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.     

Measurements on ground or sectioned otoliths: possibilities of bias

Back-calculation usually requires measurements of growth marks revealed on otoliths by specific preparations. The standardization of the grinding (or sectioning) plane is necessary, but difficult, especially along the antero-posterior axis. In order to show the importance of the grinding plane, tetracycline labelling of eel otoliths ( Anguilla anguilla L.) has been used. This marking has a calcio-traumatic effect on otoliths, which can be revealed with staining techniques. Unless the grinding plane is incorrect, the tetracycline labelling and the staining of the ground surface are then superposed.     

Marking otoliths of brown trout (Salmo trutta L.) embryos with alizarin red S

In this study a time‐ and cost effective method for marking early life stages of brown trout (Salmo trutta L.) with alizarin red S (ARS) is presented. The main intention was the development of a protocol to mark embryos. When stocking eyed‐eggs, later recognition depends on an adequate tagging method featuring reasonable expenditures, low mortality and high marking success. Best results were obtained using ARS concentrations of 500 mg/l. Consistently clearly visible marks could be detected under a fluorescence‐stereomicroscope equipped with a dsRed filter, without processing the otoliths.     

Tetracycline marks visible in Baltic cod Gadus morhua otoliths stored for 40 years

Baltic cod Gadus morhua otoliths marked with oxytetracycline (OTC) in 1974–1975 had been sectioned and the sections stored in paper envelopes at room temperature in dry, dark conditions. After 40 years of storage, the historic OTC marks were still clearly visible showing that OTC is suitable for long‐term chemical marking of otoliths. They were, however, noticeably paler than marks in recently recaptured and archived Baltic G. morhua otoliths, chemically tagged with tetracycline at similar dosage to the historic otoliths.     

Validation of daily microincrement deposition in otoliths of juvenile and adult Peruvian anchovy Engraulis ringens

Wild adult specimens of the Peruvian anchovy Engraulis ringens were captured and reared to validate the daily periodicity of otolith microincrement formation. The postcapture stress generated spontaneous spawning, making it possible to conduct a rearing trial on larvae first in an artificial nutrient‐enriched system (ANES) for 52 days followed by an artificial feeding regime in a culture tank until day 115 post‐hatch. Microincrements of the sagittal otoliths of sacrificed juveniles [mean ± s.d. total length (L_T) = 5·13 ± 0·37 cm, range 5–6 cm; c.v. = 7·5%] showed very distinct light and dark zones. The slope of the relationship between the total number of increments after the hatch check and days elapsed after hatching was not significantly different from 1. The transfer from ANES to the artificial feeding regime induced a mark in the sagittal otoliths. The number of microincrements after this induced mark coincided with the number of days elapsed after the transfer date. In parallel experiments, adult E. ringens (mean ± s.d. L_T = 14·92 ± 0·55 cm, range 13–16 cm) were exposed to one of two fluorescent marking immersion treatments with either alizarin red S (ARS; 25 mg l^?1 per 6 h) or oxytetracycline hydrochloride (OTC; 200 mg l^?1 per 10 h). The microincrements between fluorescent bands were distinct, ranging from 0·89 to 2·75 µm (mean ± s.d. =1·43 ± 0·28 µm; c.v. = 32%) and from 0·71 to 2·89 µm (1·53 ± 0·27 µm; c.v. = 35%) for ARS and OTC, respectively. The relationship between the number of microincrements between marks and the number of elapsed days for ARS and OCT treatments indicated that there was a significant correspondence between the number of increases observed and the number of days. Hence, daily microincrements of otoliths of E. ringens are likely to be formed in juveniles and adults under natural conditions.     

Batch marking of otoliths and fin spines to assess the stock enhancement of Argyrosomus japonicus

Juvenile mulloway Argyrosomus japonicus (54·6 ± 4·6 mm total length, mean ±  s . e .) were immersed in a range of oxytetracycline (OTC) solutions ranging between 0–600 mg l⁻¹ in salinities of 5 (diluted sea water) and 35 (undiluted sea water), and alizarin complexone (ALC) solutions ranging between 0–60 mg l⁻¹ in undiluted sea water, for 6, 12 and 24 h. Optimal marking conditions were 600 mg l⁻¹ OTC for 24 h in a salinity of 5, and 30 mg l⁻¹ ALC for 12 h respectively. Mark quality (MQ) was assessed using a score of 0–3 in both otoliths and anal fin spines, with a score >2 found to be acceptable for adequate mark identification. Acceptable marks were not produced using OTC in undiluted sea water. Immersion in OTC or ALC, or reduced salinity had no effect on survival relative to controls. Transverse sections of vertebrae from the ALC and OTC treatments with the highest otolith mark quality showed no discrete marks. Optimal marking techniques were used to produce double marks with a 3 day interval between marking, and marking techniques were applied to 130 000 juvenile mulloway in batch mode with minimal mortality. A numerical model of the chemical behaviour of OTC in sea water describes the decline of available OTC in increasing salinity, so that a species' salinity tolerance and successful marking can be optimized.     

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.     

Environmental effects on primary increment formation in the otoliths of newlyhatched Arctic charr

Otolith microincrements were investigated in Arctic charr Salvelinus alpinus , reared from hatching under various temperatures (1, 3, 5, 7° C) and feeding conditions (starved, fed every third day, fed daily). Larval charr otoliths were marked with oxytetracycline and alizarin complexone. Alizarin complexone was found to be 100 per cent successful in marking otoliths while oxytetracycline marks could be seen in <10 per cent of the otoliths viewed. Otolith microincrements were viewed by light and scanning electron microscopy to investigate the daily nature of increment deposition. Low temperatures (1 and 3° C) and starvation depressed daily increment formation. Increment deposition was found to be daily among the larvae reared at warmer temperatures (5 and 7° C) and fed at least every third day. Scanning electron microscopic analysis allowed us to confirm the results of light microscope increment counts from all temperatures except 1 ° C, where the number of increments enumerated were higher than the number obtained during light microscopy analyses. Increased feeding and warmer temperatures also resulted in increased increment width. The difference in increment number and width seems to be dependent upon fish growth rate which we have found to be affected by both temperature and feeding conditions.     

Tetracycline tagging in coregonid embryos and larvae

Coregonus peled (Gmelin) embryos at the eyed stage were immersed in tetracycline hydrochloride (TC) solution (600 mg l⁻¹) and fluorescent marks on the otoliths from fish were identified under a UV light microscope. Three weeks after treatment, when the larval fish had hatched, multiple primordia of the sagitta had fluorescent bands whose locations suggest that calcification begins before formation of the otolith's core. Upon hatching, increments were few or absent, and daily increments started forming after hatching. In coregonid larvae immersed in TC solution immediately after hatching, the fluorescent band was only 1 day's growth increment wide in the otolith. The age of larvae based on ring counts after marking corresponded to the actual age, but further studies are required. Triple marks due to 35-h immersions in TC solution separated by 5–7 day feeding periods were clearly readable in the otoliths. One month after treatment the fluorescent marks were identified under UV light on otoliths from every fish. In treated embryos, 38% of fish had marks 3 months after TC immersion, whereas in treated larvae 60–80% of fish grown up to 38.7 mm were marked.     

鲈鲤早期鱼苗的耳石标记研究

为研究鲈鲤早期鱼苗耳石标记的可行性,先采用高温水(20.8±0.3)℃和低温水(10.8±1.2)℃交替饲养对20日龄鲈鲤进行热标记,然后将经热标记的35和45日龄鲈鲤浸泡在50—200 mg/L的茜素络合物(AC)或茜素红S(ARS)溶液中进行荧光标记。热标记的耳石生长轮清晰可见,明显区别于其他轮纹,微耳石热标记轮轮纹宽度(IW)和高温期持续时间(T)的线性回归方程为IW=0.16462+0.24762T。经荧光物质浸泡后鲈鲤耳石在绿光下均能检测到橘红色标记; AC标记质量受溶液浓度影响显著(P<0.05),受全长的影响不显著(P>0.05),微耳石、矢耳石和星耳石间的标记质量差异显著(P<0.05); ARS标记质量受全长和溶液浓度影响不显著(P>0.05),微耳石、矢耳石和星耳石间的标记质量差异显著(P<0.05)。研究结果表明,耳石热标记、荧光标记及两者结合使用均可用于大规模标记鲈鲤早期鱼苗。     

Validation of annulus formation in otoliths of a temperate population of adult African sharptooth catfish Clarias gariepinus using fluorochrome marking of wild fish

The deposition rate of growth zones in sagittal otoliths of wild Clarias gariepinus that were fluorochrome marked with oxytetracycline and recaptured after a period of between 0·44 and 2·13 years was consistent with the deposition of one growth increment annually.     

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。       

Age estimation and validation for South Pacific albacore Thunnus alalunga

Validated estimates of age are presented for albacore Thunnus alalunga, sampled from a large part of the south‐western Pacific Ocean, based on counts of annual opaque growth zones from transverse sections of otoliths. Counts of daily increments were used to estimate the location of the first opaque growth zone, which was completed before the first assumed birthday. The periodicity of opaque zones was estimated by marginal increment analysis and an oxytetracycline mark–recapture experiment. Both validation methods indicated that opaque zones formed over the austral summer and were completed by autumn to winter (April to August). The direct comparison of age estimates obtained from otoliths and dorsal‐fin spines of the same fish indicated bias, which was assumed to be due to poor increment clarity and resorption of early growth zones in spines, resulting in imprecise age estimates. As such, age estimates from otoliths are considered to be more accurate than those from spines for T. alalunga. This is consistent with results for a growing number of tropical and temperate tuna Thunnini species. It is recommend that validated counts of annual growth zones from sectioned otoliths is used as the preferred method for estimating age‐based parameters for assessment and management advice for these important stocks.     

Tagging the next generation: validation of trans-generational chemical tagging for an endangered fish

In this study we validated marking offspring through peritoneal injection of ripe females using two concentrations of strontium (strontium chloride hexahydrate). Larvae from treatments were monitored for condition morphometrics and tested for chemical mark incorporation in their otoliths via laser ablation inductively coupled plasma mass spectrometry (LA-ICPMS) to quantify the strontium concentration (Sr/Ca) and laser ablation multi-collector inductively coupled plasma mass spectrometry (LA-MC-ICPMS) to measure the strontium isotope ratios (⁸⁷Sr:⁸⁶Sr) of otoliths. Otolith strontium concentrations and strontium isotopes ratios were elevated in the high-concentration treatment, while the low-concentration and control treatments were not significantly different from each other. Larval size and eye diameter at hatch were similar among treatments; however, yolk and oil globule diameters were significantly reduced in the high-concentration treatment. Moreover, growth rates after 60?days post-hatch were significantly reduced in the high-concentration treatment relative to the low-concentration and control treatments, suggesting trans-generational tagging can have deleterious effects on offspring. Our study provides evidence for the efficacy of artificially marking offspring via injection of strontium into ripe females and could provide new tools for managing endangered fish populations; however, careful consideration of chemical concentrations and dosages may be required prior to its application in a fisheries management experiment.     

Age,growth and reproduction of the Asian sheephead wrasse <Emphasis Type="Italic">Semicossyphus reticulatus</Emphasis> in the western Seto Inland Sea,Japan

The Asian sheephead wrasse, Semicossyphus reticulatus, is the largest labrid in temperate waters around Japan. However, the growth pattern of this wrasse remains unclear. Age, growth, and reproduction of S. reticulatus specimens were investigated by sampling in the western Seto Inland Sea, Japan. Opaque ring marks formed on the marginal zone of sectioned otoliths during February–March, suggesting their validity for age estimates. The largest individual was a male, with a standard length (SL) of 591 mm, weight of 8.5 kg, and age of 17 years. Males had significantly larger SLs and older ages than females, and male gonads were histologically confirmed to have secondary testis structures retaining central lumens, suggesting monandric protogyny. The oldest S. reticulatus individual captured was estimated to be a 31-year-old male. The von Bertalanffy growth equation calculated from estimated age and SL was L _t = 489 (1 ? e^{?0.12 (t + 1.75)}), indicating considerably slow growth, particularly after the age of 10 years, which is similar to that of the California sheephead wrasse, Semicossyphus pulcher. The humpheads of S. reticulatus enlarged gradually as their body grew, often resulting in a conspicuous humphead, even in large females.     

Otolith thermal marking in larval Chinese sucker, <Emphasis Type="Italic">Myxocyprinus asiaticus</Emphasis>

Otolith thermal marking was conducted in larval Chinese sucker, Myxocyprinus asiaticus, by exposing the larvae to water with different temperature regimes ranging from heated water (28°C) to chilled water (16°C) at different time intervals. The results showed that increment patterns differed among groups; the control of duration in the heated water could produce some increments much narrower or wider than those deposited in the natural diel water temperature fluctuation; there was a significantly positive relation between increment width and the duration reared in heated water in each cycle of water temperature fluctuation. These increments with abnormal widths were thermal marks in otoliths, and would be used as bar codes to track the released individuals, and discriminate them from the wild populations in the future. The thermal marking is suitable to mass-mark the Chinese sucker larvae.     

Age validation and seasonal growth patterns of a subtropical marsh fish: The Gulf Killifish, <Emphasis Type="Italic">Fundulus grandis</Emphasis>

Fundulus grandis (Baird and Girard), the Gulf Killifish, is an abundant species throughout the marshes of the northern Gulf of Mexico. Its wide distribution and high site fidelity makes it an ideal indicator species for brackish and salt marshes, which experience a variety of anthropogenic disturbances. Despite the ecological, commercial, and scientific importance of F. grandis, age determination methods have not been validated and little is known of its growth pattern. By combining a tag-recapture study with a chemical marker to stain otoliths, we validated an ageing method for F. grandis adults (49–128 mm TL) using whole sagittal otoliths and determined growth rates of recaptured individuals in winter (n = 58) and summer (n = 36) in Louisiana. Mean somatic growth in length was significantly greater during the winter (0.085 mm d^?1) than summer (0.054 mm d^?1). In contrast, mean otolith growth was significantly greater in summer (1.37 μm d^?1) than winter (0.826 μm d^?1). The uncoupling of somatic and otolith growth may be primarily attributed to warm summer temperatures, which led to enhanced otolith growth while simultaneously reducing somatic growth. Fundulus grandis was aged to a maximum of 2.25 years. The parameters of the von Bertalanffy growth model were estimated as: L _∞  = 87.27 mm, k = 2.43 year^?1, and t ₀ = ?0.022. These findings reveal essential age and growth information for F. grandis and provide a benchmark to evaluate responses to environmental disturbances.     

Reconstructing larval growth and habitat use in an amphidromous goby using otolith increments and microchemistry

High‐resolution analysis of growth increments, trace element chemistry and oxygen isotope ratios (δ¹⁸O) in otoliths were combined to assess larval and post‐larval habitat use and growth of Awaous stamineus, an amphidromous goby native to Hawai‘i. Otolith increment widths indicate that all individuals experience a brief period of rapid growth during early life as larvae and that the duration of this growth anomaly is negatively correlated with larval duration. A protracted high‐growth period early in larval life is associated with a lower ratio of Sr:Ca, which may reflect low salinity conditions in nearshore habitats. A distinct shift in δ¹⁸O (range: 4–5‰) is closely associated with the metamorphic mark in otoliths, indicating that larval metamorphosis occurs promptly upon return to fresh water. Strontium and other trace elements are not as tightly coupled to the metamorphosis mark, but confirm the marine‐to‐freshwater transition. Integration of microstructural and microchemical approaches reveals that larvae vary substantially in growth rate, possibly in association with habitat differences. Although time and financial costs make it difficult to achieve large sample sizes, present results show that examining even a small number of individuals can lead to novel inferences about early life history in diadromous fishes and illustrates the value of integrating analyses.     

Experimental evaluation of using calcein and alizarin red S for immersion marking of bighead carp Aristichthys nobilis (Richardson, 1845) to assess growth and identification of marks in otoliths,scales and fin rays

In order to evaluate the effects of immersion marking with calcein (CAL) and alizarin red S (ARS) on growth and mortality of juvenile bighead carp Aristichthys nobilis, and assess mark quality in otoliths, scales, and fin rays, CAL from 50 to 200 mg L^?1 and ARS from 150 to 300 mg L^?1 concentrations were used. With the exception of non‐lateral line scales from 50 mg L^?1 CAL treatments, immersion for 24 h produced detectable marks in sagittae, lateral line and non‐lateral line scales, and fin rays (dorsal, pectoral, ventral, anal, and caudal) at 100 days post‐marking. Detectable fluorescent marks in sagittae were readily observed at concentrations of 150–200 mg L^?1 CAL or 150–300 mg L^?1 ARS. Marks were poorly visible in all non‐lateral line scales from both CAL‐ and ARS‐treated groups. Fluorescent marks were readily detected in lateral line scales at 100–200 mg L^?1 CAL or 150–300 mg L^?1 ARS, and in fin rays at 150–200 mg L^?1 CAL or 150–300 mg L^?1 ARS. In particular, optimal marks were observed at the highest concentrations investigated in sagittae (300 mg L^?1 ARS), lateral line scales (150–200 mg L^?1 CAL or 250–300 mg L^?1 ARS), and fin rays (200 mg L^?1 CAL or 250–300 mg L^?1 ARS). However, fluorescent marks visible to the naked eye were not produced by any of the CAL or ARS treatments in sagittae, scales, or fin rays during this experiment. In addition, there was no significant difference on survival and growth of marked fish compared to controls throughout the experiment (P > 0.05).     

鱼类双重荧光标记研究——以中华倒刺鲃幼鱼为例

为探究盐酸四环素(TCH)和茜素红S(ARS)对中华倒刺鲃(Spinibarbus sinensis Bleeker)的影响及标记效果, 丰富鱼类荧光标记模式, 研究使用TCH(100—500 mg/L)和ARS(100—300 mg/L)对中华倒刺鲃幼鱼进行双重浸染荧光标记, 实验共设置5个处理组和1个对照组, 浸染时间均为24h。结果显示, 经90d的养殖实验后, 标记鱼的耳石(包括矢耳石和星耳石)、倒刺、鳍条和鳍棘均能检测到双重荧光标记环, 且TCH产生的黄色荧光环比ARS产生的红色荧光环更接近骨质结构内部。较高浓度处理组的矢耳石(≥300 mg/L TCH和≥150 mg/L ARS)、星耳石(≥300 mg/L TCH 和 ≥200 mg/L ARS)和倒刺(400—500 mg/L TCH 和150—300 mg/L ARS)中均能检测到明显的标记环(n≥2), 但所有处理组的侧线鳞和非侧线鳞的荧光标记不明显(0≤n≤1)。经200—500 mg/L TCH和150—300 mg/L ARS处理的鳍条, 及经300—500 mg/L TCH和200—300 mg/L ARS处理的鳍棘中可以同时检测到明显的TCH和ARS的标记环(n≥2)。此外, 在整个实验中各处理组标记鱼与对照组相比, 在生长和存活率方面均无显著性差异(P>0.05)。结果表明, 使用TCH和ARS双重标记中华倒刺鲃幼鱼是可行的。双重荧光标记方法在水生生物标记回捕实验及实验性生物学研究方面具有潜在的应用前景。