An accurate tool for ageing Two‐spined Blackfish for use in determining timing of spawning in upland streams in the Brindabella ranges,ACT, Australia

To manage populations of threatened fish species in modified habitats and regulated rivers requires an understanding of their reproductive biology and spawning cues. In particular, accurate information about early life stages in these species can be used to facilitate programmes that maximise recruitment and breeding success. This study aimed to develop methods to accurately age early juvenile freshwater native fish, Two‐spined Blackfish, (Gadopsis bispinosus), to allow the determination of spawning date. This was accomplished through the examination of otolith microstructure in early juveniles. The age at which the first ring was deposited and the relationship between days and number of rings were determined using both field and aquarium trials. Field trials of marked juvenile otoliths revealed daily deposition of rings (1.02 ± 0.02 rings per day for fish sacrificed six days postmarking). The strength of this relationship lessened slightly as juveniles aged (0.92 ± 0.02 rings per day for fish sacrificed 13 days postmarking). The first otolith ring was deposited 7.50 ± 1.09 days after spawning. The enumeration of daily rings combined with knowledge of the commencement of ring deposition enabled accurate estimation of spawning date. The current study is the first to examine otolith microstructure in juvenile Two‐spined Blackfish allowing accurate determination of spawning date. While more research is required to accurately age older juveniles, this technique has the potential to precisely correlate spawning with environmental cues, facilitating better management of temperature and flow during breeding periods, potentially increasing spawning and recruitment of this endangered species.     

Rhythmic structural and chemical patterns in otoliths of the Antarctic fish Notothenia larseni: Their application to age determination

Summary Sagittal otoliths from Notothenia larseni contain microincremental growth rings which are distinctly visible in otolith sections using Scanning Electron Microscopy. These microincrements are similar to those deposited daily in the otoliths of fishes from temperate and tropical waters. Microincrements were easily enumerated and fish length was related to increment number by a logarithmic curve. Otolith microstructure analysis appears to provide a technique to accurately determine age and growth rates in these fish. Sr/Ca ratios in otolith aragonite were analyzed along a radius from the outside edge to the core of an otolith section using an electron microprobe. The strontium/calcium (Sr/Ca) ratios varied with a cyclic periodicity apparently related to seasonal water temperature cycles and the number of cycles agreed closely with age estimated from daily microincrement counts. Sr/ca cycles can potentially be used to determine age, validate growth rates determined by other methods, and establish thermal conditions experienced during the life of a fish. Microstructural and chemical analyses of otoliths demonstrate great potential in helping to answer many questions about the growth processes and ecology of Antarctic fishes.     

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.     

大麻哈鱼胚胎耳石微结构及其群体环境标记

采用人工调控环境方法对黑龙江、绥芬河大麻哈鱼发眼期胚胎群体耳石日轮进行周期性持续标记。实验分４组: 1组为对照组, 2－3组为变温标记组, 4组为“暴气”-变温标记组, 实验用发眼卵1.2万粒。待胚胎发育至耳石日轮结构形成后实施标记。实验胚胎耳石随着标记期间环境周期性变化及其持续的时间, 形成相应变化节律的日轮标记区。获得各实验组设定环境的日轮标记图谱。人工环境标记的耳石日轮图谱, 暗带色度加深, 明带亮度增大, 并可形成生长轮距不同的标记轮, 与对照组耳石微结构有明显区别, 标记率达到100%。初步建立鱼类耳石标记及其识别技术, 适用于大麻哈鱼等鲑鳟鱼类群体标记。作为安全有效、成本低廉的群体标记技术方法, 鱼类耳石日轮标记在鱼类资源评估和增殖放流效果评价中将会得到广泛应用。       

Comparison of otolith growth and morphology with somatic growth and age in young-of-the-year bluefin tuna

Otolith morphological characteristics were studied using image analysis techniques and the relationships between otolith growth and somatic growth and age, as estimated from counting daily otolith increments, were examined in young-of-the-year (YOY) bluefin tuna Thunnus thynnus ranging in fork length ( L _F) from 8·5 to 55·5 cm. Whole otolith length, width, area and perimeter, and three shape indexes, circularity, E value and rectangularity, were extracted for each pair of sagittae. Since no statistical significant differences between left and right otolith morphometrics were found, only one otolith from each fish was used for correlations. Statistically significant relationships were observed between otoliths measurements and fish somatic growth when a linear regression was applied after logarithmic transformation of all variables tested. Among the variables, otolith length was the one that showed the highest correlation with L _F, followed by otolith area and perimeter, whereas otolith rectangularity exhibited the lowest correlation. Statistically significant relationships were also observed between the otolith variables tested and the age of the fish, which ranged from 20 to 129 days. The ages estimated using otolith mass were very close to those assessed using daily increment counts (bias ranged from 1 to 24 days). Therefore, otolith mass could represent a valuable criterion for age estimation in YOY bluefin tuna that is objective, economic and easy to perform compared to daily increment counting method.     

Immunohistochemical localization of two otolith matrix proteins in the otolith and inner ear of the rainbow trout,<Emphasis Type="Italic"> Oncorhynchus mykiss</Emphasis>: comparative aspects between the adult inner ear and embryonic otocysts

The fish otolith consists mainly of calcium carbonate and organic matrices, the latter of which may play important roles in the process of otolith formation. We previously identified two otolith matrix proteins, named otolith matrix protein-1 (OMP-1) and otolin-1, from the rainbow trout, Oncorhynchus mykiss, and the chum salmon, O. keta. In this study, recombinant proteins corresponding to OMP-1 and otolin-1 were synthesized using yeast and bacterial expression systems, respectively, to produce specific antibodies against each protein. Immunohistochemical analysis using these antisera revealed that in the otoliths of adult fish, OMP-1 and otolin-1 were colocalized along the daily rings possibly formed by alternate deposition of calcium carbonate and organic matrices. In the adult inner ear, OMP-1 was produced at most of the saccular epithelium, while otolin-1 was produced at a limited part of cylindrical cells located at the marginal zone of the sensory epithelium. In the embryonic inner ear, these proteins had already existed in the otolith primordia when calcification had commenced. In addition, otolin-1 was localized in the fibrous materials connecting otolith primordia and sensory epithelium at this stage. These results indicate that these proteins are required as essential components for otolith formation and calcification.     

The use of otoliths as indicators of Little Penguin Eudyptula minor diet

The validity of using otoliths from stomach contents quantitatively to determine the number and size of fish consumed was tested on Little Penguins. They were fed different meal sizes of known number and size of fish and the stomach contents were recovered after various time intervals. There were no differences in estimates of original fish size when calculated from otolith length or weight. Rate of digestion of otoliths tended to decrease with increased meal size but increased with time after ingestion. Digestion of otoliths proceeds rapidly and, if ignored, estimates of numbers of fish consumed and of original fish size can be significantly underestimated. This problem can be partially solved by inspection of otolith condition and restricting calculations of fish size to otoliths unaffected by digestion. Many factors introduce variations into rate of otolith degradation and further species-specific studies are required before appropriate correction factors can be applied.     

The use of otoliths as indicators of Little Penguin Eudyptula minor diet

The validity of using otoliths from stomach contents quantitatively to determine the number and size of fish consumed was tested on Little Penguins. They were fed different meal sizes of known number and size of fish and the stomach contents were recovered after various time intervals. There were no differences in estimates of original fish size when calculated from otolith length or weight. Rate of digestion of otoliths tended to decrease with increased meal size but increased with time after ingestion. Digestion of otoliths proceeds rapidly and, if ignored, estimates of numbers of fish consumed and of original fish size can be significantly underestimated. This problem can be partially solved by inspection of otolith condition and restricting calculations of fish size to otoliths unaffected by digestion. Many factors introduce variations into rate of otolith degradation and further species-specific studies are required before appropriate correction factors can be applied.     

Three-dimensional imaging of walleye pollock otoliths: reconstruction from serial sections and fluorescent laser cytometry

Two techniques have been developed to examine the three-dimensional internal structure of otoliths. In the first, otoliths were sectioned serially, images were digitized, and the otolith was reconstructed as a computer model. In the second method growth increments were marked in vivo during their formation by immersing the fish in a fluorescent dye, and then the internal structure of the otolith visualized using laser cytometry. The results are useful for evaluating the potential for bias in otolith measurements and for determining the sectional plane with the least bias.     

Age and early life history of juvenile scotia sea icefish, <Emphasis Type="Italic">Chaenocephalus aceratus</Emphasis>, from Elephant and the South Shetland Islands

The otolith microstructure of juvenile Scotia Sea icefish (Chaenocephalus aceratus) was analyzed from samples collected around Elephant and South Shetland Islands, with the aim to validate previous annual ageing and to give new insight into its early life history timings. Fish were caught by bottom trawl fishing conducted on the continental shelf between 100 and 500 m depth. To determine the timing and position of the first annulus on sagittal otoliths, microincrements were counted on juvenile otoliths previously aged 1+ year old by counting annuli in sectioned otolith. Assuming that microincrements were laid down daily, age ranged from 406 to 578 days in fish measuring 13–19 cm TL, thus corroborating previous results. The relationship between fish size and otolith size/weight was estimated using the least square linear regression method. The relationship between age and otolith size was also estimated to determine the otolith length in 1-year old fish, which was approximately 1.58 mm. In all samples the otolith core was characterized by an evident strong check, assumed to be laid down at the beginning of exogenous feeding of yolk sac larvae. The yolk sac duration estimated from hatch to the first feeding check was longer than other channichthyids, lasting 29–45 days. Hatching dates were backcalculated from the date of capture using the age estimates, indicating C. aceratus sampled off Elephant and South Shetland Islands hatched over a long period lasting from July to December, with a peak in November. As a result, the potential larval dispersion driven by local oceanographic features is discussed.     

Mediterranean juvenile bluefin tuna: life history patterns

As there is a lack of information on the growth and migrations of bluefin tuna, information about them was gathered using the structural and chemical characteristics of their otoliths and mercury levels in body tissues as indicators of physiological and habitat characteristics. The otoliths of juvenile tuna caught in the Spanish Mediterranean littoral were studied. Otolith increments, assumed to be formed daily, were enumerated. Measurements by wavelength dispersive electron microprobe confirmed the presence of strontium in otolith tissue, and an inverse relationship between strontium/calcium (Sr/Ca) concentration ratio and temperature is suggested. Electron microprobe analyses combined with daily increment analyses of otoliths provided life history profiles for individual fish. Additional Sr/Ca concentration ratio data on fish supported the idea that Sr/Ca ratios can provide information on the environmental history of individual fish. Body concentrations of mercury were related to otolith analyses to suggest age structure, critical life history periods, growth environment, stock structure, food web position, and migration history. The techniques applied present an innovative approach to management-related problems, and the combination of chemical analyses with structural analyses promises to expand our knowledge of the life history of migratory fishes.     

Assessment of growth zones on whole and thin-sectioned otoliths in <Emphasis Type="Italic">Sperata aor</Emphasis> (Bagridae) inhabiting the River Ganga,India

The present study was undertaken with the objective to assess the clarity of growth zones on whole and thin-sectioned otoliths in Sperata aor. A total of 125 sagittal otoliths of S. aor were collected monthly from the river Ganga during the period, April to December 2013 at Narora, Uttar Pradesh, India. Thin sections (approximately 0.5 mm) of one of the sagittal otoliths of each fish were cut using IsoMet® Low Speed Saw. Both whole otoliths and thin-sectioned otoliths were then examined under stereozoom microscope. Parameters of agreement on growth zones were calculated by comparing the number of growth zones obtained independentlyby the two readers (R1 and R2) from the two methods (whole otolith and thin-sectioned otolith method). Thin-sectioned otolith method exhibited higher agreement than whole otolith method based on linear regression analysis and growth zones bias plot. Between readers, higher agreement was noted for reader 1 than reader 2, plausibly due to his relatively more experience in examining the growth zones on the otoliths. However, both readers reported independently that the growth zones were clearer on thin sections than on whole otoliths especially those from older individuals. Thus, it may be concluded that the thin-sectioned otolith method should be utilized for assessment of growth zones in S. aor populations from the river Ganga.     

The use of sectioned otoliths to age barramundi (Lates calcarifer) (Bloch, 1790) [Centropomidae]

The relationship between the number of rings present in sagittal otoliths and the age of barramundi, Lates calcarifer (Bloch, 1790) [Centropomidae], was investigated by examining cross sectioned otoliths of 37 tagged fish of known age between 1 and 5 years from the Johnstone River, north Queensland. Concentric rings were clearly visible in all otolith sections and were validated as annual marks. The technique was then used to estimate the age and calculate von Bertalanffy growth parameters for 70 barramundi from the Fitzroy River, central Queensland. Growth appeared to be rapid but variable in the first year; the von Bertalanffy growth parameters for length versus age were L =690 mm, K=0.53, t ₀= 0.003 years. October 1 was designated as the birth date. Whole otolith length, width and thickness were also approximated well by the von Bertalanffy equation. We suggest that examination of otoliths is a useful technique for ageing barramundi but note that further validation of the ageing method is still needed for fish older than six years.     

Otolith growth and age estimation in the European hake

The internal sulcal rings in Merluccius merluccius otoliths cannot be considered as annuli. In the absence of a strong seasonal Zeitgeber, hake otoliths in the Mediterranean did not lay down an interpretable ring pattern that would be useful for age determination. A total of 484 sagittal otoliths from specimens ranging between 6 and 94 cm L T was studied in monthly samples from the Gulf of Lions in 1989–1990. Transverse, burnt otolith sections was analysed with an image analysis system using enhanced and filtered images and using a fast Fourier transform (FFT) function to avoid subjectivity in ring interpretation. The otolith radius-fish length relationship showed allometric growth and sexual dimorphism. The ring pattern was consistent for the sulcal rings in both sexes. Changes in the marginal increment showed the formation of multiple sulcal rings, of both environmental and physiological origin. The ring pattern depended on the sex and sexual activity.     

Forensic biological pursuits of exotic fish origins: piranha in Hawaii

Synopsis The otoliths of an adult red-bellied piranha, Pygocentrus nattereri, captured from a reservoir in Hawaii were scrutinized to determine the fish's origin and growth history. Sagittal otoliths of the piranha, P. nattereri, contained internal microincrements visible by Scanning Electron Microscopy. The medial cross sectional plane of the sagitta was resolved to provide counts for the most visible micro-increments. An opportune spawning of confiscated adults provided samples for verification of daily increment formation. Daily formation of microincrements was verified from hatched individuals, and confirmed the suitability of otoliths for revealing daily patterns in the age and growth of piranhas. The central area of the sagitta was diffuse in regards to otolith microstructure and indicated the fish was held in an unchanging environment (aquarium). Therefore, otoliths provide important life history or forensic information incorporated within their structural components. The visualization of daily microincrements in the otolith of a juvenile allowed the determination of age at and since release. Fish grew rapidly after being released into the wild. From otolith increments the date of release for an individual fish can be calculated with acceptable accuracy. As presented, otolith structural information can provide age and growth data which are essential to the management of introduced species.     

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.     

Morphometric analysis of otoliths of juvenile crucifix sea catfish Sciades proops (Valenciennes, 1840)

The objective of this study was to analyze the morphometry of otoliths for Sciades proops juveniles by testing the hypothesis of equality in morphometric relationships for the right and left otoliths, which could then be interchangeably used to estimate fish size or weight. Samples were obtained monthly directly from anglers after each event that took place off the state of Sergipe from March/2014 to April/2015. Anglers used rod and reel during these events, with no restriction on hook size or line thickness. Each fish specimen sampled had their total weight (W, g) and total length (TL, cm) measured and their lapillus otoliths removed and stored separately. Each otolith had its length (OL), width (OWi), and thickness (OT) measured (all in mm) under a stereomicroscope. Otoliths were weighed using a precision scale (OW, g). A total of 883 specimens were sampled: TL = 12.0–60.5 cm and W = 9.8–1880 g. The weight‐length relationship for the juvenile fishes was W = 0.0052TL^3.086 and for their otoliths was OW = 0.0002OL^3.177. The weight‐length and length‐length relationships fitted for each otolith (right and left) were not statistically different and thus all relations were estimated for grouped otoliths. The length‐length relationships for the otoliths were: OWi = 0.947OL?0.205 and OT = O.484OL?0.698. The relationship estimated for juvenile fish and otolith weight was Wj = 1076.1OW?9.120. For juvenile fish total length and otolith length, width and thickness, the following relationships were estimated: TLj = 4.028OL?3.199, TLj = 4.208OWi?2.091, and TLj = 7.824OT + 3.659, respectively. Relationships between fish and otolith size, and between fish and otolith weight indicated a change in slope close to L_m50, which should be better explored when more adult specimens are available.     

Otolith microstructure pattern in Oreochromis niloticus (L.)

The microstructure of otoliths from young and old Oreochromis niloticus (L.) were studied. Otoliths were prepared histologically except for those from newly hatched fish. Hatching results in the formation of a check in the otoliths, which appeared 1 day later. Other checks are rare in juvenile otoliths but common in adult otoliths. Faint and non-daily increments were observed within the hatching check. After hatching, increments were deposited daily. Sub-daily increments were faint and narrow, they were present in the area along the dorso-ventral axis of the otolith but did not continue into the lateral region. Discontinuous zones in the medial area appeared different from those in the lateral area. New growth centres were not only found in the juvenile fish otoliths, but also in adult fish otoliths.     

Within-Otolith Variability in Chemical Fingerprints: Implications for Sampling Designs and Possible Environmental Interpretation

Largely used as a natural biological tag in studies of dispersal/connectivity of fish, otolith elemental fingerprinting is usually analyzed by laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS). LA-ICP-MS produces an elemental fingerprint at a discrete time-point in the life of a fish and can generate data on within-otolith variability of that fingerprint. The presence of within-otolith variability has been previously acknowledged but not incorporated into experimental designs on the presumed, but untested, grounds of both its negligibility compared to among-otolith variability and of spatial autocorrelation among multiple ablations within an otolith. Here, using a hierarchical sampling design of spatial variation at multiple scales in otolith chemical fingerprints for two Mediterranean coastal fishes, we explore: 1) whether multiple ablations within an otolith can be used as independent replicates for significance tests among otoliths, and 2) the implications of incorporating within-otolith variability when assessing spatial variability in otolith chemistry at a hierarchy of spatial scales (different fish, from different sites, at different locations on the Apulian Adriatic coast). We find that multiple ablations along the same daily rings do not necessarily exhibit spatial dependency within the otolith and can be used to estimate residual variability in a hierarchical sampling design. Inclusion of within-otolith measurements reveals that individuals at the same site can show significant variability in elemental uptake. Within-otolith variability examined across the spatial hierarchy identifies differences between the two fish species investigated, and this finding leads to discussion of the potential for within-otolith variability to be used as a marker for fish exposure to stressful conditions. We also demonstrate that a ‘cost’-optimal allocation of sampling effort should typically include some level of within-otolith replication in the experimental design. Our findings provide novel evidence to aid the design of future sampling programs and improve our general understanding of the mechanisms regulating elemental fingerprints.     

Mn<Superscript>2+</Superscript> concentrations in coastal fish otoliths: understanding environmental and biological influences from EPR

The Mn²⁺ concentrations in the sagittae otoliths of 12 fish families (and 19 species) that co-occur in a coastal area of southeastern Brazil (~21°S) were quantified using electron paramagnetic resonance (EPR). Inferences were made about the relationship between fish habitat and trace element incorporation. Inferences were made on the relationship between trace element concentration and otolith shape. The differences in Mn²⁺ concentrations among the species suggest that habitat (and feeding habits) might drive the incorporation of this trace element into fish otoliths, with higher values in bottom-associated fish species than in surface-associated species. In surface-associated fish species, the correlation between trace element concentrations and otolith shape was stronger than in bottom-associated species. Thus, while the Mn bioavailability in a fish’s habitat, especially from feeding resources, is a local driving influence of trace element incorporation in sagittae otoliths, species-specific requirements also have an influence. Quantitative EPR is a non-destructive technique that is very useful when the available samples cannot be damaged, like with otolith collections.