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.     

Factors affecting mark quality of alizarin complexone-stained otoliths in juvenile black bream Acanthopagrus butcheri and a prescription for dosage

The relationships between otolith score, a function of increasing stain visibility, and the ratio of alizarin complexone (mg) to fish biomass (g) for 0·1, 0·6 and 7 g black bream Acanthopagrus butcheri show that acceptable marks are produced in all size classes using a stain-to-biomass ratio of 3·3 mg g⁻¹ following an immersion period of 24 h.     

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.     

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.     

Differential dispersal patterns of male and female masu salmon fry

Behavioural experiments using artificial and natural stream channels were undertaken to determine whether there were differences in dispersal between newly emerged male and female masu salmon. Eyed eggs from a cross with wild spawners were planted in the middle pool of an artificial channel. After emergence, more males than females moved into an upstream trap, while fewer males moved downstream. In a natural stream, eyed eggs were marked with alizarin complexone to distinguish them from naturally spawned eggs and these were planted into artificial redds. More newly emerged male fry remained at the planted site than female fry. In contrast, more females moved downstream than males. These results imply that differences in dispersal patterns between male and female masu salmon fry are genetically controlled.     

Validation of daily increment formation in otoliths for three Diplodus species in the Mediterranean sea

The efficacy of two fluorochromes, chlorhydrate of tetracycline (CHTC) and alizarin complexone (ALC), to induce a label on the otoliths of juvenile sparid fishes by immersion techniques was tested in different experimental conditions. CHTC did not mark otoliths in natural sea water and was toxic in divalent cation-free sea water. Immersing fish in a 50-mg 1⁻¹ ALC natural seawater solution for 24 h induced a well-defined mark on the otoliths and had no effect on survival. A daily periodicity of increment formation on otoliths was observed in captivity for Diplodus vulgaris and D. puntazzo , and was conserved in natural environment for D. sargus . The frequency of increment deposition did not vary with the age of juvenile fishes. Thus, otolith microstructure analysis will be a reliable method to give age estimates in these three sparid species during their juvenile life.     

Otolith features and growth of Malawian characid Hemigrammopetersius barnardi from the southwestern coast of Lake Malawi

Macro- and microstructures of the sagitta, asteriscus, and lapillus of juveniles of Malawian characid Hemigrammopetersius barnardi were observed. The sagittae were arrowhead shaped and showed development of rostra. Increments in the sagitta were observable until the bases of the rostra but were invisible in the rostra. The asteriscus had an irregular shape with an ambiguous core and notches at the margin. The ambiguous core of the asteriscus led to difficulty in discerning the first increment. The lapilli were round and fan shaped. Increments in the lapilli were distinct from the core to the margin. The daily increment deposition was validated by alizarin complexone treatment, and the increments increased at the rate of 1 day⁻¹ after hatching. These features suggested that in this species the lapillus is most appropriate for daily increment analysis. The hatching months were estimated based on the lapillus increment counts, and the results showed that this species continuously hatched in the rainy season for 6 months from November to April. The growth trajectory indicated that H. barnardi attains a total length of 50 mm within 4 months of hatching.     

A microbead array chemical sensor using capillary-based sample introduction: toward the development of an "electronic tongue"

The development of a micromachined fluidic structure for the introduction of liquid samples into a chip-based sensor array composed of individually addressable polymeric microbeads is presented. The micromachined structure consists of micromachined storage cavities combined with a covering glass layer that confines the microbeads and fluidic channels. In our sensor array transduction occurs via optical (colorimetric and fluorescence) changes to receptors and indicator molecules that are covalently attached to termination sites on the polymeric microbeads. Spectral data are acquired for each of the individual microbeads using a charged-coupled device (CCD) allowing for the near-real-time analysis of liquid sample. Hence the micromachined fluidic structure must allow for both optical access to the microbeads and fluid flow through the micromachined cavities that serve as the microreactors/analysis chambers. One of the key parts of the structure is a passive fluid introduction system driven only by capillary force. This simple means of fluid introduction realizes a compact device. The capillary flow on the inlet channel has been studied, and the responses of the microbeads (alizarin complexone) to a liquid sample have been characterized. The test results show that this system is useful in a micro-total-analysis-system (mu-TAS) and biomedical applications.     

Chemical marking of European glass eels Anguilla anguilla with alizarin red S and in combination with strontium: in situ evaluation of short‐term salinity effects on survival and efficient mass‐marking

This study presents a new chemical double‐marking technique for European glass eels Anguilla anguilla by combing alizarin red S (ARS) and strontium chloride hexahydrate (Sr). Marked eels (double marked with ARS and Sr, but also single marked with ARS) were exposed in situ to brackish water (15 g l^?1 artificial sea salt) for 14 days and did not exhibit increased mortalities compared with unmarked eels. Indeed, no mortality occurred in a marked group during the experiments. Moreover, an efficient mass‐marking approach with low handling effort for both single ARS and double ARS‐Sr techniques is described and was proven to be practicable for large‐scale stocking programmes.     

The healing of facial bone fractures by the process of secondary union

The mechanism of healing of facial bone fractures was investigated in a rabbit model. Twelve New Zealand white rabbits underwent surgically induced fractures of the right infraorbital rim and fracture ostectomies (4 to 5 mm) of the left infraorbital rim. Animals were sacrificed 2, 4, and 8 weeks postfracture. Bone, including periosteum, obtained from each fracture or fracture osteoctomy site was divided longitudinally for hematoxylin and eosin staining, fluorescent microscopy, microangiography, and microradiography. Sequential fluorochrome labels of oxytetracycline (30 mg/kg), alizarin complexone (30 mg/kg), DCAF (20 mg/kg), and xylenol orange (90 mg/kg) were administered 24 hours preoperatively and at 1, 2, 4, and 8 weeks postfracture. All fracture and fracture ostectomy sites demonstrated vascular ingrowth, mineralization, and woven bone formation by 2 to 4 weeks postoperatively, beginning with a cartilage precursor. Subsequently, the woven bone was replaced with remodeled lamellar bone, resulting in complete bony healing by 8 weeks postoperatively. These steps were substantiated by microscopic, microradiographic, and radiologic examination of the specimens. This study demonstrates that fractures of the facial bones in a rabbit model heal by a process of new bone formation that resembles secondary union in endochondral bones.     

An improved labelling technique for monitoring microcrack growth in compact bone

Fatigue-induced damage plays an important role in bone remodelling and in the formation of stress and fragility fractures. Recently, a technique has been developed (Lee, T.C. et al., Sequential labelling of microdamage in bone using chelating agents. Journal of Orthopedic Research, 18 (2000) 322-325) which allows microcrack growth in trabecular bone to be monitored by the application of a series of chelating fluorochromes, however, some limitations were identified with the process. The aims of this study were to refine the method of detection using these agents in order to determine the optimal sequence of application and the optimal concentrations which allowed all the agents to fluoresce equally brightly using UV epifluorescence. A chemical analysis process, ion chromatography, followed by validation tests on bone samples showed that the optimal sequence of application and concentration of each agent was alizarin complexone (0.0005 M) followed by xylenol orange (0.0005 M), calcein (0.0005 M) and calcein blue (0.0001 M). A fifth agent, oxytetracycline was excluded from the study after recurring problems were found with its ability to chelate exposed calcium when applied in sequence with the other agents. This work has developed a sequential labelling technique, which allows for microcrack propagation during fatigue testing of bone specimens to be monitored without the problem of chelating agent substitution occurring.     

Age validation and growth of three commercially important hemiramphids in south-eastern Australia

The method of using sectioned otoliths to estimate age in three species of garfishes (family Hemiramphidae) was validated by: (1) staining fishes with the vital stain alizarin complexone (ALC) and periodically examining their otolith growth, and (2) marginal increment analyses. Staining fishes with ALC indicated that opaque zones were formed during winter and spring, but did not become visible on the otolith edge until late spring and summer. Hyporhamphus australis were found to be similar to the hemiramphids of the Atlantic in having fast growth rates and a maximum observed age of 4+ years old. Hyporhamphus regularis ardelio and Arrhamphus sclerolepis krefftii were found to be more similar to the southern sea garfish, Hyporhamphus melanochir , in being moderately long-lived, with maximum observed ages of 7+ years old for both species. Females grew faster and attained greater fork lengths than males for each species. Sectioned otoliths showed large variation in the appearance of opaque zones between the three species studied, with those from the wide-ranging, oceanic H. australis appearing inconsistent and diffuse when compared to the estuarine H. r. ardelio and A. s . krefftii . This variation was also apparent from fishes kept in aquaria, suggesting that the appearance of opaque zones in otoliths of these species is largely influenced by physiology, rather than by environmental conditions.     

Growth and morphological development of sagittal otoliths of larval and early juvenile Trachurus japonicus

The daily periodicity of growth increment formation in sagittal otoliths of jack mackerel Trachurus japonicus was validated by marking otoliths with alizarin complexone (ALC). Analysis of otoliths of known‐age juveniles confirmed that the first increment formed on day 3 after hatching, and was associated with first feeding. A total of 198 specimens, ranging from 2·6 to 49·2 mm in body length (notochord length or standard length) and from 7 to 78 days in age, were collected in the East China Sea and Tosa Bay, and used to examine the association between otolith morphological development and ontogenetic development. The relationship between body length ( L ) and otolith radius ( R ) was significantly described by the linear function L  = 2·65 + 0·0425 R ( n  = 198, r ² = 0·99, P  < 0·001), indicating that somatic growth history can be reconstructed from otolith growth patterns. The otolith was primarily spherical in the preflexion larval stage, and became elongated with notochord flexion. The first secondary primordium formed at c . 25 days, during the middle postflexion stage, and was associated with metamorphosis. By c . 42 days the sagittal otolith was adult‐like in morphology, with the primary growth zone enclosed by the marginal growth zone, except in the anterior rostrum area. Thus age, growth and developmental stages were recorded in sagittal otoliths during the larval and early juvenile stages of jack mackerel.     

鳡鱼仔稚鱼耳石的标记和其日轮的确证

用茜素络合物对鳡鱼仔鱼进行了浸泡标记,耳石上能检测到橘红色荧光标记环。100mg/L茜素络合物溶液的标记效果较差;120mg/L和150mg/L溶液浸泡后微耳石的标记率很高,矢耳石和星耳石的标记率低。标记后耳石上的生长轮数与饲养天数间呈一一对应关系,相关方程为:N=0.2663 0.9276D(n=68,r~2=0.9664)。方程的斜率0.9276与1无显著差异,证明生长轮确系日轮。鱼体长与微耳石、矢耳石及星耳石的直径间呈显著的线性关系,相关方程为:BL=66.8723LD 2.7064(n=73,r~2=0.8867),BL=22.7839SD 6.6066(n=49,r~2=0.8525),BL=47.6079AD 3.5660(n=71,r~2=0.9012)。     

用荧光物质浸泡标记胭脂鱼仔、稚鱼耳石

用茜素络合物和盐酸四环素溶液分别对胭脂鱼（Myxocyprinus asiaticus）仔、稚鱼浸泡标记,结果表明,100～200mg／L的茜素络合物溶液浸泡24h对胭脂鱼仔、稚鱼耳石有很好的标记效果。相同浸泡浓度下,随着日龄的增长,耳石上的荧光反应强度降低。三对耳石中,微耳石和矢耳石对茜素络合物较敏感,星耳石敏感性则较低。盐酸四环素溶液对仔、稚鱼耳石的标记效果很差,浸泡液浓度为100mg／L和120mg/L时,仅能在微耳石上检测到较弱的荧光标记,而150mg／L及以上浓度的浸泡液对稚鱼有较高的致死作用。因此,茜素络合物是对胭脂鱼早期鱼苗进行化学标记比较合适的荧光物质,而盐酸四环素不适于标记该鱼的耳石。在对标记鱼进行荧光检测时,矢耳石和微耳石是适合的材料。     

鲫幼鱼耳石荧光标记效果

为确认茜素络合物对鲫（Carassius auratus）耳石进行有效标记的可行性,以便为鲫甚至其他鲤科鱼类标志放流技术的开发及效果评估提供一定的借鉴,本研究以孵出后90 d的鲫幼鱼为研究对象,设置单一浓度（100 mg/L）的茜素络合物浸泡标记5 d,分析茜素络合物在耳石上的沉积情况以及不同耳石在不同后续饲养天数的动态变化。结果表明,矢耳石、微耳石和星耳石上在可见光、绿色和蓝色激发光下都检测到了良好的茜素络合物标记环,标记率和存活率均为100%。但不同耳石的茜素络合物标记效果不同,荧光下,星耳石的标记效果最显著,微耳石次之;可见光下,微耳石的标记效果最好,星耳石次之。随着后续饲养天数的延长,可见光下标记逐渐减弱,至20 d时基本消失,而在绿色和蓝色激光下标记环荧光强度无减弱迹象,能长久保持,且在蓝色激发光下标记环更易被观测到。上述结果结合鲫生长、存活和行为正常等情况综合显示,在100 mg/L茜素络合物溶液中浸泡标记鲫幼鱼5 d,其耳石可以获得满意的标记效果。     

白囊耙齿菌对茜素红脱色条件优化及其毒性变化检测

白囊耙齿菌Irpex lacteus是分离自倒木上的一株可以分泌漆酶和锰过氧化物酶的白腐真菌。利用I. lacteus对固体条件下的活性黑、活性红、结晶紫、茜素红和孔雀石绿进行脱色能力的检测,通过单因素和正交试验优化I. lacteus对茜素红的脱色条件,并以3种作物发芽率为指标测定茜素红被I. lacteus脱色前后的毒性变化。结果显示,I. lacteus对5种染料均可脱色,其中对茜素红染料的脱色更为彻底;单因素和正交试验优化I. lacteus对茜素红的脱色条件为：pH 7.0、葡萄糖10.0g/L、硫酸铵0.66g/L、接种量2片（Φ=8.0mm）、100.0mL三角瓶装液20.0mL,优化条件下I. lacteus对茜素红脱色10d时的脱色率为88.26%,与未优化前的脱色率相比提高了60.50%;茜素红染料被I. lacteus脱色前后毒性大小排序为：染料原液>染料脱色后>PDB培养基处理,表明茜素红染料存在一定的毒性,I. lacteus脱色茜素红后可以使其毒性减弱。通过本研究,为I. lacteus在茜素红等染料废水脱色以及降低染料废水毒性方面的应用奠定基础。     

Alizarin and tetracycline binding by bone mineral

The nature of alizarin combination with bone is thought to be a chelation with a divalent cation on the surface of the crystal. Reactions of alizarin with bone mineral and apatite, rather than with intact bone, have been demonstrated by several investigators. In vitro titration indicates that a positive divalent or trivalent ion is capable of chelating with alizarin. Aluminum and iron form the strongest chelates, but calcium, magnesium, barium, etc. are capable of reacting. Not only does bone mineral, such as hydroxyapatite, combine with alizarin, but such other calcium-containing salts as calcium fluoride also bind the dye. The structure of alizarin that reacts with the metal ion appears to require a hydroxyquinone as the ligand. The presence of a second hydroxy group on the ring in the 2 position, or in almost any other position on the ring, facilitates the binding of the ligand to the apatite. This makes the dihydroxy molecules far more effective at low pH, and it is to this family of compounds that alizarin belongs. Alizarin red S is simply the placement of asulfonic acid group on the ring to get additional ionic structure, in order to make it soluble in water. A variety of isomers of alizarin have been studied, and all are capable of reacting with bone mineral on the same molar basis. In fact, removal of the unsubstituted ring of alizarin to form dihydroxy-naph-aquinone produces a dye still capable of reacting with apatite. The ratio of the moles of napthaquinone bound to those of anthraquinone may be explained by the relative sizes of the two dye molecules. It has been shown that phosphate competitively replaces alizarin, and that its presence in solution will prevent alizarin attachment to the surface of apatite. This result can be interpreted to mean that both the alizarin and the phosphate tend to occupy the same site when they are being adsorbed onto the surface. Tetracyclines, while more complex in structure, appear to have a binding mechanism to apatite which resembles that of alizarin.     

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.     

Polyfluorochrome marking slows growth only during the marking month in the green sea urchin Strongylocentrotus droebachiensis

Abstract. Used singly, the fluorochrome tags tetracycline and calcein have yielded important insights into sea urchin biology, especially regarding growth. We present a new method of tagging using sequential fluorochrome markers, as well as a more precise method of quantifying growth. Such polyfluorochromes enable repeated markings that allow measurement of multiple growth points and unique identification of individuals or groups. We marked sea urchins, Strongylocentrotus droebachiensis , with four fluorochromes: alizarin complexone, calcein, calcein blue, and tetracycline. All fluorochromes marked both by injection and immersion. We examined the clarity of the mark produced with low, metabolically scaled doses, and higher doses similar to those that have been previously used. We tested the effect of fluorochromes on survival, growth, jaw size, and gonad size by marking a size range (3.9–44.3 mm in diameter) of urchins with either one or all four fluorochromes. We quantified growth using a nominal diameter, that is a fitted constant, times the cube root of weight, which increased the precision of measurements by a factor of six relative to measured diameter. Growth rate was a decreasing function of diameter except for a growth lag in the smallest urchins. Growth rate data for all sizes were fitted using: gamma distributions; Tanaka functions; and, for larger sizes, straight lines (von Bertalanffy model). All treatments produced clear marks, with higher doses producing more reliably clear marks. Tetracycline marking did not affect growth; other treatments produced only transient slowing of growth in the marking month. Growth rate, survival, gonad production, and jaw weight did not differ between control and treatments during the following 6 months. Thus, polyfluorochromes produce reliable marks that do not significantly affect growth or gonad production.