Population genetic structure of Semisulcospira gottschei: simultaneous examination of mtDNA and microsatellite markers.

Semisulcospira gottschei is an Asian endemic species inhabiting Korea and China. However, genetic structure analysis of the resource management of this species has not been performed. To investigate the genetic diversity among populations, microsatellites can be used to determine the geographic origins of marine and freshwater species. This study investigated the genetic structures of the Korean and Chinese populations of S. gottschei based on mitochondrial DNA (mtDNA) Cytochrome oxidase subunit I (COI) and polymorphic microsatellite loci developed from Semisulcospira coreana. Analysis of the mtDNA COI sequence revealed 43 haplotypes, which indicated no gene flow between the Korean and Chinese populations. To further elucidate the genetic structures of the Korean and Chinese populations, the population genetics of S. gottschei were analyzed using nine microsatellite markers. The genetic diversity analysis showed an average of 5.25 alleles per locus, with an average allelic richness of 4.02. Excessive homozygosity was found at all loci, which was expected to be due to the presence of null alleles at all loci. Populations of S. gottschei formed two separate clusters according to pairwise FST and AMOVA. Also, the UPGMA tree, PCA, STRUCTURE, and GeneClass indicated separation of the 11 populations into two clusters: Korea and China. These results have potential use in the management, restoration, and distinction of the origin country of populations.

Development of novel antimicrobial peptides derived from anti-lipopolysaccharide factor of the swimming crab, Portunus trituberculatus.

Anti-lipopolysaccharide factors (ALFs) are a representative host defense protein in crustaceans. In this study, we successfully developed two novel antimicrobial peptides (AMPs), named crab-ALF2A and crab-ALF6A, which contain changes to the amino acid sequences of the lipopolysaccharide binding domain and signal peptide, respectively, of the ALF of the swimming crab Portunus trituberculatus. The crab-ALF2A peptide showed potent antimicrobial activity against the Gram-positive bacteria Bacillus cereus, Staphylococcus aureus, and Streptococcus iniae (minimal effective concentration [MEC] 1.51-1.93 µg/mL) and the Gram-negative bacteria Pseudomonas aeruginosa and Escherichia coli (MEC 1.87-1.98 µg/mL), with maximal bactericidal activity at a peptide concentration of 5 µg/mL. The crab-ALF6A peptide also showed potent antimicrobial activity against B. cereus, S. aureus, and S. iniae (MEC 1.49-2.3 µg/mL) and P. aeruginosa and E. coli (MEC 1.72-1.19 µg/mL) at a peptide concentration of 5 µg/mL. Notably, the crab-ALF2A and crab-ALF6A peptides exhibited strong activity against Candida albicans (MECs of 2.11 and 1.95 µg/mL, respectively). These activities were stable following heat treatment. Moreover, the effect of crab-ALF2A and crab-ALF6A peptide treatment on microbe cell morphology was confirmed by scanning electron microscopy. Membrane disruption and damage, and the leakage of cytoplasmic content were clearly observed. A downsizing peptide approach illustrated that the hexapeptide ALF6A8 (RVLLRL) was the shortest peptide showing significant antimicrobial activity. Our approach allows for the generation of novel antimicrobial peptides in a cost effective manner as potential next-generation antibiotics.

Two metalloenzymes from rockfish (Sebastes schligellii): Deciphering their potential involvement in redox homeostasis against oxidative stress.

Disturbance in the balance between pro-oxidants and anti-oxidants result oxidative stress in aerobic organisms. However, oxidative stress can be inhibited by enzymatic and non-enzymatic defense mechanisms. Superoxide dismutases (SODs) are well-known scavengers of superoxide radicals, and they protect cells by detoxifying hazardous reactive oxygen species. Here, we have identified and characterized two different SODs, CuZnSOD and MnSOD, from black rockfish (RfCuZnSOD and RfMnSOD, respectively). In silico analysis revealed the well-conserved molecular structures comprising all essential properties of CuZnSOD and MnSOD. Phylogenetic analysis revealed that both RfCuZnSOD and RfMnSOD cladded with their fish counterparts. The recombinant RfSOD proteins demonstrated their potential superoxide scavenging abilities through a xanthine oxidase assay. The optimum temperature and pH conditions for both rRfSODs were 25 °C and pH 8, respectively. Moreover, the potential peroxidation function of rRfCuZnSOD was observed in the presence of HCO3-. The highest peroxidation activity was observed at 100 µg/mL of rRfCuZnSOD using the MTT cell viability assay and flow cytometry. The analogous tissue-specific expression profile indicated ubiquitous expression of both RfCuZnSOD and RfMnSOD in selected tissues of healthy juvenile rockfish. An immune challenge experiment illustrated the altered expression profiles of both RfCuZnSOD and RfMnSOD against lipopolysaccharide, Streptococcus iniae, and polyinosinic-polycytidylic acid (poly I:C). Collectively, these results strengthen the general understanding of the structural and functional characteristics of SODs within the host defense system.

Molecular characterization of Rhodeus uyekii tripartite motif protein 1 (TRIM1) involved in IFN-γ/LPS-induced NF-κB signaling.

The tripartite motif-containing (TRIM) proteins are involved in a wide range of cellular processes, and the role of TRIM1 in immunity has been explored. However, fundamental studies on fish TRIM1 are lacking. In this study, we cloned and characterized TRIM1 cDNA from the Korean rose bitterling, Rhodeus uyekii (RuTRIM1). Two RuTRIM1 isoforms (RuTRIM1-X1 and RuTRIM1-X2) were identified. The coding sequence (CDS) of RuTRIM1-X1 comprised 2157 bp encoding a 718-aa protein, and the CDS of RuTRIM1-X2 comprised 1929 bp encoding a 642-aa protein. Both RuTRIM1 isoforms contained a RING finger domain, B-box 1, B-box 2, coiled-coil domain, COS box, FN3 motif, and PRY/SPRY domain. The deduced RuTRIM1-X1 and RuTRIM1-X2 proteins had high amino acid identity (76.27-98.89%) with orthologs from various other species, and a phylogenetic tree was constructed. RuTRIM1-X1 and RuTRIM1-X2 mRNA were expressed in all tissues examined, with the highest expression levels detected in the hepatopancreas. During early development, RuTRIM1-X1 and RuTRIM1-X2 mRNA levels changed differently from the gastrula period to the first feeding stage. An in vivo ubiquitination assay showed that RuTRIM1 exhibited RING-dependent E3 ubiquitin ligase activity, mainly by comparing RuTRIM1-X2 to RuTRIM1-X1. The subcellular localization of the two RuTRIM1 protein isoforms was characterized, revealing that they formed aggregates in cytoplasmic bodies in Raw264.7 cells. Interferon-γ/lipopolysaccharide-induced nuclear factor-κB signaling was negatively regulated by RuTRIM1-X1 and RuTRIM1-X2, and the negative effect was reversed in RING deletion mutants. To our knowledge, this is the first study to characterize fish TRIM1, which may play a role in the inflammatory response.

Purification and cDNA cloning of the antimicrobial peptide apMolluscidin from the pen shell, Atrina pectinata.

A 5.6 kDa antimicrobial peptide (AMP) was purified from acidified gill extract of the pen shell, Atrina pectinata, by cation exchange and C18 reversed-phase high performance liquid chromatography. Comparison of the amino acid sequences and molecular weight of this peptide with those of other known AMPs revealed that it had high sequence homology with that of cgMolluscidin or hdMolluscidin; it was designated apMolluscidin. apMolluscidin comprises 59 amino acid residues containing several dibasic residue repeats and sequence repeats such as Lys-Lys and Lys-Gly. apMolluscidin exhibited potent antimicrobial activity against both Gram-positive bacteria including Bacillus subtilis (minimal effective concentration [MEC], 2.1 µg/mL), and Gram-negative bacteria including E. coli D31 (MEC, 0.5 µg/mL), without hemolytic activity. However, it did not show any activity against fungi such as Candida albicans. Secondary structure prediction suggested that it might form two helical regions and have an amphipathic structure. Full-length apMolluscidin cDNA contained 812 base pairs (bp), including a 5'-untranslated region (UTR) of 82 bp, a 3'-UTR of 547 bp, and a coding sequence of 183 bp encoding 60 amino acids (containing Met). Furthermore, qPCR analyses revealed that the mature peptide translated from apMolluscidin mRNA is expressed in a tissue-specific manner in locations such as the gill and siphon. These results indicate that apMolluscidin might be related to the innate immune defense system of abalone and may not act directly on the bacterial membrane. This is the first report of an AMP from the pen shell with a fully identified amino acid sequence.

Purification and characterization of an antimicrobial peptide mytichitin-chitin binding domain from the hard-shelled mussel, Mytilus coruscus.

An antimicrobial peptide with 55 amino acid residues was purified by C18 reversed-phase high-performance liquid chromatography (HPLC) from foot extract of the hard-shelled mussel, Mytilus coruscus. This peptide showed strong antimicrobial activity against Gram-positive and Gram-negative bacteria, as well as fungi. The purified peptide was determined to have a molecular mass of 6202 Da by matrix-assisted laser desorption/ionization time-of-flight mass spectrophotometry (MALDI-TOF/MS). The identified 20-amino acid sequence of the purified peak by Edman degradation shared 100% identity with the N-terminal regions of mytichitin-1, mytichitin-2, mytichitin-3, mytichitin-4, mytichitin-5, and chitinase-like protein-1, and so was named mytichitin-CBD. The cDNA of mytichitin-CBD was cloned and sequenced by rapid amplification of cDNA ends (RACE). The mRNA transcripts were mainly detected in foot tissue, and they were up-regulated and peaked at 4 h after bacterial infection. We constructed and expressed recombinant mytichitin-CBD protein which displayed antimicrobial activity against Gram-negative bacteria Gram-positive bacteria and the fungus as well as anti-parasitic activity against scuticociliates. The results of this study demonstrate that the peptide isolated from M. coruscus is related to the innate immune system of this marine invertebrate and is a possible alternative to antibiotics.

Tracing the spatio-temporal dynamics of endangered fin whales (Balaenoptera physalus) within baleen whale (Mysticeti) lineages: a mitogenomic perspective.

To explore the spatio-temporal dynamics of endangered fin whales (Balaenoptera physalus) within the baleen whale (Mysticeti) lineages, we analyzed 148 published mitochondrial genome sequences of baleen whales. We used a Bayesian coalescent approach as well as Bayesian inferences and maximum likelihood methods. The results showed that the fin whales had a single maternal origin, and that there is a significant correlation between geographic location and evolution of global fin whales. The most recent common female ancestor of this species lived approximately 9.88 million years ago (Mya). Here, North Pacific fin whales first appeared about 7.48 Mya, followed by a subsequent divergence in Southern Hemisphere approximately 6.63 Mya and North Atlantic about 4.42 Mya. Relatively recently, approximately 1.76 and 1.42 Mya, there were two additional occurrences of North Pacific populations; one originated from the Southern Hemisphere and the other from an uncertain location. The evolutionary rate of this species was 1.002 × 10-3 substitutions/site/My. Our Bayesian skyline plot illustrates that the fin whale population has the rapid expansion event since ~ 2.5 Mya, during the Quaternary glaciation stage. Additionally, this study indicates that the fin whale has a sister group relationship with humpback whale (Meganoptera novaeangliae) within the baleen whale lineages. Of the 16 genomic regions, NADH5 showed the most powerful signal for baleen whale phylogenetics. Interestingly, fin whales have 16 species-specific amino acid residues in eight mitochondrial genes: NADH2, COX2, COX3, ATPase6, ATPase8, NADH4, NADH5, and Cytb.

Microarray analysis of gene expression in olive flounder liver infected with viral haemorrhagic septicaemia virus (VHSV).

The most fatal viral pathogen in olive flounder Paralichthys olivaceus, is viral hemorrhagic septicemia virus, which afflicts over 48 species of freshwater and marine fish. Here, we performed gene expression profiling on transcripts isolated from VHSV-infected olive flounder livers using a 13 K cDNA microarray chip. A total of 1832 and 1647 genes were upregulated and down-regulated over two-fold, respectively, after infection. A variety of immune-related genes showing significant changes in gene expression were identified in upregulated genes through gene ontology annotation. These genes were grouped into categories such as antibacterial peptide, antigen-recognition and adhesion molecules, apoptosis, cytokine-related pathway, immune system, stress response, and transcription factor and regulatory factors. To verify the cDNA microarray data, we performed quantitative real-time PCR, and the results were similar to the microarray data. In conclusion, these results may be useful for the identification of specific genes or for the diagnosis of VHSV infection in flounder.

Cetaceans evolution: insights from the genome sequences of common minke whales.

BACKGROUND: Whales have captivated the human imagination for millennia. These incredible cetaceans are the only mammals that have adapted to life in the open oceans and have been a source of human food, fuel and tools around the globe. The transition from land to water has led to various aquatic specializations related to hairless skin and ability to regulate their body temperature in cold water. RESULTS: We present four common minke whale (Balaenoptera acutorostrata) genomes with depth of ×13 ~ ×17 coverage and perform resequencing technology without a reference sequence. Our results indicated the time to the most recent common ancestors of common minke whales to be about 2.3574 (95% HPD, 1.1521 - 3.9212) million years ago. Further, we found that genes associated with epilation and tooth-development showed signatures of positive selection, supporting the morphological uniqueness of whales. CONCLUSIONS: This whole-genome sequencing offers a chance to better understand the evolutionary journey of one of the largest mammals on earth.

Molecular and Functional Characterization of Thioredoxin 1 from Korean Rose Bitterling (Rhodeus uyekii).

Thioredoxin is a multifunctional antioxidant enzyme that belongs to the reductase family. In this study, we cloned and characterized thioredoxin 1 cDNA from the Korean rose bitterling Rhodeus uyekii (RuTrx). The full-length RuTrx cDNA consists of 674 bp with a 324 nt open reading frame (ORF) encoding a 107 aa protein. The deduced RuTrx amino acid sequence indicated a characteristic redox active site, (31)WCGPC(35). Pairwise alignment revealed RuTrx amino acid identity (55.1%-83.2%) with orthologs from various species of mammalia, amphibia, fish and bird. Phylogenetic analysis was conducted to determine the evolutionary position of RuTrx. Expression analysis showed that RuTrx transcripts were present in all of the tissues examined, and was high in the hepatopancreas of R. uyekii. During early development, the expression of RuTrx transcripts was increased. Recombinant RuTrx protein (rRuTrx) was tested for its capacity to serve as an antioxidant enzyme using a metal-catalyzed oxidation (MCO) system. The ability of rRuTrx to protect against supercoiled DNA cleavage due to oxidative nicking increased in a dose-dependent manner. In Raw264.7 cells, Dihydroethidium (DHE) staining for ROS production indicated the antioxidant activity of rRuTrx. Together, these findings suggest that RuTrx may play a role in maintaining the redox state balance in Korean rose bitterling R. uyekii.

Rapid Origin Determination of the Northern Mauxia Shrimp (Acetes chinensis) Based on Allele Specific Polymerase Chain Reaction of Partial Mitochondrial 16S rRNA Gene.

Acetes chinensis is an economically important shrimp that belongs to the Sergestidae family; following fermentation, A. chinensis' economic value, however, is low in China, and much of the catch in China is exported to Korea at a low price, thus leading to potential false labeling. For this reason, we developed a simple method to identify A. chinensis' origin using allele-specific polymerase chain reaction (PCR). Ten single nucleotide polymorphisms (SNPs) were identified from partial (i.e., 570 bp) DNA sequence analysis of the mitochondrial 16s rRNA gene in 96 Korean and 96 Chinese individual shrimp. Among 10 SNP sites, four sites were observed in populations from both countries, and two sites located in the middle with SNP sites at their 3'-ends were used to design allele-specific primers. Among the eight internal primers, the C220F primer specific to the Chinese A. chinensis population amplified a DNA fragment of 364 bp only from that population. We were able to identify the A. chinensis population origin with 100% accuracy using multiplex PCR performed with two external primers and C220F primers. These results show that the 16S rRNA gene that is generally used for the identification of species can be used for the identification of the origin within species of A. chinensis, which is an important finding for the fair trade of the species between Korea and China.

A universal spring-probe system for reliable probing of electrochemical lab-on-a-chip devices.

For achieve sensitivity in lab-on-a-chip electrochemical detection, more reliable probing methods are required, especially for repeated measurements. Spring-probes are a promising candidate method which can replace needle-like probes and alligator clips that usually produce scratches on the surface of gold electrodes due to the strong physical contacts needed for electrochemical measurements. The superior reliability of amperometric measurements by a spring-probe system was compared with results by conventional probing methods. We demonstrated that a universal spring-probe system would be potentially suitable to achieve high performance in lab-on-a-chip devices using electrochemical detection.

Characterization, expression profile, and promoter analysis of the Rhodeus uyekii vitellogenin Ao1 gene.

The fish Vitellogenin (Vg) gene has been applied as a biomarker for exposure to estrogenic compounds in the aquatic environment. In this study, we cloned and characterized Vg cDNA from the Korean rose bitterling Rhodeus uyekii (Ru-Vg). The Ru-Vg cDNA encodes a 1424-amino-acid polypeptide that belongs to the VgAo1 family and contains a putative signal peptide, lipovitellin I, phosvitin, and lipovitellin II, but does not contain the vWFD domain or the C-terminal peptide. The deduced Ru-Vg protein has high amino acid identity (73.97%-32.17%) with fish Vg proteins. Pairwise alignment and phylogenetic analysis revealed that Ru-Vg is most closely related to Acheilognathus yamatsutae Vg. Ru-Vg transcripts were detected using quantitative polymerase chain reaction in all tissues tested, with the highest level of expression observed in the ovary. Ru-Vg mRNA was upregulated in R. uyekii hepatopancreas cells in response to treatment with 17ß-estradiol (E2) or 17α-ethinylestradiol (EE2). Luciferase reporter expression, driven by the 5'-regulatory region of the Ru-Vg gene spanning from -1020 bp to the start codon was induced by the estrogen receptor and was synergistically activated by treatment with E2 or EE2. These results suggest that R. uyekii and the Ru-Vg gene may be useful as biomarkers for exposure to E2 or EE2.

Population genetic analysis and origin discrimination of snow crab (Chionoecetes opilio) using microsatellite markers.

Major habitats for the snow crab Chionoecetes opilio are mostly found within the northwest Atlantic and North Pacific Oceans. However, the East Sea populations of C. opilio, along with its relative the red snow crab (C. japonicas), are two of the most important commercial crustacean species for fisheries on the east coast of the Korean Peninsula. The East Sea populations of C. opilio are facing declining resources due to overfishing and global climate change. Thus, an analysis of population structure is necessary for future management. Five Korean and one Russian group of C. opilio were analyzed using nine microsatellite markers that were recently developed using next-generation sequencing. No linkage disequilibrium was found between any pair of loci, indicating that the markers were independent. The number of alleles per locus varied from 4 to 18 with a mean of 12, and allelic richness per locus ranged from 4.0 to 17.1 across all populations with a mean of 9.7. The Hardy-Weinberg equilibrium test revealed significant deviation in three out of nine loci in some populations after sequential Bonferroni correction and all of them had higher expected heterozygosity than observed heterozygosity. Null alleles were presumed in four loci, which explained the homozygosity in three loci. The pairwise fixation index (F ST ) values among the five Korean snow crab populations did not differ significantly, but all of the pairwise F ST values between each of the Korean snow crab populations and the Russian snow crab population differed significantly. An UPGMA dendrogram revealed clear separation of the Russian snow crab population from the Korean snow crab populations. Assignment tests based on the allele distribution discriminated between Korean and Russian origins with 93 % accuracy. Therefore, the snow crab populations around the Korean Peninsula need to be managed separately from the populations in Bering Sea in global scale resource management. Also, this information can be used for identification of snow crab origin which is problematic in worldwide crab trade.

Organoclay-assisted interfacial polymerization for microfluidic production of monodisperse PEG-microdroplets and in situ encapsulation of E. coli.

We developed a novel one-pot synthetic strategy for preparing monodisperse polyethylene glycol diacrylate (PEGDA) microdroplets via organoclay-assisted interfacial polymerization approach for Escherichia coli encapsulation. Based on the mechanism of spontaneous and rapid polymerization of PEGDA precursor solution with Mg-organoclay, the prepared PEGDA microdroplets have uniform size and fine round shape, with size range of 74-118 µm. The size of microdroplets can be controlled through the changing continuous phase flow rate. Organoclay-assisted polymerization method provides a unique environment to produce non-toxic ways of fabricating microorganism encapsulated microdroplets and to prohibit microdroplets merge during the processes. Furthermore, we successfully carried out to entrap E. coli inside of the PEGDA microdroplets. E. coli expressing a green fluorescent protein shows a good viability inside the PEGDA microdroplets. The in situ microfluidic synthetic method provides a novel approach for the preparation of monodisperse PEGDA microdroplets via a one-pot route.

Development of microsatellite markers to genetically differentiate populations of Octopus minor from Korea and China.

Of the more than 300 octopus species, Octopus minor is one of the most popular and economically important species in Eastern Asia, including Korea, along with O. vulgaris, O. ocellatus, and O. aegina. We developed 19 microsatellite markers from Octopus minor and eight polymorphic markers were developed to analyze the genetic diversity and relationships among four octopus populations from Korea and three from China. The number of alleles per locus varied from 10 to 49, and allelic richness per locus ranged from 2 to 16.4 across all populations. The average allele number among the populations was 11.1, with a minimum of 8.3 and a maximum of 13.6. The mean allelic richness was 8.7 in all populations. The Hardy-Weinberg equilibrium (HWE) test revealed significant deviation in 19 of the 56 single-locus sites, and null alleles were presumed in five of eight loci. The pairwise F ( ST ) values between populations from Korea and China differed significantly in all pairwise comparisons. The genetic distances between the China and Korea samples ranged from 0.161 to 0.454. The genetic distances among the populations from Korea ranged from 0.033 to 0.090, with an average of 0.062; those among populations from China ranged from 0.191 to 0.316, with an average of 0.254. The populations from Korea and China formed clearly separated into clusters via an unweighted pair group method with arithmetic mean dendrogram. Furthermore, a population from muddy flats on the western coast of the Korean Peninsula and one from a rocky area on Jeju Island formed clearly separated subclusters. An assignment test based on the allele distribution discriminated between the Korean and Chinese origins with 96.9 % accuracy.

Label-free electrochemical diagnosis of viral antigens with genetically engineered fusion protein.

We have developed a simple electrochemical biosensing strategy for the label-free diagnosis of hepatitis B virus (HBV) on a gold electrode surface. Gold-binding polypeptide (GBP) fused with single-chain antibody (ScFv) against HBV surface antigen (HBsAg), in forms of genetically engineered protein, was utilized. This GBP-ScFv fusion protein can directly bind onto the gold substrate with the strong binding affinity between the GBP and the gold surface, while the recognition site orients toward the sample for target binding at the same time. Furthermore, this one-step immobilization strategy greatly simplifies a fabrication process without any chemical modification as well as maintaining activity of biological recognition elements. This system allows specific immobilization of proteins and sensitive detection of targets, which were verified by surface plasmon resonance analysis and successfully applied to electrochemical cyclic voltammetry and impedance spectroscopy upto 0.14 ng/mL HBsAg.

Synthesis of bioactive microcapsules using a microfluidic device.

Bioactive microcapsules containing Bacillus thuringiensis (BT) spores were generated by a combination of a hydro gel, microfluidic device and chemical polymerization method. As a proof-of-principle, we used BT spores displaying enhanced green fluorescent protein (EGFP) on the spore surface to spatially direct the EGFP-presenting spores within microcapsules. BT spore-encapsulated microdroplets of uniform size and shape are prepared through a flow-focusing method in a microfluidic device and converted into microcapsules through hydrogel polymerization. The size of microdroplets can be controlled by changing both the dispersion and continuous flow rate. Poly(N-isoproplyacrylamide) (PNIPAM), known as a hydrogel material, was employed as a biocompatible material for the encapsulation of BT spores and long-term storage and outstanding stability. Due to these unique properties of PNIPAM, the nutrients from Luria-Bertani complex medium diffused into the microcapsules and the microencapsulated spores germinated into vegetative cells under adequate environmental conditions. These results suggest that there is no limitation of transferring low-molecular-weight-substrates through the PNIPAM structures, and the viability of microencapsulated spores was confirmed by the culture of vegetative cells after the germinations. This microfluidic-based microencapsulation methodology provides a unique way of synthesizing bioactive microcapsules in a one-step process. This microfluidic-based strategy would be potentially suitable to produce microcapsules of various microbial spores for on-site biosensor analysis.

Development of a plastic-based microfluidic immunosensor chip for detection of H1N1 influenza.

Lab-on-a-chip can provide convenient and accurate diagnosis tools. In this paper, a plastic-based microfluidic immunosensor chip for the diagnosis of swine flu (H1N1) was developed by immobilizing hemagglutinin antigen on a gold surface using a genetically engineered polypeptide. A fluorescent dye-labeled antibody (Ab) was used for quantifying the concentration of Ab in the immunosensor chip using a fluorescent technique. For increasing the detection efficiency and reducing the errors, three chambers and three microchannels were designed in one microfluidic chip. This protocol could be applied to the diagnosis of other infectious diseases in a microfluidic device.

Rapid development of microsatellite markers with 454 pyrosequencing in a vulnerable fish, the mottled skate, Raja pulchra.

The mottled skate, Raja pulchra, is an economically valuable fish. However, due to a severe population decline, it is listed as a vulnerable species by the International Union for Conservation of Nature. To analyze its genetic structure and diversity, microsatellite markers were developed using 454 pyrosequencing. A total of 17,033 reads containing dinucleotide microsatellite repeat units (mean, 487 base pairs) were identified from 453,549 reads. Among 32 loci containing more than nine repeat units, 20 primer sets (62%) produced strong PCR products, of which 14 were polymorphic. In an analysis of 60 individuals from two R. pulchra populations, the number of alleles per locus ranged from 1-10, and the mean allelic richness was 4.7. No linkage disequilibrium was found between any pair of loci, indicating that the markers were independent. The Hardy-Weinberg equilibrium test showed significant deviation in two of the 28 single-loci after sequential Bonferroni's correction. Using 11 primer sets, cross-species amplification was demonstrated in nine related species from four families within two classes. Among the 11 loci amplified from three other Rajidae family species; three loci were polymorphic. A monomorphic locus was amplified in all three Rajidae family species and the Dasyatidae family. Two Rajidae polymorphic loci amplified monomorphic target DNAs in four species belonging to the Carcharhiniformes class, and another was polymorphic in two Carcharhiniformes species.