Spatial variation in adult sex ratio across multiple scales in the invasive golden apple snail, Pomacea canaliculata.

Adult sex ratio (ASR) has critical effects on behavior and life history and has implications for population demography, including the invasiveness of introduced species. ASR exhibits immense variation in nature, yet the scale dependence of this variation is rarely analyzed. In this study, using the generalized multilevel models, we investigated the variation in ASR across multiple nested spatial scales and analyzed the underlying causes for an invasive species, the golden apple snail Pomacea canaliculata. We partitioned the variance in ASR to describe the variations at different scales and then included the explanatory variables at the individual and group levels to analyze the potential causes driving the variation in ASR. We firstly determined there is a significant female-biased ASR for this species when accounting for the spatial and temporal autocorrelations of sampling. We found that, counter to nearly equal distributed variation at plot, habitat and region levels, ASR showed little variation at the town level. Temperature and precipitation at the region level were significantly positively associated with ASR, whereas the individual weight, the density characteristic, and sampling time were not significant factors influencing ASR. Our study suggests that offspring sex ratio of this species may shape the general pattern of ASR in the population level while the environmental variables at the region level translate the unbiased offspring sex ratio to the female-biased ASR. Future research should consider the implications of climate warming on the female-biased ASR of this invasive species and thus on invasion pattern.

The Asian arowana (Scleropages formosus) genome provides new insights into the evolution of an early lineage of teleosts.

The Asian arowana (Scleropages formosus), one of the world's most expensive cultivated ornamental fishes, is an endangered species. It represents an ancient lineage of teleosts: the Osteoglossomorpha. Here, we provide a high-quality chromosome-level reference genome of a female golden-variety arowana using a combination of deep shotgun sequencing and high-resolution linkage mapping. In addition, we have also generated two draft genome assemblies for the red and green varieties. Phylogenomic analysis supports a sister group relationship between Osteoglossomorpha (bonytongues) and Elopomorpha (eels and relatives), with the two clades together forming a sister group of Clupeocephala which includes all the remaining teleosts. The arowana genome retains the full complement of eight Hox clusters unlike the African butterfly fish (Pantodon buchholzi), another bonytongue fish, which possess only five Hox clusters. Differential gene expression among three varieties provides insights into the genetic basis of colour variation. A potential heterogametic sex chromosome is identified in the female arowana karyotype, suggesting that the sex is determined by a ZW/ZZ sex chromosomal system. The high-quality reference genome of the golden arowana and the draft assemblies of the red and green varieties are valuable resources for understanding the biology, adaptation and behaviour of Asian arowanas.

Comparative Functional Responses Predict the Invasiveness and Ecological Impacts of Alien Herbivorous Snails.

Understanding determinants of the invasiveness and ecological impacts of alien species is amongst the most sought-after and urgent research questions in ecology. Several studies have shown the value of comparing the functional responses (FRs) of alien and native predators towards native prey, however, the technique is under-explored with herbivorous alien species and as a predictor of invasiveness as distinct from ecological impact. Here, in China, we conducted a mesocosm experiment to compare the FRs among three herbivorous snail species: the golden apple snail, Pomacea canaliculata, a highly invasive and high impact alien listed in "100 of the World's Worst Invasive Alien Species"; Planorbarius corneus, a non-invasive, low impact alien; and the Chinese native snail, Bellamya aeruginosa, when feeding on four locally occurring plant species. Further, by using a numerical response equation, we modelled the population dynamics of the snail consumers. For standard FR parameters, we found that the invasive and damaging alien snail had the highest "attack rates" a, shortest "handling times" h and also the highest estimated maximum feeding rates, 1/hT, whereas the native species had the lowest attack rates, longest handling times and lowest maximum feeding rates. The non-invasive, low impact alien species had consistently intermediate FR parameters. The invasive alien species had higher population growth potential than the native snail species, whilst that of the non-invasive alien species was intermediate. Thus, while the comparative FR approach has been proposed as a reliable method for predicting the ecological impacts of invasive predators, our results further suggest that comparative FRs could extend to predict the invasiveness and ecological impacts of alien herbivores and should be explored in other taxa and trophic groups to determine the general utility of the approach.

Complete mitochondrial genome of the Red devil cichlid (Amphilophus citrinellus).

In this study, the complete mitochondrial genome sequence of Amphilophus citrinellus was firstly sequenced and determined. The total genome is 16,522 bp in length with an A + T content of 54.19%, and contained 13 protein-coding genes, 22 tRNA genes, 2 ribosomal RNA genes and two main non-coding regions. The gene composition and order is similar to that of most other vertebrates, as is base composition and codon usage. These data will provide useful molecular information for phylogenetic relationships within the family Cichlidae species.

Complete mitochondrial genome of Zebra tilapia, Tilapia buttikoferi.

We determined the complete mitochondrial genome of Tilapia buttikoferi, which was 16,577 bp in length with an A + T content of 53.0%, containing 13 protein-coding genes, 2 rRNAs, 22 tRNAs and a complete control region. The gene arrangement was similar to that of typical fishes. The total base composition of the mitogenome was 25.6% T, 30.8% C, 27.4% A and 16.2% G. Of the 13 protein-coding genes, 12 genes start with an ATG codon, except for COX1 with GTG. Seven (ND1, ND2, COX1, ATPase8, ATPase6, ND4L and ND6) used TAA or AGA as the termination codon, whereas six (COX2, COX3, ND3, ND4, ND5 and cyt b) had incomplete stop codon T. Its control region was atypical in being short at 861 bp, and contained TACAT motif and one microsatellite-like region (TA)7. This mitogenome sequence data may be useful for phylogenetic and systematic analyses within the family Cichlaidae.

An unusual mitochondrial genome structure of the tonguefish, Cynoglossus trigrammus: Control region translocation and a long additional non-coding region inversion.

Flatfishes (Pleuronectiformes) exhibit different types of large-scale gene rearrangements. In the present study, the mitochondrial (mt) genome (18,369bp) of a tonguefish, Cynoglossus trigrammus, was determined using de novo mitochondrion genome sequencing. Compared with other flatfishes, the mt genome of C. trigrammus revealed distinct mitogenome architectures that primarily included two striking findings: 1) insertion of an additional long non-coding region (1647bp) making it the second largest genome length among Pleuronectiformes and 2) the translocation of the control region. The reconstructed phylogenetic tree based on 13 mt protein-coding gene sequences recovered the monophyletic suborder Pleuronectoidei and the family Cynoglossidae. These data provide useful information for a better understanding of the mitogenomic diversities and evolution in fish as well as novel genetic markers for studying population genetics and species identification.

Transcriptome analysis between invasive Pomacea canaliculata and indigenous Cipangopaludina cahayensis reveals genomic divergence and diagnostic microsatellite/SSR markers.

BACKGROUND: Pomacea canaliculata is an important invasive species worldwide. However, little is known about the molecular mechanisms behind species displacement, adaptational abilities, and pesticide resistance, partly because of the lack of genomic information that is available for this species. Here, the transcriptome sequences for the invasive golden apple snail P. canaliculata and the native mudsnail Cipangopaludina cahayensis were obtained by next-generation-sequencing and used to compare genomic divergence and identify molecular markers. RESULTS: More than 46 million high quality sequencing reads were generated from P. canaliculata and C. cahayensis using Illumina paired-end sequencing technology. Our analysis indicated that 11,312 unigenes from P. canaliculata and C. cahayensis showed significant similarities to known proteins families, among which a total of 4,320 specific protein families were identified. KEGG pathway enrichment was analyzed for the unique unigenes with 17 pathways (p-value < 10(-5)) in P. canaliculata relating predominantly to lysosomes and vitamin digestion and absorption, and with 12 identified in C. cahayensis, including cancer and toxoplasmosis pathways, respectively. Our analysis also indicated that the comparatively high number of P450 genes in the P. canaliculata transcriptome may be associated with the pesticide resistance in this species. Additionally, 16,717 simple sequence repeats derived from expressed sequence tags (EST-SSRs) were identified from the 14,722 unigenes in P. canaliculata and 100 of them were examined by PCR, revealing a species-specific molecular marker that could distinguish between the morphologically similar P. canaliculata and C. cahayensis snails. CONCLUSIONS: Here, we present the genomic resources of P. canaliculata and C. cahayensis. Differentially expressed genes in the transcriptome of P. canaliculata compared with C. cahayensis corresponded to critical metabolic pathways, and genes specifically related to environmental stress response were detected. The CYP4 family of P450 cytochromes that may be important factors in pesticide metabolism in P. canaliculata was identified. Overall, these findings will provide valuable genetic data for the further characterization of the molecular mechanisms that support the invasive and adaptive abilities of P. canaliculata.

Characterization of the Macropodus opercularis complete mitochondrial genome and family Channidae taxonomy using Illumina-based de novo transcriptome sequencing.

In this study, the complete mitochondrial genome of Macropodus opercularis was sequenced using Illumina-based de novo transcriptome technology and annotated using bioinformatic tools. The circular mitochondrial genome was 16,496bp in length and contained two ribosomal RNAs, 13 protein-coding genes, 22 transfer RNA genes, and the control region. The gene composition and order were similar to suborder Anabantoidei. Phylogenetic analyses using concatenated amino acid and nucleotide sequences of the 13 protein-coding genes with two different methods (Neighbor-joining and Bayesian analysis) both highly supported the close relationship of M. opercularis to M. ocellatus, consistent with previous classifications based on morphological and molecular studies. Furthermore, family Channidae and Parachanna insignis were clustered in the same clade. Our results supported the inclusion of family Channidae in suborder Channoidei. The complete mitochondrial genome of M. opercularis will provide genetic markers for better understanding species identification, population genetics and phylogeographics of freshwater fishes.

Complete mitochondrial genome of northern spotted barramundi, Scleropages jardinii.

We sequenced the complete mitogenome of northern spotted barramundi Scleropages jardinii, an ancestral bonytongue with economic and conservation value. The mitogenome is 16,670 bp in length with an A + T content of 52.9%, and contains 13 protein-coding genes, 2rRNAs, 22 tRNAs and a control region. The gene order and arrangement is similar to that of other Osteoglossidae species, as is base composition and codon usage. These data will provide useful molecular information for phylogenetic relationships within the family Osteoglossidae species.

Complete mitochondrial genome of the ocellate river stingray (Potamotrygon motoro).

We determined the first complete mitochondrial genome sequence of Potamotrygon motoro from South American freshwater stingrays. The total length of P. motoro mitogenome is 17,448 bp, which consists of 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes and a control region, with the genome organization and gene order being identical to that of the typical vertebrate. The overall nucleotide composition is 32.3% A, 24.4% T, 30.5% C and 12.8% G. These data will provide useful molecular information for phylogenetic relationships within the family Potamotrygonidae species.

Molecular characteristics of the HSP70 gene and its differential expression in female and male golden apple snails (Pomacea canaliculata) under temperature stimulation.

Heat-shock protein 70 (HSP70) is one of the most important heat-shock proteins that helps organisms to modulate stress response via over-expression. The HSP70 gene from Pomacea canaliculata was cloned using the RACE approach; the gene is 2,767 bp in length and contains an open reading frame of 1,932 bp, which is encoded by a polypeptide of 643 amino acids. BLAST analysis showed that the predicted amino acid sequence of the P. canaliculata HSP70 gene shared a relatively high similarity with that of other known eukaryotic species that display conserved HSP characteristics. The phylogeny demonstrated a separate clustering of the apple snail HSP70 with other constitutive members from other mollusk species. Quantitative real-time RT-PCR was used to detect the differential expression of HSP70 in both sexes of P. canaliculata at different temperature conditions. These results showed that HSP70 transcript levels decreased slightly under cold shock and increased significantly under heat-shock conditions in both sexes compared to normal temperatures (26 °C). Under cold-shock treatment, the sex effect was not significant. With heat treatment, HSP70 expression could be induced at 36 °C in both females and males, and it peaked at 42 and 39 °C in females and males, respectively. In addition, a clear time-dependent HSP70 expression pattern of the apple snail exposed to the same high temperature (36 °C) was observed at different time points. The maximal induction of HSP70 expression appeared at 12 and 48 h in males and females after heat shock, respectively. The maximal induction in females was significantly higher compared to males under heat stimulus. Taken together, these results strongly suggested that males were more susceptible to heat than females and provided useful molecular information for the ecological adaptability of P. canaliculata against extreme environmental stress.

Mitochondrial DNA as effective molecular markers for the genetic variation and phylogeny of the family Osteoglossidae.

The present study examined the genetic variation of the family Osteoglossidae from different geographical locations based on the mitochondrial NADH dehydrogenase subunit 2 (ND2) and ATPase subunit 6 (ATPase6) genes; we then re-constructed the phylogenetic relationships using the two sequences in combination. The results showed that the partial sequences of mitochondrial ND2 and ATPase6 of the family Osteoglossidae were 813 bp and 669 bp, respectively. A total of 42 species-specific nucleotide positions of the family Osteoglossidae were found to be useful for molecular identification. The sequence variation showed greater differences (8.3%~28.1% for the combined sequences, 8.3%~26.7% for the ND2 gene, and 9.3%~28.7% for the ATPase6 gene) among the different species of Osteoglossidae, and there was a significant association between the genetic difference and geographical location. Phylogenetic analyses using neighbor-joining, Bayesian inference, and maximum parsimony (MP) methods based on the combined sequences of the two genes were able to distinguish the different species and were in agreement with the existing taxonomy based on morphological characters and in association with the geographical distribution among seven species of the family Osteoglossidae.

Molecular structure of the largemouth bass (Micropterus salmoides) Myf5 gene and its effect on skeletal muscle growth.

Myogenic Regulatory Factors (MRFs), a family of basic helix-loop-helix (bHLH) transcription factors, play important roles in regulating skeletal muscle development and growth. Myf5, the primary factor of MRFs, initiates myogenesis. Its expression pattern during somitomyogenesis in some fish has been revealed. To further study its effect on fish muscle during postembryonic growth, characterization and function analysis of myf5 cDNA were carried out in largemouth bass. The 1,093 bp cDNA sequence was identified by RT-PCR and 3'RACE, then the ORF of Myf5 cDNA was cloned into the expression vector pcDNA3.1(-)/mycHisB. The recombinant plasmid pcDNA3.1(-)/mycHisB-Myf5 was injected into the dorsal muscle of tilapias. RT-PCR and histochemical results showed that the exogenous gene was transcribed and translated in vivo. Its effect on muscle growth focused on myofiber hypertrophy in white muscle 60 days post injection. This indicated that overexpression of Myf5 can promote myogenesis during the fish muscle postembryonic growth period.

Molecular cloning and expression of grass carp MyoD in yeast Pichia pastoris.

MyoD, expressed in skeletal muscle lineages of vertebrate embryo, is one of muscle-specific basic helix-loop-helix (bHLH) transcription factors, which plays a key role in the determination and differentiation of all skeletal muscle lineages. In this study, a cDNA of grass carp MyoD was cloned and characterized from total RNA of grass carp embryos by RT-PCR. The full-length cDNA of grass carp MyoD is 1597 bp. The cDNA sequence analysis reveals an open reading frame of 825 bp coding for a protein of 275 amino acids, which includes a bHLH domain composed of basic domain (1-84(th) amino acids) and HLH domain (98-142(th) amino acids), without signal peptide. Then the MyoD cDNA of grass carp was cloned to yeast expression vector pPICZalphaA and transformed into P. pastoris GS115 strain, the recombinant MyoD protein with a molecular weight of about 31KD was obtained after inducing for 2d with 0.5% methanol in pH 8.0 BMGY medium, and the maximum yield was about 250 mg/L in shaking-flask fermentation. The results were expected to benefit for further studies on the crystal structure and physiological function of fish MyoD.

Molecular cloning, sequencing, expression of Chinese sturgeon cystatin in yeast Pichia pastoris and its proteinase inhibitory activity.

Cystatin, a superfamily of cysteine proteinase inhibitor of cathepsins and other cysteine proteinases, is widely distributed in animal tissues and body fluids. Although considerable attention has been given to mammalian and avian cystatins, little is known about cystatins from other vertebrates. In this study, a cDNA coding for Chinese sturgeon (Acipenser sinensis) cystatin was isolated and characterized. The corresponding mature cystatin peptide cDNA is 336 nucleotides long and encodes a protein of 112 amino acids. Sequence comparison showed that the cloned cystatin was a homolog of the mammalian Family II cystatin. The cystatin cDNA of Chinese sturgeon was subcloned into yeast expression vector pPICZalphaA and transformed into Pichia pastoris GS115 strain. After methanol induction, SDS-PAGE analysis of the culture supernatant indicated that the yield of recombinant cystatin was about 215 mg/l medium supernatant in shaking-flask fermentation medium, accounting for 73.6% of the total supernatant secreted proteins. Our data also showed that the recombinant cystatin is active in inhibiting the protease activity of papain and cathepsin B. Heat stability of the recombinant cystatin was also measured.

[Expression of Chinese sturgeon cystatin in yeast Pichia pastoris and its proteinase inhibitory activity analysis].

Cystatin, which widely distributed in both tissues and body fluids of animal and plant, was a superfamily of cysteine proteinase inhibitors. It could form activity-inhibitor complexes with cysteine proteinases to inhibit the hydrolytic activity of proteinases. Cystatin played important roles not only in the inhibition of the proteolytic degradation of fish muscle, but also in biological defense systems against invaders. To explore the functions of fish cystatin and the potential values in fish disease prevention and cure, as well as seafood processing, the recombinant yeast strains which could express Chinese sturgeon cystatin were constructed. First, the cystatin cDNA of Chinese sturgeon, which had been PCR modified, was subcloned into yeast integrated vector pPICZaA. After extracted and purified, the recombinant plasmids were linearized by Sac I. The yeast Pichia pastoris GS115 strain was transformed by use of the Lithium Chloride transformation method, and the recombinant cystatin yeast strains got. After 0.5% methanol induction, SDS-PAGE analysis of the culture supernatant indicated that the yield of recombinant cystatin was about 215mg x L(-1) with the percentage about 73.6%. The recombinant cystatin was purified through Q-Sepharose anion-exchange chromatography, and the purity reached about 94.2%. The inhibitory activity of recombinant cystatin was measured by inhibiting the proteinase activity of papain. The results showed that about 1 microg recombinant cystatin could inhibit the activity of 15 microg papain. Heat stability assay results showed that there was a decrease in inhibitory activity of cystatin with the increasing of temperature. When solution of recombinant cystatin was kept at 70 degrees C for 5min, the inhibitory activity reduced fast. While the recombinant cystatin was heated to 90 degrees C for 5min, the inhibitory activity of recombinant cystatin was undetected. The inhibitory activity for recombinant Chinese sturgeon cystatin was higher than that of CPI (cysteine proteinase inhibitor) from seeds of corn, that about 1 microg purified CIP could inhibited the activity of 0.278 microg papain. But the heat stability of recombinant cystatin is lower than that of the corn CPI. The expression level and the activity of recombinant cystatin from yeast Pichia pastoris were higher than those from E. coli. Moreover, recombinant cystatin from Pichia pastoris was easier to separate and purify. This paper reported that recombinant fish cystatin was produced in a highly efficient expression system based on the methylotrophic yeast, further work will focus on the function of recombinant Chinese sturgeon cystatin to resist fish disease and explore the value of cystatin as a food additive to inhibit cysteine proteinases during surimi processing.