A comparison of staining techniques to improve precision of age estimation from fish otoliths

A total of 30 pairs of sagitta otoliths of horse mackerel Trachurus trachurus were sampled from three length-classes, and five staining methods were applied to each pair. Ten age estimates were obtained per fish, method and length-class. Significant differences in age estimates were found between methods, due to the variable enhancement in the otolith pattern produced by each method. A method using amido-schwartz as the staining reagent produced the best results in terms of precision of the age estimates.     

cost-effective method of preparing larval fish otoliths for reading using enzyme digestion and staining

A fast and cost-effective method for examining otoliths in fish larvae was developed whereby the otolith remains in situ . Whole fish of the clownfish Amphiprion melanopus were enzymecleared using a laundry pre-soak and then stained using the Von Kossa silver staining method for calcium. The otolith nucleus, daily rings and the otolith edge were all clearly visible and were suitable for a variety of age and growth analyses. The total 'hands on' time required to process these otoliths was c . 3 min, and multiple samples could be processed simultaneously. The reduction in labour of this method to produce clear daily rings in the otolith lends itself to broad use in fish biology where large quantities of otoliths need to be examined in a cost- and time-efficient manner.     

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.     

Fossil fish otoliths from the Chibanian Miyata Formation,Kanagawa Prefecture,Japan, with comments on the paleoenvironment

The Paleo-Tokyo Bay (Kanto Plateau) that persisted during the transgression periods in the Middle-Late Pleistocene is known for its rich marine fauna that accommodated both the warm and cold water taxa brought by the Kuroshio and Oyashio Currents, respectively. However, little is known on the paleoichthyofauna in this area and the processes shaping the marine ichthyofauna of modern southern Kanto. We used fossil fish otoliths and teeth discovered in the Sha’ana Tuffaceous Sand Member belonging to the Chibanian Miyata Formation, exposed at Sha’ana-dai, Miura City, Kanagawa Prefecture, Japan, to reconstruct the paleoichthyofauna and its associated paleoenvironment and explore the content of fish assemblages during this period. The Sha’ana Tuffaceous Sand Member was composed of upper sandy mud and lower muddy sand horizons at the exposure. A total of 1,675 fish remains were collected from this site, of which 1,389 were assigned to 62 distinct taxa (20 orders, 31 families). The taxonomic composition revealed taxa from various environments, including shallow continental shelf, epi- to meso-pelagic settings, and the majority were from a generally temperate climatic zone. Paleoenvironmental analyses suggested that the fish assemblages were from the continental shelf (approximate depth of 100–200 m) in a temperate sea where both warm Kuroshio and cold Oyashio Currents had an influence on the biota. The analysis also showed that two boreal fishes, namely Clupea pallasii and Theragra chalcogramma, once had a wider distribution that extended to the southwestern areas of the Paleo-Tokyo Bay. This study provides a more complete view of the related paleobiogeography of marine fishes during the Chibanian and is essential for reconstructing the evolutionary processes of the rich ichthyofauna in the southern Kanto.     

Evaluation of copper, iron and lead substitution techniques in elasmobranch age determination

Copper and lead substitution reactions were found to be useful in demonstrating growth patterns in vertebrae from several elasmobranch species. In contrast, iron substitution was ineffective in detecting variations in vertebral mineralization. The present study provides two alternative methods for elasmobranch age determination that may prove useful when traditional methods are deemed ineffective. However, species-specific variations in the efficiency of these methods reemphasize the observation that different types of vertebrae often require different methods of staining.     

Using otolith weight to age fish

The problem of determining and verifying ages of fish, from populations having a considerable variation in size at age, has been investigated using the relationship between otolith size and fish size, which has been shown by several authors to be influenced by growth rate. In such a population of Sardinella aurita Val. an index of age can be obtained for individual fish by calculating the equivalent otolith weight at a particular fish length, using the otolith weight–fish length relationship determined for each age group. This statistic not only permits a much greater proportion of fish to be assigned ages than is possible with otolith reading alone, but also enables the age groups to be verified as year classes. However, it is concluded that, although appropriate models based on otolith-fish size relationships can predict age for groups of fish in which growth rates are known or can be assumed to be consistent, such techniques have a limited application in ageing fish from wild populations with highly variable growth rates.     

Effect of DNA extraction on ageing success in coral trout (Plectropomus leopardus) otoliths

To maximize the information commonly collected from otoliths, the effect of DNA extraction on the estimation of age with otoliths was evaluated by comparing sagittal otolith samples from common coral trout ( Plectropomus leopardus ) for clarity and ageing discrepancies in DNA-extracted and untreated control otoliths. The DNA extraction process had no significant effect, indicating that archived otoliths can be used as a source of DNA while retaining their utility for age estimation.     

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.     

Growth backcalculations based on otoliths incorporating an age effect: adding an interaction term

An extension, including an age × length interaction, was developed for a recent model incorporating the age effect in the backcalculation of growth in length from otoliths. The interaction was demonstrated using empirical data from Arctic charr Salvelinus alpinus .     

鱼类性别决定的影响因素

鱼类的性别决定机制极其复杂,受到外源性激素、外界环境因素和遗传因素等多方面的影响。因此,鱼类性别决定机制的研究不仅对我们控制鱼类的性别有帮助,也将使我们对脊椎动物性别决定的可塑性有一个全面的了解。该文对影响鱼类性别决定的各种因素及其研究前景和发展趋势进行综述。     

Practising methods of age determination. Comments on methods combining multiple age indicators

For some time, trends in age determination of skeletons have tended to shift from methods using single age indicators to methods combining multiple age indicators. Evidently the latter are expected to offer more balanced age assessments. If one decides to apply a method using multiple age indicators, a choice can be made between two «current» methods, both based on non-metrical age indicators: the socalled Complex Method of Acsádi and Nemeskéri, statistically elaborated by Sjøvold (WEA, 1980), and the so-called Multifactorial Method of Lovejoy et al., 1985a. Comments are given on practical applicability and supposed confidence.     

Field age determination of leopards by tooth wear

Age determination is an important tool in wildlife studies. Estimating the age of animals in the field using tooth wear criteria may be subject to error as a result of variations between individuals, habitats and populations. Data on age estimation of leopards and tooth wear characteristics are lacking. Nineteen leopards in Namibia were assessed for tooth eruption and wear. Between 1991 and 1995 leopards (including 13 individuals of known age) were monitored at one year intervals ('28 leopard years') to record age and tooth wear. At the age of two years leopards had fully developed dentition. Wear started with the incisors and canines, and spread to the premolars and molars. A chronology of tooth eruption and wear in relation to age is presented. Above the age of three years, male leopards showed higher frequencies of enamel flaking and canine fractures than females.     

Pteridine fluorescence for age determination of Anopheles mosquitoes

The age structure of mosquito populations is of great relevance to understanding the dynamics of disease transmission and in monitoring the success of control operations. Unfortunately, the ovarian dissection methods currently available for determining the age of adult mosquitoes are technically difficult, slow and may be of limited value, because the proportion of diagnostic ovarioles in the ovary declines with age. By means of reversed-phase HPLC this study investigated the malaria vectors Anopheles gambiae and An. stephensi to see if changes in fluorescent pteridine pigments, which have been used in other insects to determine the age of field-caught individuals, may be useful for age determination in mosquitoes. Whole body fluorescence was inversely proportional to age (P < 0.001, r2 > 91%) up to 30 days postemergence, with the regression values: y = 40580-706x for An. gambiae, and y = 52896-681x for An. stephensi. In both species the main pteridines were 6-biopterin, pterin-6-carboxylic acid and an unidentified fluorescent compound. An. gambiae had only 50-70% as much fluorescence as An. stephensi, and fluorescent compounds were relatively more concentrated in the head than in the thorax (ratios 1:0.8 An. gambiae; 1:0.5 An. stephensi). The results of this laboratory study are encouraging. It seems feasible that this simpler and faster technique of fluorescence quantification could yield results of equivalent accuracy to the interpretation of ovarian dissection. A double-blind field trial comparing the accuracy of this technique to marked, released and recaptured mosquitoes is required to test the usefulness of the pteridine method in the field.     

Enhancing the contrast and visibility of daily growth increments in fish otoliths etched by proteinase K buffer

The otoliths of the amphidromous gobies Stiphodon elegans and Sicyopterus japonicus , a marine eel Strophidon sathete and a freshwater fish Varicorhinus barbatulus were digested by proteinase K buffer (PKb), which removed the proteins and retained the major calcified structure to reveal conspicuous daily increments. Compared with etching by ethylenediaminetetraacetate (EDTA), etching with PKb revealed more visible daily increments and enhanced the contrast. Moreover, by using PKb, both the daily increments and annuli could be observed simultaneously. The better performance of PKb as an etching agent can be attributed to digesting of predominantly only the organic matrix and leaving the calcified structure almost intact. PKb provides another effective means to reveal otolith microstructure so as to examine and count daily growth increments.     

Comparison of two histological techniques for age determination in small cetaceans

Age estimation in odontocetes is based on counts of growth layer groups (GLGs) deposited in recording structures such as teeth. Generally, tooth sections are obtained using a cryostat microtome. However, some researchers prefer obtaining thin sections using a traditional paraffin microtome. Little information is available on the application of this technique to dolphin teeth. Our main aim was to investigate if the paraffin technique can be a viable alternative. We considered whether estimated age would be affected by preparation technique, staining method, and section thickness, while controlling for effects of species, body length, and sex. We also analyzed whether the staining method would affect readability of GLGs and age reading variability. Teeth from 86 individuals (representing seven species) were used, but not all were prepared using both techniques because sufficient teeth were not available in all cases. Although the staining method had significant effects on the estimated age using both techniques, the variability of GLG counts was small and appeared to be similar for both techniques. Using Mayer's hematoxylin stained sections of 8 μm thickness, good agreement of ages was obtained from both techniques, with more preparations classified as "good quality" for the paraffin technique. Mayer's hematoxylin provided the best contrast of the GLGs when using the paraffin technique. We conclude that the paraffin technique is viable and represents a cost-effective alternative to a cryostat microtome when preparing cetacean teeth for age determination.     

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.     

The appearance of accessory growth centres in adult whiting Merlangius merlangus otoliths

The appearance of accessory growth centres (AGCs) in the otoliths of adult whiting Merlangius merlangus reared in the laboratory for 2 years is reported. The causes and implications of AGCs appearing in otoliths of adult fish are discussed.     

The morphology of the otoliths of the bagrid catfish,chrysichthys nigrodigitatus (lacepede) and their use in age determination

Two pairs of otoliths, the saccular and sagittae are shown to be well developed in the catfish,Chrysichthys nigrodigitatus. The gross morphology of each pair is provided. Growth markings arranged in a periodic manner are demonstrated. In addition, daily growth marks are shown to be present. The growth markings on the sagittae are interpreted and used in estimating the age and growth of the species.