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.     

Daily growth increments in otoliths of wild-caught honmoroko Gnathopogon caerulescens

Otolith growth increments in wild-caught alizarin complex one (ALC)-marked honmoroko Gnathopogon caerulescens were examined to verify the veracity of the age determination method in cyprinids. ALC-marked G. caerulescens recaptured from their natural environment had lapilli increment counts outside the ALC ring mark that had formed on a daily basis during the juvenile stage. This apparently being the first direct evidence of daily periodicity of otolith increment formation in wild-caught cyprinids.     

Daily growth increments in the otoliths of the three-spined stickleback, Gasterosteus aculeatus L.

Daily increment formation was demonstrated in the sagittae of the three-spined stickleback, Gasterosieus aculeatus L., under both laboratory and field conditions. The onset of daily increment formation coincided with the day of hatching. The main sources of inaccuracy in age determination resulted from difficulties in resolving narrow increments in the sagittae of fish held at low temperatures and the increasing incidence of checks with age. The advantage of daily increments for investigating growth in this species was demonstrated from a comparison between increment-derived age-at-length data and length-at-capture data, since the latter significantly under-estimated early growth rate within a population.     

Daily growth increments in the otoliths of juvenile greenback flounder, Rhombosolea tapirina Gunther, 1862

Otolith check marks of both tetracycline-treated and control juvenile greenback flounders, Rhombosolea tapirina, were attributed to the stress of transfer between aquarium tanks. Subsequent counts showed that otolith increments were deposited daily.     

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.     

草鱼仔鱼耳石的自然标记和生长轮的清晰度

将野生和人工繁殖的草鱼仔鱼的耳石取出并置于显微镜下观察微结构特征。结果表明 :草鱼耳石一般有一个圆形或卵圆形的原基和中心核 ,但有 0 4 1%～ 4 6 7%的样本具有双原基或双中心核。343尾野生仔鱼中 ,6 71%的个体在矢耳石和微耳石上具有营养转换标记 ,而 187尾人工繁殖的仔鱼中 ,在矢耳石和微耳石上出现营养转换标记的比例分别是 6 4 17%和 5 0 80 % ;在营养转换标记处 ,矢耳石和微耳石的直径分别为 5 4 12±9 4 9μm和 4 0 4 8± 7 0 2 μm (n =5 0 ) ;133尾野生仔鱼在转入实验室饲养的过程中 ,86 4 7%的个体在耳石上形成了转移标记 ;野生仔鱼生长轮纹清晰的矢耳石 (n =5 2 1)和微耳石 (n =5 2 1)样本的比例分别低于 10 %和2 5 % ,但在人工饲养仔鱼中 ,95 0 0 %个体的矢耳石 (n =186 )和 88 0 0 %个体的微耳石 (n =184 )具有清晰的生长轮纹 ;野生仔鱼经人工饲养后 ,其耳石上在饲养期间沉积的生长轮的清晰度亦明显比在野外生存期间沉积的高 ;对比实验显示饥饿对仔鱼耳石生长轮的清晰度没有明显的影响.     

Daily growth increments in the otoliths of Atlantic salmon parr, Salmo salar L., and the influence of environmental factors on their periodicity

Daily increments were demonstrated in the sagitta otoliths of fast- and slow- growing Atlantic salmon parr, Salmo salar L., when held under natural photoperiod and temperature. Otolith increments continued to be deposited at a daily rate when fish were held under constant light and/or temperature and on single or multiple feeding regimes. However abnormally short photoperiods of 6L: 6D induced two increments per day. The results suggest that an endogenous rhythm, synchronized lo light/dark transitions within a 24 h period, controls otolith increment deposition.     

Effects of starvation on the formation of daily growth increments in the otoliths of milkfish, Chanos chanos (Forsskål), larvae

The effects of starvation on daily growth and increment formation in the otolith were examined using a double oxytetracycline-labelling method on larval milkfish, Chanos chanos (Forsskål), reared under different feeding regimes. The results indicated that the differences in body and otolith growth between the larvae fed once and three times a day were not significant, and that the otolith growth increment was deposited daily in both groups of fed larvae. In contrast, the starved larvae grew at a slower rate than fed larvae in body length and otolith dimensions, and the otolith growth increment in the starved larvae was not deposited on a daily basis. After undergoing starvation, the larvae were unable to recover their normal growth either in otolith increment deposition or in body and otolith growth even though they were fed. Therefore, the application of ageing techniques based on counting otolith growth increments seems to be inaccurate for starved larvae.     

Validation of daily increment deposition and early development in the otoliths of Sarotherodon melanotheron

A one-to-one relationship was found between days of rearing and counted daily increments in the otoliths of Sarotherodon melanotheron verifying daily increment deposition. A marked hatch check was found in all otoliths from both the reared fish and the wild specimens. Five to 10 faint prolarval increments were visible but the rate of their formation was not investigated. The rate of increment deposition of S. melanotheron is apparently independent of somatic growth and increments found in the otoliths of this species can therefore be used for ageing.     

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.     

Validation of otolith daily increment formation for two temperate syngnathid fishes: the pipefishes Stigmatopora argus and Stigmatopora nigra

Otoliths were used for the first time to successfully validate the age of members of the family Syngnathidae: the spotted pipefish Stigmatopora argus and the wide-bodied pipefish Stigmatopora nigra. Otolith increments were deposited daily in (1) known-age juveniles ranging in age from 0 to 31 days and (2) adults that had been stained with alizarin complexone, and a hatch mark was found on all otoliths which represented day 0. Otolith increment validation will allow development of growth models for S. argus and S. nigra, essential to understanding and managing these exclusive seagrass species.     

Preparing sagittae for examination of daily growth increments of young‐of‐the‐year fishes: a modification of the embed method

A modification (termed the slide‐glass‐embed‐method, SGEM) of the embed method for preparing fish sagittae is described. The SGEM is based on a very simple principle: a dome of mixed resin containing the embedded sagittae loses hardness after being heated and can be easily cut with dissecting scissors.     

Daily increments on the otoliths of larval and juvenile Coregonus spp., and their modification by environmental factors

The sagittal otoliths of pre- and posthatch embryos, larval, and juvenile coregonids (Coregonus spp.) were examined for growth increments. Under laboratory conditions, a check is formed on the day of hatching and subsequently one growth increment per day is deposited during at least 265 days. Under the experimental conditions of this study, the age of young coregonids can therefore be determined with high accuracy.In starving larvae, both increment width and ring contrast decrease during the first 10 days after hatching until daily increments are no longer recognizable. A change from one diet to another, alteration of the water temperature, or a short starvation period lead to the formation of characteristic ring patterns which appear on the otoliths within 1 to 3 days. These patterns are highly reproducible among all specimens of each treatment group and can therefore be used as intrinsic marks. They could be applied to hatchery-reared coregonids, thus providing a basis for judging the efficacy of stocking operations.     

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.     

Evaluation of some morphological characteristics of spores of two species of Microsporida by scanning electron microscope and freeze-etching techniques

Scanning electron microscopy revealed spores of Nosema apis and Thelohania fibrata to be egg-shaped, but only the mature spore of T. fibrata was shown to possess a horseshoe-like concavity at the posterior pole. Freezeetched preparations indicated that this concavity was due to a thin area of the spore coat. Freeze-etching studies also show spores of N. apis do possess an umbrella-shaped polaroplast, and a polar filament which is arranged in a double layer with over 30 coils. The spore of T. fibrata contains a pear-shaped arrangement of the polaroplast membrane, and a polar filament arranged in a single layer of 22 coils.     

Relating annual increments of the endangered Blanding's turtle plastron growth to climate

This research is the first published study to report a relationship between climate variables and plastron growth increments of turtles, in this case the endangered Nova Scotia Blanding's turtle (Emydoidea blandingii). We used techniques and software common to the discipline of dendrochronology to successfully cross‐date our growth increment data series, to detrend and average our series of 80 immature Blanding's turtles into one common chronology, and to seek correlations between the chronology and environmental temperature and precipitation variables. Our cross‐dated chronology had a series intercorrelation of 0.441 (above 99% confidence interval), an average mean sensitivity of 0.293, and an average unfiltered autocorrelation of 0.377. Our master chronology represented increments from 1975 to 2007 (33 years), with index values ranging from a low of 0.688 in 2006 to a high of 1.303 in 1977. Univariate climate response function analysis on mean monthly air temperature and precipitation values revealed a positive correlation with the previous year's May temperature and current year's August temperature; a negative correlation with the previous year's October temperature; and no significant correlation with precipitation. These techniques for determining growth increment response to environmental variables should be applicable to other turtle species and merit further exploration.     

Water temperature and fish growth: otoliths predict growth patterns of a marine fish in a changing climate

Ecological modeling shows that even small, gradual changes in body size in a fish population can have large effects on natural mortality, biomass, and catch. However, efforts to model the impact of climate change on fish growth have been hampered by a lack of long‐term (multidecadal) data needed to understand the effects of temperature on growth rates in natural environments. We used a combination of dendrochronology techniques and additive mixed‐effects modeling to examine the sensitivity of growth in a long‐lived (up to 70 years), endemic marine fish, the western blue groper (Achoerodus gouldii), to changes in water temperature. A multi‐decadal biochronology (1952–2003) of growth was constructed from the otoliths of 56 fish collected off the southwestern coast of Western Australia, and we tested for correlations between the mean index chronology and a range of potential environmental drivers. The chronology was significantly correlated with sea surface temperature in the region, but common variance among individuals was low. This suggests that this species has been relatively insensitive to past variations in climate. Growth increment and age data were also used in an additive mixed model to predict otolith growth and body size later this century. Although growth was relatively insensitive to changes in temperature, the model results suggested that a fish aged 20 in 2099 would have an otolith about 10% larger and a body size about 5% larger than a fish aged 20 in 1977. Our study shows that species or populations regarded as relatively insensitive to climate change could still undergo significant changes in growth rate and body size that are likely to have important effects on the productivity and yield of fisheries.     

Biological applications of the scanning transmission electron microscope

The STEM has the capability to acquire spatial and chemical information from almost all scattered electrons. Dedicated STEMs do not have any post-specimen lenses but many different detectors instead.The STEM axial bright field mode is quite insensitive to multiple scattering occurring while imaging thick samples such as vitrified whole cells. The blur of small gold sphere has been simulated by Monte Carlo calculations.Direct electron detectors record the rich information of microdiffraction patterns from every irradiated spot while the STEM beam raster-scans over the sample.

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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.