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To overcome some of the problems with these previous studies, the use of molecular techniques to identify salmonid prey in seal scats has been explored. Such methods have mostly involved conventional PCR amplification of cytochrome oxidase III, cytochrome b or mitochondrial 16S ribosomal RNA genes, followed by either restriction fragment length polymorphism (RFLP) (Purcell et al. 2004; Parsons et al. 2005) or denaturing gradient gel electrophoresis (Deagle et al. 2005) to identify different prey species represented.
The present study employed quantitative PCR (qPCR) to detect Atlantic salmon and sea trout DNA in seal scats. Spiked scat material was used to assess the applicability and accuracy of this technique. The field study targeted estuarine areas of the Moray Firth (North Sea, UK) that are inhabited by mixed populations of harbour and grey seals known to consume salmonids (Middlemas et al. 2006).
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Our study reports the first application of qPCR to detect marine mammal prey in scats, and reveals data previously unobtainable using conventional PCR (Purcell et al. 2004; Deagle et al. 2005; Parsons et al. 2005). Additionally, qPCR provides a more sensitive tool than conventional hard-part analysis, consistently detecting more scats containing salmonids than the traditional analyses. The Taqman assays presented here demonstrated high specificity in the detection of target DNA. The seal assay showed no amplification from salmonid-derived material, and both Atlantic salmon and sea trout assays were unable to amplify DNA extracted from 29 different fish species recorded in the diet of harbour seal and grey seal (Hammond et al. 1994a, 1994b; Thompson et al. 1996; Tollit & Thompson 1996).
In the 2003 field scat samples, high levels of PCR inhibitors hindered detection of prey DNA in the neat template in most samples. A high level of DNA degradation, shown by the failure to detect the positive control seal DNA in 17% of scat samples from 2003, might indicate that ethanol is not the best preservative for scat samples. Storage of scats at –80 °C was shown to be a better method for storage and preservation, as the presence of PCR inhibitors in the neat template of 2005 was reduced to ≈20% of scats, and detection of the control seal DNA failed in only 6% of scats. Also, the seal scat sampling protocol was shown to affect results of the qPCR assays, and might in some cases lead to underestimation of the proportion of salmonids consumed by seals. As DNA from prey species is known to be distributed unevenly within a scat, a thorough homogenization, followed by subsampling (minimum of three subsamples per scat) of scats after removal of the hard parts, is recommended to prevent underestimation of consumed prey.
The experimental study was designed to assess the potential use and accuracy of the qPCR assay to detect salmonids from seal scats, and these data were used to estimate salmonid remains in scats obtained from the Moray Firth area in 2005. The experimental trial on spiked seal scats confirmed high efficiency in detecting seal and Atlantic salmon DNA in scat material. Based on the Ct values for detection of seal DNA, similar quantities of seal DNA were present in each of the spiked scat experimental groups. This also confirms that mixing scats prior to analysis is a useful approach to avoid subsampling error, as previously stated by Deagle et al. (2005). The seal DNA in scat samples originates from the cells of the rectal lining, and is expected to be localized mainly on the surface of scats. The fact that no significant differences were found in Ct values for seal detection among the spiked scat experimental groups indicates a uniform distribution of seal DNA, and most likely prey DNA, in mixed scats. The Atlantic salmon qPCR assay performed using the spiked scat experimental material revealed high consistency in detection of the target DNA, even at concentrations as low as 5 and 1% of Atlantic salmon-fed scat in the entire scat. All 45 subsamples in each spiked experimental group showed amplification of target DNA, suggesting a uniform distribution of prey DNA in mixed spiked scats. Performing the qPCR assay on a dilution series revealed that it is possible to detect as little as 0·01% Atlantic salmon in seal scats using the qPCR assay designed in this study.
Based on Ct values of Atlantic salmon from the experimental study, an approximate calibration curve could be constructed and the concentration of Atlantic salmon in field scat samples estimated. However, estimating the quantity of prey in field scats based on this is only relative. This is because no information is available on the rate of DNA degradation for different prey species during digestion, or degradation of prey DNA in scats in the environment prior to collection. Furthermore, seals may not consume whole specimens of fish, but may only eat soft tissue (Pierce & Boyle 1991; Orr et al. 2004). The limitations of this approach mean it is impossible to accurately relate concentration of prey DNA to the number of prey specimens consumed by an individual seal. However, future application of the Taqman assays on scat material derived from a controlled seal-feeding trial might help to reduce these problems and enable more accurate quantification of prey species in seal scats.
Analysis of seal scats collected from the field in 2005 revealed that only two samples out of 49 tested positive for Atlantic salmon DNA, and one sample tested positive for sea trout. Based on the calibration curve from the experimental study, the predicted concentration of the salmonid target in whole scat samples was about 1–5 and 1% for Atlantic salmon and sea trout, respectively. This suggests that these types of prey were not common in the samples. If salmonids represent only an infrequent or ‘pulse’ component of diet, detection of target DNA using conventional PCR is restricted to scats produced within 48 h of ingestion (Deagle et al. 2005). However, the qPCR technique has been shown to detect target DNA, even in cases where conventional PCR failed (Snow et al. 2006), so using the qPCR approach a ‘pulse’ component of diet might be detectable for longer than 2 days after ingestion. A higher number of scats that were positive for salmonids was found in the 2003 sampling season. However, it was difficult to relate Ct values from these samples to values from the spiked scat experiment, as a different amount of template was used in the qPCR reactions due to the presence of PCR inhibitors. Also, only 2-g subsamples of scats were taken to be analysed in 2003, so the calibration curve from the spiked scat experiment cannot be used in this case.
A parallel conventional analysis of hard parts was performed on the scat samples collected in both years. The occurrence of salmonid hard parts in the scat samples showed a pattern similar to that of the genetic analysis. There were, however, a number of important differences, notably the ability to differentiate between Atlantic salmon and sea trout. Additionally, salmonid DNA was detected in a greater number of scats than hard parts, including positive results from areas and months where otoliths and bones were absent (Cromarty Firth, May 2003; Findhorn Bay, 2005). This shows that analysis of DNA is better for detecting rare prey species than the conventional analysis of hard parts.
The results illustrate the potential use of DNA alongside conventional analyses in providing insights into the feeding ecology of seals. For example, qPCR analyses support previous hard-part analyses and confirm that salmonids are relatively scarce prey items at these study sites. The results suggest that sea trout were more prevalent in the diet of harbour seals compared with a mixed group containing both grey and harbour seals, although definite identification of seal species would be required to confirm this finding. This result is in agreement with previous diet work (Hammond et al. 1994a, 1994b; Tollit & Thompson 1996; Middlemas et al. 2006) and observations showing that harbour seals are more common than grey seals in rivers (Carter et al. 2001; Middlemas et al. 2006). In addition, the results of this study suggest that predation on sea trout is linked to predation on flatfish rather than sandeels. The occurrence of these prey groups in the diets of harbour seals in the Moray Firth region has been shown to be linked to the seals’ use of different water depths and sea bed sediments (Tollit et al. 1998). This result therefore potentially provides information on where seals are consuming sea trout (Tollit et al. 1998; Bowen et al. 2002).
Just as the classical hard-part approach involves a bias in quantification of prey in seal scats (Pierce & Boyle 1991; Tollit et al. 1997a,b; Bowen 2000; Browne et al. 2002; Arim & Naya 2003; Orr et al. 2004), quantification based on DNA techniques is complex. Deagle et al. (2005) designed a captive feeding trial with Steller sea lions, Eumetopias jubatus, to assess the reliability of conventional PCR amplification of prey DNA from seal scats, and also tried to estimate diet composition by quantifying the amount of DNA present in scats, using library screening. They concluded that DNA techniques are accurate; however, they uncovered a possible bias when estimating quantity of prey, especially when unequal meals have been ingested during a day, and suggested pooling DNA extracted from scats with patchy diet to ensure a more accurate proportional estimate.
For field studies, the results derived from the controlled trial by Deagle et al. (2005) suggest that multiple scat samples from individual seals would provide better insight into feeding habits. Linking scats to individual seals may be possible using microsatellite analysis, as described by Reed et al. (1997). This would allow identification of specialist seals which is of importance for management of predator–prey interactions (Graham et al. 2005). Moreover, in other predator/prey systems, differences between prey consumption of an individual predator and that of the whole population were highlighted, reflecting differences in fitness and reproductive success within the predator population, or their social dominance (Fedriani & Kohn 2001; Gende & Quinn 2003).
In conclusion, an important step in the fisheries management of seal–salmonid interaction in the Moray Firth is to determine accurately the occurrence of salmonids in the seal diet (Middlemas et al. 2003). Using conventional techniques, it is not possible to determine unequivocally whether a low occurrence of salmonid remains is due to their scarcity in the diet or the fragility of their hard parts compared with those of other prey species (Boyle et al. 1990). Our DNA-based approach allows the relative contribution of Atlantic salmon and sea trout in the seal diet to be determined. With only approximately 10% of scats testing positive for salmonid DNA, our DNA analysis supports previous suggestions that salmonids are rare in the diet of seals in the Scottish estuaries (Pierce et al. 1991a, 1991b; Tollit & Thompson 1996; Middlemas et al. 2006). Consequently, these results support current management practice, which focuses on control of the small number of seals that move into key salmonid rivers, rather than targeting the larger groups of animals that haul-out in nearby estuaries (Butler et al., in press).