Effect of DNA extraction on ageing success in coral trout (Plectropomus leopardus) otoliths

To maximize the information commonly collected from otoliths, the effect of DNA extraction on the estimation of age with otoliths was evaluated by comparing sagittal otolith samples from common coral trout ( Plectropomus leopardus ) for clarity and ageing discrepancies in DNA-extracted and untreated control otoliths. The DNA extraction process had no significant effect, indicating that archived otoliths can be used as a source of DNA while retaining their utility for age estimation.     

Mediterranean juvenile bluefin tuna: life history patterns

As there is a lack of information on the growth and migrations of bluefin tuna, information about them was gathered using the structural and chemical characteristics of their otoliths and mercury levels in body tissues as indicators of physiological and habitat characteristics. The otoliths of juvenile tuna caught in the Spanish Mediterranean littoral were studied. Otolith increments, assumed to be formed daily, were enumerated. Measurements by wavelength dispersive electron microprobe confirmed the presence of strontium in otolith tissue, and an inverse relationship between strontium/calcium (Sr/Ca) concentration ratio and temperature is suggested. Electron microprobe analyses combined with daily increment analyses of otoliths provided life history profiles for individual fish. Additional Sr/Ca concentration ratio data on fish supported the idea that Sr/Ca ratios can provide information on the environmental history of individual fish. Body concentrations of mercury were related to otolith analyses to suggest age structure, critical life history periods, growth environment, stock structure, food web position, and migration history. The techniques applied present an innovative approach to management-related problems, and the combination of chemical analyses with structural analyses promises to expand our knowledge of the life history of migratory fishes.     

cost-effective method of preparing larval fish otoliths for reading using enzyme digestion and staining

A fast and cost-effective method for examining otoliths in fish larvae was developed whereby the otolith remains in situ . Whole fish of the clownfish Amphiprion melanopus were enzymecleared using a laundry pre-soak and then stained using the Von Kossa silver staining method for calcium. The otolith nucleus, daily rings and the otolith edge were all clearly visible and were suitable for a variety of age and growth analyses. The total 'hands on' time required to process these otoliths was c . 3 min, and multiple samples could be processed simultaneously. The reduction in labour of this method to produce clear daily rings in the otolith lends itself to broad use in fish biology where large quantities of otoliths need to be examined in a cost- and time-efficient manner.     

The use of otolith shape analysis for ageing juvenile red snapper, Lutjanus campechanus

Morphological changes in otolith shape with age, of young (<age 3) red snapper were examined through shape analysis and tested as an objective method for age determination. Otoliths from two collections of juvenile fish (hatchery and wild) were used in the study. First, shape analysis was applied to a series of known-age otoliths from hatchery-reared age 0, 1 and 2 fish. Multidimensional scaling and non-parametric analysis of similarities showed significant shape differences among the three age classes of fish. Discriminant function analysis and cross-validation classification showed 65.6% correct age classification based on shape variables alone, and 86.7% correct age classification with inclusion of otolith weight in the discriminant function (n?=?90). Subsequently, the method was applied to otoliths from a series of age 0, 1 and 2 wild caught red snapper. Otoliths from wild fish showed a similar age classification success rate of 68.9% based on shape variables alone and 86.7% correct age classification with the inclusion of otolith weight in the discriminant function (n?=?90). Ageing of juvenile red snapper through otolith increment counts has been difficult in past studies and this study provides an alternative, objective method of otolith shape analysis for ageing young fish of this species.     

Validation of a simple and well‐suited chemical cleaning method for fish otoliths

In marine biology, many research fields are based on use of fish otoliths. All the studies dealing with otoliths need as starting point a perfectly clean otolith. Dissection is difficult when working on small or highly jagged otoliths. A common problem is that during otolith preparation some fish tissue may remain stuck to it, even after a mechanical cleaning. Then, supplementary cleaning with chemicals is needed. Classical methods are known to possibly alter otolith's structure and/or composition. Here, we present a chemical cleaning method using only sodium hydroxide. We have validated the method on two different fish species, Oblada melanura and Dicentrarchus labrax. Observation and analyses of morphological measurements have confirmed significant suppression of residual tissues. We propose this method as a good alternative to previously published mono‐ or multichemical methods.     

The use of archived tags in retrospective genetic analysis of fish

Collections of historical tissue samples from fish (e.g. scales and otoliths) stored in museums and fisheries institutions are precious sources of DNA for conducting retrospective genetic analysis. However, in some cases, only external tags used for documentation of spatial dynamics of fish populations have been preserved. Here, we test the usefulness of fish tags as a source of DNA for genetic analysis. We extract DNA from historical tags from cod collected in Greenlandic waters between 1950 and 1968. We show that the quantity and quality of DNA recovered from tags is comparable to DNA from archived otoliths from the same individuals. Surprisingly, levels of cross‐contamination do not seem to be significantly higher in DNA from external (tag) than internal (otolith) sources. Our study therefore demonstrates that historical tags can be a highly valuable source of DNA for retrospective genetic analysis of fish.     

Age and growth information available from the otoliths of the Hawaiian snapper,Pristipomoides filamentosus

The otoliths of tropical fish may provide important life history information incorporated within their structural and chemical constituents. All three otoliths (sagitta, lapillus, asteriscus) of the tropical fish Pristipomoides filamentosus were examined internally by Scanning Electron Microscope methods to observe micro-increments and externally to determine three dimensional structure. It was discovered that the sagitta contained four cores and that the plane chosen to be sectioned for micro-increment enumeration could result in errors if more than one core were transversed. The medial cross sectional plane was consequently resolved to effer the most accurate micro-increment counts. Obserations of lapilli also revealed micro-increments and subsequent counts were closely correlated to those detected in the sagittae. The visualization of increments made it feasible to assess age and evolve a growth model. In addition, sagitta weight was found to be related to growth rate and may provide a quick estimate of relative growth. Chemical analyses of otoliths for stable isotopes and Sr/Ca ratios all suggested that an individual fish inhabited warmer waters as it became older. A combination of otolith structural and chemical information can provide age and growth data which is essential to the calculation of accurate population parameters.     

A factorial design experiment as a pilot study for noninvasive genetic sampling

Noninvasive genetic sampling has increasingly been used in ecological and conservation studies during the last decade. A major part of the noninvasive genetic literature is dedicated to the search for optimal protocols, by comparing different methods of collection, preservation and extraction of DNA from noninvasive materials. However, the lack of quantitative comparisons among these studies and the possibility that different methods are optimal for different systems make it difficult to decide which protocol to use. Moreover, most studies that have compared different methods focused on a single factor – collection, preservation or extraction – while there could be interactions between these factors. We designed a factorial experiment, as a pilot study, aimed at exploring the effect of several collection, preservation and extraction methods, and the interactions between them, on the quality and amplification success of DNA obtained from Asiatic wild ass (Equus hemionus) faeces in Israel. The amplification success rates of one mitochondrial DNA and four microsatellite markers differed substantially as a function of collection, preservation and extraction methods and their interactions. The most efficient combination for our system integrated the use of swabs as a collection method with preservation at ?20 °C and with the Qiagen DNA Stool Kit with modifications as the DNA extraction method. The significant interaction found between the collection, preservation methods and the extraction methods reinforces the importance of conducting a factorial design experiment, rather than examining each factor separately, as a pilot study before initiating a full‐scale noninvasive research project.     

Ready-to-use DNA extracted with a CTAB method adapted for herbarium specimens and mucilaginous plant tissue

This report summarizes major changes in previously published protocols for DNA extraction to improve the quality of DNA extracted from plants. Here, we highlight the critical modifications in the original protocols. The efficiency of these changes results in high-quality DNA ready to use in a variety of phytogenetically distant plant families, in particular species with mucopolysaccharides. The DNA obtained can be used without further purification in various molecular biology assays, including direct sequencing and AFLP and RAPD (random-amplified polymorphic DNA) analyses. The effectiveness of this method is proven by the amplification and sequencing of PCR products of up to 1 kb with DNA extracted from herbarium tissue ≥60 years old. This versatility is not usually found in DNA extraction protocols. In addition, this method is quick, adaptable to standard laboratories, and most important, safer and more cost-effective.     

Coupling non-destructive DNA extraction and voucher retrieval for small soft-bodied Arthropods in a high-throughput context: the example of Collembola

Here, we describe a simple method adapted for high-throughput protocols allowing voucher specimen recovery for Collembola and by extension for other soft-bodied small arthropods. A standard extraction protocol was tested to examine the effects of lysis duration (1, 2, 4, 12 h) on DNA concentration, amplification success and specimen condition. Good quality DNA was obtained after 1 h of lysis, while voucher condition was fine for up to 12 h. The lysis step substantially shortens the clearing process necessary for morphological examination.     

Forensic biological pursuits of exotic fish origins: piranha in Hawaii

Synopsis The otoliths of an adult red-bellied piranha, Pygocentrus nattereri, captured from a reservoir in Hawaii were scrutinized to determine the fish's origin and growth history. Sagittal otoliths of the piranha, P. nattereri, contained internal microincrements visible by Scanning Electron Microscopy. The medial cross sectional plane of the sagitta was resolved to provide counts for the most visible micro-increments. An opportune spawning of confiscated adults provided samples for verification of daily increment formation. Daily formation of microincrements was verified from hatched individuals, and confirmed the suitability of otoliths for revealing daily patterns in the age and growth of piranhas. The central area of the sagitta was diffuse in regards to otolith microstructure and indicated the fish was held in an unchanging environment (aquarium). Therefore, otoliths provide important life history or forensic information incorporated within their structural components. The visualization of daily microincrements in the otolith of a juvenile allowed the determination of age at and since release. Fish grew rapidly after being released into the wild. From otolith increments the date of release for an individual fish can be calculated with acceptable accuracy. As presented, otolith structural information can provide age and growth data which are essential to the management of introduced species.     

Three-dimensional imaging of walleye pollock otoliths: reconstruction from serial sections and fluorescent laser cytometry

Two techniques have been developed to examine the three-dimensional internal structure of otoliths. In the first, otoliths were sectioned serially, images were digitized, and the otolith was reconstructed as a computer model. In the second method growth increments were marked in vivo during their formation by immersing the fish in a fluorescent dye, and then the internal structure of the otolith visualized using laser cytometry. The results are useful for evaluating the potential for bias in otolith measurements and for determining the sectional plane with the least bias.     

Sectioned or whole otoliths? A global review of hard structure preparation techniques used in ageing sparid fishes

Reviews in Fish Biology and Fisheries - While otoliths are considered the most reliable structure to accurately age fish, a variety of otolith preparation techniques are available, which have...     

Seeking the true time: Exploring otolith chemistry as an age-determination tool

Fish otoliths' chronometric properties make them useful for age and growth rate estimation in fisheries management. For the Eastern Baltic Sea cod stock (Gadus morhua), unclear seasonal growth zones in otoliths have resulted in unreliable age and growth information. Here, a new age estimation method based on seasonal patterns in trace elemental otolith incorporation was tested for the first time and compared with the traditional method of visually counting growth zones, using otoliths from the Baltic and North seas. Various trace elemental ratios, linked to fish metabolic activity (higher in summer) or external environment (migration to colder, deeper habitats with higher salinity in winter), were tested for age estimation based on assessing their seasonal variations in concentration. Mg:Ca and P:Ca, both proxies for growth and metabolic activity, showed greatest seasonality and therefore have the best potential to be used as chemical clocks. Otolith image readability was significantly lower in the Baltic than in the North Sea. The chemical (novel) method had an overall greater precision and percentage agreement among readers (11.2%, 74.0%) than the visual (traditional) method (23.1%, 51.0%). Visual readers generally selected more highly contrasting zones as annuli whereas the chemical readers identified brighter regions within the first two annuli and darker zones thereafter. Visual estimates produced significantly higher, more variable ages than did the chemical ones. Based on the analyses in our study, we suggest that otolith microchemistry is a promising alternative ageing method for fish populations difficult to age, such as the Eastern Baltic cod.     

Age determination of the Antarctic fishes Champsocephalus gunnari and Notothenia rossii marmorata from South Georgia

Summary Investigations were performed on otoliths from Champsocephalus gunnari and Notothenia rossii marmorata as part of an effort to study the population dynamics of Antarctic fishes. Examination of otoliths by scanning electron microscope (SEM) revealed internal microincrements which were similar to daily increments described for other fish species. No easily identifiable annuli could be discerned and fishes were aged by counting microincrements. Multiple regression analyses relating age to otolith morphometrics and fish size, provided a simpler ageing method. It was determined with these techniques that C. gunnari grew to 50 cm in 15 yrs and N. rossii marmorata grew to 50 cm in 6.5 yrs. The lack of larger-sized individuals for N. rossii marmorata may be indicative of recent overfishing with only the younger age groups remaining. It appears that length-at-age data determined on a yearly basis, could provide valuable scientific and management information for these species.     

Rapid and Economic DNA Extraction from a Single Salmon Egg for Real-Time PCR Amplification

Salmon eggs are common in Japanese sushi and other seafood products; however, certain fish eggs are used as counterfeit salmon eggs which are found in foods and processed products. This study develops a simple, rapid, and cost-effective method for DNA extraction, filtration (FT) and dilution (DL) protocols from a single salmon egg with good DNA quality for real-time PCR amplification. The DNA amount, DNA quality, and real-time PCR performance for different dilutions and different lengths of PCR amplicons were evaluated and compared with the common Qiagen tissue kit (QTK) and Chelex-100-based (CX) protocols. The extracted DNA from a single salmon egg using the FT or DL protocol can be applied in phylogenic research, food authentication and post-marketing monitoring of genetically modified (GM) food products.     

Exposure to thyroid hormone in ovo affects otolith crystallization in rainbow trout Oncorhynchus mykiss

Calcareous otoliths in the inner ears of fishes are necessary for proper hearing and vestibular function. Sagittal otoliths are usually composed of the calcium carbonate polymorph aragonite but may contain the polymorph vaterite, a phenomenon called otolith crystallization. The causes of otolith crystallization are poorly understood. Thyroid hormone (TH) can influence the chemical microenvironment and structure of the inner ear, suggesting that TH may influence otolith crystallization. The present study examined the effect of exogenous TH treatment on sagittal otolith crystallization and growth in larval and juvenile rainbow trout, Oncorhynchus mykiss. In the first experiment, 110?C179?day-old fish raised from TH-treated oocytes had significantly fewer sagittal otoliths containing the crystalline calcium carbonate polymorph vaterite as compared to untreated fish. Vaterite-containing otoliths were significantly longer than those containing the typical polymorph aragonite, although there was no effect of TH treatment on otolith length. In the second experiment, juveniles immersed in an exogenous solution of TH for 6?weeks had slightly longer otoliths (relative to fish length) than age-matched controls, but this effect was not significant. This juvenile population had a very high percentage (88.3?%) of vaterite sagittae overall and this percentage did not change significantly with treatment, suggesting the switch from aragonite to vaterite occurred prior to inclusion of the fish in the study. These results suggest that early manipulation of TH levels may affect calcium carbonate deposition on the otolith but that later TH exposure is unable to restore typical otolith composition.     

Validation of scales and otoliths for estimating age of redband trout in high desert streams of Idaho

Studies validating aging structures for rainbow trout are sparse and none have been conducted for redband trout, a common western U.S. sub-species. Oxytetracycline mark-recapture methods (MR), marginal incremental analysis (MIA), and comparisons across multiple populations were used to evaluate the utility of two structures for aging redband trout in high desert streams. We assessed periodicity of annulus formation on scale and otolith samples from all age classes of trout residing in two streams, identified the location of the first annulus on otoliths, and compared age estimates and between-reader coefficient of variation on nine additional streams. The use of MIA successfully validated opaque zone periodicity for fish transitioning from age-0 to age-1, and from age 1 to age 2 in two streams. For fish at liberty 13 and 28 months in the same two streams, MR-derived age estimates from whole and sectioned otoliths were 94–100% accurate for fish from 2–9 years old. Scales were only 77% and 38% accurate for fish at liberty 13 and 28 months, respectively. Between-reader coefficient of variation (CV) for scales was high (11.5%), while CV for sectioned otoliths using whole otoliths as corroboratory structures averaged 2.3%. Scales were thus, an unacceptable aging structure for desert redband trout. Given the confusion in the literature, we suggest that more rigorous research should be conducted to define and explain otolith zone formation.     

用鳞片和耳石鉴定鲫年龄的比较研究

两观测者独立观测了分别采自洪湖的175尾和洞庭湖的168尾鲫样本的鲜片和耳石,结果显示鳞片适于鉴定年龄组成简单、生长较快的洞庭湖鲫种群的年龄,两观测者年轮读数的总吻合率可达90.5%,与耳石上年轮读数的吻合率也可高达91.7%;但用鳞片鉴定年龄结构复杂、生长缓慢的洪湖鲫种群年龄,两观测者总吻合率只有50.9%,各龄组吻合率随年龄上升而迅速下降。与耳石上年轮读数总吻合率也仅为56.6A%,存在比耳石低估高龄个体年龄的问题,用耳石鉴定鲫年龄具有易于识别、精确度高的优点,用之鉴定洪湖和洞庭湖鲫种群年龄,两观测者年轮读数的总吻合率都很高,分别为92.7%和97.0%,且随年龄上升,依然可保持较高的精确度,对洪湖鲫种群应用耳石为宜。