Validation of first annulus formation in red snapper otoliths with the use of an alizarin complexone fluorescent marker

The red snapper (Lutjanus campechanus) is among the most studied reef fish species in the Gulf of Mexico. Several studies have used counts of annuli in sectioned sagittal otoliths to age red snapper. However, interpretation of the putative first annulus has been a major source of debate among otolith readers throughout the Gulf of Mexico. Our objective was to use the chemical marker alizarin complexone to validate the periodicity of first opaque annulus formation in red snapper otolith sections. Juvenile red snapper were immersed in 100 mg alizarin complexone per L seawater solution in November 2005 and then reared in 6000 l circular tanks until July 2006. Otoliths were then removed from the fish and thin sectioned. All experimental otolith sections displayed a distinct fluorescent mark ranging from 0.62 to 0.96 mm from the core when viewed under the microscope with a rhodamine filter. The diffuse opaque annulus was located distally to the alizarin mark in all specimens (ranging from 0.88 to 1.51 mm). The distal position of the presumptive first annulus relative to the alizarin mark in all specimens indicates that this diffuse opaque annulus in red snapper sectioned otoliths forms during the first winter after hatching. Translucent marginal edges of all otolith sections indicate that first opaque annulus formation is completed by mid-July.     

Age validation,and comparison of otolith,vertebra and opercular bone for estimating age of <Emphasis Type="BoldItalic">Schizothorax o’connori</Emphasis> in the Yarlung Tsangpo River,Tibet

A collection of 514 Schizothorax o’connori was made between August 2008 and August 2009 from Yarlung Tsangpo River to assess the suitability of three bony structures for age estimation. The annulus characteristics of otolith, vertebra and opercular bone were described. Location of the first annulus was validated by daily growth increment (DGI) analysis in the otoliths. Annual periodicity was verified by marginal increment ratio (MIR) analysis in otoliths and edge analysis in vertebrae and opercular bones. Annuli formed, once a year, between March and May for all three bony structures. Otoliths, vertebrae and opercular bones were examined to determine which structure produced the most precise and accurate age estimates in S. o’connori. Vertebrae and otoliths matched closely for the first 21 years of life, while opercular bones appeared to underestimate age. For older fish, the counts diverged and otoliths consistently providing higher age estimates. Sectioned otoliths proved to be the most precise and accurate structure for age estimation. The oldest observed schizothoracine fish was 50, more than twice the longevity previously accepted in S. o’connori.     

A comparison of different structures and methods for estimating age of northern-form Dolly Varden <Emphasis Type="Italic">Salvelinus malma malma</Emphasis> from the Canadian Arctic

Assessment of anadromous northern-form Dolly Varden Salvelinus malma malma in the Western Canadian Arctic requires reliable methods for estimating ages. Additionally, conservation efforts warrant determining whether fin rays provide a non-lethal alternative to otoliths. Precision and bias of whole and sectioned otoliths, and sectioned pectoral and pelvic fin rays were examined. Two age readers with different levels of experience ageing this species read each structure three times. Coefficient of variation (CV) was calculated to measure precision, and age bias plots were created for each method of preparation/structure within and between readers. The experienced reader demonstrated the highest precision with sectioned otoliths (CV = 1.6 %) followed by whole otoliths (CV = 4.2 %) while pectoral and pelvic fins were the lowest, CV = 7.7 % and 7.5 %, respectively. The age bias plot showed little difference between whole and sectioned otoliths, although greater imprecision/bias was evident for whole otoliths at age ≥9. Compared to otoliths, fin rays produced younger age estimates starting at 5 years; however, pelvic fins were more biased towards younger estimates than pectoral fins. The less experienced reader had greater inconsistencies, tending to overage younger and underage older samples for all methods compared to the more experienced reader, underscoring the importance of experience when estimating age for this species. We conclude that both types of fin rays are a poor non-lethal alternative to otoliths for fish ≥5 years and recommend an experienced ager could use whole otoliths up to age 8 and sectioned otoliths for fish ≥9 years (>500 mm fork length).     

Ring formation on otoliths and scales of Pagrus pagrus: a comparative study

One annulus formed on the scales of farmed red porgy Pagrus pagrus each year during March and one opaque zone on the otoliths during June. Scales proved to be more sensitive than otoliths in recording the life history of the red porgy. A false annulus (ring of capture) was observed on the scales of the 0+ age group, but not on the otoliths. A gradual lengthening of the period of annulus formation was observed, from the younger to the older fish. The annulus formation in fish older than 2+ was less synchronized, due to the aquaculture conditions. Scales were more accurate than otoliths in annuli formation. A high percentage of missing or additional opaque zones were observed on otoliths of older fish. These irregularities were related to fish maturation and/or farming conditions. Farmed fish grew three times as fast as the wild fish.     

Validation of annual periodicity in otoliths of red snapper, <Emphasis Type="Italic">Lutjanus campechanus</Emphasis>

The periodicity of otolith growth increments (opaque and translucent zones) from adult red snapper (Lutjanus campechanus) was examined through a mark and recapture study (2005–2010), and laboratory holding of hatchery reared red snapper over a 2 year period (2002–2004). Wild red snapper (n = 295) were caught hook-and-line, marked with anchor tags, injected with oxytetracycline dihydrate (OTC), and released in the Gulf of Mexico 15–40 km south of Dauphin Island, Alabama. Marked fish were recaptured up to 2.8 years after release (n = 35) and sagittal otoliths were dissected, sectioned and examined under white and blue-violet light. The number of opaque growth zones past the OTC mark was compared to time at liberty for each fish and supported an annual periodicity of growth increment formation. Also, most (87%) of the hatchery reared fish showed two opaque zones that supported an annual increment formation rate. However, an unusual timing of opaque zone formation was shown for mark-recaptured fish. Based on known timing of OTC marking, otoliths from mark-recapture fish showed opaque zone formation from late summer (August) to early winter (December). This fall formation of opaque zones is in contrast to previous studies and its timing may relate to the end of spawning for this species.     

Age determination, bomb-radiocarbon validation and growth of Atlantic halibut (Hippoglossus hippoglossus) from the Northwest Atlantic

Atlantic halibut (Hippoglossus hippoglossus) is the largest and one of the most widely-ranging and commercially-valuable groundfish in the Atlantic Ocean. Although presumed to be long-lived, their age and growth has not been validated. Ages were estimated by counting growth increments from approximately 2400 thin-sectioned sagittal otoliths collected from the Scotian Shelf and southern Grand Banks off eastern Canada. The accuracy of age estimates made from otolith thin sections was validated using bomb-radiocarbon assays of 13 otolith cores whose year of formation ranged from 1949 to 1975, encompassing the timeframe of the global radiocarbon pulse. Known-age juvenile halibut from a culture facility were used to identify the approximate location of the first annulus. Growth rate for males and females was similar up to about 70 cm (~5 years), after which point male growth slowed, while female growth continued to an age of up to 38 years and a maximum observed size of 232 cm. Males grew to an observed maximum length of about 175 cm and a maximum age of 50 years. A comparison of age estimates for otoliths collected in a ‘historic’ time frame (1963 to 1974) with those from recent years (1997 to 2007) shows that growth rate has not changed appreciably between the two time periods. Small but significant growth differences were observed between the Scotian Shelf and southern Grand Banks for both sexes, while large differences in length at age were observed between halibut caught with longline compared to otter trawl due to differences in length-based gear selectivity. Age interpretations based on sectioned otoliths tended to be 10–15% different than those based on break and burn, although the age comparison was confounded by other variables and must be considered provisional. Atlantic halibut is a long-lived fish, living up to at least 50 years, an important consideration for the management of the fishery.     

Validation of annual growth bands in the otolith of tropical parrotfishes (Scarus schlegeli Bleeker)

The otoliths of the scarid Scarus schlegeli (Bleeker) from the Lizard Island on the northern Great Barrier Reef showed a series of clear periodic bands. The rate at which bands are deposited on the otoliths was distinguished by mark-recapture experiments using tetracycline and marginal increment analysis. Mark-recapture experiments were carried out both in the field and in a large aquarium system. One otolith band was deposited during the liberty periods of July 1990 to April and September 1991 while two similar bands were formed from November 1989 to February 1991. The marginal increment analysis indicated that annual bands were formed around the austral summer. Scales also displayed regular period check marks. These provided relatively reliable aging estimates for the younger fishes up to 5 years.     

Scanning electron microscopic analysis of annulus microstructure in otolith of European eel, Anguilla anguilla

To validate the yearly periodicity of annulus formation in the otolith of the eel, the structure of annuli in otoliths of the European eel, Anguilla anguilla , stocked for 7 and 12 years in Lake Ommen on the east coast of southern Sweden, was examined. The population was stocked from elvers imported both from France (Bay of Biscay) in April 1979 and England (River Severn) in March–April 1984. The microstructure of an annulus consisted of single, double and/or composite tings depending on the location in the otolith. The counts of annuli in otoliths of these eels were approximately consistent with the expected age. However, supernumerary false annuli and/or annulus underestimation frequently occurred. The methodology for annulus discrimination with light and scanning electron microscopes is described.     

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.     

Metabolism,swimming performance,and tissue biochemistry of high desert redband trout (Oncorhynchus mykiss ssp.): evidence for phenotypic differences in physiological function

Redband trout (Oncorhynchus mykiss ssp.) in southeastern Oregon inhabit high-elevation streams that exhibit extreme variability in seasonal flow and diel water temperature. Given the strong influence and potential limitations exerted by temperature on fish physiology, we were interested in how acute temperature change and thermal history influenced the physiological capabilities and biochemical characteristics of these trout. To this end, we studied wild redband trout inhabiting two streams with different thermal profiles by measuring (1) critical swimming speed (U(crit)) and oxygen consumption in the field at 12 degrees and 24 degrees C; (2) biochemical indices of energy metabolism in the heart, axial white skeletal muscle, and blood; and (3) temperature preference in a laboratory thermal gradient. Further, we also examined genetic and morphological characteristics of fish from these two streams. At 12 degrees C, maximum metabolic rate (Mo2max) and metabolic power were greater in Little Blitzen redband trout as compared with those from Bridge Creek (by 37% and 32%, respectively). Conversely, Bridge Creek and Little Blitzen trout had similar values for Mo2max and metabolic power at 24 degrees C. The U(crit) of Little Blitzen trout was similar at the two temperatures (61+/-3 vs. 57+/-4 cm s(-1)). However, the U(crit) for Bridge Creek trout increased from 62+/-3 cm s(-1) to 75+/-3 cm s(-1) when water temperature was raised from 12 degrees to 24 degrees C, and the U(crit) value at 24 degrees C was significantly greater than for Little Blitzen fish. Cost of transport was lower for Bridge Creek trout at both 12 degrees and 24 degrees C, indicating that these trout swim more efficiently than those from the Little Blitzen. Possible explanations for the greater metabolic power of Little Blitzen redband trout at 12 degrees C include increased relative ventricular mass (27%) and an elevation in epaxial white muscle citrate synthase activity (by 72%). Bridge Creek trout had 50% higher lactate dehydrogenase activity in white muscle and presumably a greater potential for anaerobic metabolism. Both populations exhibited a preferred temperature of approximately 13 degrees C and identical mitochondrial haplotypes and p53 gene allele frequencies. However, Bridge Creek trout had a more robust body form, with a relatively larger head and a deeper body and caudal peduncle. In summary, despite the short distance ( approximately 10 km) and genotypic similarity between study streams, our results indicate that phenotypic reorganization of anatomical characteristics, swimming ability at environmentally pertinent temperatures and white axial muscle ATP-producing pathways occurs in redband trout.     

Age validation of juvenile Notothenia rossii at Potter Cove, South Shetland Islands, using mark-recapture data

Among all validation methods of age determination in fish, release of known age and marked specimens gives the most reliable information. We carried out a tag-recapture experiment on Notothenia rossii at Potter Cove, to validate, for first time for this species using this method, the principle of annual deposition of an annulus in scales and otoliths. Of 132 juvenile specimens (TL = 22.1–38.1 cm) tagged and released in successive years from 2004 to 2010, 7 were recaptured at the same site after periods of 1–13 months. In scales of five specimens recovered after 10–13 months, one extra annulus was laid down, exhibiting an additional winter zone of closely spaced sclerites. Consistently, the same analysis in two individuals marked and recaptured during the same summer, after 1–3 months at liberty, did not show the deposition of an additional annulus. All the fish tagged or recaptured during the experiment period (December to March) showed in their scales an edge zone of widely spaced sclerites, in agreement with the known pattern of growth in summer. Likewise, an analysis in selected specimens showed good consistency between the numbers of sclerites deposited in scales and the time of fish release. The comparative analysis between scales taken at recapture and otoliths of the same individual allowed a simultaneous counting of the annuli with complete correspondence. The growth in length of fish ranged from 0.5 to 6.1 cm, depending on the time of release.     

Sympatric relationship between redband trout and non-native brook trout in the Southeastern Oregon Great Basin

Brook trout (Salvelinus fontinalis) and rainbow trout (Oncorhynchus mykiss) have been widely introduced outside their respective ranges within North America causing declines and displacement of native trout. Yet, successful coexistence of native and non-native trout has received little attention. Here we evaluated the effect of introduced brook trout on the size and density of native redband trout in two invaded sub-basins in southeastern Oregon. In a multi-year study, we investigated whether habitat and fish communities differed between streams and stream reaches where redband trout were allopatric versus where redband trout were sympatric with brook trout. We hypothesized that redband trout would be less dense and have smaller total length in sympatry with brook trout than in allopatry, but that total trout density would not differ. We investigated whether differences in habitat existed between sympatric and allopatric locations that would indicate differentiation in site level habitat preferences for each trout species. We found that sympatric locations had more wood but similar fish community structure. Mean length and densities of redband trout were higher at allopatric locations. However, in most years at sympatric locations total trout density was twice that of allopatric redband trout sites. Using comparable data from an eastern United States system where brook trout are native, sympatric sites had lower densities of brook trout; however, total trout density did not differ. We conclude that invading trout negatively impact native trout densities; but in southeastern Oregon system the negative impact is minimized.     

Otolith annulus validation and variables influencing false annuli formation in dorsal spines of saugeye

Marginal increment analysis is a common technique for validating formation of a single annual growth ring on an ageing structure. False annuli can form on ageing structures when environmental variables affect growth of a fish, potentially resulting in age estimation bias. Therefore, validating ageing structures is essential to ensure that accurate and precise age estimates are collected when assessing fish population dynamics. Saugeye (Sander vitreus, [Mitchill, 1818]) and S. Canadensis, [Griffith and Smith, 1834]) are highly valued sport fish that are stocked across the Midwest United States. Using marginal increment analysis, we confirmed that a single annulus was formed yearly in otoliths of juvenile saugeye, however two annuli formed in dorsal spines in a single year. Timing of the first annulus formation in both otoliths and dorsal spines was completed after a slow growth period during winter (otoliths forming in April; dorsal spines forming in March). The second annulus (false annulus) that formed during August in dorsal spines did not form in otoliths. To understand what environmental factors may influence the false annulus to form, we collected monthly water temperatures and percent empty stomachs of juvenile saugeye. The highest water temperatures of the year occur during July and August, which resulted in saugeye seeking thermal refuge and affecting feeding habits. Mean monthly temperature and percent empty stomachs were positively correlated, so during times of high temperatures foraging rates declined, suggesting the formation of false annulus on dorsal spines of juvenile saugeye. This study demonstrates how thermal stress affected accuracy of non‐lethal aging structures and further emphasizes the need for age validation studies prior to using non‐lethal ageing structures to estimate age for a particular species from different aquatic systems.     

Individual condition indicators of thermal habitat quality in field populations of redband trout (Oncorhynchus mykiss gairdneri)

We monitored redband trout (Oncorhynchus mykiss gairdneri) physiological status in multiple streams across eastern Oregon, USA that exhibit various summer thermal profiles. Summer river temperatures were higher overall in 2009 than in 2010. Heat shock protein 70 (hsp70) levels in both liver and fin tissue from redband trout increased with temperature regardless of location. Lipid levels were lower in fish from waters above 23 °C (in terms of degree-day exposure), and lipids were also related to food availability in 2010. Overall, fish ate more in 2009 than in 2010. Fish size increased more rapidly, and lipids were higher and hsp70 was lower in the cooler Crooked River relative to streams in the John Day River system, though the Crooked River did not have the highest food available. While food availability plays a role in physiological health, our data suggest that temperature is the most influential factor driving growth and energy reserves in stream salmonids during high temperature months; we argue that monitoring physiological indicators in individual fish can serve as a useful proxy for overall thermal habitat quality and population status.     

How old are you—Evaluation of age reading methods for the invasive round goby (Neogobius melanostomus,Pallas 1814)

In the Baltic, the first observation of the round goby (Neogobius melanostomus, Pallas 1814) was made in 1990. Within the past decade the species became invasive and spread rapidly throughout the Baltic Sea. Studies about the fishes potential impacts on resident species promote the need for an increasing knowledge of their basic stock structures such as growth rates, longevity and mortality, which all rely on accurate estimates of age. Former studies on the round goby have used several different age reading techniques. In this study, we compared three standard otolith preparation methods for ageing and present the best procedure for the invasive round goby. The results showed significant differences in age estimates of the same fish between the different preparation methods and between readers. The estimation of the first annulus, the first year, was the most problematic. The overall agreement was lowest when reading the whole otoliths while the best performance was achieved with sectioned and stained preparation method. Depending on method used the growth estimates also differed. The results question comparability between previous studies and highlight the importance of harmonised aging procedures for the round goby for obtaining correct estimates of population parameters such as growth rate, age at maturity, and longevity.     

Thermal tolerance and metabolic physiology among redband trout populations in south‐eastern Oregon

Streamside measurements of critical thermal maxima (T_crit), swimming performance (U_crit), and routine (R_r) and maximum (R_max) metabolic rates were performed on three populations of genetically distinct redband trout Oncorhynchus mykiss in the high‐desert region of south‐eastern Oregon. The T_crit values (29·4 ± 0·1° C) for small (40–140 g) redband trout from the three streams, and large (400–1400 g) redband trout at Bridge Creek were not different, and were comparable to published values for other salmonids. At high water temperatures (24–28° C), large fish incurred higher metabolic costs and were more thermally sensitive than small fish. U_crit(3·6 ± 0·1 L_F s^?1), R_r(200 ± 13 mg O₂ kg^?0·830 h^?1) and metabolic power (533 ± 22 mg O₂ kg^?0·882 h^?1) were not significantly different between populations of small redband trout at 24° C. R_max and metabolic power, however, were higher than previous measurements for rainbow trout at these temperatures. Fish from Bridge Creek had a 30% lower minimum total cost of transport (C_min), exhibited a lower refusal rate, and had smaller hearts than fish at 12‐mile or Rock Creeks. In contrast, no differences in U_crit or metabolism were observed between the two size classes of redband trout, although C_min was significantly lower for large fish at all swimming speeds. Biochemical analyses revealed that fish from 12‐mile Creek, which had the highest refusal rate (36%), were moderately hyperkalemic and had substantially lower circulating levels of free fatty acids, triglycerides and albumin. Aerobic and anaerobic enzyme activities in axial white muscle, however, were not different between populations, and morphological features were similar. Results of this study: 1) suggest that the physiological mechanisms that determine T_crit in salmonids are highly conserved; 2) show that adult (large) redband trout are more susceptible to the negative affects of elevated temperatures than small redband trout; 3) demonstrate that swimming efficiency can vary considerably between redband trout populations; 4) suggest that metabolic energy stores correlate positively with swimming behaviour of redband trout at high water temperatures; 5) question the use of T_crit for assessing physiological function and defining thermal habitat requirements of stream‐dwelling salmonids like the redband trout.     

Validation of the periodicity of increment formation in the otoliths of a cichlid fish from Lake Tanganyika, East Africa

Tetracycline was used as a chemical tag in a mark‐recapture study to examine the pattern of increment formation in the otoliths of Tropheus moorii , a rock‐dwelling cichlid from Lake Tanganyika. A total of 256 fish were captured by divers and injected with tetracycline. Of these, nine were recaptured after either 1 or 2 years at liberty and eight retained tags within their otoliths. Comparison of the number of growth increments formed after the tag and the time at liberty demonstrated that increments were deposited on an annual basis in the otoliths of this species. Furthermore, there was a strong relationship between otolith mass and age suggesting that otoliths grew at a predictable rate throughout the life of the fish. Validation of an annual pattern of increment deposition allowed age and growth information to be derived from otoliths. This showed that T. moorii grew rapidly to attain adult size by 3 years of age. Males grew faster than females and also attained a larger size than females (8·74 v . 7·91 cm L _S respectively). The longevity of some of these small freshwater fish was surprising; the oldest individual had an age of 10 years, while the average age of adults was 4 years.     

Adaptation of redband trout in desert and montane environments

Natural populations that evolve under extreme climates are likely to diverge because of selection in local environments. To explore whether local adaptation has occurred in redband trout (Oncorhynchus mykiss gairdneri) occupying differing climate regimes, we used a limited genome scan approach to test for candidate markers under selection in populations occurring in desert and montane streams. An environmental approach to identifying outlier loci, spatial analysis method and linear regression of minor allele frequency with environmental variables revealed six candidate markers (P < 0.01). Putatively neutral markers identified high genetic differentiation among desert populations relative to montane sites, likely due to intermittent flows in desert streams. Additionally, populations exhibited a highly significant pattern of isolation by temperature (P< 0.0001) and those adapted to the same environment had similar allele frequencies across candidate markers, indicating selection for differing climates. These results imply that many genes are involved in the adaptation of redband trout to differing environments, and selection acts to reinforce localization. The potential to predict genetic adaptability of individuals and populations to changing environmental conditions may have profound implications for species that face extensive anthropogenic disturbances.     

长江口刀鲚耳石年轮确证和年龄与生长研究

洄游性刀鲚是长江中下游重要的渔业资源,20世纪70年代以来,其资源量严重下降。研究以耳石为年龄鉴定材料,对长江口刀鲚渔获物的年龄结构和生长进行研究,主要目的在于确证刀鲚耳石上年轮,分析刀鲚渔获物的年龄结构和生长特征,评价刀鲚资源状况。2006年4月至2007年8月在长江口逐月采样,共采集了576尾洄游性刀鲚。对矢耳石横截面分析发现,从耳石核出发,腹侧生长轴具有周期性的直-弯生长模式,直线生长发生在4到12月,弯曲生长在10月到第二年的6月;边缘轮纹增长率分析也表明,这种直-弯生长模式具有年周期性,可以用来鉴定年龄。弯曲生长向下一个直线生长的转换发生在4月至6月间,将此转换定义为年轮标志。年龄分析表明,长江口刀鲚渔获物包括0龄至4龄共五个年龄组,以1龄和2龄个体为主,雌雄个体年龄组成相似,生长趋势也比较接近,拟合的von Bertalanffy生长方程为SL=327.691-e-0.51(t+0.28)(n=576,r2=0.71,P3.36(n=576,r2=0.95,P<0.05)。与20世纪70年代渔获物调查结果相比,现在长江口刀鲚种群明显低龄化和小型化,表明刀鲚资源已严重过度利用。降低刀鲚的捕捞强度是刀鲚资源保护和恢复的基础。       

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.