Resolution adjustable Lissajous scanning with piezoelectric MEMS mirrors.

We previously designed a dual-axis piezoelectric MEMS mirror with a low crosstalk gimbal structure, which is utilized as the key device for further research for laser beam scanning. This paper mainly focuses on studying the Lissajous scanning resolution of this MEMS mirror with frequency ratio and phase modulation. For accurately evaluating the scanning resolution, the center angular resolution of Lissajous scanning is redefined by theoretical calculation and verified with experimental measurement. Meanwhile, the scanning nonlinearity of MEMS mirror is studied carefully. Finally, the MEMS mirror works at the state of pseudo-resonance, and the center angular resolution better than 0.16° (H) × 0.03° (V) is achieved at a scanning Field of view (FoV) of 35.0° (H) × 16.5° (V). Moreover, a feasible route of resolution adjustable Lissajous scanning is provided by optimization of frequency ratio and phase modulation, which is helpful for high definition and high frame rate (HDHF) laser scanning imaging with the dual-axis mirror.

Microfabricated sensor device for CW and pulsed laser power measurements.

On-line measurement is a trend of development toward laser-based applications. We present a fiber-integrated force sensor device for laser power measurement with both CW mode and pulse mode based on laser radiometric heat and radiation force sensing simultaneously. The sensor device is fabricated using a standard microfabrication process. Laser intensity is determined through the displacement of a movable mirror measured by an integrated Fabry-Perot interferometer. Compared with the performance of the device in the ambient condition, a non-linearity error of 0.02% and measurement uncertainty of 2.06% is observed in the quasi-vacuum condition for CW laser illumination. This device can measure a CW laser power with a 46.4 µW/Hz1/2 noise floor and a minimum detection limit of 0.125 mW. For a pulsed laser, a non-linearity error of 0.37% and measurement uncertainty of 2.08% is achieved with a noise floor of 1.3 µJ/Hz1/2 and a minimum detection limit of 3 µJ.

High-Q adiabatic micro-resonators on a wafer-scale ion-sliced 4H-silicon carbide-on-insulator platform.

A 4H-silicon carbide-on-insulator (4H-SiCOI) has emerged as a prominent material contender for integrated photonics owing to its outstanding material properties such as CMOS compatibility, high refractive index, and high second- and third-order nonlinearities. Although various micro-resonators have been realized on the 4H-SiCOI platform, enabling numerous applications including frequency conversion and electro-optical modulators, they may suffer from a challenge associated with spatial mode interactions, primarily due to the widespread use of multimode waveguides. We study the suppression of spatial mode interaction with Euler bends, and demonstrate micro-resonators with improved Q values above 1 × 105 on ion-sliced 4H-SiCOI platform with a SiC thickness nonuniformity less than 1%. The spatial-mode-interaction-free micro-resonators reported on the CMOS-compatible wafer-scale 4H-SiCOI platform would constitute an important ingredient for the envisaged large-scale integrated nonlinear photonic circuits.

Antibacterial activity study of a novel piscidin 5-like type 4 from Larimichthys crocea.

As one of the key components of innate immune system, piscidins are likely to play pivotal role in the first defense line in fish. Piscidins own multiple resistance activity. A novel piscidin 5-like type 4 was excavated from Larimichthys crocea (termed Lc-P5L4) liver transcriptome immuned by Cryptocaryon irritans, and upregulated at 7 days post infection when secondary bacterial infection occurred. In the study, we characterized the antibacterial activity of Lc-P5L4. The liquid growth inhibition assay detected the recombinant Lc-P5L4 (rLc-P5L) had potent antibacterial activity to Photobacterium damselae. Scanning electron microscope (SEM) observed the cell surface of P. damselae collapsed to form pit, and membrane of some bacteria ruptured after co-incubation with rLc-P5L. Further, transmission electron microscope (TEM) was also employed to observe the intracellular microstructural damage, rLc-P5L4 caused cytoplasm contraction, pores formation and contents leakage. After knowing about its antibacterial effects, the preliminary antibacterial mechanism was also explored, western blot analysis showed rLc-P5L4 could bind to P. damselae through targeting to LPS. Agarose gel eletrophoresis analysis further showed rLc-P5L4 could also penetrate into cells and brought about genome DNA degradation. Therefore, rLc-P5L4 was of potential being a candidate to explore new antimicrobial drug or additive agent, especially to P. damselae.

Comparative analysis of transcriptomic data shows the effects of multiple evolutionary selection processes on codon usage in Marsupenaeus japonicus and Marsupenaeus pulchricaudatus.

BACKGROUND: Kuruma shrimp, a major commercial shrimp species in the world, has two cryptic or sibling species, Marsupenaeus japonicus and Marsupenaeus pulchricaudatus. Codon usage analysis would contribute to our understanding of the genetic and evolutionary characteristics of the two Marsupenaeus species. In this study, we analyzed codon usage and related indices using coding sequences (CDSs) from RNA-seq data. RESULTS: Using CodonW 1.4.2 software, we performed the codon bias analysis of transcriptomes obtained from hepatopancreas tissues, which indicated weak codon bias. Almost all parameters had similar correlations for both species. The gene expression level (FPKM) was negatively correlated with A/T3s. We determined 12 and 14 optimal codons for M. japonicus and M. pulchricaudatus, respectively, and all optimal codons have a C/G-ending. The two Marsupenaeus species had different usage frequencies of codon pairs, which contributed to further analysis of transcriptional differences between them. Orthologous genes that underwent positive selection (ω > 1) had a higher correlation coefficient than that of experienced purifying selection (ω < 1). Parity Rule 2 (PR2) and effective number of codons (ENc) plot analysis showed that the codon usage patterns of both species were influenced by both mutations and selection. Moreover, the average observed ENc value was lower than the expected value for both species, suggesting that factors other than GC may play roles in these phenomena. The results of multispecies clustering based on codon preference were consistent with traditional classification. CONCLUSIONS: This study provides a relatively comprehensive understanding of the correlations among codon usage bias, gene expression, and selection pressures of CDSs for M. japonicus and M. pulchricaudatus. The genetic evolution was driven by mutations and selection pressure. Moreover, the results point out new insights into the specificities and evolutionary characteristics of the two Marsupenaeus species.

The contributions of fliG gene to the pathogenicity of Pseudomonas plecoglossicida and pathogen-host interactions with Epinephelus coioides.

Pseudomonas plecoglossicida is a Gram-negative aerobic rod-shaped bacterium with polar flagella. It is the causative agent of visceral white spot disease in cultured fish, resulting in serious economic losses. In our previous study, RNA sequencing showed that the expression of the fliG gene in P. plecoglossicida is significantly up-regulated during infection of orange-spotted grouper (Epinephelus coioides). In this study, four P. plecoglossicida RNA interference (RNAi) mutants were successfully constructed by linking four short hairpin RNAs (shRNAs), which target different sites of the fliG gene, to pCM130/tac, respectively. The mRNA expression levels of the fliG gene in P. plecoglossicida were significantly decreased in four mutants. The shRNA-335 mutant (fliG-RNAi strain) showed the best silencing efficiency (88.2%) and was thus chosen for further analysis. Electron microscopy indicated that the flagella of the fliG-RNAi strain of P. plecoglossicida were shorter and finer than those of the wild type strain. The fliG-RNAi strain also showed significantly decreased mobility, chemotaxis, adhesion, and biofilm formation. Furthermore, compared with wild type strain infection, E. coioides infected with the fliG-RNAi strain exhibited a 0.5-d delay in the time of first death and 55% reduction in accumulated mortality, as well as milder splenic symptoms. RNAi of the fliG gene significantly affected the transcriptomes of both pathogen and host in the infected spleens of E. coioides. KEGG analysis revealed that the flagellar assembly pathway, bacterial chemotaxis pathway, and starch and sucrose metabolism pathway were significantly enriched in the pathogen at 3 days post infection (dpi). In contrast, the complement and coagulation cascade pathway and antigen processing and presentation pathway were significantly enriched in the host at 3 dpi. More immune-related pathways were enriched at 5 dpi and more differentially expressed genes were found in the complement and coagulation cascade and antigen processing and presentation pathways. Cytokine-cytokine receptor interaction, hematopoietic cell lineage, and IgA-producing intestinal immune network pathways were significantly enriched in the host at 5 dpi. These results indicate that fliG is an important virulence gene of P. plecoglossicida and contributes to the pathogenicity of P. plecoglossicida as well as pathogen-host interactions with E. coioides.

Time-resolved dual RNA-seq of tissue uncovers Pseudomonas plecoglossicida key virulence genes in host-pathogen interaction with Epinephelus coioides.

Bacterial pathogen-host interactions are highly dynamic, regulated processes that have been primarily investigated using in vitro assays. The dynamics of bacterial pathogen-host interplay in vivo are poorly understood. Using time-resolved dual RNA-seq in a Pseudomonas plecoglossicida-Epinephelus coioides infection model, we observed that bacterial genes encoding classical virulence factors and host genes involved in immune regulation were dynamically expressed during infection. Using network inferencing, we were able to predict interspecies regulatory networks linking bacterial virulence genes to host immune genes. Together with gene co-expression network analysis of the pathogen, secY was predicted to be a key virulence gene for P. plecoglossicida pathogenicity in the host, fliN was predicted to be a less important virulence gene. The results of bioinformatics prediction were confirmed by animal infection experiments. Our work provides the first paradigm to study dynamic alterations of bacterial pathogen and host interactions based on the elucidation of time-resolved interactive transcriptomes in vivo, and may be developed into a novel and universal method for revealing the true complexity of the bacterial infection process.

Identification and functional characterization of a novel C-type lectin from the kuruma shrimp, Marsupenaeus japonicus.

C-type lectins (CTLs) are immune molecules that are crucial to the invertebrate innate immune system with the primary function of recognizing invading pathogens. In the present study, a novel CTL was cloned from Marsupenaeus japonicus (MjCTL), and its tissue distribution and expression patterns over time in response to white spot syndrome virus (WSSV) and Vibrio parahaemolyticus were further investigated. The open reading frame (ORF) of MjCTL was 513 bp and encoded a polypeptide of 170 amino acids, which contained a signal peptide and a carbohydrate recognition domain (CRD) that are typical for CTLs. MjCTL was primarily expressed in the hepatopancreas and weakly expressed in hemocytes, gill, stomach, intestine, heart, muscle and eyestalk. The expression level of MjCTL in the hepatopancreas was dramatically increased at 48 h post-injection with WSSV at a dosage of 1 × 105 virions. Glutathione-S-transferase (GST) pull-down assays showed that MjCTL could directly bind to several WSSV envelope proteins, including VP19, VP24, VP26 and VP28. Moreover, MjCTL displayed antibacterial activity against V. parahaemolyticus. Our results indicated that MjCTL exhibited multiple functions in innate immune response against pathogens.

Full-length transcriptome analysis provides new insights into the innate immune system of Marsupenaeus japonicus.

As invertebrates, shrimp are generally thought to solely rely on their innate immune system to combat invading pathogens. Recently, an increasing number of studies have revealed that the innate immune response of invertebrates exhibits diversity and specificity based on their diverse immune molecules. Herein, a full-length transcriptome analysis of several immune-related tissues (hepatopancreas, gill, hemocytes, stomach and intestine) in the kuruma shrimp (Marsupenaeus japonicus) was conducted to identify immune-related molecules with a focus on transcript variations. In total, 11,222 nonredundant full-length transcripts with an N50 length of 5174 were obtained, and most of these transcripts (94.84%) were successfully annotated. In addition, a total of 147 long noncoding RNAs (lncRNAs) were also predicted. Importantly, transcript variants of several vital immune-related genes were observed, including twenty-five alpha-2-macroglobulins (α2-Ms), ten Toll-like receptors (TLRs), six C-type lectins (CTLs), five M-type lectins (MTLs) and three Down syndrome cell adhesion molecules (Dscams). Furthermore, 509 nonredundant full-length transcripts were predicted to be generated from alternative splicing (AS) events, which contribute to the diversity of immune molecules. Overall, our study provides valuable data on the full-length transcripts of M. japonicus, which will facilitate the exploration of immune molecules in this species. Moreover, numerous transcript variants of immune molecules detected in this study provide clues for further investigating the diversity and specificity of the innate immune response in shrimp.

Evolutionary adaptation analysis of immune defense and hypoxia tolerance in two closely related Marsupenaeus species based on comparative transcriptomics.

Kuruma shrimp, a major farmed shrimp species in the world, includes two cryptic or sibling species, Form I (Marsupenaeus japonicus) and Form II (Marsupenaeus pulchricaudatus). Due to the lack of genomic resources, little is known about the molecular mechanisms associated with immune defense and hypoxia tolerance. Here, we sequenced the transcriptomes of two closely related Marsupenaeus species and compared genomic divergence. This study obtained 77049 and 84561 unigenes with N50 values of 1281bp and 1244bp for M. japonicus and M. pulchricaudatus, respectively, and 5036 pairs of putative orthologs were identified between two Marsupenaeus species. Estimation of Ka/Ks ratios indicated that 165 orthologous genes may be under positive selection (Ka/Ks > 0.5), including 49 pairs with a Ka/Ks ratio >1. According to the peak of synonymous rates, the divergence time between M. japonicus and M. pulchricaudatus was about 0.26-0.69 Mya. These positively selected orthologous genes related to the immune process mainly comprised single VWC domain protein, legumain, ras-related C3 botulinum, caspase, C-type lectin and were enriched in functions related to immune (Toll-like receptor and PI3K-Akt signaling) and hypoxia signaling (HIF-1 signaling and VEGF signaling). In this study, dozens of caspase-like unigenes were screened from two Marsupenaeus transcriptomes. Among these, the PjCaspase orthologous gene was subjected to positive selection (Ka/Ks = 1.22), which had different secondary and three-dimensional structure prediction. Based on the single copy caspase gene, eight populations of Marsupenaeus species were divided into two phylogeographic lineages from the East and South China. We characterized the transcriptomes of the two Marsupenaeus species and obtained several key orthologs associated with immune defense and hypoxia tolerance, which provides new insights into the immunity and genetic divergence of the two varieties. Moreover, this study will facilitate further comparative genomic studies of the two varieties.

Dual RNA-Seq uncovers the function of an ABC transporter gene in the host-pathogen interaction between Epinephelus coioides and Pseudomonas plecoglossicida.

As an important pathogen in aquaculture, Pseudomonas plecoglossicida has caused heavy losses. The expression of an ABC transporter gene-L321_23611 of P. plecoglossicida at 18 °C was found significant higher than those at 28 °C by RNA-seq and qRT-PCR. RNAi significantly reduced the content of L321_23611 mRNA in P. plecoglossicida with a maximal decrease of 89.2%. Compared with the wild type strain, the infection of L321_23611-RNAi strain resulted in the reduction in mortality and the onset time delay of a kind of marine teleosts, Epinephelus coioides. The results of dual RNA-seq showed that the RNAi of L321_23611 resulted in a significant change in both pathogen and host transcriptome in the spleens of infected E. coioides. The result of GO and KEGG analysis from dual RNA-seq data showed both host genes of chemokine signaling pathway, coagulation and complement system, hematopoietic cell lineage pathway as well as hemoglobin complex GO term and pathogenic genes of bacterial-type flagellum-dependent cell mortality GO term and flagellar assembly, biosynthesis of amino acids and lysine biosynthesis systems pathways were mainly affected by L321_23611 gene of P. plecoglossicida. The results indicated that: 1. ABC transporter gene-L321_23611 was a virulent gene of P. plecoglossicida. 2. Both the activation of the host immune pathways and depression of pathogenic virulence-related pathways facilitated E. coioides to remove L321_23611-RNAi strain than the wild type strain of P. plecoglossicida.

Mechanistic insight into the roles of Pseudomonas plecoglossicida clpV gene in host-pathogen interactions with Larimichthys crocea by dual RNA-seq.

Large yellow croaker (Larimichthys crocea) is an economical important farmed fish in China. "Visceral White Spot Disease" caused by Pseudomonas plecoglossicida is a disease with a high mortality rate in cage-cultured L. crocea in recent years and resulted in heavy economy lossess. The dual RNA-seq results of previous study showed that the expression of clpV gene in P. plecoglossicida was significantly up-regulated during infection. RNAi significantly reduced the expression of clpV in P. plecoglossicida with maximum silencing efficiency of 96.1%. Compared with the wild type strain, infection of clpV-RNAi strain resulted in a delayed onset time and a 25% reduction in mortality of L. crocea, as well as lessening the symptoms of the spleen. The results of dual RNA-seq of L. crocea infected by clpV-RNAi strain of P. plecoglossicida changed considerably, compared with the counterpart infected with the wild strain. The KEGG enrichment analysis showed that Cytokine-cytokine receptor interaction, Toll-like receptor signaling pathway, C-type lectin receptor signaling pathway and MAPK signaling pathway of L. crocea were most affected by the silence of clpV in P. plecoglossicida. RNAi of clpV resulted in the downregulation of genes in flagella assembly pathway and a weaker immune response of host.

The effect of a LysR-type transcriptional regulator gene of Pseudomonas plecoglossicida on the immune responses of Epinephelus coioides.

As an important pathogen in aquaculture, Pseudomonas plecoglossicida has caused heavy losses. It was determined with RNA-seq that the expression of a LysR-type transcriptional regulator gene (L321_20267) of P. plecoglossicida at 18 °C was significantly higher than that at 28 °C, which was verified by quantitative real-time PCR (qRT-PCR). RNAi significantly reduced the content of L321_20267 mRNA in P. plecoglossicida, with a maximal decrease of 90.63%. Compared with the wild-type strain, infection with the L321_20267-RNAi strain resulted in a 50% reduction in mortality and an onset time delay of Epinephelus coioides, as well as alleviation of the symptoms in E. coioides spleens. Compared with the wild-type strain of P. plecoglossicida, the L321_20267-RNAi strain resulted in a significant change in the spleen transcriptome of infected E. coioides. The results of GO and KEGG analysis showed that genes of serine hydrolase activity, the antigen processing and presentation pathway, the B cell receptor signalling pathway and the chemokine signalling pathway were most affected by the L321_20267 gene of P. plecoglossicida. Meanwhile, the immune genes were related to different numbers of miRNAs and lncRNAs, and some miRNAs were related to more than one gene. The results indicated that 1. L321_20267 is a virulence gene of P. plecoglossicida; 2. the upregulation of the immune pathways facilitated E. coioides to remove the L321_20267-RNAi strain compared with the wild-type strain of P. plecoglossicida; and 3. the immune genes were regulated by miRNA and lncRNA in a complex manner.

Integrated dual RNA-seq and dual iTRAQ of infected tissue reveals the functions of a diguanylate cyclase gene of Pseudomonas plecoglossicida in host-pathogen interactions with Epinephelus coioides.

The interactions between host and pathogen is exceedingly complex, which involves alterations at multiple molecular layers. However, research to simultaneously monitor the alterations of transcriptome and proteome between a bacterial pathogen and aquatic animal host through integrated dual RNA-seq and dual iTRAQ of tissue during infection is currently lacking. The important role of a diguanylate cyclase gene (L321_RS15240) in pathogenicity of Pseudomonas plecoglossicida against Epinephelus coioides was suggested by previous dual RNA-seq of our lab. Then L321_RS15240-RNAi strains of P. plecoglossicida were constructed with pCM130/tac, and the mutant with the best silencing effect was selected for follow-up study. The RNAi of L321_RS15240 resulted in a significant decrease in bacterial virulence of P. plecoglossicida. The E. coioides spleens infected by wild type strain or L321_RS15240-RNAi strain of P. plecoglossicida were subjected to dual RNA-seq and dual iTRAQ, respectively. The results showed that: RNAi of L321_RS15240 led to 1)alterations of host transcriptome associated with complement and coagulation cascades, ribosome, arginine and proline metabolism, and oxidative phosphorylation; 2)high expression of host proteins which related to phagosome and metabolism responses (metabolism of glutathione, amino sugar and nucleotide sugar); 3)the highly differentially expression of host lncRNAs and miRNAs. The differentially expressed proteins and mRNAs of pathogen were different after infection, but the functions of these proteins and mRNAs were mainly related to metabolism and virulence. This study provides a new insight to comprehensively understand the gene functions of pathogens and hosts at multiple molecular layers during in vivo infection.

Dual RNA-seq uncovers the immune response of Larimichthys crocea to the secY gene of Pseudomonas plecoglossicida from the perspective of host-pathogen interactions.

Pseudomonas plecoglossicida is a Gram-negative aerobic bacterium that causes high mortality and serious economic losses in some commercial marine fish. Expression of secY was found to be significantly upregulated at 18 °C compared to 28 °C by RNA-seq and qRT-PCR. All five tested recombinant vectors (pCM130/tac + shRNA) significantly reduced secY mRNA levels in P. plecoglossicida. The recombinant vector encoding shRNA-1165 exhibited the best gene-silencing efficiency, 82.4% and was used to create an RNAi strain for further studies. Compared with the wildtype strain, infections of Larimichthys crocea with the RNAi strain resulted in a 2-day delay in onset time and a 35% reduction in mortality, as well as the alleviation of spleen symptoms. The spleens of L. crocea infected by the wild type or RNAi strain of P. plecoglossicida were subjected to dual RNA-seq at 2 dpi. Compared with the wildtype strain, infection of P. plecoglossicida with the RNAi strain resulted in significant changes in the transcriptomes of both host and pathogen. KEGG analysis showed that the complement and coagulation cascade and the Toll-like receptor signalling pathway were the most enriched host pathways. In the pathogen, genes of the "Sec secretion system" were significantly downregulated. This downregulation of "Sec secretion system" genes hindered the secretion of bacterial proteins and reduced the virulence of P. plecoglossicida. Thus, it was easier for L. crocea to clear the RNAi strain of P. plecoglossicida, and the immune response was similarly reduced. The results indicated that secY was a virulence gene of P. plecoglossicida and played roles in the host-pathogen interactions of L. crocea and P. plecoglossicida.

The role of rpoS in the regulation of Vibrio alginolyticus virulence and the response to diverse stresses.

Vibrio alginolyticus is a leading aquatic pathogen, causing huge losses to aquaculture. rpoS has been proven to play a variety of important roles in stress response and virulence in several bacteria. In our previous study, upon treatment with Cu2+ , Pb2+ , Hg2+ and low pH, the expression levels of rpoS were downregulated as assessed by RNA-seq, while impaired adhesion ability was observed, indicating that rpoS might play roles in the regulation of adhesion. In the present study, the RNAi technology was used to knockdown rpoS in V. alginolyticus. In comparison with wild-type V. alginolyticus, RNAi-treated bacteria showed significantly impaired abilities of adhesion, growth, haemolytic, biofilm production, movement and virulence. Meanwhile, alterations of temperature, salinity, pH and starvation starkly affected rpoS expression. The present data suggested that rpoS is a critical regulator of virulence in V. alginolyticus; in addition, rpoS regulates bacterial adhesion in response to temperature, pH and nutrient content changes. These are helpful to explore its pathogenic mechanism and provide reference for disease control.

clpV is a key virulence gene during in vivo Pseudomonas plecoglossicida infection.

Interaction between bacterial pathogen and aquatic animal host is exceedingly complex, which involves large dynamic changes in gene expression during different stages of the disease. However, research on identifying key virulence genes based on the dynamics of gene expression changes of a one-sided bacterial pathogen in tissue has not been reported so far across different stages of infectious disease. The clpV for the T6SS of Pseudomonas plecoglossicida was identified for a candidate for key virulence gene based on dynamic changes of gene expression. For the Epinephelus coioides infected using clpV-RNAi strain, no deaths were observed up to 20 dpi. The spleens, kidneys and livers of all the E. coioides that received clpV-RNAi strain failed to develop visible nodules at 5-8 dpi, with the swelling gradually disappearing. The burdens of clpV-RNAi strain in the spleen and blood were greatly reduced at most of the time points after injection, and the burdens of clpV-RNAi strain in the head kidneys and trunk kidneys also had a sharp reduction from 72 to 120 hpi. This paper provides a new insight into the discovery of key virulence genes of pathogens in infected tissue systems.

A metabolomic investigation into the temperature-dependent virulence of Pseudomonas plecoglossicida from large yellow croaker (Pseudosciaena crocea).

Pseudomonas plecoglossicida is associated with multiple fish diseases, and temperature is one of the most important environmental factors related to its outbreak. To elucidate the influence of temperature variation on the pathogen, the global metabolomics of P. plecoglossicida (NZBD9) were analysed at the virulent (18°C) and avirulent (12°C and 28°C) temperatures. The result showed that the levels of Phosphoric acid, Tyrosine, Spermidine and Sucrose were significantly reduced，while Itaconic acid, Glucaric acid and Isomaltose were increased in P. plecoglossicida at 18°C. These metabolic adjustments assist P. plecoglossicida to survive in adverse environments, proliferate in the host, colonize and resist host immune clearance during the initial steps of infection. The results suggested that L321_03626 and L321_18122 genes played a key role in the regulation of these metabolic adaptions and thus regulated P. plecoglossicida virulence at virulent temperature, which was proved by further gene silencing and artificial infection. The present study, for the first time, determines the P. plecoglossicida metabolomic responses to temperature variation, which is helpful to explore its pathogenic mechanism and provides reference for disease control.

Molecular mechanisms of an antimicrobial peptide piscidin (Lc-pis) in a parasitic protozoan, Cryptocaryon irritans.

BACKGROUND: Cryptocaryon irritans is an obligate parasitic ciliate protozoan that can infect various commercially important mariculture fish species and cause high lethality and economic loss. Current methods of controlling this parasite with chemicals or antibiotics are widely considered to be environmentally harmful. Piscidins with broad spectrum antibacterial, antifungal and antiviral activities were found to have potent activity against C. irritans. Little, however, has been understood about the killing mechanisms of piscidins in parasites. RESULTS: In total, 57.12, 50.44, 55.86 and 47.87 million raw reads were generated from untreated theront and trophont, and piscidin (Lc-pis) treated theront and trophont libraries, respectively. After de novo assembly, 966,609 unigenes were generated with an average length of 420 bp: among these, 618,629 unigenes showed identity with sequences in one or more databases, with some showing to be significantly manipulated by Lc-pis treatment. The species classification showed that more than 25.8% unigenes from trophonts were homologous to the large yellow croaker (Larimichthys crocea) and less than 3.8% unigenes from theronts were matched. The homologous unigenes demonstrated that the tissue from host could exist in trophonts and might be transported to parasite via vesicular transports. Our analysis showed that regulatory transcripts were involved in vesicular trafficking. Among transcripts induced by Lc-pis, most genes up-regulated in treated and untreated theronts were involved in cell migration and apoptosis related pathways. Few transcripts were found to be down-regulated in treated and untreated trophonts related to cell structure and migration after treatment. CONCLUSIONS: This is the first transcriptome analysis of C. irritans exposed to Lc-pis, which enhanced the genomic resources and provided novel insights into molecular mechanisms of ciliates treated by cationic antimicrobial peptide. Our comprehensive transcriptome analysis can facilitate the identification of potential drug targets and vaccines candidates for controlling this devastating fish pathogen.

Identification of microRNAs with heat stress responsive and immune properties in Marsupenaeus japonicus based on next-generation sequencing and bioinformatics analysis: Essential regulators in the heat stress-host interactions.

Summer mortality syndrome is one of the most serious issue for Marsupenaeus japonicus aquaculture in China. Since it causes massive economic loss and threatens sustainability of M. japonicus aquaculture industry, thus, there is an urgent desire to reveal the heat stress-host interactions mechanisms that lead to mass mortalities of M. japonicus in hot summer months. MicroRNAs (miRNAs) are small noncoding RNAs that involved in regulation of diverse biological processes, including stress and immune response, and might serve as potential regulators in the heat stress-host interactions. In the present study, miRNAs with heat stress responsive and immune properties were identified and characterized in M. japonicus by small RNA sequencing and bioinformatics analysis. In total, 79 host miRNAs were identified, among which 15 miRNAs were differentially expressed in response to heat stress. Target genes prediction and function annotation revealed that a variety of host cellular processes, such as signal transduction, transcription, anti-stress response, ribosomal biogenesis, lipid metabolism, cytoskeleton, etc, were potentially subject to miRNA-mediated regulation in response to heat stress. Furthermore, a total of 30 host miRNAs that potentially involved in interaction with white spot syndrome virus (WSSV) were obtained via predicting and analyzing the target genes from WSSV. The results showed that a batch of WSSV genes that code for structural proteins and enzymes that are essential for WSSV infection and proliferation, such as envelope proteins, capsid proteins, immediate-early proteins, collagen-like protein, protein kinase, thymidylate synthetase, TATA-box bind protein, etc, were predicted to be targeted by host miRNAs. Several of the host miRNAs with predicted antiviral capacity were down-regulated under heat stress, indicating a repression of host miRNA-mediated antiviral immune response. This study highlighted the essential roles of host miRNAs in the heat stress-host interactions and provided valuable information for further investigation on the mechanism of miRNA-mediated heat stress and immune response of shrimp.