A Multiplex PCR Method for Rapid Identification of Brachionus Rotifers

Cryptic species are increasingly being recognized in many organisms. In Brachionus rotifers, many morphologically similar yet genetically distinct species/biotypes have been described. A number of Brachionus cryptic species have been recognized among hatchery strains. In this study, we present a simple, one-step genetic method to detect the presence of those Brachionus sp. rotifers that have been found in hatcheries. With the proposed technique, each of the B. plicatilis sensu stricto, B. ibericus, Brachionus sp. Nevada, Brachionus sp. Austria, Brachionus sp. Manjavacas, and Brachionus sp. Cayman species and/or biotypes can be identified with polymerase chain reaction (PCR) analysis. Based on 233 cytochrome c oxidase subunit I sequences, we reviewed all the available cryptic Brachionus sp. genetic polymorphisms, and we designed six nested primers. With these primers, a specific amplicon of distinct size is produced for every one of the involved species/biotypes. Two highly sensitive protocols were developed for using the primers. Many of the primers can be combined in the same PCR. The proposed method has been found to be an effective and practical tool to investigate the presence of the above six cryptic species/biotypes in both individual and communal (bulk) rotifer deoxyribonucleic acid extractions from hatcheries. With this technique, hatchery managers could easily determine their rotifer composition at the level of cryptic species and monitor their cultures more efficiently. Kalliopi Vasileiadou and Spiros Papakostas contributed equally to this work.     

Neutral and Nonneutral Mitochondrial Genetic Variation in Deep-Sea Clams from the Family Vesicomyidae

Nucleotide sequences at two mitochondrial genes from 57 individuals representing eight species of deep-sea clams (Vesicomyidae) were examined for variation consistent with the neutral model of molecular evolution. One gene, cytochrome oxidase subunit I (COI), deviated from the expectations of neutrality by containing an excess of intraspecific nonsynonymous polymorphism. Additionally, one species, Calyptogena kilmeri, showed a significant excess of rare polymorphism specifically at the COI locus. In contrast, a second mitochondrial gene, the large-subunit 16S ribosomal RNA gene (16S), showed little deviation from neutrality either between or within species. Together, COI and 16S show no deviation from neutral expectations by the HKA test, produce congruent phylogenetic relationships between species, and show correlated numbers of fixed differences between species and polymorphism within species. These patterns of both neutral and nonneutral evolution within the mitochondrial genome are most consistent with a model where intraspecific nonsynonymous polymorphism at COI is near neutrality. In addition to examining the forces of molecular evolution, we extend hypotheses about interspecific relationships within this family for geographical locations previously unexamined by molecular methods including habitats near the Middle Atlantic, the Aleutian Trench, and Costa Rica. Received: 10 March 1999 / Accepted: 13 September 1999     

Polymorphism of mtDNA regions in brown hare (Lepus europaeus) populations from Vojvodina (Serbia and Montenegro)

It is well known that there is heterogeneity of mitochondrial DNA (mtDNA) within and among natural populations of same species. The polymorphism level of particular regions of mtDNA gives valuable results in detection of population genetic structure. The aim of this paper was to detect polymorphism of three mtDNA regions: cytochrome oxidase I (COI), Control region, and 12S/16S rRNA, by the mtDNA RFLP-PCR method, in three Lepus europaeus populations from Vojvodina province (Serbia and Montenegro). Polymorphism was detected within the two regions, COI and Control region, while 12S/16S rRNA region was monomorphic in all 77 individuals. Eight haplotypes were detected in the brown hare population in Vojvodina, and three were unique for the Srem brown hare population.     

Multigene Barcoding and Phylogeny of Geographically Widespread Muricids (Gastropoda: Neogastropoda) Along the Coast of China

The identification and phylogeny of muricids have been in a state of confusion for a long time due to the morphological convergence and plasticity. DNA-based identification and phylogeny methods often offer an analytically powerful addition or even an alternative. In this study, we employ a DNA barcoding method to identify 17 known and easily confused muricid species (120 individuals) from the whole China coast based on mitochondrial cytochrome c oxidase subunit I (COI) and 16S rRNA sequences, and nuclear ITS-1 and 28S rRNA sequences. The phylogeny of muricid subfamilies is also analysed based on all mitochondrial and nuclear sequences. The universal COI and 16S rRNA primers did not work broadly across the study group, necessitating the redesign of muricid specific COI and 16S rRNA primers in this paper. Our study demonstrates that COI gene is a suitable marker for barcoding muricids, which can distinguish all muricid species studied. Phylogenetic analysis of 16S rRNA, ITS-1 and 28S rRNA data also provide good support for the species resolution observed in COI data. The relationships of muricid subfamilies are resolved based on the separate and combined gene data that showed the monophyly of each the subfamilies Ergalataxinae, Rapaninae, Ocenebrinae and Muricinae, especially that Ergalataxinae did not fall within Rapaninae.     

Sources contribution for benthic invertebrates: an inter-lake comparison in a flood plain system

Dwarfism in males is a common phenomenon in planktonic monogonont rotifers. Considering the small body size of males, some studies suggested that the development time of male rotifers is faster than that of females. However, empirical studies associated with such a speculation are scarce. In this study, we used two common rotifer species, Brachionus calyciflorus and Brachionus plicatilis (each with two geographical populations), to test the hypothesis that the development time of dwarf males is significantly shorter than that of females. Results showed that male eggs (embryos) were deposited much faster than female eggs (embryos) in both Brachionus species. However, the embryonic development time of these small male eggs (embryos) was longer than that of large female eggs (embryos). As a result, males needed significantly longer total development time than females. The total development time of males was 2–3 h longer than that of females in both Brachionus species. Male-producing and female-producing females in the two Brachionus species did not show any difference in development time. Because sexual reproduction begins at high population densities in both species, postponement of development in males will be advantageous by decreasing mating costs in male rotifers.     

The genome of the freshwater monogonont rotifer Brachionus calyciflorus

Monogononta is the most speciose class of rotifers, with more than 2,000 species. The monogonont genus Brachionus is widely distributed at a global scale, and a few of its species are commonly used as ecological and evolutionary models to address questions related to aquatic ecology, cryptic speciation, evolutionary ecology, the evolution of sex and ecotoxicology. With the importance of Brachionus species in many areas of research, it is remarkable that the genome has not been characterized. This study aims to address this lacuna by presenting, for the first time, the whole‐genome assembly of the freshwater species Brachionus calyciflorus. The total length of the assembled genome was 129.6 Mb, with 1,041 scaffolds. The N50 value was 786.6 kb, and the GC content was 24%. A total of 16,114 genes were annotated with repeat sequences, accounting for 21% of the assembled genome. This assembled genome may form a basis for future studies addressing key questions on the evolution of monogonont rotifers. It will also provide the necessary molecular resources to mechanistically investigate ecophysiological and ecotoxicological responses.     

The Rotifera of Malaysia and Singapore with remarks on some species

Summary An intensive and extensive sampling of all types of Malaysian and Singaporean freshwaters for zooplankton gave 165 species, sub-species and varieties of Rotifera, mainly belonging to the Monogononta. Bdelloida were rarely identified. The present records bring Malaysia and Singapore in line with other well studied tropical countries as far as Rotifera records are concerned. 7 new genera and over 50 species, subspecies, varieties and forms have been added to the Malaysian and Singaporean Rotifera records and interesting and marked morphological and size variation in species from previous records, have been noted.The richest (in species) fauna of Rotifera occurs in rice fields and ponds which have a seasonal succession of dry to wet conditions and vegetation which shelters the rotifers.The Malaysian and Singaporean Rotifera fauna is typically tropical with a dominance of the genus Brachionus over temperate genera like Keratella. Also very rare or missing are typical temperate genera like Ploesoma, Notholca and Synchaeta. The dominance of Brachionus in Malaysia is less evident than expected because only a very small proportion of samples were taken in limnetic situations where Brachionus clearly dominates in the tropics.     

Molecular evolution of the leptin exon 3 in some species of the family Canidae

The structure of the leptin gene seems to be well conserved. The polymorphism of this gene in four species belonging to the Canidae family (the dog (Canis familiaris) – 16 different breeds, the Chinese racoon dog (Nyctereutes procyonoides procyonoides), the red fox (Vulpes vulpes) and the arctic fox (Alopex lagopus)) were studied with the use of single strand conformation polymorphism (SSCP), restriction fragment length polymorphism (RFLP) and DNA sequencing techniques. For exon 2, all species presented the same SSCP pattern, while in exon 3 some differences were found. DNA sequencing of exon 3 revealed the presence of six nucleotide substitutions, differentiating the studied species. Three of them cause amino acid substitutions as well. For all dog breeds studied, SSCP patterns were identical.     

Systematics,reproductive isolation and species boundaries in monogonont rotifers

The typological concept of rotifer species and the morphological basis of rotifer systematics is reviewed and alternatives proposed. Occasional sexuality in the cyclical parthenogenetic life cycle of monogononts permits application of the biological species concept to this group. Data from cross-mating experiments with Asplanchna, Brachionus and Epiphanes illustrate the usefulness of reproductive isolation as a criterion for species boundaries. Populations from different geographic regions are often interfertile indicating that rotifer species are genetically integrated over wide areas. The main types of isolating mechanisms operating in monogononts are reviewed. The role of behavioral reproductive isolation in maintaining species boundaries is examined. The use of a mate recognition bioassay which estimates the probability of copulation and quantifies the degree of isolation is described. Recent work of the mechanism of mate recognition is reviewed. It is concluded that the biological species concept is applicable to rotifers and that a more experimental approach to determining species boundaries is both feasible and desirable.     

Genetic structure and diversity of the Diachasmimorpha longicaudata species complex in Thailand: SSCP analysis of mitochondrial 16S rDNA and COI DNA sequences

Genetic variation among 20 populations of Diachasmimorpha longicaudata (Ashmead) (Hymenoptera: Braconidae) in Thailand was investigated using single strand conformation polymorphism (SSCP) analysis of mitochondrial DNA sequences. From a total 641 individual parasitoids, seven distinct haplotypes containing a total of 32 polymorphic sites were observed from cytochrome oxidase subunit I (COI) sequences along with five distinct haplotypes containing a total 16 polymorphic sites from 16S rDNA sequences. Values obtained through pairwise F_ST comparisons and analysis of molecular variance (AMOVA) indicated significant genetic differentiation among D. longicaudata populations in Thailand. Congruent relationships showing separation of these populations into three groups were obtained from Neighbor joining and Bayesian phylogenetic tree analyses along with the use of haplotype networks. This SSCP analysis of populations of the D. longicaudata species complex is the first report using molecular population genetic methods to analyze the structure of this parasitoid species in Thailand. This may provide useful information for release of parasitoid strains to maximize their benefit in biological control programs.     

New records of two deep-sea eels collected from the Western Pacific Ocean based on COI and 16S rRNA genes

Background

Two deep-sea eels collected from the Western Pacific Ocean are described in this study. Based on their morphological characteristics, the two deep-sea eel specimens were assumed to belong to the cusk-eel family Ophidiidae and the cutthroat eel family Synaphobranchidae.

Methods and results

To accurately identify the species of the deep-sea eel specimens, we sequenced the mitochondrial genes (cytochrome c oxidase subunit I [COI] and 16S ribosomal RNA [16S rRNA]). Through molecular phylogenetic analysis based on mtDNA COI and 16S rRNA gene sequences, these species clustered with the genera Bassozetus and Synaphobranchus, suggesting that the deep-sea eel specimens collected are two species from the genera Bassozetus and Synaphobranchus in the Western Pacific Ocean, respectively.

Conclusions

This is the first study to report new records of the genera Bassozetus and Synaphobranchus from the Western Pacific Ocean based on COI and 16S rRNA genes

     

The rotifer fauna of the River Loire (France), at the level of the nuclear power plants

In the heated affluents of nuclear power plants of the river Loire, rotifers are abundant. Cosmopolitan species are numerically dominant, but a tropical fauna is also present, among which the genus Brachionus, representing 20% of the total species, is best represented. From a comparison between water upstream and downstream of the power plants, it further appears that in downstream warmed-up waters, some species show an important development, but not in colder upstream waters.     

Competition between two planktonic rotifer species at different temperatures: an experimental test

1. The effect of temperature on the outcome of resource competition between two planktonic rotifers (Synchaeta pectinata and Brachionus calyciflorus) was investigated in laboratory experiments. In addition to the competition experiments, several physiological variables and their temperature‐dependence were characterised, including ingestion rate and starvation tolerance. 2. Because of a lower threshold food level (TFL) for population growth for the food algae Cryptomonas erosa, Synchaeta was predicted to be the superior competitor at low temperatures (12 °C). In contrast, Brachionus had a lower TFL at 20 °C and was predicted to be competitively superior at this temperature. 3. In both rotifer species, ingestion rates increased with temperature, but the increase was much more pronounced in Brachionus. Ingestion rates of Brachionus at temperatures from 8 to 24 °C were always higher than in Synchaeta (up to 4.6‐fold). 4. Starvation resistance reduced with temperature in both rotifer species. At all temperatures investigated (12, 16 and 20 °C) Brachionus could survive starvation for longer than Synchaeta. This difference was strongest at 12 °C (5.8 days versus 2.5 days). 5. In the first competition experiment, food was supplied at 48 h‐intervals. Brachionus displaced Synchaeta at both experimental temperatures (12 and 20 °C). Competitive exclusion of Synchaeta at the lower temperature was probably because of large fluctuations in algal densities that resulted from the long intervals between feeding, a condition that favoured Brachionus because of its higher starvation resistance. 6. In the second competition experiment, one third of the food suspension was renewed every 8 h, resulting in a much better approximation to a continuous resource supply. At 12 °C Synchaeta and Brachionus coexisted for more than 1 month and the densities of both rotifer species were significantly lower in the presence of their competitor. In contrast to expectations, Brachionus was able to persist even when Cryptomonas concentrations fell below its TFL. This was probably because Brachionus was using detritus and associated bacteria as additional food sources, which were present in the cultures during the later phase of the experiment. 7. Autocorrelation analysis of the temporal changes in egg ratios revealed significant periodic cycles in Synchaeta during the second competition experiment. A possible explanation for this is the fecundity schedule of Synchaeta, in which reproduction is highly concentrated in a few age classes. According to demographic theory, such a life cycle feature can cause slower convergence to a stable age distribution.     

Phylogenetic and systematic study of Korean <Emphasis Type="Italic">Orius</Emphasis> species (Heteroptera: Anthocoridae) on the basis of molecular and morphological data

A phylogenetic and systematic study of Orius species (Heteroptera: Anthocoridae) from Korea has been conducted using both morphological and molecular characters. Thirty morphological character states were coded for 10 strains of 9 species. Five molecular markers, partial cytochrome c oxidase I (COI), cytochrome b (CytB), 16S rRNA (16S), 18S rRNA (18S), and 28S rRNA (28S), from mitochondrial and nuclear genes, were tested. Phylogenetic analyses based on molecular data were conducted by minimum evolution, maximum parsimony, maximum likelihood, and Bayesian phylogenetic (BP) analyses. Analysis of morphological data was performed using the parsimony programs NONA, and the combined dataset of morphological and molecular data was analyzed using BP analyses. The results of this study indicate that use of COI and CytB enabled relatively effective identification of species, whereas the sequences of 16S, 18S and 28S did not enable identification of closely related species such as Orius minutus and O. strigicollis. We discuss the usefulness of the five molecular markers for determining phylogenetic relationships and identifying the species.     

The phylogeny of rotifers: molecular,ultrastructural and behavioural data

This work discusses the nature and significance of molecular, ultrastructural, and behavioural characters that can be used in phylogenetic analyses of rotifers.Recent molecular research has demonstrated the presence of very small amounts of 4-hydroxyproline in rotifers, probably arising from acetyl-cholinesterase or glycoproteins. Thus, rotifers appear to be the first known Metazoa without collagen.Ultrastructural work also has made some interesting discoveries. (1) The myelinic cuticle of the integument and pharynx of gastrotrichs is present in the pharynx of at least two rotifers (Philodina and Brachionus) and some Annelida. (2) The intracytoplasmic lamina (IL) of the syncytial ingestive integument of Acanthocephala is similar to the IL of the syncytial stomach of Bdelloidea. (3) The fibrous terminal web of primitive epidermal ciliated cells may have evolved in the skeletal IL of the syncytial, aciliated integument of rotifers. (4) Using the ultrastructural features of the skeletal, IL of the integument, I derived two possible dendograms of rotifer evolution. (5) These models and other ultrastructural data predict that Bdelloidea should be separated from Monogononta, while Seison has several characters which suggest that it should be more closely aligned to the Monogononta than previously proposed.Molecular and ultrastructural data suggest that rotifers are primitive Metazoa, probably derived by neoteny from ancestral, ciliated larvae. Finally, I argue that information on sensory organs and the behaviour of rotifers may offer unique insights into the evolution of the phylum.     

Swimming speed and Reynolds numbers of eleven freshwater rotifer species

We obtained data on the swimming speed of 11 freshwater rotifer species. These data were analyzed in terms of Reynolds numbers given the fact that rotifers are in the evolutionary boundary between the use of cilia and swimming appendages. Swimming speed ranged from 0.174 to 0.542 mm ^–1. Philodina acuticornis odiosa was the fastest rotifer in terms of absolute speed. However, Gastropus hyptopus, the smallest of the rotifers analyzed, was the fastest (2.849) in terms of body-lengths s^–1. Reynolds numbers (Re) among the 11 species analyzed varied from 0.023 to 0.301. Lecane furcata had the smallest Re, while Epiphanes senta had the highest Re. The two bdelloid rotifers analyzed in this work swam five times faster than they crept. The importance of Reynolds numbers and drag coefficients are discussed in view of the present results and data found in the literature.     

Deoxyribonucleic acid reassociation in the classification of coagulase-positive staphylococci

DNA-DNA reassociation studies were performed with coagulase-positive staphylococci belonging to the biotypes A, B, C, D, E and F. These studies present genetic evidence for the existence of at least two distinct species within this group of organisms. The common Staphylococcus aureus strains were represented by organisms from biotypes A to D, and their DNA revealed over 80% nucleotide sequence homology under restrictive conditions. Less than 15% DNA homology was detected between strains from biotypes A to D (S. aureus) and those from biotypes E and F. The DNA of organisms from either the biotypes E or F displayed over 70% homology. Together, both biotypes are considered to represent the species S. intermedius. However, DNA homology values dropped to 50–65% between strains from different biotypes. This may justify the separation of S. intermedius biotypes E and F on a subspecies level.Abbreviations O.D. optical density - SSC standard saline citrate buffer (0.15 M NaCl, 0.015 M sodium citrate, pH 7.0) This work was supported by Deutsche Forschungsgemeinschaft     

RFLP Analysis of Mitochondrial DNA to Evaluate Genetic Variation in Striped Red Mullet (Mullus surmuletus L.) and Red Mullet (Mullus barbatus L.) Populations

The genetic differentiation of striped red mullet (Mullus surmuletus) and red mullet (Mullus barbatus) was investigated in 6 Mediterranean populations of each species by means of restriction fragment length polymorphism analysis of mitochondrial DNA. Three segments amplified by polymerase chain reaction (control region, COI, and 12S–16S ribosomal RNA) were digested with 20 restriction endonucleases, revealing 71 haplotypes for M. surmuletus and 30 for M. barbatus. For the two species nucleotide diversity was equally distributed within and among populations, leading to N _ST values of 0.545 and 0.500 for M. surmuletus and M. barbatus, respectively. However, intrapopulation and interpopulation genetic structuring appeared to be much higher for M. surmuletus than for M. barbatus (1.88% vs. 0.46% of mean intrapopulation nucleotide diversity; 1.94% vs. 0.47% of mean interpopulation nucleotide diversity; 0.055% vs. 0.002% of net interpopulation divergence). Furthermore, 81.69% of the haplotypes observed for M. surmuletus were unique, whereas 70.29% of M. barbatus individuals were grouped in 3 common haplotypes. Given that fishing pressure and population sizes are similar for both species, this differentiation could be attributed to differences in biological parameters and life histories between the two species, coupled with oceanographic conditions prevailing in the studied area. Received July 25, 2000; accepted December 29, 2000     

Associations of planktonic and periphytic rotifers in a Malaysian estuary and two nearby ponds

Samples were taken from a Malaysian estuary along a salinity gradient from full strength sea water down to a conductivity of 27 µS cm^–1. In a region where the conductivity fluctuated between 6500 and 14000 µS only empty loricas of Keratella cochlearis and Lecane bulla were found. Where the salinity reached a maximum of 3800 µS Lecane bulla was the dominant rotifer, and 17 other species were found. Where the conductivity remained below 1000 µS at all stages of the tidal cycle there were more species of rotifers, up to 33 at one station.The rotifer fauna of the estuary was compared to the assemblage found in two local ponds. Of the 63 species found in the estuary only 29 were also found in the ponds. Compared to the total known rotifer fauna of Malaysia the estuary has a higher proportion of Lecane species (40%), and the genera Brachionus and Trichocerca are under-represented.Species associated with inland saline waters, such as Brachionus plicatilis, B. dimidiatus and Hexarthra jenkinae were not found in the estuary.     

人工神经原网络处理PCR数据在细菌鉴定中的应用

目的16SrRNA和16S-23SrRNA间区片段是常用细菌分类鉴定靶点,本研究探讨人工神经原网络（ANN）对上述位点PCR扩增产物数据分析在细菌快速鉴定方面的价值。方法2对15SrRNA基因荧光引物和1对16S-23SrRNA区间基因引物用于扩增血液标本中分离出的317株细菌。相关毛细管电泳（CE）限制性片段长度多态性（RFLP）和单链构象多态性（SSCP）数据进行人工神经原网络分析。结果16S-23SrRNA基因的RFLP数据对未知菌鉴定的准确率高于16SrRNA基因的SSCP数据,分别为98．0％和79．6％。结论实验证明了人工神经原网络作为一种模式识别方法对于简化细菌鉴定十分有价值。