Influence of DNA isolation from historical otoliths on nuclear-mitochondrial marker amplification and age determination in an overexploited fish, the common sole (Solea solea L.)

Historical otolith collections are crucial in assessing the evolutionary consequences of natural and anthropogenic changes on the demography and connectivity of commercially important fish species. Hence, it is important to define optimal protocols for purifying DNA from such valuable information sources while avoiding any damage to the physical structure of the otolith. Before being able to conclude on the harmlessness of a method, it is important to validate protocols on different kinds of otoliths by testing purification methodologies under standardized conditions. Here we compare the effect of two DNA extraction methods on the success in identifying the age in an overexploited marine fish, the common sole (Solea solea L.). To ensure optimal future population genetic and demographic analyses, we assessed DNA quantity and tested the DNA quality by investigating the amplification success of a mitochondrial and nuclear marker. Our results show that the choice of the DNA extraction method had a significant effect on the success of using these otoliths in age and growth analyses. Standard commercial and published protocols resulted in a severe damaging of the otolith structure, hampering accurate preparation and analyses of the morphological structures of the otoliths. Shortening the lysis time and lowering the EDTA (ethylene diamine tetraacetic acid) and SDS (sodium dodecylsulphate) concentration turned out to be beneficial for the stability of otolith structure, while maintaining an overall high DNA quality measured through polymerase chain reaction amplification success. We therefore recommend that care should be taken when choosing the extraction method for a molecular study on archived samples, in order to enable the maximal use of information embedded in historical material.     

Age validation,and comparison of otolith,vertebra and opercular bone for estimating age of <Emphasis Type="BoldItalic">Schizothorax o’connori</Emphasis> in the Yarlung Tsangpo River,Tibet

A collection of 514 Schizothorax o’connori was made between August 2008 and August 2009 from Yarlung Tsangpo River to assess the suitability of three bony structures for age estimation. The annulus characteristics of otolith, vertebra and opercular bone were described. Location of the first annulus was validated by daily growth increment (DGI) analysis in the otoliths. Annual periodicity was verified by marginal increment ratio (MIR) analysis in otoliths and edge analysis in vertebrae and opercular bones. Annuli formed, once a year, between March and May for all three bony structures. Otoliths, vertebrae and opercular bones were examined to determine which structure produced the most precise and accurate age estimates in S. o’connori. Vertebrae and otoliths matched closely for the first 21 years of life, while opercular bones appeared to underestimate age. For older fish, the counts diverged and otoliths consistently providing higher age estimates. Sectioned otoliths proved to be the most precise and accurate structure for age estimation. The oldest observed schizothoracine fish was 50, more than twice the longevity previously accepted in S. o’connori.     

Comparison of otolith and scale readings for age and growth estimation of common dentex Dentex dentex

Three methods of age estimation were compared for Dentex dentex. Based on sectioned otoliths, scales appeared to be relevant only up to 5 years and whole otoliths up to 12 years. The maximum estimated age was 36 years, which constitutes to date the oldest age reported.     

Accuracy and precision in eel age estimation, using otoliths of known and unknown age

Using otoliths of eels Anguilla anguilla of known and unknown age from fresh, brackish and marine waters, the accuracy of age estimation was uncertain and deviations from the correct age were dependent on reader, locality and fish age.     

Evaluating the effectiveness of teeth and dorsal fin spines for non‐lethal age estimation of a tropical reef fish,coral trout Plectropomus leopardus

This study investigated whether teeth and dorsal fin spines could be used as non‐lethal methods of age estimation for a vulnerable and highly valued tropical fisheries species, coral trout Plectropomus leopardus. Age estimation of individuals from 2 to 9 years old revealed that dorsal spines represent an accurate ageing method (90% agreement with otoliths) that was more precise [average per cent error (APE) = 4·1, coefficient of variation (c.v .) = 5·8%] than otoliths (APE = 6·2, c.v . = 8·7%). Of the three methods for age estimation (otoliths, dorsal spines and teeth), spines were the most time and cost efficient. An aquarium‐based study also found that removing a dorsal spine or tooth did not affect survivorship or growth of P. leopardus. No annuli were visible in teeth despite taking transverse and longitudinal sections throughout the tooth and trialling several different laboratory methods. Although teeth may not be suitable for estimating age of P. leopardus, dorsal spines appear to be an acceptably accurate, precise and efficient method for non‐lethal ageing of individuals from 2 to 9 years old in this tropical species.     

The growth ofOreochromis andersonii (Pisces: Cichlidae) from the Okavango Delta,Botswana, and a comparison of the scale and otolith methods of ageing

Synopsis Otoliths and scales were used for age and growth determination ofOreochromis andersonii from the Okavango Delta, Botswana. Marginal increment analysis showed that an annulus was formed in both the scales and otoliths during the dry summer period. Using scales, the growth ofO. andersonii was described by L_t = 285.27(1-e^{-0.26(t+2.02)}) mm SL and using otoliths by the equation L_t = 267.48(1-e^{-0.25(t+2.18)}) mm SL. Maximum age estimates of 10 years using scales and 13 years using otoliths were obtained and the growth curves were significantly different (p < 0.01). Age estimation using scales tended to over-emphasise growth inO. andersonii resulting in larger predicted lengths-at-age. For this reason, otoliths are considered to be more reliable and suitable than scales in determining the age and growth of this species.     

Suitability of glycerin-preserved otoliths for age validation using bomb radiocarbon

The effect of glycerin storage on the bomb radiocarbon content of otoliths was determined experimentally. Storage in either pre- or post-bomb glycerin had no detectable effect on the bomb radiocarbon content of either pre- or post-bomb otoliths. Therefore bomb-dated age validation studies need not be restricted to freshly-collected samples or dry otoliths, implying that the large numbers of glycerin-archived otoliths around the world are suitable for age validation studies using bomb radiocarbon.     

Seeking the true time: Exploring otolith chemistry as an age-determination tool

Fish otoliths' chronometric properties make them useful for age and growth rate estimation in fisheries management. For the Eastern Baltic Sea cod stock (Gadus morhua), unclear seasonal growth zones in otoliths have resulted in unreliable age and growth information. Here, a new age estimation method based on seasonal patterns in trace elemental otolith incorporation was tested for the first time and compared with the traditional method of visually counting growth zones, using otoliths from the Baltic and North seas. Various trace elemental ratios, linked to fish metabolic activity (higher in summer) or external environment (migration to colder, deeper habitats with higher salinity in winter), were tested for age estimation based on assessing their seasonal variations in concentration. Mg:Ca and P:Ca, both proxies for growth and metabolic activity, showed greatest seasonality and therefore have the best potential to be used as chemical clocks. Otolith image readability was significantly lower in the Baltic than in the North Sea. The chemical (novel) method had an overall greater precision and percentage agreement among readers (11.2%, 74.0%) than the visual (traditional) method (23.1%, 51.0%). Visual readers generally selected more highly contrasting zones as annuli whereas the chemical readers identified brighter regions within the first two annuli and darker zones thereafter. Visual estimates produced significantly higher, more variable ages than did the chemical ones. Based on the analyses in our study, we suggest that otolith microchemistry is a promising alternative ageing method for fish populations difficult to age, such as the Eastern Baltic cod.     

An assessment of otoliths,dorsal spines and scales to age the long‐finned gurnard,Lepidotrigla argus,Ogilby, 1910 (Family: Triglidae)

Sagittal otoliths, dorsal spines and scales were critically assessed as structures to potentially determine the age of the long‐finned gurnard, Lepidotrigla argus. Counts were made of opaque growth increments and a readability score was assigned to each structure. Comparisons of growth increment counts were made between structures and between readings. All three structures showed some degree of readability and quantifiable growth increments, but this varied within fishes and between structures. Initial results showed that whole otoliths were more suitable to determine age estimates than dorsal spines and scales. Scales were considered unsuitable due to between reading ageing bias, variation in age estimates between structures, low precision and poor readability for this species. Dorsal spines showed evidence of loss of growth increments due to hollowing of the vascular core, which resulted in underestimation of older individuals in comparison to whole otoliths. Further analysis showed that growth increment counts from whole otoliths were lower for older individuals in comparison to sectioned otoliths. It is suggested that this is because of decreased clarity of growth increments towards the outer margin of whole otoliths in older individuals; this problem was not present with sectioned otoliths. It was concluded that sectioned otoliths were a more suitable structure from which to estimate age of L. argus than were whole otoliths, dorsal spines and/or scales.     

New data on the longevity of coastal cod Gadus morhua Linnaeus, 1758 in the White Sea

The cod, Gadus morhua, is a common and abundant demersal fish in the White Sea coastal zone. The published data on the maximum age of White Sea cod still requires additional documentation, because some authors have noted difficulties in age determination by otoliths of large specimens. To obtain accurate data on the longevity of this species in the White Sea, an image analysis of thin‐sections of otoliths was made for age estimation. Research surveys in the Chupa Inlet and adjacent waters of the Kandalaksha Bay were conducted in June to August from 2007 to 2013. The five largest specimens of a total of 3564 captured fish were selected for age determination. Age of these largest individuals ranged between 7 and 12 years, total length and weight varied from 60.2 to 77.0 cm and from 2.4 to 6.1 kg, respectively. According to the data, maximal White Sea cod longevity is greater than in previously published data, and demonstrates similar longevity to conspecifics from the Baltic and North seas, whereas its life cycle is much shorter than cod from the Northeast Arctic, Iceland, Greenland, Newfoundland and Labrador stocks.     

An evaluation of the otolith characteristics of Conger conger during metamorphosis

A sample of 20 metamorphosing conger eel Conger conger leptocephali were collected from the Minho River, Portugal, in February 1999 and their sagittal otoliths were analysed by scanning electron microscopy. Four different etching agents were applied along both sagittal and frontal sections during otolith preparation to examine the microstructural growth in this species. Otolith growth increments were visible throughout the increment countable zone using all four treatments, but a permanent peripheral diffuse zone, where the daily increments were unclear, appeared on all otoliths, preventing accurate age estimation. To understand more about the nature of the diffuse zone, otoliths of 10 other metamorphosing leptocephali reared in aquaria were marked by immersion in tetracycline hydrochloride. The distance between the fluorescent marks and otolith edge, measured over a fixed period of time, was used to estimate the otolith growth rate. The application of this technique led to an anomalously high estimated otolith growth rate, probably as a result of the capture, marking and handling stress.     

Otolith annulus validation and variables influencing false annuli formation in dorsal spines of saugeye

Marginal increment analysis is a common technique for validating formation of a single annual growth ring on an ageing structure. False annuli can form on ageing structures when environmental variables affect growth of a fish, potentially resulting in age estimation bias. Therefore, validating ageing structures is essential to ensure that accurate and precise age estimates are collected when assessing fish population dynamics. Saugeye (Sander vitreus, [Mitchill, 1818]) and S. Canadensis, [Griffith and Smith, 1834]) are highly valued sport fish that are stocked across the Midwest United States. Using marginal increment analysis, we confirmed that a single annulus was formed yearly in otoliths of juvenile saugeye, however two annuli formed in dorsal spines in a single year. Timing of the first annulus formation in both otoliths and dorsal spines was completed after a slow growth period during winter (otoliths forming in April; dorsal spines forming in March). The second annulus (false annulus) that formed during August in dorsal spines did not form in otoliths. To understand what environmental factors may influence the false annulus to form, we collected monthly water temperatures and percent empty stomachs of juvenile saugeye. The highest water temperatures of the year occur during July and August, which resulted in saugeye seeking thermal refuge and affecting feeding habits. Mean monthly temperature and percent empty stomachs were positively correlated, so during times of high temperatures foraging rates declined, suggesting the formation of false annulus on dorsal spines of juvenile saugeye. This study demonstrates how thermal stress affected accuracy of non‐lethal aging structures and further emphasizes the need for age validation studies prior to using non‐lethal ageing structures to estimate age for a particular species from different aquatic systems.     

Analysis of the soluble matrix of vaterite otoliths of juvenile herring (Clupea harengus): do crystalline otoliths have less protein?

Otoliths are calcium carbonate concretions laid down in the inner ear of fish and used in fish age estimation. Otoliths precipitate in the form of aragonite but aberrant precipitation may result in vaterite formation instead of aragonite. Vaterite otoliths are more translucent than aragonite. The quantity of HCl-soluble proteins (SP) was measured in the vaterite otoliths and their aragonite pairs of one year old reared herring Clupea harengus to assess the changes induced by the precipitation of vaterite in the amount of soluble proteins in the otolith. Results showed that vaterite otoliths had as much soluble proteins as their aragonite pairs (p>0.05). Due to the lower density of the vaterite, vaterite otoliths were lighter than their aragonite pairs (p<0.05) which explained that protein concentrations were significantly higher (p<0.05) than in aragonite otoliths. These results indicate that the precipitation of vaterite in otoliths did not affect the inclusion of soluble proteins. Furthermore, they suggest that soluble proteins do not affect the translucent or opaque appearance of otoliths. Differences in translucency may instead be caused by the amounts of insoluble proteins or by differences in the physical properties of proteins. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of the otolith proteins revealed two bands at 50 and 62 kDa in both aragonite and vaterite otoliths suggesting that the precipitation of vaterite in the otolith is not controlled by either of these two proteins present in the otolith.     

Validation of daily increment formation in otoliths for three Diplodus species in the Mediterranean sea

The efficacy of two fluorochromes, chlorhydrate of tetracycline (CHTC) and alizarin complexone (ALC), to induce a label on the otoliths of juvenile sparid fishes by immersion techniques was tested in different experimental conditions. CHTC did not mark otoliths in natural sea water and was toxic in divalent cation-free sea water. Immersing fish in a 50-mg 1⁻¹ ALC natural seawater solution for 24 h induced a well-defined mark on the otoliths and had no effect on survival. A daily periodicity of increment formation on otoliths was observed in captivity for Diplodus vulgaris and D. puntazzo , and was conserved in natural environment for D. sargus . The frequency of increment deposition did not vary with the age of juvenile fishes. Thus, otolith microstructure analysis will be a reliable method to give age estimates in these three sparid species during their juvenile life.     

Aging three-spined sticklebacks Gasterosteus aculeatus: comparison of estimates from three structures

In order to assess the accuracy and reliability of age estimates from calcified structures in the three-spined stickleback Gasterosteus aculeatus, we evaluated intra and inter-reader repeatability from three structures: otoliths, gill covers and pelvic spines). Average age estimates were also compared between the structures. The overall intra-reader repeatability of age estimates were highest for otoliths (69%), lowest for gill covers (53%) and intermediate for spine cross-sections (63%). Although four of the seven readers had the highest intra-reader repeatability score for spine cross-sections, the inter-reader variance in this structure was much higher than in others. Otoliths were the easiest in terms of their pre-analysis treatment and exchange of materials (as digital images) between readers. In addition, otoliths are more well-studied compared with the other structures with respect to their development through ontogenesis; hence, age estimates based on otoliths should be the most reliable. Therefore, our recommendation is that whenever possible, analysis of otoliths should be the preferred approach for aging G. aculeatus.     

A reliable method for ageing of whiting (Merlangius merlangus) for use in stock assessment and management

Accurate age estimation is important for stock assessment and management. The importance of reliable ageing is emphasized by the impending analytical assessment of whiting (Merlangius merlangus) in the Baltic Sea. Whiting is a top predator in the western Baltic Sea, where it is fished commercially although less extensively compared to the North Sea. Although the species is considered one of the most difficult gadoids to age, few efforts have been made to shed light on the ageing problems. The aim of the present study was to identify and validate the 1st winter ring and to examine the visibility of the subsequent winter rings. Microstructure analysis was used to confirm the 1st winter ring. Additionally, otolith growth trajectories were obtained, confirming the allometric growth as seen in many fish species. The method for ageing of whole otoliths presented in this study can be directly implemented in future ageing of whiting otoliths from the Baltic Sea – and potentially also adjacent areas where the conspecifics have similar growth rates.     

Using the otolith sulcus to aid in prey identification and improve estimates of prey size in diet studies of a piscivorous predator

Diet studies are fundamental for understanding trophic connections in marine ecosystems. In the southeastern US, the common bottlenose dolphin Tursiops truncatus is the predominant marine mammal in coastal waters, but its role as a top predator has received little attention. Diet studies of piscivorous predators, like bottlenose dolphins, start with assessing prey otoliths recovered from stomachs or feces, but digestive erosion hampers species identification and underestimates fish weight (FW). To compensate, FW is often estimated from the least affected otoliths and scaled to other otoliths, which also introduces bias. The sulcus, an otolith surface feature, has a species‐specific shape of its ostium and caudal extents, which is within the otolith edge for some species. We explored whether the sulcus could improve species identification and estimation of prey size using a case study of four sciaenid species targeted by fisheries and bottlenose dolphins in North Carolina. Methods were assessed first on otoliths from a reference collection (n = 421) and applied to prey otoliths (n = 5,308) recovered from 120 stomachs of dead stranded dolphins. We demonstrated in reference‐collection otoliths that cauda to sulcus length (CL:SL) could discriminate between spotted seatrout (Cynoscion nebulosus) and weakfish (Cynoscion regalis) (classification accuracy = 0.98). This method confirmed for the first time predation of spotted seatrout by bottlenose dolphins in North Carolina. Using predictive models developed from reference‐collection otoliths, we provided evidence that digestion affects otolith length more than sulcus or cauda length, making the latter better predictors. Lastly, we explored scenarios of calculating total consumed biomass across degrees of digestion. A suggested approach was for the least digested otoliths to be scaled to other otoliths iteratively from within the same stomach, month, or season as samples allow. Using the otolith sulcus helped overcome challenges of species identification and fish size estimation, indicating their potential use in other diet studies.     

Otolith size and location in digestive tracts of northern fur seals (Callorhinus ursinus): Implications for dietary interpretations

Walleye pollock (Theragra chalcogramma) otoliths (n= 2,706) recovered from stomachs, small intestines, and colons of 43 northern fur seals (Callorhinus ursinus) were evaluated for size and wear by location in the digestive tract. Pollock fork length was regressed on otolith length after correction for erosion, and age was estimated from the calculated body size. Age‐1+ pollock otoliths (≥6.3‐mm length) were concentrated in stomachs while age‐0 otoliths (≤6.2‐mm length) were concentrated in colons. Less than 10% of otoliths were found in the small intestines. Pollock age decreased with progression along seal gastrointestinal tracts. Otolith quality increased along gastrointestinal tracts in numbers ≥20, which was typical of age‐0 otoliths recovered from colons. Otolith distribution by age and quality along gastrointestinal tracts suggests that small (≤12 cm) schooling prey are consumed in large volume and passed as a bolus rapidly through the digestive tract before significant erosion of bony remains occurs; while larger prey are eaten in smaller volume and subjected to otolith erosion due to longer retention in the stomach. Our results illustrate the importance of multiple sampling strategies to comprehensively represent prey size in pinniped diet.     

用鳞片和耳石鉴定鲫年龄的比较研究

两观测者独立观测了分别采自洪湖的175尾和洞庭湖的168尾鲫样本的鲜片和耳石,结果显示鳞片适于鉴定年龄组成简单、生长较快的洞庭湖鲫种群的年龄,两观测者年轮读数的总吻合率可达90.5%,与耳石上年轮读数的吻合率也可高达91.7%;但用鳞片鉴定年龄结构复杂、生长缓慢的洪湖鲫种群年龄,两观测者总吻合率只有50.9%,各龄组吻合率随年龄上升而迅速下降。与耳石上年轮读数总吻合率也仅为56.6A%,存在比耳石低估高龄个体年龄的问题,用耳石鉴定鲫年龄具有易于识别、精确度高的优点,用之鉴定洪湖和洞庭湖鲫种群年龄,两观测者年轮读数的总吻合率都很高,分别为92.7%和97.0%,且随年龄上升,依然可保持较高的精确度,对洪湖鲫种群应用耳石为宜。     

The use of archived tags in retrospective genetic analysis of fish

Collections of historical tissue samples from fish (e.g. scales and otoliths) stored in museums and fisheries institutions are precious sources of DNA for conducting retrospective genetic analysis. However, in some cases, only external tags used for documentation of spatial dynamics of fish populations have been preserved. Here, we test the usefulness of fish tags as a source of DNA for genetic analysis. We extract DNA from historical tags from cod collected in Greenlandic waters between 1950 and 1968. We show that the quantity and quality of DNA recovered from tags is comparable to DNA from archived otoliths from the same individuals. Surprisingly, levels of cross‐contamination do not seem to be significantly higher in DNA from external (tag) than internal (otolith) sources. Our study therefore demonstrates that historical tags can be a highly valuable source of DNA for retrospective genetic analysis of fish.