Trondheim, Norway 4 Nofima Marin, Muninbakken 9-13, P.O. Box 6122, N-9291 Tromsø, Norway 1 2
INTRODUCTION To map the distribution and possible ecological impacts of escaped farmed cod, escapees need to be traced in the wild. Simple, reliable, and fast methods for determining the origin of cod are required. The importance of reliable determination of the origin of cod caught in the wild is illustrated by frequent reports in Norwegian media during recent years regarding catches of abnormal and assumed escaped farmed cod. In many of these cases, it has hitherto been difficult to verify if such fish were of farm origin, because genetic samples were not taken. Identification of escapees from fish farms may be done in several ways. For instance, recent developments within genetics have led to increasingly more efficient and less costly ways for distinguishing between farmed and wild cod (e.g. Glover 2010). In addition, analyses of scales and body morphology can distinguish farmed and wild salmonids with a high degree of certainty (e.g. Fiske et al. 2006). Trace element compositions in scales and otoliths are also effective in distinguishing between wild and farmed salmon (Adey et al. 2009). Furthermore, variation in fatty acid composition in body tissues has been suggested as a tool for determining
wild or farmed origin because commercial fish feeds used in aquaculture affect the fatty acid composition of farmed fish compared to wild fish which feed on natural organisms (Fernandez-Jover et al. 2007).
The Atlantic cod is an epibenthic-pelagic species, which is widely distributed in a variety of habitats, from the shoreline down to the continental shelf. Cod are distributed along the North American coast; around Greenland and Iceland, and along the coasts of Europe from the Bay of Biscay to the Barents Sea. Cod may grow up to 2 m in length and more than 90 kg, although this is extremely seldom today. In commercial fisheries, sizes typically range from 2 to 20 kg. Cod are omnivorous; they feed at dawn or dusk on invertebrates and fish, including young cod. They spawn pelagic eggs in batches once a year, usually during late winter and early spring. A 5 kg wild female may spawn around 2.5 million eggs during a spawning season. The most important stocks are the Norwegian Arctic stock in the Barents Sea and the Icelandic stock. Populations around Greenland and Newfoundland have declined dramatically, whereas the stock in the Barents Sea remains healthy. The Atlantic cod is en economically important species and it is marketed fresh, dried or salted, smoked and frozen.
OBJECTIVE We evaluated whether analyses of scales, body morphology, fatty acids and trace elements have the potential to rapidly and accurately separate escapees from wild Atlantic cod. We did not focus on genetic methods, as reliable and effective methods to separate escaped and wild cod based on genetic variation already exist (Glover 2010).
METHODS We evaluated several methods for identifying escaped cod by sampling fish from several commercial cod farms, situated along the Norwegian coast, and wild cod caught in the vicinity of these farms. First, the potential for using scales and fish morphology to separate between escapees and wild fish was assessed by analysing digital images of fish and their scales using computer-based image
analyses. Three measurements of the fish scales were used to separate wild and farmed cod; mean circuli breadth, length-adjusted scale radius and length-adjusted circuli number per scale (see Uglem et al. 2011 for details). The radius was measured from the centre to the edge of the scale, and the distances between individual circuli were measured along the same axis (Figure 4.2.1). We selected morphological features on the basis that they would: 1) clearly differentiate between farmed and wild fish; and 2) be easy to measure in the field. Morphological codes were then assigned to these features (see Figure 4.2.1 for a clear illustration). Fatty acid variation was examined in samples from two commercial cod farms and also in a sample of wild cod captured at a spawning ground 80 km away from the nearest fish farm. Fatty acids were measured in ovary and liver tissue using a capillary gas chromatograph (Perkin Elmer, Autosystem XL, USA), with methods according to KjĂ¸rsvik et al. (2009). In addition, trace elements were measured in fish scales by ICPMS, after cleaning and complete dissolution, using the method of Adey et al. (2009). Samples were analysed together with certified reference materials and blanks. The applicability of morphological, scale, fatty acid and trace element measurements for identifying escapees was analysed using multivariate statistical analyses (Uglem et al. 2011), and the results presented as MDS plots (R open source statistical software).
Figure 4.2.1. Principle sketch showing morphological measures and scale measures. The abbreviations are described in Table 3. The area from where the scale samples were taken is indicated under the last dorsal fin.
RESULTS Analysis showed that the mean scale circuli breadth and the length-adjusted scale radius measurements differed significantly between wild and farmed cod, whereas the lengthadjusted circuli number did not. Discriminant analyses indicated that the mean scale circuli breadth measurement correctly classified wild and farmed fish 86% of the time, while the length-adjusted scale radius measurement was successful in 80% of cases (Table 4.2.1). Correct classified (%) Model
Mean circuli breadth and length-adjusted 1 scale radius
Morphology, PC1, 2, 3
Morphology, LJ, HA, and FA
Fatty acids, Ovaries, PC1, 2, 3, 4
Fatty acids, Liver PC1, 2, 3
Table 4.2.1. Data from discriminant analysis for scale and morphological parameters, and also fatty acids, including the proportion of wild and farmed Atlantic cod being correctly classified. Original classification and cross-validation is identical. The codes for the morphological parameters are described in detail in Figure 4.2.1.
The extent to which morphological variation could be used to separate between escapees and wild cod was assessed in two ways. Firstly, principal component analysis (PCA) was used to reduce the variation among the morphological features (size-adjusted) into three principal components (PCs), and these explained 73% of the variation. A discriminant analysis of individual PC scores showed that 97% of the wild and 96% of the farmed fish were classified correctly (Table 4.2.1). Subsequently, three morphological features were selected to assess the viability of using a few simple measurements to discriminate between farmed and wild cod. The primary selection criterion was that these parameters would be easy to measure in the field, and the parameters FA, LJ and HA were thus selected (Figure 4.2.1). A discriminant analysis with FA, LJ, and HA showed that 100% of the wild fish were classified correctly, while 95% of the farmed fish were classified correctly (Table 4.2.1). Fatty acids profiles varied among wild and farmed fish and between farms (Figure 4.2.2, 4.2.3) PCA reduced the number of variables that represented the variation in fatty acids between farmed and wild fish. Discriminant analysis of the significant PCs, for both the liver and the ovary samples, showed that the fish could be correctly classified with respect to origin 100% of the time using this technique (Table 4.2.1).
The metal profiles from farmed and wild fish from TromsĂ¸ (Figure 4.2.4b) and Trondheimsfjord (Figure 4.2.4c) can be distinguished with very high confidence. When plotted together (Figure 4.2.4a) it is clear that the 95% confidence limits from some populations overlap. For example, this reveals that wild TromsĂ¸ scales are much more similar to farmed Trondheim fish than to farmed fish from the same fjord. Farmed fish from the three farms are separated with high confidence as do the two wild populations. These results indicate that fish scale chemistry has the potential to discriminate between farmed fish and local wild populations of Norwegian cod with high confidence, and could provide a useful tool in determining the origin of suspected escapees. However, at present, we do not know whether the significant difference found in the metal profiles of wild and farmed fish would diminish with increasing time after escape.
Figure 4.2.2. Individual factor scores and loadings for ovaries samples for PC1 and PC2 in relation to Atlantic cod (Gadus morhua) origin.
Figure 4.2.3. Individual factor scores and loadings for liver samples for PC1 and PC2 in relation to Atlantic cod (Gadus morhua) origin.
Figure 4.2.4. MDS plots of cod scale chemistry profiles from (right to left) a) Trondheimsfjord (farmed and wild), TromsĂ¸ (farmed and wild), and Tysfjord (farmed); b) TromsĂ¸ (farmed and wild) and; c) Trondheimsfjord (farmed and wild). Ellipses indicate 95% confidence intervals.
DISCUSSION We have shown that analyses of scales, morphology, fatty acid profiles and trace metals all have the potential to distinguish between wild and farmed Atlantic cod, and that in the future these tests might be useful as management tools. For instance, our results indicate that a high proportion of Atlantic cod can be correctly classified as farmed or wild, based on three simple morphological features, which can be measured either from digital images or from live fish, following anaesthesia. Often a field-based determination of fish origin is an advantage over more labour and time intensive laboratory techniques, such as genetic discrimination. A standardized methodology based on a few simple traits for identifying escaped farmed cod, would represent a cheap and simple approach for evaluating the origin of cod, complementing more advanced methods. This series of experiments can be regarded as a proof-of-concept study. We recommend that the methods tested be verified through blind tests before they are applied in a real life situation, i.e. testing datasets not originally used to develop the statistical models. Furthermore, there is a need to examine additional farmed and wild fish populations, as well as several year classes, ages and diets, before functional and reliable methodologies for discrimination between wild and farmed cod can be developed. The precision of the various measures could be verified by simultaneously carrying out genetic analyses.
RECOMMENDATIONS s We have shown that analyses of scales, morphology, fatty acid profiles and trace metals all have the potential to distinguish between wild and farmed Atlantic cod s However, our results are based on proof-of-concept studies and considerable efforts are needed to develop standardized, reliable and functional methods that can be used as operational management tools
TO PUBLISHED DOCUMENTS:
Uglem I, Berg M, Varne R, Nilsen R, Mork J, Bjørn PA (2011) Discrimination of wild and farmed Atlantic cod (Gadus morhua) based on morphology and scale-circuli pattern. ICES Journal of Marine Science 68, 1928-1936, DOI: 10.1093/icesjms/fsr120
Adey EA, Black KD, Sawyer T (2009) Scale microchemistry as a tool to investigate the origin of wild and farmed Salmo salar. Ma Ecol Prog Ser 390: 225-235 Fernandez-Jover D, Jimenez JAL, Sanchez-Jerez P (2007) Changes in body condition and fatty acid composition of wild Mediterranean horse mackerel (Trachurus mediterraneus, Steindachner, 1868) associated to sea cage fish farms. Mar Environ Res 63:1-18 Fiske P, Lund RA, Hansen LP (2006) Relationships between the frequency of farmed Atlantic salmon, Salmo salar (L.), in wild salmon populations and fish farming activity in Norway, 19892004. ICES J Mar Sci 63:1182-1189 Glover KA, Dahle G, Westgaard JI, Johansen T, Knutsen H, Jørstad KE (2010) Genetic diversity within and among Atlantic cod (Gadus morhua) farmed in marine cages: a proof-of-concept study for the identification of escapees. Anim Genet 41, 515-522 Kjorsvik E, Olsen C, Wold PA, Hoehne-Reitan K, Cahu CL, Rainuzzo J, Olsen AI, Oie G, Olsen Y (2009) Comparison of dietary phospholipids and neutral lipids on skeletal development and fatty acid composition in Atlantic cod (Gadus morhua). Aquaculture 294:246-255 Uglem I, Berg M, Varne R, Nilsen R, Mork J, Bjørn PA (2011) Discrimination of wild and farmed Atlantic cod (Gadus morhua) based on morphology and scale-circuli pattern. ICES J Mar Sci 68:928-1936