Optimizing the mass marking of fish with alizarin red S: an example with glass eels

Fish marking is an essential tool for fisheries management, especially for evaluating the stocking of endangered fish species to support conservation and sustainable use of fish stocks. Batch marking of young European eels Anguilla anguilla (L.) prior to stocking is recommended as the benefits of stocking for the spawning stock can be evaluated by recapturing marked fish over time, therefore mass marking of young eels with substances such as alizarin red S (ARS) is becoming increasingly important. To improve the marking method and reduce marking costs when immersing glass eels in an ARS solution, eight laboratory experiments under varying conditions (e.g., temperature, ARS concentration, immersion time, osmotic induction, fish density) and with ARS from different suppliers were carried out. The results show that optimal marking of glass eels can be carried out in the field or during transport by putting approximately 50 g of glass eels per liter in 150 mg L⁻¹ ARS solution for 3 h at 10–15°C. Lower concentrations did not result in reliable marking. Water temperatures of 5°C and below can have a stunning effect on the eels and increase mortality significantly, regardless of the concentration of ARS. Glass eel densities below 50 g L⁻¹ in the marking bath increase marking costs unnecessarily, while a higher density of 100 g L⁻¹ resulted in significantly higher mortality and lower marking success. A somewhat more difficult but less expensive alternative is to bathe the fish in a saline solution of 1% (10 PSU) of 80 mg L⁻¹ ARS for 3 h at 10°C. Costs can also be significantly reduced by choice of supplier for ARS, but care should be taken as the quality of the powder appears to vary (mean percentage of sufficiently marked eels ranged from 59% to 91% among suppliers in the present study) and can lead to marking failure. The optimal marking conditions can help ensure that stocked glass eels can be reliably identified in future studies to assess stocking benefits while reducing costs.     

Mass‐marking of farmed European eels (Anguilla anguilla (Linnaeus, 1758)) with alizarin red S

The Working Group on Eel of the International Council for the Exploration of the Sea (ICES) regularly reports that a significant amount of stocked eels in Europe was pre‐grown in aquaculture farms prior to stocking—so called “farmed eels.” The ICES advices chemical marking of stocked recruits to ensure their traceability throughout all life stages. To date, however, there was a lack of knowledge concerning the most suitable chemical substance and its application on farmed eels. The aim of this study was to fill this gap by presenting successful attempts of marking those eels with alizarin red S (ARS). An ARS concentration of 150 mg L^?1 buffered with 150 mg L^?1 Tris(hydroxymethyl)aminomethane applied as an immersion bath over 9 h was sufficient to mark a total of 3572 kg of farmed eels (6.5–8.0 g mean body weight). The marking success was 100% on otoliths and highest stocking density of up to 67.1 kg m^?3 (corresponding 54.0 kg m^?2) turned out to have no effect on mortality which was consistently below 1%.     

The use of live and frozen Artemia salina nauplii enriched with fluorochromes for mass‐marking vendace Coregonus albula (L.) larvae

The aim of this study was to investigate the use of Artemia salina nauplii enriched with chosen fluorochromes for mass marking vendace Coregonus albula (L.) larvae. In the experiment, vendace larvae (6 DPH) were fed with live or frozen A. salina nauplii immersed in tetracycline hydrochloride (TC), calcein (Cal) or alizarin red S (ARS) for four subsequent days. More successful effects (marking otoliths) were obtained by feeding the fish with live nauplii enriched with fluorochromes, regardless of the dye type. The highest percentage of marked otoliths (100%) was recorded in the group fed with live nauplii immersed in TC. In the groups fed live or frozen nauplii enriched with Cal and ARS, a lower percentage of marked individuals (63.3%–73.7% and 56.7%–63.3%, respectively) were recorded. The survival rate of vendace larvae in particular groups oscillated between 93.7% and 95.7%. There were no significant differences in the total body length and body weight of the reared vendace larvae among different groups. In conclusion, for mass marking of vendace larvae using a feeding method, fish fed A. salinalive nauplii enriched with TC at a dose of 600 ppm is recommended for fishery practice.     

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).     

Mass-marking of brown trout (Salmo trutta L.) larvae by alizarin: method and evaluation of stocking

This study describes otolith marking of brown trout (Salmo trutta L.) larvae by immersion in different solutions of alizarin red S (ARS). The best results were obtained after marking with ARS at a concentration of 150 mg L^?1. To evaluate the efficiency of stocking with brown trout fry, 10 000 20‐day‐old larvae were marked in years 2002 and 2003 with ARS and released 2 weeks later into sections of a river with natural brown trout reproduction. Electro‐fishing surveys carried out 2 months after stocking in 2002 revealed that only 4.8% of all caught young‐of‐the‐year trout originated from stocking; in 2003 the percentage was 8.9%. Based on the substantial natural reproduction and the low ratio of stocked to wild trout, it was recommended to discontinue stocking.     

Experimental evaluation of calcein and alizarin red S for immersion marking of silver carp Hypophthalmichthys molitrix (Valenciennes, 1844)

Calcein (CAL) and alizarin red S (ARS) at concentrations of 50–200 and 150–300 mg/L, respectively, were used for immersion marking of juvenile silver carp Hypophthalmichthys molitrix (79.65 ± 2.11 mm total length, mean ± SD). The marked fish were kept in separate tanks (0.012 individuals per litre, rearing temperature 18.4–25.7°C) for 360 days. Each experimental treatment group consisted of three replicates (16 individuals per replicate). Immersion for 24 h produced detectable marks in the sagittae, lateral line and non‐lateral line scales, and fin rays (dorsal, pectoral, ventral, anal, and caudal) at 360 days post‐marking. Acceptable marks in the sagittae were observed for CAL at concentrations of 150–200 mg/L and for ARS at concentrations of 200–300 mg/L. Fluorescent marks were poorly visible in all non‐lateral line scales from both the CAL‐ and ARS‐treated groups. Acceptable fluorescent marks in the lateral line scales and fin rays were detected for CAL at concentrations of 150–200 and 100–200 mg/L, respectively, and for ARS at concentrations of 200–300 and 150–300 mg/L, respectively. In particular, optimal marks were observed at the highest concentrations investigated in lateral line scales (200 mg/L CAL, 300 mg/L ARS) and fin rays (200 mg/L CAL, 200–300 mg/L ARS). There was no significant difference in the survival or growth of marked fish compared to controls throughout the experiment (P > 0.05).     

Strontium metabolism in the juvenile Lake Sturgeon,Acipenser fulvescens (Rafinesque, 1817), and further evaluation of the isotope as a marking tool for stock discrimination

Stock enhancement efforts have been used to aid in increasing dwindling population levels of Lake Sturgeon, Acipenser fulvescens, that are currently of conservation concern. Consistent monitoring of stock enhanced fisheries requires a reliable and reproducible method to mark stocked fish such that the success of such programs can be determined. Recently, stable isotopic marking of hard structures in fish has been developed in a variety of species, however, optimal marking conditions are less well understood. In this study the metabolism of strontium in juvenile Lake Sturgeon and determined parameters for optimisation of marking success in the fin ray were examined. Using radioactive strontium (⁸⁵Sr), whole body influx and efflux rates of strontium were 3.02 ± 0.364 pmol h^?1 g^?1 (mean ± standard error) and 0.04 ± 0.007 pmol h^?1 g^?1, respectively. Furthermore, short‐term accumulation of ⁸⁵Sr in a variety of tissues was assessed and found to be greatest in the fin ray. Biological half‐life of the stable isotope ⁸⁶Sr was shortest in muscle and longest in the fin ray tissue. Immersion and length of immersion timing had positive relationships with marking success. Additionally, the signature remained in 75% of fish analysed 550 days post‐mark. Data suggest that marking success was determined by duration of immersion in the mark and concentration of the mark introduced to the water.     

Validation and efficacy of transgenerational mass marking of otoliths in viviparous fish larvae

Transgenerational mass marking of viviparous fish larvae in vivo was validated by intra‐muscular injection of elemental strontium chloride (SrCl₂) in gestating females and detection of the Sr in the otoliths of developing larvae. All otoliths of brown rockfish Sebastes auriculatus larvae produced from SrCl₂‐injected females showed enriched Sr:Ca ratios near the otolith edges, and the signatures did not appear to be affected by the anterior, centre and posterior positions of larvae within the ovary. Results from the present study indicate that transgenerational marking is a highly reliable technique for marking large numbers of extremely small viviparous fish larvae.     

Improved methods for color‐marking hummingbirds

ABSTRACT Individual color‐marking is an essential tool for studying the behavior of free‐living birds. Hummingbirds represent a particular challenge for traditional avian color‐marking techniques because of their small size and short tarsi. Although several techniques have been successfully used, the retention time of color‐markers and their safety and ease of construction could be improved. I developed two new color‐marking techniques for marking Little Hermits (Phaethornis longuemareus): (1) a plastic back tag constructed by fusing colored beads, and (2) a leg tag attached to a metal band fitted around the tarsus. Both tag designs were visible in field conditions, and neither appeared to adversely affect behavior. Both back and leg tags had high retention rates within seasons, but back tags had a poor retention rate between years. Although these marking techniques were designed for use on one species of hummingbird, they would likely also be useful with other species of hummingbirds.     

Wolf scent marking with raised-leg urination

Raised-leg urinations were performed almost exclusively by dominant male and female wolves (Canis lupus). The alpha male and female in two captive groups showed full raised-leg urinations (RLUs) throughout the year. Each alpha male and female sniffed and marked on one another's marks frequently; however, the pair that bred did more marking and more double marking. They increased their marking frequency prior to and during the breeding season, decreased it at the time of parturition, while the female was in the den, and then increased to a moderate level when the female took part in pack activities again. Because the alpha pair was the principal sender and receiver of the message within the group, it is likely that double marks function to maintain and advertise the pair bond. Because the group that did not breed also did not double mark frequently, the presence of double marking in a captive group may be diagnostic of the viability of the group as a pack.     

Critical swimming speed of yellow‐ and silver‐phase European eel (Anguilla anguilla,L.)

The prime objective of this study was to evaluate differences between the swimming performance of two distinct life stages of European eels. The critical swimming speed (U_crit) of 29 yellow‐ and 33 silver‐phase eels was evaluated in a swim tunnel. Silver‐phase eels showed a better swimming performance (U_crit = 0.66 ms^?1) than yellow individuals (U_crit = 0.43 ms^?1). Male and female silver eels reached an identical U_crit despite their different sizes, which may be a strategy to increase the synchronization of arrival at the spawning grounds.     

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.     

lac‐1 and lag‐1 with ras‐1 affect aging and the biological clock in Neurospora crassa

Using an automated cell counting technique developed previously (Case et al., Ecology and Evolution 2014; 4: 3494), we explore the lifespan effects of lac‐1, a ceramide synthase gene paralogous to lag‐1 in Neurospora crassa in conjunction with the band bd (ras‐1) gene. We find that the replicative lifespan of a lac‐1^KO bd double mutants is short, about one race tube cycle, and this double mutant lacks a strong ~21‐hr clock cycle as shown by race tube and fluorometer analysis of fluorescent strains including lac‐1^KO. This short replicative lifespan phenotype is contrasted with a very long estimated chronological lifespan for lac‐1^KO bd double mutants from 247 to 462 days based on our regression analyses on log viability, and for the single mutant lac‐1^KO, 161 days. Both of these estimated lifespans are much higher than that of previously studied WT and bd single mutant strains. In a lac‐1 rescue and induction experiment, the expression of lac‐1⁺ as driven by a quinic acid‐dependent promoter actually decreases the median chronological lifespan of cells down to only 7 days, much lower than the 34‐day median lifespan found in control bd conidia also grown on quinic acid media, which we interpret as an effect of balancing selection acting on ceramide levels based on previous findings from the literature. Prior work has shown phytoceramides can act as a signal for apoptosis in stressed N. crassa cells. To test this hypothesis of balancing selection on phytoceramide levels, we examine the viability of WT, lag‐1^KO bd, and lac‐1^KO bd strains following the dual stresses of heat and glycolysis inhibition, along with phytoceramide treatments of different dosages. We find that the phytoceramide dosage–response curve is altered in the lag‐1^KO bd mutant, but not in the lac‐1^KO bd mutant. We conclude that phytoceramide production is responsible for the previously reported longevity effects in the lag‐1^KO bd mutant, but a different ceramide may be responsible for the longevity effect observed in the lac‐1^KO bd mutant.     

Application of otolith microchemistry to estimate the migratory history of Japanese eel Anguilla japonica on the Sanriku Coast of Japan

The age and migratory history of the Japanese eel, Anguilla japonica Temminck & Schlegel, collected in Miyako Bay along the Sanriku coast of Japan, was examined using the otolith microstructure and analysis of strontium (Sr) and calcium (Ca) concentrations conducted with wavelength dispersive X‐ray spectrometry by an electron microprobe. The line analysis of Sr : Ca ratios along the life history transect of each otolith showed a peak (ca. 15–17 × 10^?3) which corresponded with the period of their leptocephalus and early glass eel stages in the ocean. The mean Sr : Ca ratios from the elver mark to the otolith edge indicated that there were eels with several general categories of migratory history, including sea eels that never entered freshwater (average Sr : Ca ratios, ≥6.0 × 10^?3), and others that entered freshwater for brief periods but returned to the estuary or bay. This evidence of the occurrence of sea eels in this northern area indicates that Japanese eels of the Sanriku coast do not necessarily migrate into freshwater rivers during recruitment as do glass eels at the beginning of their growth phase; even those that do enter freshwater may later return to the marine environment. Thus, anguillid eel migrations into freshwater are clearly not an obligatory migratory pathway, but rather a facultative catadromy with seawater or estuarine residents as an ecophenotype.     

Vertical habitat shift of viviparous and oviparous deep‐sea cusk eels revealed by otolith microstructure and stable‐isotope composition

Otolith stable‐oxygen‐isotope composition and microstructure were analysed in order to investigate the vertical habitat shift of deep‐sea cusk eels (Ophidiiformes). Otolith δ¹⁸O profiles suggested that both viviparous blind cusk eels and oviparous cusk eels experienced a pelagic larval stage and then settled to the deep‐sea floor over a vertical distance that ranged among individuals from 200 to >1000 m. This result shows that the larvae of viviparous Barathronus maculatus undertake an ontogenetic vertical migration after a period of larval drift that may facilitate their wide distribution on the sea floor.     

Field‐readable alphanumeric flags are valuable markers for shorebirds: use of double‐marking to identify cases of misidentification

Implicit assumptions for most mark‐recapture studies are that individuals do not lose their markers and all observed markers are correctly recorded. If these assumptions are violated, e.g., due to loss or extreme wear of markers, estimates of population size and vital rates will be biased. Double‐marking experiments have been widely used to estimate rates of marker loss and adjust for associated bias, and we extended this approach to estimate rates of recording errors. We double‐marked 309 Piping Plovers (Charadrius melodus) with unique combinations of color bands and alphanumeric flags and used multi‐state mark recapture models to estimate the frequency with which plovers were misidentified. Observers were twice as likely to read and report an invalid color‐band combination (2.4% of the time) as an invalid alphanumeric code (1.0%). Observers failed to read matching band combinations or alphanumeric flag codes 4.5% of the time. Unlike previous band resighting studies, use of two resightable markers allowed us to identify when resighting errors resulted in reports of combinations or codes that were valid, but still incorrect; our results suggest this may be a largely unappreciated problem in mark‐resight studies. Field‐readable alphanumeric flags offer a promising auxiliary marker for identifying and potentially adjusting for false‐positive resighting errors that may otherwise bias demographic estimates.     

Adverse effects of fluorescent dust marking on the behavior of western black widow spiderlings

Fluorescent dust marking is commonly employed to identify and track small arthropods for studies of ecology, demography, and behavior. Despite its widespread use, no study to date has empirically tested the suitability of dust marking for studies of spider behavior. Here, we test the effects of fluorescent dust marking on proximity of cohabitation, sibling cannibalism, and non‐cannibalistic mortality of western black widow spiderlings, Latrodectus hesperus Chamberlin & Ivie (Araneae: Theridiidae). Results indicate that dust‐marked spiderlings cohabitated at closer proximities and died sooner than undusted spiderlings due to a greater incidence of cannibalism in the dust‐marked group. Thus, we conclude that fluorescent dust marking significantly affected the cohabitation and cannibalistic behavior of L. hesperus spiderlings. Although few studies have reported adverse effects of dust marking on arthropods, our results should serve as a warning to future studies that normal behavior may be disrupted by the use of these fluorescent dust markers. Therefore, preliminary testing should be routine when determining the suitability of any marking technique for not only new species, but also new life stages and behaviors.     

Genome‐wide single‐generation signatures of local selection in the panmictic European eel

Next‐generation sequencing and the collection of genome‐wide data allow identifying adaptive variation and footprints of directional selection. Using a large SNP data set from 259 RAD‐sequenced European eel individuals (glass eels) from eight locations between 34 and 64^oN, we examined the patterns of genome‐wide genetic diversity across locations. We tested for local selection by searching for increased population differentiation using F_ST‐based outlier tests and by testing for significant associations between allele frequencies and environmental variables. The overall low genetic differentiation found (F_ST = 0.0007) indicates that most of the genome is homogenized by gene flow, providing further evidence for genomic panmixia in the European eel. The lack of genetic substructuring was consistent at both nuclear and mitochondrial SNPs. Using an extensive number of diagnostic SNPs, results showed a low occurrence of hybrids between European and American eel, mainly limited to Iceland (5.9%), although individuals with signatures of introgression several generations back in time were found in mainland Europe. Despite panmixia, a small set of SNPs showed high genetic differentiation consistent with single‐generation signatures of spatially varying selection acting on glass eels. After screening 50 354 SNPs, a total of 754 potentially locally selected SNPs were identified. Candidate genes for local selection constituted a wide array of functions, including calcium signalling, neuroactive ligand–receptor interaction and circadian rhythm. Remarkably, one of the candidate genes identified is PERIOD, possibly related to differences in local photoperiod associated with the >30° difference in latitude between locations. Genes under selection were spread across the genome, and there were no large regions of increased differentiation as expected when selection occurs within just a single generation due to panmixia. This supports the conclusion that most of the genome is homogenized by gene flow that removes any effects of diversifying selection from each new generation.     

A new red fluorescent protein that allows efficient marking of murine hematopoietic stem cells

Background

Genetic marking of hematopoietic stem cells (HSCs) with multiple fluorescent proteins (FPs) would allow analysis of their features, including interaction with adjacent cells. However, there are few red FPs that are comparable to green FPs in terms of low toxicity and high fluorescent intensity. This study has evaluated the usefulness of Kusabira Orange (KO) originated from the coral stone Fungia concinna as a red FP for marking of HSCs

Methods

A vector used was the MSCV‐type retroviral vector, DΔNsap that has the PCC4 cell‐passaged myeloproliferative sarcoma virus derived long terminal repeat devoid of a binding site for YY1 and the primer‐binding site derived from the dl587rev, respectively. The vector was cloned with the codon‐optimized KO cDNA for higher expression in mammalian cells (huKO) and converted to the corresponding retroviruses pseudotyped with the vesicular stomatitis virus G envelope protein, then transduced into c‐KIT⁺Sca‐1⁺Lineage^? cells obtained from C57BL/6 (Ly5.1) mice followed by transplantation into lethally irradiated Ly5.2 mice.

Results

Approximately 70% of donor‐derived cells highly expressed huKO at 16 weeks post‐transplantation. Furthermore, the high expression of huKO was also detected in serially transplanted mice, suggesting that expression of huKO per se had little deleterious effect on murine hematopoiesis. In double marking experiments, huKO‐expressing hematopoietic cells were easily distinguished from those expressing EGFP by flow cytometery and fluorescent microscope analysis.

Conclusions

Overall, the results obtained from the present study suggest that huKO can be used as a valuable and versatile red fluorescent marker for HSCs. Copyright © 2008 John Wiley & Sons, Ltd.