Introduction. Shortraker rockfish (Sebastes borealis) occur over the continental slope .... The shortraker rockfish otolith sections contain many fine dark growth.
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Using Radiometric Ages to Develop Conventional Ageing Methods for Shortraker Rockfish (Sebastes borealis) Charles E. Hutchinson, Craig R. Kastelle, and Daniel K. Kimura
NOAA Fisheries, Alaska Fisheries Science Center, Seattle, Washington
Donald R. Gunderson
University of Washington, School of Aquatic and Fisheries Sciences, Seattle, Washington
Abstract Whole otoliths from a previous radiometric ageing study were thin sectioned using a modified preparation technique. Growth rings on the thin sections were counted using three different ageing strategies with different levels of banding (i.e., grouping fine growth zones). Ages generated from the different ageing strategies were compared with the radiometric ages to select the “best” ageing strategy. A comparison of ageing strategies and radiometric ages suggests that the “best” strategy had a transition age of approximately 20 years below which fine growth zones should be grouped into bands, and above which finer marks should be counted. Otoliths from additional specimens were thin sectioned and aged using this “best” strategy, and also radiometrically aged. Using the “best” ageing strategy, von Bertalanffy growth parameters for female shortraker rockfish were estimated to be (L ∞ = 83.2 cm., K = 0.04, and t0 = 4.68 years). These parameters, plus the length at maturity were used to estimate an age range at 50% sexual maturity of between 18 to 28 years which is similar to the probable transition age of 20 years.
Introduction Shortraker rockfish (Sebastes borealis) occur over the continental slope of the North Pacific ocean, from Japan, the Okhotsk Sea, southeast-
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Hutchinson et al.—Ageing Methods for Shortraker Rockfish
ern Kamchatka, to the Bering Sea and Aleutian Islands, and south to California, at depths of 25 to 875 m. Rockfish (Sebastes spp.) are an important commercial species in the North Pacific because of their high market value with multiple species being harvested either directly or indirectly as retainable incidental catch. To ensure better management, it is important to understand rockfish life history characteristics such as longevity, growth, and age at maturity. Otoliths are the most common structure used for fish ageing. Otoliths are good structures to use because they are made of a hard material called aragonite, a form of calcium carbonate crystal, which stands up to different preparation techniques and the deposition of otolith material appears to occur each year without resorption (Chilton and Beamish 1982). A standard preparation for reading otoliths is the break and burn method (Christensen 1964). In this method the otolith is snapped or sawed through its core on the transverse plane and then burned over an alcohol flame. The burning darkens the slow growth (dark translucent) zone distinguishing it from the fast growth (light opaque) zone when viewed under a dissecting microscope with reflected light. Ageing is accomplished by counting these growth zones. A set of guidelines or ageing criteria is used to help age the fish. Radiometric ageing has been used to validate ageing criteria for rockfish species such as splitnose rockfish (S. diploproa), rougheye rockfish (S. aleutianus), Pacific ocean perch (S. alutus), northern rockfish (S. polyspinus), and yelloweye rockfish (S. ruberrimus) (Bennett et al. 1982, Kastelle et al. 2000, Andrews et al. 2002). This method measures the disequilibrium of daughter/parent isotope pairs found in otolith cores to independently determine age. The Alaska Fisheries Science Center’s (AFSC) Age and Growth Program has been able to age a number of different species using the break and burn method. However, shortraker rockfish has not been aged because of the difficulty of interpreting the growth zones and the presence of “glassy areas” (Fig. 1) on the burnt half of the otolith. In the “glassy areas” the growth zone are obscured. Therefore no ageing criteria have been set for shortraker rockfish at the AFSC. The goal of this study was to obtain an otolith preparation method to elucidate growth patterns, to examine three very different ageing strategies, and determine the best ageing criteria from these strategies. This was accomplished by modifying an existing thin section technique to generate ages that could be compared to 210Pb /226 Ra radiometric ages. Validated ages generated by the thin section method were then used to estimate von Bertalanffy growth parameters which were applied to length at maturity data (McDermott 1994) to estimate age at maturity for female shortraker rockfish. We provide detailed information of the methods used because these are new methods for obtaining ageing criteria using radiometric results.
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Figure 1. Photograph of a shortraker rockfish break and burn otolith half with presence of a “glassy area” highlighted within the box outline. In the “glassy areas” the growth zones are obscured.
Materials and methods Shortraker rockfish thin sectioning A production thin sectioning method used at the Central Ageing Facility in Queenscliff, Victoria, Australia was adapted for use with shortraker rockfish otoliths (Smith et al. 1995). Using a pencil, otoliths were lightly marked transversely through the middle of the first year. Otoliths were embedded in resin blocks using a polyester resin (i.e., Artificial Water [trade name not necessarily endorsed by NOAA]). A bottom layer of resin for the blocks was poured into silicone molds and allowed to cure for 40 minutes. Marked otoliths were then placed on the semi-cured resin which had been scored. In this way the otoliths were lined up evenly in a row and did not lose alignment by sinking into the resin. Another layer of resin was applied encasing the otoliths in resin and allowed to cure for 2 days. The block was cut using a high speed Tyslide saw that made thin sections approximately 0.4 mm thick. Three to five thin sections were cut from each specimen to ensure that a thin section contained a cross section of the first year. Next the thin sections were mounted on
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a glass slide using a UV curing adhesive, Loctite 349®. The adhesive was cured by exposure to UV light for 20 minutes. Finally the mounted thin sections were reduced in thickness by grinding on a Hillquist® grinding wheel to a thickness of 0.2 mm.
Development of age determination strategies The shortraker rockfish otolith sections contain many fine dark growth zones punctuated by occasional major or thick dark growth zones when viewed under reflected light. Thus, the question for the age reader was to determine which zones represent yearly growth. Previous results for orange roughy (Hopostethus atlanticus) indicate that a transition age occurs when a fish’s somatic growth slows due to the production of reproductive material as a fish matures, and is usually close to the age at 50% maturity (Francis and Horn 1997). Three age determination strategies were developed: strategy 1 focused on counting major growth zones or lumping together of finer growth zones. If only the major growth zones were counted, the age reader obtained a young age. Strategy 2 focused on counting fine growth zones and if all of the fine growth zones were counted, the age reader obtained a much greater age. Strategy 3 was a combination of strategies 1 and 2 and included a “transition age,” below which fine growth zones should be grouped into bands, and above which finer growth zones should be counted. We used radiometric ages to determine which strategy was best.
First radiometric experiment: determination of transition age The first experiment involved ageing the remaining whole otoliths from a radiometric ageing study conducted by Kastelle et al. (2000). The otoliths were collected from the 1993 AFSC summer trawl surveys. Kastelle et al. (2000) used radiometrics to age seven length categories of shortraker rockfish since no ages (i.e., ages that were generated by the break and burn method) were available. To help establish the ageing criteria for shortraker rockfish, the remaining otoliths from that radiometric study were thin sectioned as previously described. The thin sections from each of these length categories were then aged (i.e., counting growth zones) using strategy 1 and strategy 2 and the ages averaged for each length category used in the radiometric study. The averaged thin section age was compared to the average radiometric age for each length category (Table 1). To compare the previous radiometric ages (Kastelle et al. 2000) with the possible strategy 3 ageing criteria, we posited that a transition age occurred at each of the strategy 1 ages for each length category (Table 1). A matrix of estimated strategy 3 ages, for each assumed transition age and length category combination, was calculated using the follow-
36.0
30.8
51.3
57.3
56.2
92.6
61.0
76.0
81.7
83.7
25.3
23.2
22.6
13.7
117.0
79.6
58.8
59.1
63.1
51.2
65.0
Strategy 2 average age
37.4
20.8
-0.3
-4.0
11.9
-13.8
0.0
Kastelle et al. (2000).
3,308
79.5
42.1
21.3
21.6
25.6
13.7
8.5
23.2
8.5
3,942
76.5
39.1
18.3
18.6
22.6
13.7
8.5
3,060
81.1
43.7
22.9
23.2
22.6
13.7
2,796
83.5
46.1
25.3
23.2
22.6
13.7
8.5
c
25.3
Estimated strategy 3 age
22.6
4,364
63.5
26.1
25.3
23.2
22.6
13.7
8.5
26.1
Posited transition age for strategy 3 13.7
6,721
36.0
26.1
25.3
23.2
22.6
13.7
8.5
36.0
c
Estimated ages for the length groups for a given transition age are derived by adding the strategy 2 incremental increases to the transition age starting with next larger length group after the transition age.
b
a
60.5
23.1
2.3
2.6
6.6
-5.3
8.5
8.5
10,080
Sum of squared (SS) residuals between radiometric ages and posited transition ages.
SS residuals a
97.0
26.1
36.6
43.0
8.5
22.4
40.0
Strategy 1 average age
Radiometric ageb
Ave length (cm)
Incremental increase in strategy 2 ages
Table 1. Radiometric age, strategy 1 and 2 ages, and strategy 3 ages based on different posited transition ages. Arranged by length groups used by Kastelle et al. (2000).
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Hutchinson et al.—Ageing Methods for Shortraker Rockfish
ing procedure: first, an incremental increase in strategy 2 ages was calculated for each length category. Next, from the diagonal the lower left half of the matrix was filled by adding the incremental increase to the assumed transition age and then to each following strategy 3 age. In this way, the strategy 3 ages in successive length categories, once above the assumed transition age, were increased by an amount equal to the incremental increase of strategy 2 ages. Finally, for the upper right half of the matrix, the estimated strategy 3 age can not be above the strategy 1 age by definition when using the strategy 1 age as the assumed transition ages. For example, for the 43 cm length grouping, these specimens were found to have an average of 13.7 major growth zones using strategy 1 and an average of 51.2 fine growth zones using strategy 2. For the next length category 61 cm, strategy 2 gave an average fine growth zone count of 63.1. For strategy 3, if we posited a transition age of 13.7 years, the estimated average age for the 61 cm category fish would be 13.7 + (63.1 − 51.2) = 25.6 years. The value 63.1 − 51.2 = 11.9 shows up in Table 1 under the category “incremental increase in strategy 2 ages,” and 25.6 shows up under the strategy 3 ages with an assumed 13.7 year transition age for the 61 cm length category. Negative incremental ages used in the calculations were the result of sampling variability, averaging the strategy 2 ages from each group of fish, and ageing error. The sum of squared residuals was calculated between the radiometric and the estimated strategy 3 ages for each of the assumed transition ages (Table 1). A parabola was fit to the sum of squares with the assumed transition age as the independent variable. The lowest point on the parabola was solved using calculus to obtain a least squares estimate of the transition age, which yielded thin section ages closest to the radiometric ages. Using the least squares estimate of the transition age as a guideline, the original radiometric sample was reaged. Prior to reaging the thin sections, the specimen identification numbers were randomly rearranged to prevent the reader’s knowledge of previous ages obtained using strategies 1 and 2 from affecting results. The reader did not have access to any sample data, including fish size. Apparent transition ages were recorded. Upon completion of re-ageing the radiometric sample, recorded apparent transition ages were averaged.
Second radiometric experiment: testing strategy 3 A new set of otoliths was used to test strategy 3 with the averaged apparent transition age. For this part of the experiment, otoliths from summer trawl survey cruises were used with samples collected from areas around the Aleutian Islands. Otolith data collection and storage methods followed standard AFSC procedures. One otolith from each fish was thin sectioned and aged using strategy 3. The remaining otoliths were used for radiometric analysis.
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In this radiometric analysis, we used otolith material laid down in the first 3 years of the fish’s life (cored otoliths). Previous radiometric studies have shown that young fish or cored otoliths are less likely to violate assumptions associated with radiometric ageing. Because approximately 1 gm of otolith material was needed for each sample, this meant that otoliths of the same nominal age needed to be grouped into pools. Samples were chosen for three pools (designated SR1, SR2, and SR3) based on age availability, experimental design, and time concerns. The initial step in the radiometric work for the pooled otoliths was to core (i.e., remove all material beyond the third year) each otolith. This was accomplished by mechanically removing the material using a Buehler® low speed cutting saw and grinding wheel. The cored otolith size was determined by taking measurements of the first 3 years on whole and thin-sectioned otoliths from samples being processed for radiometric analysis. Early growth zones were distinguishable in shortraker rockfish otoliths, especially when layers were first ground off of the distal side in the coring process. The visual location of the third year served as a coring guide in addition to the third year measurements. Cores were rinsed with distilled water, dried, and weighed. Cores were then stored in a clean, acid-washed vials containing 60% ethanol until further processing. The samples were cleaned and processed to measure the activity of 226Ra and 210Pb as described in Kastelle et al. (2000). The measured 210Pb/226Ra in the otoliths can be used to predict a radiometric age from the curve: A2 = 1 − exp ( − λ2t + R* exp ( − λ2t A1
)
)
Here A1 is the activity for 226Ra, A2 and λ2 are the activity and decay constant, respectively, for 210Pb, t is time (age in years), and R* is the initial ratio of 210Pb/226Ra incorporated into the otolith cores. In this study, two R* values were used: an assumed value of R* = 0.0, and from rougheye rockfish, R* = 0.0636 (Kastelle et al. 2000). Sources of error associated with the radiometric ages came from counting statistics involved in measuring decays of radionuclides, reagent blanks, background measurements, and yield tracers. Errors were propagated through all calculations to estimate errors in radiometric age. The radiometric ages from the pooled otoliths were adjusted to take into account the time between date of capture and date of analysis and average age of material in the 3 year core.
Estimation of age at 50% sexual maturity All female shortraker rockfish from the 1993 radiometric study and females (37 cm or less) from the 2000 AFSC cruises were used to estimate age at 50% sexual maturity. Otoliths were aged using strategy 3
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Hutchinson et al.—Ageing Methods for Shortraker Rockfish
with the averaged apparent transition age. A total of 150 specimens were used: 60 from the 1993 cruise and 90 from the 2000 cruise. Female shortraker growth parameters were determined from the von Bertalanffy growth equation:
{
(
Lt = L∞ 1 − exp −k t − t 0
)}
Here Lt is the length at age t, L ∞ is the theoretical length at age infinity, t0 is the theoretical age at length zero and k is the growth rate. The von Bertalanffy curve was solved for t to estimate age at 50 % sexual maturity: L t50 = t 0 − (1 / k ln 1 − 50 L∞
)
Here, t50 is the estimated age at 50% sexual maturity using the length at 50% sexual maturity (L50), previously estimated to be 44.9 cm by McDermott (1994). The likelihood method (Kimura 1980) was used to estimate von Bertalanffy growth parameters, and to estimate the covariance matrix of parameter estimates. The delta method (Seber 1973) was used to construct 95% confidence levels for age at 50% female sexual maturity.
Results First radiometric experiment: determination of transition age Comparison of ageing strategies to the radiometric ages in the first radiometric experiment yielded the following results: strategy 1 (major or thick growth zones) gave ages that were younger than the radiometric ages. Strategy 2 (fine growth zones) gave ages that were older. Strategy 3 produced ages closest to the radiometric ages. Strategy 3 ages included the averaged apparent transition age. The least squares estimate of the transition age was obtained from the calculated sum of squared residuals between the radiometric and the strategy 3 ages (Table 1) was 23±3.2 years (±2 SE). The average apparent transition age was 20.7 years.
Second radiometric experiment: testing strategy 3 Estimated radiometric ages for pooled sample SR1, for both initial ratios, were younger than average thin section ages. Estimated radiometric ages for pooled samples SR2 and SR3, for both initial ratios, were older than average thin section ages (Table 2). The 95% confidence intervals for mean radiometric age ranges for both initial ratios included the mean thin section ages for all three samples (Fig. 2) except for SR3 with the initial ratio of 0.0.
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Table 2. Comparison of average ages for SR1, SR2, and SR3 samples from the thin section (using strategy 3) and radiometric ageing methods. Ninety-five percent confidence intervals are shown for radiometric ages. Ave thin section age (yr)
Initial ratio
SR1
20
0.0000
SR2
30
SR3
20
Pools
Radiometric age (yr) 18.3
95% Cl ±5.9
0.0636
16.2
±5.9
0.0000
59.8
±36.4
0.0636
57.7
±36.4
0.0000
33.2
±12.9
0.0636
31.1
±12.9
Estimation of age at 50% sexual maturity Growth parameters and age at 50% sexual maturity were estimated for female shortraker rockfish (Table 3). The age at 50% sexual maturity of female shortraker rockfish ranged from 21.4 to 23.2 years.
Discussion Growth zones in the otoliths of shortraker rockfish are difficult to interpret. The light and dark growth zones may have irregular spatial patterns that bring into question whether they are annual marks. The otoliths exhibit major or thick dark growth zones that can be split into finer dark growth zones. This is seen from the nucleus to the edge on the reading surface of a shortraker rockfish otolith. Experienced age readers know that for short-lived species such as Pacific cod, only major marks need to be counted (Roberson et al. 2002). However, for long-lived species such as shortraker rockfish, counting only major marks may not be the correct strategy because once growth slows fine marks could represent annual zones (Beamish 1979). The “correct strategy” might be to count major marks up to a “transition age,” and then count fine marks past this point. A unique (and difficult) aspect of shortraker rockfish otoliths is that occasionally major marks are visible past a transition age. The problem for the age reader is to determine the location of this transition age when there are several apparent possibilities in the pattern of growth zones. The comparison between thin section ages and radiometric ages from a previous radiometric study dismissed strategy 1 (counting only major marks) and strategy 2 (counting all fine marks) and found strategy 3 to be best (incorporating an averaged apparent transition zone around 20 years).
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Table 3. Von Bertalanffy growth parameters and age at 50% sexual maturity for female shortraker rockfish. Samples
N
L ∞ cm
k
t0
50% maturitya
95% CI
1993 sample
60
83.2
0.042
4.68
23.22
±4.40
2000 sample
90
69.1
0.043
–2.89
21.55
±4.12
150
84.6
0.030
–3.62
21.42
±3.60
Combined
All samples were aged using the thin section method and strategy 3 with the averaged apparent transition age.
a
The second radiometric experiment performed on new samples was designed to test the observed transition age. The agreement between the radiometric ages and the thin section ages supported the hypothesis of a transition age of around 20 years. The pool of older fish (SR2) strengthened the ageing criteria by extending ages past the transition age and confirming that all growth zones should be counted after the transition age. Although there was fair agreement between radiometric and thin section ages using strategy 3, with the transition age of around 20 years, caution should be observed. Confidence intervals for radiometric ages can encompass a wide age range. The radiometric analysis that tested the transition age was based on only three pools and limited age ranges were used. More testing is needed to examine the accuracy of the ageing criteria over a wider age range and to study the possibility of underestimating ages. One limitation of radiometric ageing is that otolith cores need to be pooled and these results yield an average age rather than an individual age. As fish age, the variability in length at age may increase and length may become less correlated with age. Therefore, the pooled fish analyzed by Kastelle et al. (2000), which were length-based, may represent a broad age range. This study suggests that the initial ratio (R*) of 0.0 produced slightly better agreement than 0.0636. The use of an initial ratio of 0.0 can be rationalized because previous rockfish radiometric studies have found initial ratios that were close to 0.0 or assumed a value of 0.0 (Bennett et al. 1982, Campana et al. 1990, Fenton and Short 1995, Kastelle et al. 2000, Andrews et al. 2002). Age at 50% maturity was determined from the 1993 and 2000 samples and also from the samples combined, indicating that age at maturity is approximately 22 years. The reduction in somatic growth is related to narrower spacing between translucent growth zones on the otolith. This study used ages generated by strategy 3 to estimate age at 50% sexual maturity. Although this is somewhat circular, the estimated
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100
SR1 90
SR1 SR2 SR2
80
SR3 SR3
Radiometric ages
70
60
50
40
30
20
10
0 0
10
20
30
40
Thin section ages
Figure 2. Radiometric ages for SR1, SR2, and SR3 for initial ratios (R*) of 0.0 (shown in clear symbols) and 0.0636 (shown in solid symbols) plotted against thin sections ages. The error bars on the x-axis represent thin section age range in the samples. The y-axis error bars represent two standard errors for the radiometric ages. Point estimators for each sample on the x axis are offset (by 1 year) for a clearer view of error bars. Dark solid line represents line of agreement.
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Hutchinson et al.—Ageing Methods for Shortraker Rockfish
Figure 3. Thin section of shortraker rockfish aged using Strategy 3. First small dot represents transition age. Large dots indicate major or banded zones and small dots indicate fine growth zones.
age at 50% sexual maturity is consistent with the averaged apparent transition age of 20.7 years. Under strategy 3 ageing, the transition age in the otolith growth pattern signals a shift in otolith growth indicated by a reduced distance between dark or translucent growth zones. The comparison between radiometric and thin section ages indicated that a transition zone was present around the age of 20 years. This was useful because a clear demarcation between fast and slow growth patterns was lacking in many otolith specimens. This result provided a rationale for using a 20 year transition age when the transition age was not apparent. Although specimens could be found that exhibited apparent transition zone ages between 10 to 30 years (Fig. 3), most transition zone ages occurred around 20 years. The criteria became more subjective when the transition year was not apparent. This will lead to a lower precision between age readers. Munk (2001) aged shortraker rockfish up to 157 years, while the oldest age we generated using strategy 3 was 102 years. A future goal will be to work with other age readers to establish a set of ageing criteria that will produce ages with reasonable precision.
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