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.     

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.     

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.     

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.     

Daily growth increments in otoliths of milkfish, Chanos chanos (Forsskål), larvae

The formation of daily growth increments of otoliths was studied in the reared larvae of milkfish. The first growth increment was formed during the yolk–sac reabsorption period c .2 days after hatching, and increment formation continued on a daily schedule regardless of growth rate. The initial incremental zone was of amorphous structure, and the subsequent incremental zone was of needle–shaped crystalline structure; the former structure was formed in the yolk–sac reabsorption period of the larva and the latter in the exogenous feeding period. Accordingly, ageing of milkfish larvae is possible by counting growth increments, and timings within the developmental stage of the larvae can be understood by examining the otolith microstructure.     

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.     

Development of fluctuating asymmetry in tail feathers of the barn swallow Hirundo rustica

Fluctuating asymmetry represents usually small, random deviations from symmetry in bilateral morphological characters. The ontogeny of asymmetry in morphological characters may reveal information about developmental processes in a general sense. I studied the development of fluctuating asymmetry in feather characters of the barn swallow Hirundo rustica, that are developed repeatedly during the single annual moult, with the following results. First, the side developing a larger feather was found to be partially biased, as demonstrated by one side consistently developing a larger feather under natural and experimentally induced growth episode events. Second, asymmetric feathers were found to consist of asymmetric daily growth increments, and the size of the increments developing under different environmental conditions were positively correlated. Third, fluctuating asymmetries of feathers developing under different environmental conditions were positively correlated, although the level of asymmetry was larger under adverse environmental conditions. Fourth, individual asymmetries in tail length and growth bar length were unrelated to the duration of the developmental period, although late growth increments were smaller and more symmetric than early increments. These observations suggest that fluctuating asymmetry partially arises as a consequence of a random bias in the feather follicles and differences in environmental conditions during ontogeny of feathers.     

Does water calcium content influence the distinctness of daily growth increments in the otoliths of larval whitefish (Coregonus lavaretus L.)?

In larval and juvenile whitefish ( Coregonus lavaretus L.) from Lake Constance, Germany, the otolith increments are deposited daily, whereas daily deposition could not be confirmed in larval whitefish from Lake Pyhäselkä, Finland. The calcium concentration in Lake Constance is high (around 1.3 m m ), while calcium deficiency is typical for Finnish lakes (around 0.15 m m ). Therefore, the hypothesis that the distinctness of daily otolith increments in whitefish is related to water calcium content was tested by rearing three groups of Lake Constance whitefish in water of 0.2, 1.3 and 4.7 m m Ca. The eggs were incubated in lake water (1.3 m m Ca), and the larvae were acclimated to the experimental calcium concentrations on the day of hatching. After 39 days of ad libitum- feeding with Artemia nauplii, the three groups did not differ significantly in total length, wet and dry weight, and otolith length and width. The daily increments were easily recognizable, and contrast between dark (D)- and light (L)-zones was the same in the fish of all test groups. For the experimental set-up of this study, and particularly the range of calcium concentrations tested, the hypothesis that water calcium content influences the distinctness of daily otolith increments was rejected.     

Comparison of growth rate and formation of otolith increments in age-0 red snapper

For wild red snapper Lutjanus campechanus , mean otolith increment deposition rate after marking with oxytetracycline dihydrate (OTC) was daily (0.97 increments day⁻¹) when growth rates were fast (0.63 mm fork length, L _F day⁻¹), but were not daily (0.82 increments day⁻¹) when somatic growth was slow (0.2 mm L _F day⁻¹). For reared larvae ( n =8), increment deposition rates were daily (0.99–1.03 increments day⁻¹), and growth rates ranged from 0.6 to 0.9 mm L _F day⁻¹. Growth rate affected increment deposition rate as a threshold function, i.e. when growth rate was <0.3 mm L _F day⁻¹, deposition was less than daily, but above this level increment deposition did not exceed a daily rate. As growth rates increased increment widths increased. Examination of a sub-sample ( n =8) of the otoliths from the slowest growing wild fish by scanning electron microscopy did not increase increment counts. Because L. campechanus are late spring-early summer spawners, young fish can expect maximum growth due to warm summer temperatures. Thus, daily ageing methods should be well suited to this species.     

Histological structure of the cancellous bone layer in Bothriolepis canadensis (Antiarchi, Placodermi)

The Placodermi are extinct basal gnathostomes which had extensive dermal and perichondral bone, but which lacked the endochondral bone which characterizes the more derived bony fishes. Thin sections of bone from a specimen of the antiarch placoderm Bothriolepis canadensis, from the Escuminac Formation (Frasnian, Upper Devonian), Québec, Canada, reveal that part of the cancellous layer in its dermal and endoskeletal bone formed from perichondral bone trabeculae growing around cartilage spheres. The resultant structure mimics that of osteichthyan endochondral bone. The layout and dimensions of this polygonal mosaic patterning of the bone trabeculae and flattened cartilage spheres resemble those of the prismatic layers of calcified cartilage in chondrichthyans. If the lack of endoskeletal bone in chondrichthyans is a derived character, then the structure identified in B. canadensis could represent a 'template' for the formation of prismatic calcified cartilage in the absence of bone.     

Structural study of the calcifying collagen in turkey leg tendons

The calcified turkey leg tendon represents a simple bone-like tissue that is ideally suited to analysis by diffraction methods. In this paper we report some structural studies of the tendon collagen in the uncalcified, fully calcified and partially calcified states. The low-angle meridional X-ray pattern from the uncalcified tendon is very similar to that of the rat tail tendon, and the resulting one-dimensional structure of the collagen fibril exhibits no feature that could be related to its eventual calcification. The structure of the fully calcified tendon, as determined by a combination of X-ray and neutron diffraction analyses, shows that the mineral is associated with the collagen at the level of the hole or gap region. In the calcifying tendon, increases in the amplitudes of the first and second X-ray meridional reflections are correlated with an increase in the mineral content of the collagen. On the basis of simple models, it is shown that this change in the pattern can be explained by a nucleation mechanism of calcification. It is concluded that when collagen becomes calcified the mineral penetrates throughout the fibril and is crystalline in the hole region but amorphous between the collagen molecules. The mechanism of calcification and the mechanical implications of the fully calcified structure are also discussed.     

Three-dimensional reconstruction of fracture callus morphogenesis.

Rat and mouse femur and tibia fracture calluses were collected over various time increments of healing. Serial sections were produced at spatial segments across the fracture callus. Standard histological methods and in situ hybridization to col1a1 and col2a1 mRNAs were used to define areas of cartilage and bone formation as well as tissue areas undergoing remodeling. Computer-assisted reconstructions of histological sections were used to generate three-dimensional images of the spatial morphogenesis of the fracture calluses. Endochondral bone formation occurred in an asymmetrical manner in both the femur and tibia, with cartilage tissues seen primarily proximal or distal to the fractures in the respective calluses of these bones. Remodeling of the calcified cartilage proceeded from the edges of the callus inward toward the fracture producing an inner-supporting trabecular structure over which a thin outer cortical shell forms. These data suggest that the specific developmental mechanisms that control the asymmetrical pattern of endochondral bone formation in fracture healing recapitulated the original asymmetry of development of a given bone because femur and tibia grow predominantly from their respective distal and proximal physis. These data further show that remodeling of the calcified cartilage produces a trabecular bone structure unique to fracture healing that provides the rapid regain in weight-bearing capacity to the injured bone.     

Limits for the detection of daily otolith increments in whitefish (Coregonus lavaretus L.) larvae

The formation of daily otolith increments in European lake white-fish was evaluated in the laboratory during 40 days at 4, 6, and 8 °C under limited and ad libitum food supply. Daily increments were easily identified in larvae reared at 8 °C. At 6 °C and, more, at 4 °C, an unstructured perinuclear zone was formed, and daily increments could only be recognized in the light microscope starting from 10 to 35 days after hatching. In some larvae from the 4 °C groups, no increments at all were found. Scanning electron microscopy (SEM) could not improve increment resolution. Only those increments could be viewed by SEM which were visible in the light microscope as well. We conclude that whitefish larvae which experience low (4–6 °C) temperatures during their first weeks of life, hence those which hatch in the lake, cannot be aged by the currently employed preparation techniques.     

Validation of daily otolith increments in larval and juvenile Chinese sucker, <Emphasis Type="Italic">Myxocyprinus asiaticus</Emphasis>

Otolith development was observed and the formation of daily growth increments in otoliths of Chinese sucker, Myxocyprinus asiaticus, was validated by monitoring known-age larvae and juveniles in the laboratory from 2003 to 2005. Otolith shape changed with larval and juvenile development, and there was an exponential relationship until a body length of 16 mm or so, and a linear relationship after a body length of 16 mm between otolith size and fish size. The first increment was identified in larvae 1 day after hatching. The regressed equations between daily age (D) and increment number in otoliths (N) were N = −0.64 + 0.96D in lapillus, and N = −0.31 + 0.98D in sagitta. The slopes were not significantly different than 1.0. This demonstrated that otolith increments in this species were formed daily and can be used for daily age determination.     

Otolith microstructure and daily age of Anguilla japonica, Temminck & Schlegel elvers from the estuaries of Taiwan with reference to unit stock and larval migration

To test the hypothesis that the Japanese eel, Anguillu japonica , elvers in the eastern and western coasts of Taiwan are recruited from two different spawning grounds and to increase the knowledge of the early life history of the eel, the otolith microstructure and daily age of elvers collected from five estuaries in the coast of Taiwan during December 1989 through February 1990 were examined by scanning electron microscopy. The total length of elvers at arrival in the estuaries was similar among estuaries, averaging c . 56.0 mm and showed a seasonal decrease. The maximum radius of the sagittal otolith of elvers ranged from 124.46 to 181.82μm with a mean of 143.15 ± 12.72 μm. The otolith from centre to edge included an organic-rich primordium (9.20 ± 2.02 μm in diameter), a diffusively calcified core (20.94 ± 1.99 μm), and the daily growth increments; these three layers were probably deposited during the embryonic, yolk sac and feeding period respectively. The growth rate of the otolith was higher at the beginning of early life (0.5−1.0 μm day ⁻¹), lowest at approximately 100-days old (<0.5 μm day⁻¹), and highest 1 month before arrival at the estuary (> 1.0 pm day⁻¹). The mean age for elvers arriving in the estuaries of the coasts of Taiwan was approximately 170.4 ± 21.02 days. Neither the growth pattern of the otolith nor the age of elvers arriving in the estuary were significantly different among estuaries, indicating that the elvers in both eastern and western Taiwan were probably recruited from the same spawning ground. The growth pattern of the otolith in relation to larval migration was analysed.     

Growth increments and biomineralization process in cephalopod statoliths

A study on morphological, structural and biochemical composition of Sepia officinalis and Loligo vulgaris statoliths and statocyst endolymph was undertaken with the aim of determining the major factors affecting the deposition process of statolith formation and to clarify the cause for the poor definition of the growth increments in S. officinalis statoliths. It is suggested that the different biochemical composition of the statocyst endolymph found in the two species accounts for distinct statolith crystallisation processes, which results in a different microstructure. This explains the better definition of growth increments in L. vulgaris statoliths comparing with those of S. officinalis. The protein content as well as Ca(2+) and Mg(2+) concentrations in the endolymph are more implicated in growth increments formation than Sr(2+) ion concentration. Moreover, the daily variations of the three factors mentioned, allowed us to formulate a working hypothesis to explain the daily deposition of growth increments: a dark ring (rich in organic matter) is deposited during daylight whereas a light ring (rich in CaCO(3)) during darkness. These results are discussed in the light of alternative hypotheses explaining the deposition mechanisms in statoliths.     

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.     

Daily growth increments in the larval otolith of the Japanese eel,Anguilla japonica

Early formation of otolith was studied on artificially hatched larvae of the Japanese eel,Anguilla japonica. Newly hatched larvae had a pair of sagittae which were flat and subelliptical with 8.3 μm in mean diameter. The diameter of the sagitta increased linearly with age. No growth increments were observed in the sagitta at hatching, while larvae which were 2, 4 and 6 days old had on average 2.1, 3.6 and 6.0 increments, respectively. The number of the increments (Y) and the age in days after hatching (X) showed a close linear relationship (Y=0.96X+ 0.06, r = 0.913, n = 40), suggesting daily deposition of sagittal increments. In 95 % of the field-caught elvers of this species, a distinct dark ring (check) with the diameter of 6–12 μm was found around the nucleus of the sagitta. This seems to be a “hatch check” deposited at hatching, since its diameter roughly agreed with that of the sagitta in the newly-hatched larvae. Possibly, the number of the increments outside the hatch check represents the age of the fish in days.     

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.