Efficacy of metabarcoding for identification of fish eggs evaluated with mock communities

There is urgent need for effective and efficient monitoring of marine fish populations. Monitoring eggs and larval fish may be more informative than that traditional fish surveys since ichthyoplankton surveys reveal the reproductive activities of fish populations, which directly impact their population trajectories. Ichthyoplankton surveys have turned to molecular methods (DNA barcoding & metabarcoding) for identification of eggs and larval fish due to challenges of morphological identification. In this study, we examine the effectiveness of using metabarcoding methods on mock communities of known fish egg DNA. We constructed six mock communities with known ratios of species. In addition, we analyzed two samples from a large field collection of fish eggs and compared metabarcoding results with traditional DNA barcoding results. We examine the ability of our metabarcoding methods to detect species and relative proportion of species identified in each mock community. We found that our metabarcoding methods were able to detect species at very low input proportions; however, levels of successful detection depended on the markers used in amplification, suggesting that the use of multiple markers is desirable. Variability in our quantitative results may result from amplification bias as well as interspecific variation in mitochondrial DNA copy number. Our results demonstrate that there remain significant challenges to using metabarcoding for estimating proportional species composition; however, the results provide important insights into understanding how to interpret metabarcoding data. This study will aid in the continuing development of efficient molecular methods of biological monitoring for fisheries management.     

Identification of larval fish in mangrove areas of Peninsular Malaysia using morphology and DNA barcoding methods

The identification of larval fish has been an important morphological issue in marine biology due to the dramatic transformations that most species undergo from early larval stages to adulthood. Insufficient morphological diagnostic characters in larval fishes made it easy to misidentify them and a difficult process to key to genus and species level. The experiment aims to find out, by applying DNA barcoding, how consistent the morphological identifications can be among larval fish. Larval fish were mainly collected using plankton nets around mangrove areas in Pendas (Johor), Setiu (Terengganu), Pekan (Pahang) and Matang (Perak) Malaysia between April 2015 and October 2015. A total of 354 samples were morphologically identified, mostly to the family level and a few to the genus level. Larval fish ranged from 1.5 mm to 31 mm of total length, with the most abundant individuals being <3 mm. Among them, a total of 177 individuals were selected for DNA barcoding analyses. Molecular works involved polymerase chain reaction (PCR) and sequencing of mitochondrial Cytochrome c Oxidase I (COI) gene fragment (655 base pairs) methods. DNA barcoding enabled all samples to be identified down to species level. The overall genetic identities ranged from 91% to 100%. Morphological identification classified the specimens into 19 families and 11 genera while DNA barcoding identified them into 19 families 33 genera and 40 species. A comparison between the two methods showed a mismatched identification of 42.6% where the accuracy percentage for morphological identification was moderate for the family level (67.8%) but was low for genus level identification (30%). The DNA barcoding method also managed to successfully identify 86.4% of the samples up to their species level where morphological method has failed to do so. The most misidentified families in the study were Blenniidae, Sparidae, Apogonidae Ambassidae and Monachantidae while almost all samples from the family Gobiidae and Engraulidae were correctly identified to family level because of their distinct morphology. In conclusion, taxonomic studies of larval fish should continue using combination of both morphology and DNA barcoding methods. Morphological identification should be more conservative i.e., when in doubt, it is better to key only to family and not to the genus and species level. DNA barcoding is a better method for deeper taxonomic levels identification with the existence of robust sequence reference libraries and should be able to validate the accuracy of traditional larval fish identification.     

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     

Noninvasive molecular methods to identify live scarab larvae: an example of sympatric pest and nonpest species in New Zealand

Despite the negative impact that many scarab larvae have on agro-ecosystems, very little attention has been paid to their taxonomy. Their often extremely similar morphological characteristics have probably contributed to this impediment, which has also meant that they are very difficult to identify in the field. Molecular methods can overcome this challenge and are particularly useful for the identification of larvae to enable management of pest species occurring sympatrically with nonpest species. However, the invasive collection of DNA samples for such molecular methods is not compatible with subsequent behavioural, developmental or fitness studies. Two noninvasive DNA sampling and DNA analysis methods suitable for the identification of larvae from closely related scarab species were developed here. Using the frass and larval exuviae as sources of DNA, field-collected larvae of Costelytra zealandica (White) and Costelytra brunneum (Broun) (Scarabaeidae: Melolonthinae) were identified by multiplex PCR based on the difference in size of the resulting PCR products. This study also showed that small quantities of frass can be used reliably even 7 days after excretion. This stability of the DNA is of major importance in ecological studies where timeframes rarely allow daily monitoring. The approach developed here is readily transferable to the study of any holometabolous insect species for which morphological identification of larval stages is difficult.     

Diet analysis in piscivorous birds: What can the addition of molecular tools offer?

In trophic studies on piscivorous birds, it is vital to know which kind of dietary sample provides the information of interest and how the prey can be identified reliably and efficiently. Often, noninvasively obtained dietary samples such as regurgitated pellets, feces, and regurgitated fish samples are the preferred source of information. Fish prey has usually been identified via morphological analysis of undigested hard parts, but molecular approaches are being increasingly used for this purpose. What remains unknown, however, is which dietary sample type is best suited for molecular diet analysis and how the molecular results compare to those obtained by morphological analysis. Pellets, feces, and regurgitated fish samples of Great Cormorants (Phalacrocorax carbo sinensis) were examined for prey using both morphological hard part analysis and molecular prey identification. The sample types and methods were compared regarding number of species detected (overall and per sample) as well as the prey species composition and its variability among individual samples. Via molecular analysis, significantly higher numbers of prey species were detected in pellets, feces, and fish samples. Of the three sample types, pellets contained the most comprehensive trophic information and could be obtained with the lowest sampling effort. Contrastingly, dietary information obtained from feces was least informative and most variable. For all sample types, the molecular approach outperformed morphological hard part identification regarding the detectable prey spectrum and prey species composition. We recommend the use of pellets in combination with molecular prey identification to study the diet of piscivorous birds.     

Using DNA barcoding and phylogenetics to identify Antarctic invertebrate larvae: Lessons from a large scale study

Ecological studies of the diversity and distribution of marine planktonic larvae are increasingly depending on molecular methods for accurate taxonomic identification. The greater coverage of reference marine species on genetic databases such as GenBank and BoLD (Barcoding of Life Data Systems; www.boldystems.org); together with the decreasing costs for DNA sequencing have made large scale larval identification studies using molecular methods more feasible. Here, we present the development and implementation of a practical molecular approach to identify over 2000 individual marine invertebrate larvae that were collected in the Ross Sea, Antarctica, during the austral summer over five years (2002-2007) as part of the LGP (Latitudinal Gradient Project). Larvae for molecular ID were morphologically identified to belong to the Phyla Mollusca, Echinodermata, Nemertea and Annelida (Class Polychaeta), but also included unidentified early developmental stages which could not be assigned a specific taxon (e.g., eggs, blastulae). The use of a 100μm mesh plankton net makes this one of the first larval identification studies to simultaneously consider both embryos and larvae. Molecular identification methods included amplification of up to three molecular loci for each specimen, a pre-identification step using BLAST with GenBank, phylogenetic reconstructions and cross-validation of assigned Molecular Operational Taxonomic Units (MOTUs). This combined approach of morphological and molecular methods assigned about 700 individuals to 53 MOTUs, which were identified to the lowest possible taxonomic level. During the course of this long-term study we identified several procedural difficulties, including issues with the collection of larvae, locus amplification, contamination, assignment and validation of MOTUs. The practical guidelines that we describe here should greatly assist other researchers to conduct reliable molecular identification studies of larvae in the future.     

High-throughput molecular identification of fish eggs using multiplex suspension bead arrays

The location and abundance of fish eggs provide information concerning the timing and location of spawning activities and can provide fishery-independent estimates of spawning biomass. However, the full value of egg and larval surveys is severely restricted because many species' eggs and larvae are morphologically similar, making species-level identification difficult. Recent efforts have shown that nearly all species of fish may be identified by mitochondrial DNA (mtDNA) sequences (e.g. via 'DNA barcoding'). By taking advantage of a DNA barcode database, we have developed oligonucleotide probes for 23 marine fish species that produce pelagic eggs commonly found in California waters. Probes were coupled to fluorescent microspheres to create a suspension bead array. Biotin-labelled primers were used to amplify the mitochondrial cytochrome oxidase subunit I (COI) and 16S ribosomal rRNA genes from individual fish eggs. The amplicons were then hybridized to the bead array, and after the addition of a reporter fluorophore, samples were analysed by flow cytometry with Luminex 100 instrumentation. Probes specifically targeted eggs that are abundant and/or from morphologically indistinguishable species pairs. Results showed that the 33 different probes designed for this study accurately identified all samples when PCR was successful. Suspension bead arrays have a number of benefits over other methods of molecular identification; these arrays permit high multiplexing, simple addition of new probes, high throughput and lower cost than DNA sequencing. The increasing availability of DNA barcode data for numerous fish faunas worldwide suggests that bead arrays could be developed and widely used for fish egg, larval and tissue identifications.     

Characterization of the complete mitochondrial genome of Diphyllobothrium nihonkaiense (Diphyllobothriidae: Cestoda), and development of molecular markers for differentiating fish tapeworms

We sequenced and characterized the complete mitochondrial genome of the Japanese fish tapeworm D. nihonkaiense. The genome is a circular-DNA molecule of 13607 bp (one nucleotide shorter than that of D. latum mtDNA) containing 12 protein-coding genes (lacking atp8), 22 tRNA genes and two rRNA genes. Gene order and genome content are identical to those of the other cestodes reported thus far, including its congener D. latum. The only exception is Hymenolepis diminuta in which the positions of trnS2 and trnL1 are switched. We tested a PCR-based molecular assay designed to rapidly and accurately differentiate between D. nihonkaiense and D. latum using species-specific primers based on a comparison of their mtDNA sequences. We found the PCR-based system to be very reliable and specific, and suggest that PCR-based identification methods using mtDNA sequences could contribute to the study of the epidemiology and larval ecology of Diphyllobothrium species.     

Identifying freshwater mussels (Unionoida) and parasitic glochidia larvae from host fish gills: a molecular key to the North and Central European species

Freshwater mussels (order Unionoida) represent one of the most severely endangered groups of animals due to habitat destruction, introduction of nonnative species, and loss of host fishes, which their larvae (glochidia) are obligate parasites on. Conservation efforts such as habitat restoration or restocking of host populations are currently hampered by difficulties in unionoid species identification by morphological means. Here we present the first complete molecular identification key for all seven indigenous North and Central European unionoid species and the nonnative Sinanodonta woodiana, facilitating quick, low-cost, and reliable identification of adult and larval specimens. Application of this restriction fragment length polymorphisms (RFLP) key resulted in 100% accurate assignment of 90 adult specimens from across the region by digestion of partial ITS-1 (where ITS is internal transcribed spacer) polymerase chain reaction (PCR) products in two to four single digestions with five restriction endonucleases. In addition, we provide protocols for quick and reliable extraction and amplification of larval mussel DNA from complete host fish gill arches. Our results indicate that this new method can be applied on infection rates as low as three glochidia per gill arch and enables, for the first time, comprehensive, large-scale assessments of the relative importance of different host species for given unionoid populations.     

Zebrafish Danio rerio shows behavioural cross-context consistency at larval and juvenile stages but no consistency between stages

Coping style is defined as a set of individual physiological and behavioural characteristics that are consistent across time and context. In the zebrafish Danio rerio, as well as in many other animals, several covariations have been established among behavioural, physiological and molecular responses. Nonetheless, not many studies have addressed the consistency in behavioural responses over time starting at the larval stage. Therefore, this study aimed to improve the understanding of behavioural consistency across contexts and over time in zebrafish from the larval to juvenile stages. Two distinct experiments were conducted: a larval stage experiment (from 8 to 21 days post fertilization, dpf) and a juvenile stage experiment (from 21 to 60 dpf). On one hand, the larval experiment allows to focus on the transition between 8 and 21 dpf, marked by significant morphological changes related to the end of larval stage and initiation of metamorphosis. On the other hand, the juvenile experiment allows to properly cover the period extending from the end of larval stage to the juvenile stage (60 dpf), including metamorphosis which is itself completed around 45 dpf. Within each experiment, boldness was determined using a group risk-taking test to identify bold and shy individuals. A novel environment test was then performed at the same age to evaluate consistency across contexts. Groups of fish (either bold or shy) were bathed in an alizarin red S solution for later identification of their initially determined coping style to evaluate behavioural consistency over time. Fish were then reared under common garden conditions and challenged again with the same behavioural tests at a later age (21 and 60 dpf in the larval and juvenile experiments, respectively). Behavioural consistency was observed across contexts, with bold fish being more active and expressing higher thigmotaxis regardless of age. There was, however, little behavioural consistency over age, suggesting behavioural plasticity during development. Moreover, the use of alizarin red S to conduct this experiment provides new perspectives for the further study of the longitudinal evolution of various traits, including behaviour, over life stages in fish.     

Evolution of cannibalism in the larval stage of pelagic fish

Larvae of several ocean pelagic fish species, such as tunas and marlins, have been known to have large jaws, but the ecological significance of this unique morphological character has been hardly analyzed in evolutionary ecology. Pelagic spawners produce small and nutrition-poor ova, and spawning and nursery grounds of the open ocean migratory fishes are oligotrophic. We hypothesize that cannibalism would be a possible life style in the larval period and the large mouth gape would be an adaptive morphological characteristic for a cannibal in the oligotrophic pelagic environment. We showed that mouth gape size of the open ocean pelagic fish is significantly larger than that of offshore/coastal pelagic fish in larval period. A mathematical model demonstrated that cannibalism would tend to evolve in high sea environment. Our findings suggest an evolutionary pattern of cannibalism trait in the larval stage of pelagic fishes. This revised version was published online in July 2006 with corrections to the Cover Date.     

Molecular identification of the hybrid between the catfish species Pseudoplatystoma corruscans and Pseudoplatystoma reticulatum using a set of eight microsatellite markers

This study distinguished hybrids of surubim or pintado Pseudoplatystoma corruscans and cachara Pseudoplatystoma reticulatum from pure strains using a set of eight microsatellite markers and population assignment methods. Applications of this molecular tool range from certification of hybrid‐free breeders in restocking conservation programmes to the identification of fish products lacking traditional morphological characteristics.     

Maximizing dietary information retrievable from carcasses of Great Cormorants Phalacrocorax carbo using a combined morphological and molecular analytical approach

Avian carcasses can provide important information on the trophic ecology of birds. Usually, the number of carcasses available for examination is limited and therefore it is important to gain as much dietary information per specimen as possible. In piscivorous birds and raptors, the stomach has been the primary source of dietary information, whereas the gut (intestine) has so far been neglected as it usually contains only a few morphologically identifiable hard parts of prey. Molecular approaches have the potential to retrieve dietary information from the gut, although this has not yet been verified. As well as identifying the prey, it is important to estimate any secondary predation to avoid food web errors in dietary analyses. The assignment of accidentally consumed prey is notoriously difficult regardless of the prey identification approach used. In the present study, morphological and molecular analyses were, for the first time, combined to maximize the dietary information retrievable from the complete digestive tract of Great Cormorants Phalacrocorax carbo sinensis. Moreover, a novel approach based on predator–prey size ratios was applied to these piscivorous birds to minimize the number of samples that might contain secondarily predated prey. The stomach contents of the examined birds were found to provide the most dietary information when morphological and molecular analyses were used in combination. However, compared with the morphological approach, the molecular analysis increased the number of fish species detected by 39%. The molecular approach also permitted the identification of fish DNA in the Cormorant guts. Predator–prey size ratios derived from morphological analysis of fish hard parts can reduce the incidence of potential confounding influence of secondarily predated prey by 80%. Our findings demonstrate that a combination of morphological and molecular approaches maximizes the trophic information retrievable from bird carcasses.     

Molecular identification of three Ompok species using mitochondrial COI gene

A DNA-based barcode identification system that is applicable to all animal species will provide a simple, universal tool for the identification of fish species. The barcode system is based on sequence diversity in subunit 1 cytochrome c oxidase (COI) gene. Identification and characterization of fish species based on morphological characters are sometimes found to be erroneous and environmentally affected. There are no studies on the genus Ompok in India at molecular level and species identification of the Ompok is usually carried out through morphological features. A total of 106 samples from three species Ompok pabda, O. pabo and O. bimaculatus were collected from eight sampling sites of seven Indian rivers. One hundred and six sequences were generated from COI region of three Ompok species and 21 haplotypes were observed. The sequence analysis of COI gene revealed three genetically distinct Ompok species and exhibited identical phylogenetic resolution among them. The partial COI gene sequence can be used as a diagnostic molecular marker for identification and resolution of taxonomic ambiguity of Ompok species.     

Identifying the ichthyoplankton of a coral reef using DNA barcodes

Marine fishes exhibit spectacular phenotypic changes during their ontogeny, and the identification of their early stages is challenging due to the paucity of diagnostic morphological characters at the species level. Meanwhile, the importance of early life stages in dispersal and connectivity has recently experienced an increasing interest in conservation programmes for coral reef fishes. This study aims at assessing the effectiveness of DNA barcoding for the automated identification of coral reef fish larvae through large‐scale ecosystemic sampling. Fish larvae were mainly collected using bongo nets and light traps around Moorea between September 2008 and August 2010 in 10 sites distributed in open waters. Fish larvae ranged from 2 to 100 mm of total length, with the most abundant individuals being <5 mm. Among the 505 individuals DNA barcoded, 373 larvae (i.e. 75%) were identified to the species level. A total of 106 species were detected, among which 11 corresponded to pelagic and bathypelagic species, while 95 corresponded to species observed at the adult stage on neighbouring reefs. This study highlights the benefits and pitfalls of using standardized molecular systems for species identification and illustrates the new possibilities enabled by DNA barcoding for future work on coral reef fish larval ecology.     

Relationship between swimming capacities and morphological traits of fish larvae at settlement stage: a study of several coastal Mediterranean species

Experimental measurements were made in the laboratory to determine the swimming capacities of settlement-stage fish larvae of several Mediterranean coastal species collected from the nearshore waters of Corsica, France. Critical swimming speed (U_crit, cm s⁻¹) was measured to provide a realistic laboratory estimate of in situ swimming speed. Morphometric traits were measured to assess potential predictors of a species’ swimming ability and, when possible, daily otolith increments were used to estimate age. Observed swimming speeds were consistent with other temperate species and demonstrated that the tested species are competent swimmers and not passive components of their environment. Morphological traits varied in their correlation with U_crit across groups and species. Direct measurements of morphological traits were better predictors than calculated ratios. Pelagic larval duration had little relationship with swimming speed among species for which daily otolith increments were counted. In addition to expanding the database on swimming capacities of settlement-stage fish larvae in the Mediterranean Sea, this study also developed methods that simplify the assessment of larval fish swimming ability. Swimming speed data are essential for improving larval dispersal models and for predicting recruitment rates in coastal fish populations.     

Developmental responses to predation risk in morphologically defended mayflies

Densities and species composition of predators could affect morphological defences, larval development and the timing of emergence of their prey. To address this issue we studied the morphology and life history of an ephemerellid mayfly, Ephemerella invaria, from two streams in a deciduous forested drainage basin in central New York. Both streams contained predatory fish, but densities and species composition of fish differed. A field survey provided evidence that Ephemerella inhabiting a stream with 10 fish species and high relative densities of fish emerged several weeks earlier and at smaller sizes than Ephemerella inhabiting a nearby tributary with ~2 fish species and low relative densities of fish. However, the two populations of mayflies showed no differences in defensive morphology or growth rates. In laboratory rearing experiments, we exposed Ephemerella larvae from these two locations to fish chemical cues or control water (no fish) over 2 months to test whether differences in life histories could be attributed to fish. Fish cues induced faster larval development, but also smaller size of mature Ephemerella individuals from both high and low predator locations. Although shorter development times in more dangerous environments could increase larval survival, smaller size of females results in a fecundity cost associated with this life history shift. Consistent with the field studies, laboratory rearing experiments revealed no effects of fish cues on Ephemerella's morphological defences. These data suggest that variation in the density or species composition of predators may favour the evolution of developmental plasticity to reduce mortality in the larval environment.     

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.