Molecular identification of prey in the stomach contents of Harp Seals (Pagophilus groenlandicus) using species-specific oligonucleotides

All methods of diet analysis in marine mammals, including hard part analysis (HPA), have biases affecting the accuracy of prey-species identification and frequency in the estimated diet due to differential consumption, digestion and retention. Using PCR amplification of specific prey DNA with species-specific primers, we developed a DNA-based method that complements HPA and provides an alternative means to detect prey from stomach contents of Harp Seals (Pagophilus groenlandicus). The target size that could be reliably amplified was determined using a digestion time-series of Atlantic Cod (Gadus morhua) tissue in simulated seal stomachs. Various target lengths were trialed using general teleost primers; amplicons of approximately 800 bp or less were consistently obtained. Prey species-specific PCR primers for Atlantic Cod, Arctic Cod (Boreogadus saida) and Capelin (Mallotus villosus) were designed and tested with DNA from the stomach contents of 31 Harp Seals. Amplicons were obtained for all three species-specific primer sets. Amplification results compared with HPA revealed: (i) Atlantic Cod hard parts were found in five stomachs where no Atlantic Cod DNA amplified, suggesting that Atlantic Cod may be over-represented in the estimated diet, (ii) amplification of Arctic Cod DNA occurred for 17 stomachs, including all 12 stomachs with, and five stomachs without, Arctic Cod hard parts, and (iii) Capelin DNA amplified for four of five stomachs with Capelin hard parts and for one stomach without Capelin hard parts. We conclude that PCR amplification of specific prey DNA provides a viable means to complement Harp Seal diet analysis by HPA, but suggest that valuable information for quantitative diet analysis rests in a quantitative PCR approach.     

Mitochondrial functional complementation in mitochondrial DNA-based diseases

Mitochondria exist in networks that are continuously remodeled through fusion and fission. Why do individual mitochondria in living cells fuse and divide continuously? Protein machinery and molecular mechanism for the dynamic nature of mitochondria have been almost clarified. However, the biological significance of the mitochondrial fusion and fission events has been poorly understood, although there is a possibility that mitochondrial fusion and fission are concerned with quality controls of mitochondria. trans-mitochondrial cell and mouse models possessing heteroplasmic populations of mitochondrial DNA (mtDNA) haplotypes are quite efficient for answering this question, and one of the answers is “mitochondrial functional complementation” that is able to regulate respiratory function of individual mitochondria according to “one for all, all for one” principle. In this review, we summarize the observations about mitochondrial functional complementation in mammals and discuss its biological significance in pathogeneses of mtDNA-based diseases.     

DNA identification of gut contents of large pelagic fishes

Partially digested prey items taken from the gut contents of seven species of large pelagic fishes were identified by DNA sequencing of part of the cytochrome b gene. Sequences matched against those of five pelagic prey species held in GenBank® with 99–100% identities. The method is applicable to most gut content identifications, the only limiting factor being the range of control species sequences held in the reference database.     

The feeding habits of a group of herbivorous rock-dwelling cichlid fishes (Cichlidae: Perciformes) from Lake Malawi,Africa

Synopsis The feeding; habits of a group of tropical herbivorous rock-dwelling cichlid fishes from Lake Malawi, Africa, are investigated using stomach content analyses. The various species fed selectively on the periphyton of the rocky shores. Blue-green alga of the genus Calothrix was the most common item ingested by the group. Diatoms (Chrysophyta) also were abundant food items. Discriminant analysis showed that dietary items were good variables to identify species. Interspecific dietary differences showed a continuum from those species feeding primarily on Calothrix to those feeding primarily on diatoms. Algal resources exhibit distinct patterns of spatial variation. Diet was correlated with foraging behavior and trophic morphology. Interspecific differences in diet could possibly facilitate ecological coexistence among various species. Such coexistence would contribute to the maintenance of the high diversity fish faunas characteristic of the Great Rift Lakes of Africa.     

Effectiveness of TaqMan probes for detection of fish eggs and larvae in the stomach contents of a teleost predator

Experiments were conducted on the ability of TaqMan molecular probes to detect plaice Pleuronectes platessa DNA from eggs, and cod Gadus morhua DNA from eggs and larvae following ingestion by a teleost predator, whiting Merlangius merlangus. Estimated half-life detection rate (T50) for eggs was 31 h, and 26 h for larvae, with some positive detections occurring even after visual inspection indicated complete gut clearance. Because TaqMan probes are taxon specific, the results presented demonstrate that this technique can provide a means of rapid and unambiguous detection of predation by teleosts on fish eggs and larvae.     

Molecular identification methods of fish species: reassessment and possible applications

Fish species identification is traditionally based on external morphological features. Yet, in many cases fishes and especially their diverse developmental stages are difficult to identify by morphological characters. DNA-based identification methods offer an analytically powerful addition or even an alternative. This work intends to provide an updated and extensive overview on the PCR-methods for fish species identification. Among the ten main methods developed, three PCR-RFLP, PCR-FINS and PCR-specific primers have been the most used. Two other emerging methods, namely real-time PCR and microarray technology, offer new potential for quantification of DNA and simultaneous detection of numerous species, respectively. Almost 500 species have been targeted in the past decade, among which the most studied belong to gadoids, scombroids, and salmonids. The mitochondrial cytochrome b gene was by far the most targeted DNA markers. The most common applications belonged to the forensic, taxonomic, and ecological fields. At last, some key problems, such as the degradation of DNA, the reliability of sequences, and the use of scientific names, likely to be encountered during the development of molecular identification methods are described. In conclusion, the tremendous advances in molecular biology in the past 10 years has rendered possible the study of DNA from virtually any substrates, offering new perspectives for the development of various applications, which will likely continue to increase in the future.     

DNA技术在海洋食品物种鉴定中的应用

随着分子生物学技术的发展, 海洋食品物种鉴定方法由原来的蛋白质水平深入到了DNA水平。目前应用于海洋食品物种鉴定的DNA技术主要是FINS(Forensically informative nucleotide sequencing)、PCR-RFLP和物种特异性PCR标记技术等, 能够实现对新鲜、冰冻、腌制或灌装食品物种进行鉴定, 而对混合样本的鉴定及量化分析是尚待解决的一个问题。基因数据库对物种鉴定的影响也越来越大, 是海洋加工食品物种鉴定可利用的另一种重要信息资源。文章综述了DNA技术在海洋食品物种鉴定中的应用研究进展, 并展望DNA技术在海洋食品检测中的发展趋势。     

An apparatus for sampling gut contents of large,living fishes

Synopsis Apparatus and methods are described and illustrated for flushing and retaining gut contents from large, living fishes with water supplied by a 12 volt portable pump.     

Single molecule PCR in mtDNA mutational analysis: Genuine mutations vs. damage bypass-derived artifacts

The area of somatic mtDNA mutation measurement is in a crisis because the methods used to quantify mtDNA mutations produce results varying by multiple orders of magnitude. The reason for these discrepancies is not clear, but given that most methods involve PCR, the prime suspect is PCR artifacts (e.g. spontaneous errors by the DNA polymerases used). In addition to simple misincorporation, another important source of artificial mutations is the conversion of chemically modified (e.g. damaged) nucleotides into mutations when bypassed by a thermostable DNA polymerase. These latter mutations are particularly difficult to account for because appropriate controls are not available. Here, we argue that single molecule PCR (smPCR) is uniquely positioned to account for these bypass-related artificial mutations and discuss the methodology involved in employing this technique.     

Quantitative evaluation of the point method for fish stomach contents analysis

To test the accuracy of the point methods a simulation was conducted using simulated stomachs. Results from seven participants told to analyse the stomachs by the point and percentage methods differed considerably among subjects and from the control.     

Immunohistochemical and electron-microscopic identification of neuroendocrine cells in the stomach of uremic rats

Many disturbances in electrolyte and hormonal balance in the body induced by functional impairment of renal parenchyma may affect the activity of amine precursor uptake and decarboxylation (APUD) cells, which constitute a very important link in the regulation of homeostasis. The aim of the present study was the morphological, immunohistochemical and ultrastructural estimation of enteroendocrine cells in the stomach of uremic rats. Fragments of gastric pylorus were collected 1, 2 and 4 weeks after nephrectomy. Paraffin embedded sections were stained with H + E and by silver impregnation. For identification of neuroendocrine cells, immunohistochemical reactions were performed using specific antibodies against somatostatin, synaptophysin, neuron-specific enolase and anti-calcitonin gene related peptide. The analysis showed an increased number of APUD cells in the stomach of uremic rats compared to control rats, which may be a morphological expression of their hyperfunction in the functional impairment of renal parenchyma. These results suggest that chronic renal failure can modulate the secretory processes of APUD cells.     

Short-term foraging patterns of individual cornetfish, Fistularia commersonii, based on stomach content analysis

Stomach contents of 75 specimens of the cornetfish Fistularia commersonii, collected in shallow water off Kuchinoerabu-jima Island, southern Japan were analyzed. Many fish contained multiple prey. The prey were mostly fish, grouped into two types, pelagic and reef fishes. The size of prey increased as the size of F. commersonii increased. All the small individuals (<50 cm SL) had fed on only small reef fish. However, most of the large individuals (>50 cm SL) had fed on either prey type. Both pelagic and reef fishes usually occurred simultaneously in shallow water, suggesting that most of the large cornetfish may selectively hunt either type of prey. Received: February 27, 2001 / Revised: September 11, 2001 / Accepted: October 10, 2001     

The stomach contents of Patagonian toothfish around South Georgia (South Atlantic)

The proportion of Patagonian toothfish Dissostichus eleginoides , caught around South Georgia in the south-west Atlantic, with empty stomachs was much lower in fish caught in pots compared to longlines (28 and 91%, respectively, of examined individuals). It is hypothesized that pots caused reduced levels of stress on capture. Stomach content data examined from pot-caught fish will probably therefore be more comprehensive than that from fish caught using longlines. A wide range of prey items was identified in the stomachs of D. eleginoides and stomach contents of individuals caught using the two fishing methods were significantly different. The most common prey item for D. eleginoides caught using pots was the decapod prawn Nauticaris sp., which was restricted in location and depth. However, prawns were not common in the stomachs of D. eleginoides caught from the same location using longlines. Stomach contents from the two fishing methods remained significantly different when Nauticaris sp. were eliminated from the assessment, although fishes then dominated the diet of D. eleginoides caught using either fishing gear. The study confirms that D. eleginoides is an opportunistic carnivore, and indicates that feeding habits depend on the local availability of food items, as well as factors such as depth and predator size. The potential ecological impacts of fishing for D. eleginoides on the South Atlantic ecosystem are discussed.     

Quantitative analysis of total mitochondrial DNA: competitive polymerase chain reaction versus real-time polymerase chain reaction

An efficient and effective method for quantification of small amounts of nucleic acids contained within a sample specimen would be an important diagnostic tool for determining the content of mitochondrial DNA (mtDNA) in situations where the depletion thereof may be a contributing factor to the exhibited pathology phenotype. This study compares two quantification assays for calculating the total mtDNA molecule number per nanogram of total genomic DNA isolated from human blood, through the amplification of a 613-bp region on the mtDNA molecule. In one case, the mtDNA copy number was calculated by standard competitive polymerase chain reaction (PCR) technique that involves co-amplification of target DNA with various dilutions of a nonhomologous internal competitor that has the same primer binding sites as the target sequence, and subsequent determination of an equivalence point of target and competitor concentrations. In the second method, the calculation of copy number involved extrapolation from the fluorescence versus copy number standard curve generated by real-time PCR using various dilutions of the target amplicon sequence. While the mtDNA copy number was comparable using the two methods (4.92 +/- 1.01 x 10(4) molecules/ng total genomic DNA using competitive PCR vs 4.90 +/- 0.84 x 10(4) molecules/ng total genomic DNA using real-time PCR), both inter- and intraexperimental variance were significantly lower using the real-time PCR analysis. On the basis of reproducibility, assay complexity, and overall efficiency, including the time requirement and number of PCR reactions necessary for the analysis of a single sample, we recommend the real-time PCR quantification method described here, as its versatility and effectiveness will undoubtedly be of great use in various kinds of research related to mitochondrial DNA damage- and depletion-associated disorders.     

Utilization of stomach content DNA to determine diet diversity in piscivorous fishes

The objective of the study was to validate and apply DNA-based approaches to describe fish diets. Laboratory experiments were performed to determine the number of hours after ingestion that DNA could be reliably isolated from stomach content residues, particularly with small prey fishes (c. 1 cm, <0·75 g). Additionally, experiments were conducted at different temperatures to resolve temperature effects on digestion rate and DNA viability. The molecular protocol of cloning and sequencing was then applied to the analysis of stomach contents of wild fishes collected from the western basin of Lake Erie, Canada-U.S.A. The results showed that molecular techniques were more precise than traditional visual inspection and could provide insight into diet preferences for even highly digested prey that have lost all physical characteristics.     

Immunological detection of winter flounder (Pseudopleuronectes americanus) eggs and juveniles in the stomach contents of crustacean predators

Predation on the early life history of fish is an important factor regulating year-class strength. Verifying predation events, however, is difficult when analyses rely on visually identifying the remnants of partially digested fish in the stomachs of suspected predators. The objective of this study was to assess the utility of using immunological assays to detect the presence of winter flounder eggs and juveniles (Pseudopleuronectes americanus) in the gut contents of sand shrimp (Crangon septemspinosa) and green crab (Carcinus maenas). After defining assay capabilities, the stomach contents of field-collected shrimp and crabs were examined to determine if these predator-prey relationships occur under natural conditions. Winter flounder-specific antisera developed and used in this study successfully identified homologous antigens (egg or juvenile flounder extracts) without appreciably cross-reacting with antigenic material from predators or nontarget prey. Moreover, antisera detected flounder eggs 10.8-16.4 h after initial feeding by various sized shrimp, and identified juvenile flounder 9.4 and 7.8 h after initial ingestion by shrimp and crabs, respectively. Immuonological dietary analysis of decapod crustaceans collected from Niantic River, Connecticut, revealed that C. septemspinosa and C. maenas are potentially important predators on the early life stages of winter flounder. The temporal trends and magnitude of flounder predator-induced mortality was affected primarily by the spatial and temporal overlap between predator and prey (egg mortality), and the size-dependent relationships underlying crustacean and flatfish predator-prey interactions (juvenile mortality).     

Changes in digestive rate of a predatory beetle over its larval stage: Implications for dietary breadth

Prey and non-prey foods differ substantially in their suitability for zoophytophagous omnivores, but the relative quality of these foods depends on the stage-specific digestive capabilities of the organism in question. Quantitative (or real-time) PCR was used to amplify food-specific DNA and measure consumption rates and digestion efficiencies of four foods - two prey (Aphis glycines and Leptinotarsa decemlineata eggs) and two non-prey (Zea mays pollen and the yeast Saccharomyces cerevisiae) species - over different larval stages of Coleomegilla maculata. The amount of Z. mays pollen consumed increased as larvae aged, but not proportionately with larval size, such that consumption rates decreased uniformly with insect age. While aging larvae fed A. glycines had a similar pattern in their diminishing consumption rates, they consumed similar amounts of A. glycines regardless of age, suggesting a negative feedback mechanism for consumption of this species of aphids. Older larvae digested three of the four foods significantly more efficiently than younger larvae, the exception being larvae fed A. glycines which was digested at a similar rate throughout the larval stage. There was a significant effect of time on food quantity detected for all four species of food. We conclude that C. maculata expands its physiological capacity for digesting prey and non-prey foods as they age in order to better accommodate the increased nutritional needs of the older larvae. This strategy has important implications for the life history strategies of zoophytophagous insects and how they function within foods webs.     

地高辛标记探针检测Vero细胞狂犬病疫苗残余Vero细胞DNA含量

地高辛标记Vero细胞DNA,并制备成DNA探针,以此探针进行点杂交,确定探针的工作浓度,特异性及灵敏度,并用此法监测Vero细胞狂犬病疫苗浓缩原液纯化工艺的Vero细胞DNA去除率,测定精制Vero细胞狂犬病疫苗成品中残余Vero细胞DNA含量,结果明显,该法特异性强,重复性好,快速简便,灵敏度高,检测值可达5pg/剂量,可用于精制ero细胞狂犬病疫苗纯化工艺的质量控制和半成品检定。     

A ribosomal DNA-based framework for the detection and quantification of stress-sensitive nematode families in terrestrial habitats

Indigenous communities of soil‐resident nematodes have a high potential for soil health assessment as nematodes are diverse, abundant, trophically heterogeneous and easily extractable from soil. The conserved morphology of nematodes is the main operational reason for their under‐exploitation as soil health indicators, and a user‐friendly biosensor system should preferably be based on nonmorphological traits. More than 80% of the most environmental stress‐sensitive nematode families belong to the orders Mononchida and Dorylaimida. The phylogenetic resolution offered by full‐length small subunit ribosomal DNA (SSU rDNA) sequences within these two orders is highly different. Notwithstanding several discrepancies between morphology and SSU rDNA‐based systematics, Mononchida families (indicated here as M1–M5) are relatively well‐supported and, consequently, family‐specific DNA sequences signatures could be defined. Apart from Nygolaimidae and Longidoridae, the resolution among Dorylaimida families was poor. Therefore, a part of the more variable large subunit rDNA (≈ 1000 bp from the 5′‐end) was sequenced for 72 Dorylaimida species. Sequence analysis revealed a subclade division among Dorylaimida (here defined as D1–D9, PP1–PP3) that shows only distant similarity with ‘classical’ Dorylaimid systematics. Most subclades were trophically homogeneous, and — in most cases — specific morphological characteristics could be pinpointed that support the proposed division. To illustrate the practicability of the proposed molecular framework, we designed primers for the detection of individual subclades within the order Mononchida in a complex DNA background (viz. in terrestrial or freshwater nematode communities) and tested them in quantitative assays (real‐time polymerase chain reaction). Our results constitute proof‐of‐principle for the concept of DNA sequence signatures‐based monitoring of stress sensitive nematode families in environmental samples.     

基于胃含物分析和稳定同位素技术研究鳀的摄食生态

鳀是重要的渔业资源捕捞对象,同时也是生态系统营养动力学研究的关键种.基于2020年和2008-2009年东海区采集的鳀样品,结合胃含物分析和肌肉碳、氮稳定同位素技术,分析了鳀的食物组成、食性昼夜差异、不同发育阶段的食性转变及其营养级,研究鳀的摄食生态.胃含物分析显示,鳀主要摄食浮游甲壳类和小型鱼类,优势饵料依次为太平洋...