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.     

Notes on age determination,size and age structure,longevity and growth of co-occurring macrourid fishes

Nineteen species of the deep-water fish family Macrouridae inhabit the Mid-Atlantic Ridge (MAR) of the North Atlantic. Size and age structure, longevity and length at age were explored for seven of the more abundant species: Coryphaenoides armatus, Coryphaenoides brevibarbis, Coryphaenoides carapinus, Coryphaenoides guentheri, Coryphaenoides mediterraneus, Coryphaenoides rupestris and Macrourus berglax. The otoliths from these species had growth increments resembling those accepted as annuli in other deep-water species for which validation experiments have been conducted. Based on the counts of these growth increments, age estimates were derived as the basis for studies of age structure, growth and longevity. All the species appeared to have relatively low growth rates, but the interspecific variation in longevity illustrates that there is a considerable variation within the family and among species inhabiting the same deep-sea environment. Most of the species likely complete full life cycles on the MAR, but it is probable that both the youngest and the largest-oldest individuals of some of the species were not sampled adequately.     

Otolith characteristics and age determination of an endemic Ptychobarbus dipogon (Regan, 1905) (Cyprinidae: Schizothoracinae) in the Yarlung Tsangpo River, Tibet

We describe the microincrements, checks and annuli in the lapilli of the schizothoracine Ptychobarbus dipogon, an endemic species of the Tibetan plateau. We collected samples in the Yarlung Tsangpo River and its tributaries on a monthly basis (from April 2004 to August 2006). We describe the shape features of the three pairs of otoliths and document the full trajectory of lapillus development. We found that five to seven checks were clearly visible in the opaque zone of the first annulus. The pattern of 21–23 daily growth increments within each check might be explained as a lunar-induced deposition. We counted between 137 and 154 increments within the first annulus. Annuli appeared as a sequence of gradually declining increment widths, whereas false rings were characterized by abrupt checks. Our oldest estimates were 23⁺years for males and 44⁺ for females. The time of annulus completion was clearly between March and April each year using monthly marginal increments analysis. We consider the factors responsible for daily increment formation as an endogenous circadian rhythm. Environmental information, such as strong sunlight and cold water temperatures in the Tibetan Plateau, could reinforce the endogenous daily cycle. Our results provided important data addressing the ecology and population dynamics of P. dipogon.     

Age and growth rate determinations of an intertidal bivalve, Phacosoma japonicum, using internal shell increments

A venerid bivalve Phacosoma japonicum (Reeve) occurring commonly in the Japanese coastal area preserves periodic growth lines in the shell cross-section. Long-term shell growth patterns of this species have been traced for many individuals on the intertidal flat of the Seto Inland Sea, west Japan. Sclerochronological analysis of these individuals and specimens collected monthly shows that several growth cessation marks within their shells are formed during the winter of each year prior to spawning. Hence the marks were used for age and growth rate determinations. As large individuals showed little shell growth for more than two years after the formation of 7 or 8 annual increments, this species probably has a lifespan of more than ten years. Shell growth patterns of this species based on annual increments can be accurately approximated by a von Bertalanffy curve. The number of microgrowth increments formed during a year tends to decrease with age, although it varies markedly among specimens of the same age. Furthermore, even in summer during rapid shell growth, the microgrowth increments do not represent daily and/or sub-daily tidal rhythms in many specimens. The results of this study and those by several authors strongly suggest that the annual increments are the key for age and growth rate determinations of both living and fossil bivalve species.     

Late Devonian dimpled phosphatic microspherules associated with conodonts are possible otoliths of short‐lived and opportunistic organisms

Dimpled phosphatic microspherules, contradictorily associated with conodonts, are widely distributed in strata ranging in age from the Cambrian to Carboniferous. These microspherules have attracted much attention from palaeobiologists and were suggested to be ‘conodont pearls’, ‘conodont otoliths’ or ‘fish otoliths’ due to their similar chemical composition and co‐occurrence with conodonts or fish teeth. However, these hypotheses are still highly controversial. Here, we report ‘checks’, ‘rhythmic growth patterns’ and ‘sub‐diurnal increments’ from growth annuli of the Late Devonian phosphatic microspherules from South China, on the basis of quantitative microstructure analysis. The annulus width of phosphatic microspherules becomes narrower with increasing radius. These microstructural characteristics of growth annuli are most typical indicators of modern animal otoliths. In addition, a maximum value of about 90 annuli is encountered from all the specimens. We propose that these microspherules are essentially phosphatic otoliths, which might have been secreted by a specific kind of marine organisms with very short lifespans (less than 90 days). Furthermore, the sudden enrichment of phosphatic microspherules in the late Frasnian may represent a biological response of short‐lived animals to ecological crisis such as ocean eutrophication.     

Growth of North Alboran Sea sardine larvae estimated by otolith microstructure, nucleic acids and protein content

Wet mass and DNA, RNA and protein content increased significantly with standard length ( L _S) of sardine Sardina pilchardus larvae, collected in January 1995, in the Bay of Málaga, North Alboran Sea. L _S, wet mass and DNA, RNA and protein content were closely related allometrically to otolith radius ( R ). Larval daily length increments decreased but DNA, protein and wet mass daily increments increased with larval age. Daily length increments showed a negative and poor relationship with long-term otolith growth. In contrast, DNA, protein and wet mass daily increments were positively correlated. Differences between observed and back-calculated otolith radius-at-age indicated that larvae with slow otolith growth were under represented in older age groups, suggesting the existence of growth-selective mortality. Recent otolith growth, estimated from the mean widths of the last six increments, increased with age and R . Individual RNA: DNA and protein: DNA ratios were correlated significantly, although weakly, with L _S and larval growth.     

Distribution, population biology, and trophic ecology of the deepwater demersal fish Halosauropsis macrochir (Pisces: Halosauridae) on the mid-Atlantic Ridge

Halosauropsis macrochir ranked amongst the most abundant and widespread demersal fishes on the mid-Atlantic Ridge of the North Atlantic (Iceland-Azores) with greatest abundance at 1700-3500 m. All sizes, ranging from 10-76 cm total length, occurred in the area without any apparent spatial pattern or depth trend. Using otolith sections displaying growth increments assumed to represent annuli, the age range recorded was 2-36 years, but most individuals were <20 years. Length and weight at age data were used to fit growth models. No differences between sexes in length and weight at age were observed. The majority of samples had a surplus of males. Diet analysis showed that H. macrochir feeds on Crustacea, Teleostei, Polychaeta, and Cephalopoda, but few prey could be identified to lower taxonomical levels. The mid-Atlantic Ridge constitutes a major portion of the North Atlantic living space of the abyssal halosaur where it completes its full life cycle, primarily as an actively foraging euryophagous micronekton/epibenthos and infauna feeder, becoming a partial piscivore with increasing size.     

Validation of daily sagittal increments in the golden-spotted rabbitfish Siganus guttatus (Bloch) using known-age larvae and juveniles

The study presents a first report on the validation of otolith increment in the golden-spotted rabbitfish Siganus guttatus . The formation of daily sagittal growth increments in S. guttatus was validated using hatchery-reared larvae and juveniles collected from day of hatching to day 35. Mean increments were 5.6, 13.1, 24.5 and 34.8 for the 7, 15, 25 and 35 day-old fish, respectively. The results demonstrated that sagittal increments were formed daily and thus can be used to determine the age in days of the species.     

Assessing the validity of stylets as ageing tools in Octopus pallidus

A method to age octopus was determined through the quantification of growth increments within the stylet structure (highly reduced internal shells). To validate the periodicity of the increments, stylets were analysed from known-age laboratory-raised Octopus pallidus. The animals, which ranged from 3 to 8 months old, were exposed either to a simulated natural or to constant temperature regime. Transverse stylet sections were embedded in thermo-plastic cement and ground until a thin section was achieved. A pre-hatch region and first post-hatch increment was identified in the stylet microstructure. The number of increments, across all ages and temperature treatments, was in remarkably close agreement to age (number of days), clearly demonstrating that stylet increments are deposited daily throughout the lifecycle of O. pallidus. Morphometric analyses of the stylet indicated that increments were laid down regularly during stylet growth and that stylets grew in concert with body size, further supporting daily periodicity. This study successfully validates daily increments in stylets and thus demonstrate a method to age octopus, therefore, potentially having critical implications for future octopus research and the effective management of stocks worldwide.     

Age determination of the Antarctic fishes Champsocephalus gunnari and Notothenia rossii marmorata from South Georgia

Summary Investigations were performed on otoliths from Champsocephalus gunnari and Notothenia rossii marmorata as part of an effort to study the population dynamics of Antarctic fishes. Examination of otoliths by scanning electron microscope (SEM) revealed internal microincrements which were similar to daily increments described for other fish species. No easily identifiable annuli could be discerned and fishes were aged by counting microincrements. Multiple regression analyses relating age to otolith morphometrics and fish size, provided a simpler ageing method. It was determined with these techniques that C. gunnari grew to 50 cm in 15 yrs and N. rossii marmorata grew to 50 cm in 6.5 yrs. The lack of larger-sized individuals for N. rossii marmorata may be indicative of recent overfishing with only the younger age groups remaining. It appears that length-at-age data determined on a yearly basis, could provide valuable scientific and management information for these species.     

Unravelling growth trajectories from complicated otoliths – the case of Brazilian codling Urophycis brasiliensis

Uncertainty regarding the age determination of the Brazilian codling Urophycis brasiliensis has hampered its stock assessment. Transverse sections of otoliths displayed up to seven (in males) and 12 (in females) alternate opaque and translucent bands that could not be conclusively validated as annuli, resulting in unrealistically high ages of first maturity (A₅₀) (A_50male = 4·5 years and A_50female = 6 years). Therefore, growth was described by the von Bertalanffy (VB) model using an alternative approach that combined microstructure data (daily growth increments) and a fixed asymptotic total length (L_∞). This approach was supported by applying it to two other co‐occurring species, the whitemouth croaker Micropogonias furnieri and the king weakfish Macrodon atricauda, for which daily and annual ring formation has previously been validated. The sensitivity to realistic errors associated with the choice of the L_∞ and the daily increment readings was shown to be low. The results show that U. brasiliensis has a fast growth rate (K_male = 1·19 year^?1, K_female = 0·71 year^?1) and early maturation (A_50male = 1·1–1·5 years, A_50female = 1·6–1·8 years); typical life‐history traits for a sub‐tropical coastal gadiform. This novel study offers an alternative approach for age and growth reconstruction for species with complex patterns of opaque and translucent bands provided that daily growth increments in the yearlings can be counted and L_∞ reliably estimated.     

Otolith formation,microstructure and daily increment validation in juvenile perch Perca fluviatilis

The otoliths of laboratory‐reared larval and juvenile perch Perca fluviatilis of known age were analysed to determine the age of otolith formation and validate the formation of daily increments. There was a linear relationship between number of increments and age in days post‐hatching, although by 82 days post‐hatching daily increment counts underestimated actual age by an average of 5 days. Otolith dimensions in relation to standard length indicated allometric growth of otoliths until completion of yolk absorption, and isometric growth thereafter, up to 82 days post‐hatching.     

Dynamics of incisor growth and daily increments on the incisor surface in three species of small rodents

The lower and upper incisors has been studied in striped field mice (Apodemus agrarius), yellow-necked mice (A. flavicollis), and migratory hamsters (Cricetulus migratorius) trapped in the Volgograd Region from April to March. The incisors of all animals have been found to have a striated surface, with series of slight ridges and grooves. As shown by analyzing the incisors of animals labeled with tetracycline, these are daily increments of dentin. The total numbers of increments (the period of complete tooth renewal) in the upper and lower incisors of the same animal are similar, but the degrees of their distinctness can be different. The number of daily increments increases but their width (daily growth rate) decreases with age even in adult animals and, in addition, varies by seasons. These two trends—the decrease in tooth increment with age and its seasonal variation—have proved to mask each other when an uneven-aged sample of animals trapped over several months is studied. Therefore, the season of death of an animal cannot be reliably estimated from the width of daily tooth increment unless the age of this animal is taken into account. The increment width in young animals in autumn may be the same as in older animals in spring and summer. In attempts to use the incisor surface sculpture for determining the season of animal death, it is necessary to separate the young from adults (at least by the criterion of incisor length). The decrease of increment width with age and its seasonal variation in the absence of obvious changes in the diet and pattern of incisor attrition indicate that the incisor growth rate is subject to age-dependent and seasonal changes, as is the growth rate of the organism as a whole. The distinctness and internal structure of daily increments also vary depending on animal age and species, which is due apparently to differences in the circadian rhythms of incisor growth. The presence of one or, less frequently, several grooves within a daily increment, which has occurred in all species studied, is evidence for not only circadian but also ultradian rhythms in the growth of incisors.     

The use of otolith microstructure to estimate age in adult Atlantic cod Gadus morhua

Transverse sections of otoliths from Atlantic cod Gadus morhua from the Baltic Sea revealed narrow growth increments. The widths of these increments corresponded to daily increments from fish with known otolith growth rates and were therefore assumed to be daily increments. They exhibited a distinct pattern with increasing distance from the primary primordium. A series of zones with clearly distinguishable increments, first with increasing then with decreasing widths in a dome‐shaped pattern, were separated by zones where no regular increment structure was visible. Increment width seemed to be tightly coupled to the annual cycle in environmental temperature at a depth of 30–60 m, where G. morhua predominantly reside. Between 135 and 200 increments occurred within the different zones, with a non‐significant trend towards lower increment numbers and widths with distance from the primary primordium of the otolith. Increment formation apparently ceased at temperatures < 5–6° C, but growth during the cold months corresponded closely with estimated growth rates. The increment patterns seemed to reflect annual cycles in environmental temperature, and the count of the increment cycles may thus be a promising tool for the determination of the true age of Baltic G. morhua.     

The Morphology and Periodic Structures of the Otolith of the Chinook Salmon (Oncorhynchus tshawytscha), and Temperature-dependent Variation in Otolith Microscopic Growth Increment Width

The otolith (sagitta) of the chinook salmon (Oncorhynchus tshawytscha) has a variable external crystalline morphology which is related to differences in the growth rate of crystals in different parts of the otolith. The internal crystal structure of the otolith is complex with different mineral phases in different growth fields of the otolith and a well-defined series of microscopic growth increments in both the dorsal and the ventral parts (orientation in situ) of the otolith. The period of the microscopic growth increments was shown, by both a rearing experiment and interpolation from fish of known age, to be daily. By rearing sibling chinook salmon at different temperatures (while still maintaining them on the same diet) it was shown that the width of the daily growth increment varies with temperature, but neither multi- nor sub-daily increments appear in the area of regular, daily growth incrementation. Inclusions of the vaterite morph of calcium carbonate crystallizing in the botryoidal habit occur in the otherwise aragonitic otolith. Regularly spaced check rings in the sulcul part of the otolith are homologous with those occurring in the dorsal and ventral growth axis and are. on average, separated by about 28 microincrements.     

Validation of a technique for reconstructing daily patterns in the recruitment of coral reef damselfish

Counting growth increments in otoliths recently has become an accepted method of ageing tropical fishes, however, verification is essential for each new species. In this study, growth increments in otoliths of the juveniles of several coral reef damselfishes (Pomacentridae) were deposited daily and a distinct transition from wide to narrow increments coincided with settlement from the pelagic larval phase into the demersal habitat. Thus, the data of settlement for each individual fish could be calculated with acceptable accuracy. The daily pattern of recruitment to a coral reef was successfully reconstructed using the otoliths from a large sample of juvenile fish collected at the end of the breeding season. This was because the original pattern of settlement was preserved in the age distribution for at least 4 to 5 months. This application of otolith ageing techniques may be extended to reveal the temporal patterns of recruitment to many localities encompassing spatial scales larger than would be logistically possible using visual censuses.     

Growth of the clupeid fishes, Stolothrissa tanganicae and Limnothrissa miodon, in the Zambian waters of Lake Tanganyika

The age and growth of two endemic Lake Tanganyikan clupeid fishes, Stolothrissa tanganicae and Limnothrissa miodon , were estimated from analysis of otolith microstructure in specimens collected in Zambian waters from October to December 1990. Both species had relatively clear, concentric otolith increments which were believed to represent daily increments based on their similarity to daily increments found in some marine clupeid species. The growth rates estimated from length-at-age data were: for S. tanganicae, L_t = 104·0 (1 – exp (– 0.00512 ( t – 5·8))); for L. miodon, L_t = 155.4 (1 – exp (– 0.00236 ( t +8.1))). In most cases, the growth parameters obtained from otolith analyses generally agreed with those previously estimated from analyses of length-frequency distributions.     

Linking larval history to juvenile demography in the bicolor damselfish Stegastes partitus (Perciformes: Pomacentridae)

Otolith-based reconstructions of daily larval growth increments were used to examine the effect of variation in larval growth on size and age at settlement and post-settlement growth, survival and habitat preferences of juvenile bicolor damselfish (Stegastes partitus Poey). During August 1992 and 1994, newly settled S. partitus were collected from Montastraea coral heads and Porites rubble piles in Tague Bay, St. Croix, U.S. Virgin Islands (17 degrees 45'N, 64 degres 42' W). Daily lapillar otolith increments from each fish were counted and measured with Optimas image analysis software. S. partitus pelagic larval duration was 23.7 d in 1992 (n = 70) and 24.6 d in 1994 (n = 38) and larval age at settlement averaged 13.0 mm total length both years. Analysis of daily otolith increments demonstrated that variation in larval growth rates and size and age at settlement had no detectable effect on post-settlement survivorship but that larger larvae showed a preference for Montastraea coral at settlement. Late larval and early juvenile growth rates showed a significant positive relationship indicating that growth patterns established during the planktonic stage can span metamorphosis and continue into the benthic juvenile phase. Larval growth rates during the first two weeks post-hatching also had a strong effect on age to developmental competence (ability to undergo metamorphosis) in both 1992 and 1994 with the fastest growing larvae being 8 d younger and 0.8 mm smaller at settlement than the slowest growing larvae. These differential growth rates in early stage larvae established trajectories toward larval developmental competence and may prove important in biogeographical studies of larval dispersal.     

Age and growth of the spotted goatfish, Pseudupeneus maculatus (Bloch, 1793) in Brazil, validated through marginal increment and oxytetracycline dyes in the sagittae

A sample composed of 4973 specimens of the spotted goatfish, Pseudupeneus maculatus (4.5–29.2 cm fork length) was collected off northeastern Brazil from 1999 to 2002. Sagittae (398) were analyzed for age and growth estimation using both annuli and microincrements. For the first approach, the mean monthly marginal increment ratio analysis suggested that one band is formed annually from May to June. With the microincrements analysis, 15 specimens were reared (3–71 days) for validation of periodicity using oxytetracycline. Fluorescent dyes were observed in 10 specimens (7.5–16.9 cm). After testing, one microincrement was found to form daily. Otolith sections were polished and analyzed using a light microscope (100–1200× magnifications). Individuals larger than 22.6 cm (4 years) showed heavily overlaid increments on the otolith edge, which hampered the counts. There was agreement on the size from counts on an annual and daily basis. The von Bertalanffy function provided the following parameters: L_∞ = 33.23 cm; K = 0.265; t₀ = 0.0883 year. The sample was composed of 0⁺ to >5‐year‐old specimens. Age at first maturity was 3.6 years (20 cm), whereas the recruitment age for fisheries was 3.9 years. Juveniles represented about half of the catch (48.7%), which may have implications for population equilibrium.