NORTH PACIFIC RESEARCH BOARD FINAL REPORT
Pilot-scale development of laser ablation – isotope ratio mass spectrometry (LA-IRMS) for use in
retrospective studies of marine productivity in the North Pacific Ocean
NPRB Project 1223 Final Report
James Moran, Timothy Linley, and Megan Nims
Pacific Northwest National Laboratory, Post Office Box 999, Richland, Washington 99352,
(509) 371-6910, Timothy.Linley@pnnl.gov
September 2014
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Abstract
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Muscle and scales of Pacific herring (Clupea pallasi) from Prince William Sound (PWS) were analyzed
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by conventional and laser ablation isotope ratio mass spectrometry (IRMS) to measure seasonal and inter-
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annual variation in stable isotope ratios of carbon (δ13C) and nitrogen (δ15N). Stable isotope analysis is
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often employed to characterize energy flow and trophic relationships in aquatic ecosystems, and can
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potentially be used to identify factors that influence long-term changes in stock abundance from archived
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samples of calcified tissue. Our objective was to determine if there was a relationship between muscle and
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scale δ13C and δ15N in PWS herring that would support a retrospective analysis of the scale archive of this
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population. Conventional IRMS revealed significant relationships between muscle and scale for δ13C and
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δ15N, although these relationships were affected by both the age of the herring analyzed and their season
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of capture. By contrast, laser ablation IRMS showed limited variation in scale δ13C among age classes or
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seasons, but wide variation in δ13C within scales, suggesting that scale architecture and processes such as
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maturation and winter dietary stress may influence scale δ13C. The results support the use of conventional
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IRMS to analyze the PWS scale archive for evidence of long-term changes in feeding ecology within this
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population.
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Keywords
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Pacific herring, scales, stable isotope, mass spectrometry, laser ablation
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Citation
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Moran, J., T. Linley, and M. Nims. 2014. Pilot-scale development of laser ablation – isotope ratio mass
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spectrometry (LA-IRMS) for use in retrospective studies of marine productivity in the North Pacific
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Ocean. NPRB Project 1223 Final Report. 25 p.
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Table of Contents
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Abstract .................................................................................................................................................
1
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Introduction ...........................................................................................................................................
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Project Objectives .................................................................................................................................
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Methods ................................................................................................................................................
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Sample Preparation .......................................................................................................................
Isotopic Analysis ..........................................................................................................................
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10
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Results ...................................................................................................................................................
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Conventional IRMS in Muscle and Scales ...................................................................................
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LA-IRMS in Scales ......................................................................................................................
Discussion .............................................................................................................................................
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Conclusions ...........................................................................................................................................
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Management Implications .....................................................................................................................
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Publications ...........................................................................................................................................
Outreach ................................................................................................................................................
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References .............................................................................................................................................
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Figures
1.
Scanning electron microscope images of non-acid-treated and acid-treated scales analyzed
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for δ13C by laser ablation. Panel A. A non-acid-treated scale at 2,000x. The laser has failed
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to penetrate through the apatite layer and into the collagen layer, except for one small spot.
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Panel B. Enlarged image (8,000x) of the same ablation shows the collagen matrix visible
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underneath the apatite layer. Panel C. Acid-treated scale at 2,000x. The collagen matrix is
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visible on the surface of the scale because the acid treatment has removed the apatite. Panel
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D. A finer scale image (10,000x) of the same ablation spot showing the collagen matrix. ..........
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2.
The mean (± SD) 13C (left panel) and 15N (right panel) in the muscle of age-2 (white bar)
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and age-3 (black bar) herring in the fall compared to age-3 (white bar) and age-4 (black bar)
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herring in the spring. Bars without letters in common are significantly different from each
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other (P  0.05). Letters within bars indicate significant differences between age classes
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within seasons, whereas letters above bars indicate significant differences within age classes
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between seasons. ...........................................................................................................................
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3.
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Comparison of the mean (± SD) 13C (left panel) and 15N (right panel) in the scales of age2 (white bar) and age-3 (black bar) herring sampled in the fall to age-3 (white bar) and age-4
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(black bar) herring in the spring. Bars without letters in common are significantly (P  0.05)
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different. Letters within bars indicate significant differences between age classes within
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seasons, whereas letters above bars indicate significant differences within age classes
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between seasons. ...........................................................................................................................
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4.
The relationship between muscle and scale δ13C for age-2 () and age-3 (▲) herring
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collected in the fall (left panel) and age-3 () and age-4 (■) herring in spring (right panel) in
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PWS. .............................................................................................................................................
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5.
collected in the fall (left panel) and age-3 () and age-4 (■) herring in spring (right panel) in
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PWS. .............................................................................................................................................
6.
caught in April 2012. The light and dark bands are collagen layers deposited over the life of
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the fish, 1 band per year/annulus. The entire scale cross section length is 25 μm. Each
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collagen layer is approximately 5 μm wide. The dark grey curved lines are artifacts of the
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sectioning process. ........................................................................................................................
7.
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A scanning electron micrograph showing the cross section of a 6-year-old herring scale
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The relationship between muscle and scale δ15N for age-2 () and age-3 (▲) herring
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Depth profile of δ13C in herring scales measured by LA-IRMS. (N) indicates the number of
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laser pulses in each scale at a single location (distal to the first annulus) and is a measure of
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the relative depth in the scale. The left panel shows age-2 herring in the fall () and age-3
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herring in the spring (■). The right panel shows age-3 herring in the fall () and age-4
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herring in the spring (■). ...............................................................................................................
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Tables
1.
The mean (± SD) of 13C in the scales of PWS herring measured by LA-IRMS at three scale
surface locations. ..........................................................................................................................
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Study Chronology
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The original timeline for the study was November 2012 to April 2014. We requested a no-cost extension
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for the project through September 2014 because of technical problems with the LA-IRMS. Progress
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reports July 2013, January 2014, and July 2014 contributed to the final report.
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Introduction
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Stable isotope analysis (SIA) is an important and widely used technique for studying energy flow and
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describing trophic relationships in biological communities. The most common approach involves
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determining the ratio between rare and abundant isotopes of carbon (C) and nitrogen (N) because these
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follow predictable pathways from primary producers to consumers (Peterson and Fry 1987). In general,
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the isotope ratio for 13C/12C (a commonly expressed ratio in terms of its difference from a known standard
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in δ-notation) in consumers approximates that of their diet, with a range of enrichment of ~ 0−1‰
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(DeNiro and Epstein 1978), whereas N (15N/14N) is enriched by about 3.4‰ at each trophic level
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(Minagawa and Wada 1984; Owens 1987). Hence, δ13C has been most often used to identify diet sources
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while δ15N provides an estimation of an animal’s trophic level along a food chain.
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In aquatic ecosystems, SIA has proven useful in determining how natural and fishing-related shifts in
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food webs can affect productivity and stock abundance. The Newfoundland and Labrador marine
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ecosystem, for example, supported important fisheries such as Atlantic cod (Gadus morhua) for nearly
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400 years before a dramatic collapse in late the 1980s and early 1990s (Sherwood et al. 2007; Sherwood
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and Rose 2005). Despite a fishing moratorium, the northern segment of this stock has yet to recover. The
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SIA data indicate the problem is at least partly related to a similar decline in a major prey species, capelin
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(Mallotus villosus), and greater reliance on benthic prey (e.g., shrimp). Moreover, the SIA data revealed
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that the effect on cod recruitment was specific to medium-sized fish, resulting in slower growth, lower
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condition factor, and reduced reproductive potential. Similarly, Jennings et al. (2002) used contemporary
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δ15N data to show how fishing-induced trophic level changes in the North Sea fish community depend
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more on the differential effect on size composition rather than species composition. However, because
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their stable isotope data were contemporary, their use of the relationship between body mass and δ15N to
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characterize the long-term trophic-level trends relied on a number of assumptions, several of which could
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not be subsequently verified. By contrast, Wainright et al. (1993) had access to archived scales for
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retrospective analysis of long-term food web dynamics in the Georges Bank and were able to relate
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isotopic changes δ13C and δ15N for 13 fish species to variation in primary production, particularly the
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importance of diatoms that have a relatively enriched δ13C signal. They were also able to show that
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isotopic variation for some fish species was significantly correlated with both physical factors (North
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Atlantic Oscillation Index, Greenland Regional Pressure Anomaly) and fish mortality. Satterfield and
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Finney (2002) used a similar approach to describe interspecific trophic relationships among five species
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of Pacific salmon and characterize the long-term variation in δ13C and δ15N for one of these species—
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sockeye salmon (Oncorhynchus nerka). Their results showed that the combined δ13C and δ15N values for
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five species clustered into three distinct groups, indicating clear dietary differences. They also found
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highly significant correlations between muscle and scale for δ13C and δ15N (R2 ≥ 0.95), which allowed for
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accurate estimates of δ13C and δ15N over the ~ 35 years of the scale archive. The δ13C data showed only
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minor change over the time period, whereas δ15N was more pronounced with clear inter-decadal variation,
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suggesting changes in primary production and nitrate utilization, or shifts in feeding location. Similar
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relationships have been shown for striped bass (Pruell et al. 2003), whitefish (Perga and Gerdeaux 2003)
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and Atlantic salmon (MacKenzie et al. 2012; Trueman et al. 2012).
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These studies illustrate the value of using SIA to improve understanding of complex ecosystem processes,
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particularly when archive samples, such as scales, are available to examine population dynamics over an
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extended period of time. Laser ablation isotope ratio mass spectrometry (LA-IRMS) offers a means to
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further refine SIA by being able to potentially measure both seasonal and inter-annual stable isotope
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variation within a single scale sample and among individual fish. Among techniques used for sample
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introduction for mass spectrometry, laser ablation has become a standard for high sample throughput,
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multi-elemental and, more recently, light element SIA (Moran et al. 2011). The accuracy and precision of
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determining elemental and isotopic concentrations in fish scales using laser ablation is, however, partly
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confounded by scale architecture (Hutchinson and Trueman 2006). For elemental analyses, this is less
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problematic because the data are ideally obtained from the thin, outer osseous layer composed of
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hydroxapatite, and proper depth profiling with the laser can usually resolve the issue (Hola et al. 2011).
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By contrast, C and N isotopic data are derived from the collagen that forms the basal plate, which grows
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down and outward from the scale focus as a series of concentric overlapping layers. This structure
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increases uncertainty in resolving seasonal and inter-annual isotopic signatures using laser ablation
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because of the potential for the laser to penetrate multiple layers and thereby sample material from
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different time periods. Courtemanche et al. (2005) showed that sagittal sectioning of scales significantly
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improves signal accuracy and precision, but at the cost of added sample preparation. Nevertheless, the use
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of LA-IRMS has the potential to improve understanding of energy flow and trophic-level relationships
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within marine fish populations by enabling more detailed measurement of seasonal and inter-annual
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variation in δ13C and δ15N.
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We tested this technique using scales and muscle from Pacific herring from Prince William Sound
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(PWS). The abundance of Pacific herring in PWS increased in synchrony along with other Alaskan
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herring stocks beginning in the late 1970s before peaking at more than 100,000 tons annually between
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1989 and 1993 (Williams and Quinn 2000; Gray et al. 2002). Although estimates differ, the herring
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population declined significantly thereafter and is presently at levels similar to those observed in the
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1970s. Early studies suggested that the Exxon Valdez oil spill impacted recruitment of the 1989 year class,
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but its effect on the longer term population decline is unclear (Carls et al. 1998, 2001). Other stressors,
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including predation, poor overwinter rearing conditions, and disease within the population, also appear to
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have been important (Norcross et al. 2001; Rice and Carls 2007) and may still be acting as major
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pressures contributing to persistent low population abundance. Among the suspected causes, poor forage
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conditions in the fall may be particularly important because food is generally limited in winter and herring
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rely heavily on stored energy reserves to survive until spring (Foy and Paul 1999). These effects are also
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greater for younger age classes (Paul et al. 1998), indicating that population recovery has been
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constrained, in part, by poor recruitment of juvenile fish. Whether this reflects a persistent, long-term
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change in prey type or abundance is not clear, but studies have suggested that a major factor contributing
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to the lack of recovery in PWS herring is competition or predation by hatchery pink salmon
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(Oncorhynchus gorbuscha) (Deriso et al. 2008; Pearson et al. 2012). More specifically, Pearson et al.
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(2012) noted that although oceanic factors in the Gulf of Alaska (GOA) influence biomass levels of adult
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herring in PWS, the lack of improvement in the condition of these fish at low biomass levels suggests
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other factors are involved, and because juvenile pink salmon and juvenile herring exhibit wide overlap in
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early rearing habitat and food sources, competition could potentially compromise overwintering ability
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and year class strength.
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Interestingly, foraging dynamics for both PWS Pacific herring and pink salmon may be driven by
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mesoscale eddies in the GOA. Kline (2009) identified distinct δ13C signatures in the copepod Neocalanus
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cristatus, indicative of their origins either in the GOA or PWS. Neocalanus is an abundant and ubiquitous
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zooplankter and serves as an important forage species for marine and anadromous fish in the northern
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GOA and PWS. The observed δ13C differences in Neocalanus relate to δ13C values in the diatoms that
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dominate the phytoplankton in PWS (high δ13C) versus the diatom-free, iron-limited species found in the
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GOA (low δ13C). Shifts in the δ13C values in PWS fish from high to low appear to reflect subsidies from
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oceanic production (Kline 2007). Importantly, these shifts have been linked to marine survival for pink
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salmon (Oncorhynchus gorbuscha) in PWS and may be affecting other species such as Pacific herring as
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well, either directly through the food chain, or indirectly by drawing predators away from PWS to more
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productive mesoscale eddies in the GOA (Kline 2010).
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We hypothesized that if Pacific herring abundance was related to productivity associated with GOA
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mesoscale eddies, or alternatively competition with juvenile pink salmon, or both, the signal could be
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detectable through long-term changes in δ13C and possibly δ15N in the scale archive and associated
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historical data that have been collected on stock abundance, age structure, length and weight at age, etc.
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since the early 1970s. To test this hypothesis, however, we needed to establish a calibration metric
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between the isotope values in scales and those in tissues such as muscle that are physiologically relevant.
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For example, seasonal as well as inter-annual differences in δ13C and δ15N may reflect both dietary
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influences as well as nutritional stress. In the fall, δ13C probably depends mostly on the food available
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during the summer from either PWS or oceanic sources because the fish will have spent the last 4–5
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months actively foraging, growing, and storing surplus energy. Moreover, dietary influences on δ13C and
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δ15N have been shown to be affected by both food quantity (Gaye-Siessegger et al. 2004a) and quality
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(Gaye-Siessegger et al. 2004b). By contrast, spring δ13C (and possibly δ15N) values are more likely to be a
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function of their nutritional stress through the winter when food is either absent or greatly reduced
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because lipid and protein catabolism result in δ13C and δ15N enrichment, respectively (Doucett et al.
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1999). Given the high degree of correspondence shown for δ13C and δ15N in scales and muscle in salmon
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(Satterfield and Finney 2002) and haddock (Melanogrammus aeglefinus) (Wainwright et al. 1993), we
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expected to find a similar association in PWS Pacific herring and possibly detect seasonal changes in
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these values using a combined conventional and LA-IRMS approach. These calibration data would then
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provide the basis for a retrospective analysis of herring scales from the Alaska Department of Fish and
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Game’s (ADF&G’s) archive to help identify processes and functional responses that drive long-term food
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web relationships for this species in PWS.
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Project Objectives
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1. Determine the carbon (δ13C) and nitrogen (δ15N) isotope fractionation between the scales (in the
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last year of growth) and muscle of herring (age 2−4) collected during the fall of 2012 and spring
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of 2013 in Prince William Sound by conventional IRMS.
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Muscle and scale samples were obtained from Pacific herring collected by the ADF&G in Port
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Gravina in eastern PWS (60.7 ⁰N, 146.3 ⁰W) during November 2012 and from late March to
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early April 2013. Lipids were removed from muscle samples before analysis, while scales were
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washed in solutions of hydrogen peroxide and deionized water. Samples of both were weighed
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into tin capsules and analyzed for isotopic ratios for δ13C and δ15N with a Thermo Fisher Delta V
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isotope ratio mass spectrometer (see Methods section page 11). We found significant differences
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in muscle δ13C between seasons and for muscle δ15N between age classes. Muscle and scale δ15N
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were significantly correlated for herring collected in both seasons, whereas muscle and scale δ13C
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were correlated only for herring collected in the fall (see Results pages 12–14 and Figures 1–5).
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2. Determine the seasonal (~ spring and fall) variation in δ13C values in scales from these same fish
from the focus to the penultimate annulus using LA-IRMS.
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Annuli on scales used for LA-IRMS were first marked with a micro-scalpel so they could be
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identified with the laser camera after demineralization. The scales were demineralize by washing
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with hydrochloric acid (1.2 M) for 6 minutes, then rinsed with deionized water to remove the
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outer mineralized plate and expose the collagen matrix. The scales were ablated in triplicate at
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three locations on the surface to determine δ13C using a Cetac LSX-too Nd:YAG laser coupled to
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a Thermo Fish Delta V Plus isotope ratio mass spectrometer (see Methods section page 11).
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Additional de-mineralized scales were sampled with the same system to measure δ13C through the
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depth of the scale (i.e., depth profiling). We found a slight but non-significant trend in δ13C
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between the outer margin of the scale and the first annulus in each age class. There were also no
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significant differences within scale locations between herring of different ages or caught in
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different seasons.
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3. Apply the calibration data obtained from Objective 1 to δ13C values from Objective 2 and
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retrospectively estimate (i.e., back-calculate) the seasonal δ13C values in the muscle of herring
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(age 2−4) from 2008 onward.
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We were unable to complete this objective because of the overall lack of variation in scale surface
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δ13C determined by LA-IRMS. Back calculating muscle δ13C from scale δ13C would have resulted
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in equally invariant values for muscle δ13C.
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Methods
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Sample Preparation
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Pacific herring collected by the ADF&G in the fall (November) of 2012 and spring (March–April) of
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2013 were sampled for muscle and scales from a total of N = 100 fish representing two cohorts (brood
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years 2009 and 2010) and two age classes (age-2 and age-3 herring collected in the fall and age-3 and
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age-4 herring collected in the spring). The length and weight of each fish was measured and the sex, if it
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could be determined, was recorded. Muscle samples were taken from the left side, above the lateral line,
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anterior to the dorsal fin. The wet weight of the muscle was recorded and the samples were placed in 50-
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mL Falcon tubes (Corning, Tewksbury, MA, USA), covered with parafilm, and freeze dried.
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Lipids were removed from muscle samples prior to analysis to account for nutritional stress and
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differences in lipid content between herring collected during the spring and fall (Paul et al. 1998). Lipids
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were extracted by chloroform-methanol extraction described by Bligh and Dyer (1959). Briefly, muscle
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samples were placed in glass vials with 2 mL of 10:5:4 methanol:chloroform:water and agitated with a
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glass pipette. The samples were centrifuged for 1 minute and supernatant was then discarded. This
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procedure was repeated a total of three times, after which 2 mL of methanol was added and the sample
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was again centrifuged for 1 minute. The supernatant was then removed and samples were left to dry
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overnight in a fume hood and re-homogenized the following day.
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Scale samples were taken from the left side of each fish from one or both of the preferred areas used by
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the ADF&G for aging. These areas are (1) posterior to the gill plates and dorsal to the pectoral fin, at or
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below the lateral line, and (2) immediately below the dorsal fin at or just below the lateral line. The scales
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were sonicated in 3% hydrogen peroxide for 6 minutes then each was scrubbed for approximately 20
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seconds with an electric brush and triple rinsed with deionized water. After rinsing, the scales were placed
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between glass slides and dried flat.
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For analysis of 13C and 15N on the scale margin, the scale material between the last annulus and outside
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margin was sectioned using a micro-scalpel blade and separated from the rest of the scale. The outer
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margin material from approximately 10 individual scales was needed to obtain enough material for
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analysis. For analysis of wholes scales, each scale was considered a replicated sample. We conducted a
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preliminary test to determine if 13C and 15N on the scale margin were correlated with the respective
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ratios in the whole scale. There was a significant correlation between the scale margin and whole scale for
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13C (P < 0.001, R2 = 0.3) as well as for 15N (P < 0.001, R2 = 0.41), indicating a substantial contribution
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to whole scale 13C and 15N from the most recent period of growth. As a result, the remaining
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comparisons between scale and muscle stable isotopes were conducted with whole scales. In addition,
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because scales are composed of both organic (e.g., collagen) and inorganic (e.g., hydroxyapatite)
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materials, there is the potential for the latter to influence the isotopic composition due to the presence of
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residual amounts of carbonate (Elliot 2002). Studies have shown, however, that the effects of carbonate
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(primarily dissolved inorganic carbon [DIC]) on scale δ13C and 15N are equivocal and appear to depend
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on the species and environment (Sinnatamby et al. 2007; Blanco et al. 2009; Ventura and Jeppesen 2010).
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To account for this potential source of variation, we analyzed both acid-treated and non-treated scales.
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Acid treatment removes the layer of apatite from the surface of the scale. We performed a preliminary test
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on scales from N = 38 fish (9−10 from each age group and season) that were sectioned in half, and half of
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which were demineralized by immersion in 1.2M HCl for 5 min, then analyzed these samples for δ13C
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using conventional IRMS. We found no significant difference in δ13C between the two groups (t = 0.65,
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df = 74, P = 0.52), and the slope (b = 0.61) and intercept ( = -6.24) did not differ significantly from 1.0
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(t = -1.85, df = 36, P =0.07) and 0 (t = -1.83, df = 36, P = 0.08), respectively. This indicated that the δ13C
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contribution from the inorganic carbon fraction in scales was probably limited and not likely to affect
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bulk δ13C analysis, thus all remaining isotope analyses for whole scales were conducted on non-treated
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scales. We also conducted a preliminary analysis of acid-treated and non-treated scales used for LA-
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IRMS. By contrast, the results of this test indicated that the mineralized apatite layer did influence the
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δ13C content of scale by preventing the laser from penetrating to the collagen underneath. This was
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verified by examining acid-treated and non-acid-treated scales with scanning electron microscopy, as
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shown in Figure 1. As a result, all subsequent scales used for LA-IRMS were acid-treated.
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Figure 1. Scanning electron microscope images of non-acid-treated and acid-treated scales analyzed for
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δ13C by laser ablation. Panel A. A non-acid-treated scale at 2,000x. The laser has failed to penetrate
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through the apatite layer and into the collagen layer, except for one small spot. Panel B. Enlarged image
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(8,000x) of the same ablation shows the collagen matrix visible underneath the apatite layer. Panel C.
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Acid-treated scale at 2,000x. The collagen matrix is visible on the surface of the scale because the acid
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treatment has removed the apatite. Panel D. A finer scale image (10,000x) of the same ablation spot
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showing the collagen matrix.
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Isotopic Analysis
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Muscle and bulk-scale samples were analyzed for δ13C and 15N using a Costech Instruments Elemental
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Combustion System (Valencia, CA), coupled to a Thermo Scientific Delta V Plus Isotope Ratio Mass
10
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Spectrometer. The samples were weighed (~ 400 ug) into tin capsules and N = 8 were analyzed in
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triplicate in each analytical run, along with randomly placed replicates of in-house glutamic acid isotope
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standards (PNNL LOW δ13C = -11.09 ‰, δ15N = -8.58 ‰; PNNL MED = δ13C = 16.73 ‰, δ15N =73.58
315
‰). These glutamic isotope standards have been previously standardized for carbon and nitrogen isotope
316
analysis using two glutamic acid isotope standards, USGS40 (δ13C = -26.39 ‰, δ15N = -4.50 ‰) and
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USGS 41 (δ13C = 37.63 ‰, δ15N = 47.60 ‰) (National Institute of Standards and Technology,
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Gaithersburg, MD, USA). A two-point linear correction was applied to the measured δ13C and δ15N
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values. An acetanilide standard (Costech Analytical Technologies, Inc, Valencia, CA, USA; δ13C = -33.77
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‰, δ15N = -1.11 ‰) was used as a secondary standard to examine the effectiveness of the linear
321
correction. Samples were analyzed for both carbon and nitrogen in the same analytical run. Isotope ratio
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values for δ13C and δ15N are reported in per mil notation (‰) and are referenced to the Vienna Peedee
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Belemnite (δ13C) and air N2 (δ15N):
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δR (‰) = [Rsample/Rstandard – 1] x 1000
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Scales for laser ablation were mounted on a glass coverslip with double-sided Scotch tape (3M, St. Paul,
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MN, USA). Four scales were mounted per slide. Samples were ablated using a Cetac LSX-500 Nd:YAG
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laser (Omaha, NE, USA). The ablation system (described in detail in Moran et al. 2011) consisted of the
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laser unit coupled to a micro combustion reactor (Thermo Fisher Scientific, Waltham, MA, USA),
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cryofocusing unit, and a Thermo Fisher Delta V Plus isotope ratio mass spectrometer. Helium was used
330
as a carrier gas to transport the sample to the combustion reactor, to convert organic carbon into CO2.
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Following combustion, the CO2 was trapped with a cryofocusing unit that consisted of a 0.53-mm internal
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diameter fused-silica capillary immersed in a liquid nitrogen bath and a 6-port valve (Valco Instruments
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Co. Inc., Houston, TX, USA). The capillary was immersed in the liquid nitrogen bath for 60 seconds to
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trap the sample before removing the capillary and allowing the sample to flow to the mass spectrometer.
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A total of 10 demineralized scales from each age class were analyzed by LA-IRMS. The scales were
337
ablated in triplicate at three locations on the scale surface: at the outer margin, between the outer margin
338
and last annulus, and distal to first annulus. Samples were bracketed with measurements of a blank and a
339
of 15# test fishing line (Premium Plus 7 monofilament, Danielson, Auburn, WA, USA), which was used
340
as a standard. The value for the 15# test standard (δ13C = -27.71 ‰) was established by Moran et al.
341
(2011). Measured δ13C values were corrected using blank measurements and 15# test values. Nylon
342
thread (Wonder Invisible Threads, YLI Corp, Rock Hills, SC, USA) was used as a secondary standard to
343
examine the efficacy of the correction, the isotopic value of which was established by Moran et al. (2010)
344
(δ13C = -28.71 ‰). The scales and standards were ablated using a 50-µm spot at a frequency of 10 Hz,
345
and power settings of 40% and 80%, respectively.
11
346
347
Isotope values for both conventional and LA-IRMS data were checked for normality and equality of
348
variances for each sample site, sex and age class. Analysis of variance (ANOVA) was used to test for
349
differences in δ13C and δ15N between seasons, sexes, and age classes, or in the case of non-parametric
350
data, ANOVA by ranks (Kruskal-Wallis). Season and age class were treated as fixed effects. Significant
351
differences among age classes were tested by Tukey-Kramer honestly significant differences (HSD) post
352
hoc test to determine which means differed. The potential effect of fish length on δ13C and δ15N was
353
tested by least squares regression. For significant regressions the residuals were used in place of the
354
observed isotopic ratios.
355
356
Results
357
Conventional IRMS in Muscle and Scales
358
Lipid extraction resulted in significant (P ≤ 0.001) enrichment in muscle δ13C and δ15N in herring
359
collected in both spring and fall. Muscle δ13C increased by ~ 0.5‰ and muscle δ15N by ~ 1.0‰ (data not
360
shown). Two-way ANOVA of this lipid-extracted muscle indicated that season, but not age, had a
361
significant effect on muscle δ13C (P ≤ 0.001). However, there was also a significant interaction between
362
season and age (P = 0.034). Overall, herring collected in the spring were more enriched in muscle δ13C
363
than herring collected in the fall of the year (Figure 2), and age-3 fish in the fall were more enriched in
364
δ13C than age-2 fish. By contrast, age did have a significant effect on muscle δ15N (P = 0.007), whereas
365
season did not (P = 0.98). Age-3 herring in the fall were significantly enriched (P ≤ 0.045) in muscle δ15N
366
compared to younger fish in the same season. We also tested for differences in muscle δ15N between male
367
and female fish in the spring, but found no effect due to sex (P = 0.84). Moreover, although there were
368
significant differences in length among age groups, linear regression indicated that length did not have a
369
significant effect on muscle δ15N within (P ≥ 0.13) or across age groups (P = 0.57).
12
-18.0
-18.5
-19.0
-19.5
a
a
b
-20.0
-20.5
Muscle  15N (‰)
Muscle δ13C (‰)
14.5
b
b
a
b
13.5
a
13.0
-21.0
Fall
370
14.0
Fall
Spring
Spring
371
Figure 2. The mean (± SD) 13C (left panel) and 15N (right panel) in the muscle of age-2 (white bar) and
372
age-3 (black bar) herring in the fall compared to age-3 (white bar) and age-4 (black bar) herring in the
373
spring. Bars without letters in common are significantly different from each other (P  0.05). Letters
374
within bars indicate significant differences between age classes within seasons, whereas letters above bars
375
indicate significant differences within age classes between seasons.
376
377
Scale 13C did not differ between seasons or age classes (Figure 3), whereas age had a significant effect
378
on scale 15N (P = 0.03). Scales were more enriched in δ15N for older herring compared to younger fish in
379
both seasons even after accounting for the effect of length on scale δ15N (P < 0.001, R2 = 0.24).
1.0
-15.8
b
-16.0
-16.2
-16.4
Resideual Scale  15N
Scale  13C (‰)
-15.6
-16.6
0.5
0.0
b
a
a
b
-0.5
-1.0
Fall
Spring
Fall
Spring
380
381
Figure 3. Comparison of the mean (± SD) 13C (left panel) and 15N (right panel) in the scales of age-2
382
(white bar) and age-3 (black bar) herring sampled in the fall to age-3 (white bar) and age-4 (black bar)
383
herring in the spring. Bars without letters in common are significantly (P  0.05) different. Values for
384
scale 15N are residuals after regression on length.
385
13
The relationship between muscle and scale δ13C is shown in Figure 4. Muscle and scale δ13C were
387
significantly correlated (P = 0.006, R2 = 0.18) for herring collected in the fall, but not for herring collected
388
in the spring (P = 0.28, R2 = 0.02). Carbon was enriched in scales compared to muscle by ~ 2‰. A similar
389
significant relationship shown in Figure 5 was found for muscle and scale δ15N for herring collected in the
390
fall (P = 0.002, R2 = 0.18), but in contrast to δ13C, muscle and scale δ15N were also correlated for herring
391
collected in the spring (P = 0.002, R2 = 0.18). Moreover, in both seasons muscle δ15N was enriched
392
compared to scale δ15N by ~ 2 ‰.
-17.5
-17.5
-18.5
-18.5
Muscle δ13C (‰)
Muscle δ13C (‰)
386
-19.5
-20.5
-21.5
-18
-17
-16
13
Scale δ C (‰)
-15
-19.5
-20.5
-21.5
-18
-17
-16
Scale δ13C (‰)
-15
393
394
Figure 4. The relationship between muscle and scale δ13C for age-2 () and age-3 (▲) herring collected in
395
the fall (left panel) and age-3 () and age-4 (■) herring in spring (right panel) in PWS.
16
Muscle  15N (‰)
Muscle  15N (‰)
16
15
14
13
11
12
396
13
14
Scale 15N (‰)
15
14
13
11
15
12
13
14
Scale  15N (‰)
15
397
Figure 5. The relationship between muscle and scale δ15N for age-2 () and age-3 (▲) herring collected
398
in the fall (left panel) and age-3 () and age-4 (■) herring in spring (right panel) in PWS.
399
400
LA-IRMS in Scales
401
Studies have shown that scale architecture can confound time-resolved analyses of δ13C because the data
402
are derived from the collagen that forms the basal plate, which grows down and outward from the scale
403
focus as a series of concentric overlapping layers (Hutchinson and Trueman 2006). For LA-IRMS this can
404
increase the uncertainty in obtaining accurate and precise δ13C measurements in scales because of the
14
405
potential for the laser to penetrate multiple layers and thereby sample material from different time
406
periods. A potential solution is to measure δ13C in a transverse section of the scale in a manner analogous
407
to that described by Courtemanche et al. (2005). As a preliminary test of this approach, we embedded,
408
cut, and measured the architecture of a scale in cross section using electron microscopy. We found that
409
the scale from a 6-year-old herring captured in April 2012 had a cross section thickness 25 μm (Figure 6),
410
and an approximate width of an individual collagen layer of 5 μm. Moreover, during method
411
development, we determined that a laser beam diameter of 50 μm is needed to ensure enough material is
412
ablated to provide an adequate IRMS signal size. Based on these findings, we concluded that a transverse
413
section of the scale would not provide sufficient resolution to measure δ13C by laser ablation and thus all
414
remaining scales were ablated on the surface plane.
415
416
Figure 6. A scanning electron micrograph showing the cross section of a 6-year-old herring scale caught
417
in April 2012. The light and dark bands are collagen layers deposited over the life of the fish, 1 band per
418
year/annulus. The entire scale cross section length is 25 μm. Each collagen layer is approximately 5 μm
419
wide. The dark grey curved lines are artifacts of the sectioning process.
420
421
Analysis of discrete locations on the surface of demineralized herring scales by season revealed little
422
variation in δ13C between age classes, seasons, or scale locations (Table 2). Although there was a slight
423
trend toward enrichment of δ13C from the outer margin to the first annulus, differences within age classes
424
and (or) seasons were not significant (P ≥ 0.44). In fact, the range in δ13C across both seasons and age
15
425
groups was only 0.6‰ or about 20−25% of the range observed in δ13C for scales and muscle measured by
426
conventional IRMS.
427
428
Table 1. The mean (± SD) of 13C in the scales of PWS herring measured by LA-IRMS at three scale
429
surface locations.
Season
Fall
Spring
430
431
Scale location
Age class Outer margin Outer margin-last annulus Distal to 1st annulus
2
-17.7 (0.6)
-17.5 (0.5)
-17.4 (0.7)
3
-17.7 (1.5)
-17.4 (1.4)
-17.1 (1.2)
3
-17.4 (0.5)
-17.4 (0.7)
-17.4 (1.0)
4
-17.6 (0.8)
-17.2 (0.8)
-17.1 (1.0)
By contrast, depth profiling of scales from each age class and season indicated wide variation in δ13C
433
(Figure 7). Although δ13C increases by ~ 1−2‰ from the scale surface (Laser pulse 1) through pulses 5-6,
434
it becomes rapidly depleted as material is obtained at greater depths in the scale. The highly depleted
435
values near the basal portion of the scale may reflect the influence of the adhesive used to mount the scale
436
for ablation, which we measured at ~ -30‰. Interestingly, however, the herring collected in the fall
437
appear more enriched in δ13C than the herring collected in the spring, particularly as the depth of the δ13C
438
measurement increases.
-15
-15
-17
-17
-19
-19
δ13C ‰
δ13C ‰
432
-21
-23
-23
-25
-25
0
439
-21
2
4
6
Laser pulse (N)
8
10
0
2
4
6
Laser pulse (N)
8
10
440
Figure 7. Depth profile of δ13C in herring scales measured by LA-IRMS. (N) indicates the number of laser
441
pulses in each scale at a single location (distal to the first annulus) and is a measure of the relative depth
442
in the scale. The left panel shows an age-2 herring in the fall () and an age-3 herring in the spring (■).
443
The right panel shows an age-3 herring in the fall () and an age-4 herring in the spring (■).
444
16
445
Discussion
446
The goal of this project was to determine if carbon (δ13C) and nitrogen (δ15N) isotope ratios in the muscle
447
and scales of PWS Pacific herring co-varied in a manner that would permit a retrospective analysis of the
448
ADF&G herring scale archive to identify potential processes that drive long-term food web relationships
449
for this population. We employed conventional IRMS and LA-IRMS to respectively measure isotopic
450
variation among and within individual herring. Conventional IRMS revealed significant seasonal and age
451
class differences in both muscle (δ13C and δ15N) and scale (δ15N only) isotope ratios and a significant
452
correlation between these tissues for both δ13C and δ15N. LA-IRMS, however, indicated only minor and
453
non-significant inter-annual variation in δ13C within individual herring, but potentially large δ13C
454
variation linked to scale architecture and (or) seasonal feeding dynamics. Herring caught in the spring
455
during the spawning season were enriched in muscle δ13C by ~ 1‰ compared to herring in the fall,
456
despite the fact that lipids were extracted from the muscle prior to analysis. Lipids are depleted in δ13C
457
compared to other tissues (DeNiro and Epstein 1977), and thus spawning or otherwise nutritionally
458
stressed herring should be enriched in δ13C assuming this tissue fraction is the primary source of energy at
459
this time. The enrichment in δ13C in the muscle of spawning herring in the spring compared to non-
460
spawning fish in the fall is consistent with results reported for other species, e.g., Atlantic salmon, during
461
their spawning migration (Doucett et al. 1999; Sinnatamby et al. 2008), as are seasonal changes in δ13C
462
that have been observed in young-of-the-year and age-1 herring in PWS (T. Kline, personal
463
communication). Equally interesting, however, is that age-3 herring in the fall were more enriched than
464
age-2 herring. Although this may also be due to differences in nutritional status, it may similarly reflect
465
variation in the relative contribution of differing dietary sources. The whole body energy content of age-2
466
herring in PWS in the fall increases significantly from that of age-1 herring, despite the fact that these fish
467
exhibit limited growth during their third year (Paul et al. 1998). This suggests a possible transition in diet
468
from that younger herring to age-3 fish. Age-3 herring may have access to a wider range of prey with
469
differing δ13C than that available to age-2 herring, and if these sources are more enriched in δ13C this
470
could account for observed differences between age-2 and age-3 herring in the fall. This suggestion is
471
supported by the similar difference in muscle δ15N between age-2 and age-3 herring in the fall. In contrast
472
to δ13C, which is generally used to identify dietary sources, δ15N provides an estimation of an animal’s
473
trophic level along a food chain, as well as other nutritional effects (Doucett et al. 1999). Differences in
474
δ15N between age classes in the fall could indicate different sources of food, possibly a shift from
475
herbivorous to carnivorous zooplankton. Kline (1999) found that carnivorous zooplankton in PWS had
476
the highest δ15N, which is consistent with access to a wider range of prey in age-3 herring.
477
17
478
Scales also differed significantly in relation to season and age, but only for δ15N. The scales of age-4
479
herring in the spring were enriched with 15N by ~ 0.5‰ compared to age-3 herring in the fall, but scales
480
were depleted by ~ 1.0‰ in both seasons and age classes compared to muscle. This result has been
481
generally observed in other studies (Satterfield and Finney 2002; Perga and Gerdeaux 2003; Kelly et al.
482
2006; Sinnatamby et al. 2008) and presumably reflects the high glycine content of collagen in scales
483
(Ikoma et al. 2003), which is depleted in 15N relative to bulk food by ~ 1.4‰ (Hare et al. 1991). The
484
glycine content in scales also affects 13C and likely contributes to the difference of ~ 3.5‰ that we found
485
between scale and muscle δ13C. These offsets between scale and muscle δ13C and δ15N were relatively
486
uniform among individual herring for both age classes and seasons and thus provided a significant
487
correlation between scale and muscle isotope ratios. These correlations are important because they will
488
allow us to retrospectively extrapolate muscle δ13C and δ15N from the scale archive for PWS herring held
489
by ADF&G. Related studies have shown similar although generally higher correlations between scale and
490
muscle δ13C and δ15N (R2 > 0.5) (Satterfield and Finney 2002; Perga and Gerdeaux 2003; Pruell et al.
491
2003), but the range for δ13C and δ15N was either much greater (~ 10−20 ‰) or conducted on species such
492
as striped bass (Pruell et al. 2003) that would not likely experience the nutritional stress reported for PWS
493
herring (Paul et al. 1998). The results of these studies illustrate the utility of using scale archives to
494
identify long-term trends in feeding ecology by demonstrating either cyclic or directional changes in scale
495
δ13C and δ15N over 20−30 year time frames. Whether similar trends are apparent in the scales of herring
496
from PWS remains to be seen, but annual and age-specific sample sizes with sufficient statistical power
497
should allow for identifying such trends if they exist.
498
499
In contrast to the scale δ13C results derived from conventional IRMS, those obtained by LA-IRMS were
500
equivocal. Scales grow by underplating, whereby the collagen that forms the basal plate grows down and
501
outward from the scale focus to form a series of concentric overlapping layers (Hutchison and Trueman
502
2006). Each of these layers extends to just below the surface of the scale where it comes in contact with
503
the outer mineralized layer. Acid treatment removes this mineralized layer (Ventura and Jeppesen 2010),
504
which presumably would expose the underlying concentric layers and make them available for LA-IRMS.
505
Our results showed limited variation in δ13C across the scale surfaces regardless of age class (< 0.5 ‰),
506
whereas depth profiling revealed variation of δ13C within scales of ~ 8−10 ‰, which was 4-5 times the
507
range of δ13C we observed in bulk analyses by conventional IRMS. As noted above, the highly depleted
508
values probably result from the influence of the adhesive used to mount the scale. This is partly because
509
of the close agreement in conventional (mean = -16.7 ‰, SD = 0.5) and laser ablation (mean = -17.6 ‰,
510
SD = 0.9) IRMS analyses for δ13C in the outer margin of the scales, and because the outer margin and
511
basal portion of the scale presumably reflect the most recent growth and thus should have similar δ13C.
18
512
We had also shown in a previous report (January 2014) that reversing the scale orientation did not
513
materially affect the depth profile δ13C result. Interestingly, however, the older herring in the spring from
514
both cohorts are generally more depleted in δ 13C with increasing scale depth (i.e., laser pulse), which
515
suggests that scales in these fish may be undergoing some resorption. Scales serve as reservoirs for
516
calcium and are resorbed during periods of high calcium demand or dietary restriction (Carragher and
517
Sumpter 1991; Armour et al. 1997). Moreover, if lipids cannot entirely supply the overwintering and
518
spawning energy requirements in PWS herring, it is possible that protein, including scale collagen, could
519
also be metabolized to meet these demands. Protein catabolism during spawning or periods of dietary
520
stress should result in δ15N enrichment (Doucett et al. 1999), and although this did not occur in muscle,
521
there was a significant increase in scale δ15N between fall age-3 and spring age-4 herring (Figure 3). This
522
supports the suggestion that herring scales may undergo resorption during the winter and spring because
523
of dietary stress, which would also account for the general lack of δ 13C variation across discrete time
524
periods of scale formation.
525
526
Conclusions
527
In summary, our study provides evidence that muscle and scale δ13C and δ15N differ in relation to age and
528
season in PWS herring when measured by conventional IRMS, and that these isotopic ratios are also
529
correlated between the tissue types. These correlations are a necessary condition for examining the
530
ADF&G 40-year scale archive to determine if PWS herring experienced a major shift in their feeding
531
ecology during this time period that might account for the lack of recovery in the biomass of this
532
population. Conversely, we found limited variation in δ13C in scales measured by LA-IRMS. This
533
finding, along with the observed enrichment in scale δ15N in the spring, suggests that herring scales may
534
undergo resorption during periods of dietary restriction or spawning, obviating the utility of LA-IRMS to
535
identify discrete life history shifts in δ13C within scales of individual fish. As a result, we did not attempt
536
to back-calculate muscle δ13C from scale δ13C in the herring analyzed by LA-IRMS. Additional research
537
is needed to determine if dietary stress from spawning or overwinter fasting leads to resorption and
538
remodeling of δ13C within scales.
539
540
Management Implications
541
The abundance of Pacific herring in PWS remains near historic lows despite a fishing moratorium for
542
more than 20 years. Several hypotheses have been advanced to account for the lack of recovery including
543
potential competition from juvenile pink salmon. In addition to natural production of pink salmon in
544
PWS, hatcheries annually release approximately 700 million juvenile pink salmon that may compete for
545
food with young-of-the-year herring. If these releases are shown to be acting as a constraint on herring
19
546
recruitment, stakeholders and resource managers may need to weigh the costs and benefits of these
547
programs and determine if current levels of salmon production are congruent with the potential recovery
548
of the herring population.
549
550
Publications
551
In preparation: Age and season-dependent effects on stable isotope ratios in the muscle and scales of
552
Pacific herring (Clupea pallasi) from Prince William Sound, Alaska. Proposed journal: Fisheries
553
Management and Ecology. Projected date of submission: November 2014.
554
555
Outreach
556
Conference Presentations
557

558
Fact sheet
559

560
Poster at the Alaska Marine Science Symposium 2013, by J. Moran, and T. Linley
Informational pamphlet produced for distribution at presentations in Cordova (attached)
Community Presentations
561

Cordova High School (2) September 2014, by M. Nims
562

Prince William Sound Science Center (PWSSC) September 2014, by M. Nims
563
564
Radio broadcast

Interview at the PWSSC for Field Notes September 2014, by M. Nims
565
566
Acknowledgements
567
We thank Krystin Riha and Kathleen Carter for their support and assistance with the IRMS, the Alaska
568
Department of Fish and Game (Rich Brenner, Steve Moffitt) for providing the herring samples, and the
569
Prince William Sound Science Center for their help with community outreach. Funding for this study
570
was provided by the North Pacific Research Board.
571
572
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573
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