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Otolith age validation

Best methods for insuring ageing accuracy in otoliths, whether in support of large-scale production ageing or a small-scale research project.

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Releasing known-age and marked fish

Releasing known-age and marked fish into the wild is a rigorous age validation methods, as the absolute age of the recaptured fish is known without error. Since the released fish are generally less than 1 year old, recaptured fish will have spent the majority of their lives in natural surroundings.

You can mark fish:

We’ve used this method to successfully confirm absolute age and growth increment formation at both the daily and the yearly scale. However, this approach isn’t well suited to long-lived species, since recapture rates of old fish tend to be minimal. Also, this method can’t be used on species which can’t be reared in captivity prior to release.

There are 2 variations on this method which make it more widely available at the expense of relatively minor assumptions.

Determining age with scale annuli

The first variation involves removing scales when tagging and releasing wild fish.

You can estimate the absolute age of the fish by estimating the age at tagging (using the removed scale) added to the time at liberty. This works in situations when:

Where the age at tagging is short compared to the time at liberty, this approach allows us to know the wild tagged fish’s age at release. Therefore, we don’t need to rear them in captivity.

Estimating age with fish size

The second variation involves tagging young fish and approximating age based on fish size.

This approach was used to study bluefin tuna. The tuna were estimated to be 1 to 3 years old at the time of tagging and were subsequently recaptured up to 15 years later. There was more or less than a 1 year margin of error around the age estimate at the time of tagging. However, that margin was too small to change the conclusion that vertebral growth marks were formed annually after tagging.

Bomb radiocarbon

Bomb radiocarbon derived from nuclear testing provides one of the best age validation approaches available for long-lived fish.

The onset of nuclear testing in the late 1950s resulted in an abrupt increase in atmospheric carbon-14. This was soon incorporated into organisms that were growing at the time, including:

Thus, the period is analogous to a large-scale chemical tagging experiment where all otolith cores of fish hatched:

Interpretation of carbon-14 chronology

The interpretation of the carbon-14 chronology in a sample of otolith cores is relatively simple. The otolith-based carbon-14 chronology spanning the 1960s should match other published carbon-14 chronologies for the region:

Any under-ageing would phase shift the otolith carbon-14 chronology towards more recent years. Over-ageing would phase shift it towards earlier years. Otolith contamination with material of more recent origin can only increase the D carbon-14 value, not decrease it.

The otolith D carbon-14 value sets a minimum age to the sample, and the years 1958 to 1965 become the most sensitive years for D carbon-14-based ageing. You can use bomb radiocarbon to confirm the accuracy of more traditional ageing approaches with an accuracy of at least more or less 1 to 3 years.

The discriminatory power of samples born before or after this period is more than an order of magnitude lower. The carbon-14 signal recorded in deep-sea and freshwater environments is different from that of surface marine waters. Deep-sea signal is delayed and fresh water is advanced. Therefore, you must use reference of carbon-14 chronologies appropriate to the environment experienced during the period of otolith core formation.

Disadvantages and advantages

This approach isn’t well suited:

However, the low radioactive decay rate of carbon-14 implies that both archived and recent collections are appropriate for assay.

Mark-recapture of chemically-tagged wild fish

Mark-recapture of chemically-tagged wild fish is one of the best methods available for validating the periodicity of growth increment formation. The method is based on rapid incorporation of calcium-binding chemicals into fish:

The chemicals used when tagging include:

You can apply the tag through:

However, injection is the most practical method for tagging studies of wild fish.

The result is a permanent mark, visible under fluorescent light (except strontium), in the growth increment being formed at the time of tagging. The number of growth increments formed distal to the chemical mark is then compared to the time at liberty after tagging.

This approach has been used to validate annulus formation in a wide variety of structures and species, including:

We’ve also successfully used this approach at the microstructural level, validating daily increment formation in a variety of tuna species.

Advantages and disadvantages

A major advantage of this approach is that the growth increments being validated are formed while the fish is growing in a natural environment. Experiments in which fish are chemically-tagged and then reared in the laboratory or an outside enclosure are less optimal, although they’re logistically easier to carry out.

A disadvantage of the chemical tagging approach is that the number of increments formed after tagging is often low. This results in a potentially large relative error if an increment (such as that at the growing edge) is misinterpreted. For example misinterpretation of a:

Long-term mark-recaptures have detected problems with annulus identification that weren’t evident from short-term recaptures. For this reason, fish tagged at a young age and recaptured at an old age provide the most robust validation results.

With this method you can only validate growth increment formation for the size/age of fish tagged. However, it’s still a powerful method and one of the few you can use with adult wild fish.

Radiochemical dating

Radiochemical dating of otoliths is based on the radioactive decay of naturally occurring radioisotopes which are incorporated into the otolith during its growth.

When you incorporate radioisotopes into the otolith they decay into radioactive daughter products. These are themselves retained within the acellular crystalline structure.

The ratio between the known (and fixed) half-lives of the parent and daughter isotopes is an index of elapsed time since incorporation of the parent isotope into the otolith.

By restricting the assay to the extracted otolith core (as opposed to the whole otolith) you can make objective and accurate estimates of absolute age.

The isotopic concentrations requiring measurement are exceedingly low. This results in assay precisions which are often less than optimal, although recent methodological changes have substantially improved precision.

Current discriminatory power is on the order of 5 year for 210Pb:226Ra and 1 to 2 year for 228Th:228Ra. This is over age ranges of 0 to 40 and 0 to 8 years, respectively.

This approach is best suited to long-lived species where the candidate age interpretations are widely divergent, such as in sebastes or hoplostethus.

Progression of discrete length modes sampled for age structures

We haven’t rigorously applied progression of discrete length modes sampled for age structures. However, it’s a reasonably robust approach for validating the interpretation of annuli in young fish. If you monitoring the progression of discrete length modes across months within a year, it’s relatively straightforward to determine if the modes correspond to age classes.

Absolute age is confirmed in instances where:

You can then use the examination of the ageing structures sampled from those same modes to test the validity of the presumed annuli as age indicators. This was the basis of the approach that found good correspondence between the:

This approach isn’t equal to the more common practice of observing discrete length modes in a single sample and assuming each to correspond to an age class. While such an approach provides corroboration for an age interpretation, there isn’t any independent evidence that the length modes represent age classes. Therefore, an approach that doesn’t track modal progression through the year doesn’t validate either absolute age or annulus periodicity.

In principle, sampled modal progression should also be applicable to daily age validation. However, in practice size-selective mortality and/or migration is often pronounced in young fish, thus invalidating the assumption that a distinct cohort is being tracked.

Capture of wild fish with natural, date-specific markers

Capturing wild fish with natural, date-specific markers is an approach that has many of the same advantages and disadvantages of bomb radiocarbon dating. They both rely on large-scale events that apply dated marks to all fish in a population.

In the specific (and rare) instances where you can apply this method, you can validate growth increment formation over a substantial portion of a fish's life history. This method has been used to determine:

However, such natural marks would generally:

Physiologically-generated marks

A related but different approach is to take advantage of physiologically-generated marks or checks on salmonid ageing structure, such as:

This can be a powerful validation method of either absolute age or increment periodicity, as long as the:

With the salmonid experiment, an observer would note the date of emergence of a specific fish from the gravel. This emergence check became a dated mark on the otolith of that fish. We could then use the mark to validate both the absolute age and the frequency of formation of the daily increments formed until the capture date.

This method is probably better suited to daily increment validation than to annulus validation, since hatch checks and settlement marks are common in some groups of fish. Nevertheless, analogous marks do exist in many older fish, such as the otolith transition zone associated with the onset of sexual maturity.

In all cases, a key requirement is the independent observation of the date of the physiological event. Without it, the check is associated with an age, but not a date, of formation.

Marginal increment analysis

Marginal increment analysis is the most commonly used, and the most likely to be abused, of age validation methods.

The underlying premise as a method for validating increment periodicity is sound. It states that if a growth increment is formed on a yearly (daily) cycle, the average state of completion of the outermost increment should display a yearly (daily) sinusoidal cycle when plotted against season (time of day).

The popularity of this method can be attributed to its modest sampling requirements and low cost. However, in many ways, it’s one of the most difficult validation methods to carry out properly. This is due to the technical difficulties associated with viewing a partial increment affected by variable light:

The absence of an objective means of interpreting the data further complicates the situation. In a review of 104 annulus seasonality studies, about 30% of the species from a given region formed annuli at times different than that of the other species. It’s possible that annuli didn’t form in all of these species, or that the time of opaque zone formation varied widely among species.

We have a lack of understanding of the mechanisms underlying annulus formation. However, a more likely explanation is that this technique itself was of low resolving power.

Even more problematic are studies that attempt to validate daily increment formation with marginal increment analysis, working near the resolution limit of light. This is confounded by the presence of subdaily increments.

There is some merit to daily marginal increment analysis based on transmission electron microscopy or using otoliths with unusually broad increments. However, in general, studies of daily increments are of questionable value.

Differentiation from edge analysis

Marginal increment analysis is sometimes differentiated from edge analysis, but when used as a validation method, has similar properties.

The marginal increment is usually calculated as a proportional state of completion, ranging from:

When you plot it as a function of month or season, the mean marginal increment should describe a sinusoidal cycle with a frequency of 1 year in true annuli.

Edge analysis doesn’t assign a state of completion to the marginal increment, but rather records its presence as either an opaque or translucent zone. The change in relative frequency of each edge zone is plotted across months or seasons. As with marginal increment analysis, the cycle frequency should equal 1 year in true annuli.

In both marginal increment analysis and edge analysis, a yearly cycle of formation can be difficult to distinguish from other frequencies. This contributes to their poor performance as validation methods. Changes in the seasonal timing of the marginal increment with age or location undoubtedly contribute to the problem. Significant and unexplained differences among years have also been noted.

Despite their problems, both are well suited for determining the month or season of formation of the opaque or translucent zone once annulus formation has been validated through independent means.

Reasons for misleading results

There are several reasons why marginal increment analysis may provide misleading results. Prominent among these is the fact that the marginal increment is most easily discerned in young, fast-growing fish. They’re at a life history stage where the marginal increment may accurately confirm the formation of annual increments.

The problem arises later, when you apply these validation results to older fish contrary to the assumptions of all age validation methods. You can use marginal increment analysis and achieve age validation on of young fish but find incorrect ages in older fish.

More troublesome is validating age based on marginal increment analysis of young fish and using this method for routine ageing of the species across all age groups. This happened when using scales in young snapper quickly became the basis for all scale ageing of the species in several countries. The mark-recapture results later showed that scale ages underestimated true age in older fish.

This also happened in the northeast Pacific, where all routine ageing of sablefish by several countries was based on scales validated with marginal increment analysis. When the otolith mark-recapture studies were completed, scientists realized that scale ages were underestimating the age of older fish by up to a factor of 4.

Marginal increment analysis misuse isn’t restricted to scale ageing. Annuli in whole otoliths of redfish were validated this way, and subsequently became an accepted procedure of many organizations for ageing these long-lived fish. Subsequent validations have demonstrated that whole otoliths grossly underestimate age in older fish.

The conclusion is clear. When proper age validation studies are lacking, researchers will often seize upon any available studies which can corroborate their age interpretations. Since marginal increment analysis is restricted to young, fast-growing fish, it’s also the most likely to lead to serious ageing error when applied blindly.

Validation protocol

Marginal increment analysis results are valid if done with sufficient rigor. Aspects of a rigorous protocol include:

In other words, the validation results should be considered to be age-specific.

Captive rearing

Captive rearing is generally discounted as a reliable means of validating annulus formation, but maintains some utility at the daily level.

Laboratory environments are seldom able to mimic natural environments, due to their:

Since annulus formation is strongly influenced by the environment, an artificial environment is likely to produce artificial annuli.

Daily growth increments are much less affected by environmental conditions, due to the endocrine-driven endogenous rhythm which controls their formation. Laboratory environments are well known for resulting in daily increments of altered appearance. However, the frequency of their formation isn’t an issue unless the rate of growth is unnaturally low.

For this reason, laboratory experiments to confirm daily increment formation of known-age or chemically-marked fish are common.

Providing more natural rearing environments

You can provide improved and more natural rearing environments for validation studies, including:

For otolith microstructure studies in particular, outdoor rearing can be expected to produce daily increments which are quasi-natural in appearance and frequency. However, growth rates can be artificially high in hatchery operations.

At the annual level, outdoor rearing can also be expected to produce more natural-looking growth structures. However, we haven’t determined if annuli produced under such conditions are equivalent to those of wild fish.

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