A non-invasive technique for rapid extraction of DNA from fish scales

DNA markers are being increasingly used in studies related to population genetics and conservation biology of endangered species. DNA isolation for such studies requires a source of biological material that is easy to collect, non-bulky and reliable. Further, the sampling strategies based on non-invasive procedures are desirable, especially for the endangered fish species. In view of above, a rapid DNA extraction method from fish scales has been developed with the use of a modified lysis buffer that require about 2 hr duration. This methodology is non-invasive, less expensive and reproducible with high efficiency of DNA recovery. The DNA extracted by this technique, have been found suitable for performing restriction enzyme digestion and PCR amplification. Therefore, the present DNA extraction procedure can be used as an alternative technique in population genetic studies pertaining to endangered fish species. The technique was also found equally effective for DNA isolation from fresh, dried and ethanol preserved scales.     

Non-destructive genetic sampling in fish. An improved method for DNA extraction from fish fins and scales

DNA-based studies have been one of the major interests in conservation biology of endangered species and in population genetics. As species and population genetic assessment requires a source of biological material, the sampling strategy can be overcome by non-destructive procedures for DNA isolation. An improved method for obtaining DNA from fish fins and scales with the use of an extraction buffer containing urea and further DNA purification with phenol-chloroform is described. The methodology combines the benefits of a non-destructive DNA sampling and its high efficiency. In addition, comparisons with other methodologies for isolating DNA from fish demonstrated that the present procedure also becomes a very attractive alternative to obtain large amounts of high-quality DNA for use in different molecular analyses. The DNA samples, isolated from different fish species, have been successfully used on random amplified polymorphic DNA (RAPD) experiments, as well as on amplification of specific ribosomal and mitochondrial DNA sequences. The present DNA extraction procedure represents an alternative for population approaches and genetic studies on rare or endangered taxa.     

Spatial Scaling of Environmental Variables Improves Species-Habitat Models of Fishes in a Small,Sand-Bed Lowland River

Habitat suitability and the distinct mobility of species depict fundamental keys for explaining and understanding the distribution of river fishes. In recent years, comprehensive data on river hydromorphology has been mapped at spatial scales down to 100 m, potentially serving high resolution species-habitat models, e.g., for fish. However, the relative importance of specific hydromorphological and in-stream habitat variables and their spatial scales of influence is poorly understood. Applying boosted regression trees, we developed species-habitat models for 13 fish species in a sand-bed lowland river based on river morphological and in-stream habitat data. First, we calculated mean values for the predictor variables in five distance classes (from the sampling site up to 4000 m up- and downstream) to identify the spatial scale that best predicts the presence of fish species. Second, we compared the suitability of measured variables and assessment scores related to natural reference conditions. Third, we identified variables which best explained the presence of fish species. The mean model quality (AUC = 0.78, area under the receiver operating characteristic curve) significantly increased when information on the habitat conditions up- and downstream of a sampling site (maximum AUC at 2500 m distance class, +0.049) and topological variables (e.g., stream order) were included (AUC = +0.014). Both measured and assessed variables were similarly well suited to predict species’ presence. Stream order variables and measured cross section features (e.g., width, depth, velocity) were best-suited predictors. In addition, measured channel-bed characteristics (e.g., substrate types) and assessed longitudinal channel features (e.g., naturalness of river planform) were also good predictors. These findings demonstrate (i) the applicability of high resolution river morphological and instream-habitat data (measured and assessed variables) to predict fish presence, (ii) the importance of considering habitat at spatial scales larger than the sampling site, and (iii) that the importance of (river morphological) habitat characteristics differs depending on the spatial scale.     

Standardization of Genomic DNA Isolation from Minute Quantities of Fish Scales and Fins Amenable to RAPD-PCR

The main focus of this study was to standardize a non-destructive procedure for extraction of genomic DNA (gDNA) from minute quantities of scales and fins of two commonly available fishes Labeo bata and Heteropneustes fossilis and also to compare the gDNA yields from live and as well from frozen samples. The spectrophotometric and electrophoretic analyses revealed a significant difference in the DNA yields from live and frozen samples. The isolated gDNAs were used as templates for RAPD-PCR. The quality and consistency of banding pattern showed that gDNA templates from live tissues performed better than that from frozen tissue samples. It was also found that the minute quantities of fresh scales or fin tissues from live fish provided satisfactory quantity and quality of gDNAs that could support several rounds of RAPD-PCR. This non-destructive sampling has a great implication in gDNA based population genetic studies in endangered and vulnerable species of fishes, where killing or sacrificing is an ethical issue.     

Spatial Scaling of Non-Native Fish Richness across the United States

A major goal and challenge of invasion ecology is to describe and interpret spatial and temporal patterns of species invasions. Here, we examined fish invasion patterns at four spatially structured and hierarchically nested scales across the contiguous United States (i.e., from large to small: region, basin, watershed, and sub-watershed). All spatial relationships in both richness and fraction between species groups (e.g., natives vs. exotics) were positive at large scales. However, contrary to predictions using null/neutral models, the patterns at small scales were hump-shaped (unimodal), not simply negative. The fractions of both domestic (introduced among watersheds within the USA) and foreign (introduced from abroad) exotics increased with area across scales but decreased within each scale. The foreign exotics exhibited the highest dominance (lowest evenness) and spatial variation in distribution, followed by domestic exotics and natives, although on average natives still occupy larger areas than domestic and foreign exotics. The results provide new insight into patterns and mechanisms of fish species invasions at multiple spatial scales in the United States.     

核糖体转录间隔子2应用于鱼类种属的鉴别

为了防止珍稀鱼类的非法捕捞和销售, 鱼类种属的鉴别就成为非常关键的问题, 特别是形态学方法无法区分的样品(如鱼苗、鱼鳞、鱼卵、鱼肉及其加工产品等)。为了帮助珍稀鱼类资源的管理和保护, 文章报道了一种利用核糖体基因的转录间隔子2鉴别鱼类种属的分子遗传学方法: (1) 利用同一目鱼类5.8S rRNA和28S rRNA基因的保守性, 设计出扩增鲤形目鱼类这两个基因间转录间隔子2 DNA片段, 测序获得它们的碱基排列顺序; (2) 再根据不同鱼类转录间隔子2序列的差异, 设计出每种鱼的种属特异引物、种属鉴别标准物, 构建鱼类分子分类图谱, 利用PCR复合扩增技术鉴别鱼类种属。通过对国内不同地方采集的5种鲤形目鱼类的210个单一品种样本和40个混合样本的鉴别检验, 该方法能够准确、灵敏和快速鉴别这5种鱼, 可用于鱼类资源保护和评估、管理和开发, 特别是在渔业管理人员渔业执法、海关打击珍稀鱼类走私、防止商业欺诈和外来有害生物入侵等方面非常有用     

A preliminary study of parasite diversity in the anadromous Kamchatkan mikizha Parasalmo mykiss mykiss (Osteichthyes: Salmoniformes: Salmonidae)

The study of parasites associated with the anadromous mikizha from several rivers of the Western Kamchatka has revealed 44 parasite species. The majority of parasites (59%) belong to marine and estuarine-marine species. Lecithophyllum bothryophorum, Echinorhynchus leidyi and plerocercoids of the family Litobothriidae were found for the first time in anadromous fish entering into Asian continental watersheds. It has been revealed that only a few specimens of the anadromous mikizha feed in fresh waters. Most of feeding fish consume a minor amount of food, and this feeding is occasional. Young forms of intestinal parasites of the freshwater and estuarine-freshwater groups (Neoechinorhynchus spp., Crepidostomum spp., Cucullanus truttae, Eubothrium salvelinf) come into anadromous fishes mainly before their entering in fresh waters.     

Inhibition of whole body Ca2+ uptake in fresh water teleosts, Channa punctatus and Cyprinus carpio in response to salmon calcitonin

In many fish, ultimobranchial-derived calcitonin (CT) has been shown to be a potent hypocalcemic regulator. But an equal number of studies failed to show any correlation between CT and plasma calcium levels. Again, in fish, where CT has been shown to function as a hypocalcemic hormone, the way this is brought about is not well known. While the regulatory role of many hormones e.g., stanniocalcin, pituitary-derived prolactin and cortisol on gill calcium (Ca2+) transport (GCAT) has been well established, very few studies have been done to examine the effects of CT on GCAT in fish. In the present study we examined the effects of synthetic salmon calcitonin (sCT) in vivo on GCAT in two distinctly different species of fresh water teleost, Channa punctatus (partially air breathing) and Cyprinus carpio (fully gill breathing). Whole body calcium uptake, a measure of GCAT, was lower in the partial air breathing fish. We found that salmon CT had significant inhibitory effect on GCAT in both the fish species, kept either in normal tap water or low-calcium water. Fish, kept in high-calcium water, showed little response. In parallel studies we also observed that inhibition of GCAT was correlated with simultaneous changes in plasma calcium levels in response to exogenous administration of sCT. The present findings therefore suggest that CT in fresh water teleosts regulate its hypocalcemic action through inhibition of GCAT.     

Metabarcoding for stomach‐content analyses of Pygmy devil ray (Mobula kuhlii cf. eregoodootenkee): Comparing tissue and ethanol preservative‐derived DNA

The application of high‐throughput sequencing to retrieve multi‐taxon DNA from different substrates such as water, soil, and stomach contents has enabled species identification without prior knowledge of taxon compositions. Here we used three minibarcodes designed to target mitochondrial COI in plankton, 16S in fish, and 16S in crustaceans, to compare ethanol‐ and tissue‐derived DNA extraction methodologies for metabarcoding. The stomach contents of pygmy devilrays (Mobula kuhlii cf. eregoodootenkee) were used to test whether ethanol‐derived DNA would provide a suitable substrate for metabarcoding. The DNA barcoding assays indicated that tissue‐derived operational taxonomic units (OTUs) were greater compared to those from extractions performed directly on the ethanol preservative. Tissue‐derived DNA extraction is therefore recommended for broader taxonomic coverage. Metabarcoding applications should consider including the following: (i) multiple barcodes, both taxon specific (e.g., 12S or 16S) and more universal (e.g., COI or 18S) to overcome bias and taxon misidentification and (ii) PCR inhibitor removal steps that will likely enhance amplification yields. However, where tissue is limited or no longer available, but the ethanol‐preservative medium is still available, metabarcoding directly from ethanol does recover the majority of common OTUs, suggesting the ethanol‐retrieval method could be applicable for dietary studies. Metabarcoding directly from preservative ethanol may also be useful where tissue samples are limited or highly valued; bulk samples are collected, such as for rapid species inventories; or mixed‐voucher sampling is conducted (e.g., for plankton, insects, and crustaceans).     

A Simple and Rapid Procedure for Isolation of Total DNA Suitable for Fingerprint Analysis from shape Amaranthus

A simple, efficient and reliable method is described for isolation of total DNA from young leaves of Amaranthus species. This procedure yields a high amount (600–800 µg DNA/g fresh leaf tissue) of good quality DNA free from contaminating proteins, polysaccharides, and coloured pigments. The DNA is suitable for digestion with several restriction endonucleases, preparation of Southern blots, and PCR amplification. The DNA has been successfully used for generating DNA fingerprint profiles and RAPD banding patterns in two species of Amaranthus. The procedure is suitable for processing of a large number of samples simultaneously.     

Single-layer centrifugation separates spermatozoa from diploid cells in epididymal samples from gray wolves, Canis lupus (L.)

Sperm samples may be used for assisted reproductive technologies (e.g., farmed or endangered species) or as a source of haploid DNA or sperm-specific RNA. When ejaculated spermatozoa are not available or are very difficult to obtain, as is the case for most wild endangered species, the epididymides of dead animals (e.g., animals that have been found dead, shot by hunters or poachers, or that that require euthanasia in zoological collections) can be used as a source of sperm. Such epididymal sperm samples are usually contaminated with cellular debris, erythrocytes, leukocytes, and sometimes also bacteria. These contaminants may be sources of reactive oxygen species that damage spermatozoa during freezing or contribute undesired genetic material from diploid cells. We used single-layer centrifugation through a colloid formulation, Androcoll-C, to successfully separate wolf epididymal spermatozoa from contaminating cells and cellular debris in epididymal samples harvested from carcasses. Such a procedure may potentially be applied to epididymal sperm samples from other species.     

Contrasting patterns of density‐dependent selection at different life stages can create more than one fast–slow axis of life‐history variation

There has been much recent research interest in the existence of a major axis of life‐history variation along a fast–slow continuum within almost all major taxonomic groups. Eco‐evolutionary models of density‐dependent selection provide a general explanation for such observations of interspecific variation in the "pace of life." One issue, however, is that some large‐bodied long‐lived “slow” species (e.g., trees and large fish) often show an explosive “fast” type of reproduction with many small offspring, and species with “fast” adult life stages can have comparatively “slow” offspring life stages (e.g., mayflies). We attempt to explain such life‐history evolution using the same eco‐evolutionary modeling approach but with two life stages, separating adult reproductive strategies from offspring survival strategies. When the population dynamics in the two life stages are closely linked and affect each other, density‐dependent selection occurs in parallel on both reproduction and survival, producing the usual one‐dimensional fast–slow continuum (e.g., houseflies to blue whales). However, strong density dependence at either the adult reproduction or offspring survival life stage creates quasi‐independent population dynamics, allowing fast‐type reproduction alongside slow‐type survival (e.g., trees and large fish), or the perhaps rarer slow‐type reproduction alongside fast‐type survival (e.g., mayflies—short‐lived adults producing few long‐lived offspring). Therefore, most types of species life histories in nature can potentially be explained via the eco‐evolutionary consequences of density‐dependent selection given the possible separation of demographic effects at different life stages.     

The influence of variability in species trait data on community‐level ecological prediction and inference

Species trait data have been used to predict and infer ecological processes and the responses of biological communities to environmental changes. It has also been suggested that, in lieu of trait, data niche differences can be inferred from phylogenetic distance. It remains unclear how variation in trait data may influence the strength and character of ecological inference. Using species‐level trait data in community ecology assumes intraspecific variation is small in comparison with interspecific variation. Intraspecific variation across species ranges or within populations may lead to variability in trait data derived from different scales (i.e., local or regional) and methods (i.e., mean or maximum values). Variation in trait data across species can affect community‐level relationships. I examined variability in body size, a key trait often measured across taxa. I collected 12 metrics of fish species length (including common and maximum values) for 40 species from literature, online databases, museum collections, and field data. I then tested whether different metrics of fish length could consistently predict observed species range boundary shifts and the impacts of an introduced predator on inland lake fish communities across Ontario, Canada. I also investigated whether phylogenetic signal, an indicator of niche‐conservativism, changed among measures. I found strong correlations between length metrics and limited variation across metrics. Accordingly, length was a consistently significant predictor of the response of fish communities to environmental change. Additionally, I found significant evidence of phylogenetic signal in fish length across metrics. Limited variation in length across metrics (within species), in comparison with variation within metrics (across species), made fish species length a reliable predictor at a community‐level. When considering species‐level trait data from different sources, researchers should examine the potential influence of intraspecific trait variation on data derived by different metrics and at different scales.     

Marking zebrafish, Danio rerio (cyprinidae), using scale regeneration

Tagging or marking small laboratory-bred fish species is not an easy task. This also holds for the zebrafish, Danio rerio, which is widely used throughout the world as a model organism for genetics, developmental biology, etc. We present a simple marking technique based on scale regeneration. A comparative morphological study of various types of zebrafish scales indeed shows that regenerated scales are easily distinguishable from nonregenerated ones. We propose to take advantage of this typical morphology to mark a single or several individuals. This technique, based on a natural biological process, is easy to perform and does not enhance fish mortality in laboratory breeding conditions. It permits assembly of several specimens in a single tank with the possibility of identifying each of them by regenerated-scale coding. Nevertheless, a prerequisite is that the species does not lose and regenerate scales in large numbers in laboratory breeding conditions. To check this, 5,200 scales were removed from a large region of the left flank in 100 zebrafish and the number and position of regenerated scales were statistically analysed. Our results indicate that (1) laboratory-bred zebrafish have only a few regenerated scales (7.48%), (2) the probability of finding a regenerated scale is similar whatever its position in a row (antero-posterior axis), but (3) it differs from one row to another (scales from the back are more frequently lost than those from the pectoral region). This paper presents a procedure to mark small breeding colonies of zebrafish using scale regeneration with the number and position of the scales to be removed with high probability of marking success. J. Exp. Zool. 286:297-304, 2000.     

A national approach for mapping and quantifying habitat-based biodiversity metrics across multiple spatial scales

Ecosystem services, i.e., services provided to humans from ecological systems have become a key issue of this century in resource management, conservation planning, and environmental decision analysis. Mapping and quantifying ecosystem services have become strategic national interests for integrating ecology with economics to help understand the effects of human policies and actions and their subsequent impacts on both ecosystem function and human well-being. Some aspects of biodiversity are valued by humans in varied ways, and thus are important to include in any assessment that seeks to identify and quantify the benefits of ecosystems to humans. Some biodiversity metrics clearly reflect ecosystem services (e.g., abundance and diversity of harvestable species), whereas others may reflect indirect and difficult to quantify relationships to services (e.g., relevance of species diversity to ecosystem resilience, cultural value of native species). Wildlife habitat has been modeled at broad spatial scales and can be used to map a number of biodiversity metrics. In the present study, we present an approach that (1) identifies mappable biodiversity metrics that are related to ecosystem services or other stakeholder concerns, (2) maps these metrics throughout a large multi-state region, and (3) compares the metric values obtained for selected watersheds within the regional context. The broader focus is to design a flexible approach for mapping metrics to produce a national-scale product. We map 20 biodiversity metrics reflecting ecosystem services or other aspects of biodiversity for all vertebrate species except fish. Metrics include species richness for all vertebrates, specific taxon groups, harvestable species (i.e., upland game, waterfowl, furbearers, small game, and big game), threatened and endangered species, and state-designated species of greatest conservation need, and also a metric for ecosystem (i.e., land cover) diversity. The project is being conducted at multiple scales in a phased approach, starting with place-based studies, then multi-state regional areas, culminating into a national-level atlas. As an example of this incremental approach, we provide results for the southwestern United States (i.e., states of Arizona, New Mexico, Nevada, Utah, and Colorado) and portions of two watersheds within this region: the San Pedro River (Arizona) and Rio Grande River (New Mexico). Geographic patterns differed considerably among metrics across the southwestern study area, but metric values for the two watershed study areas were generally greater than those for the southwestern region as a whole.     

Species introduced from nearby sources have a more homogenizing effect than species from distant sources: evidence from plants and fishes in the USA

Biotic homogenization is occurring in many biota as widespread introduced species are replacing unique native species. Although efforts to document homogenization have increased, no studies have explicitly compared the homogenizing effects of species introduced from distant areas to the homogenizing effects of species introduced from more proximate areas. The author analysed three data sets, at different scales and in different taxa, that distinguish species introduced from distant sources (e.g. outside the US) from species introduced from less distant sources (e.g. within the United States). These data include: plant introductions among eight major US cities and fish introductions among 12 US states and among 10 watersheds from New York state. The authors found that, for all data sets, species introduced from less distant sources (within the US) have a greater homogenizing effect on community composition than species from more distant sources (outside the US). In agreement with other studies, the author also find that, in terms of absolute numbers, introductions from nearby sources are far more frequent than introduction of species from distant sources. While tentative, these findings point out the importance of considering species introduced from nearby areas (e.g., extralimital native species) when discussing biotic homogenization from human activities.     

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.     

Infectious disease in fish: global risk of viral hemorrhagic septicemia virus

As the global human population continues to increase and become more industrialized, the need for safe, secure, and sustainable protein production is critical. One sector of particular importance is seafood production, where capture fishery and aquaculture industries provide 15–20% of the global protein supply. However, fish production can be severely affected by diseases. Notably, viral hemorrhagic septicemia, caused by the viral hemorrhagic septicemia virus (VHSv; Rhabdoviridae), may be one of the most devastating viral diseases of fishes worldwide. We explored the ecology and epidemiology of VHSv using an ecological niche modeling approach to identify vulnerable disease-free regions. Results showed an impressive ecological plasticity of VHSv. The virus was found in >?140 fish species in marine and freshwater ecosystems, with high diversity of lineages in Eurasia. Sub-genotypes from marine and fresh waters were ecologically similar, suggesting broad ecological niches, rather than rapid evolutive adaptation to novel environments. Ecological niche models predicted that VHSv may have favorable physical (e.g., temperature, runoff), chemical (e.g., salinity, pH, phosphate), and biotic (i.e., chlorophyll) conditions for establishing into areas with important fish industries that, so far, are believed to be disease-free (i.e., freshwater and marine ecosystems of Africa, Latin America, Australia, and inland China). The model and our review suggest fish species from the Perciformes, Salmoniformes, and Gadiformes orders are likely to be infected with VHSv in novel regions as the virus expands its range to areas predicted to be at risk. In conclusion, VHSv remains an emerging disease threat to global food security and aquatic biodiversity.     

Patterns of variability in early-life traits of fishes depend on spatial scale of analysis

Estimates of early-life traits of fishes (e.g. pelagic larval duration (PLD) and spawning date) are essential for investigating and assessing patterns of population connectivity. Such estimates are available for a large number of both tropical and temperate fish species, but few studies have assessed their variability in space, especially across multiple scales. The present study, where a Mediterranean fish (i.e. the white seabream Diplodus sargus sargus) was used as a model, shows that spawning date and PLD are spatially more variable at a scale of kilometres than at a scale of tens to hundreds of kilometres. This study indicates the importance of considering spatial variability of early-life traits of fishes in order to properly delineate connectivity patterns at larval stages (e.g. by means of Lagrangian simulations), thus providing strategically useful information on connectivity and relevant management goals (e.g. the creation of networks of marine reserves).