Water-Immersible MEMS Mirror with a Large Optical Aperture.

This paper presents a two-axis AlScN-based water-immersible MEMS mirror fabricated in an 8-inch MEMS process. Compared with other studies, this device has a larger optical aperture 10 mm in diameter. The resonant frequencies of the device are 1011 Hz in air and 342 Hz in water. The scanning angle reaches ±5° and ±2° at resonant frequencies in air and water, respectively. The cavitation phenomenon is observed when the device is operating in water, which leads the device to electrical failure. To address this issue, a device with reduced resonant frequencies-246 Hz and 152 Hz in air and water-is characterized, through which the bubbles can be effectively prohibited. This MEMS mirror could potentially be used in ultrasound and photoacoustic microscopy applications.

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.

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.

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.

AlScN Piezoelectric MEMS Mirrors with Large Field of View for LiDAR Application.

This paper presents AlScN piezoelectric two-axis MEMS mirrors with gimbal-less and gimbaled designs fabricated in a CMOS-compatible manner. Integrated piezoelectric sensors provided feedback signals of the actual mirror positions. The mirror with a diameter of 1.5 mm possessed adjustable optical tilt angles of up to 22.6° @ 30 V, with a high resonance frequency of about 8.2 kHz, while the 3 mm mirror reached 48.5° @ 41 V. The mirror with the gimbaled structure exhibited an excellent field of view and good mechanical decoupling. Additionally, a significant improvement in mirror scanning performance was observed in a vacuum (4 Pa), proving that the optical field of view was magnified by more than a factor of 10.

Process Optimization and Performance Evaluation of TSV Arrays for High Voltage Application.

In order to obtain high-quality through-silicon via (TSV) arrays for high voltage applications, we optimized the fabrication processes of the Si holes, evaluated the dielectric layers, carried out hole filling by Cu plating, and detected the final structure and electric properties of the TSVs. The Si through-hole array was fabricated in an 8-inch Si substrate as follows: First, a blind Si hole array was formed by the Si deep reactive etching (DRIE) technique using the Bosch process, but with the largest width of the top scallops reduced to 540 nm and the largest notch elimidiameternated by backside grinding, which also opens the bottom ends of the Si blind holes and forms 500-µm-deep Si through holes. Then, the sidewalls of the Si holes were further smoothed by a combination of thermal oxidation and wet etching of the thermal oxide. The insulating capability of the dielectric layers was evaluated prior to metal filling by using a test kit. The metal filling of the through holes was carried out by bottom-up Cu electroplating and followed by annealing at 300 °C for 1 h to release the electroplating stress and to prevent possible large metal thermal expansion in subsequent high-temperature processes. The TSV arrays with different hole diameters and spacing were detected: no visible defects or structure peeling was found by scanning electron microscopy (SEM) observations, and no detectable interdiffusion between Cu and the dielectric layers was detected by energy dispersive X-ray (EDX) analyses. Electric tests indicated that the leakage currents between two adjacent TSVs were as low as 6.80 × 10-10 A when a DC voltage was ramped up from 0 to 350 V, and 2.86 × 10-9 A after a DC voltage was kept at 100 V for 200 s.

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.

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.

Antibacterial activity of four recombinant carbohydrate recognition domain proteins identified from the kuruma shrimp Marsupenaeus japonicus.

The carbohydrate recognition domain (CRD) is the key component of C-type lectins (CTLs) with the capacity to recognize and eliminate invading pathogens. Herein, the recombinant proteins of four CRDs identified from the kuruma shrimp, Marsupenaeus japonicus, were produced and purified by an Escherichia coli expression system and affinity chromatography. Bacterial binding and antibacterial assays showed that the four CRDs displayed various bacterial binding and antibacterial activities against different bacteria. Among the four recombinant CRDs, His-CRD2-3 exhibited the broadest spectrum of bacterial binding and antibacterial activities against gram-negative bacteria (Vibrio parahaemolyticus, V. alginolyticus and V. harveyi) and gram-positive bacteria (Staphylococcus aureus and Micrococcus lysodeikticus). Moreover, the four recombinant CRDs showed different capacities to regulate the expression of several immune effector genes (MjCTL3, MjCTL4, MjCTL, Mjily and Mjsty), among which His-CRD2-3 displayed broader and stronger inductive effects on these immune effector genes. This study indicated that the four CRDs participated in immune defense by binding and killing bacteria and regulating the transcription of other immune effector genes. In addition, our results suggested that His-CRD2-3 might be a promising agent for the prevention and treatment of bacteriosis.

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.

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.

Dual RNA-seq provides novel insight into the roles of dksA from Pseudomonas plecoglossicida in pathogen-host interactions with large yellow croakers ( Larimichthys crocea).

Pseudomonas plecoglossicida is a rod-shaped, gram-negative bacterium with flagella. It causes visceral white spot disease and high mortality in Larimichthys crocea during culture, resulting in serious economic loss. Analysis of transcriptome and quantitative real-time polymerase chain reaction (PCR) data showed that dksA gene expression was significantly up-regulated after 48 h of infection with Epinephelus coioides (log 2FC=3.12, P<0.001). RNAi of five shRNAs significantly reduced the expression of dksA in P. plecoglossicida, and the optimal silencing efficiency was 96.23%. Compared with wild-type strains, the symptoms of visceral white spot disease in L. crocea infected with RNAi strains were reduced, with time of death delayed by 48 h and mortality reduced by 25%. The dksA silencing led to a substantial down-regulation in cellular component-, flagellum-, and ribosome assembly-related genes in P. plecoglossicida, and the significant up-regulation of fliC may be a way in which virulence is maintained in P. plecoglossicida. The GO and KEGG results showed that RNAi strain infection in L. crocea led to the down-regulation of inflammatory factor genes in immune-related pathways, which were associated with multiple immune response processes. Results also showed that dksA was a virulence gene in P. plecoglossicida. Compared with the wild-type strains, RNAi strain infection induced a weaker immune response in L. crocea.

Novel insights into host-pathogen interactions of large yellow croakers ( Larimichthys crocea) and pathogenic bacterium Pseudomonas plecoglossicida using time-resolved dual RNA-seq of infected spleens.

Host-pathogen interactions are highly complex, involving large dynamic changes in gene expression during infection. These interactions are fundamental to understanding anti-infection immunity of hosts, as well as the pathogenesis of pathogens. For bacterial pathogens interacting with animal hosts, time-resolved dual RNA-seq of infected tissue is difficult to perform due to low pathogen load in infected tissue. In this study, an acute infection model of Larimichthys crocea infected by Pseudomonas plecoglossicida was established. The spleens of infected fish exhibited typical symptoms, with a maximum bacterial load at two days post-injection (dpi). Time-resolved dual RNA-seq of infected spleens was successfully applied to study host-pathogen interactions between L. crocea and P. plecoglossicida. The spleens of infected L. crocea were subjected to dual RNA-seq, and transcriptome data were compared with those of noninfected spleens or in vitro cultured bacteria. Results showed that pathogen-host interactions were highly dynamically regulated, with corresponding fluctuations in host and pathogen transcriptomes during infection. The expression levels of many immunogenes involved in cytokine-cytokine receptor, Toll-like receptor signaling, and other immune-related pathways were significantly up-regulated during the infection period. Furthermore, metabolic processes and the use of oxygen in L. crocea were strongly affected by P. plecoglossicida infection. The WGCNA results showed that the metabolic process was strongly related to the entire immune process. For P. plecoglossicida, the expression levels of motility-related genes and flagellum assembly-related genes were significantly up-regulated. The results of this study may help to elucidate the interactions between L. crocea and P. plecoglossicida.

Air Exposure Affects Physiological Responses, Innate Immunity, Apoptosis and DNA Methylation of Kuruma Shrimp, Marsupenaeus japonicus.

Air exposure stress is a common phenomenon for commercial crustacean species in aquaculture and during waterless transportation. However, the antioxidant responses to air exposure discussed in previous studies may be insufficient to present the complexities involved in this process. The comprehensive immune responses, especially considering the immune genes, cell apoptosis, and epigenetic changes, are still unknown. Accordingly, we investigated the multifaceted responses of Marsupenaeus japonicus to air exposure. The results showed that the expression profiles of the apoptosis genes (e.g., IAP, TXNIP, caspase, and caspase-3) and the hypoxia-related genes (e.g., hsp70, hif-1α, and HcY) were all dramatically induced in the hepatopancreas and gills of M. japonicus. Heart rates, T-AOC (total antioxidant capacity) and lactate contents showed time-dependent changes upon air exposure. Air exposure significantly induced apoptosis in hepatopancreas and gills. Compared with the control group, the apoptosis index (AI) of the 12.5 h experimental group increased significantly (p < 0.05) in the hepatopancreas and gills. Most individuals in the experimental group (EG, 12.5 h) had lower methylation ratios than the control group (CG). Air exposure markedly reduced the full-methylation and total-methylation ratios (31.39% for the CG and 26.46% for the EG). This study provided a comprehensive understanding of the antioxidant responses of M. japonicus considering its physiology, innate immunity, apoptosis, and DNA methylation levels, and provided theoretical guidance for waterless transportation.

Fine-Scale Population Genetic Structure and Parapatric Cryptic Species of Kuruma Shrimp (Marsupenaeus japonicus), Along the Northwestern Pacific Coast of China.

The kuruma shrimp (Marsupenaeus japonicus) includes two cryptic species, which are distributed mostly allopatrically but co-occur in the northern South China Sea (from Huilai to Beihai). To obtain a better understanding of the fine-scale genetic structure and parapatric diversification of these two varieties in the northwestern Pacific region, we used a genotyping-by-sequencing (GBS) and comparative transcriptomics approach to establish their phylogenetic relationships. Using the GBS technique, we genotyped 28891 SNPs in 160 individuals in the Northwest Pacific. The results supported two highly diverged evolutionary lineages of kuruma shrimp (var. I and II). The ND and XM populations showed complex genetic patterns, which might be affected by the complex environment of the Taiwan Strait. In addition, the migration rates and inbreeding coefficients of XM and BH were much lower than those of the other populations, which might be related to the land-sea changes and complex ocean currents in the Taiwan Strait and Qiongzhou Strait. Based on the synonymous substitution rates (ds) of 2,491 candidate orthologs, we estimated that the divergence time between the two varieties was 0.26~0.69 Mya. Choice and no-choice interbreeding experiments provided support for the biological species concept, by showing the existence of reproductive isolation or incompatibility. In view of these differences between the two Marsupenaeus species, we believe that it is essential and urgent to establish a genetic database for each and reevaluate their ecological suitable conditions in order to improve species-specific culturing techniques. Moreover, this research can serve as a case study for future research on speciation and hybridization.

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.

Cross talk between heat shock protein 10 and a heat shock factor identified from Marsupenaeus japonicus.

Heat shock factors (HSFs) and heat shock proteins (HSPs) are crucial regulators and effectors of the heat shock response (HSR). In this study, the full-length cDNA sequences of MjHSP10 and MjHSF1 were cloned by rapid amplification of cDNA ends (RACE). The deduced MjHSP10 and MjHSF1 amino acid (aa) sequences exhibited conserved structures and the functional features of HSP10 and HSF1, respectively. The tissue distributions and mRNA expression profiles of the two genes in response to heat stress were analyzed by quantitative real-time PCR (qRT-PCR). MjHSP10 and MjHSF1 were ubiquitously expressed in various tissues. Heat stress induced a significant increase in MjHSP10 expression that tend to positively correlate with temperature. Additionally, MjHSF1 transcription was up-regulated less than MjHSP10 transcription under heat stress. MjHSF1 expression in the hepatopancreas was up-regulated under only long-term (48 h) heat stress, and MjHSF1 transcription in the gill increased under only acute (34 °C) heat stress. MjHSF1 knockdown by RNA interference (RNAi) down-regulated MjHSP10 expression. Glutathione-S-transferase (GST) pull-down assays showed an interaction between MjHSP10 and the DNA-binding domain (DBD) of MjHSF1. This study provided new insights into cross talk between HSP10 and HSF1 in Marsupenaeus japonicus.

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.