New species of redbait from the Philippines (Teleostei, Emmelichthyidae, Emmelichthys).

We describe a new species of redbait in the genus Emmelichthys collected from fish markets on Panay and Cebu islands in the Visayas region of the Philippines. The species is externally similar to E.struhsakeri but is diagnosable by two prominent fleshy papillae associated with the cleithrum and fewer pectoral-fin rays (18-19 vs. 19-21) and gill rakers (30-33 vs. 34-41). Additionally, mitochondrial DNA differentiates this taxon from other species of Emmelichthys. We generate mitochondrial genomes for two of the three type specimens and several other emmelichthyids to place the new taxon in a phylogenetic context. Analysis of the protein-coding mitochondrial loci calls into question the monophyly of two emmelichthyid genera (Emmelichthys and Erythrocles) and highlights the need for subsequent analyses targeting the intrarelationships of the Emmelichthyidae.

The Complete Genome Sequence of Portunus pelagicus (Portunidae, Decapoda), the Blue Swimming Crab.

Portunus pelagicus is a large crab found in the Indo-Pacific. We present the whole genome sequence of this species. Illumina paired-end reads were assembled by a de novo method followed by a finishing step. The raw and assembled data are publicly available via GenBank: Sequence Read Archive (SRR25655135) and assembled genome (JAWWMC000000000).

Biodiversity of Philippine marine fishes: A DNA barcode reference library based on voucher specimens.

Accurate identification of fishes is essential for understanding their biology and to ensure food safety for consumers. DNA barcoding is an important tool because it can verify identifications of both whole and processed fishes that have had key morphological characters removed (e.g., filets, fish meal); however, DNA reference libraries are incomplete, and public repositories for sequence data contain incorrectly identified sequences. During a nine-year sampling program in the Philippines, a global biodiversity hotspot for marine fishes, we developed a verified reference library of cytochrome c oxidase subunit I (COI) sequences for 2,525 specimens representing 984 species. Specimens were primarily purchased from markets, with additional diversity collected using rotenone or fishing gear. Species identifications were verified based on taxonomic, phenotypic, and genotypic data, and sequences are associated with voucher specimens, live-color photographs, and genetic samples catalogued at Smithsonian Institution, National Museum of Natural History. The Biodiversity of Philippine Marine Fishes dataset is released herein to increase knowledge of species diversity and distributions and to facilitate accurate identification of market fishes.

The Complete Genome Sequences of 5 Species of Sardinella (Clupeidae, Clupeiformes).

We present the complete genome sequences of 5 species of Sardinella. Illumina sequencing was performed on genetic material from wild-caught specimens. The reads were assembled using a de novo method followed by a finishing step. The raw and assembled data are publicly available via Genbank.

Testing the Utility of Alternative Metrics of Branch Support to Address the Ancient Evolutionary Radiation of Tunas, Stromateoids, and Allies (Teleostei: Pelagiaria).

The use of high-throughput sequencing technologies to produce genome-scale data sets was expected to settle some long-standing controversies across the Tree of Life, particularly in areas where short branches occur at deep timescales. Instead, these data sets have often yielded many well-supported but conflicting topologies, and highly variable gene-tree distributions. A variety of branch-support metrics beyond the nonparametric bootstrap are now available to assess how robust a phylogenetic hypothesis may be, as well as new methods to quantify gene-tree discordance. We applied multiple branch-support metrics to a study of an ancient group of marine fishes (Teleostei: Pelagiaria) whose interfamilial relationships have proven difficult to resolve due to a rapid accumulation of lineages very early in its history. We analyzed hundreds of loci including published ultraconserved elements and newly generated exonic data along with their flanking regions to represent all 16 extant families for more than 150 out of 284 valid species in the group. Branch support was typically lower at inter- than intra-familial relationships regardless of the type of marker used. Several nodes that were highly supported with bootstrap had a very low site and gene-tree concordance, revealing underlying conflict. Despite this conflict, we were able to identify four consistent interfamilial clades, each comprised of two or three families. Combining exons with their flanking regions also produced increased branch lengths at the deep branches of the pelagiarian tree. Our results demonstrate the limitations of employing current metrics of branch support and species-tree estimation when assessing the confidence of ancient evolutionary radiations and emphasize the necessity to embrace alternative measurements to explore phylogenetic uncertainty and discordance in phylogenomic data sets.[Concatenation; exons; introns; phylogenomics; species-tree methods; target capture.].

DNA barcoding cannot discriminate between Sardinella tawilis and S. hualiensis (Clupeiformes: Clupeidae).

Sardinella tawilis, the only known freshwater sardinella in the world, is endemic to Taal Lake, Philippines. Previous studies found the Taiwan sardinella, S. hualiensis, to be morphologically very similar to S. tawilis and identified it as the marine sister species of S. tawilis. In this study, DNA barcoding using the mitochondrial cytochrome c oxidase I (COI) gene was carried out to analyze species demarcation in the Sardinella genus, focusing primarily on the relationship between S. tawilis and S. hualiensis. The neighbour-joining (NJ) tree that was constructed using Kimura 2-parameter (K2P) model showed a single clade for the two species with 100% bootstrap support. K2P interspecific genetic divergence ranged from 0% to 0.522%, which is clearly below the suggested 3-3.5% cutoff for species discrimination. Recombination activating gene 1 (RAG1), mitochondrial control region (CR), cytochrome b, 16S rRNA, and S7 markers were used to further validate the results. Sardinella tawilis and S. hualiensis clustered together with a bootstrap support of 99-100% in each of the NJ trees. Low interspecific genetic distances between S. tawilis and S. hualiensis for all the markers except CR could be attributed to incipient allopatric speciation.

Genetic patterns in peripheral marine populations of the fusilier fish Caesio cuning within the Kuroshio Current.

AIM: Mayr's central-peripheral population model (CCPM) describes the marked differences between central and peripheral populations in genetic diversity, gene flow, and census size. When isolation leads to genetic divergence, these peripheral populations have high evolutionary value and can influence biogeographic patterns. In tropical marine species with pelagic larvae, powerful western-boundary currents have great potential to shape the genetic characteristics of peripheral populations at latitudinal extremes. We tested for the genetic patterns expected by the CCPM in peripheral populations that are located within the Kuroshio Current for the Indo-Pacific reef fish, Caesio cuning. METHODS: We used a panel of 2,677 SNPs generated from restriction site-associated DNA (RAD) sequencing to investigate genetic diversity, relatedness, effective population size, and spatial patterns of population connectivity from central to peripheral populations of C. cuning along the Kuroshio Current. RESULTS: Principal component and cluster analyses indicated a genetically distinct lineage at the periphery of the C. cuning species range and examination of SNPs putatively under divergent selection suggested potential for local adaptation in this region. We found signatures of isolation-by-distance and significant genetic differences between nearly all sites. Sites closest to the periphery exhibited increased within-population relatedness and decreased effective population size. MAIN CONCLUSIONS: Despite the potential for homogenizing gene flow along the Kuroshio Current, peripheral populations in C. cuning conform to the predictions of the CCPM. While oceanography, habitat availability, and dispersal ability are all likely to shape the patterns found in C. cuning across this central-peripheral junction, the impacts of genetic drift and natural selection in increasing smaller peripheral populations appear to be probable influences on the lineage divergence found in the Ryukyu Islands.

Maternal mismatches in farmed tilapia strains (Oreochromis spp.) in the Philippines as revealed by mitochondrial COI gene.

The introduction of genetically enhanced tilapia has significantly boosted the performance of Philippine aquaculture industry. While enhanced strains contribute to the increase in tilapia production, genetic characterization of present tilapia stocks is critical to maintain their quality and to ensure the genetic gains are sustained. To understand and determine the genetic relationship of the genetically enhanced strains produced in the Philippines, mitochondrial cytochrome oxidase subunit I (COI) gene using DNA barcoding approach was analyzed. Specimens representing 10 genetically enhanced strains (GIFT, FaST, GET-EXCEL, GST, SST, COLD, YY-male, GMT, Molobicus, and BEST), three red tilapia (Taiwan red, Florida red, and FAC-red), and two pure lines (initially identified as O. aureus and O. spilurus) were collected, sequenced, and identified using DNA barcoding. Results revealed that farmed tilapias consisted of four different Oreochromis species. As expected, COI could not distinguish individuals at the strain level but surprisingly, mismatch between the species of maternal origin and present-day offspring was observed. This particular result may pose a question on the genetic purity and integrity of the strains being distributed to farmers and suggests a re-evaluation of the effectiveness of major tilapia breeding centers in maintaining their stocks.

Molecular cloning and expression of chitin deacetylase 1 gene from the gills of Penaeus monodon (black tiger shrimp).

Chitin deacetylases have been identified and studied in several fungi and insects but not in crustaceans. These glycoproteins function in catalyzing the conversion of chitin to chitosan by the hydrolysis of N-acetamido bonds of chitin. Here, for the first time, the full length cDNA of chitin deacetylase (CDA) gene from crustaceans was fully cloned using a partial fragment obtained from a transcriptome database of the gills of black tiger shrimp Penaeus monodon that survived White Spot Syndrome Virus (WSSV) infection employing Rapid Amplification of cDNA Ends (RACE) PCR. The shrimp CDA, named PmCDA1, was further characterized by in silico analysis, and its constitutive expression determined in apparently healthy shrimp through reverse transcription PCR (RT-PCR). Results revealed that the P. monodon chitin deacetylase (PmCDA1) is 2176 bp-long gene with an open reading frame (ORF) of 1596 bp encoding for 532 amino acids. Phylogenetic analysis revealed that PmCDA1 belongs to Group I CDAs together with CDA1 and CDA2 proteins found in insects. Moreover, PmCDA1 is composed of a conserved chitin-binding peritrophin-A domain (CBD), a low-density lipoprotein receptor class A domain (LDL-A) and a catalytic domain that is part of CE4 superfamily, all found in group I CDAs, which are known to serve critical immune function against WSSV. Finally, high expression of PmCDA1 gene in the gills of apparently healthy P. monodon was observed suggesting important basal function of the gene in this tissue. Taken together, this is a first report of the full chitin deacetylase 1 (CDA1) gene in crustaceans particularly in shrimp that exhibits putative immune function against WSSV and is distinctly highly expressed in the gills of shrimp.

Species composition of by-catch from milkfish (Chanos chanos) fry fishery in selected sites in the Philippines as determined by DNA barcodes.

Milkfish fry fishery, an important industry in the Philippines, uses non-selective fishing gears and push nets in coastal areas which lead to the capture of other non-targeted juvenile aquatic species. Unfortunately, information on the amount and the identity of by-catch species is lacking thus the extent of impact of the fry fishery is not known. In this study, the species composition of milkfish fry fishery by-catch sampled from selected coastal areas that are known to be fry collection sites in the country were identified and assessed through the use of DNA barcoding. Analyses revealed that by-catch fish species of the milkfish fry industry included Black Tiger shrimp (Peneaus monodon), Tarpon (Megalops cyprinoides), Glass perchlets (Ambasis gymnocephalus and Ambasis buruensis), Ladyfish (Elops hawaiensis), Snapper (Lutjanus fulviflamma), Cardinal fishes (Apogon hyalosoma and Sphaeramia orbicularis), Whipfin siver biddy (Gerres filamentosus), Mullet (Liza sp.), Anchovy (Encrasicholina heteroloba), and Tiger perch (Terapon jarbua), almost all of which are potential marketable food fish and culture species. The results of the study provide preliminary information, as well as awareness, on the species composition of milkfish fry by-catch.

Illegal trade of regulated and protected aquatic species in the Philippines detected by DNA barcoding.

Illegal trade has greatly affected marine fish stocks, decreasing fish populations worldwide. Despite having a number of aquatic species being regulated, illegal trade still persists through the transport of dried or processed products and juvenile species trafficking. In this regard, accurate species identification of illegally traded marine fish stocks by DNA barcoding is deemed to be a more efficient method in regulating and monitoring trade than by morphological means which is very difficult due to the absence of key morphological characters in juveniles and processed products. Here, live juvenile eels (elvers) and dried products of sharks and rays confiscated for illegal trade were identified. Twenty out of 23 (87%) randomly selected "elvers" were identified as Anguilla bicolor pacifica and 3 (13%) samples as Anguilla marmorata. On the other hand, 4 out of 11 (36%) of the randomly selected dried samples of sharks and rays were Manta birostris. The rest of the samples were identified as Alopias pelagicus, Taeniura meyeni, Carcharhinus falciformis, Himantura fai and Mobula japonica. These results confirm that wild juvenile eels and species of manta rays are still being caught in the country regardless of its protected status under Philippine and international laws. It is evident that the illegal trade of protected aquatic species is happening in the guise of dried or processed products thus the need to put emphasis on strengthening conservation measures. This study aims to underscore the importance of accurate species identification in such cases of illegal trade and the effectivity of DNA barcoding as a tool to do this.

Distinct Yellowfin Tuna (Thunnus albacares) Stocks Detected in Western and Central Pacific Ocean (WCPO) Using DNA Microsatellites.

The yellowfin tuna, Thunnus albacares (Bonnaterre, 1788), covers majority of the Philippines' tuna catch, one of the major fisheries commodities in the country. Due to its high economic importance sustainable management of these tunas has become an imperative measure to prevent stock depletion. Currently, the Philippine yellowfin tuna is believed to be part of a single stock of the greater WCPO though some reports suggest otherwise. This study therefore aims to establish the genetic stock structure of the said species in the Philippines as compared to Bismarck Sea, Papua New Guinea using nine (9) DNA microsatellite markers. DNA microsatellite data revealed significant genetic differentiation between the Philippine and Bismarck Sea, Papua New Guinea yellowfin tuna samples. (FST = 0.034, P = 0.016), which is further supported by multilocus distance matrix testing (PCoA) and model-based clustering (STRUCTURE 2.2).With these findings, this study posits that the yellowfin tuna population in the Philippines is a separate stock from the Bismarck Sea population. These findings add evidence to the alternative hypothesis of having at least 2 subpopulations of yellowfin tuna in the WCPO and calls for additional scientific studies using other parameters to investigate this. Accurate population information is necessary in formulating a more appropriate management strategy for the sustainability of the yellowfin tuna not only in the Philippines but also in the WCPO.

Hidden diversity in sardines: genetic and morphological evidence for cryptic species in the goldstripe sardinella, Sardinella gibbosa (Bleeker, 1849).

Cryptic species continue to be uncovered in many fish taxa, posing challenges for fisheries conservation and management. In Sardinella gibbosa, previous investigations revealed subtle intra-species variations, resulting in numerous synonyms and a controversial taxonomy for this sardine. Here, we tested for cryptic diversity within S. gibbosa using genetic data from two mitochondrial and one nuclear gene regions of 248 individuals of S. gibbosa, collected from eight locations across the Philippine archipelago. Deep genetic divergence and subsequent clustering was consistent across both mitochondrial and nuclear markers. Clade distribution is geographically limited: Clade 1 is widely distributed in the central Philippines, while Clade 2 is limited to the northernmost sampling site. In addition, morphometric analyses revealed a unique head shape that characterized each genetic clade. Hence, both genetic and morphological evidence strongly suggests a hidden diversity within this common and commercially-important sardine.

Anisakis species (Nematoda: Anisakidae) of Dwarf Sperm Whale Kogia sima (Owen, 1866) stranded off the Pacific coast of southern Philippine archipelago.

Anisakid nematodes in the Pacific region of the Philippine archipelago still remain unexplored. This study was carried out to identify anisakid species from one of their final hosts, the Kogiid whale (Dwarf Sperm Whale, Kogia sima) stranded off the southern part (Davao Gulf) of the Philippine archipelago. Anisakid worms were initially identified morphologically using light and scanning electron microscopy, whereas identification to species level was carried out molecularly using PCR-RFLP and sequencing of the ITS (ITS1-5.8s rRNA-ITS2) and mtDNA cox2 regions. Parasitological study revealed new geographical records for the presence of two Anisakis species (A. brevispiculata and A. typica) and two unknown Anisakis species that are genetically close, at mtDNA cox2 region, to A. paggiae and A. ziphidarum. Based on the molecular data on both genes, the current findings suggest possible occurrence of local variations or sibling species of A. paggiae and A. ziphidarum in the region. Given that Anisakis species have not been reported in the Philippine archipelago, their presence in the Dwarf Sperm Whale inhabiting this region indicates high possibility of Anisakis infections in the marine fishes, cephalopods and other intermediate hosts within the Philippine waters.

RNA aptamers inhibit the growth of the fish pathogen viral hemorrhagic septicemia virus (VHSV).

Viral hemorrhagic septicemia virus (VHSV) is a serious disease impacting wild and cultured fish worldwide. Hence, an effective therapeutic method against VHSV infection needs to be developed. Aptamer technology is a new and promising method for diagnostics and therapeutics. It revolves around the use of an aptamer molecule, an artificial ligand (nucleic acid or protein), which has the capacity to recognize target molecules with high affinity and specificity. Here, we aimed at selecting RNA aptamers that can specifically bind to and inhibit the growth of a strain of fish VHSV both in vitro and in vivo. Three VHSV-specific RNA aptamers (F1, F2, and C6) were selected from a pool of artificially and randomly produced oligonucleotides using systematic evolution of ligands by exponential enrichment. The three RNA aptamers showed obvious binding to VHSV in an electrophoretic mobility shift assay but not to other tested viruses. The RNA aptamers were tested for their ability to inhibit VHSV in vitro using hirame natural embryo (HINAE) cells. Cytopathic effect and plaque assays showed that all aptamers inhibited the growth of VHSV in HINAE cells. In vivo tests using RNA aptamers produced by Rhodovulum sulfidophilum showed that extracellular RNA aptamers inhibited VHSV infection in Japanese flounder. These results suggest that the RNA aptamers are a useful tool for protection against VHSV infection in Japanese flounder.

Discrimination of juvenile yellowfin (Thunnus albacares) and bigeye (T. obesus) Tunas using mitochondrial DNA control region and liver morphology.

Yellowfin tuna, Thunnus albacares (Bonnaterre, 1788) and bigeye tuna, Thunnus obesus (Lowe, 1839) are two of the most economically important tuna species in the world. However, identification of their juveniles, especially at sizes less than 40 cm, is very difficult, often leading to misidentification and miscalculation of their catch estimates. Here, we applied the mitochondrial DNA control region D-loop, a recently validated genetic marker used for identifying tuna species (Genus Thunnus), to discriminate juvenile tunas caught by purse seine and ringnet sets around fish aggregating devices (FADs) off the Southern Iloilo Peninsula in Central Philippines. We checked individual identifications using the Neighbor-Joining Method and compared results with morphometric analyses and the liver phenotype. We tested 48 specimens ranging from 13 to 31 cm fork length. Morpho-meristic analyses suggested that 12 specimens (25%) were bigeye tuna and 36 specimens (75%) were yellowfin tuna. In contrast, the genetic and liver analyses both showed that 5 specimens (10%) were bigeye tuna and 43 (90%) yellowfin tuna. This suggests that misidentification can occur even with highly stringent morpho-meristic characters and that the mtDNA control region and liver phenotype are excellent markers to discriminate juveniles of yellowfin and bigeye tunas.

Molecular characterization, expression and functional analysis of a nuclear oligomerization domain proteins subfamily C (NLRC) in Japanese flounder (Paralichthys olivaceus).

Pattern recognition receptors (PRRs) are involved in the effective innate defense against several microbes. Here, we identified a nucleotide-oligomerization domain (NOD)-like receptor subfamily C (NLRC) from Japanese flounder (Paralichthys olivaceus). Full-length transcript of JfNLRC is composed of 3976 bp encoding a protein of 1175 deduced amino acid residues. The presence of a signature nucleotide-binding domain (NACHT) and leucine-rich repeated domain (LRR) suggested that the protein is a member of the NLR family. Interestingly, its C-terminus presents an extra PRY/SPRY (B30.2) domain similar to fish in the Trim (finTrim) family. A phylogenic tree of JfNLRC revealed that full-length JfNLRC diverged from the NOD1 and NOD2 clusters, and the NACHT domain in JfNLRC was clustered within the NLRC3 group. Stimulation by formalin-killed Edwardsiella tarda, Streptococcus iniae, and lipopolysaccharide (LPS) showed that the JfNLRC expression was raised a few hours after stimulation, suggesting this novel protein is involved in the immediate response against both Gram-positive and Gram-negative bacteria. Furthermore, the IL-1ß mRNA expression level in JfNLRC-over-expressing HINAE cells was significantly increased, when compared to a control, after LPS-stimulation and E. tarda infection. These results suggested that JfNLRC probably induced IL-1ß gene expression mediated by LPS-stimulation.

Microarray analyses of shrimp immune responses.

Shrimp aquaculture is one of the major foodproducing industries in the world. However, it is being impacted by several problems including diseases, antibiotic use, and environmental factors. The extent of the effects of these problems in the immune system of the shrimp at the molecular level is just beginning to be understood. Here, we review the gene expression profile of shrimp in response to some of these problems using the high-throughput microarray analysis, including white spot syndrome virus, yellow head virus, Vibrio spp., peptidoglycan, oxytetracycline, oxolinic acid, salinity, and temperature.