The complete mitochondrial genome of a wild-collected Kappaphycus malesianus (Solieriaceae, Rhodophyta).

Kappaphycus malesianus is a red seaweed farmed primarily for its carrageenan, a polysaccharide important in the food and pharmaceutical industries. Among the commercially cultivated Kappaphycus species, only K. malesianus has no mitogenome data available. Here, we assembled the mitochondrial genome of K. malesianus from next-generation sequencing data. The circular mitogenome consisted of 25,250 base pairs (bp) with a GC content of 30.25%. These values were comparable to previously sequenced solieriacean mitogenomes. Structural features, such as the stem-loop and hairpin, which were previously reported in other rhodophytes mitochondrial DNA, were also identified. The annotated genes (24 protein-coding genes, 24 tRNA genes, and 2 rRNA genes) were arranged in an order similar to the other available solieriacean mitogenomes. Lastly, phylogenetic analysis using 23 predicted protein domains showed the sister relationship of K. malesianus with other Kappaphycus species.

Novel and Broadly Applicable Microsatellite Markers in Identified Chromosomes of the Philippine Dengue Mosquitoes, Aedes aegypti (Diptera: Culicidae).

Dengue is the leading arboviral infection in the Philippines. Its endemicity in the country is due to the presence of its primary mosquito vector, Aedes aegypti (L.). This species has limited microsatellite markers. This study characterized microsatellite markers screened in silico from intergenic regions of the updated reference genome of Ae. aegypti from Liverpool, U.K. Criteria for good markers are: polymorphic, inherited in a Mendelian codominant manner, no null alleles, selectively neutral, randomly associated, and broadly applicable across different regions. Genotypes were scored using ABI Peak Scanner and were screened for the presence of null alleles. Hardy-Weinberg equilibrium, linkage disequilibrium, and robustness of the markers were determined by GENEPOP using Ae. aegypti samples from selected highland and lowland sites (n = 30 each) in the Philippines and outgroups (Thailand and Vietnam). Mendelian codominant inheritance was examined using F1 offspring of Ae. aegypti family (n = 30 each) derived from samples collected from Cebu city highlands and Maramag, Bukidnon. From the 63 randomly selected markers, nine were polymorphic. Two markers (Aaeg1-3D of chromosome 1 and Aaeg3-4C of chromosome 3) satisfied all criteria, hence, are good broadly useful microsatellite markers. Two other markers (Aaeg2-2E of chromosome 2 and Aaeg3-2A of chromosome 3) met all criteria but deviated from Mendelian codominant inheritance. These new markers of the Philippine Ae. aegypti with their chromosomal locations relative to the other published markers are presented, and will ultimately be useful in a variety of population genetic studies of Ae. aegypti to protect the public health.

Biochemical Mapping of Pyrodinium bahamense Unveils Molecular Underpinnings behind Organismal Processes.

Proteins, lipids, and carbohydrates from the harmful algal bloom (HAB)-causing organism Pyrodinium bahamense were characterized to obtain insights into the biochemical processes in this environmentally relevant dinoflagellate. Shotgun proteomics using label-free quantitation followed by proteome mapping using the P. bahamense transcriptome and translated protein databases of Marinovum algicola, Alexandrium sp., Cylindrospermopsis raciborskii, and Symbiodinium kawagutii for annotation enabled the characterization of the proteins in P. bahamense. The highest number of annotated hits were obtained from M. algicola and highlighted the contribution of microorganisms associated with P. bahamense. Proteins involved in dimethylsulfoniopropionate (DMSP) degradation such as propionyl CoA synthethase and acryloyl-CoA reductase were identified, suggesting the DMSP cleavage pathway as the preferred route in this dinoflagellate. Most of the annotated proteins were involved in amino acid biosynthesis and carbohydrate degradation and metabolism, indicating the active roles of these molecules in the vegetative stage of P. bahamense. This characterization provides baseline information on the cellular machinery and the molecular basis of the ecophysiology of P. bahamense.

Differentiating Two Closely Related Alexandrium Species Using Comparative Quantitative Proteomics.

Alexandrium minutum and Alexandrium tamutum are two closely related harmful algal bloom (HAB)-causing species with different toxicity. Using isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomics and two-dimensional differential gel electrophoresis (2D-DIGE), a comprehensive characterization of the proteomes of A. minutum and A. tamutum was performed to identify the cellular and molecular underpinnings for the dissimilarity between these two species. A total of 1436 proteins and 420 protein spots were identified using iTRAQ-based proteomics and 2D-DIGE, respectively. Both methods revealed little difference (10-12%) between the proteomes of A. minutum and A. tamutum, highlighting that these organisms follow similar cellular and biological processes at the exponential stage. Toxin biosynthetic enzymes were present in both organisms. However, the gonyautoxin-producing A. minutum showed higher levels of osmotic growth proteins, Zn-dependent alcohol dehydrogenase and type-I polyketide synthase compared to the non-toxic A. tamutum. Further, A. tamutum had increased S-adenosylmethionine transferase that may potentially have a negative feedback mechanism to toxin biosynthesis. The complementary proteomics approach provided insights into the biochemistry of these two closely related HAB-causing organisms. The identified proteins are potential biomarkers for organismal toxicity and could be explored for environmental monitoring.

Conomarphins cause paralysis in mollusk: Critical and tunable structural elements for bioactivity.

Two conomarphins were purified as the major component of the venom of Conus eburneus. Conomarphins Eb1 and Eb2 showed biological activity in the mollusk Pomacea padulosa, causing sluggishness and retraction of siphon, foot, and cephalic tentacles. To further probe the effects of conserved amino acids and posttranslational modifications in conomarphins, we prepared four synthetic analogues: conomarphin Eb1 Hyp10Pro, Hyp10Ala, d-Phe13Ala, and l-Phe13 variants. Structure-activity relationship analysis indicated that d-Phe13 is critical to the biological activity of conomarphins. In contrast, amino acid changes at position 10 and removal of posttranslational modification in Hyp10Pro can be tolerated. The high expression level and observed mollusk activity of conomarphins may suggest their potential role as defensive arsenal of Conoidean snails against other predatory gastropods.

Whole-genome sequencing and single nucleotide polymorphisms in multidrug-resistant clinical isolates of Mycobacterium tuberculosis from the Philippines.

OBJECTIVES: Thousands of cases of multidrug-resistant tuberculosis (TB) have been observed in the Philippines, but studies on the Mycobacterium tuberculosis (MTB) genotypes that underlie the observed drug resistance profiles are lacking. This study aimed to analyse the whole genomes of clinical MTB isolates representing various resistance profiles to identify single nucleotide polymorphisms (SNPs) in resistance-associated genes. METHODS: The genomes of ten MTB isolates cultured from banked sputum sources were sequenced. Bioinformatics analysis consisted of assembly, annotation and SNP identification in genes reported to be associated with resistance to isoniazid (INH), rifampicin (RIF), ethambutol (ETH), streptomycin, pyrazinamide (PZA) and fluoroquinolones (FQs). RESULTS: The draft assemblies covered an average of 97.08% of the expected genome size. Seven of the ten isolates belonged to the Indo-Oceanic lineage/EA12-Manila clade. Two isolates were classified into the Euro-American lineage, whilst the pre-XDR (pre-extensively drug-resistant) isolate was classified under the East Asian/Beijing clade. The SNPs katG Ser315Thr, rpoB Ser450Leu and embB Met306Val were found in INH- (4/7), RIF- (3/6) and ETH-resistant (2/6) isolates, respectively, but not in susceptible isolates. Mutations in the inhA promoter and in the pncA and gyrA genes known to be involved in resistance to INH, PZA and FQs, respectively, were also identified. CONCLUSIONS: This study represents the first effort to investigate the whole genomes of Philippine clinical strains of MTB exhibiting various multidrug resistance profiles. Whole-genome data can provide valuable insights to the mechanistic and epidemiological qualities of TB in a high-burden setting such as the Philippines.

A CAZyme-Rich Genome of a Taxonomically Novel Rhodophyte-Associated Carrageenolytic Marine Bacterium.

Carbohydrate-active enzymes (CAZymes) have significant biotechnological potential as agents for degradation or modification of polysaccharides/glycans. As marine macroalgae are known to be rich in various types of polysaccharides, seaweed-associated bacteria are likely to be a good source of these CAZymes. A genomics approach can be used to explore CAZyme abundance and diversity, but it can also provide deep insights into the biology of CAZyme producers and, in particular, into molecular mechanisms that mediate their interaction with their hosts. In this study, a Gram-negative, aerobic, rod-shaped, carrageenolytic, and culturable marine bacterium designated as AOL6 was isolated from a diseased thallus of a carrageenan-producing farmed rhodophyte, Kappaphycus alvarezii (Gigartinales, Rhodophyta). The whole genome of this bacterium was sequenced and characterized. Sequence reads were assembled producing a high-quality genome assembly. The estimated genome size of the bacterium is 4.4 Mb and a G+C content of 52%. Molecular phylogenetic analysis based on a complete sequence of 16S rRNA, rpoB, and a set of 38 single-copy genes suggests that the bacterium is an unknown species and represents a novel genus in the family Cellvibrionaceae that is most closely related to the genera Teredinibacter and Saccharophagus. Genome comparison with T. turnerae T7901 and S. degradans 2-40 reveals several features shared by the three species, including a large number of CAZymes that comprised > 5% of the total number of protein-coding genes. The high proportion of CAZymes found in the AOL6 genome exceeds that of other known carbohydrate degraders, suggesting a significant capacity to degrade a range of polysaccharides including κ-carrageenan; 34% of these CAZymes have signal peptide sequences for secretion. Three putative κ-carrageenase-encoding genes were identified from the genome of the bacterium via in silico analysis, consistent with the results of the zymography assay (with κ-carrageenan as substrate). Genome analysis also indicated that AOL6 relies exclusively on type 2 secretion system (T2SS) for secreting proteins (possibly including glycoside hydrolases). In relation to T2SS, the product of the pilZ gene was predicted to be highly expressed, suggesting specialization for cell adhesion and secretion of virulence factors. The assignment of proteins to clusters of orthologous groups (COGs) revealed a pattern characteristic of r-strategists. Majority of two-component system proteins identified in the AOL6 genome were also predicted to be involved in chemotaxis and surface colonization. These genomic features suggest that AOL6 is an opportunistic pathogen, adapted to colonizing polysaccharide-rich hosts, including carrageenophytes.

Divergence of the Venom Exogene Repertoire in Two Sister Species of Turriconus.

The genus Conus comprises approximately 700 species of venomous marine cone snails that are highly efficient predators of worms, snails, and fish. In evolutionary terms, cone snails are relatively young with the earliest fossil records occurring in the Lower Eocene, 55 Ma. The rapid radiation of cone snail species has been accompanied by remarkably high rates of toxin diversification. To shed light on the molecular mechanisms that accompany speciation, we investigated the toxin repertoire of two sister species, Conus andremenezi and Conus praecellens, that were until recently considered a single variable species. A total of 196 and 250 toxin sequences were identified in the venom gland transcriptomes of C. andremenezi and C. praecellens belonging to 25 and 29 putative toxin gene superfamilies, respectively. Comparative analysis with closely (Conus tribblei and Conus lenavati) and more distantly related species (Conus geographus) suggests that speciation is associated with significant diversification of individual toxin genes (exogenes) whereas the expression pattern of toxin gene superfamilies within lineages remains largely conserved. Thus, changes within individual toxin sequences can serve as a sensitive indicator for recent speciation whereas changes in the expression pattern of gene superfamilies are likely to reflect more dramatic differences in a species' interaction with its prey, predators, and competitors.

Prevalence, diversity and co-occurrence of the white spot syndrome virus, monodon baculovirus and Penaeus stylirostris densovirus in wild populations of Penaeus monodon in the Philippines.

The farming of the black tiger shrimp Penaeus monodon in the Philippines relies on wild broodstock. PCR was thus used to determine the prevalence of white spot syndrome virus (WSSV), monodon baculovirus (MBV) and Penaeus stylirostris densovirus (PstDV) in a total of 178 shrimp from 6 geographically disparate locations where broodstock are captured for use in hatcheries. PCR amplicons were also sequenced to identify phylogenetic relationships of the virus haplotypes detected. Shrimp from southeastern Luzon (Camarines Norte) had the highest prevalence of each of the 3 viruses and were frequently co-infected with 2 or more viruses. No viruses were detected in shrimp from northwestern Luzon (Pangasinan). MBV was most prevalent and PstDV strains displayed the most genetic diversity. WSSV was detected at 3 sites, and a VP28 gene sequence examined was invariant and consistent with strains found in many countries, including Thailand, China, Japan, Korea, Indonesia, Iran, Brazil and Mexico. WSSV open reading frame 94 gene sequence analysis identified location-specific repeat types. MBV sequences were dissimilar to haplotypes detected in India. PstDV sequences were diverse and included 2 lineages detected either in Australia or in the United States, Ecuador, Taiwan, China and Vietnam. The PCR data confirmed that WSSV, MBV and PstDV are endemic in P. monodon in the Philippines but that populations at some locations might remain free of infection.

Variation in virome diversity in wild populations of Penaeus monodon (Fabricius 1798) with emphasis on pathogenic viruses.

Marine animals typically harbor a community of viruses, a number of which are known to cause diseases. In shrimp aquaculture, viral pathogens are the principal causes of major economic losses. However, the composition of the viral load of shrimps in wild population is poorly known. In this study, we explored the viral diversity in the microbiome of wild Penaeus monodon collected from six sites in the Philippines, with a view to detecting pathogenic forms. We employed a metagenomic approach via particle-associated nucleic acid isolation, sequence-independent single primer amplification, and pyrosequencing. Virome analysis of shrimp samples from different sites revealed distinct virome profiles, and hence significant differences in diversity, among the various sites based on number of OTUs, Shannon-Weaver Index, and Inverse Simpson Index. Sequences of key shrimp pathogens were detected such as the white spot syndrome virus (WSSV), and Penaeus stylirostris densovirus (PstDV). However, the patterns of distribution of the pathogenic viruses varied; whereas WSSV was found only in three out of six sites and PstDV were found in all but one site. The results also revealed shrimp-associated viruses that have not yet been observed in P. monodon such as avian virus-like, insect virus-like, plankton virus-like and bacteriophage-like sequences. Despite the diverse array of viruses detected in the study, a large proportion remains unidentified (i.e., similarity to sequences in the database was lower than the threshold required for definitive identification), and therefore could represent unexplored virus sequences and viral genomes in the environment.

Structural features of conopeptide genes inferred from partial sequences of the Conus tribblei genome.

The evolvability of venom components (in particular, the gene-encoded peptide toxins) in venomous species serves as an adaptive strategy allowing them to target new prey types or respond to changes in the prey field. The structure, organization, and expression of the venom peptide genes may provide insights into the molecular mechanisms that drive the evolution of such genes. Conus is a particularly interesting group given the high chemical diversity of their venom peptides, and the rapid evolution of the conopeptide-encoding genes. Conus genomes, however, are large and characterized by a high proportion of repetitive sequences. As a result, the structure and organization of conopeptide genes have remained poorly known. In this study, a survey of the genome of Conus tribblei was undertaken to address this gap. A partial assembly of C. tribblei genome was generated; the assembly, though consisting of a large number of fragments, accounted for 2160.5 Mb of sequence. A large number of repetitive genomic elements consisting of 642.6 Mb of retrotransposable elements, simple repeats, and novel interspersed repeats were observed. We characterized the structural organization and distribution of conotoxin genes in the genome. A significant number of conopeptide genes (estimated to be between 148 and 193) belonging to different superfamilies with complete or nearly complete exon regions were observed, ~60 % of which were expressed. The unexpressed conopeptide genes represent hidden but significant conotoxin diversity. The conotoxin genes also differed in the frequency and length of the introns. The interruption of exons by long introns in the conopeptide genes and the presence of repeats in the introns may indicate the importance of introns in facilitating recombination, evolution and diversification of conotoxins. These findings advance our understanding of the structural framework that promotes the gene-level molecular evolution of venom peptides.

Characterization of the complete mitochondrial genome of Conus tribblei Walls, 1977.

The genus Conus sensu lato consists of 500-700 species. However, the mitochondrial genomes of only few species have been fully sequenced and reported so far. In this study, the complete mitochondrial genome of Conus tribblei, a member of the poorly known subgenus Splinoconus is sequenced with the mean coverage of 604×. The mitochondrial genome is 15 570 bp long and consists of genes encoding for 13 respiratory chain proteins, 22 tRNA and 2 rRNA. The gene organization is highly conserved among the Conus species. The longest intergenic region between tRNA-Phe and cytochrome c oxidase subunit III (cox3), which in C. tribblei is 169 bp long and contains a 112 bp long segment of inverted repeat, represents the putative control region. The control regions of Conus species exhibited variability in the length and position of the inverted repeats. Therefore, this region may have the potential to be used as a genetic marker for species discrimination.

Potential DMSP-degrading Roseobacter clade dominates endosymbiotic microflora of Pyrodinium bahamense var. compressum (Dinophyceae) in vitro.

Many aspects of the biology and ecology of the toxic dinoflagellate Pyrodinium bahamense var. compressum are still poorly understood. In this brief note, we present identification of its associated intracellular bacteria or endosymbionts via PCR cloning and 16s rRNA gene sequencing and their localization by confocal microscopy, a first for Pyrodinium. The most frequently observed species in the endosymbiotic microflora were from Roseobacter clade (Alphaproteobacteria, 68%) and Gilvibacter sediminis (Flavobacteriaceae, 20%). Roseobacter lineage, the most abundant taxa in this study, is known to be involved in dimethylsulfoniopropionate metabolism which is highly produced in dinoflagellates-a possible strong factor shaping the structure of the associated bacterial community.

Comparison of the Venom Peptides and Their Expression in Closely Related Conus Species: Insights into Adaptive Post-speciation Evolution of Conus Exogenomes.

Genes that encode products with exogenous targets, which comprise an organism's "exogenome," typically exhibit high rates of evolution. The genes encoding the venom peptides (conotoxins or conopeptides) in Conus sensu lato exemplify this class of genes. Their rapid diversification has been established and is believed to be linked to the high speciation rate in this genus. However, the molecular mechanisms that underlie venom peptide diversification and ultimately emergence of new species remain poorly understood. In this study, the sequences and expression levels of conotoxins from several specimens of two closely related worm-hunting species, Conus tribblei and Conus lenavati, were compared through transcriptome analysis. Majority of the identified putative conopeptides were novel, and their diversity, even in each specimen, was remarkably high suggesting a wide range of prey targets for these species. Comparison of the interspecific expression patterns of conopeptides at the superfamily level resulted in the discovery of both conserved as well as species-specific expression patterns, indicating divergence in the regulatory network affecting conotoxin gene expression. Comparison of the transcriptomes of the individual snails revealed that each specimen produces a distinct set of highly expressed conopeptides, reflecting the capability of individual snails to fine-tune the composition of their venoms. These observations reflect the role of sequence divergence and divergence in the control of expression for specific conopeptides in the evolution of the exogenome and hence venom composition in Conus.

High conopeptide diversity in Conus tribblei revealed through analysis of venom duct transcriptome using two high-throughput sequencing platforms.

The venom of each species of Conus contains different kinds of pharmacologically active peptides which are mostly unique to that species. Collectively, the ~500-700 species of Conus produce a large number of these peptides, perhaps exceeding 140,000 different types in total. To date, however, only a small fraction of this diversity has been characterized via transcriptome sequencing. In addition, the sampling of this chemical diversity has not been uniform across the different lineages in the genus. In this study, we used high-throughput transcriptome sequencing approach to further investigate the diversity of Conus venom peptides. We chose a species, Conus tribblei, as a representative of a poorly studied clade of Conus. Using the Roche 454 and Illumina platforms, we discovered 136 unique and novel putative conopeptides belonging to 30 known gene superfamilies and 6 new conopeptide groups, the greatest diversity so far observed from a transcriptome. Most of the identified peptides exhibited divergence from the known conopeptides, and some contained cysteine frameworks observed for the first time in cone snails. In addition, several enzymes involved in posttranslational modification of conopeptides and also some proteins involved in efficient delivery of the conopeptides to prey were identified as well. Interestingly, a number of conopeptides highly similar to the conopeptides identified in a phylogenetically distant species, the generalist feeder Conus californicus, were observed. The high diversity of conopeptides and the presence of conopeptides similar to those in C. californicus suggest that C. tribblei may have a broad range of prey preferences.

The mitochondrial genome of the red alga Kappaphycus striatus ("Green Sacol" variety): complete nucleotide sequence, genome structure and organization, and comparative analysis.

The complete mitochondrial (mt) DNA sequence of the rhodophyte Kappaphycus striatus ("Green Sacol" variety) was determined. The mtDNA is circular, 25,242 bases long (A+T content: 69.94%), and contains 50 densely packed genes comprising 93.22% of the mitochondrial genome, with genes encoded on both strands. Through comparative analysis, the overall sequence, genome structure, and organization of K. striatus mtDNA were seen to be highly similar with other fully sequenced mitochondrial genomes of the class Florideophyceae. On the other hand, certain degrees of genome rearrangements and greater sequence dissimilarities were observed for the mtDNAs of other evolutionarily distant red algae, such as those from the class Bangiophyceae and Cyanidiophyceae, compared to that of K. striatus. Furthermore, a trend was observed wherein the red algal mtDNAs tend to encode lesser number of protein-coding genes, albeit not necessarily shorter, as the organism becomes more morphologically complex. This trend is supported by the phylogenetic tree inferred from the concatenated amino acid sequences of the deduced protein products of cytochrome c oxidase subunit genes (cox1, 2, and 3).

Novel venom peptides from the cone snail Conus pulicarius discovered through next-generation sequencing of its venom duct transcriptome.

The venom peptides (i.e., conotoxins or conopeptides) that species in the genus Conus collectively produce are remarkably diverse, estimated to be around 50,000 to 140,000, but the pace of discovery and characterization of these peptides have been rather slow. To date, only a minor fraction have been identified and studied. However, the advent of next-generation DNA sequencing technologies has opened up opportunities for expediting the exploration of this diversity. The whole transcriptome of a venom duct from the vermivorous marine snail C. pulicarius was sequenced using the 454 sequencing platform. Analysis of the data set resulted in the identification of over eighty unique putative conopeptide sequences, the highest number discovered so far from a Conus venom duct transcriptome. More importantly, majority of the sequences were potentially novel, many with unexpected structural features, hinting at the vastness of the diversity of Conus venom peptides that remains to be explored. The sequences represented at least 14 major superfamilies/types (disulfide- and non-disulfide-rich), indicating the structural and functional diversity of conotoxins in the venom of C. pulicarius. In addition, the contryphans were surprisingly more diverse than what is currently known. Comparative analysis of the O-superfamily sequences also revealed insights into the complexity of the processes that drive the evolution and diversification of conotoxins.

THE INDO-PACIFIC GEMMULA SPECIES IN THE SUBFAMILY TURRINAE: ASPECTS OF FIELD DISTRIBUTION, MOLECULAR PHYLOGENY, RADULAR ANATOMY AND FEEDING ECOLOGY.

The biology, feeding ecology and phylogenetic relationships of marine snails in the family Turridae remain poorly understood. Here we report our study on four deep-water species in the genus Gemmula, a major group in this family. The four species G. speciosa (Reeve 1843), G. sogodensis (Olivera 2005), G. kieneri (Doumet 1940) and G. diomedea (Powell 1964) were collected at five different sites in the Philippines, and their pattern of distribution in the sites, their feeding behaviour as well as their phylogenetic relationships with each other and with other members of the subfamily Turrinae were investigated. The radular morphology (of two Gemmula species) and potential prey (for one Gemmula species) were also examined. Actual feeding observations were also conducted for Gemmula speciosa and compared with two turrids from other genera.All four Gemmula species showed strikingly different patterns of distribution; each species was found to be relatively much more abundant at one site but not at the other sites. Molecular phylogenetic analysis based on 16S sequences correlated with previously reported 12S sequences and revealed that the four species all belong to a well-supported Gemmula clade within the subfamily Turrinae; and that this clade appeared more closely related to the clades Xenuroturris, Turris and Lophiotoma than to the other clades in the subfamily (i.e., Turridrupa, Unedogemmula and Polystira). Morphological analysis of the radula of both G. speciosa and G. sogodensis revealed that the radulae of the two species were similar but differed from the other turrids, Lophiotoma acuta and Unedogemmula bisaya, by the absence of central teeth, consistent with the separation of the Gemmula clade from the Lophiotoma and Unedogemmula clade.To identify the polychaete group that is targeted as prey by species of Gemmula, analysis of regurgitated food fragments was made; phylogenetic analysis of an mtCOI gene fragment that was PCR-amplified from the regurgitated tissue of one specimen (G. diomedea) indicated close affinity of the prey to the terebellid polychaete Amphitritides. Specimens of Gemmula speciosa, when challenged with the terebellid polychaete Loimia sp., were observed to attack the worm suggesting that Gemmula species feed on terebellid polychaetes. Lophiotoma acuta were also observed to feed on the same species of terebellid but were usually group-feeding in contrast to the solitary feeding of G. speciosa. Unedogemmula bisaya did not feed on the terebellid which also supports the separation of the Gemmula and Unedogemmula clade.Two lines of proof (i.e. the molecular phylogenetic analysis and the feeding challenge) supporting the toxin homology findings previously reported, provide consistent evidence that Gemmula is a distinct clade of worm-hunting Turrinae that feeds on Terebellidae.

Characterization of a novel psi-conotoxin from Conus parius Reeve.

The M-superfamily of conotoxins currently comprises three major groups of peptides (the mu-, kappaMu-, and psi-families) that share a key structural characteristic, the six-cysteine motif CC-C-C-CC, but differ with respect to their molecular targets. The psi-family consists of M-superfamily conotoxins that are nicotinic acetylcholine receptor (nAChR) antagonists. To date, only two psi-conotoxins, PIIIE and PIIIF, are known, both of which were isolated from a single Conus species, Conus purpurascens. In this paper, we report the discovery and initial characterization of a psi-conotoxin from another Conus species, Conus parius, which we designated as PrIIIE. Its amino acid sequence, inferred from a cloned cDNA, differed significantly from those of PIIIE and PIIIF. Its bioactivity was investigated by using the synthetic form of the peptide in mice and fish bioassays. At 2.5 nmol, the synthetic peptide induced flaccid paralysis in goldfish in ca. 4 min but did not induce any remarkable behavior in mice (after i.c. and i.p. injection of up to 10 nmol of peptide) and did not block action potential in directly stimulated frog muscle preparations. Electrophysiological experiments carried out to measure inhibition of ion currents through mouse nAChR receptors expressed in oocytes revealed that PrIIIE (IC(50) approximately 250 nM) was significantly more potent than PIIIE (IC(50) approximately 7000 nM) and that PrIIIE showed higher inhibition potency against the adult-type than the fetal-type nAChR. In similar electrophysiological assays, PrIIIE showed no inhibitory effects against the mouse muscle subtype Na(+) channel isoform Na(v) 1.4. The discovery of this psi-conotoxin from a Conus species that belongs to the subgenus Phasmoconus, which is distinct from and larger than the clade in which C. purpurascens belongs, suggests that greater structural and functional diversity of psi-conotoxins remains to be discovered from the members of this subgenus.