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.     

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).     

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.     

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.     

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).     

Quantification of alizarin red S uptake in coregonid eggs after mass-marking

An easy method to measure the uptake rate of the persistent dye alizarin red S (ARS) during marking of whitefish eggs was established and used to measure the ARS content in three different whitefish species during and at the end of the marking procedure. Those values show that only 6–10% of the ARS in the marking solution will be absorbed by the eggs (0.0061–0.0119 mg per egg). Additional analyses 6, 15 and 36 months after marking showed ARS levels below the response level (<6.9 μg kg^–1).     

Comparative study of clinical characteristics of amniotic rupture sequence with and without body wall defect: Further evidence for separation

BACKGROUND: Amniotic rupture sequence (ARS) is a disruption sequence presenting with fibrous bands, possibly emerging as a result of amniotic tear in the first trimester of gestation. Our comparative study aims to assess whether there is a difference in the clinical pattern of congenital limb and internal organ anomalies between ARS with body wall defect (ARS‐BWD) and ARS without BWD (ARS‐L). METHODS: Among 1,706,639 births recorded between 1998 and 2006, 50 infants with a diagnosis of ARS were reported to the Polish Registry of Congenital Malformations. The information on 3 infants was incomplete, thus only 47 cases were analyzed. These infants were classified into groups of ARS‐L (38 infants) and ARS‐BWD (9 infants). RESULTS: The ARS‐BWD cases were more frequently affected by various congenital defects (overall p < 0.0001), and in particular by urogenital malformations (p = 0.003). In both groups, limb reduction defects occurred in approximately 80% of cases; however, minor and distal limb defects (phalangeal or digital amputation, pseudosyndactyly, constriction rings) predominated in the ARS‐L group (p = 0.0008). The ARS‐L group also had a higher frequency of hand and upper limb involvement. CONCLUSIONS: This observation suggests that amniotic band adhesion in ARS‐L takes place at a later development stage. Although limited by a small sample size, our study contributes to the growing evidence that both ARS entities represent two nosologically distinct conditions. Birth Defects Research (Part A), 2009. © 2009 Wiley‐Liss, Inc.     

Growth,mortality and tag retention of small Anguilla anguilla marked with visible implant elastomer tags and coded wire tags under laboratory conditions

Growth, and potential marking‐related mortality of small European eel Anguilla anguilla (L.) after marking with visible implant elastomer tags (VIE) and coded wire tags (CWT) were investigated over a 6‐month period in a laboratory experiment. In addition, mark retention after marking was examined for another 16.5 months. Neither marking method had a significant effect on growth (P > 0.05) or mortality (P > 0.05). After 32 days detection of the VIE marks implanted on the ventral surface and along the base of the ventral tail fin margin was 98 and 100%, respectively, but decreased to 9 and 66% after 512 days. Retention of the CWT implanted in the dorsal musculature of A. anguilla was 99% after 32 days and did not change by day 512. It was therefore concluded that marking‐induced mortality was nil for both marking types over the 6‐month period. Generally, both methods are found to be suitable for marking young A. Anguilla. VIE tags, however, should be used for short‐time mark‐recapture experiments only, and should be injected at the base of the ventral fin margin. The use of CWTs seems to be the more suitable marking method for long‐term mark‐recapture experiments. Nevertheless, field tests are necessary to evaluate these marking methods.     

Factors influencing double brooding in Eurasian Hoopoes Upupa epops

Double brooding may be a good strategy for short‐lived species to maximize annual and lifetime reproductive success (ARS and LRS, respectively). Nevertheless, there is typically individual variation in the probability of producing a second clutch. Here we evaluate factors that influence the decision to double brood in the Eurasian Hoopoe Upupa epops. Analyses of an 11‐year dataset showed that 36% of the females and 21% of the males produced a second clutch after successfully raising a first clutch. Double‐brooded females had higher ARS (9.1 ± 1.9 fledglings; mean ± se) and LRS (0.93 ± 0.08 recruits) than single‐brooded females (ARS: 4.5 ± 2.1 fledglings; LRS: 0.36 ± 0.03 recruits). This suggests that double brooding is adaptive in Hoopoes, and raises the question of why most individuals only produce one clutch per season. The probability of double brooding varied only slightly between years, suggesting that it is influenced by individual characteristics rather than by external, population‐level environmental factors. In both sexes, the probability of double brooding increased with earlier timing of the first clutch, and the timing of reproduction was the most important factor influencing reproductive success. The latter is likely to be mediated by changes in resources during the season. The probability of double brooding also increased slightly with female age, due to differences in intrinsic quality among females rather than to a gain in experience. In contrast to many other studies, the probability of double brooding increased with an increasing number of fledglings from the first clutch, suggesting that it is a strategy of individuals of high quality. Taken together, we show that the individual quality of the breeder and the timing of their first clutches are key factors influencing the decision to double brood, and thereby that they are important determinants of reproductive performance in Eurasian Hoopoes.     

Differential targeting of Tetrahymena ORC to ribosomal DNA and non‐rDNA replication origins

The Tetrahymena thermophila origin recognition complex (ORC) contains an integral RNA subunit, 26T RNA, which confers specificity to the amplified ribosomal DNA (rDNA) origin by base pairing with an essential cis‐acting replication determinant—the type I element. Using a plasmid maintenance assay, we identified a 6.7 kb non‐rDNA fragment containing two closely associated replicators, ARS1‐A (0.8 kb) and ARS1‐B (1.2 kb). Both replicators lack type I elements and hence complementarity to 26T RNA, suggesting that ORC is recruited to these sites by an RNA‐independent mechanism. Consistent with this prediction, although ORC associated exclusively with origin sequences in the 21 kb rDNA minichromosome, the interaction between ORC and the non‐rDNA ARS1 chromosome changed across the cell cycle. In G₂ phase, ORC bound to all tested sequences in a 60 kb interval spanning ARS1‐A/B. Remarkably, ORC and Mcm6 associated with just the ARS1‐A replicator in G₁ phase when pre‐replicative complexes assemble. We propose that ORC is stochastically deposited onto newly replicated non‐rDNA chromosomes and subsequently targeted to preferred initiation sites prior to the next S phase.     

Is conspecific substrate marking a long‐term external memory of previously colonized overwintering sites in Harmonia axyridis?

The multicoloured Asian ladybeetle, Harmonia axyridis (Pallas), aggregates inside dwellings during winters to survive the cold. This beetle uses chemical cues coming from congeners to select an overwintering site. Recent research has shown that they preferentially gather at places where conspecifics previously laid a substrate marking made up of saturated and unsaturated hydrocarbons. Some authors have reported that H. axyridis colonizes the same overwintering sites from 1 year to another. Herein, the hypothesis that this substrate marking is used by H. axyridis to settle in the same aggregation sites from one winter to another was tested. To this aim, the temporal modification in the chemical profile of the hydrocarbon marking was studied by performing chromatographic analyses. After 1 year, the overall profile was modified qualitatively and quantitatively: the unsaturated hydrocarbons were no longer detected while some saturated hydrocarbons were still present in large quantities. In a behavioural assay conducted in the laboratory, the 12‐month‐old marking did not induce the aggregation of H. axyridis. This result indicates that the chemical markings left by conspecifics during a previous aggregation period in an overwintering site are not sufficient to induce the gathering of the newly arriving individuals.     

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。     

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%.     

A comparison of abundance estimates from extended batch‐marking and Jolly–Seber‐type experiments

Little attention has been paid to the use of multi‐sample batch‐marking studies, as it is generally assumed that an individual's capture history is necessary for fully efficient estimates. However, recently, Huggins et al. ( 2010 ) present a pseudo‐likelihood for a multi‐sample batch‐marking study where they used estimating equations to solve for survival and capture probabilities and then derived abundance estimates using a Horvitz–Thompson‐type estimator. We have developed and maximized the likelihood for batch‐marking studies. We use data simulated from a Jolly–Seber‐type study and convert this to what would have been obtained from an extended batch‐marking study. We compare our abundance estimates obtained from the Crosbie–Manly–Arnason–Schwarz (CMAS) model with those of the extended batch‐marking model to determine the efficiency of collecting and analyzing batch‐marking data. We found that estimates of abundance were similar for all three estimators: CMAS, Huggins, and our likelihood. Gains are made when using unique identifiers and employing the CMAS model in terms of precision; however, the likelihood typically had lower mean square error than the pseudo‐likelihood method of Huggins et al. ( 2010 ). When faced with designing a batch‐marking study, researchers can be confident in obtaining unbiased abundance estimators. Furthermore, they can design studies in order to reduce mean square error by manipulating capture probabilities and sample size.     

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.     

Externally visible fluorochrome marks and allometries of growing sea urchins

Externally visible, growing calcitic structures can be marked using fluorochromes. Such marks are useful for field recapture studies in ecology, evolution, and aquaculture as well as for studies on mechanisms of growth and development. We marked 2‐month‐old sea urchins (Strongylocentrotus droebachiensis) with the fluorochromes calcein, calcein blue, and tetracycline by batch‐marking via immersion. Neither growth nor survival was affected by marking. Marks were externally visible on the skeletal plates, demipyramids, and spines of 100% of the marked sea urchins up to 63 d post‐marking. After 342 d, marks were still externally visible on 100% of calcein‐marked, 98% of calcein blue‐marked, and 22% of tetracycline‐marked sea urchins. Marks were brightest on calcein‐marked and faintest on tetracycline‐marked sea urchins, in correspondence to the fluorochrome dose. Growth marks in the aboral oculogenital ring, followed for 333 d, showed that genital plate growth in the hoop direction was greatest adjacent to the anal suture and that both the oculogenital ring and periproct grew less than isometrically. Internal marks (not externally visible) were subsequently seen on 99% of the demipyramids at 342 d post‐marking. Such fluorochrome marks on demipyramids have previously been used to measure aboral and oral‐end demipyramid growth to allometrically calibrate diametrical growth rates of field sea urchins. We found that although aboral demipyramid marks were always clear, 13% of marks on the oral end were obscured. However, we show that measuring only aboral end growth is sufficient for allometric calibration. In a separate experiment, multiple marks of the above three fluorochromes plus alizarin complexone, administered by injection to larger (12.9–37.1 mm diameter) sea urchins, persisted internally for at least 2 years. Multi‐color, internally and externally visible, persistent marks will enhance experimental designs in laboratory, field, and common garden experiments.     

Does dimethyl sulfoxide increase protein immunomarking efficiency for dispersal and predation studies?

Marking biological control agents facilitates studies of dispersal and predation. This study examines the effect of a biological solvent, dimethyl sulfoxide (DMSO), on retention of immunoglobulin G (IgG) protein solutions applied to Diorhabda carinulata (Desbrochers) (Coleoptera: Chrysomelidae), an important biological control agent of saltcedar, either internally by feeding them protein‐labeled foliage or externally by immersing them in a protein solution. In addition, we determined whether internally or externally marked DMSO‐IgG labels could be transferred via feeding from marked D. carinulata to its predator, Perillus bioculatus (Fabricius) (Heteroptera: Pentatomidae). The presence of rabbit and chicken IgG proteins was detected by IgG‐specific enzyme‐linked immunosorbent assays (ELISA). DMSO‐IgG treatments showed greater label retention than IgG treatments alone, and this effect was stronger for rabbit IgG than for chicken IgG. Fourteen days after marking, beetles immersed in rabbit IgG showed 100% internal retention of label, whereas beetles immersed in chicken IgG showed 65% internal retention. Immersion led to greater initial (time 0) label values, and longer label retention, than feeding beetles labeled foliage. The DMSO‐IgG label was readily transferred to P. bioculatus after feeding on a single marked prey insect. This investigation shows that addition of DMSO enhances retention of IgG labels, and demonstrates that protein marking technology has potential for use in dispersal and predator–prey studies with D. carinulata. Moreover, our observation of P. bioculatus feeding on D. carinulata is, to our knowledge, a new predator–prey association for the stink bug.     

How area restricted search of a pelagic seabird changes while performing a dual foraging strategy

Movements and foraging strategies of marine predators should cope with the hierarchical spatial distribution of resources. Therefore, in order to predict the at‐sea distribution of aerial predators, it is crucial to understand the factors governing trajectory decisions at different scales. Using first passage time (FPT) analysis on precision tracking information (GPS‐loggers data) we were able to examine the foraging strategy of Cory's shearwaters Calonectris diomedea and to detect the adoption of area‐restricted search (ARS), measuring the scale and duration of this behaviour. Data were collected from three different populations foraging in different oceanographic conditions. During long excursions birds only commuted between their colony and prey patches, while on their short movements birds increased the amount of looping movements. On short trips, birds addopted ARS behaviour at an average scale of 18 km and at a second nested scale of around 2 km. When engaging in long trips, first scale of ARS occurred on average at about 67 km of radii and than a second nested scale at a radii of 24 km. Overall, the different populations showed foraging patterns matching the habitats exploited: a) at smaller scales of ARS, sea‐surface temperature, chlorophyll‐a concentration and depth influenced the time of residence (i.e. FPT) of birds (with variations at a population level); b) at larger scales of ARS, FPT increased within regions of higher gradients of sea‐surface temperature, chlorophyl‐a concentration and depth. This study demonstrates that Cory's shearwaters adopt scale‐dependent adjustments of movement in relation to the hierarchical distribution of the environment they exploit, matching the scale and duration of ARS with the hierarchical distribution of the environmental features.