Advances in Chemical Engng. Van Landeghem,H. Hanson Editors. Symposium Ser. Pure Appl. Solvent Extraction Chemistry. Dyrssen, North-Holland, Amsterdam, p. Dyrssen, North-Holland, Amsterdam p. Academic Press, New York, Hydrometallurgy 3 Solvent Extraction Conf. Series No: 42, The other phase is the solvent. The extraction is understood to be a transfer of the solute from the feed to the solvent. During and at the end of the extraction process, the feed deprived of solute becomes a raffinate and the solvent turns into extract.
Extraction is a separation process aiming to purify the feed or to recover one or more compounds from it. Request changes or add full text files to a record. The diagonal lines represents identical measurements before and after DNA extraction, indicating no effect of the treatment.
Visual inspection of the pairs of images of each cross-sectioned otolith suggested that no reduction in the contrast of annual growth increments had resulted from DNA extraction Figure 2. The effect of DNA extraction on contrast of annual growth increments: a photograph of the cross section of an otolith taken before DNA extraction.
The transect line used to measure greyscale values is depicted by a black line; b same otolith cross section after DNA extraction; c measured greyscale values along the transect of the otolith cross section photographed in a dotted lines and b solid line ; d correlation between the average contrast over annual growth increments of individual otoliths dots before and after DNA extraction.
The open triangles represent control otoliths that were not exposed to DNA extraction. The diagonal line represents identical measurements before and after DNA extraction, indicating no effect of the treatment. The measured elemental concentrations in the otoliths are summarized in Table 2 and Figure 3. Not all samples could be processed because of small sample volumes , but there were at least 23 otolith pairs for each element.
Of these, Li, Na, Ca, and Sr varied only slightly and randomly within pairs, but for Cu and Pb, there was a clear trend of a higher concentration in the otoliths exposed to DNA extraction. However, for those two elements, there was generally a very large variation in concentration measurements within otolith pairs, even for the controls Table 2 , Figure 3.
Summary statistics on the relative difference in element concentrations between the two otoliths of each pair. The difference was calculated as the concentration in the treated otolith A minus the concentration in the untreated otolith B , divided by the concentration in the untreated otolith B to give the percentage difference from the untreated otolith.
A positive value indicates that the concentration was highest in the otolith subjected to DNA extraction for the control pairs, both otoliths A and B were untreated. The p -values are from the paired t -test comparing concentrations in the treated and untreated otoliths of each pair.
The horizontal band in each box represents the median, the bottom and top of the boxes represent the 25th and 75th percentiles, and the error bars define the 5th and the 95th percentiles. Outlier data points are marked by dots. Three extreme outliers for the Pb isotopes are not shown asterisk, the light isotope; double-asterisk, the heavy isotope. However, post hoc tests revealed that this effect was mostly caused by the group exposed to lysis for 5 h having a significantly smaller difference between pairs than the groups exposed for 3 and 8 h, and in most cases none of the groups were significantly different from the control group.
This study has demonstrated that DNA can be extracted successfully from otoliths with several different methods and that the otoliths can still be used for other purposes following the DNA extraction.
There was no difference in amplification success for the DNA extracted with the three protocols tested, nor did the protocols have different effects on otolith composition, shape, or visual appearance.
Therefore, any of the protocols could be used in future studies based on combining different types of information that can be obtained from otoliths. There was no evidence of longer lysis times producing a better quality DNA product, but also no indication that longer lysis times maximally 8 h would result in more damage to the otoliths. The PCR amplification success rate was in general very high for the short fragments.
Hence, although extraction from otoliths only provides a small amount of DNA, which is often degraded, it is sufficient for a relatively consistent amplification of many genetic markers. However, the DNA extracted from juvenile otoliths exhibited a much lower success rate for the short fragments more attempts needed to get a successful amplification; data not shown , and amplification almost always failed for the longest fragment.
These otoliths were up to 50 times smaller by weight than the adult otoliths used in this study, and perhaps they simply did not contain enough tissue on the surface to provide a reasonable quantity of DNA although small stains from dried blood or mucus were visible on some of them.
Adult otoliths provided a much better source for DNA in this study. This result was expected, because DNA becomes fragmented and degraded over time, and the relationship between fragment size and amplification success in historical DNA is consistent with that reported in other studies Nielsen and Hansen, Using large otoliths and genetic markers with alleles shorter than a few hundred base pairs is likely to improve the genotyping success and decrease the risk of errors attributable to large allele dropout with historical samples.
DNA extraction did not appear to change the physical properties measured in this study. The similarity in weight, silhouette area, and perimeter suggests that the extraction solution does not dissolve the otolith, at least not within the 8-h lysis time tested here. The only observed alteration of otolith intactness was that the lobes were chipped off five of the whole otoliths. Such physical breakage could most likely be avoided by using gentle rocking or rotation during lysis, rather than vortexing as was done here.
There was no reduction in the clarity of annual growth increments; if anything, the DNA extraction may have caused a slight though not significant improvement in clarity. This result is consistent with a recent study that found that DNA extraction did not influence age-determination success in coral trout otoliths Heath et al. In contrast, however, the core area of sole otoliths became less visible when they were exposed to DNA extraction with the Hutchinson et al.
Perhaps sole otoliths, being thin and flat, are more susceptible to damage by the extraction procedure than the thicker, rounder cod otoliths. The difference could also, in part, be methodological, because the sole study assessed the visibility of the nucleus in the intact otolith. Any slight etching of the outer surface may have had a greater impact on this than on the visibility of annual growth increments in a cross-sectioned otolith, as tested here and in the study with coral trout otoliths.
In this respect, the current study may be viewed as conservative, because the reading surface on the cross section was directly exposed to the digestive solution, and DNA is often extracted from whole otoliths so the cross section is typically not directly exposed to the lysing solution. Therefore, our results clearly demonstrate that otoliths, at least from cod, can be used for both morphometric and growth-based studies after DNA extraction.
Our results also suggest that otoliths can be used for microchemical analyses after DNA extraction, because the concentrations of most elements were similar or varied only slightly between treated and untreated otoliths of matching pairs, except for Mg and Rb. An underlying assumption of these experiments is, of course, that the two otoliths of a fish have similar elemental concentrations.
These differences may represent natural variation between the two otoliths of an individual i. The variation observed here between the left and the right otoliths is generally comparable with that reported in other studies e.
Proctor and Thresher, ; Campana et al. The variation within the control otolith pairs, whether natural or a result of analytical protocol, could define the functional detection limit or a baseline for evaluating the effect of DNA extraction. Although there was a significant trend for higher concentrations in the otolith subjected to DNA extraction for Mn and Ba, these differences were so small that they fell well within the variation observed in the control otolith pairs. Therefore, perhaps otoliths subjected to DNA extraction could be used for microchemical studies with these elements without significantly compromising the accuracy of stock-structure delineation, although the possible bias resulting from slightly greater concentrations of these elements should be kept in mind.
Most notably, there was no evidence of changes in Ca or Sr concentrations as a result of DNA extraction. This finding is reassuring, because these two elements are crucial for otolith microchemistry studies. Sr concentrations are related to temperature, salinity, and fish growth characteristics Townsend et al.
The only elements that showed differences exceeding the variation in the control otolith pairs was Mg, which appeared to increase with DNA extraction, and Rb, which appeared to decrease. It is unclear what mechanism caused these changes. Mg belongs to the group 2 metals that can replace Ca in the CaCO 3 matrix of the otolith, so is expected to be fairly stable. However, Mg concentration in otoliths appears to be much more susceptible to alteration than other group 2 elements, and experiments have shown that it might be influenced by a number of frequently used handling and storage methods, such as freezing and storage in ethanol Milton and Chenery, ; Hedges et al.
It has therefore been suggested that some of the Mg is bound in the proteinaceous matrix of otoliths and hence is more labile Hedges et al. This could also be an explanation for the changes observed in Rb. In the chemistry lab, it is most common to use liquid-liquid extraction, a process that occurs in a separatory funnel. A solution containing dissolved components is placed in the funnel and an immiscible solvent is added, resulting in two layers that are shaken together.
It is most common for one layer to be aqueous and the other an organic solvent. Components are "extracted" when they move from one layer to the other.
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