Signatures of Divergence, Invasiveness, and Terrestrialization Revealed by Four Apple Snail Genomes.

The family Ampullariidae includes both aquatic and amphibious apple snails. They are an emerging model for evolutionary studies due to the high diversity, ancient history, and wide geographical distribution. Insight into drivers of ampullariid evolution is hampered, however, by the lack of genomic resources. Here, we report the genomes of four ampullariids spanning the Old World (Lanistes nyassanus) and New World (Pomacea canaliculata, P. maculata, and Marisa cornuarietis) clades. The ampullariid genomes have conserved ancient bilaterial karyotype features and a novel Hox gene cluster rearrangement, making them valuable in comparative genomic studies. They have expanded gene families related to environmental sensing and cellulose digestion, which may have facilitated some ampullarids to become notorious invasive pests. In the amphibious Pomacea, novel acquisition of an egg neurotoxin and a protein for making the calcareous eggshell may have been key adaptations enabling their transition from underwater to terrestrial egg deposition.

Egg perivitelline fluid proteome of a freshwater snail: Insight into the transition from aquatic to terrestrial egg deposition.

RATIONALE: Proteins from the egg perivitelline fluid (PVF) are assumed to play critical roles in embryonic development, but for many groups of animals their identities remain unknown. Identifying egg PVF proteins is a critical step towards understanding their functions including their roles in evolutionary transition in habitats. METHODS: We applied proteomic and transcriptomic analysis to investigate the PVF proteome of the eggs of Pomacea diffusa, an aerial ovipositing freshwater snail in the family Ampullariidae. The PVF proteins were separated with the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) method, and proteomic analysis was conducted using an LTQ Velos ion trap mass spectrometer coupled with liquid chromatography. Comparison of PVF proteomes and evolution analyses was performed between P. diffusa and other ampullariids. RESULTS: In total, 32 egg PVF proteins were identified from P. diffusa. They were categorized as PV1-like subunits, immune-responsive proteins, protein degradation, signaling and binding, transcription and translation, metabolism, oxidation-reduction and proteins with unknown function. Interestingly, the proteome includes a calcium-binding protein important in forming the hard eggshell that enabled the terrestrial transition. However, it does not include PV2, a neurotoxic protein that was assumed to be present in all Pomacea species. CONCLUSIONS: The PVF proteome data from P. diffusa can help us better understand the roles that reproductive proteins played during the transition from underwater to terrestrial egg deposition. Moreover, they could be useful in comparative studies of the terrestrialization in several groups of animals that occurred independently during their evolution.

AmpuBase: a transcriptome database for eight species of apple snails (Gastropoda: Ampullariidae).

BACKGROUND: Gastropoda, with approximately 80,000 living species, is the largest class of Mollusca. Among gastropods, apple snails (family Ampullariidae) are globally distributed in tropical and subtropical freshwater ecosystems and many species are ecologically and economically important. Ampullariids exhibit various morphological and physiological adaptations to their respective habitats, which make them ideal candidates for studying adaptation, population divergence, speciation, and larger-scale patterns of diversity, including the biogeography of native and invasive populations. The limited availability of genomic data, however, hinders in-depth ecological and evolutionary studies of these non-model organisms. RESULTS: Using Illumina Hiseq platforms, we sequenced 1220 million reads for seven species of apple snails. Together with the previously published RNA-Seq data of two apple snails, we conducted de novo transcriptome assembly of eight species that belong to five genera of Ampullariidae, two of which represent Old World lineages and the other three New World lineages. There were 20,730 to 35,828 unigenes with predicted open reading frames for the eight species, with N50 (shortest sequence length at 50% of the unigenes) ranging from 1320 to 1803 bp. 69.7% to 80.2% of these unigenes were functionally annotated by searching against NCBI's non-redundant, Gene Ontology database and the Kyoto Encyclopaedia of Genes and Genomes. With these data we developed AmpuBase, a relational database that features online BLAST functionality for DNA/protein sequences, keyword searching for unigenes/functional terms, and download functions for sequences and whole transcriptomes. CONCLUSIONS: In summary, we have generated comprehensive transcriptome data for multiple ampullariid genera and species, and created a publicly accessible database with a user-friendly interface to facilitate future basic and applied studies on ampullariids, and comparative molecular studies with other invertebrates.

De novo transcriptome analysis of Perna viridis highlights tissue-specific patterns for environmental studies.

BACKGROUND: The tropical green-lipped mussel Perna viridis is a common biomonitor throughout the Indo-Pacific region that is used for environmental monitoring and ecotoxicological investigations. However, there is limited molecular data available regarding this species. We sought to establish a global transcriptome database from the tissues of adductor muscle, gills and the hepatopancreas of P. viridis in an effort to advance our understanding of the molecular aspects involved during specific toxicity responses in this sentinel species. RESULTS: Illumina sequencing results yielded 544,272,542 high-quality filtered reads. After de novo assembly using Trinity, 233,257 contigs were generated with an average length of 1,264 bp and an N50 length of 2,868 bp; 192,879 assembled transcripts and 150,111 assembled unigenes were obtained after clustering. A total of 93,668 assembled transcripts (66,692 assembled genes) with putative functions for protein domains were predicted based on InterProScan analysis. Based on similarity searches, 44,713 assembled transcripts and 25,319 assembled unigenes were annotated with at least one BLAST hit. A total of 21,262 assembled transcripts (11,947 assembled genes) were annotated with at least one well-defined Gene Ontology (GO) and 5,131 assembled transcripts (3,181 assembled unigenes) were assigned to 329 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The quantity of assembled unigenes and transcripts obtained from male and female mussels were similar but varied among the three studied tissues, with the highest numbers recorded in the gills, followed by the hepatopancreas, and then the adductor muscle. Multivariate analyses revealed strong tissue-specific patterns among the three different tissues, but not between sexes in terms of expression profiles for annotated genes in various GO terms, and genes associated with stress responses and degradation of xenobiotics. The expression profiles of certain selected genes in each tissue type were further validated using real-time quantitative polymerase chain reaction assays and a similar tissue-specific trend was seen. CONCLUSIONS: The extensive sequence data generated from this study will provide a valuable molecular resource for facilitating environmental studies with P. viridis, and highlight the importance of tissue-specific approaches in the future.

Multi-omic approach provides insights into osmoregulation and osmoconformation of the crab Scylla paramamosain.

Osmoregulation and osmoconformation are two mechanisms through which aquatic animals adapt to salinity fluctuations. The euryhaline crab Scylla paramamosain, being both an osmoconformer and osmoregulator, is an excellent model organism to investigate salinity adaptation mechanisms in brachyurans. In the present study, we used transcriptomic and proteomic approaches to investigate the response of S. paramamosain to salinity stress. Crabs were transferred from a salinity of 25 ppt to salinities of 5 ppt or 33 ppt for 6 h and 10 days. Data from both approaches revealed that exposure to 5 ppt resulted in upregulation of ion transport and energy metabolism associated genes. Notably, acclimation to low salinity was associated with early changes in gene expression for signal transduction and stress response. In contrast, exposure to 33 ppt resulted in upregulation of genes related to amino acid metabolism, and amino acid transport genes were upregulated only at the early stage of acclimation to this salinity. Our study reveals contrasting mechanisms underlying osmoregulation and osmoconformation within the salinity range of 5-33 ppt in the mud crab, and provides novel candidate genes for osmotic signal transduction, thereby providing insights on understanding the salinity adaptation mechanisms of brachyuran crabs.

The Scaly-foot Snail genome and implications for the origins of biomineralised armour.

The Scaly-foot Snail, Chrysomallon squamiferum, presents a combination of biomineralised features, reminiscent of enigmatic early fossil taxa with complex shells and sclerites such as sachtids, but in a recently-diverged living species which even has iron-infused hard parts. Thus the Scaly-foot Snail is an ideal model to study the genomic mechanisms underlying the evolutionary diversification of biomineralised armour. Here, we present a high-quality whole-genome assembly and tissue-specific transcriptomic data, and show that scale and shell formation in the Scaly-foot Snail employ independent subsets of 25 highly-expressed transcription factors. Comparisons with other lophotrochozoan genomes imply that this biomineralisation toolkit is ancient, though expression patterns differ across major lineages. We suggest that the ability of lophotrochozoan lineages to generate a wide range of hard parts, exemplified by the remarkable morphological disparity in Mollusca, draws on a capacity for dynamic modification of the expression and positioning of toolkit elements across the genome.

Understanding the transition from water to land: Insights from multi-omic analyses of the perivitelline fluid of apple snail eggs.

Unlike most of the freshwater gastropod families, the family Ampullariidae includes members that exhibit both underwater and aerial oviposition, making it an ideal model for understanding mechanisms underlying the evolutionary transition from water to land. We applied SDS-PAGE and LC-MS/MS to analyse the proteome of the egg perivitelline fluid (PVF) of Marisa cornuarietis - an aquatic ovipositing ampullariid. Comparison with the reported PVF proteomes of two aerial ovipositing ampullariids (Pomacea canaliculata and P. maculata) showed that the three species all contain several major perivitellins that nourish the embryos. However, M. cornuarietis invests more heavily on immune-related proteins, which might be due to exposure to aquatic pathogens. Interestingly, only the PVF of out-of-water egg laying species have PV2 - a neurotoxin lethal to mice, and a calcium-binding protein which might be involved in the formation of calcareous eggshell. Integrated phylogenetic, evolutionary and gene expressional analyses detected the involvement of gene duplication, positive selection and neofunctionalisation in the formation of several major PVF proteins. Overall, our study provides multiple lines of evidence of adaptive evolution in the PVF proteins, and contributes to a better understanding of how aquatic gastropod ancestors invaded terrestrial habitats. SIGNIFICANCE: Aerial egg deposition has evolved in several groups of animals, but except for Vertebrata little is known about the mechanisms underlying this critical evolution process. We compared aquatic and aerial egg laying apple snails to understand the molecular mechanisms enabling such a transition in egg laying habitat. We found that the composition of perivitelline fluid proteomes of underwater and aerial egg depositors was remarkably different, and then gene duplication and positive selection were responsible for the formation of such novel proteins than enabled the evolutionary transition.

Developmental toxicity and molecular responses of marine medaka (Oryzias melastigma) embryos to ciguatoxin P-CTX-1 exposure.

Ciguatoxins are produced by toxic benthic dinoflagellates and cause ciguatera fish poisoning worldwide, but the toxic effects on developing marine fish have not been well investigated. The Pacific ciguatoxin (P-CTX-1), is a potent sodium channel agonist, which is one of the most toxic members among all CTXs. This study evaluated the toxic effects of microinjecting purified Pacific ciguatoxin-1 (P-CTX-1) on embryonic development of marine medaka Oryzias melastigma. A lower 96h-LD50 value was estimated for eleuthero-embryos (1.32ngg-1) than that for embryos (1.71ngg-1), indicating that P-CTX-1 is more lethal to newly hatched medaka larvae. P-CTX-1 induced detrimental effects during embryonic development, including hatching failure, abnormalities in physical development (caudal fin malformation and spinal deformities), internal damage (green coloration of the gall bladder and hemorrhaging), immune dysfunction, and altered muscle physiology (bradycardia and hyperkinetic twitching). The results of a transcriptional expression analysis of genes related to the stress/immune responses, cardiac and bone development, and apoptosis supported the observed developmental abnormalities. This study advanced the understanding of P-CTX-1 mediated toxic mechanisms in the development of early life stages of a fish, and thus contributed to the toxicity assessment of CTXs in marine ecosystems.

Photosynthetic and transcriptional responses of the marine diatom Thalassiosira pseudonana to the combined effect of temperature stress and copper exposure.

A 96-h exposure experiment was conducted to elucidate the toxicity responses of the marine diatom Thalassiosira pseudonana upon exposure to different temperatures and copper (Cu) concentrations. Three Cu treatments (seawater control; 200µg/L Cu, EC50 for the yield at 25°C; and 1000µg/L Cu, EC50 for growth inhibition at 25°C) were conducted against four temperatures (10°C, 15°C, 25°C and 30°C). Growth rate and photosynthetic responses showed a significant interacting thermal-chemical effect with strong synergistic responses observed at 30°C treatments. Expression of heat shock protein (hsp) was positively modulated by increasing temperatures. Hsp 90, hsp90-2 and sit1 (related to silica shell formation) were highly expressed at 30°C under 1000µg/L Cu, while the genes encoding light harvesting proteins (3HfcpA and 3HfcpB) and silaffin precursor sil3 were significantly up-regulated at 15°C under 200µg/L Cu. Our results indicated an increase Cu toxicity to T. pseudonana under high temperature and Cu dose.

Molecular phylogeny and toxicity of harmful benthic dinoflagellates Coolia (Ostreopsidaceae, Dinophyceae) in a sub-tropical marine ecosystem: The first record from Hong Kong.

Coolia are marine benthic dinoflagellates which are globally distributed and potentially toxic. This study provides the first investigation of species diversity and toxicity assessment of Coolia in Hong Kong waters. Fifty-one strains of four Coolia species, including C. malayensis, C. canariensis, C. tropicalis, and C. palmyrensis, were isolated from twelve sub-tidal habitats, and identified phylogenetically using 28S rDNA sequences. Exposure experiments (48-hour) demonstrated that the algal lysates extracted from the four Coolia species exhibited different toxic effects on the lethality and abnormality of two invertebrate larvae, i.e., brine shrimp Artemia franciscana and sea urchin Heliocidaris crassispina. Heliocidaris crassispina was more sensitive to the toxic effects of Coolia species than A. franciscana. Toxicity tests from both larvae revealed that C. malayensis was generally more toxic, and caused higher mortality rates when compared with the other three species. The emerging threat of harmful benthic dinoflagellates to marine environments and sensitive biota is discussed.

De novo transcriptome assembly of the marine gastropod Reishia clavigera for supporting toxic mechanism studies.

The intertidal whelk Reishia clavigera is commonly used as a biomonitor of chemical contamination in the marine environment along Western Pacific region, and as a model for mechanistic studies of organotin-mediated imposex development. However, limited genomic resources of R. clavigera have restricted its role for the investigation of molecular mechanisms of such endocrine disruptions. This study, therefore, aimed to establish tissue-specific transcriptomes of the digestive gland, gonad, head ganglia, penis and the remaining body part of the male and female R. clavigera. By combining the results, a global transcriptome was obtained. A total of 578,134,720 high-quality filtered reads were obtained using Illumina sequencing. The R. clavigera transcriptome comprised of 38,466 transcripts and 32,798 unigenes with predicted open reading frames. The average length of transcripts was 1,709bp with N50 of 2,236bp. Based on sequence similarity searches against public databases, 28,657 transcripts and 24,403 unigenes had at least one BLAST hit. There were 17,530 transcripts and 14,897 unigenes annotated with at least one Gene Ontology (GO) term. Moreover, 5,776 transcripts and 5,137 unigenes were associated with 333 Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathways. The numbers of unigenes were similar among the five target tissues and between sexes, but tissue-specific expression profiles were revealed by multivariate analyses. Based on the functional annotation, putative steroid hormone-associated unigenes were identified. In particular, we highlighted the presence of steroid hormone receptor homologues that could be the targets for mechanistic studies of the organotin-mediated imposex development in marine gastropods. This newly generated transcriptome assembly of R. clavigera provides a valuable molecular resource for ecotoxicological and environmental genomic studies.

De novo transcriptomic profile in the gonadal tissues of the intertidal whelk Reishia clavigera.

The intertidal whelk Reishia clavigera (formerly named as Thais clavigera) is one of the most sensitive species to organotin-associated imposex. However, the limited information on mRNA transcriptome of the species has restricted the molecular investigation on such endocrine disruption. By means of Illumina sequencing, we obtained a global de novo transcriptome from the gonadal tissues of both male and female R. clavigera, with 197,324 assembled transcripts and 151,684 condensed non-redundant transcripts. Blast hit results from the NCBI's non-redundant molluscan database showed that 28,948 transcripts were successfully annotated with significant matches at an e-value of ⩽1e(-6). Among them, 1108 transcripts were assigned a well-defined gene ontology term. As the first transcriptomic study on the gonadal tissues of R. clavigera, this study has enhanced the information of mRNA transcriptome on this species and will thus facilitate mechanistic studies of chemical contaminants (e.g., organotins) on this common biomonitor species.